WO2021211631A2

WO2021211631A2 - Single molecule n-terminal sequencing using electrical signals

Info

Publication number: WO2021211631A2
Application number: PCT/US2021/027155
Authority: WO
Inventors: Tal SOMEKH; Zachary Booth SIMPSON
Original assignee: Erisyon Inc.
Priority date: 2020-04-13
Filing date: 2021-04-13
Publication date: 2021-10-21
Also published as: WO2021211631A3; GB2610078A; GB202215304D0

Abstract

The present disclosure provides methods and systems for polypeptide (e.g., protein) sequencing or identification using field effect transistor (FET) arrays.

Description

SINGLE MOLECULE N-TERMINAL SEQUENCING USING ELECTRICAL SIGNALS

CROSS REFERENCE

[0001] The present application claims priority to and benefit from U.S. Provisional Application No 63/009,240, filed April 13, 2020, the entire contents of which are herein incorporated by reference.

BACKGROUND

[0002] Proteomics, the large-scale study of proteins in biological systems, can provide important insights into the biology and biochemistry of an organism . Proteomics has been applied to a variety of areas with clinical and biochemical interest, such as, for example pathogenesis, development, prevention, and treatment of a wide range of diseases. Protein identification can be used for drug development, proteomic discovery and application, and understanding the biology of systems of interest. Proteome quantifications may be performed with, for example, mass spectrometry techniques and optical techniques. For mass spectrometry techniques, proteins may be degraded into a collection of peptides, which may then be charged, separated, and measured based on their mass to charge number ratio. For optical techniques, a fluorescent reporter may be bonded to an amino acid side chain, and, upon cleavage, a light microscope may detect emitted photons.

SUMMARY

[0003] As recognized herein, an increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. Advancements in high-throughput technologies have enabled rapid and parallel sequencing of genomes and transcriptomes. Significant progress has been made improving deoxyribonucleic acid (DNA) sequencing to single-molecule sensitivity using, for example, next generation sequencing technologies, permitting high throughput whole genome measurements of individual cells and tissues. In contrast, proteomic analysis has lagged, bottlenecking cellular characterization. For example, mass spectrometry or optical techniques, both of which may require a prior knowledge of the polypeptide sample, may utilize ensemble measurements of protein sequence information from many cells, masking cell- to-cell variations, or measure abundant proteins in single cell measurements, diminishing the identification of low-copy number proteins. Further, mass spectrometry detection may have a deficient dynamic range, for example, spanning four orders of magnitude while expression levels for a typical mammalian proteome span seven orders of magnitude. [0004] An unbiased protein sequencing method with a dynamic range that covers the full range of protein concentrations in proteomes may allow for improved identification and characterization of gene products and subcellular complexes. Electrochemical sequencing may improve the efficiency of rapid single molecule sequencing in polypeptides.

[0005] Methods and systems of the present disclosure may advance current polypeptide or protein sequencing methods using electrochemical techniques. Methods and systems of the present disclosure may overcome or alleviate at least some of the disadvantages of other polypeptide sequencing methods by increasing sequencing efficiency. This may be used, for example, in cancer diagnostics.

[0006] In some aspects, the present disclosure provides a method for polypeptide sequencing, comprising providing an array having a polypeptide immobilized thereto, wherein the polypeptide is adjacent to a sensor; subjecting the polypeptide to conditions sufficient to remove an amino acid from the polypeptide in a solution; using the sensor to measure a charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of the amino acid from the polypeptide; and using at least the charge, conductivity, or impedance, or change thereof, to identify a sequence of the polypeptide.

[0007] In some embodiments, subjecting the polypeptide to conditions sufficient to remove the amino acid from the polypeptide in the solution comprises subjecting the polypeptide to Edman degradation. In some embodiments, subjecting the polypeptide to conditions sufficient to remove an amino acid from the polypeptide in a solution comprises mixing the polypeptide with a diactivated phosphate or phosphonate to form a reaction mixture, and mixing the reaction mixture with an acid to remove the amino acid. In some embodiments, the diactivated phosphate or phosphonate is a dihalophosphate ester.

[0008] In some embodiments, subjecting the polypeptide to conditions sufficient to remove an amino acid from the polypeptide in a solution and using the sensor to measure a charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of the amino acid from the polypeptide are repeated to measure an additional charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of an additional amino acid from the polypeptide.

[0009] In some embodiments, providing an array having a polypeptide immobilized thereto, wherein the polypeptide is adjacent to a sensor comprises immobilizing another polypeptide to the support, thereby providing the polypeptide to the support. In some embodiments, the polypeptide is derived from a plurality of polypeptides or a protein. In some embodiments, the plurality of polypeptides are provided to the array in a Poisson distribution. In some embodiments the plurality of polypeptides are provided to the array in a super-Poisson distribution.

[0010] In some embodiments, the sensor is a field effect transistor (FET). In some embodiments, the FET is selected from a group consisting of ion-sensitive field effect transistor (ISFET), metal-oxide-semiconductor field effect transistor (MOSFET), enzyme field effect transistor (EnFET), chemically-sensitive field effect transistor (ChemFET), a carbon nanotube field effect transistor (CNFET), immuno-field effect transistor (ImmunoFET), or a biologically sensitive field effect transistor (BioFET). In some embodiments, the FET comprises a floating gate.

[0011] In some embodiments, the floating gate has a size greater than 1 nm² having a trapped charge of less than 240 volts (V). In some embodiments, the FET occupies an area of up to 1 millimeters² (mm²). In some embodiments, the sensor measures the charge or change thereof. In some embodiments, the sensor measures the conductivity or change thereof. In some embodiments, the sensor measures the impedance or change thereof.

[0012] In some embodiments, the array comprises a support wherein the polypeptide is immobilized to the support. In some embodiments, the support is a bead. In some embodiments, the support is a surface of a well. In some embodiments, the well is among a plurality of wells.

In some embodiments, the plurality of wells comprises at least two wells. In some embodiments, the plurality of wells comprises at least 1,000 wells. In some embodiments, the plurality of wells comprises at least 5,000 wells. In some embodiments, the plurality of wells comprises at least 10,000 wells. In some embodiments, the plurality of wells comprises at least 50,000 wells. In some embodiments, the plurality of wells comprises at least 100,000 wells. In some embodiments, the plurality of wells comprises at least 500,000 wells. In some embodiments, the plurality of wells comprises at least 1,000,000 wells. In some embodiments, a well is a microwell (e.g., comprises a volume of less than a mL. In some embodiments, a well is a nanowell (e.g., comprises a volume of less than 1 pL).

[0013] In some embodiments, the polypeptide is coupled to a capture moiety coupled to the array. In some embodiments, the array comprises a plurality of individually addressable sites, and wherein the polypeptide is immobilized to an individually addressable site of the plurality of individually addressable sites. In some embodiments, the polypeptide is covalently coupled to the array. In some embodiments, the polypeptide is ionically coupled to the array.

[0014] In some embodiments, the sensor comprises a carbon nanotube transistor. In some embodiments, the array comprises a plurality of sites wherein the polypeptide is immobilized to a single site of the plurality of sites adjacent to a sensor. [0015] In some embodiments, subjecting the polypeptide to conditions sufficient to remove an amino acid from the polypeptide in a solution and using the sensor to measure a charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of the amino acid from the polypeptide are performed in substantially real time. In some embodiments, the polypeptide is coupled to an engineered side chain coupled to the array. In some embodiments, the engineered side chain comprises a covalent bond between a post translational modification on an amino acid residue of the polypeptide and a labeling reagent. In some embodiments, the engineered side chain comprises an ionic bond between the post translational modification on the amino acid residue of the peptide or protein and a labeling reagent. In some embodiments, the post translational modification on the amino acid residue is phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, methylation, acylation, alkylation, amidation, amination, hydroxylation, carboxylation, decarboxylation, halogenation, nitrosylation, succinylation, sulfation, cyclization, prenylation, flavination, trimethylation, or any combination thereof.

[0016] In some embodiments, the engineered side chain is a spectrally activated side chain and a spectral signature of the attached labelled peptide is recorded. In some embodiments, the spectrally activated side chain is stimulated by FETs. In some embodiments, the stimulation is pulsed. In some embodiments, the stimulation is constant. In some embodiments, the spectrally activated side chain is stimulated by ISFET. In some embodiments, the spectrally activated side chain identifies a unique spectral signature upon stimulation. In some embodiments, the unique spectral signature that occurs from each recording device is analyzed to determine its origin. [0017] In some embodiments, the spectral analysis is used to estimate concentrations of individual species of peptide present in the original mixture. In some embodiments, the entire collection of polypeptides loses one amino acid from the polypeptide to detect an additional signal indicative of a change in spectra subsequent to removal of the additional amino acid from the polypeptide. In some embodiments, the unique spectral signature(s) over all addressable units are collected between peptide degradation cycles.

[0018] In some aspects, the present disclosure provides a method for polypeptide sequencing, comprising: providing an array having a polypeptide immobilized thereto, wherein the polypeptide is adjacent to a sensor, subjecting the polypeptide to conditions sufficient to remove an amino acid from the polypeptide, using the sensor to measure a non-optical signal in the solution subsequent to removal of the amino acid from the polypeptide, and using at least the non-optical signal to identify a sequence of the polypeptide. [0019] In another aspect, the present disclosure provides a method for determining a polypeptide sequence, comprising performing a sequence of reactions without a label, thereby determining the polypeptide sequence.

[0020] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0021] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0022] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS [0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0025] FIG. 1 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.

[0026] FIG. 2 illustrates a cross-section diagram of a field effect transistor (FET) array configured for analyte measurement provided by aspects of the present disclosure. [0027] FIG. 3 illustrates a illustrates a schematic of a peptide processing system comprising a large-scale FET array, according to one inventive embodiment of the present disclosure.

[0028] FIG. 4 illustrates a schematic of a peptide processing system comprising a large-scale optical FET array, according to one inventive embodiment of the present disclosure.

[0029] FIG. 5 illustrates an example of a non-optical method of peptide sequencing provided by aspects of the present disclosure.

[0030] FIG. 6 illustrates an example of an optical method of peptide sequencing provided by aspects of the present disclosure.

DETAILED DESCRIPTION

[0031] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0032] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0033] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0034] The term “analyte” or “analytes,” as used herein, generally refers to a molecule whose presence or absence is measured or identified. An analyte can be a molecule for which a detectable probe or assay exists or can be produced. For example, an analyte can be a macromolecule, such as, for example, a nucleic acid, a polypeptide, a carbohydrate, a small organic, an inorganic compound, or an element, for example, gold, iron, or lead. An analyte can be part of a sample that contains other components, or can be the sole or the major component of the sample. An analyte can be a component of a whole cell or tissue, a cell or tissue extract, a fractionated lysate thereof or a substantially purified molecule. In some embodiments, the target analyte is a polypeptide. [0035] The terms “polypeptide” and “peptide” generally to refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (i.e., amino acid analogue). The polymer can be linear or branched and can include modified amino acids, and/or may be interrupted by non-amino acids. Polypeptides can occur as single chains or associated chains. The polymer may include a plurality of amino acids and may have a secondary and tertiary structure (i.e., protein). In some examples, the polymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 10,000, or more amino acids.

[0036] The term “amino acid,” as used herein, generally refers to a naturally occurring or non- naturally occurring amino acid (amino acid analogue). The non-naturally occurring amino acid may be a synthesized amino acid.

[0037] As used herein, the terms “amino acid sequence,” “peptide sequence,” and “polypeptide sequence,” as used herein, generally refer to at least two amino acids or amino acid analogs that are covalently linked by a peptide (amide) bond or an analog of a peptide bond. The term peptide includes oligomers and polymers of amino acids or amino acid analogs. The amino acids of the peptide may be L-amino acids or D-amino acids. A peptide, polypeptide, or protein may be synthetic, recombinant, or naturally occurring. A synthetic peptide may be a peptide that is produced by artificial approaches in vitro.

[0038] As used herein, the term “side chains” or “R” generally refers to unique structures attached to the alpha carbon (attaching the amine and carboxylic acid groups of the amino acid) that render uniqueness to each type of amino acid. R groups have a variety of shapes, sizes, charges, and reactivities, such as charged polar side chains, either positively or negatively charged, such as lysine (+), arginine (+), histidine (+), aspartate (-), and glutamate (-); amino acids can also be basic, such as lysine, or acidic, such as glutamic acid; uncharged polar side chains have hydroxyl, amide, or thiol groups, such as cysteine having a chemically reactive side chain, i.e., a thiol group that can form bonds with another cysteine, serine (Ser) and threonine (Thr), that have hydroxylic R side chains of different sizes; asparagine (Asn), glutamine (Gin), and tyrosine (Tyr); non-polar hydrophobic amino acid side chains include the amino acid glycine, alanine, valine, leucine, and isoleucine having aliphatic hydrocarbon side chains ranging in size from a methyl group for alanine to isomeric butyl groups for leucine and isoleucine; methionine (Met) has a thiol ether side chain; proline (Pro) has a cyclic pyrrolidine side group. Phenylalanine (with its phenyl moiety) (Phe) and tryptophan (Trp) (with its indole group) contain aromatic side chains, which are characterized by bulk as well as lack of polarity. [0039] The term “cleavable unit,” as used herein, generally refers to a molecule that can be split into at least two molecules. Non-limiting examples of cleavage reagents and conditions to split a cleavable unit include: enzymes, nucleophilic or basic reagents, reducing agents, photo irradiation, electrophilic or acidic reagents, organometallic or metal reagents, and oxidizing reagents.

[0040] The term “organophosphorus compound,” as used herein, generally refers to a molecule that comprises carbon (C) and phosphorous (P) atoms. In some embodiments, the organophosphorus compound contains a carboxylic acid (-COOH) and may be referred to as a “phosphoric acid compound.” The organophosphorus compound may be described generally as

X'

II

, 1 p ?· R

X , where R is hydrogen or a carbon-containing substituent, X and X are each leaving groups, and X’ is O or S.

[0041] The term “sample,” as used herein, generally refers to a sample containing or suspected of containing a polypeptide. For example, a sample can be a biological sample containing one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In some examples, the sample is obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In some examples, the sample can include circulating tumor cells. In some examples, the sample is an environmental sample (e.g., soil, waste, ambient air), industrial sample (e.g., samples from any industrial processes), and food samples (e.g., dairy products, vegetable products, and meat products). The sample may be processed prior to loading into a microfluidic device. For example, the sample may be processed to purify the polypeptides and/or to include reagents.

[0042] As used herein, the term “subset” generally refers to the L -ter inal amino acid residue of an individual peptide molecule. A “subset” of individual peptide molecules with an N- terminal lysine residue is distinguished from a “subset” of individual peptide molecules with an L -ter inal residue that is not lysine.

[0043] As used herein, sequencing of peptides “at the single molecule level” generally refers to amino acid sequence information obtained from individual (i.e., single) peptide molecules in a mixture of diverse peptide molecules. The amino acid sequence information may be obtained from an entirety of an individual peptide molecule or one or more portion of the individual peptide molecule, such as a contiguous amino acid sequence of at least a portion of the individual peptide molecule. Alternatively, partial amino acid sequence information may be obtained, which may allow for identification of the peptide or protein. Partial amino acid sequence information, including for example, the pattern of a specific amino acid residue (i.e., lysine) within individual peptide molecules, may be sufficient to uniquely identify an individual peptide molecule. For example, a pattern of amino acids may comprise a plurality of identified positions (e.g., identified as a particular amino acid type, such as lysine, or identified as a particular set of amino acids, such as the set of carboxylate side chain-containing amino acids), and a plurality of unidentified positions. The sequence of identified positions may be searched against a known proteome of a given organism to identify the individual peptide molecule. In some examples, sequencing of a peptide at the single molecule level may identify a pattern of a certain type of amino acid (e.g., lysine) in an individual peptide molecule. Such information may be used to identify a macromolecule (e.g., protein) from which the peptide was derived.

This may advantageously preclude the need to identify all amino acids of the peptide.

[0044] As used herein, the term “single molecule sensitivity” generally refers to the ability to acquire data (including, for example, amino acid sequence information) from individual peptide molecules in a mixture of diverse peptide molecules. In one non-limiting example, the mixture of diverse peptide molecules may be immobilized on a solid surface (including, for example, a glass slide, or a glass slide whose surface has been chemically modified). This may include the ability to simultaneously record the fluorescent intensity of multiple individual (i.e., single) peptide molecules distributed across the glass surface. Optical devices are commercially available that can be applied in this manner. For example, a conventional microscope equipped with total internal reflection illumination and an intensified charge-couple device (CCD) detector is available. Imaging with a high sensitivity CCD camera allows the instrument to simultaneously record the fluorescent intensity of multiple individual (i.e., single) peptide molecules distributed across a surface. Image collection may be performed using an image splitter that directs light through two band pass filters (one suitable for each fluorescent molecule) to be recorded as two side-by-side images on the CCD surface. Using a motorized microscope stage with automated focus control to image multiple stage positions in the flow cell may allow millions of individual single peptides (or more) to be sequenced in one experiment. [0045] As used herein, the term “Poisson Distribution” provides a spatial, temporal, or topological probability distribution for a set of species or occurrences. In some embodiments, an occurrence is independent of each other occurrence. For example, during protein cleavage or sequencing, the target proteins may be spread across a number of partitions, and the average number of molecules per partition may be estimated using a Poisson distribution. A Poisson distribution may be subjected to a variety of transforms to identify tangible metrics for a system (e.g., molecular concentrations).

[0046] As used herein, the term “electrical signal” may refer to a voltage, current, or electromagnetic wave. An electrical signal may comprise a time resolvable profile (e.g., a frequency) or an amplitude that conveys information about an electrical phenomenon. An electrical signal may be classified into criteria, such as, for example, analog, digital, continuous, discrete, deterministic, random, energy, power, even, odd, or periodic signals.

[0047] As used herein, the term “charge” generally refers to a physical property of matter that causes it to experience a force when placed in an electromagnetic field. Charge (or electric charge) may be positive or negative, and carried by protons and electrons, respectively. A molecule may have a net positive or negative charge. In some embodiments, like charges repel each other and unlike charges attract each other due to their Coulomb force. A movement of charged species may generate a current.

[0048] As used herein, the term “impedance” may denote a measure of resistance against current flow through a material or medium. Impedance may possess a phase and/or a magnitude. Impedance may denote or relate to resistance to alternating and/or direct currents. In a number of aspects provided herein, electrochemical impedance may be used in the detection of biomolecules, such as, for example nucleic acids, polypeptides, or amino acids, where a current (e.g., the frequency or magnitude of a current), voltage, impedance, conductance, inductance, resistance, capacitance, or any combination thereof may be utilized for molecular and supramolecular level detection or identification. For example, the conductance of a semiconductor, oxide, or electrochemical system may be responsive to the proximity and electronic structure of various biomolecules. Modification of a surface (e.g., an oxide surface) through immobilization, adsorption, intercalation, or hybridization of a molecule may cause a change in the charge distribution within (and thus an electrical property of) a electrical device (e.g., a transistor or a semiconductor).

[0049] As used herein, the term “conductivity” generally refers to a material’s ability to carry or transmit an electric current. A conductive material may provide a low resistance to the flow of an electric current. Conductivity may be used in the detection of biomolecules such as, for example, nucleic acid, polypeptides, or amino acids. The conductivity of a biomolecule may relate to the time it takes the biomolecule to move through a conductive channel or past a conductive material. The conductivity of a biomolecule may relate to the strength of the biomolecule’s adherence to a conductive material. A change in conductivity may comprise a proportionality to a charge of a molecule adjacent or adhering to a conductive material. [0050] As used herein, the term “Edman degradation” generally refers to methods comprising chemical removal of amino acids from peptides or proteins. In some cases, Edman degradation denotes terminal (e.g., N- or C-terminal) amino acid removal. In specific cases, Edman degradation refers to N-terminal amino acid removal through isothiocyanate (e.g., phenyl isothiocyanate) coupling and cyclization with the terminal amine group of an N-terminal residue, such that the N-terminal amino acid is removed from a peptide. In some cases, Edman degradation broadly encompasses N-terminal amino acid functionalizations leading to N- terminal amino acid removal. In some cases, Edman degradation encompasses C-terminal amino acid removal. In some cases, Edman degradation comprises terminal amino acid functionalization (e.g., N-terminal amino acid isothiocyanate functionalization) followed by enzymatic removal (e.g., by an ‘Edmanase’ with specificity for chemically derivatized N- terminal amino acids).

[0051] As used herein, the term “organophospho-degradation” generally refers to peptide cleavage by an organophosphate compound. In some embodiments, organophospho-degradation is an alternative to Edman degradation. An organophospho-degradation reaction may cleave the N-terminal amino acid of a peptide, yielding a new N-terminus that is compatible to another cycle of degradation.

[0052] As used herein, the term “array” generally refers to a plurality of sites with defined locations. In some cases, an array refers to a plurality of sites on a single surface. In some cases, an array refers to a plurality of sites on a plurality of surfaces. In some cases, an array refers to a plurality of sites within a 3D space (e.g., interstitial spaces within a lattice or locations within a polymer matrix). In some cases, two sites of an array can be differentiated from each other according to relative location. Accordingly, in many cases different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single polypeptide having a particular sequence or a site can include several polypeptides having identical or different sequences. The sites of an array can be different features located on a single substrate. In an example, features include, without limitation, wells or channels in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be disposed on or within separate substrates each bearing a different molecule.

[0053] The term “field effect transistor” (FET), as used herein, generally refers to a voltage- responsive or electric field-responsive transistor. A FET may comprise a ‘gate’, which may mediate current from a ‘source’ to a ‘drain’ of the FET. In many cases, a gate may comprise an el ectric field-dependent or a voltage-dependent conductivity. In many cases, a FET comprises an ability to change a gate conductivity by applying a voltage. In many cases, current through a gate may also be responsive to local environment or field effects. For example, a biomolecule bound or positioned adjacent to a FET may increase or decrease the conductance of a gate of the FET, thereby generating a detectable signal. A FET may comprise a metal-oxide-semiconductor transistor, which may be an insulated gate field effect transistor fabricated by the controlled oxidation of a semiconductor, such as, for example, silicon.

[0054] As used herein, the term “ion-sensitive field effect transistor” (ISFET)” or “pH field effect transistor” (pHFET), generally refers to field effect transistors that are sensitive to, and therefore able to measure, ion concentration or activity in a solution (e.g., hydrogen ions in the solution are the “analyte”).

[0055] As used herein, the term “floating gate” generally refers to a gate of a transistor that is electrically isolated form other elements of the transistor. The floating gate may be a gate of a semiconducting device having at least one dielectric layer, fabricated and assembled in a manner that may limit process-induced trapped charge to the point where its presence may not cause threshold voltages of transistors in an array in said device to vary beyond a nominal amount. [0056] As used herein, the term “carbon nanotube transistor” generally refers to a field effect transistor that utilizes a single carbon nanotube or an array of carbon nanotubes as a conductive material (e.g., as a gate material) in a FET structure. Carbon nanotube transistors can have high conductance relative to silicon transistors, and they may be useful in applications where a relatively high current may need to flow through a relatively small area.

[0057] As used herein, the term “spectral signature” generally refers to a variation of reflectance or emittance of a material with respect to wavelengths. The spectral signature of an object is a function of the incidental electromagnetic wavelength and material interaction with that section of the electromagnetic spectrum. In one example, two surfaces can be differentiated from each other as radiation from a source may reflect radiation as a function of the wavelength differently in various channels off of these surfaces.

[0058] Proteomics can denote the large-scale study of the peptides and proteins produced by an organism or system in a specific biological context or under a specific set of conditions. Proteins are quintessential components of organisms, carrying out a wide array of functions requisite for life. Accordingly, identifying a population of proteins from an organism or population of organisms can provide a wealth of information regarding the activity, behavior, and health of the organism or organisms. Peptide sequencing has the potential to enable a wide variety of proteomic endeavors. Given the density of a protein in size, amount, and information, a stopgap in polypeptide sequencing may be the throughput of polypeptide sequencing techniques.

[0059] Electrosequencing can provide a method for increasing throughput, allowing the rapid sequencing of a polypeptide without optical labelling. The present disclosure aims to overcome deficiencies in, for example, mass spectroscopy and optical peptide sequencing techniques by increasing efficiency in detecting and measuring one or more analytes via electronic sensors. [0060] The present disclosure provides methods and systems for peptide (e.g., protein) sequencing. Methods of the present disclosure may permit a peptide (e.g., protein) to be sequenced in a manner that provides various non-limiting benefits, such as, for example, (i) sequencing a protein or a peptide comprising a chemically modified N-terminal amino acid (e.g., ADP-ribosylation, fluorophores, etc.), (ii) sequencing a protein or peptide comprising a non natural or non-proteinogenic amino acid residue (e.g., b-amino acid, peptoid, PNA, etc.), (iii) identifying a position of a disulfide bridge in a peptide or protein, or (iv) sequencing a protein or peptide, even at concentrations of less than or equal to about 1 picomolar (pM). Peptide sequencing may be used to reveal novel biomarkers for the diagnosis of cancer and other diseases or in understanding the function of healthy cells. Peptide sequencing may also be used to identify or quantify a peptide (e.g., a protein) or a plurality of peptides from a biological sample. Peptides produced by cells or tissues may act as unique biomarkers. Enhanced detection of these biomarkers through polypeptide sequencing may provide earlier, more accurate diagnoses of disease.

[0061] An aspect of the present disclosure provides a method for polypeptide sequencing, comprising (i) providing an array having a polypeptide immobilized to it, where the polypeptide is adjacent to a sensor, (ii) subjecting the sensor to conditions sufficient to remove an amino acid from the polypeptide, (iii) using the sensor to measure a charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of the amino acid from the polypeptide, and (iv) using at least the charge, conductivity, or impedance, or change thereof, to identify a sequence of the polypeptide.

[0062] A peptide may be obtained from a biological sample. The biological sample may be obtained from an animal, a microorganism, a plant, or any derivative thereof. The animal may be, for example, a mammal, such as, for example, a human. The biological sample may comprise a cell-free sample. A cell-free sample may be a sample which is free of cells, substantially free of cells, or essentially free of cells. A cell-free biological sample may include a protein, a peptide, an amino acid, a nucleic acid molecule (e.g., ribonucleic acid molecule and/or deoxyribonucleic acid molecule), or a plurality thereof. While a sample may be denoted as cell-free, the sample may contain a small number of cells or cell debris while still being considered cell-free.

[0063] A peptide may comprise a peptide-conjugate, a peptoid-conjugate, a protein-conjugate, or any combination thereof. A peptide may comprise an a-peptide, a b-peptide, a peptide aptamer, a peptoid residue, a peptide nucleic acid (PNA), or any combination thereof. The peptide may comprise a plurality of peptides. A peptide may be a peptidomimetic. A peptide subunit of the plurality of polypeptides may comprise an a-amino acid, a b-amino acid, a protein, a peptide nucleic acid (PNA), a peptoid residue, or any combination thereof. The peptide may be derived from a plurality of polypeptides or a protein.

[0064] A peptide may comprise a natural amino acid sub-unit. A peptide may comprise an unnatural amino acid sub-unit. The peptide may be produced from a mRNA template. The peptide may be produced naturally, synthetically, or a combination thereof. For example, the peptide may be purified from a wild-type or recombinant organism and then subjected to in vitro chemical modification. The amino acid sub-units of the peptide may be proteinogenic amino acids or non-proteinogenic amino acids (e.g., post-translationally modified amino acids, carnitine, levothyroxine, etc.). The proteinogenic amino acids may be, for example, lysine, cystine, selenocysteine, pyrrolysine, glycine, glutamic acid, tryptophan, alanine, arginine, asparagine, aspartic acid, glutamine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, or valine. The a-amino acid may comprise natural amino acids. A b-peptide may be comprised of at least one b-amino acid (e.g., b-alanine). A peptoid residue may be N-substituted glycine. A PNA may be comprise repeating N-(2-aminoethyl)- glycine units linked by peptide bonds. A terminal amide residue of a polymer may be linked to the polymer by a carboxyl terminus.

[0065] The peptide may have a concentration of at least about 0.001 nanograms (ng)/microliter (pL), 0.005 ng/pL, 0.01 ng/pL, 0.05 ng/pL, 0.1 ng/pL, 0.2 ng/pL, 0.3 ng/pL, 0.4 ng/pL, 0.5 ng/pL, 0.6 ng/pL, 0.7 ng/pL, 0.8 ng/pL, 0.9 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 4 ng/pL, 5 ng/pL, 6 ng/pL, 7 ng/pL, 8 ng/pL, 9 ng/pL, 10 ng/pL, 11 ng/pL, 12 ng/pL, 13 ng/pL, 14 ng/pL, 15 ng/pL, 16 ng/pL, 17 ng/pL, 18 ng/pL, 19 ng/pL, 20 ng/pL, 25 ng/pL, 30 ng/pL, 35 ng/pL, 40 ng/pL, 45 ng/pL, 50 ng/pL, 55 ng/pL, 60 ng/pL, 65 ng/pL, 70 ng/pL, 80 ng/pL, 85 ng/pL, 90 ng/pL, 95 ng/pL, 100 ng/pL, 110 ng/pL, 120 ng/pL, 130 ng/pL, 140 ng/pL, 150 ng/pL, 160ng/pL, 170ng/pL, 180 ng/pL, 190 ng/pL, 200 ng/pL, 225 ng/pL, 250 ng/pL, 275 ng/pL, 300 ng/pL, 350 ng/pL, 400 ng/pL, 450 ng/pL, 500 ng/pL, 550 ng/pL, 600 ng/pL, 700 ng/pL, 800 ng/pL, 900 ng/pL, 1,000 ng/pL, or more nanograms per microliter in a sample. The peptide may have a concentration of at most about 1,000 ng/pL, 900 ng/pL, 800 ng/pL, 700 ng/pL, 600 ng/mL, 550 ng/mL, 500 ng/mL, 450 ng/gL, 400 ng/gL, 350 ng/gL, 300 ng/gL, 275 ng/gL, 250 ng/gL, 225 ng/gL, 200 ng/gL, 190 ng/gL, 180 ng/gL, 170 ng/gL, 160 ng/gL, 150 ng/gL, 140 ng/gL, 130 ng/gL, 120 ng/gL, 110 (ng/gL, 100 ng/gL, 95 ng/gL, 90 ng/gL, 85 ng/gL, 80 ng/gL, 75 ng/gL, 70 ng/gL, 65 ng/gL, 60 ng/gL, 55 ng/gL, 50 ng/gL, 45 ng/gL, 40 ng/gL, 35 ng/gL, 30 ng/gL, 25 ng/gL, 20 ng/gL, 19 ng/gL, 18 ng/gL, 17 ng/gL, 16 ng/gL, 15 ng/gL, 14 ng/gL,

13 (ng/gL, 12 ng/gL, 11 ng/gL, 10 ng/gL, 9 ng/gL, 8 ng/gL, 7 ng/gL, 6 ng/gL, 5 ng/gL, 4 ng/gL, 3 ng/gL, 2 ng/gL, 1 ng/gL, 0.9 ng/gL, 0.8 ng/gL, 0.7 ng/gL, 0.6 ng/gL, 0.5 ng/gL, 0.4 ng/gL, 0.3 ng/gL, 0.2 ng/gL, 0.1 ng/gL, 0.05 ng/gL, 0.01 ng/gL, 0.005 ng/gL, 0.001 ng/gL, or less nanograms per microliter in a sample. The peptide may have a concentration range as defined by any two of the previous values. For example, the peptide may have a concentration from 0.4 nanograms per microliter to 4 nanograms per microliter in a sample.

[0066] A power of the present methods lies not only in elucidating peptide structures and sequences, but also in identifying secondary, tertiary, and quaternary peptide structural features. In many instances, the present methods may identify protein and peptide complexes, aggregates, and multimeric structures. A protein or peptide complex may comprise a plurality of protein or peptide sub-units. The protein or peptide complex may comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 20, at least about 25, or at least about 30 protein or peptide subunits. A method of the present disclosure may identify a protein or peptide complex by identifying a subunit of the protein or peptide complex. A method of the present disclosure may distinguish between multiple forms or conformers of a multi subunit protein or peptide complex. A method of the present disclosure may identify a biological or disease state by identifying or quantifying a protein or peptide complex in a biological sample. For example, monomer-dimer ratios among the interleukin-1 cytokine family (e.g., interleukin- 37) may evidence innate immune system suppression, and thus be used as a marker for a variety of disease state progressions. .

[0067] A peptide may comprise a plurality of amine residues. The peptide may comprise a terminal amine residue and an internal amine residue. The peptide may comprise a terminal amine residue and a plurality of internal amine residues. The peptide may comprise an N- terminal amine residue, a C-terminal amine residue, and an internal amine residue. The peptide may comprise an N-terminal amine residue, a C-terminal amine residue (e.g., a C-terminal lysine), and a plurality of internal amine residues. The peptide may comprise an amine residue coupled directly to the terminal amine residue. The peptide may comprise an internal amine residue coupled directly to a terminal amine residue. The peptide may comprise an internal amine residue coupled directly to the N-terminal amine residue. Upon cleavage of the terminal amine residue of the peptide, the internal amine residue that was previously coupled directly to the terminal amine residue may be the next terminal amine residue of the peptide. The internal amino acid residue coupled directly to the terminal amine residue may become the next terminal amine residue after the terminal amine residue is cleaved. The internal amino acid residue coupled directly to the terminal amine residue may become the next terminal amine residue after the terminal amine residue is cleaved until the next amine residue is the C-terminal amine residue.

ARRAYS

[0068] Methods of sequencing or analyzing a peptide may comprise immobilizing the peptide on a support. The peptide may be immobilized using an amino acid that has a reactive moiety such as, for example, a cysteine residue, a lysine, the N-terminus, or the C-terminus. In some embodiments, the peptide is immobilized by reacting the cysteine residue with the support. A peptide may be immobilized to an array comprising a support or a plurality of supports. A peptide may be disposed adjacent to a sensor on the array. A peptide may be immobilized to a support through chemical cross-linking with glutaraldehyde (or a similar reagent), spontaneous adsorption (physical adsorption), by coupling to reactive groups on a surface (for example amine-reactive groups), by forming a protein monolayer, using a Langmuir-Blodgett technique, in which molecules are mechanically forced together by compression on the surface of water and then transferred onto a support, with biotinylation, with fusion peptides, with adhesion peptides, through an antigen, with hydrophobins, through covalent interactions such as the reaction of lysine side-chain amino groups with DITC-glass, by carbodiimide-mediated reaction of carboxyl groups with AP -glass, or by reaction of homoserine lactone groups with AEAP-glass, through noncovalent attachments, or through the use of an electric field. A peptide can be immobilized while a peptide is being cleaved or sequenced, and then the excess peptide can be washed away. A peptide may be coupled to disposed within a pore (e.g., a protein pore complex) disposed within or adjacent to said support. A plurality of peptides coupled to a support (e.g., a surface of a glass slide or a bead) may herein be referred to as an array.

[0069] A substrate or support may space a molecule from a FET by a defined distance. A FET may be disposed on or within a substrate. A FET may be disposed on an opposite side of a substrate from a sample (e.g., positioned below a bottom surface of a well comprising a liquid biological sample). A substrate may space a FET by at most 50 nm, at most 100 nm, at most 200 nm, at most 300 nm, at most 500 nm, at most 800 nm, at most 1000 nm, at most 1500 nm, at most 2000 nm, at most 4000 nm, at most 5000 nm, at most 8000 nm, at most 10000 nm, at most 12000 nm, at most 15000 nm, at most 20000 nm, at most 25000 nm, at most 30000 nm, at most 50000 nm, or at most 100000 nm from an immobilized biomolecule or a biological sample. A substrate may space a FET by at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 500 nm, at least 800 nm, at least 1000 nm, at least 1500 nm, at least 2000 nm, at least 4000 nm, at least 5000 nm, at least 8000 nm, at least 10000 nm, at least 12000 nm, at least 15000 nm, at least 20000 nm, at least 25000 nm, at least 30000 nm, at least 50000 nm, or at least 100000 nm from an immobilized biomolecule or a biological sample.

[0070] An array may comprise proteins or peptides bound to a planar surface in a random or in an organized and predetermined manner. A protein array may comprise a plurality of different proteins (or other amino acid containing biological moieties). Each protein or peptide, or a plurality thereof, may be present in a predetermined region or “cell” of the array. The regions (or cells) may be aligned with sensors in a sensor array such that there is one sensor for each region. The plurality of proteins in a single region may vary depending on the size of the protein and the size of the region and may be but is not limited to at least 2, 10, 50, 100, 500, 10³, 10⁴ or more proteins. The array itself may have any number of cells, including but not limited to at least 2, 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or more cells. For example, the array can be exposed to a sample that contains or is suspected of containing an analyte that binds to the protein. The analyte may be a molecule that binds to the protein, including, for example, another protein or peptide, a nucleic acid, a chemical species (whether synthetic or naturally occurring), and the like. The protein array may comprise a plurality of identical proteins (or other amino acid containing biological moieties). The plurality of proteins may be uniformly distributed on a planar surface or they may be organized into discrete regions on the surface. The regions can be aligned with the sensors in the sensor array such that there is one sensor for each region.

PEPTIDE DEGRADATION

[0071] Chemical techniques that allow for the mild and sequential protein degradation conditions can be important for proteomics. Degradation can be used as a method to sequence polymers (e.g., proteins or peptides) to determine the order and identity of the amino acids of a polymer. A peptide or protein may be subsequently subjected to additional cleavage conditions until the sequence of at least a portion of the peptide or protein is identified. The entire sequence of a peptide or a protein may be determined using the methods and compositions described herein. Removal of each amino acid residue may be carried out through a variety of techniques including, for example, Edman degradation, organophosphate degradation, or proteolytic cleavage. In some aspects, Edman degradation may be used to remove a terminal amino acid residue. These terminal amino acid residues may be removed from either the C-terminus or the N-terminus of the peptide chain. In some instances, the amino acid residue at the N-terminus of the peptide chain may be removed. A chemical or enzymatic technique for removing a terminal amino acid may remove a defined number of (e.g., exactly one) amino acid. Accordingly, a method for analyzing a peptide may comprise successive degradation and analysis steps, such that the removal of a defined number of amino acids from an N-terminus or C-terminus per step provides position and sequence specific amino acid identifications during analysis. A chemical or enzymatic technique for removing a terminal amino acid may cleave a peptide at a defined location (e.g., only in between two alanine residues).

[0072] An Edman degradation method may comprise chemically functionalizing a peptide N- terminus or C-terminus (e.g., to form a thiourea or a guanidinium derivative of an N-terminal amine), and then contacting the functionalized terminal amino acid with a reagent (e.g., a hydrazine), a condition (e.g., a high or low pH or temperature), or an enzyme (e.g., an Edmanase with specificity for the functionalized terminal amino acid) to remove the functionalized terminal amino acid.

[0073] A diactivated phosphate or phosphonate may be used for peptide cleavage. Such a method may utilize an acid to remove a functionalized amino acid. The diactivated phosphate or phosphonate may be a dihalophosphate ester. In other embodiments, the techniques involve using an enzyme to remove the terminal amino acid residue, such as, for example, an exopeptidase or an Edmanase. For example, a method may comprise derivatizing an N-terminal amino acid of a peptide with a diactivated phosphate, and contacting the peptide with an Edmanase with cleavage activity toward phosphate-functionalized N-terminal amino acids.

[0074] Peptide cleavage conditions may be achieved with a solvent. The solvent may be an aqueous solvent, organic solvent, or a combination thereof. The solvent may be a mixture of solvents. The solvent may be an organic solvent. The organic solvent may be anhydrous. The solvent may be a non-polar solvent (e.g., hexane, dichloromethane (DCM), diethyl ether, etc.), a polar aprotic solvent (e.g., tetrahydrofuran (THF), ethyl acetate, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), etc.), or a polar protic solvent (e.g., isopropanol (IP A), ethanol, methanol, acetic acid, water, etc.). The solvent may be a polar aprotic solvent. The solvent may be DMF. The solvent may be a Ci-Cohaloalkane. The Ci- Ciihaloalkane may be DCM. The solvent may be a mixture of two or more solvents. The mixture of two or more solvents may be a mixture of a polar aprotic solvent and a Ci-Cohaloalkane. The mixture of two or more solvents may be a mixture of DMF and DCM. The mixture of solvents may be any combination thereof. [0075] A degradation process may comprise a plurality of steps. For example, a method may comprise an initial step for derivatizing a terminal amino acid of a peptide, and a subsequent step for cleaving the derivatized terminal amino acid from the peptide. One such method comprises organophosphorus compound-mediated N-terminal functionalization and removal, and thus provides an alternative to the isothiocyanate (e.g., phenyl isothiocyanate) based processes of some Edman degradation schemes.

[0076] An organophosphate-based degradation scheme may comprise dissolving the peptide in an organic solvent or organic solvent mixture (e.g., a mixture of dichloromethane and dimethylformamide) in the presence of an organic base (e.g., triethylamine, N, N- diisopropylethylamine (DIPEA), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine, 1,5- diazabicyclo(4.3.0)non-5-ene, 2,6-di-tert-butylpyridine, imidazole, histidine, sodium carbonate, etc.). The peptide may then be contacted with at least one organophosphorus compound. The cleavage of the peptide or protein N-terminus may be initiated through the addition of a weak acid (e.g., formic acid in water). The cleavage of the peptide or protein N-terminus may also be initiated with water. The resulting products may include the terminal amino acid of the peptide or protein released from the peptide as a phosphoramide and the peptide or protein that is shortened by the terminal amino acid residue, which comprises a free N-terminus that can be used to perform a subsequent cleavage reaction.

[0077] The reaction mixture may comprise a stoichiometric or an excess concentration of the cleavage compound (e.g., relative to the concentration of peptides to be cleaved). The reaction mixture may comprise at least about 0.001% v/v, about 0.01% v/v, about 0.1% v/v, about 1% v/v, about 5% v/v, about 10% v/v, about 15% v/v, about 20% v/v, about 30% v/v, about 40% v/v, about 50% v/v, or more of the cleavage compound. The reaction mixture may comprise at most about 50% v/v, about 40% v/v, about 30% v/v, about 20% v/v, about 15% v/v, about 10% v/v, about 5% v/v, about 1% v/v, about 0.1% v/v, about 0.01% v/v, about 0.001% v/v, or less of the cleavage compound. The reaction mixture may comprise from about 0.1% v/v to about 20% v/v, about 0.5% v/v to about 10% v/v, or about 1% v/v to about 10% v/v of the cleavage compound. The reaction mixture may comprise about 5% v/v of the cleavage compound.

[0078] The reaction may be performed at a temperature of at least about 0 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 40 °C, about 50 °C, about 60 °C, about 70 °C, or more. The reaction may be performed at a temperature of at most about 70 °C, about 60 °C, about 50 °C, about 40 °C, about 30 °C, about 25 °C, about 20 °C, about 15 °C, about 10 °C, about 5 °C, about 0 °C, or less. The reaction may be performed at a temperature from about 0 °C to about 70 °C, about 10 °C to about 50 °C, about 20 °C to about 40 °C, or about 20 °C to about 30 °C. The reaction may be performed at a temperature above room temperature (e.g., about 22 °C to about 27 °C). The reaction may be performed at room temperature.

[0079] The peptide and the cleavage compound may be mixed or incubated for at least about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, or more. The peptide and the cleavage compound may be mixed or incubated for at most about 24 hours, about 20 hours, about 16 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, about 1 minute, or less. The peptide and the cleavage compound may be mixed or incubated from about 1 minute to about 24 hours, 5 minutes to about 6 hours, 5 minutes to about 2 hours, or 5 minutes to about 30 minutes.

Electrosequencing

[0080] A peptide may be subjected to conditions sufficient to remove an amino acid from a peptide and a sensor may measure a charge, conductivity, or impedance, or change thereof, in a solution subsequent to removal of said amino acid from a peptide. The removal of amino acids from a peptide may be repeated to measure additional charge, conductivity, impedance, or change thereof, in a solution subsequent to removal of an additional amino acid from a peptide.

A peptide may be in sensory communication with a sensor. A sensor may be configured to detect a signal indicative of a reaction associated with a peptide, peptide, or amino acid. The sensor may be an optical sensor, an electrical sensor, an ion sensor (e.g., a pH sensor), or any combination thereof. The sensor may comprise an electrode. The electrode may be a metal electrode (e.g., gold, copper, an alloy), a semiconductor electrode (e.g., silicon, gallium arsenide, an organic semiconductor), or a combination thereof. The sensor may comprise a plurality of electrodes. The plurality of electrodes may comprise at least about 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 1,000,000, or more electrodes. The plurality of electrodes may comprise at most about 1,000,000, 750,000, 500,000, 250,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, or less electrodes. The sensor may be among an array of sensors. The array of sensors may comprise sensors of one or more types. For example, an array of sensor may comprise an optical sensor and an electrical sensor. The sensors of the array of sensors may be individually addressable. For example, each electrode of an array of 1,000,000 electrodes can be measured independently of each other electrode. An electronic sensor may detect a change in electrical charge, current, impedance, or conductivity of one or more electrodes and use these electric differences to identify specific peptides, amino acids, amino acid subunits, or signifiers of the peptide of interest during sequencing.

[0081] An electric field may be applied to the array of peptides. The electric field may be at least about 0.001 volts (V), 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, or more. The electric field may be at most about 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V,

0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, 0.001 V, or less volts. The electric field may be applied through a metal electrode (e.g., gold, platinum, copper, silver), a semiconductor electrode (e.g., silicon, gallium arsenide), an organic semiconductor electrode (e.g., poly(3,4- ethylenedioxythiophene)-polystyrene sulfonate (PDOT:PSS), fullerene doped polymers), or any combination thereof. The electric field may be applied over a distance of at least about 0.1 micrometers (pm), 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm,

400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers. The electric field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm,

150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm,

5 pm, 1 pm, 0.1 pm, or less micrometers. For example, a pair of gold electrodes 100 pm apart can be used to apply a 0.5 V potential to the array. A magnetic field may be applied to the array. The magnetic field may be at least about 1 x 10⁶ Tesla (IE-6 T, IE-5 T, IE-4 T, IE-3 T, IE-2 T, IE-1 T, 1E0 T, 1E1 T, or more. The magnetic field may be at most about 10 T, 1 T, 10 ¹ T, 10² T, 10³ T, 10⁴, 10⁵ T, 10⁶ T, or less. The magnetic field may be applied using a permanent magnet (e.g., a Samarium Cobalt magnet, a Neodymium Iron Boron magnet), a superconducting magnet (e.g., a niobium-titanium superconducting magnet in liquid helium), or an electromagnet (e.g., a solenoid). The magnetic field may be applied over a distance of at least about 0.1 micrometers (pm), 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm,

400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers. The magnetic field may be applied over a distance of at most about 1,000 pm, 900 pm, 800 pm, 700 mih, 600 mih, 500 mih, 400 mih, 300 mih, 250 mih, 200 mih, 190 mih, 180 mih, 170 mih, 160 mih, 150 mih, 140 mih, 130 mih, 120 mih, 110 mih, 100 mih, 95 mih, 90 mih, 85 mih, 80 mih, 75 mih, 70 mih, 65 mih, 60 mih, 55 mih, 50 mih, 45 mih, 40 mih, 35 mih, 30 mih, 25 mih, 20 mih, 15 mih, 10 mih, 5 mih, 1 mih, 0.1 mih, or less micrometers. A magnetic field may comprise a substantially homogeneous strength and direction over an area comprising a sample. For example, a solenoid coil can be placed 500 pm behind the array and used to apply a substantially uniform 0.3 Tesla magnetic field over a volume of the array occupied by a sample.

[0082] Peptides partitioned to individual sites on an array may be targeted to the sites through dilution and statistical probability predicted by a Poisson distribution, which may describe patterns of low particle numbers in a volume. During cleavage or sequencing, a sample may be divided into multiple independent partitions such that each partition contains a small amount of peptides or contains no peptides. These partitions may act as an individual microreactors containing peptide sequences able to be detected in them and thus determining the concentration of the peptide in the sample. In the amplification of large peptides, statistical variance may not follow a Poisson distribution. In some instances, the fraction of sites in an array can exceed the fraction predicted by the Poisson distribution resulting in a super-Poisson distribution where there is more variance with the same mean as a Poisson distribution.

[0083] One aspect of the disclosure may encompass providing a solid support, contacting the solid support to a partitioned peptide (e.g., immobilizing or disposing the peptide adjacent to the support), thereby forming a contacted solid support, associating the contacted solid support with a field effect transistor (FET) or a FET array and measuring an electrical property of the FET or the FET array, thereby detecting the peptide or a property of the peptide (e.g., detecting the presence of a chemical label coupled to the peptide or detecting the presence of a disulfide bond of the peptide). FETs may have sequencing and sensing advantages in certain dimensions, responses, and integrations into arrays. A FET may comprise a gate, a drain, and a source. A voltage applied between the gate and the source terminals may modulate the current between the source and drain terminals. A small change in the gate voltage can cause variation in the current from the source to the drain. A field-effect transistor may be gated by changes in the surface potential induced by the binding or proximity of a biomolecule (e.g., a peptide). Upon said binding or said proximity of said biomolecule (e.g., said peptide) to said FET gate, a charge redistribution of the underlying material may result in a change in FET conductance, which may be measured to detect the presence of the biomolecule, determine the identity of the biomolecule, or identify a feature of the biomolecule. A FET may couple a transistor device, such as a semiconducting field effect transistor that acts as a transducer separated from the sample by an insulator layer. Upon binding of the peptide to the recognition element, a charge distribution at the surface changes with a corresponding change in the electrostatic surface potential of the semiconductor. This change in the surface potential of the semiconductor acts like a gate voltage, changing the amount of current that can flow between the source and drain electrodes. This change in current, impedance, or conductance can be measured, thus detecting the association (e.g., binding, covalent coupling, or adsorption) of the peptide with the solid support. The semiconductors may be organic semiconductors (e.g., C₆o, phenyl-C61 -butyric acid methyl ester, poly(3,4-ethylenedioxythiophene)-poly styrene sulfonate, or fullerene doped polymers), inorganic semiconductors (e.g., silicon, cadmium telluride, indium tin oxide, gallium arsenide), or a combination thereof.

[0084] A peptide may be adjacent to a FET. The FETs may be arranged into arrays, such as, for example, an arrangement of locations on a substrate. The locations may be arranged in two- dimensional arrays or three-dimensional arrays. The number of locations can range from several to at least hundreds of thousands. The array pattern and density of locations can vary on the array. The FET may be an ion-sensitive field effect transistor (ISFET). The ISFET may be an array of ISFETs. ISFET arrays may facilitate peptide sequencing techniques based on monitoring changes in current, impedance, or conductivity. An ISFETs may measure the hydrogen ion concentration (i.e., the pH) of a solution. More specifically, an ISFET may be an impedance transformation device that operates in a manner similar to that of a MOSFET (Metal Oxide Semiconductor Field Effect Transistor) and may be particularly configured to selectively measure ion activity in a solution (e.g., hydrogen ion concentration). An ISFET may may measure a change in local ion concentration as a change in capacitance between a source and a drain. A negative voltage may be applied across a gate and a source region to create a “p- channel” at the interface of the region and the gate. This p-channel may extend between a source and a drain, and electric current may be conducted through the p-channel when the gate-source potential VGS is sufficiently negative to attract holes from the source into the channel. The potential at which the channel begins to conduct current may be referred to as the transistor’s threshold voltage VTH (the transistor conducts when VGS has an absolute value greater than the threshold voltage VTH). This may be a source of the charge carriers that flow through the channel. The drain may be where the charge carriers leave the channel. An electric potential difference, or a “surface potential,” may arise at a solid/liquid interface between the FET and a solution. The surface potential may comprise a dependence on an ion concentration of the solution. Thus, two solutions comprising different concentrations of an ion may generate different surface potentials at a FET-solution interface. This surface potential may affect the threshold voltage VTH of the ISFET. Accordingly, the threshold voltage VTH of an ISFET may be sensitive to (and therefore capable of measuring) changes in ion concentration in the analyte solution.

[0085] Some aspects of the disclosure may use an ISFET to detect the number or concentration of ions, such as, for example, hydrogen ions. An ISFET may be configured for sensitivity to static and/or dynamic ion concentration, including but not limited to hydrogen ions. Detecting the number or concentration of ions may be used to detect pH changes. pH change may be detected in an environment with buffering capacity or in an environment with no or limited buffering capacity. An ISFET may detect pH changes on the order of 0.01, .05, 0. 1, 0.2, 0.3, ,4, 0.5, 0.6. 0.7, 0.8, 0.9, 1 pH, or more units. A sensor array may comprise a plurality of ISFETs (e.g., arranged as a two-dimensional array).

[0086] A FET may be a chemically-sensitive field effect transistor (a chemFET). A sensor array may comprise a plurality of chemically-sensitive field effect transistors (chemFETs). A chemFET may be configured to detect the presence or absence of an analyte, a concentration or abundance of the analyte or chemical and/or biological processes (e.g., a pyrophosphate generating process within a defined proximity of the chemFET). A FET or a chemFET may be an enzyme-sensitive field effect transistor (ENFETs), a carbon nanotube field effect transistor (CNFET), an immuno-field effect transistor (ImmunoFET), or a biologically sensitive field effect transistor (BioFET).

[0087] A FET array may be organized in a multidimensional pattern (e.g., a two-dimensional design using rows and columns or a three-dimensional design using rows, columns, and tiers). For example, a FET array may comprise a plurality of rows and columns of pixels, wherein each pixel comprises a FET or a plurality of FETs. Each pixel of a column or row may derive current from a common source and be measured by a common detector. In such a case, an individual pixel may be addressed by selection of a single row and a single column. Conversely, a pixel or a plurality of pixels may comprise a unique current source or detector. Any number of FETs of an array (e.g., all FETs of an array) may be configured to separately measure an analyte in contact with or adjacent to the array. A plurality of FETs array may be located within a plurality of reaction chambers (e.g., a FET or plurality of FETs may be disposed below each of a plurality of reaction chambers).

[0088] An array may comprise a range of pixel densities, for example at least 100 pixels per mm², at least 500 pixels per mm², at least 1000 pixels per mm², at least 5000 pixels per mm², at least 10000 pixels per mm², or at least 50000 pixels per mm². High pixel density may enable highly parallelized and high throughput peptide analysis. As an example, an array pitch of approximately 9 micrometers may allow an ISFET array including over 256,000 pixels (i.e., a 512 by 512 array), together with associated row and column select and bias/readout electronics, to be fabricated on a 7 millimeter by 7 millimeter semiconductor die, and a similar sensor array including over four million pixels (i.e., a 2048 by 2048 array, over 4 Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die. As many cells comprise fewer than 4 million proteins (e.g., an average E. coli cell comprises about 2 million proteins), such an array may be configured to rapidly and comprehensively analyze the proteome of an isolated single cell. Accordingly, a method consistent with the present disclosure may comprise isolating and lysing a cell, optionally separating proteins from other cell lysate components, optionally fragmenting peptides from the cell, and analyzing cell-derived peptides on a FET array.

[0089] One or more microfluidic structures may be fabricated above or between FETs of an array to provide for containment, confinement, or transient passage past FETs of the array. Such a system may be used, for example, to monitor a biological or chemical reaction in which an analyte of interest is produced or consumed. In one implementation, the microfluidic structure(s) may comprise one or more wells (e.g., small reaction chambers or “reaction wells”) containing analytes of interest, and one or more microfluidic channels to provide and remove analytes and reagents from a well or plurality of wells. In such a case, a well may be disposed above a FET or a plurality of FETs of the array, such that the FET or the plurality of FETs over which a given well is disposed detect and/or measure analytes within the well.

[0090] The FET or ISFET may comprise a floating gate. A floating gate may have an electrically isolated gate, creating a floating node in direct current where a number of secondary gates or inputs may be deposited above the floating gate and may be electrically isolated from it. A floating gate may have an area greater than about 1 nanometer squared (nm²), 1 nm², 2 nm², 3 nm², 4 nm², 5 nm², 6 nm², 7 nm², 8 nm², 9 nm², 10 nm², 15 nm², 20 nm², 25 nm², 30 nm², 35 nm², 40 nm², 45 nm², 50 nm², 60 nm², 70 nm², 80 nm², 90 nm², 100 nm², 150 nm², 200 nm², 250 nm², 300 nm², 350 nm², 400 nm², 450 nm², 500 nm², 600 nm², 700 nm², 800 nm², 900 nm², 1000 nm², 10, 000 nm², or more than 10,000 nm². A floating gate may have an area less than about 10,000 nm², 900 nm², 800 nm², 700 nm², 600 nm², 500 nm², 450 nm², 400 nm², 350 nm², 300 nm², 250 nm², 200 nm², 150 nm², 100 nm², 90 nm², 80 nm², 70 nm², 60 nm², 50 nm², 45 nm², 40 nm², 35 nm², 30 nm², 25 nm², 20 nm², 15 nm², 10 nm², 9 nm², 8 nm², 7 nm², 6 nm², 5 nm², 4 nm², 3 nm², 2 nm², 1 nm², or less than 1 nm².The floating gate may have a trapped charge or less than about 10,000 Volts (V), 5,000 V, 1,000 V, 240 V, 120 V, 50 V, 20 V, 15 V, 12 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, 1 V, 0.9 V, 0.8 V, 0.7 V, 0.6 V, 0.5 V, 0.4 V, 0.3 V, 0.2 V, 0.1 V, 0.05 V, 0.01 V, 0.005 V, 0.001 V or less volts. The floating gate may have a trapped charge of greater than about 0.001 V, 0.005 V, 0.01 V, 0.05 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 12 V, 15 V, 20 V, 50 V, 120 V, 240 V, 1,000 V, 5,000 V, 10,000 V, or more.

[0091] An array of FETs can have an area that is larger than about 100 nanometers squared (nm²), 250 nm², 500 nm², 1 micrometers squared (qm²), 2.5 qm², 5 qm², 10 qm², 100 qm², 500 qm², 1 millimeter (mm²), or larger than 1 mm². Alternatively or additionally, features of an array can each have an area that is smaller than about 1 mm², 500 qm²,100 qm², 25 qm², 10 qm², 5 qm², 1 qm², 500 nm², or 100 nm². An array can have a size that is in a range between an upper and lower limit selected from those provided herein.

[0092] An array of FET sensors may be overlay ed with an array of reaction chambers wherein the bottom of a reaction chamber is in contact with (or capacitively coupled to) a FET sensor. Each reaction chamber bottom may be in contact with a FET sensor or each with a separate FET sensor but not all reaction chamber bottoms may be in contact with a FET sensor. Each sensor in the array may be in contact with a reaction chamber. In other aspects of the disclosure, less than all sensors may be in contact with a reaction chamber. The sensor (and/or reaction chamber) array may be comprised of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, or more FET sensors (and/or reaction chambers). The reaction well volume may range based on the well dimensions. This volume may be at or about 100 picoliter (pL), 90, 80, 70, 60, 50, 40, 30, 20, 10, or fewer pL. This volume may be at or about 100 pL, 90 pL, 80 pL, 70 pL, 60 pL, 50 pL, 40 pL, 30 pL, 20 pL, 10 pL, or greater pL. The well volume may be less than 1 pL, including equal to or less than 0.5 pL, equal to or less than. 0.1 pL. equal to or less than 0.05 pL, equal to or less than 0.01 pL, equal to or less than 0.005 pL, or equal to or less than 0.001 pL. The well volume may be greater than 1 pL, including equal to or greater than 0.5 pL, equal to or greater than. 0.1 pL. equal to or greater than 0.05 pL, equal to or greater than 0.01 pL, equal to or greater than 0.005 pL, or equal to or greater than 0.001 pL,

[0093] A microfluidic device may be coupled to the FET. The microfluidic device may provide analyte in proximity to the FET. Contact of the analyte in proximity to the FET can be performed with or without fluid flow. An array of reaction sites coupled to a FET may be contacted with a solution with analytes using fluid flow. The contact may occur contemporaneous to fluid flow of an analyte solution. The array may comprise a microfluidic device. The solution may be viscous or non-viscous. The array can include a plurality of microfluidic modules integrally arranged with each other so as to be in fluid communication. The array can include, for example, at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid, at least one main channel adapted to carry at least one continuous phase fluid. The inlet channel may be in fluid communication with the main channel at a junction.

[0094] A composition, system, device, or method of the present disclosure may comprise a peptide partitioned within a droplet. A droplet may comprise a single protein or a single peptide (e.g., a peptide generated from the cleavage of a protein). A droplet may comprise a plurality of proteins or peptides. A droplet may comprise a reagent, such as a reagent for a degradation reaction such as Edman degradation or protein digestion. Two or more droplets may be combined (e.g., at the confluence of two or more channels, by electrocoalescence, or by flow focusing) to form a single droplet, which may thereby comprise the entirety or a subset of the contents of the two or more combined droplets. A droplet may also be split into two or more droplets. A reagent or peptide may be added to a droplet subsequent to its formation. A system or device may generate a plurality of droplets each comprising a specified number of proteins or peptides. In some cases, a process for forming droplets may result in more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.75%, or more than 99.99% of droplets containing exactly 1, exactly 2, exactly 3, or more than 3 peptides. In some cases, a process for forming droplets may result in more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.75%, or more than 99.99% of droplets containing at least 1 peptide, at least 2 peptides, at least 3 peptides, at least 4 peptides, at least 5 peptides, at least 8 peptides, at least 10 peptides, at least 12 peptides, at least 15 peptides, at least 20 peptides, at least 25 peptides, at least 50 peptides, at least 100 peptides, or at least 200 peptides. In some cases, a process for forming droplets may result in more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.75%, or more than 99.99% of droplets containing at most 1 peptide, at most 2 peptides, at most 3 peptides, at most 4 peptides, at most 5 peptides, at most 8 peptides, at most 10 peptides, at most 12 peptides, at most 15 peptides, at most 20 peptides, at most 25 peptides, at most 50 peptides, at most 100 peptides, or at most 200 peptides.

[0095] A fluidic (e.g., a microfluidic) junction may include a fluidic nozzle designed for flow focusing such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in the continuous phase fluid. The flow of the dispersed phase and/or the flow of the continuous phase can be pressure driven. The dispersed phase (e.g. droplets) can be neutral or have no charge and these droplets can be manipulated (e.g., coalesced, sorted) within an electric field in the continuous phase fluid. The microfluidic array can include one or more additional modules, including but not limited to, coalescence module, detection module, sorting module, collection module, waste module, delay module, droplet spacing module, or a mixing module. There may be zero, one, or more of each of the modules. Contact between the array of reaction sites with the solution containing nucleic acid molecules may happen at the same time as amplification through the flow of fluid.

[0096] A solution (e.g., a solution comprising peptides) may flow through a microfluidic device at a rate of about 1 microliter (pL)/minute (min) to about 12 pL/min. The solution comprising the peptide molecules may be flowed at a flow rate about 1 pL/min to about 2 pL/min, about 1 pL/min to about 3 pL/min, about 1 pL/min to about 4 pL/min, about 1 pL/min to about 5 pL/min, about 1 pL/min to about 6 pL/min, about 1 pL/min to about 7 pL/min, about 1 pL/min to about 8 pL/min, about 1 pL/min to about 9 pL/min, about 1 pL/min to about 10 pL/min, about 1 pL/min to about 11 pL/min, about 1 pL/min to about 12 pL/min, about 2 pL/min to about 3 pL/min, about 2 pL/min to about 4 pL/min, about 2 pL/min to about 5 pL/min, about 2 pL/min to about 6 pL/min, about 2 pL/min to about 7 pL/min, about 2 pL/min to about 8 pL/min, about 2 pL/min to about 9 pL/min, about 2 pL/min to about 10 pL/min, about 2 pL/min to about 11 pL/min, about 2 pL/min to about 12 pL/min, about 3 pL/min to about 4 pL/min, about 3 pL/min to about 5 pL/min, about 3 pL/min to about 6 pL/min, about 3 pL/min to about 7 pL/min, about 3 pL/min to about 8 pL/min, about 3 pL/min to about 9 pL/min, about 3 pL/min to about 10 pL/min, about 3 pL/min to about 11 pL/min, about 3 pL/min to about 12 pL/min, about 4 pL/min to about 5 pL/min, about 4 pL/min to about 6 pL/min, about 4 pL/min to about 7 pL/min, about 4 pL/min to about 8 pL/min, about 4 pL/min to about 9 pL/min, about 4 pL/min to about 10 pL/min, about 4 pL/min to about 11 pL/min, about 4 pL/min to about 12 pL/min, about 5 pL/min to about 6 pL/min, about 5 pL/min to about 7 pL/min, about 5 pL/min to about 8 pL/min, about 5 pL/min to about 9 pL/min, about 5 pL/min to about 10 pL/min, about 5 pL/min to about 11 pL/min, about 5 pL/min to about 12 pL/min, about 6 pL/min to about 7 pL/min, about 6 pL/min to about 8 pL/min, about 6 pL/min to about 9 pL/min, about 6 pL/min to about 10 pL/min, about 6 pL/min to about 11 pL/min, about 6 pL/min to about 12 pL/min, about 7 pL/min to about 8 pL/min, about 7 pL/min to about 9 pL/min, about 7 pL/min to about 10 pL/min, about 7 pL/min to about 11 pL/min, about 7 pL/min to about 12 pL/min, about 8 pL/min to about 9 pL/min, about 8 pL/min to about 10 pL/min, about 8 pL/min to about 11 pL/min, about 8 pL/min to about 12 pL/min, about 9 pL/min to about 10 pL/min, about 9 pL/min to about 11 pL/min, about 9 pL/min to about 12 pL/min, about 10 pL/min to about 11 pL/min, about 10 pL/min to about 12 pL/min, or about 11 pL/min to about 12 pL/min. The solution comprising the peptide molecules may be flowed at about 1 pL/min, about 2 pL/min, about 3 pL/min, about 4 pL/min, about 5 pL/min, about 6 pL/min, about 7 pL/min, about 8 pL/min, about 9 pL/min, about 10 pL/min, about 11 pL/min, or about 12 pL/min The solution comprising the peptide molecules may be flowed at least about 1 pL/min, about 2 pL/min, about 3 pL/min, about 4 pL/min, about 5 pL/min, about 6 pL/min, about 7 pL/min, about 8 pL/min, about 9 pL/min, about 10 pL/min, or about 11 pL/min. The solution comprising the peptide molecules may be flowed at most about 2 pL/min, about 3 pL/min, about 4 pL/min, about 5 pL/min, about 6 pL/min, about 7 pL/min, about 8 pL/min, about 9 pL/min, about 10 pL/min, about 11 pL/min, or about 12 pL/min.

[0097] A solution comprising peptides may have a concentration of peptides of at least about 0.001 nanograms (ng)/microliter (pL), 0.005 ng/pL, 0.01 ng/pL, 0.05 ng/pL, 0.1 ng/pL, 0.2 ng/pL, 0.3 ng/pL, 0.4 ng/pL, 0.5 ng/pL, 0.6 ng/pL, 0.7 ng/pL, 0.8 ng/pL, 0.9 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 4 ng/pL, 5 ng/pL, 6 ng/pL, 7 ng/pL, 8 ng/pL, 9 ng/pL, 10 ng/pL, 11 ng/pL, 12 ng/pL, 13 ng/pL, 14 ng/pL, 15 ng/pL, 16 ng/pL, 17 ng/pL, 18 ng/pL, 19 ng/pL, 20 ng/pL, 25 ng/pL, 30 ng/pL, 35 ng/pL, 40 ng/pL, 45 ng/pL, 50 ng/pL, 55 ng/pL, 60 ng/pL, 65 ng/pL, 70 ng/pL, 80 ng/pL, 85 ng/pL, 90 ng/pL, 95 ng/pL, 100 ng/pL, 110 ng/pL, 120 ng/pL, 130 ng/pL, 140 ng/pL, 150 ng/pL, 160 ng/pL, 170 ng/pL, 180 ng/pL, 190 ng/pL, 200 ng/pL, 225 ng/pL,

250 ng/pL, 275 ng/pL, 300 ng/pL, 350 ng/pL, 400 ng/pL, 450 ng/pL, 500 ng/pL, 550 ng/pL,

600 ng/pL, 700 ng/pL, 800 ng/pL, 900 ng/pL, 1,000 ng/pL or more nanograms per microliter. The solution comprising peptides may have a concentration of peptides of at most about 1,000 ng/pL, 900 ng/pL, 800 ng/pL, 700 ng/pL, 600 ng/pL, 550 ng/pL, 500 ng/pL, 450 ng/pL, 400 ng/pL, 350 ng/pL, 300 ng/pL, 275 ng/pL, 250 ng/pL, 225 ng/pL, 200 ng/pL, 190 ng/pL, 180 ng/pL, 170 ng/pL, 160 ng/pL, 150 ng/pL, 140 ng/pL, 130 ng/pL, 120 ng/pL, 110 ng/pL, 100 ng/pL, 95 ng/pL, 90 ng/pL, 85 ng/pL, 80 ng/pL, 75 ng/pL, 70 ng/pL, 65 ng/pL, 60 ng/pL, 55 ng/pL, 50 ng/pL, 45 ng/pL, 40 ng/pL, 35 ng/pL, 30 ng/pL, 25 ng/pL, 20 ng/pL, 19 ng/pL, 18 ng/pL, 17 ng/pL, 16 ng/pL, 15 ng/pL, 14 ng/pL, 13 ng/pL, 12 ng/pL, 11 ng/pL, 10 ng/pL, 9 ng/pL, 8 ng/pL, 7 ng/pL, 6 ng/pL, 5 ng/pL, 4 ng/pL, 3 ng/pL, 2 ng/pL, 1 ng/pL, 0.9 ng/pL, 0.8 ng/pL, 0.7 ng/pL, 0.6 ng/pL, 0.5 ng/pL, 0.4 ng/pL, 0.3 ng/pL, 0.2 ng/pL, 0.1 ng/pL, 0.05 ng/pL, 0.01 ng/pL, 0.005 ng/pL, 0.001 ng/pL, or less nanograms per microliter. The solution may have a peptide concentration range as defined by any two of the previous values. For example, the solution may have a concentration of peptides from 0.4 ng/pL per microliter to 4 ng/pL per microliter.

[0098] An open channel may include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation), physical or chemical characteristics (hydrophobicity vs. hydrophilicity), other characteristics that can exert a force (e.g., a containing force) on a fluid, or any combination thereof. The fluid within the channel may partially or completely fill the channel. In some cases, the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (e.g., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus). [0099] The substrate may include one or plurality of channels. A channel may have various sizes. The channel may have a largest dimension perpendicular to the direction of fluid flow along the channel of less than about 5 millimeters (mm), less than about 2 mm, less than about 1 mm, less than about 500 micrometers (pm), less than about 200 pm, less than about 100 pm, less than about 60 pm, less than about 50 pm, less than about 40 pm, less than about 30 pm, less than about 25 pm, less than about 10 pm, less than about 3 pm, less than about 1 micron, less than about 300 nanometers (nm), less than about 100 nm, less than about 30 nm, or less than about 10 nm or less in some cases. In some cases, larger channels, tubes, etc. can be used to store fluids in bulk and/or deliver a fluid to the channel. The dimensions of the channel may be chosen such that fluid is able to freely flow through the channel. In some cases, more than one channel may be used.

[0100] In some cases, an electric field may be applied to fluidic droplets to cause the droplets to experience an electric force. The electric force exerted on the fluidic droplets may be, in some cases, at least about 10 ¹⁶ Newtons (N)/pm³. In certain cases, the electric force exerted on the fluidic droplets may be greater, e.g., at least about 10 ¹⁵ N/pm³, at least about 10 ¹⁴ N/pm³, at least about 10 ¹³ N/pm³, at least about 10 ¹² N/pm³, at least about 10 ¹¹ N/pm³, at least about 10

¹⁰ N/pm³, at least about 10⁹ N/pm³, at least about 10⁸ N/pm³, or at least about 10⁷ N/pm³ or more. The electric force exerted on the fluidic droplets, relative to the surface area of the fluid, may be at least about 10 ¹⁵ N/pm², and in some cases, at least about 10 ¹⁴ N/pm², at least about 10 ¹³ N/pm², at least about 10 ¹² N/pm², at least about 10 ¹¹ N/pm², at least about 10 ¹⁰ N/pm², at least about 10⁹ N/pm², at least about 10⁸ N/pm², at least about 10⁷ N/pm², or at least about 10

⁶ N/pm² or more. The electric force exerted on the fluidic droplets may be at least about 10

⁹ Newtons (N), at least about 10⁸ N, at least about 10⁷ N, at least about 10⁶ N, at least about 10 ⁵ N, or at least about 10⁴ N or more in some cases.

[0101] Fluid may flow through the microfluidic channels at a flow rate approximately about 1 microliter (pL)/minute (min) to about 15 microliters pL/min. Fluid may flow through the microfluidic channels at a flow rate of at least about 1 pL/min, 2 pL/min, 3 pL/min, 4 pL/min, 5 pL/min, 6 pL/min, 7 pL/min, 8 pL/min, 9 pL/min, 10 pL/min, 11 pL/min, 12 pL/min, 13 pL/min, 14 pL/min, 15 pL/min, or greater than 15 pL/min. Fluid may flow through the microfluidic channels at a flow rate less than about 15 pL/min, 14 pL/min, 13 pL/min, 12 pL/min, 11 pL/min, 10 pL/min, 9 pL/min, 8 pL/min, 7 pL/min, 6 pL/min, 5 pL/min, 4 pL/min,

3 pL/min, 2 pL/min, 1 pL/min, or less than 1 pL/min. The fluid may comprise a droplet comprising a peptide. The fluid may comprise a plurality of droplets comprising a plurality of peptides. Peptides may be present in a solution at such a density as to allow single peptide binding to a reaction site. This can be done using Poisson statistics. The density of a peptide in a solution may be a high density, low density, average density, or any combination thereof. The density may follow a Poisson distribution, a super-Poisson distribution, or a non-Poisson distribution. The density may be constant.

[0102] A microfluidic channel may utilize a consistent flow design or an oscillatory flow design. In a consistent flow design, nucleic acids, droplets, or solution are in continuous-flow. Peptides, droplets (e.g., a plurality of droplets comprising a plurality of peptides), or solutions may be stationary or semi-stationary. For example, a fluidic channel may comprise a reservoir configured to provide a high average residency time for droplets flowing through the channel. Peptides, droplets, or solution may be in motion. A microfluidic device may utilize oscillating or bidirectional flow. A microfluidic device may combine the cycling flexibility of a stationary chamber-based system and the fast dynamics of a continuous flow system. Peptides, droplets, or solutions may be transported back and forth through a single channel or may be transported in multiple channels or capillaries. The channel(s) may span various temperature zones. A microfluidic device or array may be attached to a pumping system such as but not limited to external pumps and integrated micropumps. There may be on board power or an external power source. Centrifugal force and/or capillary forces may be used to control the fluid flow. A compact disc format may be used to house the reaction chambers or other components. A droplet may serve as a reactor environment allowing for fast reagent mixing and minimum surface adsorption. Interfacial chemistry may be used to create such a reactor droplet (e.g. an oil-water plug may be flowed through a fluid capillary to create a water-in-oil droplet).

[0103] Peptides, droplets, or solutions in a microfluidic device, in a well, attached to a support, or in an array may be incubated, split, and merged in a microfluidic device. Droplets may vary in size. Droplets may be at least about 0.5 micrometers (pm), 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80, pm, 90 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm or more in diameter. They may be less than or greater than these diameters or any value in between. Peptide, droplet, or solution formation frequency may be at least about 0.5 Hertz (Hz), 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1,000 Hz, 2,000 Hz, 3,000 Hz, 4,000 Hz, 5,000 Hz, 6,000 Hz, 7,000 Hz, 8,000 Hz, 9,000 Hz, 10,000 Hz or more. The frequency may be less than or greater than those listed here or any value in between.

[0104] An array may comprise a support wherein a peptide, amino acid, peptide, or protein is immobilized to the support. Some aspects of present disclosure may immobilize peptides on a support such as on the surface of resins, gels, quartz particles, or combinations thereof. In some non-limiting examples, the methods contemplate using peptides that have been immobilized on the support of an aminosilane modified surface, Tentagel® beads, Tentagel® resins, or other similar beads or resins. The surface used herein may be a hydrogel, such as alginate. The surface used herein may be coated with a polymer, such as polyethylene glycol. Fluoropolymers (Teflon- AF (Dupont), Cytop® (Asahi Glass, Japan)), aromatic polymers (polyxylenes (Parylene, Kisco, Calif.), polystyrene, polymethmethylacrytate) and metal surfaces (Gold coating)), coating schemes (spin-coating, dip-coating, electron beam deposition for metals, thermal vapor deposition and plasma enhanced chemical vapor deposition) and functionalization methodologies (polyallylamine grafting, use of ammonia gas in PECVD, doping of long chain end- functionalized fluorous alkanes etc) may be used in the methods described herein as a useful surface. A solid support may be conjugated with different addressable makers and ligands.

[0105] A solid support may comprise a bead. A bead may be a polymer such as a polystyrene bead or polystyrene cross-linked with divinylbenzene. The solid support bead may comprise an iron oxide core. A bead may comprise a metal salt such as a copper salt, a magnesium salt, a calcium salt, or a manganese salt. A bead may be cellulose, cellulose derivatives, gelatin, acrylic resins, glass, silica gels, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide, polyacrylamides, latex gels, dextran, crosslinked dextrans (e.g., Sephadex™), rubber, silicon, plastics, nitrocellulose, natural sponges, metal, and agarose gel (Sepharose™). The bead diameter may depend on the density of the ISFET and well array used with larger arrays (and thus smaller sized wells) requiring smaller beads. The bead may have a diameter of at least about 1 micrometer (pm), 5 pm, 10 pm, 25 pm, 50 pm, 75 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 750 pm, 1,000 pm, or more micrometers. The bead may have a diameter of at most about 1,000 pm, 750 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 75 pm, 50 pm, 25 pm, 10 pm, 5 pm, 1 pm, or less than 1 micrometers. Ahead may be a component of a well-less sensing array. A peptide may be coupled to a functional unit on the surface of the bead.

[0106] A solid support may be a surface of a well. A solid support may be the interior of a well. The well may have a dimension of x by y by z, where x, y, and z are each independently at least about 0.1 pm, 1 pm, 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 250 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more micrometers. The well may have a dimension of x by y by z, where x, y, and z are each independently at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm, 150 pm, 140 pm, 130 pm, 120 pm, 110 pm, 100 pm, 95 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 5 pm, 1 pm, 0.1 pm, or less micrometers. For example, the well can have an x dimension of 434 pm, a y dimension of 30 pm, and a z dimension of 510 pm. In another example, the well can have an x and y dimension of 16 pm and a z dimension of 1 pm. The support may be a well among a plurality of wells. The plurality of wells may comprise at least two wells. The plurality of wells may comprise at least 1,000 wells. There may be 2, 3, 4, 5,

6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500

2000, 3000, 4000, 5000, 10,000, 100,000, 1,000,000 or more than 1,000,000 wells in a plurality of wells.

[0107] A well may comprise a single bead. A peptide loaded bead, of which there may be tens, hundreds, thousands, or more, may enter a flow cell and then individual beads may enter individual wells. The beads may enter the wells passively or otherwise. For example, the beads may enter the wells through gravity without any applied external force. The beads may enter the wells through an applied external force including but not limited to a magnetic force or a centrifugal force. In some embodiments, if an external force is applied, it is applied in a direction that is parallel to the well height/depth rather than transverse to the well height/depth, with the aim being to “capture” as many beads as possible. The wells (or well arrays) may or may not be agitated, as for example may occur through an applied external force that is perpendicular to the well height/depth.

[0108] Aspects of the present disclosure may provide a method for peptide sequencing comprising providing an array having a peptide immobilized thereto wherein the peptide is adjacent to or operably coupled to a sensor (e.g., a FET). A peptide may be coupled to a capture moiety coupled to the array such as an antigen, antibody, aptamer, immunogenic sugar, or through biotinylation. The capture moiety may be modified such that it has a detection label, for example a fluorescently labeled antibody. Binding of the capture moiety to the peptide may occur through noncovalent interactions such as electrostatic force, hydrogen bonding, Van der Waals forces, or hydrophobic forces, or through covalent bonding. The peptide may be covalently coupled to the array or may be ionically coupled to the array.

[0109] The array may comprise a support to which a peptide may be immobilized. The support may comprise a sensor. The sensor may be an electrical sensor which may comprise an electrode. The electrode may be a metal electrode, a semiconductor electrode, or a combination thereof. A peptide, amino acid, peptide, or protein may be immobilized at a reaction site which is or is coupled to an electronic sensor. A sensor may detect the binding or release of an analyte. This sensor may measure a charge, or change thereof, in a solution subsequent to removal of an amino acid from the peptide and use this charge or change thereof to identify a sequence of the peptide. A sensor may measure conductivity or a change thereof in a solution subsequent to removal of an amino acid from the peptide and use this conductivity or change thereof to identify a sequence of the peptide. A sensor may measure an impedance or change thereof in a solution subsequent to removal of an amino acid from the peptide and use this impedance or change thereof to identify a sequence of the peptide. The array may comprise a plurality of individually addressable sites wherein the peptide may be immobilized to an individually addressable site of a plurality of individually addressable sites. An addressable site may be a reaction site, a support, a bead, a well, the surface of a well, or a sensor. The sensors of the array of sensors may be individually addressable. For example, an electrode of an array of 1,000,000 can be measured independently of each other electrode.

[0110] A sensor may comprise a carbon nanotube (CNT) transistor. A single-point carbon- nanotube field-effect transistor (CNTFETs) may be used to sense conformational changes and binding events in a protein structure from intrinsic molecular charge. A CNTFET may attach a single probe molecule to an individual device and thus may comprise a single-molecule sensor.

A FET (e.g., a (CNTFET) or an array of FETS may comprise a plurality of sites configured for peptide immobilization. A CNTFET may comprise a point- functionalized single-walled carbon nanotube. A CNTFET may provide an all-electronic, label-free, single-molecule detection platform through conductance sensitivity to charges localized close to a point defect generated on a sidewall of the CNT. This functionalized point may serve as a point of attachment for the peptide or target molecule. A CNTFET may comprise a plurality of CNTFETs (e.g., arranged as an array of CNTFETs). A CNTFET may be reused by detaching an attached analyte or may utilize a permanent fixture. An analyte such as a peptide may be ionically attached to the CNT. A peptide may be covalently attached to the CNT. A covalent attachment may localize charge sensitivity at the point of attachment. A CNT may be covalently modified with aryl radical, nucleophilic, or an electrophilic addition to impart a measurable resistance change in the CNTFET, by functionalizing with diazonium, or by removing carbon atoms from the lattice thus converting the carbon bonding from sp² to sp³ configuration. This alteration in carbon bonding may reduce the conductance of the CNTFET.

[0111] A CNTFET may be comprised of an array of carbon nanotubes. There may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 10,000, 100,000, 1,000,000 or more than 1,000,000 CNTs in a plurality of CNTs. CNTs may be formed through plasma arcing, carbon arc discharge, dual pulsed laser vaporization, catalyzed chemical vapor deposition, ball milling, diffusion flame synthesis, electrolysis, heat treatment, low temperature solid pyrolysis, spin casting, or laser ablation. CNTs may have a diameter of at least 1 nanometer (nm), 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1000 nm, or more than 1000 nm. CNTs may have a diameter of less than 1000 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less than 1 nm. CNTs may have a length of 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1000 nm, .01 millimeters (mm),

.1 mm, 1 mm, or more than 1 mm. CNTs may have a length less than 1 mm, .1 mm, .01 mm, 1000 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less than 1 nm. CNTs have form at a density of at least .01 carbon nanotubes per micrometer squared (CNTs/pm²), .02 CNTs/pm², .03 CNTs/ pm², .04 CNTs/pm², .05 CNTs/pm², .1 CNTs/pm², .15 CNTs/pm², .2 CNTs/pm², .25 CNTs/pm², .3 CNTs/pm², .35 CNTs/pm², .4 CNTs/pm², .45 CNTs/pm², .5 CNTs/pm², .6 CNTs/pm², .7 CNTs/pm², .8 CNTs/pm², .9 CNTs/pm², 1 CNTs/pm², 2 CNTs/pm², 3 CNTs/pm², 4 CNTs/pm², 5 CNTs/pm², 10 CNTs/pm², 20 CNTs/pm², 30 CNTs/pm², 40 CNTs/pm², 50 CNTs/pm², 60 CNTs/pm², 70 CNTs/pm², 80 CNTs/pm², 90 CNTs/pm², 100 CNTs/pm², or more than 100 CNTs/pm². CNTs may form at a density of less than 100 CNTs/pm², 90 CNTs/pm², 80 CNTs/pm², 70 CNTs/pm², 60 CNTs/pm², 50 CNTs/pm², 40 CNTs/pm², 30 CNTs/pm², 20 CNTs/pm², 10 CNTs/pm², 5 CNTs/pm², 4 CNTs/pm², 3 CNTs/pm², 2 CNTs/pm², 1 CNTs/pm², .9 CNTs/pm², .8 CNTs/pm², .7 CNTs/pm², .6 CNTs/pm², .5 CNTs/pm², .45 CNTs/pm², .4 CNTs/pm², .35 CNTs/pm², .3 CNTs/pm², .25 CNTs/pm², .2 CNTs/pm², .15 CNTs/pm², .1 CNTs/pm², .05 CNTs/pm², .04 CNTs/pm², .03 CNTs/pm², .02 CNTs/pm², .01 CNTs/pm², or less than .01 CNTs/pm². A CNT array may comprise a degree of CNT size uniformity or nonuniformity. CNTs of an array may comprise an average of at most 50% size variation (e.g., defined as absolute range in size or as a standard deviation of CNT sizes), at most 40% size variation, at most 30% size variation, at most 25% size variation, at most 20% size variation, at most 15% size variation, at most 12% size variation, at most 10% size variation, at most 8% size variation, at most 6% size variation, at most 5% size variation, at most 4% size variation, at most 3% size variation, at most 2% size variation, at most 1.5% size variation, at most 1% size variation, at most 0.5% size variation, at most 0.25% size variation, or at most 0.1% size variation. CNTs of an array may comprise an average of at least 50% size variation, at least 40% size variation, at least 30% size variation, at least 25% size variation, at least 20% size variation, at least 15% size variation, at least 12% size variation, at least 10% size variation, at least 8% size variation, at least 6% size variation, at least 5% size variation, at least 4% size variation, at least 3% size variation, at least 2% size variation, at least 1.5% size variation, at least 1% size variation, at least 0.5% size variation, at least 0.25% size variation, or at least 0.1% size variation.

[0112] CNTs may form bridging electrode pairs with an electrode width of at least around 1 micrometer (pm), 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, or more than 100 pm. CNTs may form bridging electrode pairs with an electrode width of less than 100 pm,

90 pm, 80 pm, 70 pm, 60 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 9, pm 8, pm 7 pm, 6 pm, 5 pm, 4 pm, 3 pm, 2 pm, 1 pm, or less than 1 pm. CNT electrodes may be applied to the surface of a conductive or semi conductive wafer, for example a silicon wafer. A wafer may yield multiple chips with each chip containing multiple pairs of source-drain electrical contacts. A source drain electrical contact may have a width of at least about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm,

40 pm, 45 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm 150, pm 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm or more than 1000 pm. A source drain electrical contact may have a width of at less than about 1000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 450 pm, 400 pm, 350 pm, 300 pm, 250 pm, 200 pm, 150 pm, 100 pm, 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, 45 pm, 40 pm, 35 pm, 30 pm, 25 pm, 20 pm, 15 pm, 10 pm, 9 pm, 8 pm, 7 pm, 6 pm, 5 pm, 4 pm, 3 pm, 2 pm, 1 pm, or less than 1 pm. A source drain electrical contact may have a length of at least about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm 150, pm 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1000 pm or more than 1000 pm. A source drain electrical contact may have a length of less than about 1000 pm, 900 pm, 800 pm, 700 pm, 600 mih, 500 mih, 450 mih, 400 mih, 350 mih, 300 mih, 250 mih, 200 mih, 150 mih, 100 mih, 90 mih, 80 mih, 70 mih, 60 mih, 50 mih, 45 mih, 40 mih, 35 mih, 30 mih, 25 mih, 20 mih, 15 mih, 10 mih, 9 mih, 8 mih, 7 mih, 6 mih, 5 mih, 4 mih, 3 mih, 2 mih, 1 mih, or less than 1 mih. A source drain electrical contact may have a height of at least about 1 mih, 2 mih, 3 mih, 4 mih, 5 mm, 6 mih, 7 mm, 8 mih, 9 mm, 10 mm, 15 mih, 20 mm, 25 mih, 30 mm, 35 mm, 40 mih, 45 mm, 50 mih, 60 mm, 70 mm, 80 mih, 90 mm, 100 mih 150, mih 200 mih, 250 mih, 300 mm, 350 mih, 400 mm, 450 mm, 500 mih, 600 mm, 700 mih, 800 mm, 900 mm, 1000 mih or more than 1000 mih. A source drain electrical contact may have a height of less than about 1000 mih, 900 mih, 800 mih, 700 mih, 600 mih, 500 mih, 450 mih, 400 pm, 350 mih, 300 pm, 250 mih, 200 pm, 150 pm, 100 mih, 90 pm, 80 mih, 70 pm, 60 pm, 50 mih, 45 pm, 40 mih, 35 pm, 30 pm, 25 mih, 20 pm, 15 mih, 10 pm, 9 pm, 8 mih, 7 pm, 6 mih, 5 pm, 4 pm, 3 mih, 2 pm, 1 mih, or less than 1 mih.

[0113] A back-gated voltage sweep (Vbg) may be applied to the underlying wafer substrate, for example from -10 to 10 V, to determine conductivity. A back-gated voltage sweep may comprise a range of at least 30 V, at least 25 V, at least 20 V, at least 15 V, at least 12 V, at least 10 V, at least 8 V, at least 6 V, at least 5 V, at least 4 V, at least 3 V, at least 2 V, at least 1.5 V, at least 1 V, at least 0.5 V, at least 0.25 V, or at least 0.1 V. A back-gated voltage sweep may comprise a range of at most 30 V, at most 25 V, at most 20 V, at most 15 V, at most 12 V, at most 10 V, at most 8 V, at most 6 V, at most 5 V, at most 4 V, at most 3 V, at most 2 V, at most 1.5 V, at most 1 V, at most 0.5 V, at most 0.25 V, or at most 0.1 V. Since solution-processed CNTFETs may be composed of mixtures of metallic and p-type semiconducting nanotubes, conductive devices may be determined based on measured on-current (I_on) at Vbg. For example, if a back-gated voltage sweep is applied from -10V to 10V, the conductivity may be determined based on measured on-current at Vbg = -10 V and a source-to-drain (V_Sd) of 100 mV with conductive CNTFETs having an I_on of at least 1.0 nA. A chip may contain multiple working CNTFET devices which may be bonded to a land-grid array package to interface with a circuit board. A microfluidic chamber may be operably coupled to the surface of a chip.

[0114] Further aspects of the present disclosure provide a method for peptide sequencing wherein a single molecule probe using a field-effect transistor can be used to observe and characterize consecutive chemical reactions, molecular reactions, molecular composition, molecular structure (e.g., sequence information), and molecular conformational changes. A single molecule probe may utilize quantized fluctuations in electrical signals on an electrically active and addressable surface to observe and characterize consecutive individual chemical reactions, molecular interactions, and molecular conformational changes. An electrically active and addressable surface may be electrically stimulated, and a spectral signature of the attached peptide may be recorded. Such a change in spectral signatures over all addressable units are collected between peptide degradation cycles and used to estimate the concentrations of the individual species of peptide present in the original mixture.

[0115] A point-functionalized carbon nanotube transistor may be utilized as a single-molecule FET by harnessing electrostatics to control molecular binding. Such an approach may offer a bioelectronics alternative to traditional optical labelling based on intrinsic molecular charge and may offer higher signal levels for detection. An electrically active and addressable surface may comprise a CNTFET.

Nanowells

[0116] An electrically active and addressable surface may comprise a patterned nanowell with electrical connections. A FET (e.g., a CNTFET) may be operably coupled to a patterned nanowell. A nanowell may be used to isolate a single molecule and facilitate single-molecule electrochemical reactions. A nanowell may be used to confine a chemical or electrical reaction to a single point on an individual carbon nanotube. A nanowell may be configured to adopt a plurality of temperatures (e.g., for a specific Edman degradation method, a nanowell may be configured to adopt a first temperature sufficient for phenylisothiocyanate coupling to peptide N- terminal amino acids, and a second temperature sufficient for cleavage of thiourea derivatized N- terminal amino acids from peptides). A nanowell confining electrochemical reactions to a single FET may comprise a single-molecule probe. Nanowells may be patterned using etching, lithography, photolithography, chemical etching, electron beam lithography, X-ray lithography, or replica molding.

[0117] A nanowell may be configured (e.g., comprise sufficient dimensions) to accept a droplet or a plurality of droplets. A device may be configured to deliver a droplet comprising a protein or a peptide to a nanowell, wherein the peptide or protein may couple to a peptide capture site or moiety (e.g., a peptide capture site of a CNTFET). A device may be configured to generate a plurality of droplets comprising a plurality of peptides, and deliver an average of one, at least one, or at most one droplet to each of a plurality of nanowells.

[0118] A nanowell may comprise one or a plurality of materials. For example, a nanowell may comprise an amine functionalized silicon dioxide surface and a gold base. A nanowell may comprise an aminosilane modified surface, silicon dioxide, a hydrogel, a polymer, such as polyethylene glycol, fluoropolymer (Teflon-AF (Dupont), Cytop® (Asahi Glass, Japan)), aromatic polymers (polyxylenes (Parylene, Kisco, Calif.), polystyrene, polymethmethylacrytate), metal (Gold coating), a metal salt such as a copper salt, a magnesium salt, a calcium salt, or a manganese salt. A bead may be cellulose, cellulose derivatives, gelatin, acrylic resins, glass, silica gels, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide, polyacrylamides, latex gels, dextran, crosslinked dextrans, rubber, silicon, plastics, nitrocellulose, natural sponges, chitosan, or agarose gel. The sidewall face of a nanowell can be hydrophilic, such as a sidewall comprising silicon, silica, metal, or metal oxide, and the bottom surface of the nanowell can be hydrophobic. If the bottom surface of the nanowell is made of a material having hydrophilic properties, it can be modified to be hydrophobic. For example, if the bottom surface of the nanowell is made of a hydrophilic silicate or metal, it can be modified to be hydrophobic, such as R1 _x -Si(0-R2)_4-X (where R1 is a hydrophobic group such as an alkyl chain -(CFh )_n - CFE , and R2 is C _n H 2n+i , wherein x and n are integers, and 1 x 3), or use, for example, a polymer having a functional group selected from the group consisting of: -COOH, -PO3 Fh , - SH, or -NFh . In another embodiment, if the bottom surface of the nanowell 120 is made of a hydrophilic metal oxide, it can be modified to be hydrophobic, such as Rl_x -Si(0-R2) 4- _x (wherein R1 is a hydrophobic group such as an alkyl chain -(CFh )_n -CFh , and R2 is C_n H 2n+i , wherein x and n are integers, and 1 x 3), or using, for example, a polymer having a functional group selected from the group consisting of: -COOH, -PO3 ¾ , -SH, or -NH2 .

[0119] There may be one electrically active and addressable nanowell or there may be a plurality of electrically active and addressable nanowells in an array of nanowells. There may be

2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 10,000, 100,000, 1,000,000 or more than 1,000,000 nanowells in an array of nanowells. Peptides may be affixed to the electrically active and addressable surface with a bond, such as being ionically, covalently, or electrostatically coupled to a plurality of wells. The bond may bind one peptide per nanowell or FET or may bind a plurality of peptides per nanowell or FET. There may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,

35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000

10,000, 100,000, 1,000,000 or more than 1,000,000 peptides bound to a nanowell or FET. A nanowell may be formed in a coating layer using lithography. A nanowell may include an upper opening and a bottom surface, wherein the upper opening may be larger than the bottom surface. The nanowell may extend through a thickness of a portion of the at least one coating layer, a total thickness of the at least one coating layer, a total degree of the at least one coating layer, a partial thickness of the core layer, and a total of the at least one coating layer.

[0120] A nanowell may be formed using lithography and etching. For example, an oxide film may be formed on a substrate, such as a silicon dioxide substrate, with a process such as plasma chemical vapor deposition or thermal oxidation. A metal layer, such as a gold, platinum, silver, palladium, copper, or nickel layer, may be deposited on the oxide layer using a deposition method such as sputtering, evaporation, or ion beam deposition. A photosensitive layer, such as photoresist, may be applied to the metal layer by coating a photosensitive agent on the metal layer and forming a fine pattern, a mask, through a photolithography process using a stepper exposure equipment. The metal layer may be etched into a fine pattern shape by removing the photosensitive layer and depositing an insulating layer. When an etching is finished, the photosensitive layer may be removed. A subsequent photosensitive layer may be formed by coating a photosensitive agent on the insulating layer and forming a nanopattem through a photolithography process using a stepper exposure equipment. The insulating layer may be etched in a nanopatterned shape.

[0121] A microfluidic device may be operably coupled to the array of nanowells. One or a plurality of CNTFETs may be housed in a nanowell. One or a plurality of peptides may be contained to a single point and bind to the one or plurality of CNTFETs housed in a nanowell. The electrochemical reactivity of a CNT to the binding a peptide may be analyzed using a back- gated voltage sweep (V_bg) applied to the nanowell substrate, for example by determining conductivity after applying a voltage from -10 to 10 V. A change in charge, conductance, or impedance after binding of the peptide to a functionalized point on a CNTFET may provide a consistent signature for consecutive chemical reactions (e.g., N-terminal amino acid removal), molecular reactions, or molecular conformational changes.

[0122] A system may be configured to deliver an average of one peptide to each of a plurality of nanowells or microwells each containing or disposed adjacent to a FET. The FET may measure a label coupled to a peptide (e.g., an amino acid type specific label such as a histidine- specific epoxide label). For example, a label may comprise a moiety that generates a detectable change in FET conductance. A method for using the system may comprise determining whether a label is lost from a peptide upon cleavage (e.g., terminal amino acid removal or internal cleavage, such as with a protease or a chemical cleavage agent such as cyanogen bromide). A series of cleavage steps may be performed to obtain extended sequence information for the peptide. For example, 10 rounds of N-terminal amino acid removal on a peptide disposed within a nanowell may be coupled to FET detection of no label loss, no label loss, no label loss, histidine label loss, tyrosine label loss, cysteine label loss, no label loss, histidine label loss, no label loss, and no label loss, thus resolving the positions of histidine, tyrosine, and cysteine within the N-terminal sequence of the peptide. A method for using the system may also comprise identifying a peptide denaturation event. For example, a method may comprise disposing a peptide within a well (e.g., a microwell) adjacent to a FET, and continuously measuring the conductance of the FET as a condition is changed (e.g., temperature is raised or a reagent is titrated into solution with the peptide) to identify a signal change indicative of denaturation of the peptide (e.g., a drop or increase in FET conductance). A method for using the system may comprise detecting a disulfide bond in a peptide, for example by disposing the peptide within a well adjacent to a FET, adding a reductant configured for disulfide bond cleavage to the well, and measuring for a change in FET conductance associated with cleavage of a disulfide bond within the peptide. A method for using the system may comprise detecting a change in peptide charge upon cleavage. Such a method may comprise disposing a peptide within a well adjacent to (e.g., coupled to) a charge-sensitive FET, cleaving the peptide, and identifying whether a neutral, positive, or negative amino acid or peptide fragment was cleaved from the peptide. For example, five rounds of N-terminal amino acid removal may identify that a positively charged amino acid is removed following a first round of cleave, a neutral amino acid is removed after the second, fourth, and fifth rounds of cleavage, and that a negatively charged amino acid is removed after the third round of cleavage, thus providing a sequence of charged, neutral, and negatively charged amino acid residues within the peptide.

Methods For Peptide Electrosequencing

[0123] Another aspect of the present disclosure may provide a method for peptide sequencing where the peptide may be subjected to conditions sufficient to remove an amino acid from the peptide in a solution and using a sensor to measure a charge, conductivity, or impedance, or change thereof, in the solution subsequent to removal of said amino acid from said peptide performed in substantially real time. Determining a change in charge, conductivity, impedance, or change thereof may be applicable in determining consecutive chemical reactions, molecular reactions, or molecular conformational changes of a peptide and thus sequencing one or a plurality of peptides. A plurality of peptides deposited on, immobilized to, or disposed adjacent to an array may be sequenced and a plurality of identical sequencing reactions may occur in each occupied well simultaneously. By performing sequencing reactions in a plurality of wells simultaneously, a plurality of different sequencing reactions may also be performed simultaneously. A sequencing reaction can be run at a range of temperatures. A reaction may be run in the range of 30-60 °C, 35-55 °C, or 40-45 °C. The reaction may be performed at a temperature that prevent or limits the degradation of the protein or peptide. A suitable temperature may be about 41 °C. The solutions, including wash buffers or degradation enzymes, may be warmed to a suitable temperature in order not to alter the temperature in the wells.

[0124] Peptide sequencing may be carried out at a rate of at approximately at least one peptide per second. Peptide sequencing may be carried out at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 10,000, 100,000, 1,000,000, or more peptides per second. Peptide sequencing may be carried out at a rate of from about 1 to about 1,000,000 peptides per second, 10 to about 10,000 peptides per second, or about 100 to about 1,000 peptides per second. Peptide sequencing may be carried out at a rate of less than one peptide per second. The results of peptide sequencing may be read by a remote system of a user (e.g., a cellular network). Examples of remote systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), microscopes, optical sequencers, imaging platforms, a breadboard, chip, circuit board, telephones, smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. A remote system may be a remote computer system which may be accessed via a network. The readout of peptide sequencing may be carried out at a rate of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,

100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 10,000, 100,000, 1,000,000, or more sequenced peptides per second. The readout of peptide sequencing may be carried out at a rate of less than one peptide per second.

[0125] An electrosequencing method may comprise providing a peptide adjacent to a sensor (e.g., a FET), measuring a first electrical signal with the sensor, subjecting the peptide to conditions sufficient to remove an amino acid from the peptide, measuring a second electrical signal with the sensor, and determining a change between the first and the second electrical signals. In some cases, the change between the first and the second electrical signals (e.g., a change in current between the first and the second electrical signals) may identify a change in charge, conductivity, impedance, or any combination thereof. In some cases, the change between the first and the second electrical signals identifies an amino acid of the peptide. In some cases, the change between the first and the second electrical signals identifies the removed amino acid. In some cases, the change between the first and the second electrical signals identifies a label coupled to an amino acid removed from the peptide. In some cases, the change between the first and the second electrical signals identifies a sequence of the peptide. In some cases, the change between the first and the second electrical signals identifies a conformational change of the peptide. In some cases, the change between the first and the second electrical signals identifies a disulfide bond formation or a disulfide bond cleavage in the peptide. In some cases, the change between the first and the second electrical signals identifies a chemical modification (e.g., a post- translational modification or dephosphorylation) of the peptide.

[0126] In some cases, the change between the first and the second electrical signals identifies a set of potential identities for the removed amino acid. For example, an electrosequencing method may generate five distinguishable types of changes in electrical signals, wherein a first type of change identifies that an amino acid with an aliphatic side chain was removed from a subject peptide, a second type of change identifies that an amino acid with an aromatic side chain was removed from the subject peptide, a third type of change identifies that an amino acid with a carboxylate side chain was removed from the subject peptide, a fourth type of change identifies that a guanidium or amine side chain-containing amino acid was removed from the subject peptide, and a fifth type of change identifies that a thiol-, thioether, hydroxyl, or amide side chain-containing amino acid was removed from the subject peptide. A method may generate at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 types of signals corresponding to removal of different amino acids. In some cases, an additional type of signal identifies removal of a post-translationally modified amino acid, such as a citrullinated amino acid, a succinylated amino acid, a methylated amino acid, or a phosphorylated amino acid.

[0127] In some cases, the peptide is disposed within a well. In some cases, the peptide is the only peptide within the well. In some cases, the method comprises analyzing a plurality of peptides disposed in a plurality of wells. In some cases, at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, or at least 10⁷ peptides are analyzed within wells of an array of wells.

[0128] In some cases, the peptide comprises a labeled amino acid. The amino acid label may comprise a specificity for a type of amino acid (e.g., be configured to only couple to one type of amino acid, such as lysine). The peptide may comprise a label on each amino acid of a specific type. The peptide may comprise a plurality of amino acid-type specific labels. For example, the peptide may comprise a first type of label coupled to each lysine residue, a second type of label coupled to each cysteine residue, and a third type of label coupled to each glutamic acid and each aspartic acid residue. A label may comprise or generate an identifiable electrical signal, or may generate an identifiable change in an electrical signal (e.g., a change in conductance or impedance). Accordingly, a method may comprise identifying a removed amino acid by identifying an electrical signal or a change in electrical signal associated with a particular type of amino acid label.

[0129] In some cases, a change between a first and a second electrical signal is sufficient for identifying an amino acid type of a peptide (e.g., identifying a signal change following the loss of an amino acid or identifying a label coupled to an amino acid) when said measuring said first signal and said measuring said second signal are performed for less than 1 second (e.g., said measuring said first signal and said measuring said second signal each comprise less than 1 second of signal averaging), less than 2 seconds, less than 3 seconds, less than 4 seconds, less than 5 seconds, less than 6 seconds, less than 8 seconds, less than 10 seconds, less than 12 seconds, less than 15 seconds, less than 20 seconds, less than 30 seconds, less than 60 seconds, less than 90 seconds, less than 120 seconds, less than 150 seconds, less than 180 seconds, less than 300 seconds, or less than 600 seconds. In some cases, said change between said first and said second electrical signals is sufficient for distinguishing at least two groups of amino acids (e.g., a first group comprising amino acids with neutral side chains and a second group comprising amino acids with charged side chains) of said peptide when said measuring said first signal and said measuring said second signal are performed for less than 1 second, less than 2 seconds, less than 3 seconds, less than 4 seconds, less than 5 seconds, less than 6 seconds, less than 8 seconds, less than 10 seconds, less than 12 seconds, less than 15 seconds, less than 20 seconds, less than 30 seconds, less than 60 seconds, less than 90 seconds, less than 120 seconds, less than 150 seconds, less than 180 seconds, less than 300 seconds, or less than 600 seconds. [0130] An electrosequencing method may comprise an optical detection component. For example, fluorosequencing and electrosequencing may be performed in tandem on a peptide, wherein a sensor adjacent to the peptide measures an electrical signal and a fluorimeter measures a change in fluorescence from the peptide following sequential rounds of amino acid removal from the peptide.

Peptide Labeling

[0131] Various methods of the present disclosure comprise labeling one or more amino acid residues of a subject peptide or protein. A label may comprise an optically detectable moiety (e.g., a fluorescent dye). A label may comprise an electrochemically detectable moiety (e.g., a redox active moiety with a characteristic oxidation or reduction potential, such as ferrocene). A label may comprise an electrically detectable moiety (e.g., a moiety which affects a detectable change in FET conductance, such as a highly polarizable organobromine complex). A method may comprise labeling a single type of amino acid (e.g., every lysine or every cysteine) in the subject protein or peptide. A method may comprise labeling a plurality of types of amino acids in the subject protein or peptide (e.g., labeling lysine and tyrosine). A method may comprise labeling one, two, three, four, five, six, or more different types of amino acids residues in a subject peptide or protein. The labeling moieties that may be used include, for example, fluorophores, chromophores, and quenchers. A plurality of amino acid residues may include, for example, an N-terminal amino acid, cysteine, lysine, glutamic acid, aspartic acid, tryptophan, tyrosine, serine, threonine, arginine, histidine, methionine, or any combination thereof. Each type of labeled amino acid may be labeled with a different labeling moiety. For example, lysine, cysteine, histidine, tryptophan, and tyrosine may each be labeled with a different type of label. Alternatively, multiple amino acid residues may be labeled with the same labeling moiety such as aspartic acid and glutamic acid or asparagine and glutamine.

[0132] Labeling specificity can be a major challenge in some methods. In many cases, a label may comprise reactivity toward a plurality of amino acid types, and thus may need to be added at a specific point in a labeling scheme. For example, some maleimide labels can react with cysteine, lysine, and N-terminal amines. Discriminating between similarly reactive amino acid residues can require precise ordering of labeling steps. In the above maleimide example, lysine may be discriminated from cysteine by first reacting cysteine with a cysteine specific labeling step (e.g., iodoacetamide coupling at pH 7-8), thereby preventing further cysteine labeling in a subsequent lysine labeling step. A method may comprise cysteine labeling prior to lysine labeling. A method may comprise cysteine labeling prior to glutamate labeling. A method may comprise cysteine labeling prior to aspartate labeling. A method may comprise cysteine labeling prior to tryptophan labeling. A method may comprise cysteine labeling prior to tyrosine labeling. A method may comprise cysteine labeling prior to serine labeling. A method may comprise cysteine labeling prior to threonine labeling. A method may comprise cysteine labeling prior to histidine labeling. A method may comprise cysteine labeling prior to arginine labeling. A method may comprise lysine labeling prior to glutamate labeling. A method may comprise lysine labeling prior to aspartate labeling. A method may comprise lysine labeling prior to tryptophan labeling. A method may comprise lysine labeling prior to tyrosine labeling. A method may comprise lysine labeling prior to serine labeling. A method may comprise lysine labeling prior to threonine labeling. A method may comprise lysine labeling prior to arginine labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to tryptophan labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to tyrosine labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to serine labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to threonine labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to histidine labeling. A method may comprise carboxylate side chain (e.g., glutamate and aspartate side chain) labeling prior to arginine labeling. A method may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 amino acid labeling steps performed in a sequence configured to minimize or prevent label cross-reactivity (e.g., labeling more than the intended type or types of amino acids). Spectral Sequencing

[0133] In some aspects, the present disclosure may provide a method of identifying single molecule peptides using single-molecule spectroscopy, derivatized or labeled side chains comprising optically detectable signals, and peptide degradation. A single molecule peptide may be identified by the presence of certain spectroscopically active side chains that are affixed to the peptides, sequentially removed by peptide degradation, and then identified from a database using the unique signature of spectral changes. A spectroscopically active side chain may provide a spectral fingerprint of a post translational modification on an amino acid residue of a peptide or protein of interest. Such a spectral fingerprint may identify consecutive chemical reactions, molecular reactions, or molecular conformational changes of a peptide, thus sequencing one or a plurality of peptides.

[0134] A spectral sequencing method of the present disclosure may comprise fluorosequencing. Fluorosequencing may comprise removing peptides through techniques such as Edman degradation and subsequent visualization of labels coupled to the peptides. Sequential peptide removal may generate sequence or position-specific information by identifying the types and positions (e.g., within a peptide sequence) of labeled amino acids. For example, a reduction in fluorescence following an N-terminal amino acid removal step may indicate that a labeled amino acid, and thus that a specific type of amino acid, was disposed at a peptide N-terminal. Removal of each amino acid residue can carried out with a variety of different techniques including Edman degradation and proteolytic cleavage. The techniques may include using Edman degradation to remove the terminal amino acid residue. Alternatively, the techniques may involve using an enzyme to remove the terminal amino acid residue. These terminal amino acid residues may be removed from either the C-terminus or the N-terminus of the peptide chain. In situations where Edman degradation is used, the amino acid residue at the N-terminus of the peptide chain is removed.

[0135] The methods of sequencing or imaging the peptide sequence may comprise immobilizing the peptide on a surface. The peptide may be immobilized to the surface by coupling a peptide-derived cysteine residue, the peptide N terminus, or the peptide C terminus with the surface or with a reagent coupled to the surface. The peptide may be immobilized by reacting the cysteine residue with the surface or with a capture reagent coupled to the surface.

The peptide may be immobilized by coupling the peptide C-terminus with a C-terminal coupling reagent (e.g., a capture reagent comprising Formula (I)), and coupling the C-terminal coupling reagent to the surface or to a reagent coupled to the surface. The peptide may be immobilized on a surface. The surface may be optically transparent across the visible spectrum and/or the infrared spectmm. The surface may possesses a low refractive index (e.g., a refractive index between 1.3 and 1.6). The surface may be between 10 to 50 nm thick, between 20 and 80 nm thick, between 50 and 200 nm thick, between 100 and 500 nm thick, between 200 and 800 nm thick, between 500 nm and 1 pm thick, between 1 and 5 pm thick, between 2 and 10 pm thick, between 5 and 20 pm thick, between 20 and 50 pm thick, between 50 and 200 pm thick, between 200 and 500 pm thick, or greater than 500 pm in thickness. The surface may be chemically resistant to organic solvents. The surface may be chemically resistant to strong acids such as trifluoroacetic acid or sulfuric acid. A large range of substrates (like fluoropolymers (Teflon-AF (Dupont), Cytop® (Asahi Glass, Japan)), aromatic polymers (polyxylenes (Parylene, Kisco, Calif.), polystyrene, polymethmethylacrytate) and metal surfaces (Gold coating)), coating schemes (spin-coating, dip-coating, electron beam deposition for metals, thermal vapor deposition and plasma enhanced chemical vapor deposition) and functionalization methodologies (polyallylamine grafting, use of ammonia gas in PECVD, doping of long chain end- functionalized fluoroalkanes etc.) may be used in the methods described herein as a useful surface. A 20 nm thick, optically transparent fluoropolymer surface made of Cytop® may be used in the methods described herein. The surfaces used herein may be further derivatized with a variety of fluoroalkanes that will sequester peptides for sequencing and modified targets for selection. Alternatively, an aminosilane modified surfaces may be used in the methods described herein. The methods may comprise immobilizing the peptides on the surface of beads, resins, gels, quartz particles, glass beads, or combinations thereof. In some non-limiting examples, the methods contemplate using peptides that have been immobilized on the surface of Tentagel® beads, Tentagel® resins, or other similar beads or resins. The surface used herein may be coated with a polymer, such as polyethylene glycol. The surface may be amine functionalized or thiol functionalized.

[0136] A sequencing technique described herein involve imaging the peptide or protein to determine the presence of one or more labeling moieties (e.g., amino acid labels) coupled to the peptide. The sequencing technique may comprise imaging a plurality of peptides or proteins to determine the presence of one or more labeling moieties on individual peptides from among the plurality of peptides. The sequencing technique may comprise imaging at least 103, at least 104, at least 105, at least 106, at least 107, at least 108 or more proteins or peptides (e.g., imaging a portion of a surface comprising at least 103 to at least 108 proteins or peptides). These images may be taken after each removal of an amino acid residue and thus may enable determination of the location of the specific amino acid in the peptide sequence. A method of the present disclosure can identify the position of a specific amino acid in a peptide sequence. A method may be used to determine the locations of specific amino acid residues in the peptide sequence or these results may be used to determine the entire list of amino acid residues in the peptide sequence. A method may involve determining the location of one or more amino acid residues in the peptide sequence and comparing these locations to known peptide sequences, which may identify the entire list of amino acid residues in the peptide sequence. For example, identifying the positions of the lysines and cysteines in a 40 amino acid fragment of a human protein may uniquely identify the protein (e.g., only one human protin contains the specific pattern of lysine and cysteine residues identified in the 40 amino acid fragment).

[0137] An imaging method may involve a variety of different spectrophotometric and microscopy methods, such as fluorimetry, diffuse reflectance, interferometric scattering, Raman, resonance enhanced Raman, infrared absorbance, visible light absorbance, ultraviolet absorbance, and fluorescence. An imaging method may be performed in tandem with other methods of the present disclosure, such as electrosequencing. The fluorescent methods may employ such fluorescent techniques, such as fluorescence polarization, Forster resonance energy transfer (FRET), or time-resolved fluorescence. A spectrophotometric or microscopy method may be used to determine the presence of one or more fluorophores coupled to a single peptide. Such imaging methods may be used to determine the presence or absence of a label on a specific peptide sequence. After repeated cycles of removing an amino acid residue and imaging a subject peptide, the position of the labeled amino acid residue can be determined in the peptide. [0138] A spectroscopically active side chain affixed to a peptide may comprise a peptide coupled to an engineered side chain coupled to an array. An engineered side chain may comprise a functional group installed on a CNTFET, peptide, peptide, or molecule of interest. An engineered side chain may comprise an ionic bond between a post translational modification on an amino acid residue of the peptide or protein and a labeling reagent. An engineered side chain may comprise a covalent bond between a post translational modification on an amino acid residue of the peptide and a labeling reagent. The post translation modification may be on an amino acid residue of a protein. A labeling reagent may comprise a thiol group. The labeling reagent may comprise two or more thiol groups. The labeling reagent may be a fluorophore, oligonucleotide, or peptide-nucleic acid.

[0139] Post-translational modifications (PTMs) of proteins may be covalent attachments of chemical moieties on the side chains of select amino acids or the N and C termini of a peptide or a protein. The activity and functions of many proteins may be modulated by the nature of their PTMs. Some non-limiting examples of PTMs may include phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, trimethylation, alkylation, acylation, hydroxylation, or the attachment of a cofactor or nucleotide. A post translational modification on an amino acid may be phosphorylation on tyrosine, serine, or threonine such as when the post translational modification on the amino acid is phosphorylation on a serine. Alternatively, the post translational modification on the amino acid may be phosphorylation on a threonine. The post translational modification on the amino acid may be an N-glycosylation such as glycosylation of asparagine or arginine. The post translational modification on the amino acid may be an O- glycosylation such as glycosylation of serine, threonine, or tyrosine. The post translational modification on the amino acid may be trimethylation such as trimethylation of lysine.

[0140] In some aspects, the present disclosure may provide a method of identifying a post translational modification on an amino acid residue of a peptide or protein of interest comprising obtaining the peptide or protein of interest and a labeling reagent, reacting the peptide or protein of interest under conditions such that the post translational modification on the peptide or protein of interest may form a covalent bond between the amino acid residue to which the post translational modification was present and the labeling reagent to form a labeled peptide or protein of interest, and sequencing the labeled peptide or protein of interest. A reactive peptide or protein of interest may be formed by reacting the peptide or protein of interest with a phosphorylation post translational modification with a base. The base may be a rare earth metal hydroxide such as Ba(OH)2. The reactive peptide or protein of interest may be formed by reacting the peptide or protein of interest with a trimethyl post translational modification with silver oxide (Ag₂0) such as reacted with silver oxide in the presence of heat. The reactive peptide or protein of interest may be formed by reacting the peptide or protein of interest with a trimethyl post translational modification with a base. The base may be a nitrogenous base such as diisopropylethylamine or trimethylamine. The reactive peptide or protein of interest may be formed by reacting the peptide or protein of interest with a glycosylation post translational modification with an oxidizing agent. An oxidizing agent may be a hypervalent iodide reagent such as sodium periodate. The reactive group on the reactive peptide or protein of interest may be a double bond. The reactive peptide or protein of interest may be reacted with a labeling reagent through a thiolene-click reaction to form a labeled peptide or protein of interest. The reactive peptide or protein of interest may be reacted with the labeling reagent with a double bond in the presence of an olefin metathesis reagent to form a labeled peptide or protein of interest. The reactive peptide or protein of interest may be reacted with the labeling reagent through a cycloaddition reaction to form a labeled peptide or protein of interest.

[0141] A labeling reagent may be reacted with the reactive peptide or protein of interest to form a labeled peptide or protein of interest. The N-terminal amino acid may be labelled by replacing a post translational modification with a labeling moiety and the peptide may be sequenced to obtain the location of an amino acid residue and the identity of the post translational modification. A labeled peptide may be removed through a technique such as, for example, Edman degradation and subsequently analyzed to detect an alteration in an electric signal, indicating a specific amino acid with a particular modification has been cleaved. After repeated cycles of removing an amino acid residue and analyzing the peptide sequence, the position of the amino acid residue may be determined in the peptide.

[0142] An engineered side chain may be spectrally activated, and the spectral signal of the attached labelled peptide may be recorded and analyzed to determine its origin. A spectral signal of a hypothetical engineered side chain may be designed for the explicit purpose of having a signature in the stimulation band. A spectrally activated side chain may identify a unique spectral signature upon stimulation. A spectrally activated side chains may be affixed to the side chain of a peptide onto a single molecule FET, such as a CNTFET. A spectrally activated side chain may be stimulated with any relevant electromagnetic signal. A spectrally activated side chain may be stimulated with either constant, pulsed, or a mixture thereof stimulation. A spectrally activated side chain may be stimulated with a FET. The FET may be an ion-sensitive field effect transistor (ISFET). The ISFET may be an array of ISFETs. These ISFET arrays may facilitate peptide sequencing techniques based on monitoring changes in current, impedance, change in conductivity or by providing a spectral stimulation to a spectrally activated sidechain. The ISFET may be but is not limited to an enzyme-sensitive field effect transistors (ENFETs), a carbon nanotube field effect transistor (CNFET), an immuno-field effect transistor (ImmunoFET), or a biologically sensitive field effect transistor (BioFET).

[0143] A spectral signature may be recorded and analyzed with a remote system of a user. A remote system may include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), microscopes, optical sequencers, imaging platforms, a breadboard, chip, circuit board, telephones, cellular networks, smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), personal digital assistants, or any relevant software. A remote system may be a remote computer system which may be accessed via a network. Multiple remote systems may be employed to record and analyze a spectral signature. Such a spectral analysis may be used to estimate the concentrations of an individual species of peptide present in the original mixture. After detecting one signal indicative of a change in spectra from one peptide or portion of peptide, the entire collection of peptides may lose subsequent amino acids from the peptide to detect additional signals indicative of a change in spectra after removal of additional amino acids from the peptide. The entire collection of peptides may lose one amino acid through a degradation process, such as through Edman or organophosphate degradation. Degradation may employ a cyclical and processive method. The unique spectral signature(s) over all addressable units may be collected between peptide degradation cycles.

[0144] Another aspect of the present disclosure provides a method of peptide sequencing comprising providing an array having a peptide immobilized thereto, wherein the peptide is adjacent to a sensor, subjecting the peptide to conditions sufficient to remove an amino acid from the peptide, using the sensor to measure a non-optical signal in the solution subsequent to removal of an amino acid from the peptide, and using a non-optical signal to identify a sequence of the peptide. A non-optical signal may comprise a spectral signal, electrical, gamma ray, X-ray, ultraviolet, infrared, radio signal, or other electromagnetic signal. Peptide sequencing comprising a non-optical signal may provide a method for determining a peptide sequence using a sequence of reactions without a label. A label-free method may provide a faster, more efficient sequencing process using electrosequencing or spectral sequencing.

Sample Types

[0145] The methods described herein may comprise analyzing a biological sample. A biological sample may be derived from a subject (e.g., a patient or a participant in a study), from a tissue sample (e.g., an engineered tissue sample), from a cell culture (e.g., a human cell line or a bacterial colony), from a cell (e.g., a cell isolated during a single cell sorting assay), or a portion thereof (e.g., an organelle from a cell or an exosome from a blood sample). A biological sample may be synthetic, such as a composition of synthetic peptides. A sample may comprise a single species or a mixture of species. A biological sample may comprise biomaterial from a single organism, from a colony of genetically near-identical organisms, or from multiple organisms (e.g., enterocytes and microbiota from a human digestive tract). A biological sample may be fractionated (e.g., plasma separated from whole blood), filtered, or depleted (e.g., high abundance proteins such as albumin and ceruloplasmin removed from plasma). A biological sample may be partitioned into droplets or into wells (e.g., wells of a wellplate or nanowells of a nanowell array).

[0146] A sample may comprise all or a subset of the biomolecules from the subject, tissue sample, cell culture, cell, or portion thereof. For example, a sample from a subject may comprise the majority of proteins present in that subject, or may comprise a small subset of the proteins from that subject. A biological sample may comprise a bodily fluid such as cerebral spinal fluid, saliva, urine, tears, blood, plasma, serum, breast aspirate, prostate fluid, seminal fluid, stool, amniotic fluid, intraocular fluid, mucous, or any combination thereof. A biological sample may comprise a tissue culture, for example a tumor sample, or tissue from a kidney, liver, lung, pancreas, stomach, intestine, bladder, ovary, testis, skin, colorectal, breast, brain, esophagus,, placenta, or prostate.

[0147] The biological sample may comprise a molecule whose presence or absence may be measured or identified. The biological sample may comprise a macromolecule, such as, for example, a polypeptide or a protein. The macromolecule may be isolated (e.g., separated from other components from which it was sourced) or purified, such that the macromolecule comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of a composition by weight (e.g., by dry weight or including solvent). The biological sample may be complex, and may comprise a plurality of components (e.g., different polypeptides, heterogenous sample from a CSF of a proteopathy patient). The biological sample may comprise a component of a cell or tissue, a cell or tissue extract, or a fractionated lysate thereof. The biological sample may be substantially purified to contain molecules of a single type (peptides, nucleic acids, lipids, small molecules). A biological sample may comprise a plurality of peptides configured for a method of the present disclosure (e.g., digestion, C- terminal labeling, or fluorosequencing).

[0148] Methods consistent with the present disclosure may comprise isolating, enriching, or purifying a biomolecule, biomacromolecular structure (e.g., an organelle or a ribosome), a cell, or tissue from a biological sample. A method may utilize a biological sample as a source for a biological species of interest. For example, an assay may derive a protein, such as alpha synuclein, a cell, such as a circulating tumor cell (CTC), or a nucleic acid, such as cell-free DNA, from a blood or plasma sample. A method may derive multiple, distinct biological species from a biological sample, such as two separate types of cells. In such cases, the distinct biological species may be separated for different analyses (e.g., CTC lysate and huffy coat proteins may be partitioned and separately analyzed) or pooled for common analysis. A biological species may be homogenized, fragmented, or lysed prior to analysis. In particular instances, a species or plurality of species from among the homogenate, fragmentation products, or lysate may be collected for analysis. For example, a method may comprise collecting circulating tumor cells during a liquid biopsy, optionally isolating individual circulating tumor cells, lysing the circulating tumor cells, isolating peptides from the resulting lysate, and analyzing the peptides by a fluorosequencing method of the present disclosure. A method may comprise partitioning a sample (or a purified or isolated portion of a sample) into a plurality of droplets, and analyzing the sample with a method disclosed herein (e.g., peptide electrosequencing).

[0149] Methods consistent with the present disclosure may comprise nucleic acid analysis, such as sequencing, southern blot, or epigenetic analysis. Nucleic acid analysis may be performed in parallel with a second analytical method, such as a fluorosequencing method of the present disclosure. The nucleic acid and the subject of the second analytical method may be derived from the same subject or the same sample. For example, a method may comprise collecting cell free DNA and a peptides from a human plasma sample, sequencing the cell free DNA (e.g., to identify a cancer marker), and performing proteomic analysis on the plasma proteins.

Computer Systems

[0150] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 1 shows a computer system 101 that is programmed or otherwise configured to perform the methods described herein. The computer system 101 can regulate various aspects of the present disclosure, such as, for example, determining the ratio of peptides immobilized to a FET array. The computer system 101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0151] The computer system 101 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 105, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 101 also includes memory or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage and/or electronic display adapters. The memory 110, storage unit 115, interface 120 and peripheral devices 125 are in communication with the CPU 105 through a communication bus (solid lines), such as a motherboard. The storage unit 115 can be a data storage unit (or data repository) for storing data. The computer system 101 can be operatively coupled to a computer network (“network”) 130 with the aid of the communication interface 120. The network 130 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 130 in some cases is a telecommunication and/or data network. The network 130 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1130, in some cases with the aid of the computer system 101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server. [0152] The CPU 105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 110. The instructions can be directed to the CPU 105, which can subsequently program or otherwise configure the CPU 105 to implement methods of the present disclosure. Examples of operations performed by the CPU 105 can include fetch, decode, execute, and writeback.

[0153] The CPU 105 can be part of a circuit, such as an integrated circuit. One or more other components of the system 101 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0154] The storage unit 115 can store files, such as drivers, libraries and saved programs. The storage unit 115 can store user data, e.g., user preferences and user programs. The computer system 101 in some cases can include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet.

[0155] The computer system 101 can communicate with one or more remote computer systems through the network 130. For instance, the computer system 101 can communicate with a remote computer system of a user (e.g., a cellular network). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 101 via the network 130.

[0156] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 105. In some cases, the code can be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105. In some situations, the electronic storage unit 115 can be precluded, and machine-executable instructions are stored on memory 110.

[0157] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre compiled or as-compiled fashion.

[0158] Aspects of the systems and methods provided herein, such as the computer system 1101, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0159] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0160] The computer system 101 can include or be in communication with an electronic display 135 that comprises a user interface (UI) 140, for example, determining the ratio of peptides immobilized to a FET array or the flow rate of the analyte solution comprising a peptide. Examples of UFs include, without limitation, a graphical user interface (GET) and web- based user interface.

[0161] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 105. The algorithm can, for example, determine the ratio of peptides immobilized to a FET array or the flow rate of the analyte solution comprising a peptide.

Field Effect Transistors

[0162] The present disclosure provides field effect transistors that may be employed for use with methods, devices and systems disclosed herein. FIG. 2 depicts a cross-section of an example of a portion of a p-type silicon substrate (207) consistent with the present disclosure. The p-type silicon substrate may be one pixel (200) of a plurality of pixels, in which the n-type nanowell (206) containing source (201), drain (203), and top gate (202) of the FET is shown.

The illustration of FIG. 2 is not to scale and may not exactly represent the actual layout of a particular pixel in the design, rather these figures are conceptual in nature and are provided primarily to illustrate the requirements of multiple n-wells, and separate n-channel FETs fabricated within the p-type substrate. A carbon nanotube (204) housed within the nanowell may be functionalized to facilitate analyte (205) binding at a single point, such as the binding of a peptide. The carbon nanotube lies above a silicon dioxide bulk dielectric layer (208) and below a gate oxide layer. The gate oxide layer may extend over the source and drain (210). Each well acts as a sensor to detect in real-time through quantized changes in conductance, charge, impedance, or pH, single point binding of an analyte to the nanotube as well as consecutive chemical reactions, molecular interactions, or conformational changes occurring on the single molecule FET probe.

[0163] A CNTFET may be a back gated CNTFET, comprising patterned (e.g., parallel) metal depositions across a conductor or semiconductor substrate. Two strips may constitute a source and a drain contact for a FET, while the substrate may be a gate of the FET. A CNTFET may be top-gated, comprising a gate contact deposited on a gate dielectric. A CNTFET may comprise a wrap-around gate CNTFET, in which a CNT is wrapped around a gate contact using a method such as atomic layer deposition. A CNTFET may also comprise a suspended CNTFET.

[0164] FIG. 3 illustrates a cross-section of a p-type FET 301 consistent with the present disclosure. A p-type FET may comprise a p-type semiconductor 302 and an n-type nanowell

303. Source 304 and drain 305 p-type semiconductors may be disposed within an n-type nanowell 303, with a defined voltage of V_DS between them. An n-type semiconductor 306 may contact the n-type nanowell. A passivating or semiconductor layer 307 may be disposed above

304, 305, and 306, and may comprise openings through which electrical connections (e.g., contacts such as 308) may provide connection to the n-type nanowell. A further semiconductor

310 may serve as a gate material between 304 and 305.

[0165] A FET may be responsive to the surrounding chemical environment. Buildup of charged or polarizable materials (e.g., dissolved analytes) may generate a measurable change in conductivity and/or capacitance of the gate 310, such that a negative voltage applied across the gate may generate a “p-channel” 311. The p-channel 311 carry a current through 304 and 305 upon application of sufficient voltage, hereinafter referred to as the threshold voltage (VTH). The metal contact 309 may hold multiple components, such as the n-type well and the source, at a common potential.

[0166] The polysilicon gate 310 of the FET 301 may be coupled to multiple metal layers disposed within one or more additional oxide layers disposed above the gate oxide 307 to form a “floating gate” structure 312. The floating gate structure may be electrically isolated from other conductors associated with the FET; namely, it may lie between the gate oxide 307 and a passivation layer 313. In the FET 301, the passivation layer 313 may constitute an ion-sensitive membrane that gives rise to the ion-sensitivity of the device; for example, the presence of ions in an “analyte solution” 314 (a solution containing ions of interest) in contact with the passivation layer 313, particularly in a sensitive area above the floating gate structure 312, may alter the electrical characteristics of the FET so as to modulate a current flowing through the p-channel

311 between the source 304 and the drain 305.

[0167] Charge buildup at an interface between the passivation layer 313 and the sample 314 may generate a surface potential. The surface potential may depend on pH, temperature, or ion concentration in the sample. The surface potential may depend on and affect the VTH of the FET, and thus may be detected by potentiometric measurement performed by the system. In some cases, a system may comprise a first FET for reference measurements (e.g., exposed to a standard with known analyte concentrations) and a second FET for sample measurements (e.g., in contact with a sample). The reference electrode(s) 315 may be electrically coupled to the array, an array controller, or directly to a computer to facilitate analyte measurements based on voltage signals obtained from the array. The reference electrode may serve as an internal calibrant for a measurement system. A system may also comprise a plurality of reference electrodes exposed to a plurality of solutions or conditions, which may increase measurement accuracy or precision. A reference electrode may be coupled to or disposed near a label or a plurality of labels, such as amino acid specific labels.

[0168] FIG. 4 illustrates a peptide processing system 400 comprising a large-scale FET array, according to one inventive embodiment of the present disclosure. In one aspect, the system 400 includes a semiconductor/microfluidics hybrid structure comprising sensor array 404 and a microfluidics flow cell 403. In another aspect, the flow cell 403 is configured to facilitate the sequencing of an analyte 401 disposed in the flow cell via the controlled admission to the flow cell of a number of sequencing reagents 407. As illustrated in FIG. 4, the admission of the sequencing reagents to the flow cell 403 may be accomplished via one or more valves 402 and one or more pumps that are controlled by computer system 406. Via an array controller 405 (also under operation of the computer system 406), the FET array may be controlled so as to acquire data relating to analyte measurements, and collected data may be processed by the computer 405 to yield information associated with the processing of the analyte.

Examples

Peptide Preparation

Peptide synthesis

[0169] Test peptides are synthesized using a Liberty Blue Microwave Peptide Synthesizer (CEM Corporation). Amino acids are incorporated as common Fmoc protected derivatives (P3 Biosystems), using DIC/Oxyma coupling strategies using dimethylformamide (DMF) as a solvent (1:1: 1). The peptides are coupled for 120 seconds at 90°C. The Fmoc group is removed with 20% piperidine at 90°C for 60 seconds. Peptides are cleaved from the resin using a standard cocktail containing trifluoroacetic acid, triisopropylsilane, and FhO (95:2.5:2.5 eq) for 2.5 hours at room temperature, afterwards the peptide mixture was concentrated under a nitrogen stream, the sample is precipitated by adding 10 volumes of diethyl ether and collected by centrifuging at 7,000 gravity of the Earth (g) for 10 minutes. The peptides are purified using reverse phase high- pressure liquid chromatography (RP-HPLC) using a Grace- Vydac C18 column (4.6x250mm) and a 0-50% acetonitrile (0.1% formic acid) over 60 minutes. The fractions are analyzed by mass spectrometry and pure peptide was lyophilized to dryness. Organophosphate-mediated in-solution cleavage

[0170] Peptides are suspended in an organic solvent (DCM, DMF, or a 1 : 1 mixture of DCM/DMF) and incubated for 10 minutes with 5% DBU (v/v, final concentration) at room temperature. Organophosphate dichloride (5% v/v, final concentration) is then added and incubated with the peptide for 45 minutes at room temperature. The peptide mixture is then concentrated using N2 to remove DCM. The cleavage reaction is completed by adding in 10% formic acid and incubating for 45 minutes. When performed in solution, this reaction is also complete after 45 minutes with the addition of water, as the residual acid contained in the EDCP is sufficient for the reaction. The reaction is analyzed immediately using a liquid chromatography-mass spectrometry (Agilent). Or the peptide can be precipitated using a TCA precipitation protocol if the mass spectrometry data is difficult to interpret. For TCA precipitation, peptide solutions are supplemented with TCA to a final concentration of 50%. This is incubated on ice for 10 minutes. The sample is centrifuged at 14,000 g for 10 minutes to pellet the peptide. The pellet is carefully washed with acetone twice to remove residual TCA. Non-Optical Sequencing Example

[0171] Non-optical sequencing of peptides may occur using either a spectral or electromagnetic signature (FIG. 5). Peptides in a peptide mixture (such as from a cell lysate or from synthesis) may be either labelled with an electrical or spectrally relevant label or may use no label at all. The labelled or non labelled peptides are diluted and coupled onto an electrically active and addressable surface (500) where that surface has Field Effect Transistors (FETs), such as a carbon nanotube transistor, or is a patterned nanowell with electrical connections (510). The labelled or non-labelled peptides are directed to the electrically active surface with a bond such that there is one peptide per sensor (520). Each addressable sensor is electrically stimulated (530), and an electrical or spectral signature recorded (540) due to the adjacent attached peptide. Peptide degradation (550) is then subsequently performed over the surface whereby the entire collection of peptides loses one amino acid and an affiliated label that was affixed to it; thus, potentially changing the local spectroscopic signal. The spectral signatures over the addressable units between peptide degradation cycles are collected (560) and the unique spectral changed that occur from each recording device are analyzed to determine the set of possible peptides each spectral signature corresponds (570). Thus, a spectral analysis is used to estimate the concentrations of the individual species of peptide present in the original mixture (580).

CNTFET Formation

[0172] Carbon nanotube FET (CNTFET) devices are constructed in the following manner. First, nanotubes are grown at 890° C on the surface of 1 ^c 1 cm² bare Si (500 pm)/Si0₂ (285 nm) die via chemical vapor deposition. The average spacing between grown nanotubes is — 1 nanotube per 100 pm. Second, 64 source and drain electrodes (each 8 mm ^c 15 pm, segmented into 16 blocks of four pairs) are patterned orthogonal to the growth direction of nanotubes using a bilayer-resist photolithography process. The gap between electrodes is 4 pm, defining the nanotube channel length.

[0173] Titanium metal (100 nm) is deposited via electron-beam deposition, and the photoresist stack is lifted off. Large rectangular bars (8 mm ^c 100 pm) are photolithographically defined above and below the electrode pattern, and e-beam platinum (100 nm) is deposited to act as a pseudo-reference gate electrode. Following SEM inspection, nanotubes that bridge source-drain electrode pairs are identified. Those that transit the electrode gaps and are likely single-walled (diameter <2 nm, as confirmed via Raman spectroscopy and AFM characterization) are protected with a photoresist mask. All other nanotubes are etched with oxygen plasma in a Technics RIE tool (250 mtorr O2, 50 W, 12 s).

Nanowell Formation

[0174] Nanowells are patterned in a thin layer of poly(methyl methacrylate) (PMMA A2 950k), spincoated at 5000 rpm for 60s. The PMMA thickness is measured as approximately 70 nm using AFM. Writing is done using a high-resolution electron beam lithography writer (NanoBeam nB4). To maximize the resolution, writing is done a low current (1 nA) and patterns developed in a 4 °C solution of isopropyl alcohol and deionized water (3:1 IPA: H20). Nanowells size and full-depth development are assessed using AFM and by evaporating a thin metal layer (Ti, 8 nm) inside the nanowells followed by lift-off. Single-point functionalization is obtained inside the smallest 20 nm wide nanowells, using aryldiazonium chemistry. The 4- carboxybenzenediazonium tetrafluorob orate (CBDT) reagent is synthesized according to McNab et al. Functionalization is done by dissolving lOmM CBDT in aqueous phosphate buffer (100 mM, pH 8) and immediately immersing mask-covered devices in the solution. Devices are incubated in the solution at room temperature for 24 h to saturate the yield of attached functional groups. Although this chemistry can usually be done in a variety of solvents, aqueous conditions are necessary here to prevent dissolution of the PMMA mask.

Functionalization and Peptide Delivery

[0175] Devices are exposed to 10 mM 4-formylbenzene diazonium hexafluorophosphate dissolved in 100 mM sodium phosphate buffer solution with pH 8.0 overnight on a shaking tray and in the dark to functionalize them using diazonium. Afterwards, the thin PMMA layer is removed in heated acetone (55° C) for 2 h, rendering the surface of the chip clean again. Chips are wirebonded to ball-grid array packages using an automated wirebonder, and subsequently placed onto a custom-made circuit board described below.

[0176] After the functionalization stage, and after being mounted onto the circuit board, wirebonded chips are exposed to 10 mM of peptide solution in a 100 mM sodium phosphate buffer solution with pH 8.0, with added 200 mM sodium cyanoborohydride (NaBH^CN) dissolved in 1 N NaOH, which is used to reduce the Schiff base formed between the amine and aldehyde, converting into a stable secondary amine. Peptides are synthesized with standard fmoc chemistry using an automated solid-phase peptide synthesizer (Liberty blue microwave peptide synthesizer; CEM Corporation).

[0177] A PDMS microfluidic channel is used for interfacing solution with the fully fabricated CNTFET -nanowell devices. The PDMS microfluidic mold is constructed from a pattern drawn on a thick SU-8 layer. Such microfluidic channels have the following dimensions: 7-mm long, 750-pm wide and roughly 500-pm tall. Inlet and outlet holes are punched into the channel, and two sterile tubing segments are inserted. A syringe pump connected to the outlet terminal withdraws fluid exiting the channel, thus allowing full control over flow rates.

Computer Interfacing

[0178] A custom-made printed circuit board for data acquisition and a temperature sensor/controller for fixing and modulating the temperature in the vicinity of the chip surface also comprise the set up. The circuit board contains independently addressable measurement channels that are simultaneously interrogated in real-time. The circuitry for each channel incorporates tunable drain and source potentials and is composed of two mutable gain stages: a front-end transimpedance amplification stage with a fixed resistive gain of 1 MW, followed by an inverting voltage amplifier with variable gain from 2 x to 200 x. Each channel, furthermore, utilizes a second-order active filter topology, limiting the signal bandwidth to 5 kHz. Readings from each channel are sampled at a rate of 25 kSps. The hardware-software interface is governed by an Opal Kelly XEM6010 FPGA module, which connects to multiplexers and analog-to-digital converters on the printed circuit board, and with the PC via a USB 2.0 connection. Temperature control is achieved by using a commercially available Thermostream unit capable of monitoring and modulating the temperature of forced air within a manufactured enclosure surrounding the fabricated chip and microfluidics. The temperature is allowed to reach steady state before an experimental condition is recorded.

Signal Post-Processing

[0179] Once acquired through the FPGA-to-PC interface, data are post-processed using customizable scripts. Local drift for 5 min of transient recording from each measurement channel is systematically removed. Resulting signals are low-pass filtered with a fourth-order Butterworth filter to 1 kHz to eliminate noise close to the cutoff frequency of the anti-aliasing filter of each channel. Every trace is further analyzed using an iterative event detection algorithm, which assumes a two-state model, with wandering baseline correction. Traditionally, this single-molecule data analysis methodology is applied to evaluate current blockades due to nanopores, but the same technique can be extended to any signature with two-state random telegraph noise.

[0180] Compared to an alternate signal processing paradigm for single-molecule trajectories, the hidden Markov model, the iterative detection algorithm utilizes rudimentary statistical metrics (for example, moving average, RMS noise level) rather than Markovian matrices and machine learning principles. Consequently, the execution speed is faster, allowing for more rapid tuning of parameters by the user. Idealized traces, resulting from fits to the raw data in the iterative detection algorithm, are used to extract single-molecule binding kinetics information. Assuming the same two-state model as before, events are classified into a Tow’- and a ‘high’- conductance state. Each idealized data trace for a given experimental condition is divided into five equal parts, from which cumulative density functions are constructed for each state. Each cumulative density function is normalized to the number of event counts, thus yielding survival probability plots. Average kinetic DNA hybridization/melting rates and associated error bars are calculated from them. Algorithms for this portion of the analysis are adapted from HaMMy scripts previously written in MATLAB.

[0181] Errors for the derived kinetic and thermodynamic parameters were calculated from the standard errors of the corresponding least-square weighted fits. The errors for the derived differences between thermodynamic values were combined in quadrature. The errors for parameters derived from quotients (that is, the E_m, calculated by dividing the intercept by the slope) were obtained by taking the root of the sum of the squares of the fractional errors in the original quantities.

Optical Sequencing Example

Labeling protocol for phosphorylation peptide synthesis and purification [0182] Optical sequencing of peptides (600) may occur by labelling amino acids within a mixture of peptides (such as from a cell lysate or from synthesis) with labels that are specific to certain amino acids (610), preparing those amino-acid labels with spectroscopically active side chains so that each labelled amino acid ends up with a unique spectroscopic signature (FIG. 6). Then performing peptide degradation (620) over the surface, whereby the entire collection of peptides loses one amino acid and any affiliated label that was affixed to it; thus, potentially changing the local spectroscopic signal (630). The spectroscopic signature may then be recorded and analyzed to determine which of a set of possible peptides each signature may have come from (640), thus estimating the concentrations of individual peptides in the original mixture (650).

[0183] All peptides are synthesized with standard fmoc chemistry using an automated solid- phase peptide synthesizer (Liberty blue microwave peptide synthesizer; CEM Corporation). The standard Fmoc-amino acid building blocks and the Fmoc-O-benzylphosphoserine (Cat #: 03734) are purchased from Chemlmpex Inc (IL, USA). The peptides are cleaved and de-protected using acid cleavage cocktail, comprising TFA: water: triisopropylsilane (9.5:0.25:0.25 v: v: v mixture). After removal of TFA by drying with nitrogen, the peptide is precipitated with cold ether and centrifuged for 10 mins at 8000 ref. The pellet is resuspended in acetonitrile/water (1:1 v: v mixture) and purified by preparative high-performance liquid chromatography (Shimadzu Inc.) with an Agilent® Zorbax® column (4.6 x 250 mm) operating at 10 mL/min flow rate with a gradient of 5-95% methanol (0.1% formic acid) over 90 minutes. The fraction containing the peptide is collected, and the volume reduced using a rotary evaporator before lyophilization. Nanowell FET Sequencing Example

[0184] A plurality of peptides is distributed across a plurality of nanowells, such that an average of one peptide is delivered to each nanowell. The sidewall of each nanowell comprises an ISFET configured to detect transient metal binding. Each peptide is labeled with a tryptophan specific label comprising a high-spin Cobalt(II) complex configured to transiently adsorb to the ISFETs of the nanowells, and a C-terminal carbodiimide linker configured to couple to the bottom of a nanowell, such that, upon diffusion into a nanowell, a peptide becomes immobilized within the nanowell. The conductances of the nanowell ISFETs are simultaneously measured over 10 seconds of signal averaging. Each nanowell signal is individually transformed into an average current level, thereby identifying the number of Cobalt(II) complexes (and correspondingly the number of tryptophan residues) in each nanowell. The peptides are then subjected to alternating rounds of N-terminal amino acid removal and tryptophan detection, until 20 sequential positions of each peptide are identified. The tryptophan sequences of each peptide are processed against a database of known proteins, thereby identifying a subset of the plurality of peptides.

Nanowell FET Denaturation Assay Example

[0185] A folded, competent enzyme is coupled to a thiol surface functionalization of a graphene channel of a first FET. The conductance of the first FET drops as a result of the enzyme binding. The first FET and a reference FET (not coupled to an enzyme) are gradually subjected to increasingly chaotropic conditions through guanidinium thiocyanate titration. The reference FET provides a benchmark conductance value for normalizing the first FET conductance. Once guanidinium concentration is increased beyond a threshold value, the enzyme denatures, generating a measurable increase in conductance of the first FET. The concentration of guanidinium thiocyanate is recorded, thereby identifying the denaturation midpoint (Cm) of the enzyme. This process is performed in parallel for at least 5% of the proteins collected from a single cell lysate. The distribution of Cm values identified for the proteins is sufficient to uniquely identify the cell.

[0186] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for polypeptide sequencing, comprising: a) providing an array having a polypeptide immobilized thereto, wherein said polypeptide is adjacent to a sensor; b) subjecting said polypeptide to conditions sufficient to remove an amino acid from said polypeptide in a solution; c) using said sensor to measure a charge, conductivity, or impedance, or change thereof, in said solution subsequent to removal of said amino acid from said polypeptide; and d) using at least said charge, conductivity, or impedance, or change thereof, to identify a sequence of said polypeptide.

2. The method of claim 1, wherein (b) comprises subjecting said polypeptide to Edman degradation.

3. The method of claim 1, wherein (b) comprises mixing said polypeptide with a diactivated phosphate or phosphonate to form a reaction mixture, and mixing said reaction mixture with an acid to remove said amino acid.

4. The method of claim 3, wherein said diactivated phosphate or phosphonate is a dihalophosphate ester.

5. The method of claim 1, further comprising repeating (b) and (c) to measure an additional charge, conductivity, or impedance, or change thereof, in said solution subsequent to removal of an additional amino acid from said polypeptide.

6. The method of claim 1, wherein (d) comprises identifying said amino acid removed in (b).

7. The method of claim 1, wherein (d) comprises identifying a chemical modification of said amino acid removed in (b).

8. The method of claim 7, wherein said chemical modification comprises a post-translational modification.

9. The method of claim 7, wherein said chemical modification comprises a chemical label.

10. The method of claim 7, wherein said chemical modification comprises a disulfide.

11. The method of claim 1, wherein (a) comprising immobilizing another polypeptide to said array, thereby providing said another polypeptide to said array.

12. The method of claim 11, wherein said another polypeptide is derived from a plurality of polypeptides or a protein.

13. The method of claim 12, wherein said plurality of polypeptides are provided to said array in a Poisson distribution.

14. The method of claim 12, wherein said plurality of polypeptides are provided to said array in a super-Poisson distribution.

15. The method of claim 1, wherein said measuring comprises at most 5 seconds of signal averaging.

16. The method of claim 1, wherein said change in said impedance is at least 1 kQ.

17. The method of claim 1, wherein said change in said conductivity is at least 10⁶ Scm ¹.

18. The method of claim 1, wherein said sensor comprises a field effect transistor (FET).

19. The method of claim 18, wherein said FET is selected from a group consisting of ion- sensitive field effect transistor (ISFET), metal-oxide-semiconductor field effect transistor (MOSFET), enzyme field effect transistor (EnFET), chemically-sensitive field effect transistor (ChemFET), a carbon nanotube field effect transistor (CNFET), immuno-field effect transistor (ImmunoFET), or a biologically sensitive field effect transistor (BioFET).

20. The method of claim 19, wherein said FET comprises a floating gate.

21. The method of claim 20, wherein said floating gate has a size greater than 1 nm² having a trapped charge of less than 240 V.

22. The method of claim 19, wherein said FET occupies an area of up to 1 mm².

23. The method of claim 1, wherein said sensor measures said charge or change thereof.

24. The method of claim 1, wherein said sensor measures said conductivity or change thereof.

25. The method of claim 1, wherein said sensor measures said impedance or change thereof.

26. The method of claim 1, wherein said array comprises a support, and wherein said polypeptide is immobilized to said support.

27. The method of claim 26, wherein said support comprises a bead.

28. The method of claim 26, wherein said support comprises a surface of a well.

29. The method of claim 28, wherein said well is among a plurality of wells.

30. The method of claim 29, wherein said plurality of wells comprises at least two wells.

31. The method of claim 29, wherein said plurality of wells comprises at least 10,000 wells.

32. The method of claim 1, wherein said polypeptide is coupled to a capture moiety coupled to said array.

33. The method of claim 32, wherein said array comprises a plurality of individually addressable sites, and wherein said polypeptide is immobilized to an individually addressable site of said plurality of individually addressable sites.

34. The method of claim 33, wherein said polypeptide is covalently coupled to said array.

35. The method of claim 33, wherein said polypeptide is ionically coupled to said array.

36. The method of claim 1, wherein said sensor comprises a carbon nanotube transistor.

37. The method of claim 1, wherein said array comprises a plurality of sites, and wherein in (a) said polypeptide is immobilized to a single site of said plurality of sites.

38. The method of claim 1, wherein (b) and (c) are performed in substantially real time.

39. The method of claim 1, wherein (d) further comprises identifying a conformation or a chemical modification of said polypeptide.

40. The method of claim 1, wherein (d) further comprises identifying a disulfide bond of said polypeptide.

41. The method of claim 1, wherein said polypeptide is coupled to an engineered side chain coupled to said array.

42. The method of claim 41, wherein said engineered side chain comprises a covalent bond between a post translational modification on an amino acid residue of said polypeptide and a labeling reagent.

43. The method of claim 42, wherein said post translational modification comprises phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, or trimethylation.

44. The method of claim 41, wherein said engineered side chain comprises an ionic bond between said post translational modification on said amino acid residue of said peptide or protein and a labeling reagent.

45. The method of claim 44, wherein said post translational modification on said amino acid residue comprises phosphorylation, glycosylation, nitrosylation, citrullination, sulfenylation, or trimethylation.

46. The method of claim 41, wherein said engineered side chain is spectrally activated and a spectral signature of said attached labelled peptide is recorded.

47. The method of claim 46, wherein said spectrally activated side chain is stimulated by a field effect transistor.

48. The method of claim 47, wherein said stimulation is pulsed.

49. The method of claim 47, wherein said stimulation is constant.

50. The method of claim 46, wherein said spectrally activated side chain is stimulated by an ion-sensitive field effect transistor.

51. The method of claim 46, wherein said spectrally activated side chain identifies a unique spectral signature upon stimulation.

52. The method of claim 46, wherein said unique spectral signature that occurs from each recording device is analyzed to determine its origin.

53. The method of claim 52, wherein said spectral analysis is used to estimate concentrations of individual species of peptide present in the original mixture.

54. The method of claim 41, wherein the entire collection of polypeptides loses one amino acid from said polypeptide to detect an additional signal indicative of a change in spectra subsequent to removal of said additional amino acid from said polypeptide.

55. The method of claim 54, wherein said unique spectral signature(s) over all addressable units are collected between peptide degradation cycles.

56. A method for polypeptide sequencing, comprising:

(a) providing an array having a polypeptide immobilized thereto, wherein said polypeptide is adjacent to a sensor;

(b) subjecting said polypeptide to conditions sufficient to remove an amino acid from said polypeptide;

(c) using said sensor to measure a non-optical signal in said solution subsequent to removal of said amino acid from said polypeptide; and

(d) using at least said non-optical signal to identify a sequence of said polypeptide.

57. A method for determining a sequence of an unlabeled peptide, said method comprising: a) removing an amino acid from said unlabeled peptide; b) identifying a change in an electrical signal of a FET disposed adjacent to said peptide; c) repeating a) and b) at least once, thereby determining said sequence of said unlabeled peptide.