CN116601493A - Optofluidic array for radical protein footprinting - Google Patents

Abstract

Systems and methods of in vivo and in vitro radical protein footprinting using a optofluidic array are presented. These teachings can be used, for example, to study three-dimensional protein structure or biological dynamics. Radical dosimetry methods including optional internal standards are used. Real-time feedback based on internal standards provides comparability between different experiments, and in vivo and in vitro analysis results representing actual biological conditions.

Description

Optofluidic array for radical protein footprinting

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/128,439, filed on 12, 21, 2020, and also a partial continuation of U.S. non-provisional application No. 17/193,913, filed on 3, 5, 2021, PCT/US20/12430, filed on 6, 1, 2020, and PCT/US19/57059, filed on 18, 10, 2019; PCT/US20/12430 and PCT/US19/57059 are both part of the continuation of U.S. non-provisional patent application No. 16/316,006, now U.S. patent No. 10,816,468, filed on 1/7 of 2019; U.S. non-provisional application 16/316,006 claims the benefit of U.S. provisional patent application No. 62/747,247 filed on 10 month 18 of 2018 and U.S. provisional patent application No. 62/788,219 filed on 1 month 4 of 2019; PCT/US19/57059 further claims directly the benefit of U.S. provisional patent application No. 62/747,247 filed on 10/18 2018; both PCT/US20/12430 and PCT/US19/57059 also claim the benefit of U.S. provisional patent application No. 62/788,219, filed on 1 month 4 2019. All of the above patent applications are incorporated herein by reference.

Government support statement

The application is completed with government support under grant R43 GM137728, R43 GM125420 and R44 GM125420 from the national institute of general medical science. The government has certain rights in this application.

Technical Field

The present application relates to an apparatus and method for high-order structural analysis of biomolecules by free radical protein footprinting. Some embodiments of the application relate to determining tertiary and quaternary structures and related conformations (conformations) of biopharmaceuticals using improved apparatus and methods for flash photooxidation of proteins processed using integrated optical and microfluidic chips to determine higher order biomolecular structures of proteins.

Background

Discussion of any work, publication, sale, or activity in this document, including any documents filed with the present application, should not be taken as an admission that any such work constitutes prior art. Discussion of any activity, work, or publication herein is not an admission that such activity, work, or publication exists or is known in any particular jurisdiction.

Biomimetic is a similar but not identical therapy to existing innovators or reference products. Unlike the case of small molecule drugs, biomimetic pharmaceuticals are not just a imitated version of the original product. Traditional imitation drugs are considered therapeutically and molecularly equivalent to their original drugs. This is not the case for biomimetic pharmaceuticals, which are complex three-dimensional biomolecules whose heterogeneity and dependence on living cell production make them distinct from classical drugs. The structural and functional activity of biological therapies is very sensitive to the environment. The intended structure of the therapeutic agent is maintained by a delicate balance of factors including protein concentration, control of post-conversion modification, pH and co-solutes in the formulation, and production/purification protocols (Gabrielson, j.p.; weiss IV, w.f., technical decisionmaking with higher order structure data: starting a new dialogue; journal of Pharmaceutical Sciences, 2015). Therefore, biopharmaceutical structures must be carefully maintained because undesirable pharmacological consequences may occur if left uncontrolled.

Adverse Drug Reactions (ADRs) of biopharmaceuticals are often due to exaggerated pharmacological and immune reactions. Adverse patient reactions range from symptomatic stimulation to morbidity and mortality. While the etiology of some ADRs can be traced to the patient's drug genome sensitivity, many are due to the inherent nature of the treatment, which leads to both pathological and fatal patient consequences and brings about a tremendous economic loss to the biotherapy industry (Giezen, t.j.; schneider, c.k.; safety assessment of biosimilars in Europe: a regulatory perspective; generics and Biosimilars Initiative Journal; 2014). Thus, the occurrence of catastrophic ADR suggests that improved biopharmaceutical development and quality control analysis is needed.

To minimize ADR and facilitate development of biomimetic pharmaceuticals, FDA, drug evaluation and research centers, biological evaluation and research centers have issued guidelines that emphasize the use of the most advanced techniques to evaluate the Higher Order Structure (HOS) of proteins (Quality considerations in demonstrating bio-similarity of a therapeutic protein product to a reference product; guidance for industry; u.s.device of Health and Human Services; food and Drug Administration; center for Drug Evaluation and Research; center for Biologies Evaluation and Research Washington, DC; 2015). HOS analysis involves determining the tertiary and quaternary structure and related conformation of a given biomolecule. Such biomolecules include proteins and protein conjugates, which may or may not be considered as biotherapeutic agents. Despite the various HOS assays currently available, their shortcomings in reliably predicting therapeutic efficacy and safety of biotherapeutics have been questioned, establishing unmet needs for new and improved HOS assays (Gabrielson, j.p.; weiss IV, w.f., technical decision-making with higher order structure data: starting anew dialogue; journal of pharmaceutical sciences; 2015).

One technique for addressing the unmet need of HOS analysis is a free radical protein footprint technique (hamly, d.m.; gross, m.l.; laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale; journal of the American Society for Mass Spectrometry; 2005) that relies on irreversible protein hydroxylation and is coupled with Mass Spectrometry (MS). This process is known as Hydroxyl Radical Protein Footprinting (HRPF). HRPF has been performed using a variety of techniques. Perhaps the most widely used method relies on rapid photochemical oxidation of proteins (FPOP), which uses a single high flux short pulse of UV light from hydrogen peroxide (H ₂ O ₂ ) Generates hydroxyl group(OH) free radicals. The reaction of OH radicals with solvent-exposed amino acids generally results in the net insertion of one oxygen atom into the amino acid. OH radicals are transient and when generated by a transient UV pulse, the reaction between amino acids and radicals can be completed before any conformational change of the labeled protein occurs (Konermann, e.; trng, x.; pan, y.; protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches; journal of mass spectrometry; 2008). The mass spectrum of the enzymatically digested peptide product shows different levels of oxidation, marked by peak shifts of 16Da, 32Da, 48Da, etc. This information can be used to infer which peptides are outside of the HOS and thus provide a better understanding of HOS.

More recently, gross and colleagues have demonstrated another radical protein footprinting technique based on trifluoromethyl (CF) formed by hydroxyl radical attack from aqueous sodium triflate ₃ ) Free radical generation (Cheng, M.et al., laser-Initiated Radical Tirfluoromethylation of Peptides and Proteins: application to Mass-Spectrometry-Based Protein Footprinting; angewandte Chemie International Edition,2017,56 (45): p.14007-14010). When the protein passes through CF ₃ When the free radical is labeled, the mass spectrum of its peptide product shifts by +69Da. CF compared to OH radical attack ₃ The radicals can be highly complementary in their amino acid reaction rate and, when used in combination with OH radicals, can provide a higher coverage of surface exposed amino acids than OH radicals alone. As in the case of HRPF, CF ₃ The tag information can be used to infer which peptides are located outside the protein and thus lead to a better understanding of the protein HOS.

Detrimental effects the technical limitations of HRPF in the comparative study stem from the reaction of OH radicals with non-analyte components in the sample, such as buffer components, initial solutes, and foreign biological products. Variability in background clearance results in irreproducibility between assays, which limits comparative studies (Niu, B.et al.; dosimetry determines the initial OH radical concentration in Fast Photochemical Oxidation of Proteins (FPOP), journal nal of the American Society for Mass Spectrometry; 2015). While OH radicals are excellent probes for protein morphology, they can also react with many compounds found in analytical formulations. There is competition for free OH radicals between the analyte protein and the background scavenger, so that it is necessary to measure the effective concentration of free radicals available for oxidizing the target protein to ensure reproducible results. Similarly, variability in background clearance also applies to CF ₃ The reproducibility of the radical footprint is adversely affected. In photochemistry, the effective radical concentration is measured using a radical dosimeter internal standard. Ideally, the dosimeter should have: a simple relationship between effective radical concentration and dosimeter response; a simple, rapid and nondestructive measurement means; and is not reactive to most proteins.

The prior art teaches radical dosimetry using internal standards of added standard peptides (Niu, b., et al, dosimetry determines the initial OH radical concentration in Fast Photochemical Oxidation of Proteins (FPOP) J Am Soc Mass Spectrom,2015.26 (5): p.843-6; niu, b., et al, incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent measures.j Am Soc Mass Spectrom, 2016.) or using UV absorbing internal standards such as adenine added to buffers and evaluated in a post-labeling manner (Xie, b.; sharp, j.s., hydroxyl Radical Dosimetry for High Flux Hydroxyl Radical Protein Foot-printing Applications Using a Simple Optical Detection methods.analytical chemistry 2015,87 (21), 10719-23.). In peptide radical dosimetry, the labeled peptide and target protein are then analyzed using LC-MS (optional proteolysis) to assess the relative ratio of oxidized peptide to target protein. If the desired peptide to protein oxidation ratio cannot be achieved, the entire experiment is repeated to adjust H as needed to increase or decrease effective OH free radical loading ₂ O ₂ Is a concentration of (3). For adenine dosimetry, the effective change in adenine UV absorbance is determined at the completion of the labeling process, and the achieved change in adenine UV absorbance is compared to the target adenine UV absorbanceThe ratio of the changes is determined. Subsequently changing H according to the desired change in UV absorbance ₂ O ₂ Concentration. Both of these methods are performed after the labeling is complete and the effective OH radical loading cannot be adjusted in real time, consuming valuable sample and unnecessarily wasting time for the researcher.

U.S. patent 10,816,468 and international application PCT/US18/34682 teach systems and methods for performing free radical dosimetry in real time because biological products are labeled during the FPOP HRPF process. While creating a real-time means to adjust and compensate for background clearance variations, the systems and methods herein require the addition of an external internal standard dosimeter (extrinsic internal standard dosimeter) to the biological mixture. Under certain conditions, extrinsic internal standards may lead to artificial changes in higher order structures of biological molecules and, therefore, are incompatible with the desired goal of providing a simple means of characterizing nascent higher order structures of biological products. The disclosures of U.S. patent 10,816,468 and PCT/US18/34682 applications are incorporated herein by reference.

U.S. provisional application No. 62/747,247 and PCT/US19/57059 describe an apparatus and method by which commonly used biological buffer systems can be used as internal standards for radical dosimeters. The photometric properties of some commonly used biological buffers change in a predictable manner upon OH radical attack. Thus, these buffers can be used as radical dosimeter internal standards, eliminating the need to add exogenous reagents, and since the solvating properties of these buffers are well established to stabilize the nascent configuration of the biomolecules, they do not alter the higher order structure of the organism.

The above-described techniques describe devices and means for performing HRPF radical dosimetry when labeling proteins or biological drugs in vitro. However, the practice of applying the results of in vitro structural experiments to real in vivo behavior has been questionedM.A.；Hakim,J.B.；Schnell,S.,Connecting the dots:the effects of macromolecular crowding on cell physiology.Biophysical Journal 2014,107 (12), 2761-2766). Because of the shortcomings of in vitro HRPF, there has recently been interest and desire to expand the use of HRPF to whole cells in an in vivo manner. For example, U.S. patent No. 10,851,335B2 describes a means and method that may be used to perform in vivo HRPF. Briefly, buffer suspension cells and H are supported using multiple fused silica capillaries and microfluidic fittings ₂ O ₂ Is a mixture of (a) and (b). As taught, H ₂ O ₂ Is rapidly taken up by cells without causing cell destruction, inducing apoptosis or inducing cell death. However, the taught systems and methods still result in a variety of drawbacks. U.S. provisional application 62/788,219 and PCT/US 2020/012630 describe an apparatus and method by which in vivo FPOP HRPF is improved by providing means for performing intracellular radical dosimetry to assess and regulate intracellular cytoplasmic or organ radical scavenging. Furthermore, it teaches a means by which the sheath flow rate and subsequent intercellular spaces can be evaluated and ultimately controlled to ensure efficient irradiation of the suspended cells during in vivo labeling.

The above-described technology describes an apparatus and means by which FPOP HRPF can be performed in vitro or in vivo to label proteins or biological drugs using fused silica capillaries as a means of transport, establishing sheath flow, chemical treatment, initiating photo-radical reactions, and further to provide an optical zone in which to detect proteins, cells, chemical reagents, and labeled products. Although fused silica capillaries can be used in these early experiments, they have several undesirable characteristics: 1) They are fragile and prone to accidental breakage, especially when the outer protective polyimide coating is removed; 2) They require removal of an Ultraviolet (UV) opaque polyimide coating to enable removal of the polyimide coating from H ₂ O ₂ Hydroxyl radical formation and consistent UV photometric dosimetry; 3) They require considerable care to ensure proper alignment with the optical components; 4) They require expensive and cumbersome microfluidic fittings to create and form fluid circuitry, such as a facilitatorFluid circuitry required for the internal FPOP, and 5) they are cumbersome to replace. It is not uncommon for small ID (. Ltoreq.100 μm) capillaries to be accidentally plugged or broken when high throughput is applied. Accordingly, the previously taught systems and methods result in various disadvantages.

Disclosure of Invention

Various embodiments of the present invention include systems and methods that address the above-described shortcomings of current in vitro radical protein footprinting by providing a means to replace fused silica capillaries with an optofluidic chip assembly that supports online mixing of target proteins with labeled reagents. The disclosed system includes a serpentine photolytic chamber (serpentine photolysis cell) that significantly increases the irradiated volume during photo-induced radical chemistry as compared to a capillary configuration. Various embodiments include an online dosimetry chamber (in-line dosimetry cell) for real-time photometric assessment of effective free radical production and adjustment for unwanted background clearance using photometric properties of an in vitro free radical dosimeter internal standard.

Various embodiments of the present invention include systems and methods that address the above-described shortcomings of current in vivo radical protein footprinting by providing a means to replace fused silica capillaries with an optofluidic chip assembly that supports in-line mixing of target cells with labeled reagents. Various embodiments support the generation of microfluidic sheath flows to enable precise control of cell-to-cell distances, allowing for efficient and consistent illumination of the exterior and intracellular compartments of each cell, such as the use of a serpentine photolysis chamber that significantly increases the illuminated volume during photo-induced radical chemistry as compared to capillary configurations. Various embodiments include an online dosimetry chamber for real-time photometric assessment of effective free radical production and adjustment for unwanted background clearance using photometric properties of internal standards of an in vivo free radical dosimeter. Various embodiments also provide means by which cell isolation and partitioning (segmentation) can be evaluated and reproducibly controlled, as well as means by which the time at which cells arrive in the HRPF photodecomposition zone can be determined.

Various embodiments of the present invention relate to systems and methods on chip for analyzing higher order structures of proteins, including improved embodiments for in vivo or in vitro flash photooxidation of proteins, to achieve advanced radical protein footprinting. In some embodiments, the present invention provides an online free radical dosimetry system wherein a closed loop control is established between a flash photolysis system and a dosimeter to control irradiance of the flash photolysis system in response to measured changes in photometric properties of an internal standard free radical dosimeter.

In some embodiments, the present invention includes an on-chip, in-line, or in-vitro radical dosimetry system wherein closed loop control is established between an automated on-line microfluidic mixing system and a dosimeter to control the concentration of a labeling agent in response to measured changes in photometric properties of an internal standard radical dosimeter.

In some embodiments, the invention includes an on-chip, in-line or in-vitro radical dosimetry system wherein a closed loop control is established between the flash photolysis system and the dosimeter to control irradiance of the flash photolysis system in response to measured variation in photometric properties of an internal standard radical dosimeter, wherein the radicals are generated by photolysis of a labeled reagent.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing the cells with biological buffers, internal standard radical dosimeters that are ultimately absorbed by the cells, and other labeling reagents, (2) introducing the cells into an optical dosimetry zone, (3) determining a nascent photometric property of the cells, (4) irradiating the cells with at least one UV irradiation in an optical photolysis zone, (5) determining a change in photometric property of the cells after irradiation, and (6) adjusting the spectral irradiance of the UV light source according to the change in the radical dosimeter photometric property.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vitro online free radical dosimetry system, comprising: (1) mixing one or more target proteins with a biological buffer, an internal standard radical dosimeter, and other labeling reagents, (2) introducing the mixture into an optical dosimetry zone, (3) determining an initial photometric property of the mixture, (4) irradiating the mixture with at least one UV irradiation in an optical photolysis zone, (5) determining a change in the photometric property of the mixture after irradiation, and (6) adjusting the spectral irradiance of the UV light source according to the change in the photometric property of the radical dosimeter.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing the cells with biological buffers, internal standard radical dosimeters that are ultimately absorbed by the cells, and other labeling reagents, (2) introducing the cells into an optical dosimetry zone, (3) determining nascent photometric properties of the cells, (4) irradiating the cells with at least one UV irradiation within an optical photolysis zone, (5) determining changes in photometric properties of the cells after irradiation, and (6) adjusting the concentration of the labeling reagents according to the changes in the radical dosimeter photometric properties using an in-line microfluidic mixer.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vitro online free radical dosimetry system, comprising: (1) mixing one or more target proteins with a biological buffer, an internal standard radical dosimeter, and other labeling reagents, (2) introducing the mixture into an optical dosimetry zone, (3) determining an initial photometric property of the mixture, (4) irradiating the mixture with at least one UV irradiation within an optical photolysis zone, (5) determining a change in the photometric property of the mixture after irradiation of the light, and (6) adjusting the concentration of the labeling reagents according to the change in the photometric property of the radical dosimeter using an in-line microfluidic mixer.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing cells with biological buffer, a labeling reagent and an internal standard radical dosimeter that is ultimately absorbed by the cells, (2) introducing the cells into an optical dosimetry zone, (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry zone, (4) determining the elapsed time between successive cell arrivals, (5) determining a cell isolation volume for each product of elapsed time and net buffer flow rate, and (6) adjusting sheath flow and buffer flow parameters to achieve a desired cell isolation volume.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing the cells with biological buffer, a labeling reagent, and an internal standard radical dosimeter that is ultimately absorbed by the cells, (2) introducing the cells into an optical dosimetry zone, (3) detecting the arrival and presence of the cells by monitoring the phase change of light exiting the dosimetry zone, (4) determining the elapsed time between successive cell arrivals, (5) determining a cell isolation volume for each product of elapsed time and net buffer flow rate, and (6) adjusting sheath flow and buffer flow parameters to achieve a desired cell isolation volume.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing the cells with biological buffers, labeling reagents and internal standard radical dosimeters that are ultimately absorbed by the cells, (2) introducing the cells into an optical dosimetry zone, (3) detecting the arrival and presence of the cells by monitoring the intensity of scattered light exiting the dosimetry zone, (4) determining the net flow rate to the cells, (5) determining the interconnected volumes extending from the photolysis zone and the dosimetry zone, (6) determining the transit time required for the cells to travel from the photolysis zone to the dosimetry zone, (7) determining the photolysis zone arrival time of the cells, and (8) triggering the photolysis system when all subsequent cells reach the photolysis zone.

In some embodiments, the invention includes a method of producing a labeled biomolecule for analysis using an on-chip in-vivo on-line radical dosimetry system, comprising: (1) mixing the cells with biological buffers, labeling reagents and internal standard radical dosimeters that are ultimately absorbed by the cells, (2) introducing the cells into an optical dosimetry zone, (3) detecting the arrival and presence of the cells by monitoring the phase leaving the dosimetry zone, (4) determining the net flow rate to the cells, (5) determining the interconnected volume extending from the photolysis zone and the dosimetry zone, (6) determining the transit time required for the cells to travel from the photolysis zone to the dosimetry zone, (7) determining the photolysis zone arrival time of the cells, and (8) triggering the photolysis system when all subsequent cells reach the photolysis zone.

After the generation of the labeled biomolecules, other methods (such as mass spectrometry or electrophoresis) can be used to identify the labeled peptides and infer information about the higher order structure of the biomolecules in vivo or in vitro.

Various embodiments of the present invention include an on-chip analysis system comprising: a sample introduction system configured to provide intact biological entities to the photolysis zone, the biological entities being isolated from each other in a focused sheath flow; a photolytic light source configured to generate light from a source of the labeling reagent to generate free radicals; a photolytic region configured to receive the sheath fluid and light for labeling an internal standard and labeling a biological compound of a biological entity in the body; a dosimetry zone configured to receive the biological entity from the photolysis zone to detect the presence of the biological entity using a scatter detector and to detect oxidation of the internal standard using a fluorescence detector; control logic configured to determine a target concentration of radicals for each of the biological entities and to adjust operation of the photolytic zone to meet the target concentration of radicals; and a reservoir configured to receive a biological entity comprising the labeled biological compound.

Various embodiments of the invention include an on-chip method of labeling a biomolecule within an intact cell, the method comprising: introducing a sample mixture comprising at least one cell into a photolysis zone, introducing a source of labeled radicals and an internal dosimeter index into the photolysis zone of a flash photolysis system; providing light to generate labeled radicals from a source of labeled radicals, the labeled radicals configured to label at least one intracellular biomolecule; waiting for a selectable predetermined time for at least one cell to reach a dosimetry zone of a radical dosimeter configured to detect photometric properties of the dosimeter internal standard resulting from reactions of the dosimeter internal standard and labeled radicals, wherein the at least one cell is detectable by light scattering within a dosimetry zone of the radical dosimeter; measuring photometric properties of a dosimeter internal standard using a free radical dosimeter when at least one cell is within a dosimetry zone; determining that a target level of marker radicals is not generated based on the measured photometric properties of the dosimeter internal standard; and adjusting the concentration of the labeled radicals within the photolytic zone by adjusting at least one of: 1) the amount of light provided to the photodecomposition area, 2) the concentration of the source of labeled radicals, 3) the flow rate of at least one cell within the photodecomposition area, or 4) the time to provide light to the photodecomposition area. It should be noted that in the foregoing embodiments, the series of light pulses provided to the photodecomposition region has both a period and a phase, and adjusting the phase of the periodic pulses changes the timing of the pulses without changing the period.

Drawings

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. Furthermore, the above objects and advantages of the present invention will become apparent to those skilled in the art from a reading of the following description of the exemplary embodiments when considered in conjunction with the accompanying drawings in which features of the present invention are incorporated. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.

Any of the methods described herein may further utilize any one or more of the following, according to particular embodiments, wherein:

fig. 1 illustrates an embodiment of a flash photolysis system.

Fig. 2 illustrates an embodiment of a microfluidic system.

FIG. 3 illustrates in detail a component embodiment of an optofluidic chip.

Figure 4 shows in detail a plot of the dosimetry photometric response of a mixture of adenine and hydrogen peroxide when irradiated with pulses of UV light at 1 second intervals.

Fig. 5 illustrates an embodiment of a microfluidic system.

Fig. 6 illustrates a preferred embodiment of an in vivo optofluidic chip.

FIG. 7 is a table listing the cell distribution and fluorescence excitation and emission wavelengths of several internal standard radical dosimeters.

FIG. 8 depicts an embodiment of a combined fluorescence and light scattering detector system of the present invention.

FIG. 9 depicts an alternative embodiment of a combined fluorescence and light scattering detector of the present invention.

FIG. 10 illustrates the use in the present inventionDeep Red and hydrogen peroxide irradiates the in vivo examples.

FIG. 11 illustrates an exemplary process for determining and controlling isolated volumes or intercellular distances in the present invention.

Fig. 12 depicts an exemplary process 1200 for cell arrival determination with consistent coordination of photolytic light triggering.

Detailed Description

Devices and methods for analyzing the higher order structure of biomolecules are provided, which are achieved by selective labeling of molecular groups exposed to solvents, catalyzed by on-chip rapid photo-oxidation in vivo or in vitro, and monitoring and controlling effective free radical concentrations in real time. In addition, devices and methods for analyzing higher order structures of biomolecules are provided by on-chip online microfluidic mixing of labeled reagents with target proteins, cells, or other in vivo embodiments. In addition, devices and methods for analyzing higher order structures of biomolecules are provided, which are implemented by on-chip microfluidic channels for generating sheath flows to support rapid photooxidation reactions in vivo. In addition, devices and methods for analyzing higher order structures of biomolecules are provided that use integrated serpentine photolysis chambers through on-chip online photochemically induced reactions.

Devices and methods for analyzing the higher order structure of biomolecules are provided, which are achieved by selective labeling of molecular groups exposed to solvents, catalyzed by on-chip rapid photo-oxidation in vivo or in vitro, and monitoring and controlling effective free radical concentrations in real time. In addition, devices and methods for analyzing higher order structures of biomolecules are provided that are implemented by on-chip in vivo rapid photooxidation, real-time monitoring and control of in vivo species isolation volumes, and subsequent flash photodissociation. The devices and methods may be applicable to a variety of light transmissive or transparent in vivo embodiments, such as, but not limited to: eukaryotic cells, prokaryotic cells, bacteria, intracellular viruses, virions, virus-like particles, unicellular organisms, eukaryotic tissues and multicellular organisms. Although the present invention refers to cells for purposes of illustration, such reference is not limiting and it should be understood that such reference is inherently applicable to all in vivo biological or non-biological entities that are translucent or transparent in luminosity.

The apparatus and method may be applicable to a variety of research fields, such as: universal protein biochemistry; diagnosis and research; infection research; biopharmaceutical development; antibody development; development of biological analogues; therapeutic antibody development; research and development of small molecule drugs; development of other therapeutic compounds and materials. Furthermore, the apparatus and method may be adapted for use in a variety of research analyses, such as: protein-ligand interaction analysis; analysis of protein-protein interactions; protein-DNA interactions; protein-RNA interactions; protein fusion product analysis; protein conformation and conformational change analysis; intercellular interactions; virus-cell interactions; analyzing the action mode of the small molecule drug; biopharmaceutical mode of action analysis; antibody antigen analysis; positioning protein epitopes; protein secondary epitope localization (protein paratope mapping); and (5) chemical reaction monitoring.

The device can receive cells for subsequent chemical labeling by manually pipetting the target protein or cells into an appropriate microcentrifuge tube or microplate placed in the sample introduction assembly of the system. Alternatively, the device may be coupled to other separation and analysis instruments (phytate) and receive target proteins or cells directly from these instruments, such as but not limited to those performing selective protein separation, cell sorting, cell counting, and isolating cells from tissue.

This section provides an overview of a flash photolytic instrument with an on-chip in-line in-vivo or in-vitro free radical dosimeter that uses the photometric properties of the internal standard dosimeter to evaluate and ultimately control the effective free radical concentration in-vivo or in-vitro. Furthermore, this section provides a general overview of the invention that uses photometric properties of in vivo embodiments to assess isolation volumes and precisely control separation of the in vivo embodiments described above within a buffer flow stream. A detailed description of each sub-assembly is provided elsewhere herein. Furthermore, methods of describing the interactions of these subassemblies are provided so that typical instrument operation can be understood.

Various embodiments of the present invention include a flash photolysis system 100, as shown in FIG. 1. The flash photolysis system 100 is configured for real-time free radical dosimetry and comprises a plurality of subassemblies. The method comprises the following steps: a sample introduction system 101; a photolysis zone 102; a flash photolysis light source 103; a radical dosimeter 104; control electronics 105; an instrument controller 106; a fluid interconnect 107 between the sample introduction system 101 and the photolysis zone 102; a fluid interconnect 108 between the photodecomposition chamber and the radical dosimeter; a photonic interconnect 109 between the flash photolysis light source 103 and the photolysis zone 102; an electronic interconnect 110 between the radical dosimeter 104 and the control electronics 105; an electrical interconnect 111 between the sample introduction system 101 and the control electronics 105; an electrical interconnect 112 between the control electronics 105 and the instrument controller 106; and an electrical interconnect 113 between the control electronics and the flash photolysis system. The photodecomposition area 102, the flash photodecomposition light source 103 and the radical dosimeter 104 together form a "flash photodecomposition system"

The photodecomposition region 102 is typically included in a photodecomposition chamber. The flash photolysis system 100 is configured to oxidize sample cells in vitro or in vivo in real time to achieve radical dosimetry. The analyte is introduced via the sample introduction system 101. Analytes can be provided using a small volume microcentrifuge tube or using a multi-well microtiter plate obtained from Eppendorf (hamburg, germany). Microfluidic circuitry is configured for analyte pumping, mixing with labeled reagents, cell hydrodynamic focusing using a sheath flow device for in vivo processing, transport to photolytic and dosimetry zones, and transport and deposition of labeled products. In some embodiments, the sample introduction system 101 is configured to provide the complete biological entity to a photolysis zone (e.g., the photolysis zone 102, the biological entity isolated from each other in a focused sheath flow). The sheath is typically configured to isolate the biological entities from each other. For example, using appropriate conditions, the biological entities are separated from each other at regular intervals.

Photooxidation occurs within photodecomposition region 102. In some embodiments, the photodecomposition region is located within the optofluidic chip. In some embodiments, optofluidic chips are fabricated using various techniques, such as lithography-assisted wet chemical etching, dry reactive ion etching, and laser ablation of microstructures, which create microfluidic channels within a silicon or quartz substrate. In some embodiments, the optofluidic chip is fabricated by embossing (embossing) fluidic channels within a plastic substrate, wherein the fluidic channels formed deliver samples into optically transparent cells created by sealing optically transparent windows to areas of the plastic substrate that have been removed. Such optically transparent windows may be provided on only one or both sides of the photolysis and dosimetry chambers disclosed herein. Exemplary plastic substrates include, but are not limited to: polycarbonates, polyethylenes, polyetheretherketones, cycloolefin polymers, cycloolefin copolymers, polytetrafluoroethylene,And polychlorotrifluoroethylene. The inner diameters of the fluid and the optical channel may be in the range of 0.1 to 5.0mm, but are not limited thereto. In some embodiments, the fluid and optical channels may have different inner diameters to desirably meet different requirements for fluid transport, fluid mixing, hydrodynamic focusing, and optical coupling. In some embodiments, the photolytic zone consists of a serpentine array of channels that juxtapose multiple fluid paths in a countercurrent fashion.

The photolytic zone 102 receives analytes from the sample introduction system 101 via the microfluidic path 107. After processing, the light illuminating the analyte within the photodecomposition region 102 is transferred into the radical dosimeter 104. The photodecomposition area 102 is in optical communication with a flash photodecomposition light source 103. The flash photolysis light source 103 is an example of a photolysis light source configured to generate light from a labeling reagent source to generate labeled radicals within the photolysis zone 102. In some embodiments, the photodecomposition region 102 is configured to receive a sheath flow comprising a biological entity, receive light from the flash photodecomposition light source 103, so as to oxidize a dosimeter internal standard, so as to oxidize biological compounds of the biological entity in vivo. For example, proteins and peptides including amino acids may be oxidized in photolytic region 102.

The photodecomposition region 102, the flash photodecomposition light source 103, and the radical dosimeter 104 comprise a flash photodecomposition system. The flash photolysis system includes: a plasma flash or other suitable light source, such as an excimer laser, a solid state laser, or a laser diode; and associated light collection/transmission optics to match the requirements of the light transmission component for the photodecomposition region.

The radical dosimeter 104 is configured to receive the labeled analyte from the photolytic zone 102, or in an alternative embodiment, the radical dosimeter 104 is incorporated into the photolytic zone 102 by employing orthogonal optical paths. The radical dosimeter may employ various photometric detection schemes to monitor relevant photometric properties of the internal standard of the dosimeter. In some embodiments, the dosimeter internal standard may be an external additive incorporated into the biological sample. In some embodiments, the intrinsic photometric properties of the biological buffer system can be used as an intrinsic dosimeter internal standard. By "intrinsic" or "buffer intrinsic" is meant that the internal standard is one of the chemicals that provide the buffer properties. The buffer is optionally a physiologically compatible buffer configured to maintain the analyte or cell at a physiological pH or ion concentration. In some embodiments, the internal standard is configured to become fluorescent due to light received from the flash photolysis light received in the photolysis zone 102. For example, in various embodiments, the dosimeter internal standard may increase fluorescence by at least a factor of 10, 100, or 1000 upon reaction with hydroxide radicals.

Photometric detection schemes include, but are not limited to: fluorescence, photometric absorbance, refractive index detection, light scattering detection, and luminescence. In some embodiments, the photometric detection scheme includes a fluorescence detector that employs an Ultraviolet (UV) light excitation source to generate UV fluorescence or emissions. In some embodiments, the fluorescence detector employs a UV excitation source to produce visible fluorescence or emission. In some embodiments, the fluorescence detector employs a visible excitation source to produce visible fluorescence or emission. In some embodiments, the fluorescence detector further comprises an integrated light scattering detector. In some embodiments, the fluorescence detector further comprises an integrated optical refractive index detector.

The functions of the control electronics 105 are: providing a Direct Current (DC) drive voltage to the peripheral component derived from a laboratory Alternating Current (AC) power source; providing analog and digital control signals to the peripheral device; receiving analog or digital information from a peripheral device; providing ADC and digital to analog conversion (DAC) functions; and provides data to the instrument controller 106 and receives commands from the instrument controller 106. In the exemplary embodiment, the control electronics include a motor controller that interfaces with a motor located within sample introduction system 101. Further, in such embodiments, the control electronics may include a Universal Serial Bus (USB) hub for digital communication with the instrument controller 106.

Instrument controller 106 is used to provide process control for various instrument peripheral devices while receiving status and data information from these devices in digital format. In some embodiments, instrument controller 106 runs a software control program having two main modules: a low-level multithreading module for instrument component control, and a high-level User Interface (UI) module. In some embodiments, the control electronics 105 include an embedded microprocessor that provides low-level instrument component control while communicating with the high-level UI control program of the instrument controller 106 via a USB or wireless interface.

Together, instrument controller 106, control electronics 105, and various interconnections represent control logic configured to control flash photolysis system 100. The control logic may be configured to perform the steps of any of the methods disclosed herein. For example, in some embodiments, the control logic is configured to determine a target concentration of labeled radicals generated for each biological entity. The target concentration is optionally selected to ensure that the labelling reaction of the protein or cellular component has sufficient labelled free radical reagent to approach the desired level of completion.

The control logic may be further configured to manage a feedback loop that includes adjusting conditions in the photolytic zone 102 to meet a target concentration of the marker radicals. The conditions in the photolytic zone 102 can be adjusted by, for example, changing the concentration of the labeled radical source, changing the flow rate of the sample introduced into the system 101, changing the amount of light received from the photolytic light source 103, changing the time the light is received from the photolytic light source, changing the separation distance/volume of the isolated cells, etc. By changing the conditions in the photolytic zone 102, the control logic can provide feedback to the sample introduction system 101 and/or the photolytic light source 103 based on the analysis of the first analyte to improve the resolution of the second analyte.

In some embodiments, the control logic is configured to normalize the oxidation from the cells and/or the quantification of the identified peptide based on fluorescent signals from internal standards within the dosimetry zone. This allows comparing the results from different experiments using different instances of the flash photolysis system 100.

The control logic is optionally further configured to determine when cells in the photodecomposition area 102 receive the photodecomposition light and/or to determine a period of time to isolate cells from entering the photodecomposition area 102. These determinations may be based on detection of fluorescence, scattered light, or phase changes of light in the dosimetry zone, and may be controlled by adjusting the flow rate and/or volume of sample introduced into system 101.

The control logic is optionally further configured to control cell flow to the labeled cell library and the analyzer. For example, the control logic may be configured to control the flow of cells such that ruptured cells are transferred from a particular container of the labeled cell reservoir or such that different oxidized cells are placed in different compartments as a function of time. In some embodiments, the control logic is configured to control any aspect of the flash photolysis system 100 using the analyte signal from the analyzer.

The instrument controller 106 and the control electronics 105 are optionally combined into a single device.

In vitro analyte photochemical labeling

Fig. 2 illustrates further details of the flash photolysis system 100 configured to support in vitro analyte processing, and in particular illustrates a microfluidic system 200 of the system. The method comprises the following steps: sample/product microplate 201; a sample inlet/product outlet port 202; a combined photolysis and dosimetry chamber 203; an in-line microfluidic mixer 204; a optofluidic chip 205; a buffer syringe pump 206; a labeled reagent syringe pump 207; a buffer reservoir 208; a waste reservoir 209; a sample storage loop 210; a labeled reagent reservoir 211; a reagent transfer line 212; a buffer syringe pump four-way valve 213; an in-line mixer gate valve 214; a reagent waste line 215; marking reagent syringe pump four-way valve 216; a reagent introduction line 217; a buffer introduction line 218; a buffer waste line 219; a sample microwell 220; waste collection microwells 221 and product collection microwells 222.

Samples to be labeled are stored in a storage container, such as sample/product microplate 201, such as a 96-well plate or 384-well plate available from thermo fisher (usa). The sample is introduced by aspiration through the sample inlet/product outlet port 202. The aspirated sample passes through the dosimetry and photolysis zone 203 of the system and is further aspirated through an in-line mixer 204. The in-line mixer 204 and the photolysis and dosimetry zone 203 are all formed within an optofluidic chip 205.

The microfluidic system 200 includes two syringe pumps, a buffer syringe pump 206 and a labeling reagent pump 207. Syringe pump 206 is in communication with buffer reservoir 208, waste reservoir 209, and sample storage loop 210. Syringe pump 207 communicates with waste reservoir 209, reagent reservoir 211, and in-line mixer 204 via reagent transfer line 212.

The microfluidic circuit is primed (primed) prior to the labeling process. Syringe pump 206 fills with buffer by switching its four-way valve 213 to communicate with buffer reservoir 208. Its plunger is withdrawn to fill the syringe to its full volume. The volume of syringe pump 206 may be in the range of 50uL, 100uL, 250uL, 500uL, 1.5mL, 2.0mL, and 2.5mL, but is not limited thereto, depending on the desired volume of sample to be labeled. The syringe pump 206 then passes through the switching valve 213 to communicate with the storage loop 210 and pumps sufficient volume to flush the system to cause the microfluidic system to be primed. During system flushing, buffer is directed to flow through the in-line mixer 204, photolysis and dosimetry zone 203, through port 202, and finally to be dispensed into designated waste wells 221 of microplate 201. During this process, valve 214 remains closed to in-line mixer 204 so that buffer is directed through chip 205, rather than exiting through mixer 204, and eventually into reagent transfer line 212.

After the chip is primed, the reagent delivery circuit is primed. When the valve 216 is connected to the reagent introduction line 217, the reagent pump 207 is completely filled with reagent drawn from the reagent reservoir 211 by pulling back its plunger. The volume of the reagent pump 207 may be in the range of 50uL, 100uL, 250uL, 500uL, 1.5mL, 2.0mL, and 2.5mL, but is not limited thereto, depending on the desired volume of the sample to be labeled. The reagent delivery line 212 is flushed by pumping with a reagent pump 207 having a four-way valve 216, the four-way valve 216 being configured to deliver the contents of the syringe 207 to the reagent delivery line 212 through a valve 214, the valve 214 being configured to direct the primed volume to the waste reservoir 209 via a reagent waste line 215. The valve 214 is located in the vicinity of the in-line mixer 204 such that the interconnection volume from the valve 214 to the mixer 204 is minimized to a range of 0.25 to 1.5 uL. Further details of the fluidic connection of the chip 205 are provided in fig. 3.

The following steps outline an exemplary cascade of operations used by the embodiments described with respect to the microfluidic system 200 to label in vitro samples using light-induced free radical reactions. Such cascading variations will be apparent to those skilled in the art and are considered functionally equivalent and are not to be inventive from the examples provided herein. The sample/product microplate 201 is arranged such that the different microwells 220 are designated to contain the sample to be labeled, and empty microwells 221 and 222 that will selectively collect labeled products, waste buffers, or labeled reagents deposited during fluid infusion of the subsystems of the microfluidic system 200. For in vitro radical protein footprinting, the microwells 220 contain proteins or other protein biological mixtures. After priming, buffer syringe pump 206 dispenses a predetermined volume of buffer by moving valve 213 to communicate with sample loop 210, pumping the buffer through optofluidic chip 205 and out through inlet outlet port 202 to designated waste micro wells 221. The volume pumped here is slightly larger than the volume of the sample to be marked. For example, if 100 μl of sample is targeted for labeling, the syringe pump 206 dispenses 120 μl of buffer into the waste microwell 221. Other different volume ratios may be employed and the examples mentioned above are for illustration and not limitation of the scope.

The microplate 201 is moved such that the ports 202 are immersed into the microwells 220 containing the sample to be labeled. Buffer syringe pump 206 aspirates a predetermined volume of sample by withdrawing its plunger and placing it in fluid communication through valve 213, in-line mixer 204, photolysis and dosimetry chamber 203 and port 202. The sample is drawn into the chip 205 through the photolysis and dosimetry chamber 203 and the in-line mixer 204 and finally into the sample storage loop 210. During this sample aspiration, the in-line mixer gate valve 214 closes the mixer so that the labeled reagent is not aspirated into the loop.

Reagent reservoir 211 contains the labeling reagent required for the protein labeling process. For HRPF, reservoir 211 contains an aqueous hydrogen peroxide solution, typically at a concentration of, for example, 20mM to 1000 mM. For trifluoromethyl footprinting, reagent reservoir 211 will comprise a mixture of hydrogen peroxide and an aqueous solution of sodium triflate. If CF is mainly required ₃ Radical labeling, then H ₂ O ₂ And sodium triflate are mixed in a 1:4 concentration ratio, typically 5mM H ₂ O ₂ And 20mM sodium triflate. If HRPF and CF are simultaneously required ₃ Footprint method H ₂ O ₂ And trifluoromethanesulfonic acid are mixed in a ratio of 5:1, respectively, typically using 50mM H ₂ O ₂ And 10mM triflic acid. The above mixtures are intended to be exemplary and should not be construed as limiting the scope, as other ratios may play a functionally equivalent role and absolute conditions may vary based on actual protein labeling conditions.

To begin the marking process, pumps 206 and 207 begin dispensing their respective solutions into the microfluidicIn the bulk circuit. The relative pumping speeds of pumps 206 and 207 depend on the desired final concentration of the labeled reagent entering the photolytic zone and the starting concentration of the labeled reagent located within reservoir 211. Taking hydroxyl radical labeling as an example, reagent reservoir 211 may contain H at a concentration of 1000mM ₂ O ₂ And the final concentration required during labelling was 100mM. In this condition, the pumping rate of pump 207 will be one tenth of the pumping rate of pump 206. The net flow rate is the sum of the pumping rates established by pumps 206 and 207.

FIG. 3 depicts a further embodiment of an exemplary optofluidic array 300 capable of being described later. The method comprises the following steps: a reagent inlet port 301; a buffer inlet port 302; a mixer 303; a photolysis chamber 304; a dosimetry chamber 305; an inlet/outlet port 306; and a detailed view of the photodecomposition chamber 307 containing the fluid channel 308. After establishing flow through optofluidic chip 205, photolytic light source 103 is triggered to illuminate photolytic zone 304 at regular flash intervals. The range of the flash frequency may be 0.25Hz to 5Hz, but is not limited thereto. The flash frequency and net flow rate are established to ensure that the sample and labeled reagent mixture is illuminated by only a single flash and that each subsequent illuminated mixture bolus is divided by a predetermined isolation volume. Thus, the illuminated volume of the photolysis chamber defines the necessary net flow rate and flash rate.

In capillary-based photo-induced radical footprinting, a photolytic light source is focused to illuminate a small volume of fluid located within the capillary lumen. Typical capillary Inner Diameters (ID) range from 50-250 μm, but are not limited thereto. Further, the irradiated region will extend axially down the capillary tube to define an irradiated axial length in the range of 2-8mm, but is not limited thereto. For a typical application, a 100 μm ID capillary with an axial distance of 6mm will include an irradiated volume of 47 nL. If 50 μl of sample is required for labeling, about 1,064 flashes will be required, which will take many minutes to handle and unnecessarily accelerate light source loss. Furthermore, under these conditions, only a small fraction of the photolytic light is focused into the capillary lumen, as the refractive focus limits of the excimer laser and plasma light source result in a waste of light beams that is greater than the inner diameter of the lumen. Thus, the previously taught capillary-based methods result in various drawbacks.

The optofluidic chip 205 includes a serpentine photodecomposition area 304, as shown by photodecomposition area 307. The fluid paths within the photodecomposition region 307 are arranged in a folded or serpentine fashion with adjacent flow channels in close juxtaposition and flow established in a countercurrent fashion. The serpentine photolysis zone enables a better optofluidic match between the photolysis light source 103 and the sample reagent mixture, as the photolysis light can now be distributed over a wider incidence area, which overcomes the refractive focus limit. The fluence required for effective photolytic labeling reagent for incident UV irradiation depends on the UV source spectral irradiance and the labeling reagent extinction coefficient. For H-based ₂ O ₂ Is generally required to be at least 3mJ/mm for UV wavelengths less than 250nm ² Is a flux of (a). For OH and CF ₃ The free radical marking, flash photolysis light source 103 has sufficient spectral irradiance to achieve up to 16mm ² Is provided. Under such conditions, the fluid channel 308 within the photolysis chamber 304 can be arranged to contain an irradiated volume of about 6.4 μl. In this way, the illuminated volume of the photolytic zone 304 is 136 times that of the 100 μm ID capillary system described above, and thus, the marking will proceed in a fraction of the time required for capillary-based marking while reducing the strain of the photolytic system by more than 99%.

The sample and labeled reagent mixture is illuminated by a photolytic light source 103 in a photolytic zone 304. After exposure, typically within a few microseconds, radicals rapidly form and covalently modify the sample. The labeled sample, buffer and modifying reagent are pumped down into the dosimetry zone 305. The contents of dosimetry region 305 are detected using photometric means such as, but not limited to, photometric absorbance, fluorescence, light scattering, refractive index detection, and/or chemiluminescence. Real-time measurements made in dosimetry zone 305 allow for a feedback loop in which the photolytic conditions can be modified to ensure that the desired amount of radical generation and the desired amount of in vitro analyte labeling occur. During initial operation of the system, baseline measurements are made of photometric properties of a dosimetry zone containing a non-irradiated sample and reagent mixture. Once a baseline measurement is made Photolysis takes place and a change in photometric properties is determined. In the case of hydroxyl radical protein footprinting, OH radicals covalently label the sample, with concomitant attack on commonly employed internal standard radical dosimeters, such as adenine. In this case, the photometric property to be evaluated is UV photometric absorbance at 265 nm. On OH radical attack adenine shows a decrease in UV 265nm absorbance in a manner proportional to the effective OH radical yield. By varying the intensity of the flash or H in response to the difference between the estimated change in the dosimetry signal and the target change during trimming ₂ O ₂ Concentration to control effective OH radical yield.

If the actual response of the internal standard radical dosimeter is less than the desired level, the flash energy of the photolytic light source 103 is increased until the desired level of dosimeter response is reached. Alternatively, the relative pumping speed of the reagent pump 207 is increased relative to the buffer pump 206, followed by an increase in the effective concentration of the labeled reagent after mixing with the sample in the in-line mixer 204. If the actual response of the internal standard radical dosimeter is greater than the desired level, the flash energy of the photolytic light source 103 is reduced until the desired level of dosimeter response is reached. Alternatively, the relative pumping speed of the reagent pump 207 is reduced relative to the buffer pump 206, followed by a reduction in the effective concentration of the labeled reagent after mixing with the sample in the in-line mixer 204.

FIG. 4 depicts the use of 100mM H in the presence of 1mM adenine aqueous solution at a 1Hz flash rate in dosimetry zone 305 during the formation of hydroxyl radicals in photolysis zone 304 ₂ O ₂ The real-time output trace 400 of UV absorbance obtained over time, with 2mM adenine water and 200mM H ₂ O ₂ Mix at 1:1 (v/v) in the microfluidic mixer 303. The method comprises the following steps: a non-exposure baseline 401; a descending shoulder 402; photometric response, or in this case peak change in UV absorbance (minimum 403); a rising shoulder 404; a re-established baseline 405, a minimum UV absorbance value 406 achieved at time T1; and a minimum UV absorbance value 407 achieved at time T2. Other adenine/H may be used as will be apparent to those skilled in the art ₂ O ₂ Mixing ratio is achievedThe target concentration is now, and a 1:1 mixing ratio is for illustration and not limitation of the range. The baseline UV photometric absorbance measurement of the above mixture without exposure is represented by the baseline 401 without exposure. In the non-exposed baseline 401, the baseline UV absorbance is taken to be about 55 milliabsorbance units (mAU), as determined in the context of a flowing buffer, which in this case is water. Upon irradiation of the mixture within the photolytic zone 304, OH radicals are formed and adenine is subsequently oxidized. The light irradiation period is usually 10 musec but is not limited thereto. The hydroxyl radical reaction then proceeds for a further about 1-5 musec, after which the reaction self-terminates. As the illuminated mixture flows from the photodecomposition region 304 and into the dosimetry region 305, the photometric absorbance signal begins to decrease, as shown by the decreasing shoulder 402. The minimum UV absorbance of the labeled sample was taken to be about 33mAU, as shown by minimum 403. Here, the effective change in adenine UV absorbance of the first irradiated mixture bolus was determined to be 22mAU by calculating the difference between the non-exposed baseline 401 and the minimum 403 (55 mAU-33 mAU). When the irradiated mixture exits dosimetry zone 305, the measured UV absorbance returns to its baseline, as indicated by rising shoulder 404, and baseline 405 is re-achieved. The determined change in UV absorbance upon light irradiation was compared to the target change of the labeling experiment. Altering the flash energy and/or H if the effective level of absorbance change deviates from the desired target ₂ O ₂ Concentration to adjust OH radical yield using the control loop logic previously described. Once the target level of internal standard dosimeter photometric property change is reached, the light irradiation/dosimeter response assessment process is repeated until the desired amount of labeled sample is processed and subsequently collected.

During the marking process, the effective variation of the dosimeter response for each flash period can be selectively accessed to determine the reproducibility and inherent quality of the overall marking process. Under these conditions, the mean, standard deviation, and relative standard deviation percentage (RSD) of the effective dosimeter response for each flash period is taken and compared to a pre-established quality index. Typical RSD quality indicators may be less than 5% as determined by the specific photochemical labeling conditions of the target protein, but are not limited thereto. If the quality index is achieved, the labeling experiment is determined to be of acceptable quality. If the quality index fails to be achieved, the labeling experiment is labeled as problematic and the experimenter is appropriately alerted.

Each exposed mass of mixture is divided by a predetermined isolation volume consisting of unirradiated mixture flowing through the photolytic zone 304 between successive flashes. The isolation volume is critical to ensure that each bolus of mixture is irradiated with only one light, by effectively acting as a buffer to mitigate unwanted back axial diffusion of the irradiated sample back into the next bolus of sample to be exposed and to accommodate laminar axial velocity differences. A useful isolation volume ensures that unnecessary double illumination is prevented while reducing unnecessary dilution of the marked product. An exemplary desired isolated volume target is taken as a volume that dilutes the sample by about 10%.

The photometric response plot 400 shown in fig. 4 can be used to calculate the effective isolation volume by determining the difference in dosimeter response minima generated during an OH radical attack of adenine. Other internal standard radical dosimeters, such as Tris or histidine buffers, will show increased photometric absorbance upon OH radical attack, and in this case, photometric response plot 400 will show local maxima. For this exemplary discussion, adenine is again used as an internal standard radical dosimeter. For adenine, two consecutive boluses of the optical radiation mixture will include two corresponding effective dosimeter response minima, as shown at 406 and 407. The time difference between 406 (T1) and 407 (T2) may be calculated. For exemplary purposes, consider the time difference between T1 and T2 to be 1.25 seconds. For the previously described flash rate of 1Hz, the flash interval is determined to be 1.0 seconds. In the adenine example, the isolation volume is determined by the product of the net flow rate and the difference between the flash interval and the dosimeter minimum interval. In the adenine example, the net flow rate is 70 μl/min or 1.17 μl/sec. Thus, the isolation volume was determined to be 1.17 μl/sec (1.25 sec) =0.29 μl. If the total exposure volume specified by the geometry of the photolysis chamber 304 is 3 μl, the isolation volume is taken to be about 10% of the illuminated volume and is thus determined to be sufficient to ensure a single flash exposure per bolus. If the isolation volume is less than the desired target, the net flow rate may be increased proportionally, or alternatively, the flash rate may be decreased proportionally to achieve the desired isolation volume. The net flow rate may be proportionally reduced if the isolation volume exceeds the desired target, or alternatively, the flash rate may be proportionally increased to achieve the desired isolation volume. In one embodiment, the control logic described above is automatically implemented by instrument controller 106.

Once the proper dosimeter response and isolation volumes are achieved, the sample mixture can be confident labeled and the labeled product collected. During the marking and product collection process, the instrument controller 106 records the time that the dosimeter and isolation volume targets have been achieved and determines the arrival time of the correctly marked product to the outlet of the inlet outlet port 202. The arrival time is determined by the quotient of the transfer volume extending from the dosimetry zone 305 to the inlet and outlet ports 202 and the net flow rate. The exiting contents of optofluidic chip 205 are dispensed through port 202 into designated waste well 221 before the appropriately marked product arrives. Under the control of the instrument controller 106, when a properly marked product reaches the outlet of the inlet-outlet port 202, the sample introduction system 101 moves the microplate 201 so that the marked product is collected in the product collection microwells 222.

In vivo photochemical marking

The foregoing discussion describes details of embodiments of the present invention for labeling in vitro samples. Here, we describe details of an embodiment supporting in vivo labelling reactions. Fig. 5 shows a microfluidic circuit 500 and optofluidic chip 501 for on-chip in vivo photochemical labeling. The method comprises the following steps: a microplate 201; a sample inlet product outlet port 202; microplate wells 220 containing cells; a waste receiving micro well 221; product collection microwells 221; the in vivo radical labeling optofluidic chip 501; a sheath flow generator 502; an in-line mixer 503; a photolytic zone and dosimetry zone array 504; sample buffer microfluidic pumping system 505; a sheath buffer microfluidic pumping system 506; a reagent microfluidic pumping system 507; sheath flow buffer syringe pump 508; a four-way microfluidic valve 509; a sheath buffer reservoir 510; a waste reservoir 511; a sheath buffer tee 512; sample buffer syringe pump 513; a four-way microfluidic valve 514; a waste reservoir 515; a buffer reservoir 516; a sample loop 517; a reagent syringe pump 518; a four-way microfluidic valve 519; a reagent reservoir 520; a reagent transfer line 521; sheath inflow port 522; and an in-line mixer reagent introduction port 523.

The optofluidic chip 501 consists of three different functional areas: a sheath flow generator 502, a labeled reagent/sample in-line mixer 503, and an array of photolysis and dosimetry chambers 504. The optofluidic chip 501 is connected to three different microfluidic circuits: a buffer and sample fluid system 505; sheath fluid system 506; reagents are introduced into the fluid system 507. All three fluid systems have been primed prior to the start of marking. The system 505 is primed by the action of a buffer syringe pump 513 and valve 514. The first valve 514 is configured to allow fluid communication between the buffer reservoir 516 and the syringe pump 513. Pump 513 is filled with flowing buffer pumped from buffer reservoir 516. After filling, valve 514 is reconfigured to allow communication between syringe pump 513 and waste reservoir 515. The contents of syringe pump 513 are pumped to waste reservoir 515. This priming cycle continues until all air within the fluid system 505 is vented and any residue of previous buffer is flushed away. In a typical case, the cycle is repeated three times, but is not limited thereto. Syringe pump 513 is then refilled with flow buffer and valve 514 is configured to enable fluid communication between syringe pump 513 and optofluidic chip 501 via sample loop 517. The flowing buffer is then pumped by syringe pump 513 through sample loop 517 and into optofluidic chip 501, and the buffer flows through sheath flow generator 502, in-line mixer 503, photolysis and dosimetry zone array 504, and out to designated waste microwells 221 in microplate 201 via inlet and outlet port 202.

After the priming system 505, the sheath fluid system 506 is primed. The valve 509 is configured to allow fluid communication between the sheath buffer syringe pump 508 and the sheath buffer reservoir 510. For in vivo photochemical labeling, reservoirs 510 and 516 comprise the same buffer. Sheath flow buffer is pumped to fill sheath buffer syringe pump 508. After filling, the valve 509 is reconfigured to allow fluid communication between the sheath buffer syringe pump 508 and the waste reservoir 511. The waste reservoir 511 may be shared with the reagent introduction system 507. Furthermore, the reagent introduction system 507, the microfluidic pumping system 506 and the system 505 may optionally use the same waste buffer reservoir. The sheath buffer syringe pump 508 empties its contents into the waste reservoir 511. This priming cycle continues until all air within the fluid system 506 has been vented and any remnants of previous buffer have been flushed away. In a typical case, the cycle is repeated three times, but is not limited thereto. The sheath buffer syringe pump 508 is then refilled with sheath buffer, and the valve 509 is configured to enable fluid communication between the sheath buffer syringe pump 508 and the optofluidic chip 501. The sheath flow buffer flow is split by coupling tee 512, which creates two sheath flow buffers that enter optofluidic chip 501 through port 522 of sheath flow generator 502. Sheath flow buffer then flows through sheath flow generator 502, in-line mixer 503, photolysis and dosimetry zone array 504, and out to designated waste microwells 221 in microplate 201 via inlet and outlet ports 202.

After the priming system 505 and the microfluidic pumping system 506, the reagent fluid system 507 is primed. Valve 519 is configured to allow fluid communication between reagent reservoir 520 and reagent syringe pump 518. The reagent solution is pumped to fill the reagent syringe pump 518. After filling, the valve 519 is reconfigured to allow fluid communication between the reagent syringe pump 518 and the waste reservoir 511. Waste reservoir 511 may be shared with sheath buffer fluid system 506. Furthermore, the reagent fluid system 507, the sheath buffer fluid system 506, and the system 505 may optionally utilize the same waste buffer reservoir. The reagent syringe pump 518 empties its contents into the waste reservoir 511. This priming cycle continues until all air within the fluid system 507 is vented and any residue of the previous reagent solution is rinsed away. In a typical case, the cycle is repeated three times, but is not limited thereto.

Then, reagent syringe pump 518 is refilled with reagent solution, and valve 519 is configured to enable fluid communication between reagent syringe pump 518 and optofluidic chip 501. Reagent solution is pumped through reagent transfer line 521 and enters optofluidic chip 501 through in-line mixer 503 reagent inlet port 523. Simultaneously with the latter, the fluidic systems 506 and 505 pump their fluid components into the optofluidic chip 501, as previously described. In this way, all fluid flow, including fluid flow from the reagent fluid system 507, is directed down the chip and eventually out the port 202 to the waste micro well 221. The system is now ready for in vivo radical protein footprinting.

For in vivo radical protein footprinting, the microwells 220 comprise cells, tissues, or organisms suspended in a buffer. While the reagent microfluidic pumping system 507 and the sheath buffer syringe pump 508 remain stationary, the buffer syringe pump 513 is connected to the sample loop 517 via valve 514. Buffer syringe pump 513 is used to aspirate cells from microwell 220. Cells are drawn up to port 202, through dosimetry zone array 504, in-line mixer 503, sheath flow generator 502, and ultimately into sample loop 517. Typically, a predetermined volume of cells suspended in buffer is aspirated and stored within the sample loop 517. Thus, cells also remain through each compartment of the optofluidic chip 501 and inside the port 202. When labeling begins, the fluidic systems 507, 506, and 505 initiate flow in a simultaneous manner, eventually pushing cells stored within the sample loop 517 into the optofluidic chip 501 where they mix with the labeling reagent and are subsequently irradiated with light to initiate the photoradical labeling process.

In order to separate cells from each other alone, rather than aggregate or aggregate together, when irradiated with light, a sheath flow is established within sheath flow generator 502 to isolate the cells by a small partitioned volume of sheath flow buffer. Under typical conditions, the sheath buffer flow rate is determined to be ten times the buffer syringe flow rate, but is not limited thereto. As described later, the flow rate ratio of the fluid systems 505 and 506 may be automatically adjusted to achieve a desired degree of cell isolation. With the pumping activity of the fluid systems 505 and 506, the reagent fluid system 507 delivers a labeled reagent to mix with the flowing cells within the in-line mixer 503. As with the in vitro labeling, the relative flow rate of the reagent fluid system 507 with respect to the net flow rates of the sheath buffer syringe pump 508 and the system 505 may be automatically adjusted based on the concentration of labeled reagent and the desired level of effective radical yield as determined by the on-line radical dosimetry system.

FIG. 6 depicts a detailed view of a preferred embodiment of an optofluidic chip 601. The method comprises the following steps: a optofluidic chip 601; a buffer syringe port 602; a reagent inlet port 603; a reagent mixer 604; sheath buffer inlet port 605; sheath flow generator 606; a photolysis zone 607; a dosimetry zone 608; and a sample inlet/product outlet port 609. In optofluidic chip 601, reagent mixer 604 precedes sheath flow generator 606. Cells enter optofluidic chip 601 via buffer syringe port 602. In this regard, they are not isolated, but may be accessed as a dense cell population or other in vivo embodiment. Upon entering the reagent mixer 604, they mix with the labeled reagent that enters the buffer syringe port 602 via the reagent inlet port 603. While it is known to those skilled in the art that a variety of different in vivo labeling reagents can be used, for exemplary purposes we describe the use of hydrogen peroxide for in vivo hydroxyl radical protein footprinting. As described by Jones et al (Espino, J.A.; mali, V.S.; jones, L.M.; in Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live cells, analytical chemistry 2015,87 (15), 7971-7978), hydrogen peroxide is rapidly and easily absorbed by cells or other in vivo entities and rapidly divided into all cell compartments. As will be described later, the final concentration of hydrogen peroxide used is determined in a substantially closed-loop manner by determining the effective hydroxyl radical yield, which is assessed by the change in photometric properties of the internal standard radical dosimeter detected in dosimetry zone 607.

Prior to deposition into the microwell 220, an internal standard radical dosimeter is added to the cell population or other in vivo embodiments, and the mixture is allowed to incubate (incubate) sufficiently so that the internal standard radical dosimeter is absorbed by all cells, and ultimately, a uniform distribution between cells and within cell compartments is achieved. Then, the cells were spun down (spin down) to form a pellet, and the supernatant was removed. The cell pellet was then resuspended in buffer and spun down again a second time. This process is repeated until all the extracellular internal standard radical dosimeters are removed. Once there is no extracellular internal standard radical dosimeter, the cells are resuspended in buffer and eventually transferred into well 220.

After passing through the reagent mixer 604, the cellular reagent mixture enters a sheath flow generator 606. Sheath flow generator 606 includes a lumen for receiving sheath flow buffer and sheath flow buffer port 605, and these components of generator 606 are shaped and arranged together to produce hydrodynamic focusing such that cells are isolated by sheath flow buffer. The relative flow rates of the microfluidic systems 505, 506, and 507 are adjusted to establish the desired intercellular separation volumes while concomitantly achieving the target intracellular hydroxyl radical yields. After passing through the sheath flow generator 606, the isolated cell population enters the photolysis zone 607 where the cells are irradiated with light that initiates intracellular marker radical reactions. After passing through the photolysis zone 607, the cells enter a dosimetry zone 608 where they are optically probed to evaluate effective radical yield, as well as intercellular distance and subsequent intercellular partition volumes.

During in vivo radical protein footprinting, the response of the intracellular standard radical dosimeter is measured photometrically when detected by dosimetry zone 608. Typical photometric measurement means include, but are not limited to, photometric absorbance, photometric luminescence, and photometric fluorescence. Since it is desirable to detect photometric signals generated in a single cell or other single in vivo embodiment, photometric fluorescence can be a preferred measurement means because its dynamic range and analytical sensitivity are generally superior to other previously mentioned methods.

For in vivo radical protein footprinting, a fluorescent internal standard radical dosimeter will change its fluorescent properties, such as excitation and emission properties, after radical attack and final covalent modification. Such a change may involve changing the excitation and emission characteristics of the dosimeter such that it exhibits an increase or decrease in fluorescence at a given excitation/emission wavelength setting. Since it is desirable to maximize the sensitivity of the radical dosimeter signal detection, detecting the presence of collected fluorescence due to radical attack is a preferred embodiment of the method, as it enables detection of small signals in dark or zero background. The free radical dosimeter fluorescence detector is configured using specific excitation and emission wavelengths of the covalently modified internal standard dosimeter. When the dosimeter is in its intrinsic state, no fluorescent signal is detected. Upon free radical attack, the fluorescent properties of the dosimeter are altered to correspond to the excitation and emission wavelengths selected, and the detected fluorescent signal intensity will be proportional to the concentration of the covalently modified internal standard dosimeter, which in turn is proportional to the effective free radical yield.

Fig. 7 depicts five different internal standard radical dosimeters that display fluorescent signals at the indicated excitation and emission wavelengths in a dose-responsive manner upon oxidation using hydroxyl radical attack. Table 700 lists the cell distribution, fluorescence excitation, and emission wavelengths of the following internal standard radical dosimeters that react to hydroxyl radical attack in a dose-responsive manner: terephthalic acid;green; dichlorofluorescein (dichlorofluoroscein); cellRox Orange; and CellRox Deep Red. The intracellular compartment of the primary distribution of each dosimeter is also shown.Dyes are available from thermo fisher (united states).

Fig. 8 depicts an embodiment of an internal standard free radical dosimeter photometric detector 800 of the invention. The method comprises the following steps: a light source 801; a focusing lens 802; excitation light 803; a dosimetry zone 608; a dichroic mirror 804; emitting light 805; a collimator 806; an interference notch filter 807; a narrow band of emitted light 808; a fluorescence/emission photodetector 809; scattered light 810; a collimator 811; a light scatter detector 812. The internal standard radical dosimeter photometric detector 800 is a combined fluorescence and light scattering detector system. It will be clear to a person skilled in the art that such a combined system may be replaced by two separate detectors (fluorescence and light scattering) and that the present embodiment is therefore illustrative in scope and not limiting. When the in-vivo components enter the dosimetry zone 608, they are illuminated by excitation light 803 from the light source 801, focused by lens 802 and directed into the dosimetry zone 608 by dichroic mirror 804. The light source 801 may be a narrow bandwidth solid state UV source such as a UV Light Emitting Diode (LED) available from Q-Photonics (anaburg, michigan), or a compact solid state laser available from Thorlabs (newton, new jersey). Alternatively, the light source 801 may be a solid state visible light source, such as a visible LED available from Q-Photonics (Annaburg, michigan), or a compact solid state visible laser available from Thorlabs (N.J.). Typical output power may be in the range of 0.1 to 10mW, but is not limited thereto, and typical bandwidth may be in the range of 1 to 15nm, but is not limited thereto. The wavelength of the light source 801 is selected to be the appropriate choice for the excitation wavelength of the internal standard radical dosimeter employed. The focusing lens assembly 802 is used to focus excitation light into a narrow beam on the order of, but not limited to, 1-200 μm within the center of the dosimetry zone 608, thereby creating a detected dosimetry zone. The dichroic mirror 804 is selected to efficiently reflect excitation light but transmit emitted light 805 generated by the covalently modified internal standard dosimeter illuminated within the dosimetry zone 608.

The emitted light 805 is finally collimated by a collimator 806 and then passed through an interference notch filter 807 to produce narrowband emitted light 808, which narrowband emitted light 808 is incident on a fluorescence emission photodetector 809. The photodetector 809 may include a silicon photodiode, such as an S1336-8BQ silicon photodiode available from Pinus maritima (Kogyo, japan). Alternatively, the photodetector 809 may include a compact photomultiplier tube (PMT), such as the micro PMT assembly H12400 available from Hamamatsu. The output current of the photodetector 809 is processed by a current-to-voltage (I-to-V) converter to provide a voltage proportional to the incident emitted light intensity of the narrowband emitted light 808. The output voltage of the photodetector 809 is transmitted to the control electronics 105 where an analog-to-digital converter (ADC) generates a digital signal that is ultimately transmitted to the instrument controller 106 where fluorescence calculations are performed.

As cells or other in vivo embodiments enter dosimetry region 608, they elastically scatter excitation light 803. Due to the size difference between the incident excitation wavelength (nm) and the physical size (μm) in the body, the scattered light 810 is preferably detected orthogonally with respect to the incident excitation light. Scattered light 810 is collimated by collimator 811 and ultimately incident on scattered photodetector 812. For a given excitation light intensity, the intensity of the scattered light measured will be proportional to the size and number of in-vivo entities located within the detection volume of dosimetry region 608. Since scattered light detection is performed in an orthogonal direction relative to the incident excitation light, background scattering is also measured. In the absence of in vivo entities, both the elastic and inelastic scattering of the contents of the detected volume, as well as the elastic scattering of the optical surface of the free radical dosimeter, are extremely low, resulting in very low background signals. The light scattering signal is uniformly generated, independent of any intrinsic fluorescence, and thus the presence of cells or other in vivo embodiments can be detected without any internal standard radical dosimeter signal.

Scatter photo detector 812 may include a silicon photodiode, such as an S1336-8BQ silicon photodiode available from Pinus maritima (Pinus maritima, japan). Alternatively, the photodetector 812 may include a compact photomultiplier tube (PMT), such as the micro PMT assembly H12400 available from Hamamatsu. The output current of scatter photodetector 812 is processed by a current-to-voltage (I-to-V) converter to provide a voltage proportional to the incident scattered light 810. The output voltage of the photodetector 812 is transmitted to the control electronics 105 where an analog-to-digital converter (ADC) generates a digital signal that is ultimately transmitted to the instrument controller 106 where light scattering calculations are performed.

FIG. 9 depicts another embodiment of an internal standard free radical dosimeter photometric detector 900 of the invention. The method comprises the following steps: a fluorescence excitation light source 901; a red diode laser interference pattern light source 902; a light collection and transmission bifurcated optical fiber 903; fiber optic out-coupling optics 904; a dosimetry chamber 608; a scattered light column 905; an optical beam splitter 906; a light scattering charged coupling device camera 907; fluorescent column 908; fluorescence collection optics 909; a fluorescence selective interference filter 910; fluorescence detector 809; a far-field interference fringe pattern 911; and the extracted spatial frequency 912.

In the combined fluorescence and light scattering detector system 900, the light sources can be interchanged to meet the requirements of a fluorescent internal standard radical dosimeter, and wherein light scattering detection is accomplished using a two-dimensional Charge Coupled Device (CCD) imaging camera that images the interference pattern produced by the dosimetry chamber. Fluorescence excitation source 901 and red diode interference pattern light source 902 are remote from optofluidic chip 601 and are coupled to dosimetry chamber 608 using bifurcated optical fiber 903 and requisite fiber coupling optics 904. The wavelength generated by the light source 901 is selected to match the excitation wavelength requirements of the internal standard radical dosimeter employed. The red diode interference pattern light source 902 uses a 650nm red diode laser to produce an optical interference pattern that is produced in the high beam field of the dosimetry chamber 608. In such a configuration, the light sources 901 and 902 may be mounted to the exterior of the instrument, providing a simple means of exchanging the light sources as needed for the radical dosimetry target.

The coherent light from sources 901 and 902 becomes uniform within single mode fiber 903 and is directed to be incident on dosimetry chamber 608. The beam splitter 906 is used to divide the light leaving the dosimetry chamber 608, creating two optical columns: a scatter column 905 and a fluorescent column 908. The light scattering column 905 is generated in the optical far field of the dosimetry chamber 608. Since the red diode interference pattern light source 902 is a coherent light source, its mode shape interacts with the optical properties of the dosimetry chamber 608 to produce a far field interference fringe pattern 911 as imaged by the charged coupled device two dimensional camera 907.

The fluorescent columns 908 are collected and collimated by collection optics 909, which collection optics 909 direct the transmission of light through interference filters 910. The bandpass characteristics of the interference filter 910 are selected to match the internal standard radical dosimeter emission spectrum. The optical bandwidth of the interference filter 910 may be 10nm, but is not limited thereto. Because the interference filter 910 and fluorescence excitation source 901 are exchanged for a matched pair to accommodate a plurality of different internal standard radical dosimeters, such as those listed in table 700, the interference filter 910 is housed in an easily removable holder accessible to the user. The fluorescence emission is detected by a fluorescence detector 809.

As the cells enter the dosimetry chamber 608, they elastically scatter the light of the red diode laser interference pattern light source 902, and this scattering changes the average polarization of the light, which in turn changes the refractive index and the synthetic phase of the light exiting the detected region. As the refractive index of the detected region moves, the imaged striations 911 move in position to the right or left depending on the change in refractive index. The fringe pattern of the optical interference pattern 912 is represented as a spatial frequency distribution by summing the intensities of each vertical pixel of the two-dimensional camera 907 for a given horizontal or x-delta. Using a Fast Fourier Transform (FFT), the phase of the dominant spatial frequency found in the optical interference pattern 912 can be calculated at fast time intervals. When a cell enters the dosimetry chamber 608, the accompanying light scattering changes the fringe position, which in turn is shown as a phase change. The phase change will be proportional to the size and number of cells within the detected region of the dosimetry chamber 608. As previously described, sheath flow generators 502 and 606 may be used to isolate incoming cells or other in vivo embodiments so that they are significantly separated from aggregation together, thereby effectively and consistently illuminating each entity within the radical marker photolysis zone. Controlled isolation of these in vivo entities is affected by the difference in pumping rates of the sheath buffer syringe pump 508 and buffer syringe pump 513. Nominally, the pumping speed of the sheath buffer syringe pump 508 is, for example, ten times the pumping speed of the buffer syringe pump 513, but is not limited thereto. As further described herein, effective isolation of each in-vivo entity may be assessed by determining a time difference in the detected light scattering signal that will be maximized when the in-vivo entity is illuminated and will be minimized in a manner similar to the signal distribution shown in fig. 4 in the absence of the above-described entity. In this embodiment, a light scattering detector is employed to assess the presence of an in vivo entity. In an alternative embodiment, the light scattering detector is replaced by a refractive index detector that determines the phase change of the detected light caused by elastic scattering.

Once the desired cellular or in vivo physical isolation is achieved, the free radical labeling proceeds very similarly to in vitro experiments. The effective free radical dose is assessed by monitoring the change in fluorescence signal measured in dosimetry zone 608 as the in vivo entity is irradiated in photolysis zone 607.

FIG. 10 depicts use ofDeep Red and 100mM hydrogen peroxide. The method comprises the following steps: baseline fluorescence level 1001; rising shoulder 1002 of dosimeter fluorescence signal; dosimeter maximum fluorescence level 1003; a falling shoulder 1004 of the dosimeter signal; a re-established baseline fluorescence level 1005; maximum fluorescence level 1006 occurring at time T1; and a maximum fluorescence level 1007 that occurs at time T2. As shown, when the mixture is pulsed, the baseline fluorescent dosimeter signal 1001 rapidly yields to the rising shoulder 1002 and maximizes at the fluorescence level 1003. As oxidized CellRox Deep Red leaves the dosimetry zone, the fluorescence signal decreases, as shown by the decreasing shoulder 1004, and eventually re-establishes its low background fluorescence baseline at 1005.

Similar to in vitro radical labeling, the time difference between two maximum fluorescence values of two consecutively exposed cell clusters, as shown at 1006 (T1) and 1007 (T2), or in vivo embodiments, can be used to evaluate the isolation volume generated by the sheath flow generator 606 as well as the intercellular separation distance. Like in vitro systems, the reproducibility of all maximum fluorescence signals can be evaluated to assess the quality and reproducibility of the resulting in vivo free radical markers.

Once the proper dosimeter response and in vivo entity isolation volume are achieved, the in vivo entity can be confident marked and the marked in vivo product can be collected. During the marking and product collection process, the instrument controller 106 records the time that the dosimeter and isolation volume targets have been achieved and determines the arrival time of the correctly marked product to the outlet of the inlet outlet port 202. The arrival time is determined by the quotient of the transfer volume extending from the dosimetry zone 608 to the inlet and outlet ports 202 and the net flow rate. The exiting contents of optofluidic chip 601 are dispensed through inlet-outlet port 202 into designated waste well 221 before the appropriately labeled product arrives. Under the control of the instrument controller 106, when a properly marked product reaches the outlet of the inlet-outlet port 202, the sample introduction system 101 moves the microplate 201 so that the marked product is collected in the product collection microwells 222.

Post-labeling analysis

For both in vivo and in vitro radical footprint experiments, post-labeling analysis is typically performed as follows, during which the collected sample is optionally analyzed using an analyzer. The analyzer is configured to perform a chemical analysis on the sample. For example, the analyzer may be configured to identify proteins, peptides, carbohydrates, metals, nucleic acids, lipids, and/or amino acids that are oxidized in the photolytic zone. For example, an analyzer may include a mass spectrometer, a scintillator, an electrophoresis device, a chromatograph, and/or any other device configured to separate and/or identify sample components based on radioactivity, mass, charge, size, or other characteristics. In some embodiments, the analyzer is configured to detect isotope or radioisotope labels within a cell or other biological entity, and optionally determine whether a component comprising such labels has been oxidized. In some embodiments, the analyzer is configured to measure a ratio of labeled/unlabeled concentrations of the particular component. In some embodiments, the analyzer is "on-line" with port 202 and is therefore configured to receive and analyze the sample in real-time. For example, the port 202 may comprise a capillary configured to provide the sample directly to the input of the mass spectrometer.

Specific control of sheath flow

Various embodiments of the invention include methods of determining and controlling intercellular isolation volumes to achieve reproducible in vivo radical dosimetry. In order to effectively achieve in vivo radical dosimetry, photometric measurements of internal standard radical dosimeters should be performed when in vivo entities are present within the dosimetry zone to achieve meaningful comparative measurements representing differences in intercellular, intracellular radical dosimeter responses.

Fig. 11 depicts an exemplary method 1100 of determining and controlling intercellular separation volumes using the invention described herein, which is also capable of detecting in vivo entities within a dosimetry zone. The method comprises the following steps: a cell introduction step 1101; a first cell detection step 1102; a second cell detection step 1103; a net flow rate determination step 1104; a cell isolation volume determination step 1105; a good isolation assessment step 1106; poor isolation and sheath flow rate adjustment step 1107; the loop 1108 is repeated.

In the introduce cells step 1101, a single file array of cells is formed and the array flows into the dosimetry zone 608. In a detect first cell step 1102, the signal from the scattered light photodetector 812 is monitored to detect when the first cell reaches the dosimetry zone 608. When a cell or other in-vivo biological entity reaches within dosimetry zone 608, the intensity of scattered excitation light 810 increases according to the size and number of in-vivo entities within the detected region. In the detect second cell step 1103, the above procedure is used to detect the arrival time of the second cell into the dosimetry zone 608. The net flow of the system is calculated 1104 by summing the pumping speeds of syringe pump 508, syringe pump 513, and syringe pump 518. The flow rate may be reduced to the point where the time between detection of the first cell and the second cell is long enough to reduce the probability that both cells are in the dosimetry zone 608 at the same time. This achieves single cell isolation, where the conditions allow one cell at a time to be irradiated and oxidized.

In calculate cell isolation volume step 1105, the cell isolation volume is determined by multiplying the time difference of arrival between the first cell and the second cell by the net flow rate 1104. If the empirically determined cell isolation volume deviates from the desired isolation volume (which is directly related to the separation distance and separation time) by less than +/-5% (or some other predetermined limit), then in a continuing step 1006 the system continues to label additional cells without further adjustment. Alternatively, if the empirically determined cell isolation volume deviates by more than +/-5%, the pumping speed of the sheath flow syringe pump 508 is varied in a regulate sheath flow rate step 1107 to achieve the desired cell isolation target volume 1107, and the process of determining is repeated 1108 until the target cell isolation volume is achieved.

Various embodiments include systems and methods for detecting the presence of an in vivo entity within a dosimetry zone 608. The time of entry and exit of the detected in vivo entity within dosimetry zone 608 may be used to determine the data acquisition cycle for photometrically determining the intracellular dosimeter internal standard free radical response. Upon entering dosimetry region 608, the in-vivo entity causes a rapid rise in the intensity of scattered light detected by scattered light detector 812. Consistently, the intensity of scattered light detected by scattered-light photodetector 812 drops sharply as the in-vivo entity exits. The time difference between the rise and fall in scattered light intensity is indicative of the dosimetry zone residence time of the in vivo entity (e.g., cell). During the dwell period, the light intensity values detected by the emission photodetectors 809 are summed and/or integrated to determine a net dosimetry signal of the in vivo entity of interest. By employing this method, photometric dosimetry can be performed by detecting signals generated from intracellular and/or extracellular photometric signals, while rejecting any intrinsic background signals generated by extracellular fluid for areas lacking in vivo entities. Thus, most of the photometric signals measured will consist of signals generated by intracellular components, since by experimental design the background monomer of extracellular fluid is significantly lower than that of intracellular fluid, and the measurement is only performed in the presence of one or more in vivo entities.

Determination of feasibility of in vivo embodiments

Some embodiments include systems and methods for determining the viability of an in vivo entity. For a given in-vivo entity, the scattered light intensity will be proportional to the size and number of in-vivo entities present within dosimetry zone 608 during photometric evaluation. Using the methods disclosed herein, means for effectively ensuring a constant arrival rate of an in vivo entity within the dosimetry zone 608 are described. In this case, the number of in-vivo entities per data acquisition cycle can be controlled continuously, so that the variation in measuring scattered light depends to a large extent on the variation in-vivo entity size. The change in the size of the entity in the body may be due to an inherent morphological change, or may be indicative of an artificial change in the morphology of the cell/species, which may be indicative of cell destruction, cell death or apoptosis. Since the inherent goal of in vivo HRPF is to evaluate the biomolecular complement HOS under viable conditions, it is desirable to detect and signal the presence of an impossible (viable) process and/or condition.

Embodiments of the invention described herein provide systems and methods for detecting potential damage to in vivo parts that may be caused by an HRPF protocol, and thus provide a means by which in vivo HOS analysis of surviving and undamaged in vivo entities may be performed. For example, the presence of destroyed cells or more than one cell within dosimetry zone 608 may be detected simultaneously based on the measured light scattering. The entities detected in these cases may be separated from entities that were not irradiated under these conditions and discarded.

Controlled triggering of flash photolytic light

Various embodiments of the present invention include systems and methods for triggering a flash photolysis light source 103 in synchronization with the arrival of an in-vivo entity at a photolysis zone 102. In vivo radical footprinting desirably involves determining and quantifying the presence of in vivo entities within the photolytic zone so as to reliably irradiate the one or more entities in a reproducible manner and to photocatalytically generate a reproducible intracellular radical load.

Fig. 12 depicts an exemplary method 1200 of determining that an in-vivo entity has reached the photolysis zone 608, while providing a system and method for precisely triggering the flashing of the flash photolysis light source 103 when the in-vivo entity has reached the photolysis zone 608. The method comprises the following steps: a cell introduction step 1201; a first cell detection step 1202; determination 1203 of net flow rate; determination 1204 of on-chip interconnect volume; determination of transition time 1205; determining 1206 the time of arrival of the photodecomposition area; activation 1207 of the photolytic trigger at cell arrival time; and continuing with processing 1208 by depositing appropriately labeled cells.

In the introduce cells step 1201, a single file array of cells is formed, as described elsewhere herein, and the first cells are introduced into the photolytic zone 102. In a detection step 1202, the output signal of the scatter photodetector 812 is monitored to detect the arrival time of the first cell into the dosimetry zone 608. When a cell or other in-vivo entity enters the dosimetry zone 608, the intensity of the scattered light increases according to the size and number of in-vivo entities within the detected region. This increase is detected by scatter photodetector 812. In determine net flow step 1203, the net flow rate of the system is calculated by summing the pumping speeds of syringe pump 508, syringe pump 513, and syringe pump 518. In a determine interconnect volume step 1204, as a direct manifestation of the microfluidic system design, the interconnect volume extending from the photolysis zone 102 and dosimetry zone 608 is determined and remains constant during in vivo free radical protein footprinting. In a determine transition time step 1205, the transition time required for the in vivo entity to travel from the photodecomposition region 102 to the dosimetry region 608 is calculated by dividing the interconnect volume by the net flow rate. In a determine photodecomposition region arrival time step 1206, a photodecomposition region arrival time is calculated by determining a difference between a dosimetry region arrival time and a transition time of the photodecomposition region to the dosimetry region. In a triggering step 1207, for subsequent cells or in-vivo entities, the photolysis system is triggered to flash at a consistent interval at or after the determined photolysis zone arrival time. In a continuing processing step 1208, additional labeled cells are deposited in the labeled cell reservoir 221 until a target number of cells are processed.

While the teachings herein describe specific uses of detection signals generated by light-scattering photodetector 812 and fluorescence/emission photodetector 809, it will be apparent to those of ordinary skill in the art that these detectors may be used for the purpose of detecting the arrival of an in vivo entity at dosimetry region 608, or for the purpose of predicting the arrival of an in vivo entity at photolysis region 102 by a variety of undescribed combinations or means. For example, the signal generated at the fluorescence/emission photodetector 809 may be coherently summed in time with the signal from the light scattering photodetector 812 to increase the overall sensitivity of detecting the presence of an in vivo entity within the dosimetry zone. Accordingly, the methods described herein are intended to be illustrative, rather than limiting in scope.

Closed loop control of free radical dosimetry

Various embodiments of the present invention include systems and methods for calibrating closed-loop controlled radical dosimetry systems. In these embodiments, the closed loop control radical dosimetry system includes calibration logic for predicting a desired change in optical flux or radical agent concentration in response to a measured change in radical dosimeter photometric fluorescence. The calibration function is empirically determined by multiple measurements, with a single flash of light at different levels of flux or free radical reagent concentration for known or control mixtures supporting buffers, in vivo entities and free radical dosimeters, for each different control aliquot. In an exemplary embodiment, a software routine running in a low level instrument control or high level user interface program (e.g., control electronics 105 and/or instrument controller 106) generates a look-up table or curve fit describing measured changes in dosimeter photometric fluorescence at each fluence or free radical agent concentration to allow for the generation of a mathematical expression or calibration function describing the relationship between applied flux and/or free radical agent concentration and measured dosimeter fluorescence changes for a single flash exposure. In another embodiment, the look-up table and subsequent calibration functions are manually generated by the user using each flash voltage value and/or the fluorescence change value of the free radical agent concentration.

Background free radical scavenging was assessed via dosimetry during in vivo free radical protein footprint treatment. The measured change in dosimeter photometric fluorescence is compared to a user specified target change. When the measured dosimeter value deviates from the target value +/-10% or more, the applied flux or radical agent concentration is changed to achieve a target change in the measured dosimeter absorbance. The calibration function described above is used to predict the required change in flux or free radical agent concentration.

It will be clear to those skilled in the art that many more modifications than those already described are possible without departing from the inventive concept. Accordingly, the inventive subject matter is not limited except for the spirit of this disclosure. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

Although biological cells are used herein to illustrate various embodiments of the invention, in alternative embodiments, the "cells" may be replaced by other entities, such as non-biological entities, viruses, multicellular organisms (e.g., fungi, spores, nanoorganisms (nanobe), mold, algae, nematodes, amoeba, protozoa, wire pallet, or yeast), or non-biological materials, in any example or claim.

Described herein are more specific embodiments of general structures and techniques, and different ways in which more general objectives can be achieved. Although only a few embodiments have been disclosed in detail above, other embodiments are possible and are considered to be included in the present specification. This specification describes specific examples for achieving a more general object that may be achieved by another means. The disclosure is intended to be exemplary, and the claims are intended to cover any modifications or alternatives that may be contemplated by those of ordinary skill in the art.

The logic discussed herein may comprise electronic circuitry, hardware, firmware, and/or software stored on non-transitory computer readable media.

Only the claims using the term "means for … …" should be interpreted according to the american society of america, volume 35, section 112, section 6. Furthermore, any limitations in the specification are not intended to be interpreted as any claims, unless such limitations are expressly included in the claims. The computer described herein may be any type of computer, either a general purpose computer, or a specific purpose computer such as a workstation or laboratory or manufacturing facility. The computer may be an Intel (e.g., pentium or Core 2duo, i3, etc.) or AMD based computer running Windows 10, 8, 7 or Linux, or a Macintosh computer. The computer may also be a handheld computer, such as a PDA, cell phone, tablet or notebook computer, running any available operating system including Android, windows Mobile, iOS, etc.

Copyright statement: according to 37c.f.r.1.71 (e), a portion of the present disclosure includes copyrighted material (such as, but not limited to, a source code listing, screen shot, user interface or user description, or any other aspect of the present filing, copyright protection is available or available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the patent and trademark office patent file or records. The applicable copyright laws prohibit all other copying, distributing, authoring derived works based on the content, publicly revealed, and publicly performed of the present application or any part thereof, keeping all other rights.

Claims (27)

1. An optofluidic chip for radical protein footprinting, comprising:

a substrate;

a first port, a second port, and a third port, each defined into the substrate;

a mixing region including a first cavity defined within the substrate and in fluid communication with both the second port and the third port;

a photolytic zone comprising a second cavity defined within the substrate and in fluid communication with the mixing zone, the photolytic zone further comprising a first optically transparent window sealed to the substrate and defining one side of the second cavity, the second cavity comprising channels configured in a serpentine pattern; the photodecomposition region is transparent on both sides.

A dosimetry zone comprising a third cavity defined within the substrate and in fluid communication with the photolytic zone and also in fluid communication with the first port, the dosimetry zone further comprising a second optically transparent window sealed to the substrate and defining one side of the third cavity.

2. The optofluidic chip of claim 1, wherein the substrate comprises plastic.

3. The optofluidic chip of claim 1 or 2, wherein the substrate comprises quartz.

4. The optofluidic chip of claim 3, wherein the substrate comprises a silicon middle layer, a quartz top layer, and a quartz bottom layer, the middle layer being disposed between the top layer and the bottom layer.

5. The optofluidic chip of claim 3, wherein the substrate comprises a silicon middle layer, a fused silica top layer, and a fused silica bottom layer, the middle layer disposed between the top layer and the bottom layer

6. The optofluidic chip of claims 1-4 or 5, further comprising a sheath flow generator comprising a fourth cavity defined in the substrate and in fluid communication with both the mixing zone and the photolysis zone, the sheath flow generator comprising two sheath flow inlet ports defined into the substrate and in fluid communication with the fourth cavity, the sheath flow inlet ports and the fourth cavity configured to produce hydrodynamic focusing.

7. The optofluidic chip of claim 6, wherein the online mixer is disposed in fluid communication between the sheath flow generator and the photolysis zone and the sheath flow generator is in fluid communication between the online mixer and the third port.

8. The optofluidic chip of claim 6, wherein the sheath flow generator is disposed in fluid communication between the photolysis zone and the in-line mixer, and the in-line mixer is in fluid communication between the sheath flow generator and both the second port and the third port.

9. An optofluidic system for radical protein footprinting, comprising:

a chip comprising a substrate, and first, second and third ports each defined into the substrate, the chip further comprising:

a mixing region including a first cavity defined within the substrate and in fluid communication with both the second port and the third port,

a photolytic zone comprising a second cavity defined within the substrate and in fluid communication with the mixing zone, the photolytic zone further comprising a first optically transparent window sealed to the substrate and defining one side of the second cavity, the second cavity comprising channels configured in a serpentine pattern,

A dosimetry zone comprising a third cavity defined within the substrate and in fluid communication with the photolysis zone and also in fluid communication with the first port, the dosimetry zone further comprising a second optically transparent window sealed to the substrate and defining one side of the third cavity; and

a microfluidic system in fluid communication with both the second port and the third port of the mixing zone.

10. The system of claim 9, further comprising a reservoir configured to receive a biological entity comprising an oxidized biological compound.

11. The system of claim 9 or 10, further comprising a light source configured to provide light to the dosimetry zone.

12. The system of claim 9, 10 or 11, further comprising a fluorescence detector configured to receive fluorescence emitted from within the dosimetry zone.

13. The system of claim 9 to 11 or 12, further comprising a light source configured to provide coherent light to the dosimetry zone.

14. The system of claim 13, further comprising a scattered light detector configured to receive light scattered from within the dosimetry zone and in a direction orthogonal to a direction of light from the light source.

15. The system of claim 13, further comprising a refractive index light detector configured to receive light scattered from within the dosimetry zone and in a direction orthogonal to a direction of light from the light source.

16. The system of claims 9-14 or 15, wherein the chip further comprises a sheath flow generator comprising a fourth cavity defined in the substrate and in fluid communication with both the mixing zone and the photolysis zone, the sheath flow generator comprising two sheath flow inlet ports defined into the substrate and in fluid communication with the fourth cavity, the sheath flow inlet ports and the fourth cavity configured to produce hydrodynamic focusing.

17. A method for use in conjunction with an optofluidic array comprising a photolysis zone, a photolysis light source, a dosimetry zone, and a dosimetry light source, the method comprising:

introducing a mixture comprising a biological entity, a compound, and a dosimeter internal standard into the photolysis zone;

providing a light pulse from the photolytic light source to the photolytic zone, wherein the light pulse is one pulse of a series of periodic light pulses to generate a concentration of free radicals from the compound, the free radicals being effective to react with the dosimeter internal standard;

Providing light from the dosimetry light source to the dosimetry zone and detecting a change in light received from within the dosimetry zone after the light from the photolysis light source is provided to the photolysis zone, the change in received light being indicative of the biological entity being within the dosimetry zone and measuring a photometric property of the dosimeter internal standard within the dosimetry zone;

determining that the measured photometric property is below a threshold; and

determining a change to be applied to the optofluidic array to bring the measured photometric property to the threshold value, the change comprising a change in the amount of light provided by the photolytic light source, a change in the concentration of the compound in the mixture, a change in the flow rate of the mixture through the photolytic zone, or adjusting the phase of the periodic pulse.

18. The method of claim 17, wherein the photolytic light source comprises a plasma flash, an excimer laser, a solid state laser, or a laser diode.

19. The method of claim 17 or 18, wherein the dosimetry light source comprises a visible light source or a UV light source.

20. The method of claim 17, 18 or 19, wherein the compound comprises hydrogen peroxide.

21. A method according to claim 17 to 19 or 20, wherein the compound comprises trifluoromethanesulfonic acid.

22. The method of claim 17 to 20 or 21, wherein the photometric property comprises UV absorbance.

23. The method of claim 17 to 21 or 22, wherein the photometric property comprises UV or visible fluorescence.

24. A method for sheath flow control in an optofluidic array comprising a dosimetry zone, a dosimetry light source, and a sheath flow generator, the method comprising:

introducing a mixture comprising a plurality of cells, a compound, and a dosimeter internal standard into the sheath flow generator, and further introducing a buffer into the sheath flow generator, wherein the sheath flow generator is effective to employ hydrodynamic focusing to pass the mixture comprising the plurality of cells therethrough, wherein cells of the plurality of cells individually pass at uniform intervals;

receiving the mixture from the sheath flow generator into the dosimetry zone and providing light from the dosimetry light source to the dosimetry zone while monitoring the light received from the dosimetry zone, the received light varying at a cycle as the cells individually pass through the dosimetry zone; and

The flow rate of the mixture or the flow rate of the buffer into the sheath flow generator is adjusted to control the cycle.

25. The method of claim 24, wherein the optofluidic array further comprises a first pump to deliver the mixture to the sheath flow generator and a second pump to deliver the buffer to the sheath flow generator.

26. The method of claim 25, wherein adjusting the flow rate of the mixture or the flow rate of the buffer into the sheath flow generator comprises: a pumping speed difference between the first pump and the second pump is adjusted.

27. The method of claim 25, wherein the pumping speed of the second pump is at least ten times greater than the pumping speed of the first pump.