CN113533275B - Solid-state nanopore-fluorescence resonance energy transfer composite detection method and system - Google Patents
Solid-state nanopore-fluorescence resonance energy transfer composite detection method and system Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
The invention discloses a solid-state nanopore-fluorescence resonance energy transfer composite detection method, which uses paired fluorescent groups to simultaneously mark analyte molecules, adopts a confocal microscope to focus on the plane where the nanopore is located, and the analyte passes through the nanopore under the drive of an electric field and also passes through the focal plane of the confocal microscope, when fluorescence resonance energy transfer occurs, an excitation light signal is captured by the confocal microscope and converted into an electric signal to be processed by a computer, and the electric signal of the analyte molecules passing through the nanopore is combined, so that the molecular conformational information is compositely analyzed. The invention also discloses a solid-state nanopore-fluorescence resonance energy transfer composite detection system. The system and the detection method can more effectively and further distinguish the micro-conformation difference inside the biological molecule or between the biological molecules, and are suitable for the independent detection of multiple analytes and the mixed detection of different analytes.
Description
Technical Field
The invention belongs to a single-molecule detection technology, and particularly relates to a solid-state nanopore-fluorescence resonance energy transfer composite detection method and system for single-molecule conformational detection.
Background
The solid nano-pore is used as a single molecular platform to detect the conformational change of biological molecules, and has important significance for researching the structure and the function of the biological molecules. The solid-state nanopore is an electrophoretic single-molecule sensor, and consists of a nanoscale ultrathin insulating film and two nanoscale chambers containing ionic solutions, which are separated by the insulating film. When appropriate voltages, pressures, or osmotic pressures are applied to the chambers on both sides, the nanopore can be probed for charged biomolecules (e.g., DNA, RNA, proteins, etc.) using detection of ionic current through the pore, and when the analyte occupies a portion of the volume in the nanopore, the solution conductivity in the pore can be altered, resulting in a measurable change in ionic current. Different conformations of the analyte may exhibit current signals of different magnitudes or different shapes, as may the duration of the current change. However, conventional nanopores can only resolve the profile, volume information of the portion of a substance within the pore. The conformational differences of biomolecules in solution tend to be complex and variable, some small changes may have important biological significance, affected by nanopore resolution, and these local small conformational changes cannot be distinguished by ion current.
Fluorescence resonance energy transfer refers to fluorescence from a donor fluorescent molecule, while the fluorescence spectrum of one donor fluorescent molecule overlaps with the excitation spectrum of another acceptor fluorescent molecule in two different fluorophores, the excitation energy of the donor fluorescent molecule induces the acceptor molecule to fluoresce, while the fluorescence intensity of the donor fluorescent molecule itself decays. The intensity of fluorescence resonance energy transfer is inversely proportional to the 6 th power of the separation distance between the donor and the acceptor, so that the change of the distance between different domains in the molecule or the inside of the molecule in the nanoscale can be detected very sensitively.
At present, the combination research and application of the solid-state nano-pore platform single molecule detection and fluorescence resonance energy transfer technology are not seen.
Disclosure of Invention
The invention aims to: aiming at the technical problems in the prior art, the invention provides a solid-state nanopore-fluorescence resonance energy transfer composite detection method, namely a system.
The technical scheme is as follows: the invention relates to a solid-state nanopore-fluorescence resonance energy transfer composite detection method, which comprises the steps of focusing a fluorescence microscope on a plane where a solid-state nanopore is located, then simultaneously marking analyte molecules by paired fluorescent groups, collecting a current signal of the via hole of the analyte molecules and fluorescence resonance energy transfer signal intensity among the fluorescent groups when the analyte molecules pass through the nanopore, matching characteristic light and electric signals in time, and compositely analyzing molecular conformational information.
The solid-state nanopore detection device used in the invention can be: the device comprises a silicon nitride solid-state nanopore chip, an Ag electrode and patch clamp equipment Axon 200B, wherein the Ag electrode is connected with an electrode clamping device of a patch clamp system, so that voltage can be applied to the nanopore chip twice.
The pore size of the nanopore chip may alternatively range from 2nm to 350nm, depending on the size of the analyte molecule.
The nanopore chip is treated with an oxygen plasma cleaner for three minutes before use. For the Ag/AgCl electrode, sand paper is used for polishing to remove surface oxides, then the Ag/AgCl electrode is soaked in ferric chloride solution, and the Ag/AgCl electrode is washed clean by pure water and then dried.
After incubation of the analyte mixed with fluorophore, centrifugation, column washing with dextran gel and elution with PBS. The sample labeled with the fluorophore is slowly pushed into the nanopore chip side chamber with a syringe. The Ag electrode is contacted and fixed with the solution and connected with the patch clamp system, and comprises a patch clamp amplifier, a digital-analog/digital signal converter and a data analysis system, wherein the signal of the analyte passing through the nanopore is represented by a current amplitude variation form.
Typically, a solid state nanopore device is fixed to the lower part of a confocal microscope objective, an excitation light system is selected according to fluorophores used by analytes, a beam emitted by a laser illumination source in the confocal microscope is reflected by a dichroic mirror, is connected to a scanning unit through a fiber coupler, enables a thin laser beam to completely fill the rear aperture of the objective through a beam expander, and is focused by a lens system to a very small spot on a focal plane, the size of the illumination spot ranges from about 0.25 to 0.8 microns in diameter, and the depth at the brightest intensity is 0.5 to 1.5 microns. The pinhole in the conjugate plane directly in front of the photomultiplier acts as a spatial filter and excited secondary fluorescence passes through the dichroic mirror and filter to the pinhole. The large amount of fluorescence emission occurring above and below the focal plane of the objective lens is not confocal with the pinhole, and these defocused beams form an extended airy disk in the aperture plane, most of the stray light being eliminated by the pinhole aperture. Only a small fraction of the out-of-focus fluorescent emission is transmitted through the pinhole aperture, so that the photomultiplier tube does not detect a large fraction of this extraneous light and does not affect the resulting image. The fluorescent emission through the pinhole is converted by the photomultiplier tube into an analog electrical signal having a continuously varying voltage (corresponding to the fluorescent intensity). The analog signal is periodically sampled and converted into pixels by an analog-to-digital (a/D) converter installed in the scanning unit or an accompanying electronic cabinet. The image information is temporarily stored in an image frame buffer card in the computer and displayed on the monitor.
When the method is applied, the adopted solid nano-pore material is one or more of graphene, molybdenum disulfide, silicon nitride and silicon dioxide. Can be prepared by wet etching, dielectric breakdown method, focused ion beam etching and electron beam etching. The size of the used nano-pores is designed according to the size of the analyte to be detected, and the equivalent pore diameter range is 20nm-350nm for being compatible with the optical system of a confocal microscope. The solid nano-pores prepared by different materials and methods have different dimensional structures and electrical properties, but are suitable for the method.
The fluorophores can be used alone or as a functional system, in the method, the types of the fluorophores used in single detection are 2 or more, the emission wavelength and the excitation wavelength peak value between different fluorophores are different by more than 10nm, and the spectra overlap. Fluorescence resonance energy transfer occurs when the excitation spectrum of one fluorophore and the emission spectrum of the other fluorophore overlap and are at the appropriate distance and orientation. The fluorophores used to label the analyte molecules may be proteins and peptides or small molecule organic compounds such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein, etc., nucleic acid dyes such as DAPI, ethidium bromide, etc. The method has strong universality, the analyte molecules are composed of the same kind of molecules or are composed of a plurality of kinds of molecules capable of generating structural interaction, the self-changeable conformations of biomolecules such as proteins, DNA, RNA and the like can be detected, or the relative positions and the conformations caused by the intermolecular interaction are changed, and some nano-scale charged particles can be applied to the method.
Molecules pass through the nanopore in a solution environment due to free diffusion or by at least one driving force of a potential difference, an osmotic pressure difference, or a pressure difference across the nanopore. When a molecule is forced through a nanopore, the primary forcing unit can be either the molecule itself or a labeling group. When the electric signals are collected, at least 2 metal detection electrodes are respectively positioned at two sides of the nano hole, current signals generated between the electrodes are led into a patch clamp, an electrochemical workstation or other current detection devices, and current signal information is recorded. The potential difference between the detection electrodes may or may not be preset. The wavelength range of the laser system for generating fluorescence resonance energy transfer is 300-800 nm, the fluorescence signals of all the fluorophores are collected through an optical system, converted into electric signals through a photomultiplier or a photosensitive imaging element, and the fluorescence resonance energy transfer intensity is obtained through calculation. The electrical signal of the analyte passing through the solid-state nanopore is aligned with the optical signal of FRET intensity according to time, and the event that both signals are acquired while fluctuation is generated is detected by a computer. And obtaining distance information between marking sites of the analyte to be detected by using fluorescence resonance energy transfer, and obtaining analyte profile information by using a via hole electric signal.
The analyte molecules consist of a homogeneous molecule or of a combination of a plurality of classes of molecules capable of undergoing structural interactions. Other substances in the solution at both sides of the nanopore than the analyte molecule to be detected include inorganic salts, water, tris-hydroxymethyl-aminomethane, and organic molecules that do not react or bind with the analyte molecule to be detected.
The beneficial effects are that: the invention combines the fluorescence resonance energy transfer technology with the solid-state nanopore platform, and the characteristic of single molecule detection of the solid-state nanopore platform enables heterogeneous groups in analytes to be quantitatively counted and analyzed, and simultaneously, compared with a single solid-state nanopore detection method which does not depend on optics, the invention can more effectively distinguish micro conformation differences inside biomolecules or among biomolecules. The combination of optical signals with electrical signals allows researchers to more precisely explore the conformational changes of analytes and their functional significance. The method is suitable for single detection of multiple analytes and mixed detection of different analytes, and different fluorophores can be selected according to the characteristics of the analytes.
Drawings
FIG. 1 is a schematic diagram of a platform constructed in example 1 for simultaneously detecting nanopore electrical signals and fluorescence resonance energy transfer optical signals in combination with a solid state nanopore and confocal microscope;
FIG. 2 is a graph showing FRET signal and nanopore current changes collected by detection of calmodulin in example 2;
FIG. 3 shows the FRET signal and nanopore current change collected by detecting the Klenow large fragment in example 3.
Detailed Description
The process according to the invention is further illustrated below with reference to specific examples.
Example 1
A method for preparing a silicon nitride solid state nanopore chip by drilling a nanoscale pore in a silicon nitride self-supporting film, comprising the steps of:
(1) For a silicon wafer having silicon dioxide coated on one surface, negative lithography is performed on the silicon dioxide surface. After photoetching of one side of the silicon dioxide is completed, immersing the silicon dioxide into hydrofluoric acid to remove the silicon dioxide layer, and exposing the middle silicon substrate; and then etching the silicon substrate by adopting a tetramethylammonium hydroxide solution under the heating condition so as to obtain the suspended silicon nitride film.
(2) Nanopores were prepared on suspended silicon nitride films using a spherical aberration correcting transmission electron microscope. Clamping the silicon nitride self-supporting film chip into a transmission electron microscope sample rod (with one surface with suspended silicon nitride facing downwards), placing into a cavity, and vacuumizing; then searching and positioning to a suspended film area through visual feedback, adjusting the focal plane position, determining the most suitable focal plane, and calculating the center point of the suspended area; and thirdly, amplifying in situ in the suspended film area, fine-adjusting focusing, irradiating by electron beams, and continuously observing the generation of the nanopores.
(3) The nano-pore chip is placed in a piranha solution (mixed by 98% concentrated sulfuric acid and 30% hydrogen peroxide according to the volume ratio of 3:1), heated in a water bath, washed by deionized water after the chip is cooled, and placed in a 50% ethanol solution for preservation until the chip is used.
Suspending the nanopore chip on a rubber gasket, placing the rubber gasket on a glass slide, wherein a lower gap is a cis side for accommodating cis cavity solution; the trans-side solution can be instilled over the nanopore. Two silver electrodes are directly fixed in the two-sided solution, amplified by an amplifier and connected to a patch clamp system (patch clamp apparatus Axon 200B). The whole glass slide is placed below the objective lens of the confocal microscope, the working distance between the nanopore chip and the confocal microscope is smaller than 0.17mm, and laser can be focused on a plane where the solid nanopore is located through the dichroic mirror, the small hole and other elements, so that a fluorescence resonance energy transfer signal of a molecule passing through the plane can be detected. Excitation light of the donor fluorescent group and the acceptor fluorescent group is detected and converted into an electrical signal via two photomultiplier tube detectors, respectively. The fluorescence resonance energy transfer signal of the analyte molecule to be detected and the electric signal of the analyte molecule passing through the solid-state nanopore are collected and processed by a data collection system, and characteristic light and electric signals are matched according to corresponding collection time, so that a platform combining the solid-state nanopore single-molecule detection technology and the fluorescence resonance energy transfer technology is jointly built. The whole platform is shown in fig. 1, and the solid nano-pore single-molecule detection technology and the fluorescence resonance energy transfer technology are combined, so that the conformational information of the analyte molecules to be detected can be analyzed in a compound mode.
Example 2
The platform constructed according to example 1 detects different conformational states of calmodulin. Potassium chloride solution was dropped into the cis chamber, and C-and N-terminal domains of calmodulin were labeled with fluorescent probes Cy3 (fluorescence donor, labeled D) and Cy5 (fluorescence acceptor, labeled A), respectively, and dispersed in potassium chloride solution, dropped into the trans side. CaM is a typical protein capable of undergoing conformational changes upon calcium binding, and Ca 2+ After binding, the CaM undergoes a conformational change, the conformation of which changes from a closed state to an open state, while exposing hydrophobic regions, the surface of which is capable of binding to a regulated target molecule, thereby playing a critical role in the regulation of metabolic processes. FRET values change significantly when calmodulin conformation changes. Positive pressure is applied to the trans-side of the solid-state nanopore chip, driving calmodulin to move through the nanopore to another chamber, presenting significant changes in ion current amplitude on the signal acquisition software in the patch clamp system. At the same time, the donor fluorophore was excited using a 532nm laser, data were acquired at 40 ms time resolution, and fluorescence emissions between 540nm and 750nm were collected. The imaging solution used contained Tris base (50 mM, pH 7.5), 2mM trolox, 0.8mg/ml glucose, 0.1mg/ml bovine serum albumin, 1mM DTT, 0.1mg/ml dextranGlucose oxidase, 0.02mg/ml catalase, 5mM MgCl 2 And 150mM KCl. Blocking currents of both magnitudes can be observed, with low FRET values corresponding to low current magnitudes and high FRET values corresponding to high current magnitudes, by pairing with FRET values over time, as shown in fig. 2. As can be seen from the graph, low FRET values indicate an increase in calmodulin C-terminal, N-terminal distance, corresponding to calmodulin in the expanded conformation, which has a smaller blocking current through the nanopore, indicating that calmodulin in the expanded conformation has a lower hydration radius; the high FRET value indicates the proximity of the C-terminal and N-terminal ends of calmodulin, corresponding to a folded conformation of calmodulin with a greater blocking current through the nanopore, indicating that the folded conformation of calmodulin has a higher hydration radius. Therefore, the solid state nanopore-fluorescence resonance energy transfer composite detection method can analyze the volume information of the analyte molecules in the solid state nanopore by combining the FRET signal intensity and the nanopore blocking current.
Example 3
The binding of the Klenow large fragment to DNA was detected as in example 1. The crystal structure shows that when Klenow large fragment binds to DNA substrate, the thumb domain undergoes conformational change, about 1.2nm back and forth. The following Klenow large fragment labeled with Cy3B donor fluorophore (labeled D) and Cy5 acceptor fluorophore (labeled A) was designed. The donor fluorophore was excited with a 550nm laser and fluorescence emission between 560nm and 750nm was collected at a time resolution of 50 ms. The imaging buffer contained 50mM Tris (pH 7.5), 10mM MgCl2, 100mM NaCl, 100. Mu.g/ml BSA, 5% glycerol, 1mM DTT, 1mM Trolox, 1% glucose oxidase. As shown in fig. 3, when the large Klenow fragment forms a polymer with DNA, the conformational change of the thumb domain decreases the distance between Cy3B and Cy5, showing a higher FRET value, which corresponds to a longer via time for the via current signal, indicating an increased interaction with the pore wall after formation of the polymer; lower FRET values represent large segments of Klenow alone, corresponding to rapid via events; a via event in which no fluorescence resonance energy transfer is observed is generated by the DNA molecule through the nanopore. Therefore, the solid state nanopore-fluorescence resonance energy transfer composite detection method can analyze the structures of a plurality of analyte molecules passing through the solid state nanopore by combining FRET signal intensity and nanopore blocking current duration.
Claims (9)
1. A solid-state nanopore-fluorescence resonance energy transfer composite detection method is characterized in that a fluorescence microscope is adopted to focus on a plane where a solid-state nanopore is located, then a pair of fluorescent groups are used for marking at different positions of an analyte molecule at the same time, when the analyte molecule passes through the nanopore, characteristic light and electric signals are matched in time through collecting a current signal of the analyte molecule via the nanopore and fluorescence resonance energy transfer signal intensity among the marking fluorescent groups, and composite analysis molecular conformational information comprising self-changeable conformations of the biomolecule or relative position and conformational changes caused by intermolecular interaction is carried out; the analyte molecules consist of only the same type of molecules or consist of a combination of a plurality of types of molecules capable of structural interactions; the biological molecule comprises protein, DNA or RNA; the types of the fluorophores used in the single detection are more than 2, the emission wavelength and the excitation wavelength peak value between different fluorophores are different by more than 10nm, and overlapping exists between spectra.
2. The solid state nanopore-fluorescence resonance energy transfer complex detection method of claim 1, wherein the fluorescent groups for labeling the analyte molecules are proteins and peptides or small molecule organic compounds.
3. The solid state nanopore-fluorescence resonance energy transfer complex detection method of claim 1, wherein the analyte molecules and/or labeled fluorophores are driven by a force caused by at least one of a potential difference across the nanopore, an osmotic pressure difference, a pressure difference, or by free diffusion movement through the nanopore in a solution environment.
4. The solid state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein the solid state nanopore material is one or more of graphene, molybdenum disulfide, silicon nitride and silicon dioxide; can be prepared by wet etching, dielectric breakdown method, focused ion beam etching and electron beam etching, and has equivalent aperture range of 2-350 nm.
5. The solid state nanopore-fluorescence resonance energy transfer composite detection method according to claim 1, wherein a wavelength range of a laser system for fluorescence resonance energy transfer is 300nm-800nm, fluorescent signals of each fluorescent group are collected through an optical system, converted into electric signals through a photomultiplier or a photosensitive imaging element, and fluorescence resonance energy transfer intensity is obtained through calculation.
6. The solid state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein at least 2 metal detection electrodes are respectively positioned at two sides of the nanopore, current signals generated between the electrodes are led into a patch clamp, an electrochemical workstation or other current detection devices, and current signal information is recorded; the potential difference between the detection electrodes may or may not be preset.
7. The solid state nanopore-fluorescence resonance energy transfer composite detection method according to claim 1, wherein the collected light and electric signals are aligned according to time, the time when the two collected signals simultaneously fluctuate is detected, the fluorescence resonance energy transfer is utilized to obtain distance information between the analyte marking sites to be detected, and the via hole electric signals are utilized to obtain profile information of the analyte.
8. The solid state nanopore-fluorescence resonance energy transfer complex detection method of claim 1, wherein the analyte molecule consists of a homogeneous molecule or a combination of multiple classes of molecules capable of structural interaction.
9. A solid state nanopore-fluorescence resonance energy transfer composite detection system using the solid state nanopore-fluorescence resonance energy transfer composite detection method according to any one of claims 1-8, characterized by comprising a silicon nitride solid state nanopore chip, a silver electrode, a patch clamp system, a confocal microscope and a multifunctional data acquisition system, wherein the silicon nitride solid state nanopore chip is placed on a glass slide, a gap below the nanopore accommodates a cis-type side solution, a trans-type side solution is instilled above the nanopore, silver electrodes are respectively fixed in the two side solutions, and the silver electrodes amplify signals through a front-end amplifier and are connected with the patch clamp system; the glass slide is placed below the objective lens of the confocal microscope, and laser energy is focused on a plane where the solid-state nanopore is located through an excitation source, so that a fluorescence resonance energy transfer signal of a molecule passing through the plane can be detected; excitation light of the donor fluorescent group and the acceptor fluorescent group is detected and converted into an electric signal through two photomultiplier tube detectors respectively; the fluorescence resonance energy transfer signal of the analyte molecule to be detected and the electric signal passing through the solid-state nanopore are collected and processed by a data collection system.
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