CN113533275A - Solid-state nanopore-fluorescence resonance energy transfer composite detection method and system - Google Patents

Abstract

Translated fromChinese

本发明公开了一种固态纳米孔‑荧光共振能量转移复合检测方法，该方法用成对的荧光基团同时标记的分析物分子，采用共聚焦显微镜聚焦于纳米孔所在的平面，分析物在电场的驱动下通过纳米孔，同时也通过共聚焦显微镜的焦平面，当发生荧光共振能量转移，激发光信号被共聚焦显微镜捕捉并转换为电信号经计算机处理，结合分析物分子通过纳米孔的电信号，从而复合解析分子构象信息。本发明还公开了一种固态纳米孔‑荧光共振能量转移复合检测系统。本发明系统和检测方法能够更有效地进一步区分生物分子内部或生物分子之间细微构象差异，适用于多种分析物单独检测和不同的分析物混合检测。The invention discloses a solid-state nanopore-fluorescence resonance energy transfer composite detection method. The method uses analyte molecules simultaneously labeled with paired fluorescent groups, and uses a confocal microscope to focus on the plane where the nanopore is located, and the analyte is placed in an electric field. Driven by the nanopore through the nanopore, and also through the focal plane of the confocal microscope, when fluorescence resonance energy transfer occurs, the excitation light signal is captured by the confocal microscope and converted into an electrical signal. signal, thereby compounding the molecular conformational information. The invention also discloses a solid-state nanopore-fluorescence resonance energy transfer composite detection system. The system and detection method of the present invention can further distinguish subtle conformational differences within biomolecules or between biomolecules more effectively, and are suitable for separate detection of multiple analytes and mixed detection of different analytes.

Description

Solid-state nanopore-fluorescence resonance energy transfer composite detection method and system

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 conformation detection.

Background

The solid-state nanopore serving as a single molecular platform can detect the conformational change of biomolecules and has important significance for researching the structure and the function of the biomolecules. The solid-state nanopore is an electrophoresis single-molecule sensor and consists of a nanometer-level ultrathin insulating film and two nanometer-level chambers which are separated by the insulating film and contain ionic solutions. When the two-sided chamber is subjected to an appropriate voltage, pressure or osmotic pressure, the nanopore can detect charged biomolecules (e.g., DNA, RNA, proteins, etc.) by detecting ionic current through the pore, and when the analyte occupies a portion of the volume in the nanopore, the conductivity of the solution within the pore is altered, resulting in a measurable change in ionic current. Different conformations of the analyte may exhibit different amplitudes or different shapes of current signal, as well as different durations of current change. However, conventional nanopores can only resolve the profile, volume information of the material within the pore. The conformational differences of biomolecules in solution are often complex and changeable, and some small changes may have important biological significance, and due to the influence of nanopore resolution, these local small conformational changes cannot be distinguished by ion current.

Fluorescence resonance energy transfer refers to the fact that in two different fluorescent groups, when the fluorescence spectrum of one donor fluorescent molecule has overlap with the excitation spectrum of the other acceptor fluorescent molecule, the excitation energy of the donor fluorescent molecule induces the acceptor molecule to emit fluorescence, and the fluorescence intensity of the donor fluorescent molecule is attenuated. 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 nano-scale distance change of different domains between molecules or in the molecule can be detected with high sensitivity.

At present, the combined research and application of the solid-state nanopore platform single-molecule detection and fluorescence resonance energy transfer technology are not available.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems in the prior art, the invention provides a solid-state nanopore-fluorescence resonance energy transfer composite detection method and 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 firstly focusing on a plane where solid-state nanopores are located by adopting a fluorescence microscope, then simultaneously marking molecules of an analyte by using paired fluorescent groups, collecting a through hole current signal of the molecules of the analyte and the intensity of a fluorescence resonance energy transfer signal among the marked fluorescent groups when the molecules of the analyte pass through the nanopores, matching characteristic light and an electric signal in time, and compositely analyzing molecular conformation information.

The solid-state nanopore detection device used by the invention can be as follows: the nano-pore chip comprises a silicon nitride solid-state nano-pore chip, an Ag electrode and a patch clamp device 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 nano-pore chip twice.

The pore size of the nanopore chip can be selected from the range of 2nm to 350nm according to the size of analyte molecules.

Three minutes of treatment with an oxygen plasma cleaner was required before the nanopore chip was used. For Ag/AgCl electrodes, sand paper is used for polishing to remove surface oxides, then the Ag/AgCl electrodes are soaked in ferric chloride solution, washed clean by pure water and dried.

The analyte was incubated with fluorophore mixed and centrifuged, and the column washed with Sephadex and eluted with PBS. The sample labeled with the fluorophore was slowly pushed into the nanopore chip side chamber with a syringe. The Ag electrode is contacted and fixed with the solution, is connected with a patch clamp system and comprises a patch clamp amplifier, a digital-analog/analog-digital signal converter and a data analysis system, and the signal of an analyte passing through the nanopore is presented in the form of current amplitude change.

Typically, the solid state nanopore device is mounted underneath the objective of a confocal microscope, the excitation light system is selected based on the fluorophore used by the analyte, the beam from the laser illumination source in the confocal microscope is reflected by a dichroic mirror, coupled to the scanning unit by a fiber optic coupler, and focused by a beam expander to a very small spot in the focal plane, the illumination spot size ranging from about 0.25 to 0.8 microns in diameter and 0.5 to 1.5 microns deep at the brightest intensity. The pinhole, located on a conjugate plane directly in front of the photomultiplier tube, acts as a spatial filter, and the excited secondary fluorescence passes through the dichroic mirror and the filter to the pinhole. The large amount of fluorescence emission that occurs above and below the objective focal plane is not confocal with the pinhole, these defocused beams form an expanded airy disk in the aperture plane, and most of the stray light is eliminated by the pinhole aperture. Only a small fraction of the defocused fluorescent emission is transmitted through the pinhole aperture, so the photomultiplier tubes do not detect most of this extraneous light and do not contribute to the resulting image. The fluorescent emission through the pinhole is converted by a 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 a 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-state nano-pore material is one or more of graphene, molybdenum disulfide, silicon nitride and silicon dioxide. The material can be prepared by wet etching, dielectric breakdown method, focused ion beam etching and electron beam etching. The size of the nanopore used is designed according to the size of the analyte to be detected, and the equivalent aperture range of the nanopore is 20nm-350nm in order to be compatible with the optical system of a confocal microscope. Solid-state nanopores prepared from different materials and by different methods have different size structures and electrical properties, but are all suitable for the method.

The fluorophores can be used independently or matched with each other as a functional system, in the method, the types of the fluorophores used in single detection are 2 or more, the difference between the emission wavelength and the excitation wavelength peak value among different fluorophores is more than 10nm, and the spectra are overlapped. Fluorescence resonance energy transfer occurs when the excitation spectrum of one fluorophore and the emission spectrum of the other fluorophore have overlap, and the two are at the proper distance and orientation. The fluorophore used to label the analyte molecules may be proteins and peptides or small 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 consist of the same kind of molecules or the molecules capable of structurally interacting together, the self-variable conformations of biomolecules such as protein, DNA, RNA and the like can be detected, or the changes of relative positions and conformations caused by the interaction among the molecules can be detected, and some charged particles with nanometer scales can also be applied to the method.

Molecules pass through the nanopore in a solution environment due to free diffusion or at least one driving force of potential difference, osmotic pressure difference and pressure difference on two sides of the nanopore. When a molecule is forced through a nanopore, the primary stressed unit can be either the molecule itself or a labeled group. When electric signals are collected, at least 2 metal detection electrodes are respectively positioned on 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 detection electrodes may or may not be preset with a potential difference. The wavelength range of a laser system for generating fluorescence resonance energy transfer is 300nm-800nm, fluorescence signals of all fluorophores are collected through an optical system, converted into electric signals through a photomultiplier tube or a photosensitive imaging element, and the fluorescence resonance energy transfer intensity is obtained through calculation. The electric signal of the analyte passing through the solid-state nanopore and the optical signal of FRET intensity are aligned according to time, and a computer detects an event that the two signals are collected and simultaneously generate fluctuation. And obtaining distance information between the marking sites of the analyte to be detected by utilizing fluorescence resonance energy transfer, and obtaining the outline information of the analyte by utilizing the electric signal of the via hole.

The analyte molecules consist of molecules of the same type or of a plurality of structurally interacting molecules. Other substances except the analyte molecules to be detected in the solution on the two sides of the nanopore comprise inorganic salt, water, tris (hydroxymethyl) aminomethane and organic molecules which do not react or combine with the analyte molecules to be detected.

Has the advantages 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 the heterogeneous population in the analyte to be quantitatively counted and analyzed, and meanwhile, compared with a single solid-state nanopore detection method which does not depend on optics, the invention can more effectively distinguish the micro conformation difference in the biomolecules or between the biomolecules. The combination of optical and electrical signals allows researchers to more accurately explore the conformational changes of the analyte and its functional significance. The method is suitable for the single detection of a plurality of analytes and the mixed detection of different analytes, and different fluorophores can be selected according to the characteristics of the analytes.

Drawings

Fig. 1 is a schematic platform diagram for simultaneously detecting nanopore electrical signals and fluorescence resonance energy transfer optical signals by combining a solid-state nanopore and a confocal microscope, which are built inembodiment 1;

FIG. 2 shows the FRET signal and nanopore current changes collected by the detection of calmodulin in example 2;

FIG. 3 shows the FRET signal and nanopore current changes detected in Klenow large fragment acquisition in example 3.

Detailed Description

The process of the present invention is further illustrated below with reference to specific examples.

Example 1

The preparation of silicon nitride solid state nanometer hole chip, it passes through the process of drilling nanometer pore on the silicon nitride self-supporting film, including the following steps:

(1) for a silicon wafer having one surface coated with silicon dioxide, negative tone lithography is performed on the silicon dioxide surface. After photoetching of one surface of the silicon dioxide, immersing the silicon dioxide into hydrofluoric acid to remove the silicon dioxide layer and expose the silicon substrate in the middle; and etching the silicon substrate by adopting a tetramethylammonium hydroxide solution under a heating condition to obtain the suspended silicon nitride film.

(2) Nanopores were prepared on the suspended silicon nitride film using a spherical aberration correcting transmission electron microscope. The silicon nitride self-supporting film chip is clamped into a transmission electron microscope sample rod (one surface with suspended silicon nitride is placed downwards), and the transmission electron microscope sample rod is placed into a cavity and vacuumized; then searching and positioning to a suspended thin film region through visual feedback, adjusting the position of a focal plane, determining an optimal focal plane, and calculating a central point of the suspended thin film region; and thirdly, amplifying in-situ in the suspended film area, finely adjusting and focusing, irradiating by electron beams, and continuously observing the generation of the nano holes.

(3) Putting the nanopore chip into piranha solution (98% concentrated sulfuric acid and 30% hydrogen peroxide are mixed according to the volume ratio of 3: 1), heating in water bath, after the chip is cooled, washing with deionized water, and storing in 50% ethanol solution until the chip is used.

Suspending the nanopore chip on a rubber gasket, placing the rubber gasket on a glass slide, wherein the lower gap is a cis-form side and contains a cis-form chamber solution; the trans-lateral solution can be instilled over the nanopore. Two silver electrodes are directly fixed in the solution at two sides, and the signals are amplified by an amplifier and connected with a patch clamp system (patch clamp equipment Axon 200B). The whole glass slide is placed below an objective lens of a confocal microscope, the working distance between the nanopore chip and the confocal microscope is less than 0.17mm, and laser can be focused on a plane where the solid-state nanopore is located through elements such as a dichroic mirror and a small hole, so that a fluorescence resonance energy transfer signal of molecules passing through the plane can be detected. The excitation light of the donor fluorophore and the acceptor fluorophore is detected by two photomultiplier detectors and converted into electrical signals, 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 acquired and processed by the data acquisition system, and the characteristic light and the electric signal are matched according to the corresponding acquisition time, so that a platform combining the solid-state nanopore monomolecular detection technology and the fluorescence resonance energy transfer technology is jointly established. The whole platform is shown in figure 1, and the solid-state nanopore single-molecule detection technology and the fluorescence resonance energy transfer technology are combined, so that the conformation information of the analyte molecule to be detected can be compositely analyzed.

Example 2

The different conformational states of calmodulin were detected according to the platform set up in example 1. A potassium chloride solution was dropped into the cis chamber, and the C-terminal 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 the potassium chloride solution, and dropped into the trans side. CaM is a typical protein capable of undergoing conformational change after calcium binding, and Ca²⁺After the bonding, the adhesive is cured,the CaM is changed in configuration, the conformation of the CaM is changed from a closed state to an open state, a hydrophobic region is exposed, and the surface of the hydrophobic region can be combined with a regulated target molecule, so that the CaM plays a vital role in regulating and controlling a metabolic process. When the calmodulin conformation changes, the FRET value changes significantly. Positive pressure is applied to the reverse side of the solid-state nanopore chip to drive calmodulin to move to the other chamber through the nanopore, and obvious change of ionic current amplitude is presented on signal acquisition software in a patch clamp system. At the same time, a 532nm laser was used to excite the donor fluorophore, data were acquired with a time resolution of 40 milliseconds, and fluorescence emission between 540nm and 750nm was collected. The imaging solution used contained Tris base (50mM, pH 7.5), 2mM trolox, 0.8mg/ml glucose, 0.1mg/ml bovine serum albumin, 1mM DTT, 0.1mg/ml glucose oxidase, 0.02mg/ml catalase, 5mM MgCl₂And 150mM KCl. The blocking currents of both amplitudes can be observed, by temporally pairing with the FRET values, it is observed that the low FRET value corresponds to the low current amplitude and the high FRET value corresponds to the high current amplitude, the results are shown in fig. 2. As can be seen from the graph, a low FRET value indicates an increased C-terminal, N-terminal distance of calmodulin, corresponding to calmodulin in the expanded conformation, with a smaller blocking current through the nanopore, indicating a calmodulin in the expanded conformation with a lower hydration radius; a high FRET value indicates proximity of the C-terminus and N-terminus of calmodulin, corresponding to a more blocking current through the nanopore in the folded conformation, indicating a higher hydration radius for calmodulin in the folded conformation. Therefore, the solid-state nanopore-fluorescence resonance energy transfer composite detection method can be used for analyzing the volume information of the analyte molecules in the solid-state nanopore by combining the FRET signal intensity and the nanopore blocking current magnitude.

Example 3

Binding of Klenow large fragment to DNA was detected as in example 1. The crystal structure indicates that when Klenow large fragment binds to DNA substrate, the thumb domain undergoes a conformational change, varying back and forth by about 1.2 nm. The large Klenow fragment labeled with the Cy3B donor fluorophore (labeled D) and the Cy5 acceptor fluorophore (labeled a) was designed as follows. The donor fluorophore was excited with a 550nm laser and fluorescence emission between 560nm and 750nm was collected with a time resolution of 50 milliseconds. 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. The FRET signal is temporally correlated with the nanopore blocking signal, and as a result, as shown in fig. 3, according to the graph, when Klenow large fragment forms a polymer with DNA, the conformation change of the thumb domain causes the distance between Cy3B and Cy5 to be reduced, and a higher FRET value is shown, and the corresponding via current signal has a longer via time, which indicates that the interaction between the polymer and the wall of the pore is increased after the polymer is formed; lower FRET values represent Klenow large fragments alone, corresponding to rapid via events; the via events where no fluorescence resonance energy transfer phenomenon is observed are generated by the DNA molecules passing through the nanopore. Therefore, the solid-state nanopore-fluorescence resonance energy transfer composite detection method can be used for analyzing the structure of a plurality of analyte molecules when the analyte molecules pass through the solid-state nanopore by combining the FRET signal intensity and the nanopore blocking current duration.

Claims (10)

1. A solid-state nanopore-fluorescence resonance energy transfer composite detection method is characterized in that a fluorescence microscope is focused on a plane where solid-state nanopores are located, paired fluorescence groups are used for marking molecules of an analyte at the same time, and when the molecules of the analyte pass through the nanopores, characteristic light and electric signals are matched in time through collecting through-hole current signals of the molecules of the analyte and the intensity of fluorescence resonance energy transfer signals among the marked fluorescence groups, so that molecular conformation information is analyzed in a composite mode.

2. The solid-state nanopore-fluorescence resonance energy transfer multiplexed detection method of claim 1, wherein the fluorophore for labeling the analyte molecule is a protein or a peptide or a small molecule organic compound.

3. The solid-state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein the types of fluorophores used in a single detection are more than 2, the difference between the emission wavelength and the excitation wavelength peak value among different fluorophores is more than 10nm, and the spectra are overlapped.

4. The solid state nanopore-fluorescence resonance energy transfer multiplexed detection method of claim 1, wherein the analyte molecule and/or labeled fluorophore is driven by a force caused by at least one of a potential difference across the nanopore, an osmotic pressure difference, a pressure difference, or due to free diffusion movement through the nanopore in a solution environment.

5. The solid-state nanopore-fluorescence resonance energy transfer composite detection method according to claim 1, wherein the adopted solid-state nanopore is made of one or more of graphene, molybdenum disulfide, silicon nitride and silicon dioxide; the material can be prepared by wet etching, dielectric breakdown method, focused ion beam etching and electron beam etching, and the equivalent aperture range is 2nm-350 nm.

6. The solid-state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein a laser system for fluorescence resonance energy transfer has a wavelength range of 300nm-800nm, fluorescence signals of each fluorophore are collected by an optical system, converted into electrical signals by a photomultiplier tube or a photosensitive imaging element, and the fluorescence resonance energy transfer intensity is obtained by calculation.

7. The solid-state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein at least 2 metal detection electrodes are respectively located at two sides of the nanopore, and a current signal generated between the electrodes is led into a patch clamp, an electrochemical workstation or other current detection devices to record current signal information; the detection electrodes may or may not be preset with a potential difference.

8. The solid-state nanopore-fluorescence resonance energy transfer composite detection method of claim 1, wherein the collected light and electric signals are aligned according to time, an event that the collected two signals fluctuate simultaneously is detected, distance information between labeled sites of an analyte to be detected is obtained by fluorescence resonance energy transfer, and outline information of the analyte is obtained by a via hole electric signal.

9. The solid-state nanopore-fluorescence resonance energy transfer multiplexed detection method of claim 1, wherein the analyte molecule consists of a same type of molecule or a plurality of structurally interacting types of molecules together.

10. A solid-state nanopore-fluorescence resonance energy transfer composite detection system is 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 arranged on a glass slide, a cis-form side solution is contained in a gap below a nanopore, a trans-form side solution is dripped above the nanopore, the silver electrodes are respectively fixed in the solutions on the two sides, 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 a confocal microscope objective, laser can be focused on a plane where the solid-state nano holes are located through an excitation source, and therefore fluorescence resonance energy transfer signals of molecules passing through the plane can be detected; the exciting light of the donor fluorophore and the acceptor fluorophore is detected by two photomultiplier detectors and converted into electric signals; 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 acquisition system.

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