Method for detecting polypeptide molecule sequence

Abstract

The invention relates to a method for detecting a polypeptide molecular sequence. The method comprises the steps of pretreating a polypeptide sample, injecting the sample, detecting and analyzing a signal and the like. The high-precision detection of the polypeptide molecule sequence can be realized by collecting and analyzing a blocking current signal generated when the polypeptide molecule passes through the silicon nitride film nanopore in the electrolyte under the driving of the voltage. The method disclosed by the invention can be used for characterizing polypeptide molecule electric signals, and has the advantages of small sample usage amount and accurate and rapid result detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting polypeptide molecule sequences. Background Art

[0002] Nanopore technology originated from the concept of Coulter counting. In 1996, Deamer et al. first reported the detection of nucleotides by protein nanopores. Nanopore technology has extremely high resolution. Currently, the technology has made remarkable progress in DNA sequencing, but its application in protein sequence detection is rarely involved. The main reason is that it is difficult to distinguish the 20 kinds of amino acids that make up proteins, and the spatial structure and charge properties of proteins also bring many difficulties to sequencing.

[0003] As an important component of life, the sequence information of peptides is of great significance for understanding biological functions, disease mechanisms and drug development. Peptide molecular detection is an important part of biomedical research and has immeasurable value in disease diagnosis, drug development, biological evolution and other fields. However, traditional peptide molecular detection methods have disadvantages such as high cost, complex operation and slow detection speed. Therefore, the development of new peptide molecular detection methods has become an urgent need in the current biotechnology field. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for detecting a polypeptide molecule sequence, wherein the method comprises detecting the electrical signal characteristics of the polypeptide molecule using a silicon nitride nanopore. Specifically, the method comprises collecting and analyzing the blocking current signal generated when the polypeptide molecule passes through the silicon nitride film nanopore in an electrolyte under voltage drive. Through the method of the present invention, high-precision detection of polypeptide molecule sequences can be achieved.

[0005] The method for detecting polypeptide molecule sequence comprises the following steps:

[0006] (1) Pretreatment of polypeptide samples: the polypeptide sample to be detected is mixed with a KCl solution, heated to 60-100° C., the pH is adjusted to 1-5 with an HCl solution, and then dithiothreitol (DTT) is added for reaction to obtain a pretreated polypeptide sample;

[0007] (2) Sample injection: the electrolyte is separated by a silicon nitride film with nanopores, and the pretreated polypeptide sample is injected into one part of the electrolyte, wherein the diameter of the nanopore is 0.35-5 nm, and the sensing area length of the nanopore is 0.3-2 nm;

[0008] According to the charge property of the injected sample, electrodes are inserted into the electrolyte on both sides of the silicon nitride film: if the sample is positively charged, the positive electrode is inserted into the sample side; if the sample is negatively charged, the negative electrode is inserted into the sample side;

[0009] (3) Signal detection and analysis: Voltage is applied to the electrolyte on both sides of the silicon nitride film through electrodes to drive the linear polypeptide molecules into the nanopore. The electrical signals generated when the polypeptide molecules pass through the nanopore are detected and analyzed to determine the characteristic patterns of the electrical signal changes of the polypeptide molecules and obtain the polypeptide molecule sequence information.

[0010] In a preferred embodiment, in step (1), the polypeptide sample is subjected to conventional purification and desalting before pretreatment to achieve a purity of more than 90%.

[0011] In a preferred embodiment, in step (1), the concentration of the KCl solution is 1 M, and the concentration of the dithiothreitol (DTT) is 1-3 mM.

[0012] In step (1), the pH of the mixed solution is adjusted by HCl to destroy the hydrogen bonds within the molecules, and the disulfide bonds (SS) are destroyed by adding the reducing agent DTT to release and linearize the polypeptide chains, thereby preparing for the migration of the molecules through the nanopores under the action of the electric field. After the pretreatment in step (1), the polypeptide sample becomes a linear chain structure.

[0013] In a preferred embodiment, in step (2), the electrolyte is a 1 M KCl solution, and the pH is adjusted to 1-5 with an HCl solution.

[0014] In a preferred embodiment, in step (2), the injection concentration of the pretreated polypeptide sample is 0.1 μg / mL.

[0015] In a preferred embodiment, in step (2), the electrode is an Ag / AgCl electrode.

[0016] In a preferred embodiment, in step (2), before injecting the sample into the electrolyte, the electrolyte is heated to a temperature for pre-treating the polypeptide sample.

[0017] In a preferred embodiment, in step (3), the voltage is 300 mV.

[0018] In a preferred embodiment, in step (3), the electrical signal includes current amplitude, frequency, signal shape and duration. Since each polypeptide molecule can generate a unique electrical signal, it is possible to determine whether the same polypeptide molecule is present by comparing the electrical signal characteristics of each batch of polypeptide samples. Furthermore, the type of polypeptide molecule can be determined based on the unique electrical signal generated by each polypeptide molecule.

[0019] In a preferred embodiment, in step (3), the analysis includes processing and / or analyzing the electrical signal using computer software.

[0020] In a preferred embodiment, in step (3), applying voltage and detecting electrical signals are performed in a Faraday cage.

[0021] Optionally, after step (3), the following steps are also included:

[0022] (4) Result output: The polypeptide molecule sequence information obtained by the detection and analysis is displayed or output in a visual form.

[0023] The beneficial effects of the present invention include:

[0024] 1. Through sample pretreatment, the polypeptide molecules can be linearized and then migrate through the nanopore under the action of the electric field. Each polypeptide can form a unique electrical signal. By comparing the electrical signal characteristics of each batch of polypeptide samples, it can be determined whether they are the same polypeptide molecules.

[0025] 2. It can be used for the characterization of electrical signals of polypeptide molecules, with the advantages of small sample usage and accurate and rapid detection results.

[0026] 3. The method includes collecting and analyzing the blocking current signal generated when the polypeptide molecule passes through the silicon nitride film nanopore in the electrolyte under voltage drive. The method of the present invention can achieve high-precision detection of polypeptide molecule sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the structure of a device for detecting electrical signal characteristics of polypeptide molecules using silicon nitride film nanopores in one embodiment of the present invention.

[0028] [Description of Reference Numerals]

[0029] 1-electrolyte; 2-silicon nitride film with nanopores; 3-electrode; 4-probe; 5-patch clamp amplifier; 6-digital-to-analog converter; 7-computer (running software); 8-water bath; 9-Faraday cage; 10-anti-vibration table. DETAILED DESCRIPTION

[0030] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings.

[0031] Although the exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0032] Example 1

[0033] This embodiment provides a method for detecting a polypeptide molecule sequence, comprising the following steps:

[0034] (1) Pretreatment of polypeptide samples: the polypeptide sample to be detected is mixed with a KCl solution, heated to 60-100° C., the pH is adjusted to 1-5 with an HCl solution, and then dithiothreitol (DTT) is added for reaction to obtain a pretreated polypeptide sample;

[0035] (2) Sample injection: the electrolyte is separated by a silicon nitride film with nanopores, and the pretreated polypeptide sample is injected into one part of the electrolyte, wherein the diameter of the nanopore is 0.35-5 nm, and the sensing area length of the nanopore is 0.3-2 nm;

[0036] According to the charge property of the injected sample, electrodes are inserted into the electrolyte on both sides of the silicon nitride film: if the sample is positively charged, the positive electrode is inserted into the sample side; if the sample is negatively charged, the negative electrode is inserted into the sample side;

[0037] (3) Signal detection and analysis: Voltage is applied to the electrolyte on both sides of the silicon nitride film through electrodes to drive the linear polypeptide molecules into the nanopore. The electrical signals generated when the polypeptide molecules pass through the nanopore are detected and analyzed to determine the characteristic patterns of the electrical signal changes of the polypeptide molecules and obtain the polypeptide molecule sequence information.

[0038] Example 2

[0039] A method for detecting a polypeptide molecule sequence, referring to Figure 1 , specifically including the following steps:

[0040] (1) Assembling the detection device: placing a water bath 8 on an anti-vibration platform 10, placing a silicon nitride film 2 with nanopores in the liquid pool, dividing the liquid pool of the water bath 8 into two parts (e.g., equally divided), wherein the diameter of the nanopore is 0.35-5 nm, and the sensing region length of the nanopore is 0.3-2 nm. Injecting an electrolyte 1 (1M KCl solution, with pH adjusted to 1-5 with HCl) into the two liquid pools.

[0041] (2) Pretreatment of polypeptide samples: The polypeptide sample to be detected is subjected to conventional purification and desalting before pretreatment to obtain a purity of more than 90%; then, it is mixed with a 1M KCl solution, heated to 60-100°C in a water bath, the pH is adjusted to 1-5 with an HCl solution, and then 1-3 mM dithiothreitol (DTT) is added for reaction to obtain a pretreated polypeptide sample.

[0042] (3) Sample injection: The electrolyte is heated to the temperature of the pretreated polypeptide sample, and the pretreated polypeptide sample is injected into the electrolyte on the sample side of the liquid pool, and the injection concentration of the pretreated polypeptide sample is 0.1 μg / mL; Ag / AgCl electrodes 3 are inserted into the electrolyte 1 on both sides of the silicon nitride film 2 according to the charge properties of the injected sample: if the sample is positively charged, the positive electrode is inserted into the sample side; if the sample is negatively charged, the negative electrode is inserted into the sample side;

[0043] The two electrodes 3 are connected to a patch clamp amplifier 5 (MultiClamp 700B) via a probe 4, and in turn connected to a digital-to-analog converter 6 (Axon Digidata 1550A) and a computer 7. The computer 7 can run detection and analysis software, such as visualization software.

[0044] To ensure the reliability and accuracy of the experimental data, the system is placed in a Faraday cage 9 during the experiment. For example, a water bath 8, a silicon nitride film 2, an electrolyte 1 (together with a pretreated polypeptide sample), an electrode 3, and a probe 4 are placed in a Faraday cage 9, and the Faraday cage 9 is placed on an anti-vibration table 10.

[0045] (4) Signal detection and analysis: A voltage of 300 mV is applied to the electrolyte on both sides of the silicon nitride film through electrodes to drive the linear polypeptide molecules into the nanopore, and the electrical signals generated when the polypeptide molecules pass through the nanopore are detected and processed and / or analyzed by computer 7 through computer software. The electrical signals include current amplitude, frequency, signal shape and duration, so as to determine the characteristic law of the electrical signal changes of the polypeptide molecules and obtain the sequence information of the polypeptide molecules.

[0046] (5) Result output: The polypeptide molecule sequence information obtained by the detection and analysis is displayed or output in a visual form, for example, through a display of a computer 7 .

[0047] Example 3

[0048] Based on Example 2, processing and / or analyzing the electrical signal using computer 7 through computer software includes the following exemplary process:

[0049] The obtained electrical signal data was first exported after preliminary screening by Clampfit software, and then the signal features were extracted using the Transalyzer-master software package. Among them, the Transalyzer-master software package includes multiple software for signal identification and extraction, such as GUI-detect and GUI-events. The GUI-detect software was used to extract the signals of different protein molecules in the same current time for comparison. This process requires that the signal is clear and identifiable, and there is a gap greater than 0.0001s between adjacent events. The GUI-events software was used to perform preliminary identification of the obtained signal to eliminate interference signals that do not meet the event characteristics. The amplitude, duration, baseline position data of each through-hole event obtained were imported into the Origin software to draw the frequency distribution of the blocking current amplitude and the histogram of the ratio of the current amplitude to the opening current ΔI / I0.

[0050] Example 4

[0051] In this embodiment, an exemplary method for preparing the silicon nitride film with nanopores is provided, comprising the following steps:

[0052] 1) Place the silicon nitride film into a scanning transmission electron microscope;

[0053] 2) Set the accelerating voltage to 300 kV and the beam stop aperture to 70 μm. By adjusting the beam intensity and reducing the magnification, the beam can be seen passing through the film.

[0054] 3) Adjust the magnification to SA34000x and adjust the Z height to eliminate the halo caused by focusing the beam;

[0055] 4) Set Eucentric Focus, switch to STEM mode, turn off Diffraction, turn on Beamshift, adjust Multifunction X / Y to move the beam to the center, and adjust Defocus to focus the beam to a small point;

[0056] 5) Click Done, turn on Diffraction, click Search, and adjust the Z Height to focus the beam on the film surface.

[0057] 6) Close Search, click Blank to block the beam and close the column valve, and wait for 20 minutes to allow the sample film to stabilize;

[0058] 7) Click Search to start preview, adjust the magnification to 320kx, further adjust the Z height (ZHeight) to focus the beam on the film surface, stop preview, and the electron beam converges to a point on the film and starts drilling; the spot size is 6, the current is 0.429-0.51nA, and the drilling time is 24-30s;

[0059] 8) Switch to TEM mode and measure the pore size using a transmission electron microscope.

[0060] The pore size of the nanopores of the silicon nitride film is in the range of 0.35-5 nm, and the length of the sensing region is in the range of 0.3-2 nm, which is similar to the size of a single amino acid molecule.

[0061] The method of the present invention includes collecting and analyzing the blocking current signal generated when the polypeptide molecule passes through the nanopore of the silicon nitride film in the electrolyte under voltage drive. The method of the present invention can achieve high-precision detection of the polypeptide molecule sequence. It can be used for the characterization of the electrical signal of the polypeptide molecule, and has the advantages of less sample usage and accurate and fast result detection.

[0062] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may alter, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for detecting a polypeptide molecule sequence, characterized in that: The following steps are involved: (1) Pretreatment of polypeptide samples: the polypeptide sample to be detected is mixed with a KCl solution, heated to 60-100° C., the pH is adjusted to 1-5 with an HCl solution, and then dithiothreitol (DTT) is added for reaction to obtain a pretreated polypeptide sample; (2) Sample injection: the electrolyte is separated by a silicon nitride film with nanopores, and the pretreated polypeptide sample is injected into one part of the electrolyte, wherein the diameter of the nanopore is 0.35-5 nm, and the sensing area length of the nanopore is 0.3-2 nm; According to the charge property of the injected sample, electrodes are inserted into the electrolyte on both sides of the silicon nitride film: if the sample is positively charged, the positive electrode is inserted into the sample side; if the sample is negatively charged, the negative electrode is inserted into the sample side; (3) Signal detection and analysis: Voltage is applied to the electrolyte on both sides of the silicon nitride film through electrodes to drive the linear polypeptide molecules into the nanopore. The electrical signals generated when the polypeptide molecules pass through the nanopore are detected and analyzed to determine the characteristic patterns of the electrical signal changes of the polypeptide molecules and obtain the polypeptide molecule sequence information.

2. The method according to claim 1, characterized in that In step (1), the concentration of the KCl solution is 1 M, and the concentration of the dithiothreitol (DTT) is 1 to 3 mM.

3. The method according to claim 1, characterized in that In step (2), the electrolyte is a 1 M KCl solution, and the pH is adjusted to 1-5 with an HCl solution.

4. The method according to claim 3, characterized in that In step (2), the injection concentration of the pretreated polypeptide sample is 0.1 μg / mL.

5. The method according to claim 4, characterized in that In step (2), the electrode is an Ag / AgCl electrode; before the sample is injected into the electrolyte, the electrolyte is heated to a temperature for pretreating the polypeptide sample.

6. The method according to claim 1, characterized in that In step (3), the voltage is 300 mV.

7. The method according to claim 6, characterized in that In step (3), the electrical signal includes current amplitude, frequency, signal shape and duration.

8. The method according to claim 7, characterized in that In step (3), the analysis includes processing and / or analyzing the electrical signal using computer software.

9. The method according to claim 8, characterized in that In step (3), applying voltage and detecting electrical signals are performed in a Faraday cage.

10. The method according to claim 1, characterized in that After step (3), the following steps are also included: (4) Result output: The polypeptide molecule sequence information obtained by the detection and analysis is displayed or output in a visual form.

Images