Method for detecting novel coronavirus n-protein
By using solid-state nanopore chips to detect the N-protein of the novel coronavirus and utilizing current signals to determine whether a sample contains the N-protein, this method solves the problem of excessively long detection times in existing methods and achieves rapid and sensitive detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACAD OF SCI
- Filing Date
- 2023-05-06
- Publication Date
- 2026-05-19
AI Technical Summary
Existing COVID-19 testing methods, such as nucleic acid testing and lung CT scans, take too long to provide rapid diagnostic results.
The detection of SARS-CoV-2 N-protein using a solid-state nanopore chip involves adding the sample to a sample cell containing first and second media, applying a voltage, and detecting the ion blocking current pulse signal generated by the nanopore. The presence of SARS-CoV-2 N-protein in the sample is then determined based on the signal.
It enables rapid and sensitive detection of the SARS-CoV-2 N-protein, shortens the detection window period, and provides rapid diagnostic results.
Smart Images
Figure CN116930263B_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of biological detection technology, and more specifically relates to a method for detecting the N-protein of the novel coronavirus. Background Technology
[0002] Currently, the most commonly used and highly accurate methods for detecting novel coronavirus infection include nucleic acid testing and lung CT scans. Nucleic acid testing offers advantages in terms of high throughput, high sensitivity, and high accuracy, but it takes a relatively long time. While lung CT scans are highly accurate, the process is cumbersome and also requires a considerable wait. Summary of the Invention
[0003] Based on the problems existing in the prior art, this application provides a method for detecting the N-protein of the novel coronavirus, aiming to solve the technical problem of long detection time for the novel coronavirus.
[0004] To achieve the above objectives, a method for detecting the N-protein of the novel coronavirus is provided, comprising the following steps: adding a sample to be tested into a sample cell, wherein the sample cell includes a cis-cavity, a trans-cavity, and a solid nanopore chip; the trans-cavity contains a first medium, the cis-cavity contains a second medium, and the trans-cavity and cis-cavity are connected through nanopores on the solid nanopore chip; the sample is added to the second medium; the first medium includes a first conductivity buffer, and the second medium includes the first conductivity buffer and negatively charged molecules, wherein the molecules include small molecules and macromolecules; applying a voltage to the first and second media and detecting the ion blocking current pulse signal generated by the nanopore; and determining whether the sample contains the N-protein of the novel coronavirus based on the ion blocking current pulse signal.
[0005] Optionally, determining whether a sample contains the SARS-CoV-2 N-protein based on the ion blocking current pulse signal includes: when the molecule is a small molecule, detecting the ion blocking current pulse signal determines that the sample contains the SARS-CoV-2 N-protein.
[0006] Optionally, determining whether a sample contains SARS-CoV-2 N-protein based on the ion blocking current pulse signal includes: when the molecule is a large molecule, when the ion blocking current pulse signal is detected, processing the ion blocking current pulse signal to obtain the change in the blocking current peak value; when the blocking current has two peak values, it is determined that the sample contains SARS-CoV-2 N-protein; when the blocking current has one peak value, it is determined that the sample does not contain SARS-CoV-2 N-protein.
[0007] Optionally, before adding the sample to be tested into the sample cell, the following steps are included: preparing a first conductivity buffer, which includes: mixing sodium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water, and adjusting the pH of the first conductivity buffer to 7.4, wherein the ratio of sodium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid is 1M:10mM:1mM, and M is mol / L.
[0008] Optionally, before adding the sample to be tested into the sample cell, the method further includes: rinsing the trans cavity and cis cavity with deionized water, then rinsing the trans cavity and cis cavity with anhydrous ethanol; adding the first conductivity buffer to the cis cavity and trans cavity respectively, and adding the molecular substance to the cis cavity.
[0009] Optionally, before applying voltage to the first and second media, the method further includes: inserting two Ag / AgCl electrodes into the cis and trans cavities, respectively; and connecting the two Ag / AgCl electrodes to the positive and negative terminals of the patch clamp detection system, respectively.
[0010] Optionally, applying a voltage to the first and second media includes applying a -100mV voltage to the cis-cavity or a +100mV voltage to the inverse-cavity.
[0011] Optionally, before adding the sample to be tested into the sample cell, the method further includes: preparing nanopores on a solid nanopore chip using a dielectric breakdown method.
[0012] Optionally, the preparation of nanopores on a solid-state nanopore chip using the dielectric breakdown method includes: mounting the solid-state nanopore chip in a sample cell, dividing the sample cell into a cis-cavity and a trans-cavity, wherein the solid-state nanopore chip is a silicon nitride thin film chip with windows; rinsing the cis-cavity and trans-cavity with anhydrous ethanol respectively; injecting a second conductivity buffer into the cis-cavity and trans-cavity respectively; inserting two Ag / AgCl electrodes into the cis-cavity and trans-cavity respectively; connecting the two Ag / AgCl electrodes to an external power supply to form a closed loop; grounding the cis-cavity; applying a current pulse to the trans-cavity to break down the silicon nitride thin film chip to form a preliminary pore, and then applying a voltage pulse to expand the preliminary pore to the target pore size.
[0013] Optionally, before mounting the solid nanoporous chip in the sample cell, the process further includes: first immersing the silicon nitride thin film chip in anhydrous ethanol for 30 minutes to treat the window surface of the silicon nitride thin film chip with hydrophilicity and hydrophobicity; then immersing it in deionized water for 10 minutes to wash away the anhydrous ethanol on the surface of the silicon nitride thin film chip and dissolve the inorganic salts on the surface; and then immersing the silicon nitride thin film chip in anhydrous ethanol for 10 minutes.
[0014] The beneficial effects of the method for detecting the N-protein of the novel coronavirus provided in this application are as follows:
[0015] The method for detecting SARS-CoV-2 N-protein provided in this application connects the trans and cis cavities of a sample cell through a nanopore. A first conductivity buffer is placed in both the trans and cis cavities, and negatively charged molecules are also placed in the cis cavities. The sample to be tested is added to the cis cavities, and a voltage is applied. The method detects whether the nanopore generates an ion-blocking current pulse signal based on the effect of the negatively charged molecules. The presence of SARS-CoV-2 N-protein in the sample is determined based on the detected ion-blocking current pulse signal. This method is convenient to operate, has high detection sensitivity, can detect low concentrations of SARS-CoV-2 N-protein, and is fast, shortening the detection window period and effectively providing treatment time for patients. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 This is a schematic flowchart of the N-protein detection method provided in the embodiments of this application.
[0018] Figure 2 This is a schematic diagram illustrating the detection principle of N-protein provided in the embodiments of this application.
[0019] Figure 3 This is a schematic diagram of the IV curve of the nanopore provided in the embodiments of this application.
[0020] Figure 4 The following are schematic diagrams of signal comparisons for control experiments provided in some embodiments of this application, wherein (a) is a signal diagram of the first conductivity buffer, (b) is a signal diagram of the first conductivity buffer + G-250, (c) is a signal diagram of the first conductivity buffer + N-protein, and (d) is a translocation signal diagram of the first conductivity buffer + G-250 + N-protein.
[0021] Figure 5 The following are schematic diagrams illustrating characteristic information for the detection of the G250-N-protein complex provided in some embodiments of this application: (a) is a scatter plot of the G250-N-protein complex; (b) is a time decay distribution plot and an exponential decay fitting plot of the G250-N-protein complex; (c) is a statistical plot and Gaussian fitting plot of the translocation current amplitude of the G250-N-protein complex; and (d) is a statistical plot and Gaussian fitting plot of the translocation current amplitude ratio of the G250-N-protein complex.
[0022] Figure 6The following are schematic diagrams of signal comparisons for control experiments provided in some other embodiments of this application, wherein (a) is a signal diagram of the first conductivity buffer, (b) is a signal diagram of the first conductivity buffer + N-protein, (c) is a signal diagram of the first conductivity buffer + RNA, and (d) is a translocation signal diagram of the first conductivity buffer + RNA + N-protein.
[0023] Figure 7 The following are schematic diagrams illustrating the characteristic information of RNA-N-protein complex detection provided in some other embodiments of this application, wherein (a) is a scatter plot of RNA-N-protein complex, (b) is a time decay distribution plot and exponential decay fitting plot of RNA-N-protein complex, (c) is a statistical plot and Gaussian fitting plot of translocation current amplitude of RNA-N-protein complex, (d) is a statistical plot and Gaussian fitting plot of translocation current amplitude ratio of RNA-N-protein complex, and (e) is a statistical plot and Gaussian fitting plot of RNA blocking current ratio.
[0024] The details of the reference numerals used in the above figures are as follows:
[0025] 10. Sample cell; 11. Cis-cavity; 12. Inverse-cavity; 13. Solid-state nanopore chip; 14. Nanopore. Detailed Implementation
[0026] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.
[0027] It should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly or indirectly on that other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to that other element. Unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0028] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.
[0029] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.
[0030] As described in the background section, the most commonly used methods for detecting novel coronavirus infection are currently nucleic acid testing and lung CT scans. These methods are highly accurate, but require long waiting times. Therefore, there is a need to develop a rapid and effective detection method. Protein detection (antigen detection) is a highly efficient and reliable method. Novel coronavirus infection is an RNA virus infection containing four structural proteins. Among them, the SARS-CoV-2 nucleocapsid protein (N-protein) is a highly immunogenic phosphorylated protein involved in viral RNA replication and transcription, and a multifunctional RNA-binding protein. Therefore, detecting the SARS-CoV-2 N-protein to determine whether someone is infected with novel coronavirus is a promising detection method. Simultaneously, it is necessary to develop a rapid, highly sensitive, and easy-to-operate detection method to shorten the window period and effectively provide patients with more time for treatment.
[0031] See Figures 1 to 2 As shown, embodiments of this application provide a method for detecting the N-protein of the novel coronavirus, comprising the following steps:
[0032] S1. The sample to be tested is added to the sample cell 10, wherein the sample cell 10 includes a cis-cavity 11, an inverse-cavity 12, and a solid-state nanoporous chip 13. The inverse-cavity 12 is filled with a first medium, and the cis-cavity 11 is filled with a second medium. The inverse-cavity 12 and the cis-cavity 11 are connected through nanopores 14 on the solid-state nanoporous chip 13. The sample is added to the second medium. The first medium includes a first conductivity buffer, and the second medium includes the first conductivity buffer and negatively charged molecules. The molecules include small molecules and macromolecules. The particle size of small molecules is less than 1 nm, and the particle size of macromolecules is 1-100 nm. Particle size refers to the diameter, size, or dimension of the particle.
[0033] S2. Apply voltage to the first medium and the second medium, and detect the ion blocking current pulse signal generated by the nanopore 14;
[0034] S3. Determine whether the sample contains the SARS-CoV-2 N-protein (hereinafter referred to as N-protein) based on the ion blocking current pulse signal.
[0035] It should be noted that the types of samples to be tested include, but are not limited to, saliva, blood, etc.; the first conductivity buffer is a conductive liquid, which can be an aqueous solution of potassium chloride (KCl), sodium chloride (NaCl), or lithium chloride (LiCl); small molecules are small in size, such as Coomassie Brilliant Blue G-250 (hereinafter referred to as G-250) and Coomassie Brilliant Blue R-250 (hereinafter referred to as R-250), while large molecules are such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
[0036] In this process, N-protein is added to a mixture of a first conductivity buffer and negatively charged molecules. The positively charged N-protein binds to the negatively charged molecules to form a molecular-N-protein complex with a negatively charged surface. Thus, when a voltage is applied to the first and second media, the molecular-N-protein complex can be driven through the nanopore 14.
[0037] Applying the above-described technical solution of this application, the cis-cavity 11 and trans-cavity 12 of the sample cell 10 are connected through a nanopore 14. A first conductivity buffer is placed in both the cis-cavity 11 and trans-cavity 12. The cis-cavity 11 also contains negatively charged molecules. The sample to be tested is added to the cis-cavity 11, and a voltage is applied. The nanopore 14 is used to detect whether an ion-blocking current pulse signal is generated based on the effect of the negatively charged molecules. If no ion-blocking current pulse signal is detected, it can be determined that there is no N-protein in the sample. If an ion-blocking current pulse signal is detected, the presence of N-protein in the sample is determined based on the detected ion-blocking current pulse signal. This method is convenient to operate, has high detection sensitivity, can detect low concentrations of N-protein, and is fast, shortening the window period for N-protein detection and effectively providing treatment time for patients.
[0038] See Figure 4 and Figure 5As shown, in some embodiments, determining whether a sample contains N-protein based on an ion blocking current pulse signal includes: when the molecule is a small molecule, detecting an ion blocking current pulse signal determines that the sample contains N-protein. For example, when the small molecule is G-250 or R-250, since G-250 or R-250 can bind to N-protein to form a G-250 / R-250-N-protein complex with a negatively charged surface, when the sample contains N-protein, applying a voltage to the first and second media can drive the negatively charged G-250 / R-250-N-protein complex through the nanopore 14, thereby detecting the ion blocking current pulse signal generated when the G-250 / R-250-N-protein complex passes through the nanopore 14, and thus determining that the sample contains N-protein. It is understandable that, due to the small size of G-250 or R-250, no ion blocking current pulse signal will be detected when passing through nanopore 14. Therefore, when the sample does not contain N-protein, no ion blocking current pulse signal will be detected when a voltage is applied to the first and second media, thus determining that the sample does not contain N-protein.
[0039] See Figure 6 and Figure 7As shown, in some optional embodiments, determining whether a sample contains N-protein based on the ion blocking current pulse signal includes: when the molecule is a large molecule, when the ion blocking current pulse signal is detected, processing the ion blocking current pulse signal to obtain the change in the blocking current peak value; when the blocking current has two peak values, it is determined that the sample contains N-protein; when the blocking current has one peak value, it is determined that the sample does not contain N-protein. For example, when the molecule is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), since ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) can bind with N-protein to form a negatively charged RNA / DNA-N-protein complex, when the sample contains N-protein, when a voltage is applied to the first medium and the second medium, the negatively charged RNA / DNA-N-protein complex can be driven through the nanopore 14, thereby detecting the ion blocking current pulse signal generated when the RNA / DNA-N-protein complex passes through the nanopore 14. However, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) is relatively large in size. When the sample passes through the nanopore 14 under voltage, an ion blocking current pulse signal is generated. However, by processing the detected ion blocking current pulse signal, it can be seen that the blocking current has two peaks, thus confirming that the sample contains N-protein. When the sample does not contain N-protein, although ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) can also generate an ion blocking current pulse signal when a voltage is applied to the first and second media, by processing the detected ion blocking current pulse signal, it can be seen that the blocking current has only one peak, thus confirming that the sample does not contain N-protein.
[0040] In some alternative embodiments, before adding the sample to be tested into the sample cell 10, the method further includes:
[0041] Preparation of the first conductivity buffer: Sodium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water were mixed and the pH of the first conductivity buffer was adjusted to 7.4, wherein the ratio of sodium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid = 1M:10mM:1mM, where M is mol / L;
[0042] Clean the trans chamber 12 and cis chamber 11 with deionized water, and then rinse the trans chamber 12 and cis chamber 11 with anhydrous ethanol to prevent air bubbles from being generated in the cis chamber 11 and trans chamber 12 during subsequent liquid addition, which would affect the detection effect.
[0043] After the cis-cavity 11 and trans-cavity 12 have been rinsed, the first conductivity buffer is added to the cis-cavity 11 and trans-cavity 12 respectively. The molecule is added to the cis-cavity 11. For example, 200 μL of the first conductivity buffer is added to the cis-cavity 11 and trans-cavity 12 respectively, and G-250 (final concentration of 0.25% mg / mL) is added to the cis-cavity 11.
[0044] Two Ag / AgCl electrodes are inserted into the cis-cavity 11 and the trans-cavity 12, respectively.
[0045] Connect the two Ag / AgCl electrodes to the positive and negative terminals of the patch-clamp detection system, respectively. Apply a voltage of -100mV to the cis-cavity 11 or a voltage of +100mV to the inverse-cavity 12. At this time, the patch-clamp detection system detects whether the nanopore 14 generates an ion blocking current pulse signal. If no ion blocking current pulse signal is detected, it can be determined that there is no N-protein in the sample. If an ion blocking current pulse signal is detected, it can be determined whether there is N-protein in the sample based on the detected ion blocking current pulse signal.
[0046] In some alternative embodiments, before adding the sample to be tested to the sample cell 10, the process further includes preparing nanopores 14. The solid-state nanopore chip 13 is a silicon nitride thin film chip with windows, specifically including the following steps:
[0047] Preparation of the second conductivity buffer: Potassium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water were mixed and the pH of the second conductivity buffer was adjusted to 8. The ratio of each component in the second conductivity buffer was potassium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid = 1M:10mM:1mM, where M is mol / L.
[0048] Pretreatment: First, soak the silicon nitride thin film chip in anhydrous ethanol for 30 minutes to treat the window surface of the silicon nitride thin film chip with hydrophilicity and hydrophobicity. Then soak it in deionized water for 10 minutes to wash away the anhydrous ethanol on the surface of the silicon nitride thin film chip and dissolve the inorganic salts on the surface. Then soak the silicon nitride thin film chip in anhydrous ethanol for 10 minutes.
[0049] Nanopores 14 were fabricated on a silicon nitride thin film chip using a dielectric breakdown method, specifically including:
[0050] The pretreated silicon nitride thin film chip is installed in the sample cell 10, so that the sample cell 10 is divided into a cis-cavity 11 and an inverse-cavity 12.
[0051] Rinse the cis-cavity 11 and trans-cavity 12 with anhydrous ethanol to prevent air bubbles from forming in the cis-cavity 11 and trans-cavity 12 during subsequent liquid addition, which would affect the preparation effect.
[0052] Then inject the second conductivity buffer solution into the cis-cavity 11 and the trans-cavity 12 respectively;
[0053] Two Ag / AgCl electrodes were inserted into the cis-cavity 11 and the trans-cavity 13, respectively.
[0054] Two Ag / AgCl electrodes are connected to an external power supply to form a closed loop; the cis-cavity 11 is grounded; a current pulse is applied to the inverse-cavity 12 to break down the silicon nitride thin film chip to form an initial aperture, and then a voltage pulse is applied to expand the initial aperture to the target aperture size, for example, the target aperture size of nanopore 14 is 16 nm. The aperture size of nanopore 14 is reconfirmed and calculated using an IV curve by connecting a patch-clamp detection system to ensure that the target aperture size is achieved. Figure 3 As shown.
[0055] See Figure 4 and Figure 5 As shown, this embodiment uses G-250 as a small molecule and N-protein with a concentration of 34.43 nmol / L as an example to illustrate the detection method, which specifically includes the following steps:
[0056] Pretreatment: First, immerse the silicon nitride thin film chip with the window in anhydrous ethanol for 30 minutes to perform hydrophilic and hydrophobic treatment on the surface of the silicon nitride thin film; then immerse it in deionized water for 10 minutes to wash away the anhydrous ethanol on the surface of the silicon nitride thin film and dissolve the inorganic salts on the surface; finally, immerse it in anhydrous ethanol for 10 minutes.
[0057] Nanopore preparation: The pretreated silicon nitride thin film chip was installed in the sample cell 10, dividing the sample cell 10 into cis-cavity 11 and trans-cavity 12. 200 μL of anhydrous ethanol was used to rinse cis-cavity 11 and trans-cavity 12 respectively to prevent air bubbles from forming in cis-cavity 11 and trans-cavity 12 during subsequent liquid addition. Then, 200 μL of second conductivity buffer was injected into cis-cavity 11 and trans-cavity 12 respectively. Two Ag / AgCl electrodes were then used to connect cis-cavity 11 and trans-cavity 12 to an external power supply, forming a closed loop. Cis-cavity 11 was grounded, and a current pulse was applied to trans-cavity 12 to break down the core and form an initial pore. A voltage pulse was then applied to expand the initial pore to the target pore size of 16 nm. A patch-clamp detection system was connected, and the pore size of nanopore 14 was confirmed and calculated again using IV curves. Figure 3 As shown, the pore size of nanopore 14 was confirmed to be 16 nm by IV curve calculation; wherein, the second conductivity buffer is composed of potassium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water, and the ratio of each component is potassium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid = 1M:10mM:1mM, and the pH value of the second conductivity buffer is 8.
[0058] Sample testing: After preparing nanopores 14 with a pore size of 16 nm, first wash the cis-cavity 11 and trans-cavity 12 with deionized water, and then rinse with anhydrous ethanol to prevent air bubbles from forming in the cis-cavity 11 and trans-cavity 12 during subsequent liquid addition; then add 200 μL of the first conductivity buffer to the cis-cavity 11 and trans-cavity 12; connect the two Ag / AgCl electrodes of the cis-cavity 11 and trans-cavity 12 to the patch-clamp detection system respectively; apply a voltage of -100 mV to the cis-cavity 11 (or apply a voltage of 100 mV to the trans-cavity 12) to measure the blank signal, such as... Figure 4 As shown in (a); then G-250 (final concentration of G-250 is 0.25% mg / mL), N-protein (final concentration is 34.43 nmol / L), and first conductivity buffer are added to cis-cavity 11 to a final volume of 200 μL. A voltage of -100 mV is applied to cis-cavity 11 (or a voltage of 100 mV is applied to trans-cavity 12) to drive the generated G-250-N-protein complex through nanopore 14, and the ion blocking current pulse signal generated by passing through nanopore 14 is recorded, such as... Figure 4 As shown in (d), the N-protein signal can be detected within 10 seconds. The first conductivity buffer is composed of sodium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and deionized water in the following ratio: sodium chloride: tris(hydroxymethyl)aminomethane: EDTA = 1M: 10mM: 1mM. The pH of the first conductivity buffer is 7.4.
[0059] Control experiment: The method is the same as that used for the above-mentioned sample testing, and the following situations are tested respectively: (1) The first conductivity buffer is injected into the cis-cavity 11 and the trans-cavity 12 respectively, such as Figure 4 As shown in (a), no ion blocking current pulse signal was detected in nanopore 14; (2) the first conductivity buffer and G-250 were injected into the cis cavity 11, and the first conductivity buffer was injected into the trans cavity 12, as shown in (a). Figure 4 As shown in (b), no ion blocking current pulse signal was detected in nanopore 14; (3) the first conductivity buffer and N-protein were injected into the cis cavity 11, and the first conductivity buffer was injected into the trans cavity 12, as shown in (b). Figure 4 As shown in (c), no ion blocking current pulse signal was detected in nanopore 14.
[0060] Data processing and analysis: such as Figure 3 As shown in the IV curve, the dielectric breakdown method produces symmetrical apertures. Figure 4 As shown, the detection method provided in this embodiment can quickly detect N-protein. Figure 5 As shown, some characteristic blocking signals and blocking times of the G-250-N-protein complex were analyzed. Figure 5As shown in (a), this illustrates the translocation of the G-250-N-protein complex through nanopore 14. Figure 5 As shown in (b), the blocking time of the G-250-N-protein complex through-pores was statistically analyzed and nonlinearly fitted, revealing an exponential decay curve, and τ was calculated to be 0.434 ms. Statistical analysis and nonlinear fitting of the blocking current plot showed a Gaussian distribution, indicating that the current concentration in the G-250-N-protein complex through-pores was 189 pA. Figure 5 As shown in (c), statistical and nonlinear fitting of the current difference amplitude revealed a Gaussian distribution, with the ratio concentrated around 0.024. Figure 5 As shown in (d), the signal of the G-250-N-protein complex can be detected very well. This illustrates that the detection method provided in this embodiment achieves the alteration of the surface charge of the N-protein, thereby enabling effective detection of the N-protein. Furthermore, this method provides clear detection results, is fast, highly sensitive, and simple to operate, and can be used to detect N-protein at different concentrations.
[0061] See Figure 6 and Figure 7 As shown, this embodiment uses an N-protein at a concentration of 80 nmol / L and a 40 nt SARS-CoV-2 RNA sequence (hereinafter referred to as RNA) at a concentration of 20 nmol / L as examples to illustrate the detection method, which specifically includes the following steps:
[0062] Pretreatment: First, immerse the silicon nitride thin film chip with the window in anhydrous ethanol for 30 minutes to perform hydrophilic and hydrophobic treatment on the surface of the silicon nitride thin film; then immerse it in deionized water for 10 minutes to wash away the anhydrous ethanol on the surface of the silicon nitride thin film and dissolve the inorganic salts on the surface; finally, immerse it in anhydrous ethanol for 10 minutes.
[0063] Nanopore preparation: The pretreated silicon nitride thin film chip was installed in the sample cell 10, dividing the sample cell 10 into cis-cavity 11 and trans-cavity 12. 200 μL of anhydrous ethanol was used to rinse cis-cavity 11 and trans-cavity 12 respectively to prevent air bubbles from forming in cis-cavity 11 and trans-cavity 12 during subsequent liquid addition. Then, 200 μL of second conductivity buffer was injected into cis-cavity 11 and trans-cavity 12 respectively. Two Ag / AgCl electrodes were then used to connect cis-cavity 11 and trans-cavity 12 to an external power supply, forming a closed loop. Cis-cavity 11 was grounded, and a current pulse was applied to trans-cavity 12 to break down the core and form an initial pore. A voltage pulse was then applied to expand the initial pore to the target pore size of 16 nm. A patch-clamp detection system was connected, and the pore size of nanopore 14 was confirmed and calculated again using IV curves. Figure 3As shown, the pore size of nanopore 14 was confirmed to be 16 nm by IV curve calculation; wherein, the second conductivity buffer is composed of potassium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water, and the ratio of each component is potassium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid = 1M:10mM:1mM, and the pH value of the second conductivity buffer is 8.
[0064] Sample testing: After preparing the 16nm nanopore 14, first clean the cis-cavity 11 and trans-cavity 12 with deionized water, and then rinse with anhydrous ethanol to prevent air bubbles from forming in the cis-cavity 11 and trans-cavity 12 during subsequent liquid addition; then add 200μL of the first conductivity buffer to the cis-cavity 11 and trans-cavity 12; connect the two Ag / AgCl electrodes of the cis-cavity 11 and trans-cavity 12 to the patch-clamp detection system respectively; apply a voltage of -150mV to the cis-cavity 11 (or apply a voltage of 150mV to the trans-cavity 12) to measure the blank signal, such as... Figure 6 As shown in (a); then, 40 nt RNA (final concentration 20 nmol / L), N-protein (final concentration 80 nmol / L), and first conductivity buffer were added to cis-cavity 11 to a final concentration of 200 μL. A voltage of 150 mV was applied to cis-cavity 11 (or a voltage of -150 mV was applied to trans-cavity 12) to drive the generated RNA-N-protein complex and RNA molecules through nanopore 14, and the ion blocking current pulse signal generated by the perforation was recorded, as shown in (a). Figure 6 As shown in (d). The first conductivity buffer is composed of sodium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and deionized water, with the ratio of sodium chloride:tris(hydroxymethyl)aminomethane:EDTA = 1M:10mM:1mM. The pH of the first conductivity buffer is adjusted to 7.4.
[0065] Control experiment: The method is the same as that used for the above-mentioned sample testing, and the following situations are tested respectively: (1) The first conductivity buffer is injected into the cis-cavity 11 and the trans-cavity 12 respectively, such as Figure 6 As shown in (a), no ion blocking current pulse signal was detected in nanopore 14; (2) the first conductivity buffer and N-protein were injected into the cis cavity 11, and the first conductivity buffer was injected into the trans cavity 12, as shown in (a). Figure 6 As shown in (b), no ion blocking current pulse signal was detected in nanopore 14; (3) the first conductivity buffer and RNA were injected into the cis cavity 11, and the first conductivity buffer was injected into the trans cavity 12, as shown in (b). Figure 6 As shown in (c), an ion-blocking current pulse signal was detected in nanopore 14, and... Figure 6As shown in the middle (d) control, it can be seen that the peak value of the ion blocking current pulse signal when only RNA is present in the first conductivity buffer is significantly smaller than the peak value of the ion blocking current pulse signal when both RNA and N-protein are present in the first conductivity buffer.
[0066] Data processing and analysis: such as Figure 3 As shown in the IV curve, the dielectric breakdown method produced symmetrical apertures. Figure 6 As shown, the detection method provided in this embodiment can quickly detect N-protein. Figure 7 As shown, some characteristic blocking signals and blocking times of the RNA-N-protein complex were analyzed. Figure 7 As shown in (a), this illustrates the translocation of the RNA-N-protein complex through nanopore 14. Figure 7 As shown in (e), the effect on RNA was also analyzed using the blocking current ratio and Gaussian fitting, revealing that the ratio was concentrated around 0.044. Furthermore, other characteristic information about the RNA-N-protein complex was detected; its blocking time was statistically analyzed and nonlinearly fitted, showing an exponential decay curve, and τ was calculated to be 0.068 ms. Figure 7 As shown in (b); statistical analysis of the blocking current plot and nonlinear fitting (Gaussian fitting) revealed two peaks: one for RNA and the other for the RNA-N-protein complex. The smaller peak indicates the RNA pore current is concentrated at 705 pA, while the larger peak indicates the RNA-N-protein complex pore current is concentrated at 1077 pA. Figure 7 As shown in (c); Figure 7 As shown in (d), statistical and nonlinear fitting (Gaussian fitting) was also performed on the current difference ratio amplitude, revealing two peaks with ratios concentrated at 0.086 and 0.056, respectively, which effectively distinguishes the signals of RNA and the RNA-N-protein complex. This demonstrates that the detection method provided in this embodiment can effectively detect N-protein molecules. The method offers clear detection results, is fast, and highly sensitive, and can be used to detect N-protein molecules of varying concentrations.
[0067] In summary, the method for detecting the N-protein of the novel coronavirus provided in this embodiment has at least the following beneficial technical effects:
[0068] The method for detecting N-protein provided in this application is based on the formation of a molecular-N-protein complex by the binding of a negatively charged molecule with the N-protein. The N-protein is then detected by applying a voltage to drive the charged molecular-N-protein complex through a nanopore. This method is not only simple to operate, but also fast, highly sensitive, easy to implement and promote. At the same time, it provides a new approach for detecting viruses.
[0069] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for detecting the N-protein of the novel coronavirus, characterized in that, Includes the following steps: The sample to be tested is added to a sample cell, which includes a cis-cavity, an inverse-cavity, and a solid nanoporous chip. The inverse-cavity is filled with a first medium, and the cis-cavity is filled with a second medium. The inverse-cavity and the cis-cavity are connected through nanopores on the solid nanoporous chip. The sample to be tested is added to the second medium. The first medium includes a first conductivity buffer, and the second medium includes the first conductivity buffer and negatively charged molecules, wherein the negatively charged molecules include small molecules and large molecules. A voltage is applied to the first and second media, and the ion blocking current pulse signal generated by the nanopore is detected; The presence of SARS-CoV-2 N-protein in the sample to be tested is determined based on the ion blocking current pulse signal. The SARS-CoV-2 N-protein is added to a mixture of a first conductivity buffer and negatively charged molecules. The positively charged SARS-CoV-2 N-protein binds to the negatively charged molecules to form a molecular-N-protein complex with a negatively charged surface. Thus, when a voltage is applied to the first and second media, the molecular-N-protein complex can be driven through the nanopore. When the molecule is small, an ion blocking current pulse signal is detected, confirming that the sample contains the N-protein of the novel coronavirus. When the sample is a large molecule, an ion blocking current pulse signal is detected. The ion blocking current pulse signal is processed to obtain the change in the blocking current peak. When the blocking current has two peaks, it is determined that the sample contains the SARS-CoV-2 N-protein; when the blocking current has one peak, it is determined that the sample does not contain the SARS-CoV-2 N-protein. A blocking current map is generated based on the ion blocking current pulse signal. Two peaks are obtained by nonlinear fitting of the blocking point flow map. The two peaks are the RNA peak and the RNA-N-protein complex peak, respectively. The RNA-N-protein complex peak is greater than the RNA peak.
2. The method for detecting the N-protein of SARS-CoV-2 according to claim 1, characterized in that, Before adding the sample to be tested into the sample cell, the following steps are taken: The preparation of the first conductivity buffer includes: mixing sodium chloride, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and deionized water, and adjusting the pH of the first conductivity buffer to 7.4, wherein the ratio of sodium chloride:tris(hydroxymethyl)aminomethane:ethylenediaminetetraacetic acid is 1 M:10 mM:1 mM, where M is mol / L.
3. The method for detecting the N-protein of the novel coronavirus according to claim 1, characterized in that, Before adding the sample to be tested into the sample cell, the following steps are taken: Clean the trans and cis chambers with deionized water, and then rinse the trans and cis chambers with anhydrous ethanol. The first conductivity buffer solution was added to the cis cavity and the trans cavity respectively, and negatively charged molecules were added to the cis cavity.
4. The method for detecting the N-protein of the novel coronavirus according to claim 1, characterized in that, Before applying voltage to the first and second dielectrics, the following steps are included: Two Ag / AgCl electrodes were inserted into the cis-cavity and trans-cavity, respectively. Connect the two Ag / AgCl electrodes to the positive and negative terminals of the patch clamp detection system, respectively.
5. The method for detecting the N-protein of SARS-CoV-2 according to claim 1, characterized in that, Applying voltage to the first and second dielectrics includes: Apply a voltage of -100 mV to the cis-cavity or a voltage of +100 mV to the inverse-cavity.
6. The method for detecting the N-protein of the novel coronavirus according to claim 1, characterized in that, Before adding the sample to be tested into the sample cell, the following steps are taken: nanopores are prepared on a solid nanopore chip using a dielectric breakdown method.
7. The method for detecting the N-protein of the novel coronavirus according to claim 6, characterized in that, Fabrication of nanopores on solid-state nanopore chips using dielectric breakdown includes: Solid nanoporous chips are installed in the sample cell, which is divided into cis-cavity and inverse-cavity. The solid nanoporous chip is a silicon nitride thin film chip with a window. Rinse the cis- and trans-cavities separately with anhydrous ethanol. Then inject the second conductivity buffer solution into the cis-cavity and trans-cavity respectively; Two Ag / AgCl electrodes were inserted into the cis-cavity and trans-cavity, respectively. Connect the two Ag / AgCl electrodes to an external power source to form a closed circuit. Ground the cis-cavity; A current pulse is applied to the inverting cavity to break down the silicon nitride thin film chip to form a preliminary hole, and then a voltage pulse is applied to expand the preliminary hole to the target aperture.
8. The method for detecting the N-protein of SARS-CoV-2 according to claim 7, characterized in that, Before mounting the solid-state nanoporous chip into the sample cell, the following steps are required: First, immerse the silicon nitride thin film chip in anhydrous ethanol for 30 minutes to perform hydrophilic and hydrophobic treatment on the window surface of the silicon nitride thin film chip; Soak in deionized water for 10 minutes to wash away the anhydrous ethanol and dissolved inorganic salts on the surface of the silicon nitride thin film chip. The silicon nitride thin film chip was then soaked in anhydrous ethanol for 10 minutes.