A rapid detection method for quantifying the purity of a circular RNA vaccine based on a nanopore

Abstract

The application discloses a kind of based on nanometer hole's circular RNA vaccine purity quantitative detection method of rapid detection, it is related to biological detection technical field.The application uses nanometer hole chip for detecting circular RNA or linear RNA, since circular RNA and linear RNA have different two-dimensional structures, two kinds of molecules will produce different over-pore electric signals when passing through nanometer hole, the current change amplitude caused by circular RNA when passing through nanometer hole is larger and over-pore time is longer, and the current change amplitude caused by linear RNA when passing through nanometer hole is smaller, by the signal of over-pore event in a certain time range is statistically analyzed, circular RNA and linear RNA can be identified, and be applied to the quantitative detection of circular RNA vaccine purity.The detection method is simple to operate, without sample pretreatment steps such as PCR amplification, can effectively realize the quantitative detection of circular RNA purity, and the sample demand is small, can obtain result in a few minutes after adding.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically, to a rapid quantitative detection method for the purity of a nanopore-based circular RNA vaccine. Background Technology

[0002] Artificially synthesized RNA vaccines are typically circular RNA molecules about 1,000 base pairs in length. However, due to purity issues in synthesis, vaccine samples may contain unsynthesized linear RNA, affecting the synthesis yield and purity of RNA vaccines.

[0003] However, vaccine purity significantly impacts its efficacy. Therefore, quantitative detection of the purity of circular RNA molecules in synthesized vaccine samples is of paramount importance in the development of RNA vaccines. Currently, commonly used methods for RNA vaccine purity analysis include chromatography or gene sequencing; however, both methods are complex, costly, and time-consuming.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid quantitative detection method for the purity of nanopore-based circular RNA vaccines.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide a method for detecting the purity of a circular RNA vaccine, comprising the following steps: using a nanopore chip to detect the sample to be tested; applying a set voltage to one side of the nanopore to drive circular RNA molecules or linear RNA molecules in the sample through the pore; extracting the number of pore-passing events of the sample to be tested within a set time and the current change amplitude caused by each pore-passing event; and based on the maximum current change amplitude ΔI of the linear RNA molecules passing through the pore under the set voltage condition. Line The via events of the sample under test within a set time range are judged, and the current change amplitude of the via events of the sample under test is greater than ΔI. Line The pore event is considered as the pore event of the circular RNA molecule; the current change amplitude in the pore event of the sample to be tested is ≤ the ΔI. Line The aforementioned pore events are pore events of linear RNA molecules.

[0008] Secondly, embodiments of the present invention provide the application of detection reagents in the detection of the purity of circular RNA vaccines or in the identification of circular RNA molecules or linear RNA molecules, wherein the detection reagents include: the nanopore chip described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide the application of detection reagents in the preparation of products for the detection or identification of circular RNA molecules or linear RNA molecules for the purity of circular RNA vaccines, wherein the detection reagents include: the nanopore chip described in the foregoing embodiments.

[0010] The present invention has the following beneficial effects:

[0011] This invention employs a nanopore chip for detecting circular or linear RNA. Because circular and linear RNA have different two-dimensional structures, they generate different permeation electrical signals when passing through the nanopore. Circular RNA causes a larger current change amplitude and a longer permeation time when passing through the nanopore, while linear RNA causes a smaller current change amplitude. By statistically analyzing the signals of permeation events within a certain time range, circular and linear RNA can be identified, and further, the purity of circular RNA vaccines can be quantitatively detected. This detection method is simple to operate, effectively achieving quantitative detection of circular RNA purity without the need for sample pretreatment steps such as PCR amplification. It also requires a small sample volume, with results obtained within minutes of sample addition. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of the method for quantitative detection of the purity of circular RNA vaccines according to the present invention;

[0014] Figure 2 The aperture current trajectory of the low-noise 10nm diameter nanopore of this invention under different voltages;

[0015] Figure 3 This invention demonstrates the detection of current trajectories of linear RNA at a voltage of 200mV using a 10nm diameter nanopore.

[0016] Figure 4 This is a scatter plot of linear RNA perforation events under a voltage of 200mV according to the present invention.

[0017] Figure 5 The current trajectory of RNA vaccine samples detected under 200mV voltage through a 10nm diameter nanopore according to the present invention;

[0018] Figure 6 This is a scatter plot of events related to the perforation of RNA vaccine samples under a voltage of 200mV according to the present invention.

[0019] Figure 7 This is a scatter plot of events related to the perforation of RNA vaccine samples under a voltage of 250mV according to the present invention.

[0020] Figure 8 This is a scatter plot of events related to the perforation of RNA vaccine samples under a voltage of 300mV according to the present invention.

[0021] Figure 9 The current trajectory of RNA vaccine samples detected by the present invention under a voltage of 200mV through a 15nm diameter nanopore;

[0022] Figure 10 This is a scatter plot of events related to the perforation of RNA vaccine samples with a diameter of 15 nm and a voltage of 200 mV under the present invention.

[0023] Figure 11 The current trajectory of the 10nm diameter electrolyte under 300mV in 1M LiCl and 10mM Tris-HCl electrolytes is shown in this invention.

[0024] Figure 12 This is a scatter plot of events for the 10nm diameter RNA vaccine sample of the present invention at 300mV under 1M LiCl and 10mM Tris-HCl electrolytes.

[0025] Figure 13 This is a comparison of the current power spectral density of the low-noise nanopore of this invention and that of conventional nanopores. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] To address the low detection efficiency of circular RNA molecules in RNA vaccine samples, the inventors of this application propose a method for quantitative detection of the purity of circular RNA vaccines based on solid-state nanopores. By applying a bias voltage across the nanopore chip, RNA molecules in the sample solution pass through the nanopores under the influence of the electric field, causing a change in the opening current of the nanopores and generating a downward current pulse signal. Since the two types of molecules in the solution, circular RNA and non-circular linear RNA, have different two-dimensional structures, they generate different shaped through-pore electrical signals when passing through the nanopores. Circular RNA, due to its larger spatial structure, causes a larger current change amplitude (ΔI) and a longer through-pore time (Δt) when passing through the nanopores; while the through-pore signal of linear RNA molecules has a smaller current change amplitude. By statistically analyzing the number of through-pore events with different current change amplitudes over a period of time under different voltages, the proportion of each type of event can be obtained, thus revealing the concentration proportion of the corresponding type of through-pore RNA molecule. Therefore, this method enables rapid quantitative detection of the purity of circular RNA vaccines.

[0028] On one hand, embodiments of the present invention provide a method for detecting the purity of a circular RNA vaccine, which includes the following steps:

[0029] The sample to be tested is detected using a nanopore chip. The sample is added to one side of the nanopore, and a set voltage is applied to the other side of the nanopore to drive circular RNA molecules or linear RNA molecules in the sample through the pore (through the nanopore). The number of pore-passing events of the sample to be tested within a set time and the amplitude of the current change caused by each pore-passing event are extracted.

[0030] Based on the maximum current change amplitude ΔI of the linear RNA molecule through the orifice at the set voltage. Line The via events of the sample under test within a set time range are judged, and the current change amplitude of the via events of the sample under test is greater than ΔI. Line The pore event is considered as the pore event of the circular RNA molecule; the current change amplitude in the pore event of the sample to be tested is ≤ the ΔI. Line The aforementioned pore events are pore events of linear RNA molecules.

[0031] In some embodiments, the set voltage is 50–300 mV. During detection, the voltage used is determined based on the sample type and the diameter of the nanopore. Nucleic acids (RNA) carry a large amount of negative charge, and under the same voltage, they experience a greater electrophoretic force, making them easier to pass through the pore. Typically, a voltage exceeding 50 mV is sufficient to produce a signal. The 50–300 mV range is suitable for the diameter of the nanopores used in this invention (5–50 nm). Increasing the voltage beyond this range may increase pore blockage, so a voltage below 300 mV is generally sufficient. Specifically, the set voltage can be any one or any combination of 50, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 300 mV.

[0032] The set time can be determined based on experimental conditions. Theoretically, the longer the recording time, the more events can be recorded. However, extending the time indefinitely will also affect the chip's performance, which will decrease as the usage time increases. In some embodiments, the set time is 1 second to 30 minutes. Specifically, the set time can be any one or any two of the following: 1 second, 2 seconds, 4 seconds, 6 seconds, 8 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 1 minute, 2 minutes, 4 minutes, 6 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, and 30 minutes.

[0033] In some embodiments, the detection method further includes: detecting the maximum current change amplitude ΔI of the linear RNA molecule through the pore at the set voltage. Line The steps can be as follows: using the nanopore chip to detect a sample containing only linear RNA molecules, obtaining the amplitude of the current change of the sample within the set voltage and the set time range, and taking the maximum value as ΔI. Line .

[0034] In some embodiments, the nanoporous chip includes a dielectric thin film, on which nanopores are formed.

[0035] In some embodiments, the dielectric film may be selected from any one of silicon nitride film, aluminum oxide film, hafnium dioxide film, and graphene film.

[0036] In some embodiments, the pore size of the nanopore is 5–50 nm. Unlike conventional RNA molecules, circular RNA has a larger spatial structure size and cannot pass through nanopores with smaller diameters. Therefore, the minimum nanopore diameter proposed in this invention is 5 nm. Furthermore, the nanopore diameter cannot be too large; when using nanopores with larger diameters for detection, linear RNA molecules with smaller spatial dimensions cannot generate a significant current change signal. Therefore, the maximum diameter of the nanopore proposed in this invention is 50 nm. Specifically, this pore size can be any one or any combination of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nm.

[0037] In some embodiments, the thickness of the dielectric film is 10–50 nm. Films thinner than 10 nm are difficult to fabricate, and excessively thin films have poor mechanical stability, leading to increased baseline noise in the current. However, excessively thick films (greater than 50 nm) result in a larger volume of the entire nanopore, reducing the detection resolution. Specifically, the thickness can be any one or a range between any two of 10, 15, 20, 25, 30, 35, 40, 45, and 50 nm.

[0038] This invention utilizes a chip made of low-noise dielectric thin film material. By optimizing the specific diameter and thickness of the dielectric film, an optimal solid-state nanopore sensor chip is obtained, which can more effectively distinguish the perforation events of circular RNA molecules and non-circular linear RNA molecules. The concentration ratio of the two RNA molecule shapes in the RNA vaccine can be obtained by simply calculating the number of signals with different current change amplitudes over a period of time. This method is simple to operate, requires no sample pretreatment steps such as PCR amplification, requires a small sample volume, and yields results within minutes of sample addition.

[0039] In some embodiments, the nanopore chip further includes a support substrate with openings connected to the lower surface of the dielectric film. The openings communicate with the nanopores and do not impede the passage of RNA molecules through the nanopores. During detection, the nanopore chip is immersed in an electrolyte solution, and the nanopores on the chip serve as the sole channel for ions to pass through in the electrolyte solution.

[0040] In some embodiments, the material of the support substrate includes silicon.

[0041] In some embodiments, the thickness of the support substrate is 100–300 μm. The thickness of the support substrate is typically less than 300 μm because an excessively thick substrate would prolong the window etching time during chip fabrication, and would also increase the overall thickness of the chip. Conversely, an excessively thin substrate (less than 100 μm) provides reduced protection for the silicon nitride film, making the chip more susceptible to breakage. Specifically, the thickness can be any one or a range between any two of the following: 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 300 μm.

[0042] In some embodiments, the outer diameter of the opening is greater than or equal to the pore diameter of the nanopore (5-50 nm).

[0043] In some embodiments, the outer diameter of the opening is 10 to 100 nm. Specifically, the outer diameter can be any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, and 100 nm.

[0044] This invention does not impose any special restrictions on the shape of the opening. In some embodiments, the cross-sectional shape of the opening includes, but is not limited to, circular, elliptical, rectangular, and rhomboid shapes, with a rectangular shape being preferred. The outer diameter mentioned in the foregoing embodiments can be understood as the maximum diameter of the opening.

[0045] In some embodiments, when a nanopore chip is used to detect a sample, an electrolyte solution is injected into both sides of the nanopore of the nanopore chip, and positive and negative electrodes are applied to both sides of the nanopore.

[0046] In some embodiments, the electrolyte solution includes any one or more of lithium chloride, sodium chloride, and potassium chloride.

[0047] In some embodiments, the electrolyte solution contains a NaCl buffer solution, specifically a Tris-HCl solution containing NaCl, wherein the final concentration of NaCl is 0.5–1.5 M, and the final concentration of Tris-HCl is 1–20 mM. The final concentration of NaCl can specifically be any one or any two of the following: 0.5, 0.6, 0.8, 1, 1.2, 1.4, and 1.5 M. The final concentration of Tris-HCl can be any one or any two of the following: 1, 5, 10, 15, and 20 mM.

[0048] In some embodiments, the electrolyte solution contains a LiCl buffer solution, specifically a LiCl-containing Tris-HCl solution, wherein the final concentration of LiCl is 0.5–1.5 M, and the final concentration of Tris-HCl is 1–20 mM. The final concentration of LiCl can specifically be any one or any two of the following: 0.5, 0.6, 0.8, 1, 1.2, 1.4, and 1.5 M. The final concentration of Tris-HCl can be any one or any two of the following: 1, 5, 10, 15, and 20 mM.

[0049] The detection method of this application does not have specific limitations on the length of circular RNA molecules and linear RNA molecules. The length of circular RNA molecules and linear RNA molecules can be from 3 nt to 2000 nt, specifically within any one or any two of the following ranges: 3, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 nt.

[0050] The sample to be tested can be a circular RNA vaccine. The sample volume can be 1–10 μL. Specifically, it can be any one or any two of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μL.

[0051] In some embodiments, the detection method further includes: calculating the ratio of the number of pore events of circular RNA molecules to the number of pore events of the sample to be tested within a set time period, so as to obtain the purity of the circular RNA molecules.

[0052] On the other hand, embodiments of the present invention also provide the application of detection reagents in the detection of the purity of circular RNA vaccines or in the identification of circular RNA molecules or linear RNA molecules, wherein the detection reagents include: the nanopore chip described in any of the foregoing embodiments.

[0053] In some embodiments, the detection reagent further includes the electrolyte solution described in any of the foregoing embodiments.

[0054] Furthermore, embodiments of the present invention also provide the application of the detection reagent in the preparation of products for detecting or identifying circular RNA molecules or linear RNA molecules for the purity of circular RNA vaccines, wherein the detection reagent includes: the nanopore chip described in any of the foregoing embodiments.

[0055] In some embodiments, the detection reagent further includes the electrolyte solution described in any of the foregoing embodiments.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1

[0058] This embodiment provides a quantitative detection method for the purity of circular RNA vaccines. The method involves detecting a sample (a vaccine sample with a circular RNA purity of 30% as identified by mass spectrometry). The steps of the detection method are as follows, and a schematic diagram can be referenced. Figure 1 .

[0059] (1) Obtaining a nanopore chip; In this embodiment, the nanopore chip includes a silicon nitride thin film and a silicon substrate; The silicon nitride thin film has a thickness of 30 nm and a nanopore with a diameter of 10 nm on the film; The silicon nitride thin film is deposited on the silicon substrate by chemical vapor deposition, the silicon substrate has a thickness of 200 μm, and a rectangular opening is also provided on the silicon substrate, which communicates with the nanopore, and the side length of the rectangular opening is greater than the diameter of the nanopore.

[0060] (2) Detection preparation: The nanopore chip was installed in the fluid cell, which was divided into two chambers; electrolyte solutions (1M NaCl, 10mM Tris-HCl) were added to the two chambers. Two Ag / AgCl electrode wires were inserted into the two chambers of the fluid cell to form positive and negative electrodes, and the other two ends of the electrodes were connected to the patch clamp current amplifier.

[0061] The aperture current was recorded at different voltages (-200 to 200 mV) to confirm that there were no false positive signals before the RNA sample was added. The results are as follows: Figure 2 As shown, 2 μL of linear RNA sample with the same number of bases as the circular RNA to be tested was added to one chamber of the fluidic cell, and a positive voltage was applied to the other side of the fluidic cell. The changes in the current baseline were observed and recorded. Under the influence of the electric field, the linear RNA passes through the nanopores, thereby generating a pulse signal of current change. Figure 3 As shown.

[0062] (3) Obtain the maximum current change amplitude (ΔI) caused by the linear RNA molecule passing through the orifice at a set voltage (200mV in this example). Line ):

[0063] By extracting the perforation events of linear RNA molecules at a voltage of 200 mV and performing data analysis, the maximum current change amplitude (ΔI) caused by the perforation of linear RNA molecules was determined. Line ) is 1nA, such as Figure 4 As shown in the scatter plot.

[0064] (4) Detect and analyze the via signal of the sample to be tested:

[0065] The nanoporous chip was removed from the fluid cell, rinsed thoroughly with ethanol and deionized water, and dried. The chip was then reinstalled into the fluid cell, and electrolyte solutions (1M NaCl, 10mM Tris-HCl) were added. After confirming that the opening current was stable, 2μL of the synthesized vaccine sample was added to one side of the fluid cell, and a positive voltage was applied to the other side of the fluid cell. The changes in the current baseline under different voltages were observed and recorded.

[0066] Under the influence of an electric field, circular RNA and linear RNA in the vaccine sample pass through the nanopores, causing current pulse signals with different current change amplitudes.

[0067] like Figure 5 As shown, I1 is the current change amplitude generated by linear RNA through the pore, and I2 is the current change amplitude generated by circular RNA through the pore.

[0068] Extract the current change amplitude (ΔI) and perforation time (Δt) of vaccine sample perforation events over 30 seconds at a set voltage (200mV), and plot a two-dimensional scatter plot, as shown below. Figure 6 As shown in Table 1, the total number of events within 30 seconds was determined to be 203. Among them, the number of events with a current change amplitude greater than 1 nA was 43. Therefore, the proportion of events involving circular RNA was 43 / 203.

[0069] Table 1 Test Results

[0070]

[0071]

[0072] Example 2

[0073] This embodiment provides a quantitative detection method for the purity of a circular RNA vaccine, which is largely the same as in Example 1, except that the set voltage during detection is 250mV. The baseline change of the current at this voltage is recorded, and the current change amplitude (ΔI) and via time (Δt) of the via event signal are extracted and plotted as a two-dimensional scatter plot, as shown below. Figure 7 As shown, the percentage of events involving circular RNA was 48 / 168.

[0074] Example 3

[0075] This embodiment provides a quantitative detection method for the purity of a circular RNA vaccine, which is largely the same as in Embodiment 1, except that the set voltage during detection is 300mV. The baseline change of the current at this voltage is recorded, and the current change amplitude (ΔI) and via time (Δt) of the via event signal are extracted and plotted as a two-dimensional scatter plot, as shown below. Figure 8 As shown, the percentage of events involving circular RNA was 160 / 594.

[0076] Example 4

[0077] This embodiment provides a method for quantitatively detecting the purity of a circular RNA vaccine, which is largely the same as in Example 1, except that the diameter of the nanopore is 15 nm. The baseline change of the current at this diameter is recorded, such as... Figure 9 As shown. The current change amplitude (ΔI) and via time (Δt) of the via signal event are extracted and plotted as a two-dimensional scatter plot, as shown. Figure 10 As shown.

[0078] Example 5

[0079] This embodiment provides a method for quantitative detection of the purity of a circular RNA vaccine, which is largely the same as that in Embodiment 1, except that:

[0080] (1) The electrolyte solution is a mixed solution of LiCl and Tris-HCl (1M LiCl, 10mM Tris-HCl);

[0081] (2) The set voltage during testing is 300mV.

[0082] Record the baseline change of current at this voltage, such as Figure 11 As shown. The current change amplitude (ΔI) and via time (Δt) of the via signal event are extracted and plotted as a two-dimensional scatter plot, as shown. Figure 12 As shown.

[0083] Example 6

[0084] Comparative analysis of the nanopore chip provided in Example 1 and conventional nanopore chips for detecting the purity of circular RNA vaccines.

[0085] The conventional nanopore chip is an in-situ chip, catalog number G20L08P004. The detection method is roughly the same as in Example 1 (opening voltage is -150 to 150 mV). A current baseline over a period of time at 100 mV is used to calculate the power spectral density, and this is compared with the current power spectral density of the low-noise nanopore chip (Example 1 of this application) at the same voltage. Figure 13 As shown in the figure, the power spectral density of the conventional nanopore current baseline is significantly higher than that of the nanopore used in this invention, indicating that the nanopore chip used in this invention has a lower noise level.

[0086] It should be noted that the test samples in Examples 2 to 6 are the same as those in Example 1.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the purity of a circular RNA vaccine, characterized in that, It includes the following steps: A nanopore chip is used to detect the sample to be tested. A set voltage is applied to one side of the nanopore to drive circular RNA molecules or linear RNA molecules in the sample through the pore. The number of pore-passing events and the current change amplitude caused by each pore-passing event in the sample to be tested are extracted within a set time. Based on the maximum current change amplitude of the through-hole under the set voltage for linear RNA molecules I Line The test sample is evaluated for via events within a set time range, and the current change amplitude in the via events of the test sample is greater than the specified value. I Line The perforation event is the perforation event of circular RNA molecules; The amplitude of the current change in the via event of the sample under test is ≤ the value of the test sample. I Line The aforementioned pore-passing events are pore-passing events of linear RNA molecules; The purity of the circular RNA is determined by the ratio of the number of perforation events of the circular RNA molecule within a set time range to the number of events of the sample under test within the set time range.

2. The detection method according to claim 1, characterized in that, The nanoporous chip contains a dielectric film, on which nanopores are formed.

3. The detection method according to claim 2, characterized in that, The dielectric film is selected from any one of silicon nitride film, aluminum oxide film, hafnium dioxide film and graphene film.

4. The detection method according to claim 2, characterized in that, The pore size of the nanopore is 5~50nm.

5. The detection method according to claim 2, characterized in that, The thickness of the dielectric film is 10~50nm.

6. The detection method according to claim 2, characterized in that, The nanopore chip also includes a support substrate with openings, the support substrate being connected to the lower surface of the dielectric film, and the openings communicating with the nanopores.

7. The detection method according to claim 6, characterized in that, The material of the supporting substrate includes silicon.

8. The detection method according to claim 6, characterized in that, The thickness of the supporting substrate is 100~300μm.

9. The detection method according to claim 6, characterized in that, The maximum diameter of the opening is greater than or equal to the pore diameter of the nanopore.

10. The detection method according to claim 6, characterized in that, The maximum diameter of the opening is 10~100nm.

11. The detection method according to claim 1, characterized in that, The set voltage is 50~300mV.

12. The detection method according to claim 1, characterized in that, The set time is 1 second to 30 minutes.

13. The detection method according to any one of claims 1 to 12, characterized in that, When a nanoporous chip is used to test a sample, an electrolyte solution is injected into both sides of the nanopore of the nanoporous chip, and positive and negative electrodes are applied to both sides of the nanopore.

14. The detection method according to claim 13, characterized in that, The electrolyte solution includes any one or more of lithium chloride, sodium chloride, and potassium chloride.

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