Single molecule detection method of anti-aging oligopeptide based on solid-state nanopore technology

Abstract

The present application belongs to the technical field of molecular detection, and particularly relates to a single molecule detection method of anti-aging oligopeptide based on solid-state nanopore technology. The method is as follows: the silicon nitride nanopore chip is pretreated to remove surface contaminants; then, the nanopore is prepared on the silicon nitride film loaded on the chip through a dielectric breakdown method; then, the nanopore chip is assembled with a customized flowcell, and the buffer solution containing oligopeptide is added to the cis side of the flowcell; the oligopeptide is driven to pass through the nanopore by applying a bias voltage, and the electrical signal generated when the oligopeptide passes through the nanopore is collected to realize qualitative and / or quantitative analysis of the anti-wrinkle oligopeptide. The method can efficiently provide information about multiple single molecule conformations and surface properties of the anti-wrinkle oligopeptide molecules, and provides a new scheme for rapid detection of products containing the anti-wrinkle oligopeptide, and has great significance for research and development and market supervision of the products.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology, specifically relating to a single-molecule detection method for anti-aging oligopeptides based on solid-state nanopore technology. Background Technology

[0002] In recent years, bioactive oligopeptides have garnered significant attention in the research and development of anti-aging cosmetics. Their functions include signal transduction, transport, and inhibition of enzymes and neurotransmitters. Argireline, a popular hexapeptide, is a prime example. Its molecule is composed of six specific amino acids (Glu-Glu-Met-Gln-Arg-Arg) and has been proven to be effective in reducing facial wrinkles. Studies have shown that hexapeptides can inhibit the release of neurotransmitters at the neuromuscular junction, producing a botulinum toxin-like effect, and exhibiting high skin permeability and low toxicity. Therefore, it is widely used as an alternative to botulinum toxin treatment in anti-wrinkle cosmetics. The widespread application of these oligopeptides has also created a demand for testing. Precise qualitative and quantitative analysis of anti-wrinkle oligopeptides in complex environments not only promotes their research and application but also plays a crucial role in the identification and regulation of related products.

[0003] Currently, common methods for detecting peptides in pharmaceuticals and cosmetics include the Kjeldahl method, fluorescence detection, chemiluminescence detection, electrophoresis, colorimetry, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and chromatography. For oligopeptides, liquid chromatography and mass spectrometry are the most frequently used methods. For example, patent CN115015369A discloses a method for detecting small molecule oligopeptides. This patent uses a matrix solution of N-phenyl-1-(5-methyl)naphthylamine dissolved in methanol to obtain a concentration of 10 mg / mL, and then uses matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALS) to detect the small molecule oligopeptides. Patent CN105823848A discloses an extraction and determination method for water-soluble oligopeptides, which utilizes high-performance liquid chromatography (HPLC) for detection. These methods have been proven to effectively qualitatively and quantitatively identify anti-wrinkle oligopeptides in various products, but they also have limitations, such as long processing times, low sensitivity, and the need for labeling. More importantly, these methods are mostly bulk detection methods, which are difficult to achieve single-molecule scale detection and cannot identify the physicochemical information of individual oligopeptide molecules.

[0004] Nanopore technology is a novel method for oligopeptide detection, offering advantages such as label-free operation, high speed, high sensitivity, and single-molecule detection. In nanopore detection platforms, both biological nanopores and solid-state nanopores have been proven capable of detecting short peptide molecules. However, biological nanopores have relatively fixed pore sizes, limiting their ability to detect various types of oligopeptides. Solid-state nanopores, on the other hand, offer adjustable pore sizes and ease of fabrication, making them more versatile for oligopeptide detection compared to biological nanopores. Currently, no single-molecule detection method for anti-aging oligopeptides based on solid-state nanopores has been reported. This patent is of significant importance for fundamental research on oligopeptide molecules. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a method for detecting oligopeptides based on solid-state nanopore single-molecule technology. The present invention uses silicon-based silicon nitride thin film as nanopore carrier material to detect the electrical signals of oligopeptide molecules that combat wrinkles.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting oligopeptides based on solid-state nanopore single-molecule technology includes the following steps:

[0008] S1: Pre-treat silicon nitride nanoporous chips and prepare solid nanopores;

[0009] S2: Assemble the nanopore detection platform and apply a bias voltage to drive the oligopeptide through the solid nanopore. Perform qualitative and / or quantitative analysis on the oligopeptide by collecting the electrical signal generated when the oligopeptide passes through the pore.

[0010] This invention utilizes nanopore single-molecule detection technology to collect the current hindrance signal generated when oligopeptides pass through nanopores. By analyzing the amplitude and residence time of the current hindrance signal, physicochemical information such as the charge, volume, and conformation of the oligopeptide can be obtained. Furthermore, by changing the pore size of the solid nanopore, it can be adapted to detect different oligopeptide molecules, exhibiting high applicability. Compared to traditional oligopeptide detection methods, the nanopore technology employed in this invention offers advantages such as label-free detection, high speed, high sensitivity, and single-molecule detection, representing a novel method for oligopeptide detection.

[0011] Furthermore, the oligopeptide includes tripeptides and / or hexapeptides.

[0012] Furthermore, the size of the solid nanopore is 1.8 nm-5 nm.

[0013] Furthermore, solid-state nanopores of 1.8nm-2.5nm were used to detect tripeptides; solid-state nanopores of 2.5nm-5nm were used to detect hexapeptides.

[0014] Furthermore, the pretreatment involves immersing the silicon nitride nanoporous chip sequentially in an aqueous ethanol solution, acetone, and isopropanol, then drying and cleaning it for later use.

[0015] Furthermore, the preprocessing specifically includes the following steps:

[0016] ① Soak the silicon nitride nanoporous chip in an ethanol aqueous solution for 20 minutes to remove inorganic impurities from the surface;

[0017] ② Soak in acetone and isopropanol for 20 minutes respectively to remove organic impurities on the surface;

[0018] ③ Finally, dry the chip at 60°C for 1 minute, and then use a plasma cleaner to clean the front side of the chip for 5 minutes to remove residual impurities and increase its surface hydrophilicity. Place it in a clean container for later use.

[0019] Furthermore, in the ethanol-water solution, the volume ratio of ethanol to water is 1:1.

[0020] Furthermore, the carrier of the solid nanopore is a silicon nitride thin film.

[0021] Furthermore, the thickness of the silicon nitride film is 20 nm.

[0022] Furthermore, the solid nanopores are prepared on the silicon nitride thin film supported on the silicon nitride nanochip using a dielectric breakdown method.

[0023] Furthermore, the specific method for preparing the solid-state nanopores is as follows:

[0024] ① The pretreated silicon nitride nanoporous chip is installed in a custom flowcell;

[0025] ②The silicon nitride nanoporous chip was sequentially impregnated with ethanol, water, and buffer solution (Buffer 1);

[0026] ③ Inject the buffer solution (Buffer 1) into both sides of the flowcell;

[0027] ④ Connect electrodes to both sides of the flowcell, and then connect a current source pulse dielectric breakdown device;

[0028] ⑤ Apply gradually increasing intermittent pulse current to the cis side of the chip using a power meter to break down the thin film and form nanopores;

[0029] ⑥ After the pores are formed, intermittent voltages are applied to both sides of the chip to obtain solid nanopores with the desired pore size.

[0030] Furthermore, specifically, in step ②, the surfaces of both sides of the chip are sequentially moistened with ethanol, water, and buffer solution (Buffer 1) to remove any air bubbles.

[0031] In step ⑤, the SiNx thin film is exposed to a strong electric field. Charge accumulation and thermal effects cause the defects in the film to be amplified, thereby breaking down the film to form nanopores.

[0032] Furthermore, the buffer solution used in preparing the nanopores was 1M KCl, 10mM Tris, 1mM EDTA, pH 8.

[0033] Further, S2 specifically involves: assembling the nanopore chip with a customized flowcell, adding an electrolyte solution to the trans cavity of the flowcell, and adding an electrolyte solution containing oligopeptides to the cis cavity; connecting the flowcell to a patch clamp, and applying a bias voltage across the flowcell via the patch clamp to achieve single-molecule detection of oligopeptides.

[0034] Furthermore, the two chambers of the flowcell are connected to the probe of the patch-clamp amplifier via Ag / AgCl electrodes. The patch-clamp is connected to a digital-to-analog converter and then to a computer. The applied bias voltage is controlled and the current changes are recorded using the Clampfit program.

[0035] Furthermore, when detecting tripeptides, the electrolyte solution used in both the cis and trans cavities of the flowcell is Buffer 2; when detecting hexapeptides, Buffer 2 is added to the trans cavity of the flowcell, and Buffer 3 containing hexapeptides is added to the cis cavity; Buffer 2 consists of 2M LiCl, 10mM Tris, and 1mM EDTA, pH 7.4; Buffer 3 consists of 0.4M LiCl, 10mM Tris, and 1mM EDTA, pH 7.4.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. This invention enables rapid, label-free single-molecule detection of anti-wrinkle oligopeptides using a nanopore detection platform, which can simultaneously acquire multiple information about a single oligopeptide molecule, and is of great significance for basic research on oligopeptide molecules;

[0038] 2. The detection technology of this invention has very high sensitivity and resolution, and can clearly reflect the single-molecule information of anti-wrinkle oligopeptides. It can not only efficiently perform label-free detection of anti-wrinkle oligopeptides, but also be used to study the conformational changes of such molecules in complex environments. It can effectively make up for the shortcomings of traditional oligopeptide detection methods such as low sensitivity and limited information. This invention proposes a novel single-molecule detection platform for the detection of several anti-wrinkle oligopeptides, and provides a solution for the efficient detection of anti-wrinkle oligopeptides.

[0039] 2. The single-molecule recognition method for anti-wrinkle oligopeptides based on solid-state nanopore electrical technology disclosed in this invention can rapidly detect oligopeptide molecules qualitatively and quantitatively, and provide single-molecule information that is difficult to provide by other detection methods. Therefore, it has broad application prospects in the fields of biomolecular science and biomedicine. It can provide experimental reference for the study of the mechanism of action of anti-wrinkle oligopeptides in the human body, and also provide a new detection method for clinical detection in oligopeptide anti-wrinkle treatment. Attached Figure Description

[0040] Figure 1 A schematic diagram illustrating the principle of detecting oligopeptide molecules using solid-state nanopores;

[0041] Figure 2 The results of applying a 350 mV blocking time to a 0.4 μM tripeptide in an electrolyte solution of 2 M LiCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) in a 1.8 nm nanopore.

[0042] Figure 3 The results of applying a blocking current of 350 mV to a 0.4 μM tripeptide in an electrolyte solution of 2 M LiCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) in a 1.8 nm nanopore.

[0043] Figure 4 A 0.4 μM tripeptide was subjected to an electrolytic voltage of 350 mV in an electrolyte solution containing 2 M LiCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) through a 1.8 nm nanopore. o Resulting image;

[0044] Figure 5 The results show the blocking time of 1 μM hexapeptide in a 2.5 nm nanopore with an ion concentration gradient of 2 M / 0.4 M LiCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) at 400 mV.

[0045] Figure 6 The results show the amplitude of a 400mV blocking current applied to a 1μM hexapeptide in a 2.5nm nanopore with an ion concentration gradient of 2M / 0.4M LiCl, 10mM Tris, and 1mM EDTA (pH 7.4).

[0046] Figure 7 A 1 μM hexapeptide was incubated in a 2.5 nm nanopore with an ion concentration gradient of 2 M / 0.4 M LiCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) at a ΔI / I ratio of 400 mV. o Resulting image;

[0047] Figure 8The blocking time of a 200 nM hexapeptide when a voltage of 300 mV is applied in a 5 nm nanopore is shown in the figure.

[0048] Figure 9 The blockage current amplitude of a 200 nM hexapeptide when a voltage of 300 mV is applied in a 5 nm nanopore is plotted.

[0049] Figure 10 The ΔI / I ratio of a 200 nM hexapeptide subjected to a 300 mV voltage in a 5 nm nanopore is measured. o Resulting image;

[0050] Figure 11 The blocking time of a 200 nM hexapeptide when a voltage of 350 mV is applied in a 5 nm nanopore is shown in the figure.

[0051] Figure 12 The blockage current amplitude of a 200 nM hexapeptide with a voltage of 350 mV applied in a 5 nm nanopore is shown in the figure.

[0052] Figure 13 The ΔI / I ratio of a 200 nM hexapeptide subjected to a 350 mV voltage in a 5 nm nanopore is measured. o Resulting image;

[0053] Figure 14 The blocking time of a 200 nM hexapeptide when a voltage of 400 mV is applied in a 5 nm nanopore is shown in the figure.

[0054] Figure 15 The blockage current amplitude of a 200 nM hexapeptide with a voltage of 400 mV applied in a 5 nm nanopore is shown in the figure.

[0055] Figure 16 The ΔI / I ratio of a 200 nM hexapeptide subjected to a 400 mV voltage in a 5 nm nanopore is... o Result image. Detailed Implementation

[0056] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0057] In Example 1 of this invention, the buffer solution Buffer 1 consists of 1M KCl, 10mM Tris, 1mM EDTA, and pH 8.

[0058] In this embodiment of the invention, Buffer 2 consists of 2M LiCl, 10mM Tris, 1mM EDTA, and pH 7.4.

[0059] In this embodiment of the invention, Buffer 3 consists of 0.4M LiCl, 10mM Tris, 1mM EDTA, and pH 7.4.

[0060] In this embodiment of the invention, the preparation method of the tripeptide sample is as follows: a 1 μM tripeptide is dissolved in Buffer 2 and shaken thoroughly; then the sample concentration is diluted to 50 nM, 100 nM and 400 nM respectively with the same buffer solution and shaken thoroughly.

[0061] In this embodiment of the invention, the preparation method of the hexapeptide sample is as follows: a 5 μM hexapeptide is dissolved in Buffer 2 and shaken thoroughly; then the sample concentration is diluted to 100 nM, 200 nM and 1 μM respectively with the same buffer solution and shaken thoroughly.

[0062] In this embodiment of the invention, the silicon nitride film used to prepare the nanopores has a thickness of 20 nm and is suspended in a 10 μm × 10 μm window in the center of the silicon substrate. It was purchased from Nanopore Solution in Portugal.

[0063] In this embodiment of the invention, all detections are performed in a dark Faraday cage.

[0064] Example 1

[0065] (1) Pretreatment of nanoporous chips

[0066] The silicon nitride nanoporous chip was placed in an ethanol-water solution (V 去离子水 V 乙醇 Soak the chip in a 1:1 solution for 20 minutes to remove inorganic impurities from the surface; then soak it in acetone and isopropanol for 20 minutes each to remove organic impurities from the surface; finally, dry the chip at 60°C for 1 minute, and then clean the front side of the chip with a plasma cleaner for 5 minutes to remove residual impurities and increase its surface hydrophilicity, and place it in a clean container for later use.

[0067] (2) Preparation of solid nanopores

[0068] The pretreated chip was installed in a custom flowcell. The surfaces of both sides of the chip were sequentially wetted with ethanol, water, and buffer 1 to remove air bubbles. Buffer 1 was then injected into both sides of the flowcell. Electrodes were connected to both sides of the flowcell, and a current source pulse dielectric breakdown device (Keithley 2450) was connected. An intermittently increasing pulse current was applied to the cis side of the chip through a power meter. The SiNx film was exposed to a strong electric field. Charge accumulation and thermal effects caused the defects in the film to be amplified, thus breaking down the film to form nanopores. After the pores were formed, intermittent voltages were continued to be applied to both sides of the chip to gradually expand the nanopores to the desired pore size.

[0069] (3) Assembly of the nanopore detection platform

[0070] After fabricating solid nanopores, the two chambers of the flowcell were connected to the probe of a patch-clamp amplifier (Axopatch 200B) via Ag / AgCl electrodes. The patch clamp was connected to a digital-to-analog converter (Digidata 1550B) and then to a computer. The applied bias voltage was controlled and the current changes were recorded using the Clampfit program.

[0071] (4) Detection of nanopore-based oligopeptides

[0072] A schematic diagram illustrating the principle of solid-state nanopore detection of oligopeptide molecules in this invention is shown below. Figure 1 As shown.

[0073] A. Detection of tripeptides

[0074] Nanopores of 1.8–2.5 nm were used to detect tripeptides. First, Buffer 2 was added to both sides of the flowcell, and the opening current of the nanopore was measured using patch clamp as a blank control. At the start of the test, the tripeptide sample dissolved in Buffer 2 was added to the cis side of the flowcell, while only Buffer 2 was added to the trans side. An appropriate bias voltage of suitable magnitude and orientation was applied to drive the tripeptide molecules through the nanopore. The magnitude of the bias voltage and the concentration of added tripeptides were optimized to ensure sufficient signal-to-noise ratio and capture rate. After optimizing the test conditions, the permeation signal of the tripeptide molecules under specific detection conditions was acquired to obtain sufficient signal for statistical analysis.

[0075] B. Detection of hexapeptides

[0076] Hexapeptides were detected using nanopores ranging from 2.5 to 5 nm. The capture rate of hexapeptides in Buffer 2 was very low, possibly due to excessively high perforation rates exceeding the instrument's detection range. Therefore, a buffer concentration gradient method was used to improve the capture rate. First, Buffer 3 was added to the cis side of the flowcell, and Buffer 2 to the trans side. The opening current of the nanopores was measured using patch clamp as a blank control. At the start of the test, the hexapeptide sample dissolved in Buffer 3 was added to the cis side of the flowcell, while only Buffer 2 was added to the trans side. The testing method was the same as for tripeptides.

[0077] (5) Analyze and compare data

[0078] The acquired oligopeptide perforation signals were processed and analyzed using the Clampfit program. Under the same test conditions (nanopore size, buffer solution, sample concentration, bias voltage), the translocation time, current signal amplitude, capture rate, and pore-blocking frequency of the oligopeptides were analyzed. Under the same test conditions (nanopore size, buffer solution), the differences in translocation time, current signal amplitude, capture rate, and pore-blocking frequency of the oligopeptides under different sample concentrations and different bias voltages were analyzed.

[0079] Results: In this embodiment, solid nanopores with different pore sizes were used to test tripeptides and hexapeptides. The results showed that tripeptides achieved a suitable signal-to-noise ratio and capture rate in a 1.8 nm nanopore, while hexapeptides achieved a suitable signal-to-noise ratio and capture rate in a 2.5 nm nanopore. Larger molecular volumes usually require larger nanopores for detection to obtain a suitable signal-to-noise ratio and capture rate, which is consistent with the fact that hexapeptides have a larger molecular volume.

[0080] Tripeptides and hexapeptides were detected using nanopores with the above-mentioned pore size, and the results are as follows: Figures 2-7 As shown, the blocking time, blocking current amplitude, and the ratio of current amplitude to orifice current ΔI / Io for more than 300 signals were statistically analyzed. Tripeptides and hexapeptides were detected under positive voltage, but no signal was detected under reverse voltage, indicating that the surface charge of tripeptides and hexapeptides is negative. Figure 2 and Figure 5 As shown, the blocking time of hexapeptide is significantly lower than that of tripeptide, corresponding to the very low capture rate of hexapeptide in Buffer 2 buffer. This is why we need to use concentration gradient buffers to slow down the permeation speed of hexapeptide. Figure 3 , Figure 4 , Figure 6 and Figure 7As shown, tripeptides and hexapeptides exhibit distinct characteristic peaks in their blocking current amplitude and ΔI / Io at specific pore sizes. However, due to the influence of pore size on the blocking current amplitude, the comparison between the blocking current amplitudes of tripeptides and hexapeptides is not very significant. The ΔI / Io of hexapeptides is much smaller than that of tripeptides, indicating that a 2.5 nm nanopore is too large for detecting hexapeptides, resulting in a lower blocking current amplitude. Besides the main characteristic peaks, another peak appears in the blocking current amplitude of tripeptides at a larger amplitude, and the blocking current amplitude of hexapeptides also shows a distribution at a larger amplitude. This may be related to the molecular pore conformation and orientation of the two peptides.

[0081] Figures 8-16 To test the performance of a 200 nm hexapeptide in a 5 nm nanopore under different voltages, bias voltages of 300 mV, 350 mV, and 400 mV were applied across the nanopore to drive the hexapeptide through it. The blocking time, blocking current amplitude, and ΔI / Io were statistically analyzed. The results show that the blocking time, blocking current amplitude, and ΔI / Io of the hexapeptide in the 5 nm nanopore are all shorter than those in the 2.5 nm nanopore, demonstrating the influence of pore size on blocking time and blocking current amplitude. Furthermore, it can be seen that the magnitude of the bias voltage has little effect on the blocking time and blocking current amplitude.

Claims

1. A method for detecting oligopeptides based on solid-state nanopore single-molecule technology, characterized in that, Includes the following steps: S1: Pre-treat silicon nitride nanoporous chips and prepare solid nanopores; S2: Assemble the nanopore detection platform and apply a bias voltage to drive the oligopeptide through the solid nanopore. Qualitative and / or quantitative analysis of the oligopeptide is performed by collecting the electrical signals generated during the passage through the pore. Specifically, this includes: assembling the nanopore chip with a customized flowcell; adding an electrolyte solution to the trans cavity of the flowcell and an electrolyte solution containing the oligopeptide to the cis cavity; connecting the flowcell to a patch clamp; and applying a bias voltage across the flowcell using the patch clamp to achieve single-molecule detection of the oligopeptide. The oligopeptides include tripeptides and / or hexapeptides; tripeptides are detected using 1.8 nm solid-state nanopores; hexapeptides are detected using 2.5 nm solid-state nanopores. When detecting tripeptides, the electrolyte solution used in both the cis and trans cavities of the flowcell is Buffer 2; when detecting hexapeptides, Buffer 2 is added to the trans cavity of the flowcell, and Buffer 3 containing hexapeptides is added to the cis cavity; Buffer 2 consists of 2M LiCl, 10mM Tris, and 1mM EDTA, pH 7.4; Buffer 3 consists of 0.4M LiCl, 10mM Tris, and 1mM EDTA, pH 7.

4.

2. The method according to claim 1, characterized in that, In S1, the pretreatment involves immersing the silicon nitride nanoporous chip in an aqueous ethanol solution, acetone, and isopropanol in sequence, drying it, and then cleaning it for later use.

3. The method according to claim 1, characterized in that, The solid nanopores are supported by silicon nitride thin films.

4. The method according to claim 1, characterized in that, The solid nanopores are prepared on the silicon nitride thin film supported on the silicon nitride nanopore chip by dielectric breakdown.

5. The method according to claim 4, characterized in that, The buffer solution used to prepare the solid nanopores was 1M KCl, 10mM Tris, 1mM EDTA, pH 8.

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