A nanopore detection probe generated by connecting a target molecule to be detected with a support molecule, and a preparation method and application thereof

Abstract

The application discloses a nanopore detection probe connected with a target molecule to be detected and a support molecule, and a preparation method and application thereof. The nanopore detection probe is obtained by coupling the target molecule to be detected and the support molecule through a bifunctional cross-linking reagent; wherein the support molecule is composed of a guest interaction molecule, a functional connecting group and a pulling sequence; the guest interaction molecule provides a specific signal for the nanopore generated in a combined detection system of host interaction molecules in a solution; the functional connecting group provides an action site of the bifunctional cross-linking reagent, so as to be combined with the target molecule to be detected; and the pulling sequence provides power for driving the whole probe molecule to enter a nanopore detection area under the action of an electrophoretic force. The target molecule to be detected is detected by using the nanopore detection probe and a nanopore single-molecule technology, so that characteristic signals are generated to perform qualitative and / or quantitative analysis. Taking an amino acid-support molecule as an example, twenty different amino acids can be distinguished by the method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of life analysis chemistry and protein sequencing technology, and particularly relates to a nanopore detection probe generated by connecting a target molecule to be detected and a loaded molecule, and a preparation method and application thereof. BACKGROUND

[0002] Proteins play a large number of functions in living organisms, including DNA replication, catalyzing metabolic reactions in living organisms, acting as messengers between cells, maintaining acid-base balance and body fluid balance in living organisms, and providing stable structure for cells and organisms. They are composed of long amino acid chains, the sequence of amino acids is determined by the nucleotide sequence of genes, and the amino acid chains are folded to form specific three-dimensional structures to determine their physiological activity. However, the gene sequence cannot directly encode information about protein abundance, post-translational modification or cleavage, and even a small change in the primary sequence of a protein can have a significant impact. Therefore, proteomics research is in great need of an efficient and economical protein sequencing and post-translational modification strategy. In recent years, only two strategies for de novo sequencing are feasible: Edman degradation and mass spectrometry. After decades of development, multi-channel parallel detection and automated detection have improved their detection efficiency, but both have obvious limitations: the dynamic range of detection is not large enough, the length of sequencing is not long enough, and the accuracy of detection is not high enough, which seriously limits the application of these two detection technologies.

[0003] In the past few years, a variety of new protein sequencing methods have been developed, such as fluorescence sequencing, single molecule polypeptide fingerprinting, using tunneling current to identify single amino acid, and nanopore protein sequencing. Among these new methods, nanopore technology has a great potential due to its successful application in DNA sequencing. Compared with DNA sequencing, protein sequencing is more challenging. Proteins are composed of 20 different amino acids, while DNA contains only four nucleotides, and it is undoubtedly a great challenge to read 20 different signals. In addition, unlike DNA molecules with unique negative charge, proteins have non-uniform charge, so it is difficult to control the orientation of the polypeptide chain through the nanopore.

[0004] Therefore, it is imperative to develop a new probe that can recognize 20 different amino acids and overcome the non-uniform charge property of amino acids to make the probe move through the nanopore in a directional manner. SUMMARY

[0005] In view of the defects of the prior art, one object of the present application is to provide a nanopore detection probe connected with a target molecule to be detected and a loading molecule and a preparation method thereof. The present application generates a detection probe by connecting a target molecule to a loading molecule by means of a bifunctional cross-linking agent through a simple and efficient click chemistry reaction, and qualitatively and / or quantitatively analyzes the probe by using the detection sensitivity of nanopore single molecule technology, and specifically relates to the analysis and detection of various biological small molecules (such as amino acids, nucleotides, neurotransmitters, etc.) and inorganic molecules, as well as DNA sequencing, protein sequencing, etc.

[0006] The nanopore detection probe connected with a target molecule to be detected and a loading molecule provided by the present application is obtained by coupling a target molecule to be detected and a loading molecule through a bifunctional cross-linking agent.

[0007] The loading molecule is composed of a guest-acting molecule, a functional connecting group and a traction sequence. The guest-acting molecule is combined with a host-acting molecule and interacts with a nanopore in a detection system, thereby generating a specific signal; the functional connecting group provides an action site of the bifunctional cross-linking agent, so as to be combined with the target molecule to be detected; and the traction sequence provides a power for driving the entire probe molecule into a nanopore detection area by an electrophoretic force.

[0008] Further, the guest-acting molecule on the loading molecule is selected from at least one of ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives.

[0009] Further, the functional connecting group on the loading molecule is selected from at least one of an allyl group, a thiol group, an amino group, a succinimidyl ester, and a maleimide ester.

[0010] Further, the traction sequence on the loading molecule is selected from at least one of a polynucleotide (such as PolyT, PolyA), a nucleotide random sequence, a charged polypeptide (such as PolyR, PolyD, PolyK, PolyE), and a polymeric macromolecule (such as (PEG) n ).

[0011] According to one embodiment of the present application, the loading molecule can be FGCD8, FGGCD8, FGGGCD8, FGGCGGD8, FGK(AZA)D8, an organic transition metal compound-amino acid-polynucleotide complex structure with a sequence of ferrocene-GC-TTTTTTTTTTT, etc.

[0012] Specifically, the FGCD8 is a polypeptide with an amino acid sequence from N-terminus to C-terminus of FGCDDDDDDDD; the C in the loading molecule provides a functional group for connection, and the G and the GG, GGG, etc. in the following loading molecules can change the position of the functional group, so as to realize the probe demand in different cases.

[0013] The FGGCDD8 is a polypeptide with an amino acid sequence from N-terminus to C-terminus of FGGCDDDDDDDD, as shown in SEQ ID No: 1.

[0014] The FGGGCD8 is a polypeptide with an amino acid sequence from N-terminus to C-terminus of FGGGDDDDDDDD, as shown in SEQ ID No: 2.

[0015] The FGGCGGD8 is a polypeptide with an amino acid sequence from N-terminus to C-terminus of FGGCGGDDDDDDDD, as shown in SEQ ID No: 3.

[0016] The FGK(AZA)D8 is a polypeptide with an amino acid sequence from N-terminus to C-terminus of FGKDDDDDDDD; wherein the K at the 3rd position is an azide-modified lysine, as shown in SEQ ID No: 4.

[0017] The loading molecule in the application can be synthesized by solid-phase synthesis technology, or purchased from a polypeptide synthesis company, such as Jiangyao Biotechnology Co., Ltd.

[0018] The charged polypeptide in the pulling sequence can be synthesized by a polypeptide synthesizer, the polynucleotide can be synthesized by a DNA synthesizer, and the polymeric macromolecule can be directly purchased. The functional connecting group on the loading molecule can be coupled to the pulling sequence by an amide reaction or the like. The guest molecule can be coupled to the loading molecule-pulling sequence by an amide reaction, a click chemistry reaction or the like.

[0019] Further, the bifunctional cross-linking reagent is at least one selected from 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimidyl ester (SPDP), trans-2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate (SMCC), 3,3'-dithiobispropionic acid bis(N-hydroxysuccinimide) ester (DSP), maleimidyl acetate succinimidyl ester (AMAS), iodoacetic acid-N succinimidyl (SIA), bromoacetic acid N-hydroxysuccinimidyl ester (SBA), disuccinimidyl carbonate (DSC).

[0020] Further, the main acting molecule is selected from at least one of a-cyclodextrin, b-cyclodextrin, g-cyclodextrin, other cyclodextrins and their derivatives, cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], cucurbituril

[10] , other cucurbituril derivatives.

[0021] Further, the target molecule can be various biological small molecules (such as amino acids, nucleotides, neurotransmitters, etc.) and inorganic molecules, DNA, proteins, etc.

[0022] The application also provides a preparation method of a nanopore detection probe connected with a target molecule and a loading molecule.

[0023] The method comprises the following steps:

[0024] 1) reacting the target molecule, the bifunctional cross-linking reagent and the organic base to form a target molecule-bifunctional cross-linking reagent complex;

[0025] 2) reacting the target molecule-bifunctional cross-linking reagent complex with the loading molecule to obtain the nanopore detection probe.

[0026] In step 1) of the above method, the organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, 2,6-Lutidine (2,6-dimethylpyridine), Imidazole (imidazole) and NMI.

[0027] In step 1) of the above method, the molar ratio of the target molecule, the bifunctional cross-linking reagent and the organic base is (1-100):1:(2-10), and the reaction time is 1-4h; specifically, the molar ratio of the target molecule, the bifunctional cross-linking reagent and the organic base is 1:1:2.

[0028] The target molecule is added in the form of an aqueous solution (the concentration can be 1mM), the bifunctional cross-linking reagent is added in the form of a DMF solution (the concentration can be 4mM), and the organic base is also added in the form of a DMF solution (the concentration can be 8mM).

[0029] In step 2) of the above method, the molar ratio of the target molecule-bifunctional cross-linking reagent complex to the loading molecule is (1-100):1, and the reaction is a normal temperature reaction for 1-8h; specifically, the molar ratio of the target molecule-bifunctional cross-linking reagent complex to the loading molecule is 1:1, and the reaction is a normal temperature reaction for 1h.

[0030] The loading molecule is added in the form of an aqueous solution (the concentration can be 1mM).

[0031] The application also provides a method for qualitatively and / or quantitatively detecting a target molecule by using the prepared nanopore detection probe.

[0032] The method for qualitatively and / or quantitatively detecting a target molecule provided by the application is a detection method using a nanopore, and comprises the following steps: first, combining a guest molecule and a host molecule in the nanopore detection probe generated by connecting a target molecule and a support molecule, and then performing nanopore single-channel recording.

[0033] Further, the nanopore single-channel recording refers to collection of characteristic electrical signals by using a nanopore.

[0034] Still further, the collection of characteristic electrical signals is performed at room temperature of 25.0±3.0℃. The signal sampling frequency is 100 kHz, and the Bessel low-pass filter cutoff frequency is 10 kHz. The characteristic current signal is collected by using Clampfit software for single-channel data analysis, and origin software is used for data statistics and analysis.

[0035] Further, the nanopore can be selected from at least one of a biological nanopore with a diameter of less than 2 nm and a solid-state nanopore with a diameter of less than 3 nm, and the nanopore is specifically selected from α-HL and a mutant thereof, MspA and a mutant thereof, Phi29, Aerolysin, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopore, glass nanopore, SiN x nanopore, Al2O3 nanopore, graphene nanopore, hybrid nanopore, carbon nanotube, DNAOrigami nanopore.

[0036] The method for qualitatively and / or quantitatively detecting a target molecule provided by the application comprises the following steps:

[0037] (a) using DPhPC for forming a synthetic lipid bilayer with a diameter of 100-150 microns on a 25-micron-thick polytetrafluoroethylene film (Goodfellow, Malvern, PA);

[0038] (b) embedding a nanopore in the phospholipid bilayer, and the phospholipid bilayer divides the sample cell into cis and trans chambers, both of which contain 1.0 mL of buffer solution; adding the nanopore detection probe generated by connecting a target molecule and a support molecule to the cis chamber, and simultaneously adding a host molecule to the cis chamber;

[0039] The above experiment is carried out in 0.8-4.0 M KCl, 10 mM citric acid buffer with pH value of 5.0-8.0 and temperature of 25.0±3.0℃;

[0040] The molar ratio of the nanopore detection probe generated by connecting the target molecule and the loaded molecule to the host molecule is greater than 1:10.

[0041] (c) applying a fixed bias voltage in the range of +80-250 mV to the nanopore single-channel test system and recording, the loaded molecule and the host interaction molecule moving to the nanopore under the driving of host-guest interaction and electrophoretic force, generating a characteristic current signal;

[0042] (d) analyzing and comparing the current blocking ratio, signal frequency and blocking time of the current signal to obtain the characteristic information and concentration information of the target molecule to be tested.

[0043] According to the embodiment of the application, the target molecule can be 20 natural amino acid molecules, the corresponding bifunctional cross-linking reagent is SPDP, the guest interaction molecule of the loaded molecule is phenylalanine (F), the functional connection group is sulfhydryl (-SH), the traction sequence can be DDDDDDDD, and the host molecule is cucurbituril [7], and the nanopore is alpha-HL (WT).

[0044] Compared with the prior art, the application has the following beneficial effects:

[0045] The method for connecting the detection target molecule and the loaded molecule to generate the nanopore detection probe provided by the application can qualitatively and quantitatively detect small molecules, has simple reaction, short reaction time, low detection limit, stable and uniform signal output, and strong discrimination ability for similar molecules. For the above-mentioned amino acid-loaded molecule, twenty different amino acids can be distinguished by this method. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The basic process of connecting the target molecule to the loaded molecule by using the bifunctional cross-linking reagent to generate the detection probe by simple and efficient click chemistry reaction;

[0047] Figure 2 HPLC-MS graphs of several target molecules prepared in Example 1;

[0048] Figure 3 Nanopore experiment records of the synthesized amino acid-loaded molecules; wherein, Figure 3 a is a schematic diagram of the experimental method, Figure 3 b is an actual current signal graph of the probe;

[0049] Figure 4 The concentration test curve of the probe formed by the amino acid molecule (glycine) and the loaded molecule in Example 1; the test sample concentrations are 40 pM, 400 pM, 4 nM, 40 nM, 400 nM, respectively, and the detection limit is 4 nM (with a signal greater than 1 per minute). DETAILED DESCRIPTION

[0050] The application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0051] The experimental methods in the following examples are all conventional methods, unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially. The room temperature referred to in the following examples is 25.0 ± 3.0 °C.

[0052] Example 1:

[0053] Experimental sample: In the following examples, the target is a natural amino acid molecule, the bifunctional cross-linking reagent is SPDP, the loaded molecule is FGGCD8, the guest molecule is phenylalanine (F), the functional linking group is sulfhydryl (-SH), the pulling sequence is DDDDDDDD, and the host molecule is cucurbituril [7], and the nanopore is a-HL (WT).

[0054] The loaded molecule FGGCD8 is a polypeptide FGGCDDDDDDDD (N-terminal to C-terminal), which is customized by Qiangyao Biotechnology Co., Ltd. and can be purchased from major polypeptide synthesis companies or synthesized by solid-phase synthesis technology.

[0055] Cross-linking of amino acid AVIL and loaded molecule:

[0056] According to the steps in Figure 1 , the amino acid molecule to be tested is coupled with the loaded molecule. The detailed method includes the following steps:

[0057] 1) Prepare solutions of the amino acid molecule to be tested (solvent water, concentration 1 mM), the loaded molecule (solvent water, concentration 1 mM), the bifunctional cross-linking reagent SPDP (solvent DMF, concentration 4 mM), and the organic base NMM (solvent DMF, concentration 8 mM).

[0058] 2) React the amino acid molecule to be tested, the bifunctional cross-linking reagent, and the organic base in a molar ratio of 1:1:2 for 2 h to form an amino acid-bifunctional reagent complex.

[0059] 3) The amino acid-bifunctional reagent complex was reacted with the support molecule at a molar ratio of 1:1 for 1 h to obtain the amino acid molecule-support molecule complex probe to be detected.

[0060] The probe prepared according to the above connection method was characterized by HPLC-MS, and the molecular purity information as shown in Figure 2 was obtained. The purity of the molecular probe obtained by the method is higher than 95%. The four target amino acid molecules can be efficiently connected to the support molecule to form the probe to be detected.

[0061] Example 2: Nanopore single molecule measurement of the probe obtained in Example 1

[0062] The specific method is as follows:

[0063] 1) DPhPc was used to form a synthetic lipid bilayer with a diameter of 100 μm on a 25 μm thick polytetrafluoroethylene film (Goodfellow, Malvern, PA).

[0064] 2) The α-HL nanopore was embedded in the phospholipid bilayer, which divided the sample cell into cis and trans chambers, both of which contained 1.0 ml of buffer solution. 2 μL of amino acid molecule-support molecule complex probe (concentration of 200 μM) was added to the cis chamber, and 10 uL of cucurbituril [7] (concentration of 5 mM) was added to the cis chamber. The experiment was carried out in 3.6 M KCl, 10 mM citric acid buffer (i.e. the buffer in the two chambers) with pH value of 5.0 and temperature of 25.0±3.0℃.

[0065] 3) The experiment was recorded at a voltage of +200 mV. The support molecule and the host molecule moved towards the nanopore under the driving of host-guest interaction and electrophoretic force, generating a characteristic current signal, as shown in Figure 3 b (the current signal generated by the probe obtained by coupling glycine with the support molecule FGGCD8). The current blocking ratio, signal frequency and blocking time of the current signal were analyzed and compared to obtain the characteristic information and concentration information of the target to be detected.

[0066] Example 3:

[0067] The concentration curve of the probe obtained in Example 1 (the probe obtained by coupling glycine with the support molecule FGGCD8) was measured, and the signal frequency was counted to obtain the number of characteristic signals per minute, and the results are shown in Figure 4 , the gradient concentrations of the sample to be detected were 40 pM, 400 pM, 4 nM, 40 nM, and 400 nM, and the detection limit was 4 nM (more than 1 signal per minute).

[0068] The determination method of the detection limit is as follows: in the nanopore single-channel detection system, 10 μL of test sample (FGGC(G)D8) with concentrations of 4 nM, 40 nM, 400 nM, 4 μM and 40 μM and 10 μL of host molecule cucurbituril [7] (concentration: 100 mM) are added respectively, a +200 mV voltage is applied for single-channel recording, and the recording time is greater than 1 h. The recording result is subjected to signal frequency analysis, and the signal frequency (min -1 ) in the recording is calculated. 4 nM with a signal frequency of 1.52 is taken as the sample detection limit. When the final concentration of the sample is lower than 4 nM, the signal frequency will be less than 1 min -1 , which cannot meet the signal statistics and processing.

[0069] The above describes the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application and without unnecessary experiments. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. A nanopore detection probe that connects a target molecule to a load molecule, obtained by coupling the target molecule to the load molecule through a bifunctional crosslinking reagent; in, The loaded molecule consists of a guest interacting molecule, a functional linking group, and a traction sequence. The guest active molecule combines with the host active molecule and interacts with the nanopores in the detection system, thereby generating a specific signal; the functional linker provides the action site of the bifunctional crosslinking reagent, enabling it to bind to the target molecule; the traction sequence provides the driving force for the entire probe molecule to enter the nanopore detection region by electrophoretic force. The guest acting molecule on the loaded molecule is selected from at least one of the following: ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives. The bifunctional crosslinking agent is selected from at least one of the following: 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidone-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane carboxylate (SMCC), di(N-hydroxysuccinimide) ester of 3,3'-dithiodipropionic acid (DSP), maleimide succinimide ester (AMAS), iodoacetic acid-N-succinimide (SIA), bromoacetic acid N-hydroxysuccinimide ester (SBA), and disuccinimide carbonate (DSC). The main active molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other types of cyclodextrin and their derivatives, cucurbita[5], cucurbita[6], cucurbita[7], cucurbita[8], cucurbita[10], and other types of cucurbita derivatives; The target molecule to be tested is selected from any one of the following: small biological molecules, inorganic molecules, DNA, and proteins.

2. The nanopore detection probe according to claim 1, characterized in that: The functional linking group on the loaded molecule is selected from at least one of allyl, mercapto, amino, succinimide ester, and maleimide ester; Alternatively, the traction sequence on the loaded molecule is selected from at least one of the following: polynucleotides, random nucleotide sequences, charged peptides, and polypolymers.

3. The method for preparing the nanopore detection probe formed by connecting the target molecule and the load molecule as described in claim 1 or 2, comprising the following steps: 1) The target molecule, bifunctional cross-linking reagent, and organic base are reacted to form a target molecule-bifunctional cross-linking reagent complex; 2) The target molecule-bifunctional cross-linking reagent complex is reacted with the loaded molecule to obtain the nanopore detection probe.

4. The preparation method according to claim 3, characterized in that: In step 1), the organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, 2,6-dimethylpyridine, imidazole and NMI; Alternatively, in step 1), the molar ratio of the target molecule, the bifunctional crosslinking reagent, and the organic base is (1-100):1:(2-10), and the reaction time is 1-4 h; the target molecule is added in the form of an aqueous solution, the bifunctional crosslinking reagent is added in the form of a DMF solution, and the organic base is added in the form of a DMF solution. Alternatively, in step 2), the molar ratio of the target molecule-bifunctional crosslinking reagent complex to the loaded molecule is (1-100):1, and the reaction is carried out at room temperature for 1-8 h; the loaded molecule is added in the form of an aqueous solution.

5. A method for qualitative and / or quantitative detection of a target molecule using the nanopore detection probe according to claim 1 or 2, wherein the detection is performed using a nanopore, comprising the following steps: first, binding the guest molecule in the nanopore detection probe formed by connecting the target molecule and the load molecule to the host molecule, and then performing single-channel recording in the nanopore. The nanopore single-channel recording refers to the acquisition of characteristic electrical signals using nanopores; The nanopores are selected from biological nanopores with a diameter of less than 2 nm or solid nanopores with a diameter of less than 3 nm.

6. The method according to claim 5, characterized in that: The characteristic electrical signals were acquired at room temperature (23 ± 0.5 ℃); the signal sampling frequency was 100 kHz, and the Bessel low-pass filter cutoff frequency was 10 kHz; the characteristic current signals were acquired using Clampfit software for single-channel data analysis, and the data were statistically analyzed using Origin software.

7. The method according to claim 5, characterized in that: The nanopores are specifically selected from α-HL and its mutants, MspA and its mutants, Phi29, Aerolysin, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopores, glass nanopores, and SiN. x At least one of the following: nanopores, Al2O3 nanopores, graphene nanopores, hybrid nanopores, carbon nanotubes, and DNA Origami nanopores.

8. The method according to any one of claims 5-7, characterized in that: The method specifically includes the following steps: (a) Using diaphytylphosphatidylcholine to form a synthetic lipid bilayer with a diameter of 100-150 micrometers on a 25-micrometer-thick polytetrafluoroethylene film; (b) The nanopore is embedded in a phospholipid bilayer, which divides the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution; the nanopore detection probe formed by connecting the target molecule and the load molecule is added to the cis chamber, and the host active molecule is added to the cis chamber at the same time; The above experiments were conducted in 0.8–4.0 MKCl, 10 mM citrate buffer at a pH of 5.0–8.0 and a temperature of 25.0 ± 3.0 ℃. The molar ratio of the nanopore detection probe formed by connecting the target molecule and the load molecule to the host molecule is less than 1:10; (c) Apply a fixed bias voltage in the range of +80 – 250 mV to the nanopore single-channel test system and record that the load molecules and host molecules move into the nanopore under the drive of host-guest interactions and electrophoretic forces, generating characteristic current signals. (d) The current blocking ratio, signal frequency and lag time of the current signal are analyzed and compared to obtain the characteristic information and concentration information of the target molecule to be tested.

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