CN108645905A - A method of hydrogen peroxide is detected based on solid nano hole - Google Patents

Abstract

The present invention relates to a kind of methods detecting hydrogen peroxide based on solid nano hole, by beating different nano-pores on 10 100nm ultra-thin silicon nitride films, with a variety of silanization treatments, realize nanoporous surface and inner wall differentiation modification, by silane coupled molecule by peroxidase conjugate in nano-pore inner wall, it constructs controllable, stable, unimolecule hydrogen peroxide sersor part in highly sensitive nano-pore, by the nano-pore ion current signal for parsing enzymatic reaction, the model mechanism and rule of single enzyme molecule enzymatic reaction Km and Kcat in nano-pore, with Real_time quantitative detection hydrogen peroxide.The present invention reaches nanomole or less by the controllable modification of differentiation modification and horseradish peroxidase unimolecule in nano-pore inside and outside nano-pore, the small molecule real-time sensing detection method of the specificity of foundation, precision.

Description

A method for detecting hydrogen peroxide based on solid-state nanopores

technical field

The invention relates to nanopore sensing and detection, in particular to a method for detecting hydrogen peroxide based on solid-state nanopores.

Background technique

Nanopore sensing detection technology has been considered as one of the powerful technologies for rapid unlabeled single molecule detection since it was proposed in 1996. Nanopores are mainly divided into solid-state nanopores and biological nanopores. Nanopore sensing technology has become a new, important and potential analysis and detection method in the fields of chemistry and biology. It has broad application prospects and great potential value in the fields of DNA sequencing research, single-molecule detection, single-molecule chemical reaction and protein folding. Nanopores originated from the phenomenon of transmembrane ion channel transport on the surface of cell membranes, using the concept of resistance effect in solution. Similar to the concept, the micropore sensor was first proposed by Coulter in the early 1950s, which enabled the Coulter counter to be produced and developed, and has become a commonly used blood cell counting instrument. With the development of research technology, this kind of small hole has been made to the nanometer level, and scientists have begun to try to carry out research at the molecular level, which has become a new type of sensor.

Due to the vigorous development of gene sequencing, scientists began to study the ability of nanopores to sense nucleic acid molecules and read the internal information of nucleic acid molecules. In the 1990s, Branton and Kasianowicz first proposed that nanopore sensing technology could be used for sequencing. They successfully drove single-stranded DNA electrophoresis through a channel protein α-hemolysin located on the phospholipid bilayer, and observed the current Blocking phenomenon, if the blocking currents of different magnitudes generated by these four kinds of detection can be detected, the sequence of DNA can be deduced. Therefore, nanopore is considered to be the most promising third-generation gene sequencing technology for rapid and low-cost DNA sequencing. In 2014, Oxford Nanopore, UK, launched the first commercial nanopore sequencer, MinION. The current market price of MinION is $1,000 each. Oxford Nanopore, UK, launched the MinION nanopore sequencer early trial program last year, demonstrating the potential of MinION to assist genome assembly and rapidly sequence microbial genomes and environmental samples outside of standard laboratories. Researchers at NASA's Johnson Space Center plan to take MinION to space in 2016 to sequence samples from the International Space Station. Some researchers believe that MinION can be used for point-of-care testing (POCT), for example, a rapid test to diagnose an unknown infection, real-time analysis of disease outbreaks such as the Ebola outbreak in West Africa. Roche also announced that it has made a strategic investment in nanopore sequencing company Stratos Genomics, and the two companies have reached a cooperation agreement to further develop single-molecule sequencing technology. In July 2012, Stratos Genomics demonstrated DNA sequencing of 36 bases, which has rapidly improved to 210 bases by September 2013. The company said it will develop a low-cost sequencing platform that combines the speed and throughput of nanopores with improved resolution and signal-to-noise ratio for targeted and whole-genome sequencing. In June 2014, Roche acquired Genia Technologies, an American nanopore sequencing company, for US$350 million; in June, Roche and venture capital jointly invested US$15 million in StratosGenomics, an American nanopore sequencing company. Roche is also co-developing solid-state nanopore technology with IBM. And Illumina and Lifetech are also focusing on developing or investing in nanopore sequencing technology. Brown's company has developed a palm-sized nanopore gene sequencer that connects to a computer with a USB cable and can detect DNA in samples. The small instrument is already being used in Africa to detect Ebola. This kind of detector is integrated and miniaturized, can be embedded in daily necessities, and wirelessly connected to the Internet to continuously monitor health status.

In addition to the great attractiveness and potential of nanopore technology in the field of gene sequencing, nanopores, as a fast, high-throughput, and highly sensitive single-molecule sensor, have been widely used in the field of single-molecule detection with high precision. , non-marked advantage. Such as the detection of heavy metal ions, the quantitative detection of small molecules and the detection of glucose content in diabetes].

In recent years, more and more researchers have begun to notice that nanopore is a nanoscale confinement space, its signal is easy to be detected, and it can also be used as a high-precision, label-free monitoring method. Moreover, in nanopore detection, the sensitive amplification characteristics combined with the small volume allow the sample to be detected only at femtomolar concentrations, which can be used to analyze biomolecular samples in physiological solutions that have been filtered but not further processed. The micro/nano detection of molecular technology provides an excellent miniaturized platform for the construction of rapid small molecule quantitative detection.

Hydrogen peroxide (hydrogen peroxide) is a strong oxidizing agent, which is widely used in pharmaceutical, clinical, environmental, mining, textile, food manufacturing, etc. In addition, hydrogen peroxide is an important chemical substance in biological systems, Has strong cytotoxicity. What's more serious is that the accumulation of hydrogen peroxide in the mitochondria may lead to cancer and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases.

However, traditional methods for detecting hydrogen peroxide usually use spectroscopic methods, chemiluminescent methods, amperometric titration methods, electrochemical methods, etc. Traditional spectroscopic and chemiluminescent methods are usually time-consuming, and they require expensive reagents (eg, fluorescent labels) and equipment. In addition, many electrochemical techniques use Pt electrodes to detect hydrogen peroxide-dependent direct oxidation of molecules. Due to its poor selectivity, low sensitivity and electrode contamination. In view of the fact that the construction of an integrated nanopore single molecule real-time rapid micro/nano hydrogen peroxide monitoring method is an urgent problem to be solved in my country's food industry and food safety. Traditional hydrogen peroxide sensors are mostly studied from a relatively macroscopic level, and the resulting parameters are often difficult to reflect the kinetic characteristics of the reaction between the enzyme and the substrate at the molecular level. Therefore, to construct an integrated single-molecule horseradish peroxidase sensor for A method for real-time quantitative detection of hydrogen peroxide is particularly important.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the object of the present invention is to construct a method for the real-time quantitative detection of hydrogen peroxide by integrating a single-molecule horseradish peroxidase sensor.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows: a method for detecting hydrogen peroxide based on solid-state nanopores, specifically comprising the following steps: a method for detecting hydrogen peroxide based on solid-state nanopores, specifically comprising the following steps:

Step A. Preparation of nanopores: drilling nanopores on an ultra-thin silicon nitride film of 10-100nm;

Step B. Modification of nanopores and construction of single-molecule enzyme sensors: Various silanization treatments are used to achieve differential modification of the surface and inner wall of nanopores, and a single horseradish peroxidase is stably assembled by silane coupling molecules fixed on the inner wall of the nanopore to obtain a nanopore sensor;

Step C. Substrate-specific sensing detection: Add different concentrations of hydrogen peroxide into the reaction system, analyze the current characteristic signal, draw a standard curve of hydrogen peroxide concentration, and use the standard curve to quantitatively detect hydrogen peroxide in real time.

In the step A, the FEI Strata 201FIB system is used to bombard the surface of the 10-100nm ultra-thin silicon nitride film with an accelerating potential of 30kV to drill nanopores.

In the step B, various silanization treatments are one or more of methylsilane, trifluoro or aminosilane, carboxylsilane.

In the step B, the surface of the nanopore is modified in a covalent or non-covalent manner by using a comb-like graft copolymer to shield the charge on the inner surface of the nanopore.

In the step B, the silane coupling molecule aminosilane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are used to realize the inner wall of the nanopore. Immobilization and site-directed assembly of oxidase molecules.

In the step B, before chemical modification, the Si3N4 chip was washed in piranha solution at 90°C for 30 min to generate hydroxyl groups on the Si3N4 surface, activated in methanol with 3-APTES for 3 h, and 1,4-phenylene 1,4-phenylene diisothiocyanate was treated with dimethyl sulfoxide for 5h, washed, and 1,4-phenylene diisothiocyanate was mixed with 1 mg/ml of horseradish peroxidase molecule HRP The horseradish peroxidase is stably assembled and fixed in the wall of the nanopore by performing a covalent coupling reaction with the primary amine group, and after 12-24 hours, the modified channel is thoroughly washed with a buffer solution to obtain a nanopore sensor.

In the step C, by analyzing the characteristic current signal, the characteristic signal generated by the enzymatic reaction is determined, and the detection limit of the substrate is measured.

The detection limit and standard curve of the nanopore detection hydrogen peroxide of the present invention have a linear range of 5-15nmol/L, a working curve: Y=-183.431X+3580.726, a correlation coefficient R^2=0.986, and a detection limit of 10pmol/L.

Compared with prior art, the beneficial effect of the present invention is:

1. Through the differential modification inside and outside the nanopore, and the controllable modification of the single molecule of horseradish peroxidase in the nanopore, the present invention establishes a new specific small molecule real-time sensing and detection method, and its precision reaches Below nanomolar. Taking advantage of the fast, non-labeling and high-sensitivity characteristics of nanopore sensing and detection, the enzymatic kinetics of a single enzyme molecule and its influencing factors were studied. Combining the advantages of solid-state nanopore stability, easy design and processing, and physical and chemical modification, a biochemical sensor was constructed. Controlled, stable, and highly sensitive in-nanopore single-molecule horseradish peroxidase sensing device.

2. Nanopore detection hydrogen peroxide detection limit and standard curve, the linear range is 5-15nmol/L, the working curve: Y=-183.431X+3580.726, the correlation coefficient R^2=0.986, the detection limit can reach 10pmol/L.

3. When the nanopore sensor constructed by the present invention detects hydrogen peroxide, when there is hydrogen peroxide, it will cause a certain degree of current reduction, and when the concentration of hydrogen peroxide gradually increases, the ion current gradually decreases, and the reduced The ion electric amplitude has a linear relationship with the increase of the concentration of hydrogen peroxide, indicating that the constructed nanopore sensor has a positive effect on the reaction catalyzed by hydrogen peroxide.

4. The silicon nitride nanopore of the present invention can also achieve covalent immobilization of proteins, aptamers, biophilins, etc. by different means, in order to study the interaction between molecules at a finer level, such as protein between proteins and nucleic acids, and between ligand-receptor interactions. Achieving real-time monitoring at the single-molecule level can use new means to conduct in-depth research on the mechanism of interactions between biomolecules and microscopic fluctuations in the conformation of biomacromolecules, and provides a new way to quantitatively detect some small molecules. The limit is nano/femtomolar.

Description of drawings

Figure 1. Nanopore imaging diagram and I-V curve;

Figure 2. (A) Schematic diagram of enzyme immobilization steps; (B) Schematic diagram of enzyme redox to produce ABTS+;

Figure 3. Nanopore characterization diagram;

(A) I–V curves of 0.1 mol/L KCl and 0.1 mol/L KCl containing 1.5 mMABT and hydrogen peroxide before and after HRP functionalization;

(B) I-V curve of 0.1mol/L KCl containing 1.5mM ABTS after HRP functionalization;

(C) I-V curve of 0.1mol/L KCl containing 1.5mM ABTS and hydrogen peroxide after HRP functionalization;

(D) I-V curves of unmodified nanopores containing 1.5mM ABTS and hydrogen peroxide at 0.1mol/L KCl and 0.1mol/L KCl;

Figure 4. The reproducibility and working curve of the enzyme-functionalized nanopore sensor;

(A) Effect of different pH on HRP-functionalized nanopores;

(B) Reproducibility of HPR-functionalized nanopore detection system;

(C) I-V curves of HPR-functionalized nanopore detection system containing 1.5mM ABTS and different concentrations of hydrogen peroxide in 0.1mol/L KCl;

(D) Linear fit of different concentrations of hydrogen peroxide and ion current.

Detailed ways

The present invention constructs an integrated single-molecule enzyme nanopore sensor for real-time quantitative detection of peroxidation by analyzing the nanopore ion current signal of the enzymatic reaction, and the model mechanism and law of the enzymatic reaction Km and Kcat of a single enzyme molecule in the nanopore. Hydrogen specifically comprises the following steps:

1. Preparation of Nanopores

The research on the process of preparing high-insulation all-solid-state silicon nitride film has successfully obtained ultra-thin silicon nitride films with various film thicknesses of 30-100nm. Through the optimization of the process, 10-100nm ultra-thin silicon nitride film that meets the drilling requirements will be obtained; through the optimization of the focused electron beam or ion beam processing conditions in the TEM, FIB, and HIM processing system platforms, various thicknesses, The pore size is the aspect ratio, the shape of the nanopore.

2. Modification of nanopores and construction of unimolecular enzyme sensors

Various silanization treatments (methylsilane, trifluorosilane, carboxysilane, etc.) are used to achieve differential modification of the nanopore surface and inner wall. The peroxidase is coupled to the inner wall of the nanopore through a silane coupling molecule. For example, comb-like graft copolymers, such as poly-L-lysine-graft-poly (ethylene glycol), can be covalently or non-covalently modified on the surface of nanopores to shield the charges on the inner surface of nanopores. Use silane coupling molecules such as aminosilane, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) to achieve nanopore inner wall Immobilization and site-directed assembly of peroxidase molecules.

Characterization of key steps: characterize nanopore morphology and structure by SEM, TEM, AFM, etc., and characterize nanopore chemical modification (such as carboxysilane hydrophilic modification, Trifluorosilane hydrophobic modification, etc.). The change of the current signal in the nanopore is used to determine the binding speed and quantity of peroxidase molecules in the hole.

3. Substrate-specific sensing assays

Add different concentrations of hydrogen peroxide into the reaction system, analyze the current characteristic signal, draw the standard curve of hydrogen peroxide concentration, and measure the detection limit of the sensor for hydrogen peroxide.

The hydrogen peroxide catalytic reaction equation is as follows:

HRP(Fe ⁴⁺ ＝O)Porp+HA→HRP(Fe ³⁺ )Porp+A ^· +H ₂ O (3)

Using 2,2'-amino-bis(3-ethyl-benzothiazolinesulfonic acid-6)ammonium salt (ABTS), in the presence of hydrogen peroxide, the product Compound I of horseradish peroxidase HRP is oxidized . The porphyrin cationic free radical compound accepts an electron from the reduced substrate molecule to generate Compound II. Then, compound II is reduced to resting enzyme through the transfer of an electron, and ABTS is used as a substrate to generate ABTS·+ product, and the reaction rate of the enzyme is detected by using the change of electrical signal during the reaction.

A stable assembly of a single horseradish peroxidase is fixed in the nanopore wall, and through the analysis of the characteristic current signal, combined with the relevant mathematical and physical models, the characteristic signal generated by the enzymatic reaction is determined, and the detection limit of the substrate is measured ; Distinguish the difference of current characteristic signals such as substrates, substrate analogs, competing substrates, etc., and use the characteristic signals to measure the enzymatic kinetic parameters.

Example

Reagents and materials: APTES, Sigma; potassium chloride (KCl), Sigma; methanol, Sigma; dimethyl sulfoxide, Hefei Xinyuan Biotechnology Co., Ltd.; horseradish peroxidase (HRP), Sigma; ABTS, 98% Sigma; Hydrogen Peroxide (H2O2), 30% SL Labor-Service; Methanol, 98% Sigma; Aminosilane, 98% Sigma;

Test equipment: field emission scanning electron microscope, Malvern particle size analyzer, analytical balance, vibration isolation table, shielding box, silver wire, pipette gun, ultrapure water, patch clamp,

Experimental solution preparation

A. Preparation of 1mol/L KCl solution: Weigh 0.7458g of KCl with an analytical balance, dissolve it, put it in a 10mL volumetric flask, shake well, filter it with an Anotop 0.02μm filter membrane, and then dilute to 0.1mol/L KCl.

B. Preparation of piranha solution: piranha solution, the ratio is 3:1 (volume ratio) of concentrated sulfuric acid (98%) and hydrogen peroxide solution (30%), prepared in a fume hood, pay attention to protection when preparing. Add the hydrogen peroxide solution to the concentrated sulfuric acid very slowly while stirring rapidly, and the order of adding must not be reversed.

C. Preparation of 0.5% p-phenylene diisothiocyanate: Weigh 0.005g of p-APTES and dissolve it in 8mL of dimethyl sulfoxide and put it in a 10mL volumetric flask to constant volume, store at -20°C until use.

Sample solution preparation: Weigh 0.001g of HRP with an analytical balance and dilute to a final concentration of 10mmol/L PBS (1mg/mL), and store in a 4°C refrigerator for later use.

All solutions prepared above were prepared from ultrapure water obtained by passing through a Milli-Q water purification system (18.2 MΩ, 25° C.) and filtering through a 0.02 μm filter (filter paper).

A method for detecting hydrogen peroxide based on solid-state nanopores, specifically comprising the following steps:

(1) Preparation of nanopores: nanopores of silicon nitride membranes were deposited on a silicon substrate with a thickness of 300 um to obtain an independent silicon nitride film (nominal thickness of 100 nm). This film is prepared by first depositing a layer of low-stress silicon nitride, then using low-pressure chemical vapor deposition (the deposition rate of the silicon wafer is 5nm/min, the combustion chamber pressure is 4mbar, and the substrate temperature is 810°C) followed by photolithography (The size of the open window of photolithography is 500um×500um), deep reactive ion etching, and tetramethylammonium hydroxide (TMAH) etching to form a 50μm×50μm film. (TMAH is used for silicon etching, and Si3N4 is used as an etching mask for TMAH etching. The etching rate is about 40 μm/h, and the Si/Si3N4 etching selectivity is greater than 1000. Etching Si3N4 (500 μm×500 μm) with DRIE , and then etched with 5% TMAH at 80°C, the silicon wafer is <100>). In the FEI Strata 201 FIB system (FEI Corporation, Hillsboro, or the United States), the surface is bombarded with an accelerating potential of 30 kV and a current value of 1 pA to drill holes on the surface of Ga+ ions. In SPOT mode, the milling time is 1.5S. The resulting nanopores of the Si3N4 thin film chip were imaged by FESEM (Fig. 1). The radius of the small opening (AS) is below FESEM resolution and determined from the current-voltage (I-V) curve (Fig. 1).

(2) Nanopore HRP enzyme functionalization: as shown in Figure 2, a schematic diagram of the experimental steps is given in Figure 2a. Before chemical modification, the Si3N4 chip was washed in piranha solution at 90 °C for 30 min to generate hydroxyl groups on the Si3N4 surface. The hydroxyl group (-OH) generated on the surface of the channel during the chemical reaction is modified on the surface of the enzyme molecule with primary amines by the following steps: First, the entire silicon nitride surface is covered on the Si3N4 surface with 3-APTES (1 %V/V in methanol) for 3h activation (Figure 2b). Subsequently, it was treated with 1,4-phenylene diisothiocyanate (0.5% W/V in DMSO) cross-linker for 5 h, followed by two washes in DMSO, and Two washes were performed in double distilled water. In the next step, 1,4-phenylene diisothiocyanate was covalently coupled to the primary amine groups present in the horseradish peroxidase molecule (HRP, 1 mg/ml) and reacted overnight. Finally, the modified channels are thoroughly washed with buffer solution.

Through controlled experiments, it is proved that the reduction of current is actually due to the change of chemical surface coefficient. First, cover the entire silicon nitride surface on a Si3N4 surface in methanol (1 ml) for 3 h at room temperature. Subsequently, the chips were treated with DMSO (1 ml) for 5 h, followed by two washes in DMSO followed by two washes in ddH2O. Finally, an overnight treatment with buffer solution (1 ml) was performed.

(3) The small molecule hydrogen peroxide (hydrogen peroxide) is detected by using the nanopore after immobilizing HRP as a sensor.

1. Nanopore Characterization:

A single cylindrical nanopore was prepared by a symmetrical method. Wash the nanopores in piranha solution at 90°C for 30 minutes to generate hydroxyl (-OH) groups on the surface of the pores. Whereas for tapered nanopores, at neutral and alkaline pH, and the nanopores in aqueous solution are mainly filled with oppositely charged ions to the immobilized groups on the pore walls, the ionized groups (-OH) towards the pore walls Apply a negative charge. The observed conductance is due to the monopolar solution of positive ions inside the nanopore after each potential difference applied externally. In contrast, cylindrical nanopores do not rectify current due to the lack of intrinsic (geometric and electrostatic) asymmetry. As shown in Figure 3, Figure 3(A) shows the theoretical and experimental I-V curves of a cylindrical pore with -COOH groups on the pore wall, for which I-V curves were measured in a 1mol/L KCl electrolyte solution at pH=7.6 curve. At this time, the electrolyte concentration in the solution is high enough that the -COO- group has almost negligible effect on the fixed charge. Therefore, only the geometry of the nanopores controls the ion flux on monoporous membranes.

2. HRP biofunctionalized nanopores:

Covalent attachment of HRP enzyme molecules to the pore walls was accomplished with the methanol-soluble reagent 3-APTES. The entire film was activated with 3-APTES, which is attached to the hydroxyl groups of the native silicon oxide layer present on the silicon nitride surface. The chip was then treated with 1,4-phenylene diisothiocyanate crosslinker. Finally, the cross-linker is covalently coupled to the primary amine on the surface of the enzyme molecule. The I/V characteristics of the HRP-modified single-porous membrane were measured under symmetric electrolyte conditions with 0.1mol/L KCl solution (Figure 3). Changes in nanopore conductance, measured from the corresponding I-V curves, are the main assays for the detection of biomolecular immobilization and supramolecular bioconjugation on the pore surface. In fact, the immobilization of HRP resulted in a significant decrease in the pore conductance at 1 V: the conductance of unmodified cylindrical pores was 10 ns, compared with 3.33 ns after HRP immobilization. The 66% of the observed drop in ion conductance most likely comes from the reduction in the effective nanopore radius. The nanopore used had a radius of 35.5 nm before HRP functionalization and the same nanopore after HRP functionalization had a radius of 20.4 nm. A reduction in nanopore radius of 15 nm is quite high compared to a protein with a molecular weight of approximately 3 nm x 3.5 nm x 6 nm. Figure 2 gives a reasonable explanation for this reduction in pore clogging: this is mainly because the immobilized enzyme molecule undergoes three steps of molecular assembly, resulting in a reduction in pore size that includes an immobilized HRP molecule, and the assembly silanization and crosslinking agent.

As shown in Fig. 3(A), the measured pH-dependent behavior of the rectification I–V curve of the HRP nanochannel system can provide evidence for the successful immobilization of enzymes on the nanochannel wall. The role of immobilized enzymes in single nanopore channels in volume-constrained redox reactions: immobilized HRP enzymes in the presence of 2,20-azabis(3-ethylbenzothiazole- 6-sulfonate) (ABTS) as the substrate was tested with hydrogen peroxide (H2O2) as the analyte, as shown in Figure 3(B). Figure 3(C) shows the transmembrane currents recorded in the presence of the substrate ABTS (1.5 mmol/L) in the presence of 0.1 mol/L KCl (pH=6.5) before and after the addition of hydrogen peroxide. In the absence of hydrogen peroxide, the I–V curves are smooth, very similar to those recorded only in 0.1 mol/L KCl solution. The addition of 0.5mmol/L hydrogen peroxide caused a significant decrease in the cathode current and also became more unstable. Thus, it is indicated that the current change is the appearance of the cationic product of the redox reaction occurring in the presence of HRP, ABTS and hydrogen peroxide.

The small molecule hydrogen peroxide (hydrogen peroxide) is detected by using the nanopore after immobilizing HRP as a sensor, and the ammonium salt (ABTS) is used as the amplification effect of the small molecule hydrogen peroxide, and the redox product ABTS+ is expected to be generated, and more Directly display the detection results of hydrogen peroxide. The current change records of hydrogen peroxide detected by HRP nanopore in ABTS, 0.1mol/L KCl and only in 0.1mol/L KCl are shown in Fig. 3(D). It can be seen from the figure that when no hydrogen peroxide is added, the I-V curve is basically unchanged from before, as shown in Figure 3(B). However, as shown in Figure 3(C), the current has decreased to a considerable extent, and it is obvious that the I-V curve becomes tortuous and the shape is very different from before, and it is no longer smooth. This is after adding 0.5mmol/L What happens after hydrogen peroxide. At the same time, nanopores with similar size and pore size, but not modified by HRP, were used to detect the current changes of hydrogen peroxide in ABTS, 0.1mol/L KCl and 0.1mol/L KCl, and record them. It is found that the I-V curve does not change substantially, including that the results are the same without adding hydrogen peroxide and adding 0.5mmol/L hydrogen peroxide. The above experiments prove that only HRP, ABTS and hydrogen peroxide are in the nanopore at the same time, which will cause the decrease of the current as shown in the figure and the change of the shape of the I-V curve, thus indicating that the enzyme catalyzes the redox reaction we expect The desired product ABTS·+ is formed.

3. Reproducibility and working curve of enzyme-functionalized nanopore sensors:

The effect of changing the pH of the solution on the rectification characteristics of the modified nanopore is shown in Figure 4(A). It can be seen from the figure that changing the pH from 2 to 9 has no effect on the shape of the I-V curve of the nanopore modified by HRP. is too large, but there are slight changes, indicating that the working stability of the nanopore at different pH is in a qualified state.

There are two more important indicators of the constructed nanopore detection system: reproducibility and sensitivity. The I-V curve of hydrogen peroxide in the presence of 0.1 mol/L KCl, 1.5 mmol/L ABTS and simultaneously in the absence was measured by multiple measurements. This measurement process is realized by changing the type and concentration of the solution in the experimental device sequentially through manual operation each time. Fig. 4(B) is the reproducibility of the ion current of the nanopore detection system under the voltage of 1000mV. It can be found that the current is stable and changes very little. The above results show that the reproducibility of the constructed nanopore sensor is qualified, the nanopore sensor has good reproducibility, and the same nanochannel can be used for multiple sensing experiments. Only one nanopore is needed to complete several actual detections with high quality.

The I-V curves shown in Figure 4(C) are drawn after measuring the HRP nanopore detection system we constructed in the state of 0.1mol/L KCl, adding 1.5mmol/L ABTS and different concentrations of hydrogen peroxide. It can be seen from the figure that when the concentration of hydrogen peroxide gradually increases, the size of the ion current gradually decreases, and the magnitude of the reduced ion current is linearly related to the concentration of hydrogen peroxide, as shown in Figure 4(D). The linear range of hydrogen peroxide is 5-15nmol/L, the obtained standard curve: Y=-183.431X+3580.726, the correlation coefficient R^2=0.986, and the lowest detection limit reaches 10pM.

Claims (9)

1. a kind of method detecting hydrogen peroxide based on solid nano hole, it is characterised in that：Specifically include following steps：

The preparation of step A. nano-pores：Nano-pore is beaten on the ultra-thin silicon nitride film of 10-100nm；

The modification of step B. nano-pores and unimolecule enzyme sensor structure：With a variety of silanization treatments, realize nanoporous surface and The assembling that single horseradish peroxidase is stablized is fixed on nanometer by inner wall differentiation modification by silane coupled molecule Hole hole inner wall obtains nanopore sensor；

Step C. substrate specificity sensing detections：Reaction system is added in various concentration hydrogen peroxide, analyzes current signature, Concentration of hydrogen peroxide standard curve is drawn, the standard curve Real_time quantitative detection hydrogen peroxide is utilized.

2. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 1, it is characterised in that：It is described In step A, using 201 FIB systems of FEI Strata, the ultra-thin silicon nitride film of 10-100nm is bombarded with the accelerating potential of 30kV Nano-pore is beaten on surface.

3. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 1 or 2, it is characterised in that： In the step B, a variety of silanization treatments are one or more in methyl-monosilane, trifluoro or amino silane, carboxy-silane.

4. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 3, it is characterised in that：It is described In step B, using comb-like graft copolymer by modifying in nanoporous surface by the way of covalently or non-covalently, with screen nano hole Inner surface institute is electrically charged.

5. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 1 or 4, it is characterised in that： In the step B, using silane coupled molecule amino silane, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, N-hydroxysuccinimide realizes fixation and the Site selective assembly of nano-pore inner wall peroxidase molecules.

6. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 5, it is characterised in that：It is described In step B, before chemical modification, Si3N4 chips are cleaned into 30min in 90 DEG C of Piranha solution, on the surfaces Si3N4 Upper generation hydroxyl, 3h is activated with 3-APTES in methyl alcohol, is handled in dimethyl sulfoxide (DMSO) with Isosorbide-5-Nitrae-phenylene diisothio-cyanate 5h, washing, after washing primary present in the horseradish peroxidase molecule HRP of Isosorbide-5-Nitrae-phenylene diisothio-cyanate and 1mg/ml Amine groups carry out covalent coupling reaction and the assembling that horseradish peroxidase is stablized are fixed in nano-pore hole wall, are used after 12-24h Buffer solution thoroughly washs modified channel, both obtains nanopore sensor.

7. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 1 or 6, it is characterised in that： In the step C, by the analysis to characteristic current signal, determines the characteristic signal generated by enzymatic reaction, measure substrate Detectable limit.

8. a kind of method detecting hydrogen peroxide based on solid nano hole according to claim 7, it is characterised in that：Nanometer Hydrogen peroxide detection limit and standard curve are detected in hole, and the range of linearity is in 5-15nmol/L, working curve：Y=-183.431X+ 3580.726 coefficient R^{^2}=0.986, detection limit reaches 10pmol/L.

9. modifying unimolecule horseradish peroxidating in a kind of nano-pore obtained by any one of claim 1,2,4,6,8 the method The nanopore sensor of object enzyme.