CN115078460B - Hydrogen peroxide detection reagent based on solid-state nanopore sensor and quantitative detection method thereof - Google Patents

Hydrogen peroxide detection reagent based on solid-state nanopore sensor and quantitative detection method thereof Download PDF

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CN115078460B
CN115078460B CN202210163804.8A CN202210163804A CN115078460B CN 115078460 B CN115078460 B CN 115078460B CN 202210163804 A CN202210163804 A CN 202210163804A CN 115078460 B CN115078460 B CN 115078460B
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武灵芝
王润雨
曾祥杰
翁丽星
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Nanjing University of Posts and Telecommunications
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Abstract

The invention providesHydrogen peroxide detection reagent based on solid-state nanopore sensor and quantitative detection method thereof are provided, and H is detected based on gold nanorod etching method mediated by enzyme catalytic reaction of solid-state nanopore 2 O 2 In particular, the use of peroxidases to catalyze H 2 O 2 The hydroxyl radical with stronger oxidability is generated by reaction to etch the gold nanorod, the product generates via hole signals with different characteristics when passing through the solid nano holes, and H based on the solid nano holes is realized by detecting the nano hole signals in the whole etching process 2 O 2 H is detected with high sensitivity, especially under low concentration and low sample condition 2 O 2 Belongs to the field of nanopore sensor detection.

Description

Hydrogen peroxide detection reagent based on solid-state nanopore sensor and quantitative detection method thereof
Technical Field
The invention provides a hydrogen peroxide detection reagent based on a solid-state nanopore sensor and a quantitative detection method thereof, which comprises the following steps of 2 O 2 The enzymatic reaction mediated change of the shape of the nano particles is combined with the nano pore sensing technology, and belongs to the detection field of nano pore sensors.
Background
H 2 O 2 The method has wide application in the fields of chemical industry, biology, pharmacy, clinic, environment and food processing, and has great significance for the sustainable development of industry and agriculture and the environmental safety in content and analysis and identification. Especially in the biomedical field, H 2 O 2 Is an important biochemical molecule, and is involved in cell proliferation, differentiation and migration and a signal pathway related to occurrence of diseases. Intracellular H under normal physiological conditions 2 O 2 Is very low (10) -8 ～10 -6 M) are involved in maintaining various functions such as intracellular signal transduction (Nat command, 2019,10,4526). Upon abnormality, H at too high a concentration 2 O 2 Can induce various biological damage, and cause aging, cancer and central nervous system diseases, such as H in cancer cells 2 O 2 Is about 1-2 orders of magnitude higher than normal cells, and thus is more sensitive to H in physiological environments 2 O 2 The accurate and sensitive detection has important significance in the biomedical field.
Different current technology is applied to H 2 O 2 The detection method of (2) has been developed, and mainly comprises a colorimetry method, a fluorescence method, a high performance liquid chromatography method, an electrochemical method and the like, wherein the colorimetry method and the electrochemical method are most widely used. However, colorimetric methods have the problems of large sample size and low sensitivity, while electrochemical methods involve the limitations of strict enzyme immobilization, strict carrier electrode materials, poor stability and the like, and are difficult to meet the requirements of the current environmental safety, food sanitation, especially H existing in physiological environments 2 O 2 Low cost, high throughput, and high throughput. Therefore, the invention develops a simple operation and high-sensitivity H based on solid-state nanopore sensing 2 O 2 Is provided.
Disclosure of Invention
The solid nano-pore has the advantages of no marking, high sensitivity and low cost, and has important application prospect in the field of single molecule sensing. In nanopore sensing detection, a single molecule passes through a nanoscale pore canal to cause instantaneous change of conductivity in the pore, so that detectable ion current change is formed, and single molecule level detection is realized by analyzing characteristic information such as amplitude, time and the like of an ion current signal. Different from the traditional method, the method is used for detecting the average behaviors of a large number of molecular measurement ensembles in a solution environment, the high resolution of the nano-pores can reveal more abundant information such as single molecules, dynamic changes of single particles, molecular fluctuation in a local environment and the like, and meanwhile, the method does not need marking, has simple device, is easy to operate and low in sample size, and provides a high-efficiency single-molecule sensing platform.
Based on the nanopore sensing single-molecule detection platform, in a first aspect, the invention provides a hydrogen peroxide detection reagent based on a solid-state nanopore sensor, wherein the reagent comprises: nano-or bio-enzymes with peroxidase-like activity, noble metal nanoparticles, cetyltrimethylammonium bromide CTAB and buffer;
wherein the biological enzyme with peroxidase-like activity is preferably horseradish peroxidase HRP; further preferably, the concentration of HRP is 1.0-5.0. Mu.M, and the optimal enzyme activity concentration is 2. Mu.M, so that the horse radish peroxidase has high enzyme activity and saves cost.
The nano enzyme with peroxidase-like activity comprises nano enzymes such as peroxidase-like enzyme, oxidase-like enzyme, catalase-like enzyme, superoxide dismutase-like nano enzyme and the like and derivatives thereof, and can be used for carrying out redox decomposition on H2O 2. Such as Fe 3 O 4 Nanoenzyme (ACS Biomaterials Science)&Engineering,2021,7 (1): 299-310), platinum nanoenzyme (ACS Nano,2021,15 (3): 5735-5751) and other similar metal oxide nanomaterials and derivatives thereof (analysis, 2021,146 (23): 7284-7293); biomacromolecules, 2021,11 (7): 1015).
Preferably, CTAB is used in a range of 1-5mM, the buffer is either a citrate buffer (0.05M, pH 3.0-5.0) or a phosphate buffer (0.01M PBS, pH 3-5.0), with a citrate buffer (0.05M, pH 4.0) being preferred, along with CTAB 2mM.
Preferably, the noble metal nanoparticle is any one or a combination of two of silver nanoparticle or gold nanoparticle, and further preferably gold nanoparticle.
Furthermore, the gold nanoparticles are preferably rod-shaped gold nanoparticles, namely gold nanorods, compared with gold nanoparticles with spherical or cubic structures, the rod-shaped gold nanoparticles have the advantages of high length-diameter ratio, obvious structural anisotropy and high signal to noise ratio of a via hole signal, and the aperture of the solid-state nano hole is 20-200nm, and the thickness is 20-100nm.
Further preferably, in order to ensure significant etching effect and high signal-to-noise ratio of the nanopore via signal and significant signal characteristic variation difference generated before and after particle etching, the optimal aspect ratio of the rod-like gold nanoparticle is about 3:1, diameter 16+ -2 nm, length 50+ -3 nm.
Preferably, the concentration range of the gold nanorods in the noble metal nanoparticle solution is 0.01-1nM, the gold nanorods with the concentration have high enough capture rate in the nanopore, the capture rate is more than or equal to 1000 per minute, the enzymatic reaction is facilitated to be rapidly carried out, and the cost is saved.
Preferably, in order to improve the enzymatic reaction efficiency, the molar concentration ratio of the noble metal nano-particles to the horseradish peroxidase is more than or equal to 1000, and the preferred molar concentration ratio is 10000.
Preferably, the horseradish peroxidase catalyzes H 2 O 2 The reaction time for generating hydroxyl free radicals to etch the gold nanorods is controlled to be about 20 minutes, so that the etching can generate a remarkable effect, and the depletion of etching products caused by overlong time is avoided.
Preferably, when the diameter of the gold nanorod is about 16nm and the length is about 50nm, the optimal diameter of the selected solid nano-pore is 60-80nm, and the thickness is 50nm, the high signal to noise ratio can be obtained in the nano-pore experiment, the capturing rate is improved, and the occurrence of the particle pore blocking phenomenon is reduced.
In a second aspect, the present invention provides a method for drawing a hydrogen peroxide quantitative detection standard curve based on a solid-state nanopore sensor, the method comprising: configuring H with different concentration gradients 2 O 2 Solutions containing H at zero concentration 2 O 2 Solution, respectively adding the detection reagents to hatch, detecting the fully reacted mixed solution by using a nanopore sensor after terminating the reaction, injecting the solution into one side of a fluid device, driving etched noble metal nanoparticles in the solution to pass through nanopores by using voltage, and counting the generated noble metal nanoparticle nanopore ion current signals to obtain peak position change values (delta Ip) and H of ion current pulse amplitude values 2 O 2 Linear relationship between concentrations, and plotting a standard curve.
Preferably, the length of the rod-like gold nanoparticles is 30-70 nm; the diameter is 15-20 nm; aspect ratio is 2: 1-4:1, the regulation of the length-diameter ratio depends on the proportion of the silver nitrate solution to the seed solution.
In a third aspect, the invention provides a method for quantitatively detecting hydrogen peroxide based on a solid-state nanopore sensor, comprising the following steps: adding a sample to be detected into the prepared detection reagent to perform enzymatic reaction, adding the reacted gold nanoparticle product into a solid-state nanopore sensor to detect, generating an ion current signal by a particle via under the drive of voltage, counting the peak position variation value (delta Ip) of the ion current amplitude, taking the peak position variation value into the standard curve, and determining a corresponding sampleH in the product 2 O 2 Concentration value, finally realize H in the sample 2 O 2 And (5) detecting concentration.
The beneficial effects are that:
as shown in figure 1, the hydrogen peroxide detection reagent based on the solid-state nanopore sensor and the quantitative detection method thereof convert a detection object into the change of a nanomaterial structure by using an etching technology, and skillfully utilize the characteristics of high sensitivity and high flux of the nanopore; the solid-state pore has controllable size, stable property and strong selectivity, is suitable for various detection environments (acid and alkali and concentration), greatly expands the application range of the nanopore, and enables the nanopore sensor to be a high-flux and low-cost detection platform;
pure H 2 O 2 The etching of gold nanoparticles requires very high concentrations and high temperatures and acidic environments, and the present invention utilizes peroxidase to react H 2 O 2 Has the advantages of high catalytic activity, can quickly reduce the catalyst into hydroxyl free radical (OH), and has higher oxidability, and the hydroxyl free radical can accelerate H 2 O 2 The speed of etching noble metal particles, thereby realizing the utilization of H under the conditions of ultra-low concentration and low sample size 2 O 2 And the noble metal particles are required to be etched efficiently.
Aiming at the problems of low resolution and the like of other detection methods, the hydrogen peroxide quantitative detection method utilizes a silicon nitride nano-pore sensing platform, utilizes noble metal particles such as gold nano-rods and the like as reaction carriers, and catalyzes H through peroxidase 2 O 2 Generates hydroxyl radical with higher oxidability, and H is 2 O 2 The concentration is converted into the difference of morphology structures in the process of nano material etching, so that H is realized 2 O 2 Is high in sensitivity;
the invention also solves the problems of large sample size, easy interference of color change, activity reduction caused by enzyme immobilization, high requirement on electrode materials, unstable performance and the like of the prior art methods such as a colorimetry method, an electrochemical method and the like, and enlarges the application range of the solid nano-pore to H 2 O 2 And the detection application range of small molecules.
Drawings
FIG. 1 shows the detection of H by the enzyme-catalyzed reaction-mediated gold nanorod etching method based on solid-state nanopore sensing according to the present invention 2 O 2 Is a schematic diagram of the principle of (a);
FIG. 2a is an ultraviolet and TEM image of gold nanorods with different aspect ratios prepared in example 1 of the present invention;
FIG. 2b is a graph of different characteristic events of electrical signals of rod-like gold nanoparticles with different aspect ratios in a nanopore;
FIG. 3 is a graph showing the comparison of characteristic events of electrical signals of the gold nanorods and the gold spheres, jin Lifang prepared in example 1 of the present invention;
FIG. 4 is an electron microscope image of nanoparticle products with different morphologies obtained by performing an enzymatic gold nanorod etching reaction in example 2 of the present invention, and an electrical signal image of characteristic events of the product passing through the nanopore;
FIG. 5 is a graph showing the time dynamics of the etching reaction of gold nanorods in example 2 of the present invention;
FIG. 6 is a graph showing the effect of different concentrations of HRP enzyme activity on the etching reaction in example 2 of the present invention;
FIG. 7 shows H concentrations in example 3 of the present invention 2 O 2 A three-dimensional statistical graph of an electric signal generated in the nanopore by a product after enzyme-catalyzed gold nanorod AuNRs etching;
FIG. 8 is a diagram of H in example 3 of the present invention 2 O 2 A concentration quantitative analysis curve;
FIG. 9 is a histogram of electrical signals for different numbers of cells in example 4 of the present invention.
Detailed description of the preferred embodiments
The technical scheme of the invention is further described below with reference to the specification, the drawings and the specific embodiments.
Example 1: preparation of gold nanorods
First, preparing a gold seed solution: 10mL of a 0.1M CTAB solution and 0.25mL of 0.01M HAuCl were taken 4 Mixing, and adding magnetons; then 0.6mL of 0.01M ice water freshly prepared NaBH was added 4 The solution is stirred vigorously for 2min while being added, and then is put into a water bath kettle at 30 ℃ for reaction for 2h;
then preparing a growth solution: 40mL of a 0.1M CTAB solution was taken, 2mL of 0.01M HAuCl 4 Solution and 350. Mu.L of 0.01M AgNO 3 The solutions were mixed and stirred with the addition of a magnet. Adding 0.76mL of 1M HCl solution to adjust the pH value to about 2; 0.32mL of a 0.1M solution of Ascorbic Acid (AA) was added and the solution was stirred until it became colorless. Adding 250 mu L of the prepared gold seed solution, stirring, uniformly mixing, and standing in a water bath kettle at 30 ℃ for growing for 8 hours;
and finally, centrifuging the gold nanorod AuNRs solution after 8h growth for 30min at 8000rpm by using a 50mL centrifuge tube, sucking the supernatant to retain precipitate, adding ultrapure water for re-dissolution, repeating for three times, adding 44mL of ultrapure water into the centrifuged AuNRs solution, and sealing and preserving by using a wide-mouth bottle.
As shown in fig. 2a, the prepared rod-like gold nanoparticles have a length of 30-70 nm; the diameter is 15-17 nm; aspect ratio is 2: 1-4:1, the regulation and control of the length-diameter ratio of the gold nanorods depends on the ratio of the silver nitrate solution to the seed solution, the amount of 250 mu L of gold seed solution is controlled to be unchanged, and when the addition amount of 0.01M silver nitrate solution is changed to be 230 mu L, 350 mu L and 470 mu L respectively, the length-diameter ratio is 2:1 (length about 31nm, diameter about 16 nm), 3:1 (length about 48nm, diameter about 16 nm), 4:1 (length about 63nm, diameter about 16 nm), wherein AR in FIG. 2a represents an aspect ratio. Fig. 2b shows different characteristic event diagrams of electrical signals of rod-shaped gold nanoparticles with different length-diameter ratios in a nanopore.
The gold nanorods are similar to a DNA linear structure and have higher resolution in the nanopores, as shown in figure 3, and the different characteristic event patterns of the electrical signals of the gold nanorods, the gold spheres and the Jin Lifang nanoparticles in the nanopores, which are prepared by adopting the method and have the length-diameter ratio of about 3:1, are adopted. The graph (a) in fig. 3 is a nanopore signal characteristic event graph of a gold nanorod, which has high amplitude and long residence time, and the graph (b) in fig. 3 is a nanopore signal characteristic event graph of a gold nanosphere prepared in a laboratory, which has smaller amplitude and short residence time, so that compared with spherical gold nanoparticles, the gold nanorod has higher signal to noise ratio in a nanopore, and when the gold nanorod is etched into a sphere, obvious signal difference is generated, and the resolution is high. FIG. 3 (c) is a graph of the signal characteristics of a laboratory prepared Jin Lifang nanopore, which also has a high signal to noise ratio, but because Jin Lifang is large in volume (. Gtoreq.50 nm) and isotropic, it is time consuming and expensive to etch, and therefore gold nanorods are preferred as the reaction carrier in the present invention.
The specific preparation process of the gold nanoparticles is as follows:
heating 50mL of 0.01% chloroauric acid solution to boil, rapidly adding 5mL of 40 mM sodium citrate solution, stirring for 10min under boiling state, turning off the heat source, stirring for 15 min, and cooling to room temperature to obtain seed solution; 2mL of seed solution and 0.5mL of hydroxylamine hydrochloride solution are added into 20mL of ultrapure water, the mixture is uniformly mixed at room temperature, 0.1% chloroauric acid solution is added dropwise, the color of the growth solution gradually deepens along with the dropwise addition of the chloroauric acid solution, and the absorption peak of the nano gold growth solution appears at the wavelength of about 520nm and is about 20nm.
The specific preparation process of the Jin Lifang nano-particles is as follows:
firstly, placing prepared 0.01M NaBH4 (0.0189 g dissolved in a 50mL volumetric flask) in an ice water domain for standby; the reaction flask was placed in a 32℃water bath, and then magneton, HAuCl4, (0.125 mL, 0.01M) CTAB (3.75 mL, 0.1M) was added, followed by stirring, and finally NaBH4 (0.3 mL, 0.1M) was added as a reducing agent. Stirring for three minutes, standing for 2 hours, wherein the reaction solution is brown, and the experiment is completed in a water bath (32 ℃); the reaction flask was placed in a 32℃water bath, and magneton, ultrapure water (8 mL), CTAB (1.6 mL, 0.1M), HAuCl4 (0.2 mL, 0.01M), AA (0.95 mL, 0.1M) were added sequentially; 10. Mu.L of seed solution was taken in 240. Mu.L of water (diluted 25-fold), 10. Mu.L of the solution was added to the growth solution, and the solution was allowed to stand overnight, the solution color being pink. Finally, the synthesized AuNC particle solution is subjected to centrifugal purification, and is centrifuged twice under the conditions of 4000 turns and 15 minutes, and finally redissolved in ultrapure water for about 50nm.
Example 2: enzyme-catalyzed gold nanorod etching reaction
Gold nanorods were prepared according to example 1, which had a length of 50nm, a diameter of 16nm, and an aspect ratio of about 3:1, then adding enzyme and hydrogen peroxide solution with different concentrations to etch the gold rod. Firstly, taking prepared gold nanorods AuNRs10 ul (2.0 nM), adding 10ul HRP solution (20 uM) and 10ul CTAB (0.02M), and adding 0ul,1ul and 6ul of H respectively 2 O 2 The solution (1M/L) was then added 70-60ul of citric acid buffer (0.05M, pH 4.0) to give 0uM,10uM,60uM H 2 O 2 The enzymatic reaction system was incubated at 25℃for 20min and finally quenched by the addition of 10uL of 5M HCl.
And (4) carrying out electron microscope observation on the reaction product and simultaneously carrying out a nano hole through hole experiment to obtain the result of fig. 4. With H 2 O 2 The increase in concentration gradually shortens the length of AuNRs while the diameter remains almost unchanged, see (a) and (b) graphs in fig. 4. At a concentration of 60uM, auNRs has been etched into a sphere in a certain period of time, see (c) diagram in fig. 4. Meanwhile, according to the characteristic event signals of the nanopores of the product, the smaller the ionic current amplitude of the product etched by the gold nanorods through the solid nanopores is along with the progress of enzyme catalytic reaction, the phenomenon completely accords with the morphology change of AuNRs in the etching process, the diameter of the AuNRs keeps unchanged along with the progress of etching, the length of the AuNRs is shorter and shorter, the size of the via hole particles is reduced, meanwhile, the charges carried by the particles are reduced, and the ionic current blocking caused by the particles passing through the solid nanopores is further reduced.
In order to improve the reaction efficiency, the time of the enzymatic reaction is optimized, as shown in fig. 5, which is a graph of the peak position variation of the characteristic signal statistics of the etched product of the 60nm gold nanorod in the nanopore at different times (0, 10min, 20min and 30 min), it can be seen that as the etching proceeds, the time required for the gold nanoparticles to pass through the solid-state nanopore is shorter and shorter, and the time of the reaction is obviously different at 20min, so that in order to improve the reaction efficiency, and meanwhile, the overetching is prevented, and 20min is taken as the reaction termination time of the enzymatic catalysis.
At the same time, the HRP concentration is optimized for saving cost, and the HRP concentration contains H 2 O 2 Adding HRP with different concentrations into (60 mu M) gold nanorod etching solution for enzyme catalysis, and adding the catalyzed product into a nanopore sensor for signal detection. As shown in FIG. 6, when HRP concentration reached 2. Mu.M, nanopore productionThe maximum change in the current peak generated indicates that 2. Mu.M HRP vs. H 2 O 2 The catalytic effect of etching is optimal, and the presence of excessive HRP can instead inhibit HRP activity, reducing reaction efficiency.
Example 3: drawing H 2 O 2 Standard curve for detection
Preparation of H at different concentrations 2 O 2 Is a gold nanorod etching reaction system. Taking gold nanorods with AuNRs final concentration of 0.2nM and length-diameter ratio of about 3:1, which has a length of 50nm and a diameter of 16nm. Adding HRP solution (final concentration of 2 uM) and CTAB (final concentration of 0.01M), adding different volumes of 1M/L H 2 O 2 Solutions were brought to final concentrations of 0. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M and 100. Mu.MH 2 O 2 And the reaction system was brought to 100ul with additional citrate buffer (0.05M, pH 4.0), incubated at 25℃for 20min, and finally quenched by the addition of 10uL of 5M HCl.
Taking 60nm silicon nitride nano-pores to perform nano-pore sensing experiments, and carrying out H with different concentrations 2 O 2 And carrying out a nanopore via experiment on the gold nanoparticle product after the enzymatic reaction. Under the drive of voltage, gold nanorods and etched products pass through the nanopore channel to cause a series of current pulse signals, the amplitude change of the electrical signals detected by the nanopores is subjected to histogram statistical analysis, the relative current peak change is obtained by fitting, and the corresponding H can be obtained by bringing hydrogen peroxide concentration into 2 O 2 A standard curve.
As shown in FIG. 7 as H 2 O 2 When the solution concentrations are respectively 0, 2 mu M,5 mu M,10 mu M,20 mu M,40 mu M,60 mu M,80 mu M and 100 mu M, the enzyme catalytic etching reaction is carried out to obtain a three-dimensional statistical graph of the electric signals detected by the gold nanorod products in the nanopores, the x-axis is the amplitude variation value of the current blocking signals, the y-axis is the hydrogen peroxide concentration, and the z-axis is the proportion of characteristic events, so that the amplitude peak position of the current signals of the products is moved and gradually becomes smaller along with the increase of the hydrogen peroxide concentration. The variation value (delta Ip) and H of the peak position of the ion current amplitude are obtained through fitting 2 O 2 Linear relationship between concentrations. As shown in fig. 8, with H 2 O 2 The concentration increases and the peak change value (delta Ip) of the nanopore electrical signal is linearly related to the concentration, and the function relation is y=0.01047+0.00141 x (R) 2 =0.996), calculated as signal-to-noise ratio S/n=3, the corresponding detection limit for this scheme is 31nM.
Example 4: h in sample to be measured 2 O 2 Quantitative detection of (2)
Hela cell culture medium incubated with DMEM medium containing 10% bovine serum and 1% penicillin-streptomycin was used, and cells were incubated in an incubator at 37 ℃ containing 5% co2/95% air for a growth cycle of approximately 36h. Collecting cells in the cell index growth period, sucking out the original culture solution in a culture bottle, repeatedly cleaning the culture bottle by using 3mL of 1 XPBS solution, sucking out PBS after cleaning, adding 2mL of trypsin solution for digestion, waiting for 3min to fall off from the wall of the culture bottle, immediately adding 4mL of DMEM culture solution for stopping digestion, then sucking out the cell solution into a centrifuge tube, centrifuging for 3min by using a centrifuge at the rotating speed of 750rpm, finally taking out the centrifuge tube, removing the supernatant, reserving the cell sediment below, adding 1mL of culture solution for redissolution, counting about 100 ten thousand/mL by using a counting plate, and vibrating uniformly. Then, the mixture was sonicated (20% power, sonicated for 3s at intervals of 10s and repeated 30 times), and the supernatant was centrifuged (8000 g, 10min at 4 ℃) and placed on ice to be tested.
Samples to be tested (HeLa cell suspension) were taken separately, gold nanorods (0.2 nM), HRP (2 uM) and CTAB (0.01M) were added, and citric acid buffer (0.05M, pH 4.0) was added, mixed well and incubated at 25℃for 20min, and finally 10uL 5M HCl was added to terminate the reaction.
Adding the reaction product into a solid state nanopore sensor, wherein the diameter of a nanopore is 60nm and the thickness of the nanopore is 50nm, analyzing nanoparticle via hole signals by applying voltage of 600mV, counting the amplitude and time information of characteristic events of the generated electric signals, fitting the amplitude of the events, bringing the variation value of the peak position into a standard curve y=0.01047+0.00141 x (R2=0.996), and determining H in the corresponding sample 2 O 2 Concentration of H in the sample is finally realized 2 O 2 And (5) detecting concentration. As shown in FIG. 9, the cell count and testRelationship between measured concentrations. When compared to no cells, the number was 10 6 Δip≡ 0.1048, about 0.1. Mu.M, when the number is 10 7 At this point Δip≡ 0.1294, about 1.7uM.
In conclusion, all test results show that the silicon nitride nano-pore sensor designed by the invention can realize the measurement of H 2 O 2 The quantitative test of the method solves the problems of low sensitivity, poor stability and the like in the traditional method, combines the nano-pore monomolecular sensing and the enzyme catalysis etching reaction, realizes the high resolution of the nano-pore to the appearance change of the gold nanorods, and realizes the solid nano-pore to H 2 O 2 The quantitative analysis of small molecules is simple, and the method is suitable for various environmental reactions, in particular H 2 O 2 Low sample, high sensitivity quantitative analysis.

Claims (1)

1. The hydrogen peroxide quantitative detection method based on the solid-state nanopore sensor is characterized by comprising the following steps of:
adding a sample to be detected into a detection reagent to perform enzymatic reaction, adding the reacted gold nanorods into a solid-state nanopore sensor to perform detection, generating an ion current signal through the gold nanorod via holes under voltage drive, counting the peak position change value delta Ip of the ion current amplitude, introducing into a hydrogen peroxide quantitative detection standard curve based on the solid-state nanopore sensor, and determining H in the corresponding sample 2 O 2 Concentration value, finally realize H in the sample 2 O 2 Detecting the concentration; wherein the detection reagent comprises: 0.2nM gold nanorod solution, 2. Mu.M HRP and 0.01M CTAB,0.05M citric acid buffer pH 4.0;
the step of adding a sample to be tested into a detection reagent for enzymatic reaction comprises the following steps: taking a sample to be detected, adding 0.2nM gold nanorod solution, 2 mu M HRP and 0.01M CTAB, adding 0.05M citric acid buffer solution with pH of 4.0, uniformly mixing, incubating for 20min at 25 ℃, and finally adding 10 mu L5M HCl to terminate the reaction;
the diameter of the gold nanorod is 16+/-2 nM, the length of the gold nanorod is 50+/-3 nM, the concentration of the gold nanorod is 0.2nM, the aperture of a nanopore in the solid-state nanopore sensor is 60nM, and the thickness of the gold nanorod is 50nM;
the method for drawing the hydrogen peroxide quantitative detection standard curve based on the solid-state nanopore sensor comprises the following steps: configuring H with different concentration gradients 2 O 2 Solutions containing H at zero concentration 2 O 2 The solution is respectively added with the detection reagent for incubation, a solid nano-pore sensor is used for detecting the mixed solution after the reaction is stopped, the mixed solution is injected into one side of a fluid device during detection, the etched gold nano-rods in the solution are driven by voltage to pass through nano-pores, and the generated gold nano-rod through-pore ion current signals are counted to obtain peak position change values delta Ip and H of ion current amplitude values 2 O 2 Linear relationship between concentrations, and plotting a standard curve.
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