CN114295819A - Label-free high-flux quantitative detection method for glucose - Google Patents
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Abstract
The application discloses a label-free high-flux glucose quantitative detection method, and belongs to the technical field of nanopore sensing. According to the invention, the glucose concentration is detected by the solid-state nanopore sensor by utilizing the catalytic reaction of glucose oxidase on glucose, the generated product etches the nanoparticles under the assistance of enzyme, so that the morphology of the nanoparticles is changed, and the nanoparticles before and after etching are translocated in the solid-state nanopore to generate differential electric signals. The invention overcomes the limit that the sizes of the nano-pores and detection molecules are close in the solid nano-pore sensing technology, realizes the high-sensitivity detection of glucose and other small molecules through the change of the shapes of the nano-particles, and enlarges the detection range of the nano-pore field for glucose and other small molecules.
Description
Technical Field
The invention belongs to the technical field of nanopore sensor detection, and particularly relates to a label-free high-flux glucose quantitative detection method.
Background
Glucose is an important energy substance of a human body, and the glucose in blood is called blood sugar, so that the dynamic balance and stability of blood sugar concentration under the regulation and control of nerves and hormones in the body are important for the energy supply of the human body. Therefore, glucose detection and analysis have been the focus of attention. In the last decades, people develop various technical means such as electrochemistry, spectroscopy and the like to detect glucose, the electrochemical detection method is divided into enzyme-based and enzyme-free electrochemical sensing detection, the electrode modified by enzyme has poor stability, and glucose oxidase is easy to denature in the electrode structure, storage and use processes, so that the stability and the high efficiency of the enzyme are difficult to meet, and the enzyme is difficult to fall off and inactivate; the specificity of the enzyme-free electrochemistry is poor, and corresponding current can be detected by the large amount of ascorbic acid and uric acid in the sample; the spectrum detection method has the problems of complex sample pretreatment and difficulty in accurate distinguishing at low concentration, and the practicability is limited. In conclusion, the existing solutions cannot stably and accurately detect the glucose concentration, and therefore, there is a need to continuously find a stable, fast, efficient, reliable and economical detection and analysis method for realizing highly sensitive detection of the glucose concentration.
The solid-state nanopore sensor is used as a new single-molecule detection tool, is simple and rapid, does not need to be marked, has low cost and high flux, and is an ideal high-sensitivity biomolecule detection platform. By means of a solid-state nanopore platform, hydrogen peroxide is generated by using glucose and glucose oxidase, the morphology of the gold nanoparticles is regulated and controlled by the hydrogen peroxide and bromide ions under the catalytic action of horseradish peroxidase, and the etched nanoparticle product can be subjected to statistical analysis through signals of the nanopore to realize the quantitative detection of the glucose. The method does not need to pretreat the sample, has extremely high specificity and sensitivity, wide detection range and strong stability, and is suitable for different body fluid environments.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects in the prior art, the application provides a label-free high-flux glucose quantitative detection method, and aims to solve the technical problems that other detection methods in the prior art are low in resolution, unstable in fluorescent agent, unobvious in color difference and difficult to distinguish by naked eyes, detection results of different detection instruments are different, and the like.
The technical scheme is as follows:
a label-free high-flux quantitative detection method for glucose uses glucose as a reaction main body, glucose oxidase is used for carrying out catalytic reaction on the glucose to generate hydrogen peroxide, the hydrogen peroxide enables gold simple substances to become gold ions under the catalytic action of horseradish peroxidase and sodium bromide, the shape of gold nanoparticles is regulated, and the gold nanoparticles with different sizes and shapes are finally obtained through reaction and are subjected to signal detection through a nanopore sensor; the detection of glucose micromolecules is converted into translocation signals of the nano particles in the nano holes, the size of the electric signals is in direct proportion to the appearance of the nano particles, and the higher signal-to-noise ratio is realized by regulating and controlling the size of the gold nano particles, so that the high-sensitivity detection of the glucose concentration is realized.
As a preferred technical scheme of the application: the gold nanoparticles are carriers, and the components include but are not limited to metals, alloys and materials which react with glucose mediated reaction to change the shape and/or size; the gold nanoparticles are prepared by a chemical or physical method, and the shapes of the gold nanoparticles comprise a two-dimensional or three-dimensional structure, a polygonal sheet with high specific surface area and a polyhedron; the size of the gold nanoparticles is 5-100 nm.
As a preferred technical scheme of the application: the size of the gold nanoparticles is 40-100 nm; when the gold nanoparticles are gold nanopyramids (AuTNs), the size is 50 +/-3 nm.
As a preferred technical scheme of the application: the diameter of the solid-state nano-pores is 60-600 nm, and the thickness of the solid-state nano-pores is 20-100 nm.
As a preferred technical scheme of the application: the diameter of the solid-state nano-pores is 100-160 nm; the thickness is 50-100 nm; when the gold nanoparticles are AuTNs, the diameter of the solid nanopore is 120 +/-5 nm, and the thickness of the solid nanopore is 50 nm.
As a preferred technical scheme of the application: the catalytic reaction temperature of the glucose oxidase on glucose is 37 ℃, and the catalytic reaction temperature of horseradish peroxidase and sodium bromide is 25 ℃.
As a preferred technical scheme of the application: the detection method comprises the following specific steps:
step a, synthesizing gold nano triangular plates: adding 42.5 microliter of 1% chloroauric acid into 4.7 mL of 0.1M hexadecyl trimethyl ammonium chloride, adding magnetons, and stirring; adding 300 mu L of 0.01M sodium borohydride solution, stirring for 2 minutes, standing in a water bath kettle at 25 ℃ for 2 hours to react to form 5-7 nm seed solution, taking 1.6 mL of 0.1M hexadecyltrimethylammonium chloride solution, adding into 8 mL of deionized water, and adding 68 mu L of 1% chloroauric acid and 15 mu L of 0.01M potassium iodide solution to prepare growth solution 1; adding 170 mu L of 1% chloroauric acid into 8 mL of 0.05M hexadecyl trimethyl ammonium chloride solution, and adding 60 mu L of 0.01M potassium iodide solution to prepare growth solution 2; diluting the seed solution 10 times with 0.1M cetyltrimethylammonium chloride; adding 40 μ L of 0.1M ascorbic acid into growth liquid 1, adding 80 μ L of 0.1M ascorbic acid into growth liquid 2, and stirring growth liquids 1, 2 to colorless; adding 110 mu L of diluted seed solution into the growth solution 1 added with the perascorbic acid, shaking up, immediately adding 640 mu L of diluted seed solution into the growth solution 2 added with the perascorbic acid, shaking up the mixed solution for 1 minute, and standing and reacting at room temperature for 1 hour to obtain an unpurified gold nanoparticle triangular plate solution; subpackaging the solution of the unpurified gold nanoplatelets (1 ml for each centrifuge tube), adding hexadecyltrimethylammonium chloride (0.78M, 345 mu L) into each centrifuge tube until the final concentration is 0.2M, shaking uniformly, mixing, standing overnight for 16 hours, absorbing the red liquid on the upper layer by using a liquid transfer gun, adding 400 mu L of water and 50 mu L of hexadecyltrimethylammonium chloride (0.1M) into the sediment at the bottom, shaking uniformly and mixing to obtain the purified gold nanoplatelets;
step b, reaction condition optimization: diluting 10 mg/mL glucose oxidase and 10 mM glucose solution with 0.01M PBS (pH 7.4) to obtain glucose solutions (0, 10, 100, 1000, 2000 mu M) with different concentrations, simultaneously diluting to obtain 0.5-5 mg/mL glucose oxidase solution, incubating the diluted glucose solution and glucose oxidase solution in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, adding 110 mM horseradish peroxidase (HRP), 0.1M sodium bromide (NaBr) and 1.5 nM AuTNs solution, adding 0.01M PBS (pH 4), allowing the final solution to reach 500 mu L, allowing the final concentration of HRP in the solution to be 1-3 mM, the final concentration of sodium bromide in the solution to be 5.5-7 mM, and the final concentration of AuTNs in the solution to be 0.5-1.5 nM, incubating for 15 minutes at 25 ℃, and then completing the reaction, observing the shape change of AuTNs through a transmission electron microscope;
step c, drawing a standard curve: preparing glucose reaction systems (0 mu M, 5 mu M, 10 mu M, 20 mu M, 50 mu M, 100 mu M, 200 mu M, 1000 mu M, 2000 mu M, 5 mM and 10 mM) with different concentrations, respectively adding 0.5-5 mg/ml glucose oxidase solution, incubating in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, then adding 110 mM HRP, 0.1M NaBr and 1.5 nM AuTNs solution, adding 0.01M PBS (pH 4), wherein the final solution reaches 500 mu L, the final concentration of HRP in the solution is 1-3 mM, the final concentration of sodium bromide in the solution is 5.5-7 mM, the final concentration of AuTNs in the solution is 0.5-1.5 nM, after incubating for 15 minutes at 25 ℃, completing the reaction, finally obtaining gold nanoparticle products with different morphologies, and detecting nanopore signals of the final products; performing characteristic extraction on electric signals of glucose reaction system products with different concentrations through a nanopore translocation experiment, performing histogram statistical chart on the extracted relative current signals, fitting to obtain a peak diagram, and drawing a standard curve according to the change of the peak position value under different concentrations;
d, quantitative detection of glucose: incubating a sample to be tested with 0.5-5 mg/ml glucose oxidase at 37 ℃ for 10 minutes, then adding 110 mM HRP, 0.1M NaBr, 1.5 nM AuTNs solution and 0.01M PBS (pH 4) solution, wherein the total solution is 500 muL, the final concentration of the HRP in the solution is 1-3 mM, the final concentration of sodium bromide in the solution is 5.5-7 mM, and the final concentration of the AuTNs in the solution is 0.5-1.5 nM, incubating for 15 minutes at room temperature, adding the solution into a nanopore detection device, and obtaining a series of electric signals under voltage driving; the residence time and the relative current of the electric signal generated by the nanopore are subjected to histogram statistical analysis, the peak value change of the relative current is obtained by fitting and is brought into a standard curve, so that the glucose concentration contained in the corresponding sample can be obtained, and the quantitative detection of the glucose concentration is finally realized.
As a preferred technical scheme of the application: the concentration of the glucose oxidase is 0.8mg/mL, the final concentration of the HRP in the solution is 1.2 mM, the final concentration of the sodium bromide in the solution is 6 mM, and the final concentration of the AuTNs in the solution is 0.6 nM; the electrical signal of the feature extraction in step c comprises a relative current value and a retention time.
Explanation of the shape regulation principle of the gold nanometer triangular plate: the hydrogen peroxide enables gold simple substances to become gold ions under the catalytic action of horseradish peroxidase and sodium bromide, the gold nanoparticles are etched, and by combining the characteristics that the chemical energy sharp angle, the edge and the plane of a gold nanometer triangular plate are combined, the etching starts from the corner of the triangular plate, a 40 +/-2 nm disc is finally formed, and the nanostructure in the whole process is changed.
Has the advantages that:
1. the invention provides a label-free high-flux quantitative glucose detection method, which combines enzyme cascade reaction and nanoparticle etching technology to convert the concentration of glucose into the morphology difference of gold nanoparticle etching process, and performs signal detection through a nanopore sensor, thereby realizing the high-sensitivity detection of solid-state nanopores on glucose, and belonging to the field of nanopore sensor detection.
2. The scheme ingeniously converts the glucose concentration into the shape change of gold nanoparticles, and the nanopore sensor with stable property and controllable size is used as a detection tool, so that the solid-state nanopore sensor has the advantages of universality, low cost, high flux and the like for the glucose level in different environments, and the application range and the development potential of the solid-state nanopore sensor are expanded.
3. According to the gold triangular nanosheet prepared by the invention, through observation of a transmission electron microscope, different degrees of etching can be observed after glucose with different concentrations is added, so that the appearance of the gold triangular nanosheet is verified to be successfully regulated and controlled by the designed glucose mixed liquid.
4. The glucose designed by the invention reacts with the glucose oxidase to generate hydrogen peroxide, so that the gold nanoparticles are etched, and the size and shape change is generated.
5. The molecular weight of glucose is 180.16, the size is below 0.8 nm, and according to the principle that the size of a detection molecule is similar to that of a nanopore, a nanopore with the size less than 1 nm can obtain a high signal-to-noise ratio; however, the existing preparation process of the solid-state hole with the diameter of less than 10 nm is difficult, the cost is high, and high-flux detection is difficult to realize; the glucose reaction system designed by the invention converts the detection of the biomolecular into the change of the shape of the nano particles, breaks through the limitation of the size of the nano holes, and realizes the high-sensitivity high-flux detection of glucose by utilizing the solid nano holes with large size and low cost.
6. The solid-state nanopore is used, the material is not limited, the size is controllable, and the high-flux detection of the large-aperture solid-state nanopore sensor on the gold nanoparticles is realized by combining the nanoparticle etching technology.
7. The solid-state nanopore sensor designed by the invention can realize quantitative detection of glucose, solves the problems of great environmental influence, difficult temperature control and unstable fluorescent substance in the detection process of electrochemistry and spectrometry, and has the characteristics of high sensitivity, high resolution and high flux of the nanopore for responding to the change of the morphology of the gold nanoparticle. 8. The solid-state nanopore sensor designed by the invention breaks through the limitation of nanopore size and realizes high-sensitivity detection of small biological molecules. The method is based on a solid-state nanopore sensing platform, takes glucose as a reaction main body and gold nanoparticles as a reaction medium, etches the gold nanoparticles through glucose oxidase cascade reaction, realizes high-flux and high-sensitivity detection on the glucose through the shape change of the gold nanoparticles, and has very important application prospect.
9. The solid-state nanopore sensor designed by the invention utilizes a nanopore sensing platform, and designs a set of feasible glucose detection scheme, which is an important potential for realizing the development of a nanopore monomolecular technology. Aiming at the problems of low resolution and the like of other detection methods, the invention utilizes a solid-state nano-pore sensing platform, takes gold nano-particles as materials, utilizes glucose to generate hydrogen peroxide, converts the concentration of the glucose into the difference of structures in the etching process of nano-materials, is favorable for realizing high-flux and high-sensitivity detection of the glucose, and has very important research value.
10. The nanopore sensor designed by the invention has specificity only for glucose, can realize high-sensitivity quantitative detection of glucose, has wide detection range and strong stability, is suitable for detection requirements of different blood and body fluid environments, particularly carries out non-invasive detection in a low-concentration body fluid environment, and better carries out real-time monitoring on the glucose concentration.
Drawings
Fig. 1 is a schematic diagram of the present application.
FIG. 2 is a TEM image of a 50 + -3 nm gold nanoplatelet used in the present application and a TEM image of a gold nanoplatelet obtained in a glucose reaction system, wherein FIG. a is the gold nanoplatelet and FIG. b is the gold nanoplatelet.
Fig. 3 is electron microscope images of gold nanoparticle triangular plate morphology obtained before and after the glucose reaction system used in the present application and characteristic event electrical signal images recorded by corresponding nanopores, where fig. a is a TEM image of gold nanoparticles before reaction, fig. b is a TEM image of gold nanoparticles in reaction, fig. c is a TEM image of gold nanoparticles after reaction, and fig. d, fig. e, and fig. f are characteristic event electrical signal images of nanopores corresponding to fig. a, fig. b, and fig. c, respectively.
FIG. 4 is a graph of the present application for the quantitative analysis of glucose concentration.
FIG. 5 is a glucose specificity profile of the present application.
Detailed Description
The technical scheme of the invention is further explained by combining the drawings and the specific embodiments in the specification.
Example 1:
a label-free high-flux glucose quantitative detection method comprises the following specific steps:
step a, synthesizing gold nano triangular plates: adding 42.5 microliter of 1% chloroauric acid into 4.7 mL of 0.1M hexadecyl trimethyl ammonium chloride, adding magnetons, and stirring; adding 300 mu L of 0.01M sodium borohydride solution, stirring for 2 minutes, standing in a water bath kettle at 25 ℃ for 2 hours to react to form 5-7 nm seed solution, taking 1.6 mL of 0.1M hexadecyltrimethylammonium chloride solution, adding into 8 mL of deionized water, and adding 68 mu L of 1% chloroauric acid and 15 mu L of 0.01M potassium iodide solution to prepare growth solution 1; adding 170 mu L of 1% chloroauric acid into 8 mL of 0.05M hexadecyl trimethyl ammonium chloride solution, and adding 60 mu L of 0.01M potassium iodide solution to prepare growth solution 2; diluting the seed solution 10 times with 0.1M cetyltrimethylammonium chloride; adding 40 μ L of 0.1M ascorbic acid into growth liquid 1, adding 80 μ L of 0.1M ascorbic acid into growth liquid 2, and stirring growth liquids 1, 2 to colorless; adding 110 mu L of diluted seed solution into the growth solution 1 added with the perascorbic acid, shaking up, immediately adding 640 mu L of diluted seed solution into the growth solution 2 added with the perascorbic acid, shaking up the mixed solution for 1 minute, and standing and reacting at room temperature for 1 hour to obtain an unpurified gold nanoparticle triangular plate solution; subpackaging the solution of the unpurified gold nanoplatelets (1 ml for each centrifuge tube), adding hexadecyltrimethylammonium chloride (0.78M, 345 mu L) into each centrifuge tube until the final concentration is 0.2M, shaking uniformly, mixing, standing overnight for 16 hours, absorbing the red liquid on the upper layer by using a liquid transfer gun, adding 400 mu L of water and 50 mu L of hexadecyltrimethylammonium chloride (0.1M) into the sediment at the bottom, shaking uniformly and mixing to obtain the purified gold nanoplatelets;
step b, reaction condition optimization: diluting 10 mg/mL glucose oxidase and 10 mM glucose solution with 0.01M PBS (pH 7.4), obtaining glucose solutions (0, 10, 100, 1000, 2000. mu.M) with different concentrations by dilution, simultaneously obtaining 0.8mg/mL glucose oxidase solution by dilution, incubating the diluted glucose solution and glucose oxidase solution in a constant temperature water bath at 37 ℃ for 10 minutes, adding 5.5. mu.L of 110 mM horseradish peroxidase (HRP), 30. mu.L of 0.1M sodium bromide (NaBr), 200. mu.L of 1.5 nM AuTNs solution, further adding 0.01M PBS (pH 4), and finally incubating the solution for 500. mu.L, with HRP at a final concentration of 1.2 mM in solution, sodium bromide at a final concentration of 6 mM in solution, AuTNs at a final concentration of 0.6 nM in solution, and completing the reaction after 15 minutes at 25 ℃, observing the shape change of AuTNs through a transmission electron microscope;
step c, drawing a standard curve: preparing glucose reaction systems (0, 5, 10, 20, 50, 100, 200, 1000, 2000 mu M, 5 mM and 10 mM) with different concentrations, respectively adding 0.5-5 mg/ml glucose oxidase solution, incubating in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, then adding 110 mM HRP 5 mu L, 0.1M NaBr 30 mu L and 1.5 nM AuTNs solution 200 mu L, adding 0.01M PBS (pH 4), wherein the final solution reaches 500 mu L, the final concentration of HRP in the solution is 1.2 mM, the final concentration of sodium bromide in the solution is 6 mM, the final concentration of AuTNs in the solution is 0.6 nM, completing the reaction after incubating for 15 minutes at 25 ℃, finally obtaining gold nanoparticle products with different morphologies, and detecting nanopore signals of the final products; performing characteristic extraction on electric signals of glucose reaction system products with different concentrations through a nanopore translocation experiment, performing histogram statistical chart on the extracted relative current signals, fitting to obtain a peak diagram, and drawing a standard curve according to the change of the peak position value under different concentrations;
step d: mixing 0.8mg/mL glucose oxidase with the actual sample, placing the mixture in a constant-temperature water bath kettle at 37 ℃ for incubation for 10 min, then adding 5.5 mu L of HRP (110 mM), 30 mu L of NaBr (0.1M), 200 mu L of AuTNs solution (1.5 nM) and 0.01M PBS (pH 4) solution, wherein the total solution is 500 mu L, the final concentration of HRP in the solution is 1.2 mM, the final concentration of sodium bromide in the solution is 6 mM, the final concentration of AuTNs in the solution is 0.6 nM, and incubating the mixture at room temperature for 15 min;
the method comprises the steps of detecting mixed liquid containing glucose with different concentrations by using a nanopore sensor with a pore diameter of 120 nm, injecting the solution into one side of a nanopore sensor fluid device, driving small molecules in the mixed liquid to pass through a nanopore by using voltage, carrying out statistical analysis and drawing on the residence time, the base line current and the amplitude of an electric signal generated by the nanopore, bringing peak value changes obtained by fitting into a standard curve, and distinguishing the etching degree of a gold triangular nanosheet by detecting the size of a blocking signal, thereby realizing the detection of the glucose.
According to the gold nanoparticle triangular plate prepared by the application, the etching of different degrees can be observed after glucose with different concentrations is added through observation of a transmission electron microscope, so that the gold nanoparticle triangular plate is verified to be successfully etched by the glucose mixed liquid designed by people.
Glucose can react with glucose oxidase to generate hydrogen peroxide, under the catalytic action of bromide ions and horseradish peroxidase, the gold nanoparticle triangular plate is etched to become a disc gradually, the condition of single-molecule via holes is detected at high sensitivity through the nano holes, the form of gold nanoparticles is analyzed according to signals, the signals are counted and analyzed, and the concentration of the glucose is quantitatively estimated. As shown in fig. 1, which is a schematic diagram of the present application, glucose generates hydrogen peroxide under the catalysis of glucose oxidase, and the hydrogen peroxide changes a gold simple substance into gold ions under the catalytic action of NaBr and HRP, so that the gold nanoparticles are subjected to nanopore signal detection after appearance regulation.
The enzyme cascade catalytic reaction of glucose oxidase to glucose is utilized, and the gold triangular plate is etched by the generated product under the assistance of enzyme, so that the appearance of the nano-particles is changed, the nano-particles with different sizes are obtained after etching, translocation research is carried out in the solid nano-holes, unique electric signals are generated, and the high-sensitivity detection of the solid nano-hole sensor to the glucose concentration is realized.
As shown in FIG. 2, the synthesized material is a 50 +/-3 nm gold nanometer triangular plate and a 40 +/-2 nm disk TEM image after etching, the morphology change before and after etching is obvious, the property is stable, the size is uniform, and the dispersibility in the solution is good.
As shown in fig. 3, TEM images of three stages in the gold nanoparticle etching process and corresponding nanopore characteristic signal images are shown, wherein an image a is a TEM image of the gold nanoparticle, an image b is a TEM image of the nanoparticle in the etching process, an image c is a TEM characterization image of a disk after etching, and images d, e and f are respectively characteristic signal images corresponding to the images a, b and c, so that it can be clearly seen that the differences of the three processes in relative current are very obvious, and nanopore characteristic signals in different stages can also distinguish the changes of particle morphology.
FIG. 4 is a line graph showing the quantitative analysis of glucose concentration according to the present invention, wherein the difference between the peak positions at different concentrations and 0 mM is plotted as scatter plots, and finally the line is fitted to obtain the function equation y =1.61x +0.001 (R) of the line2= 0.9958), y is the difference of the relative current of the signal generated by the nanoparticle via hole in the glucose reaction system, x is the final concentration of glucose in the reaction system, R2Is a correlation coefficient, represents the coincidence degree of experimental data and a fitting function, and simultaneously calculates the detection limit to be 2.07 mu M.
As shown in fig. 5, the present application was conducted to study the specificity of a blank sample, ascorbic acid, sucrose, lactose, glycine, cysteine, fructose, maltose, uric acid, and glucose, and the above samples were mixed with glucose oxidase and PBS, incubated at 37 ℃ for 10 minutes, then sequentially added with 2 mM HRP, 6 mM NaBr, and 0.6 nM AuNTs solutions, and after 15 minutes, the uv spectrum was measured on the mixed sample.
In conclusion, all test results show that the solid-state nanopore sensor designed by the application can realize quantitative detection of glucose, solves the problems of instability of fluorescent substances and unclear color resolution in the detection process of colorimetry and spectrometry, and simultaneously reflects the etching degree by the change of the shape and size of the gold nanoparticles so as to reflect the concentration of the glucose, and has the characteristics of high sensitivity, high resolution and high flux of the nanopore to the change of the shape of the gold nanoparticles, so that the application has important research value on the quantitative detection of the glucose.
Example 2:
a label-free high-flux glucose quantitative detection method comprises the following specific steps:
step a, synthesizing gold nano triangular plates: adding 42.5 microliter of 1% chloroauric acid into 4.7 mL of 0.1M hexadecyl trimethyl ammonium chloride, adding magnetons, and stirring; adding 300 mu L of 0.01M sodium borohydride solution, stirring for 2 minutes, standing in a water bath kettle at 25 ℃ for 2 hours to react to form 5-7 nm seed solution, taking 1.6 mL of 0.1M hexadecyltrimethylammonium chloride solution, adding into 8 mL of deionized water, and adding 68 mu L of 1% chloroauric acid and 15 mu L of 0.01M potassium iodide solution to prepare growth solution 1; adding 170 mu L of 1% chloroauric acid into 8 mL of 0.05M hexadecyl trimethyl ammonium chloride solution, and adding 60 mu L of 0.01M potassium iodide solution to prepare growth solution 2; diluting the seed solution 10 times with 0.1M cetyltrimethylammonium chloride; adding 40 μ L of 0.1M ascorbic acid into growth liquid 1, adding 80 μ L of 0.1M ascorbic acid into growth liquid 2, and stirring growth liquids 1, 2 to colorless; adding 110 mu L of diluted seed solution into the growth solution 1 added with the perascorbic acid, shaking up, immediately adding 640 mu L of diluted seed solution into the growth solution 2 added with the perascorbic acid, shaking up the mixed solution for 1 minute, and standing and reacting at room temperature for 1 hour to obtain an unpurified gold nanoparticle triangular plate solution; subpackaging the solution of the unpurified gold nanoplatelets (1 ml for each centrifuge tube), adding hexadecyltrimethylammonium chloride (0.78M, 345 mu L) into each centrifuge tube until the final concentration is 0.2M, shaking uniformly, mixing, standing overnight for 16 hours, absorbing the red liquid on the upper layer by using a liquid transfer gun, adding 400 mu L of water and 50 mu L of hexadecyltrimethylammonium chloride (0.1M) into the sediment at the bottom, shaking uniformly and mixing to obtain the purified gold nanoplatelets;
step b, reaction condition optimization: diluting 10 mg/mL glucose oxidase and 10 mM glucose solution with 0.01M PBS (pH 7.4), obtaining glucose solutions (0, 10, 100, 1000, 2000. mu.M) with different concentrations by dilution, simultaneously obtaining 0.5 mg/mL glucose oxidase solution by dilution, incubating the diluted glucose solution and glucose oxidase solution in a constant temperature water bath at 37 ℃ for 10 minutes, adding 110 mM horseradish peroxidase (HRP) 4.5. mu.L, 0.1M sodium bromide (NaBr) 27.5. mu.L, 1.5 nM AuTNs solution 166.7. mu.L, further adding 0.01M PBS (pH 4), and finally obtaining 500. mu.L solution, wherein the final concentration of HRP in the solution is 1 mM, the final concentration of sodium bromide in the solution is 5.5 mM, the final concentration of AuTNs in the solution is 0.5 nM, and the reaction is completed after 15 minutes at 25 ℃, observing the shape change of AuTNs through a transmission electron microscope;
step c, drawing a standard curve: preparing glucose reaction systems (0, 5, 10, 20, 50, 100, 200, 1000, 2000 mu M, 5 mM and 10 mM) with different concentrations, respectively adding 0.5-5 mg/ml glucose oxidase solution, incubating in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, then adding 110 mM HRP 4.5 mu L, 0.1M NaBr 27.5 mu L and 1.5 nM AuTNs solution 166.7 mu L, adding 0.01M PBS (pH 4), and finally completing the reaction after the solution reaches 500 mu L, wherein the final concentration of the HRP in the solution is 1 mM, the final concentration of the sodium bromide in the solution is 5.5 mM, the final concentration of the AuTNs in the solution is 0.5 nM, and incubating for 15 minutes at 25 ℃ to obtain gold nanoparticle products with different morphologies, and detecting a nanopore signal of the final product; performing characteristic extraction on electric signals of glucose reaction system products with different concentrations through a nanopore translocation experiment, performing histogram statistical chart on the extracted relative current signals, fitting to obtain a peak diagram, and drawing a standard curve according to the change of the peak position value under different concentrations;
step d: mixing 0.5 mg/mL glucose oxidase with the actual sample, placing the mixture in a constant-temperature water bath kettle at 37 ℃ for incubation for 10 min, then adding 4.5 mu L of HRP (110 mM), 27.5 mu L of NaBr (0.1M), 166.7 mu L of AuTNs solution (1.5 nM) and 0.01M PBS (pH 4) solution, wherein the total solution is 500 mu L, the final concentration of HRP in the solution is 1 mM, the final concentration of sodium bromide in the solution is 5.5 mM, and the final concentration of AuTNs in the solution is 0.5 nM, and incubating the mixture at room temperature for 15 min;
the method comprises the steps of detecting mixed liquid containing glucose with different concentrations by using a nanopore sensor with the aperture of 115 nm, injecting the solution into one side of a nanopore sensor fluid device, driving small molecules in the mixed liquid to pass through a nanopore by using voltage, carrying out statistical analysis and drawing on the residence time, the base line current and the amplitude of an electric signal generated by the nanopore, bringing peak value change obtained by fitting into a standard curve, and distinguishing the etching degree of a gold triangular nanosheet by detecting the size of a blocking signal, thereby realizing the detection of the glucose.
Example 3:
a label-free high-flux glucose quantitative detection method comprises the following specific steps:
step a, synthesizing gold nano triangular plates: adding 42.5 microliter of 1% chloroauric acid into 4.7 mL of 0.1M hexadecyl trimethyl ammonium chloride, adding magnetons, and stirring; adding 300 mu L of 0.01M sodium borohydride solution, stirring for 2 minutes, standing in a water bath kettle at 25 ℃ for 2 hours to react to form 5-7 nm seed solution, taking 1.6 mL of 0.1M hexadecyltrimethylammonium chloride solution, adding into 8 mL of deionized water, and adding 68 mu L of 1% chloroauric acid and 15 mu L of 0.01M potassium iodide solution to prepare growth solution 1; adding 170 mu L of 1% chloroauric acid into 8 mL of 0.05M hexadecyl trimethyl ammonium chloride solution, and adding 60 mu L of 0.01M potassium iodide solution to prepare growth solution 2; diluting the seed solution 10 times with 0.1M cetyltrimethylammonium chloride; adding 40 μ L of 0.1M ascorbic acid into growth liquid 1, adding 80 μ L of 0.1M ascorbic acid into growth liquid 2, and stirring growth liquids 1, 2 to colorless; adding 110 mu L of diluted seed solution into the growth solution 1 added with the perascorbic acid, shaking up, immediately adding 640 mu L of diluted seed solution into the growth solution 2 added with the perascorbic acid, shaking up the mixed solution for 1 minute, and standing and reacting at room temperature for 1 hour to obtain an unpurified gold nanoparticle triangular plate solution; subpackaging the solution of the unpurified gold nanoplatelets (1 ml for each centrifuge tube), adding hexadecyltrimethylammonium chloride (0.78M, 345 mu L) into each centrifuge tube until the final concentration is 0.2M, shaking uniformly, mixing, standing overnight for 16 hours, absorbing the red liquid on the upper layer by using a liquid transfer gun, adding 400 mu L of water and 50 mu L of hexadecyltrimethylammonium chloride (0.1M) into the sediment at the bottom, shaking uniformly and mixing to obtain the purified gold nanoplatelets;
step b, reaction condition optimization: glucose solutions of 10 mg/mL glucose oxidase and 10 mM were diluted with 0.01M PBS (pH 7.4) to give glucose solutions of various concentrations (0, 10, 100, 1000, 2000. mu.M), diluting to obtain 5 mg/ml glucose oxidase solution, incubating the diluted glucose solution and glucose oxidase solution in a constant temperature water bath kettle at 37 ℃ for 10 minutes, adding 13.6 mu L of 110 mM horseradish peroxidase (HRP), 35 mu L of 0.1M sodium bromide (NaBr) and 1.5 nM AuTNs solution to 500 mu L respectively, wherein the final concentration of the HRP in the solution is 3 mM, the final concentration of the sodium bromide in the solution is 7 mM, and the final concentration of the AuTNs in the solution is 1.5 nM, after incubation for 15 minutes at 25 ℃, the reaction is completed, and the morphology change of AuTNs is observed by a transmission electron microscope;
step c, drawing a standard curve: preparing glucose reaction systems (0, 5, 10, 20, 50, 100, 200, 1000, 2000 mu M, 5 mM and 10 mM) with different concentrations, respectively adding 0.5-5 mg/ml glucose oxidase solution, incubating in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, then adding 110 mM HRP 13.6 mu L, 0.1M NaBr 35 mu L and 1.5 nM AuTNs solution to 500 mu L, wherein the final concentration of the HRP in the solution is 3 mM, the final concentration of the sodium bromide in the solution is 7 mM, the final concentration of the AuTNs in the solution is 1.5 nM, completing the reaction after incubating for 15 minutes at 25 ℃, finally obtaining gold nanoparticle products with different morphologies, and detecting nanopore signals of the final products; performing characteristic extraction on electric signals of glucose reaction system products with different concentrations through a nanopore translocation experiment, performing histogram statistical chart on the extracted relative current signals, and fitting to obtain a peak bitmap;
step d: mixing 5 mg/mL glucose oxidase with an actual sample, placing the mixture in a constant-temperature water bath kettle at 37 ℃ for incubation for 10 min, then adding 110 mM HRP (13.6 mu L), 0.1M NaBr (35 mu L) and 1.5 nM AuTNs solution to 500 mu L, wherein the final concentration of the HRP in the solution is 3 mM, the final concentration of the sodium bromide in the solution is 7 mM, the final concentration of the AuTNs in the solution is 1.5 nM, and incubating the mixture for 15 min at room temperature;
the method comprises the steps of detecting mixed liquid containing glucose with different concentrations by using a nanopore sensor with a pore diameter of 125 nm, injecting the solution into one side of a nanopore sensor fluid device, driving small molecules in the mixed liquid to pass through a nanopore by using voltage, carrying out statistical analysis and drawing on the residence time, the base line current and the amplitude of an electric signal generated by the nanopore, bringing peak value changes obtained by fitting into a standard curve, and distinguishing the etching degree of a gold triangular nanosheet by detecting the size of a blocking signal, thereby realizing the detection of the glucose.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (8)
1. A label-free high-flux glucose quantitative detection method is characterized in that: the method comprises the following steps of taking glucose as a reaction main body, utilizing glucose oxidase to perform catalytic reaction on the glucose to generate hydrogen peroxide, converting a gold simple substance into gold ions by the hydrogen peroxide under the catalytic action of horseradish peroxidase and sodium bromide, realizing shape regulation of gold nanoparticles, and performing signal detection on the gold nanoparticles with different sizes and shapes finally obtained by reaction through a nanopore sensor; the detection of glucose micromolecules is converted into translocation signals of the nano particles in the nano holes, the size of the electric signals is in direct proportion to the appearance of the nano particles, and the higher signal-to-noise ratio is realized by regulating and controlling the size of the gold nano particles, so that the high-sensitivity detection of the glucose concentration is realized.
2. The label-free high-throughput quantitative glucose detection method according to claim 1, wherein: the gold nanoparticles are carriers, and the components include but are not limited to metals, alloys and materials which react with glucose mediated reaction to change the shape and/or size; the gold nanoparticles are prepared by a chemical or physical method, and the shapes of the gold nanoparticles comprise a two-dimensional or three-dimensional structure, a polygonal sheet with high specific surface area and a polyhedron; the size of the gold nanoparticles is 5-100 nm.
3. The label-free high-throughput quantitative glucose detection method according to claim 2, wherein: the size of the gold nanoparticles is 40-100 nm; when the gold nanoparticles are gold nanopyramids (AuTNs), the size is 50 +/-3 nm.
4. The label-free high-throughput quantitative glucose detection method according to claim 1, wherein: the diameter of the solid-state nano-pores is 60-600 nm, and the thickness of the solid-state nano-pores is 20-100 nm.
5. The label-free high-throughput quantitative glucose detection method according to claim 4, wherein: the diameter of the solid-state nano-pores is 100-160 nm; the thickness is 50-100 nm; when the gold nanoparticles are AuTNs, the diameter of the solid nanopore is 120 +/-5 nm, and the thickness of the solid nanopore is 50 nm.
6. The label-free high-throughput quantitative glucose detection method according to claim 1, wherein: the catalytic reaction temperature of the glucose oxidase on glucose is 37 ℃, and the catalytic reaction temperature of horseradish peroxidase and sodium bromide is 25 ℃.
7. The label-free high-throughput quantitative detection method for glucose, according to claim 1, is characterized in that the detection method comprises the following specific steps:
step a, synthesizing gold nano triangular plates: adding 42.5 microliter of 1% chloroauric acid into 4.7 mL of 0.1M hexadecyl trimethyl ammonium chloride, adding magnetons, and stirring; adding 300 mu L of 0.01M sodium borohydride solution, stirring for 2 minutes, standing in a water bath kettle at 25 ℃ for 2 hours to react to form 5-7 nm seed solution, taking 1.6 mL of 0.1M hexadecyltrimethylammonium chloride solution, adding into 8 mL of deionized water, and adding 68 mu L of 1% chloroauric acid and 15 mu L of 0.01M potassium iodide solution to prepare growth solution 1; adding 170 mu L of 1% chloroauric acid into 8 mL of 0.05M hexadecyl trimethyl ammonium chloride solution, and adding 60 mu L of 0.01M potassium iodide solution to prepare growth solution 2; diluting the seed solution 10 times with 0.1M cetyltrimethylammonium chloride; adding 40 μ L of 0.1M ascorbic acid into growth liquid 1, adding 80 μ L of 0.1M ascorbic acid into growth liquid 2, and stirring growth liquids 1, 2 to colorless; adding 110 mu L of diluted seed solution into the growth solution 1 added with the perascorbic acid, shaking up, immediately adding 640 mu L of diluted seed solution into the growth solution 2 added with the perascorbic acid, shaking up the mixed solution for 1 minute, and standing and reacting at room temperature for 1 hour to obtain an unpurified gold nanoparticle triangular plate solution; subpackaging the solution of the unpurified gold nanoplatelets (1 ml for each centrifuge tube), adding hexadecyltrimethylammonium chloride (0.78M, 345 mu L) into each centrifuge tube until the final concentration is 0.2M, shaking uniformly, mixing, standing overnight for 16 hours, absorbing the red liquid on the upper layer by using a liquid transfer gun, adding 400 mu L of water and 50 mu L of hexadecyltrimethylammonium chloride (0.1M) into the sediment at the bottom, shaking uniformly and mixing to obtain the purified gold nanoplatelets;
step b, reaction condition optimization: diluting 10 mg/mL glucose oxidase and 10 mM glucose solution with 0.01M PBS (pH 7.4) to obtain glucose solutions (0, 10, 100, 1000, 2000 mu M) with different concentrations, simultaneously diluting to obtain 0.5-5 mg/mL glucose oxidase solution, incubating the diluted glucose solution and glucose oxidase solution in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, adding 110 mM horseradish peroxidase (HRP), 0.1M sodium bromide (NaBr) and 1.5 nM AuTNs solution, adding 0.01M PBS (pH 4), allowing the final solution to reach 500 mu L, allowing the final concentration of HRP in the solution to be 1-3 mM, the final concentration of sodium bromide in the solution to be 5.5-7 mM, and the final concentration of AuTNs in the solution to be 0.5-1.5 nM, incubating for 15 minutes at 25 ℃, and then completing the reaction, observing the shape change of AuTNs through a transmission electron microscope;
step c, drawing a standard curve: preparing glucose reaction systems (0 mu M, 5 mu M, 10 mu M, 20 mu M, 50 mu M, 100 mu M, 200 mu M, 1000 mu M, 2000 mu M, 5 mM and 10 mM) with different concentrations, respectively adding 0.5-5 mg/ml glucose oxidase solution, incubating in a constant-temperature water bath kettle at 37 ℃ for 10 minutes, then adding 110 mM HRP, 0.1M NaBr and 1.5 nM AuTNs solution, adding 0.01M PBS (pH 4), wherein the final solution reaches 500 mu L, the final concentration of HRP in the solution is 1-3 mM, the final concentration of sodium bromide in the solution is 5.5-7 mM, the final concentration of AuTNs in the solution is 0.5-1.5 nM, after incubating for 15 minutes at 25 ℃, completing the reaction, finally obtaining gold nanoparticle products with different morphologies, and detecting nanopore signals of the final products; performing characteristic extraction on electric signals of glucose reaction system products with different concentrations through a nanopore translocation experiment, performing histogram statistical chart on the extracted relative current signals, fitting to obtain a peak diagram, and drawing a standard curve according to the change of the peak position value under different concentrations;
d, quantitative detection of glucose: incubating a sample to be tested with 0.5-5 mg/ml glucose oxidase at 37 ℃ for 10 minutes, then adding 110 mM HRP, 0.1M NaBr, 1.5 nM AuTNs solution and 0.01M PBS (pH 4) solution, wherein the total solution is 500 muL, the final concentration of the HRP in the solution is 1-3 mM, the final concentration of sodium bromide in the solution is 5.5-7 mM, and the final concentration of the AuTNs in the solution is 0.5-1.5 nM, incubating for 15 minutes at room temperature, adding the solution into a nanopore detection device, and obtaining a series of electric signals under voltage driving; the residence time and the relative current of the electric signal generated by the nanopore are subjected to histogram statistical analysis, the peak value change of the relative current is obtained by fitting and is brought into a standard curve, so that the glucose concentration contained in the corresponding sample can be obtained, and the quantitative detection of the glucose concentration is finally realized.
8. The label-free high-throughput quantitative glucose detection method according to claim 7, wherein: the concentration of the glucose oxidase is 0.8mg/mL, the final concentration of the HRP in the solution is 1.2 mM, the final concentration of the sodium bromide in the solution is 6 mM, and the final concentration of the AuTNs in the solution is 0.6 nM; the electrical signal of the feature extraction in step c comprises a relative current value and a retention time.
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| CN105424690A (en) * | 2015-11-24 | 2016-03-23 | 鲁东大学 | Colorimetric method for detecting glucose concentration based on triangular silver nanosheets |
| CN109342529A (en) * | 2018-09-19 | 2019-02-15 | 上海大学 | Non-enzymatic catalysis sensor of glucose and preparation method thereof |
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| CN105424690A (en) * | 2015-11-24 | 2016-03-23 | 鲁东大学 | Colorimetric method for detecting glucose concentration based on triangular silver nanosheets |
| CN109342529A (en) * | 2018-09-19 | 2019-02-15 | 上海大学 | Non-enzymatic catalysis sensor of glucose and preparation method thereof |
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| Title |
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| 江丽萍等: "基于金纳米的第3代平面型葡萄糖传感器的研究" * |
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