Electrochemical sensor based on porous silicon-platinum nanoflower-DNA composite material and preparation method and application thereof

Abstract

The invention belongs to the field of nano sensors, and discloses an electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material and a preparation method and application thereof. A porous silicon layer is prepared on a polished surface of a silicon wafer, then platinum nanoflowers and a T-rich sulfhydrylation DNA probe are modified, a metal film is plated on the other surface of the silicon wafer, and the electrochemical sensor is prepared and applied to melamine detection. The DNA probe can adsorb melamine on the surface of a material through a hydrogen bond and generate current response linearly related to the concentration of the melamine; hydroxyl on the surface of the porous silicon can enhance the adsorption effect on melamine; the platinum nanomaterial with the flower-shaped structure can enhance the sensitivity of the sensor; in addition, negative charges carried on the surface of the porous silicon and common coexisting substances of melamine have an electrostatic repulsion effect, so that the sensor has good selectivity. The high-performance nano sensor provides a novel method with practicability for on-site rapid detection of melamine.

Description

Technical Field

[0001] The present invention belongs to the field of electrochemical biosensors, and relates to an electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material, a preparation method thereof, and an application thereof. Background Art

[0002] Melamine is a nitrogen-containing organic compound that is often illegally added to food to increase the apparent protein content. However, its ingestion can cause serious health problems such as kidney stones and renal failure. Therefore, the rapid and accurate detection of melamine in food is a key link in ensuring food safety. At present, the conventional detection methods for melamine include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). Although these methods have high accuracy, they rely on large-scale instrument equipment, have complex operation processes, long detection cycles, and high costs, and are difficult to meet the needs of rapid on-site detection in the food supply chain. Therefore, the development of new analytical methods with simple operation, high sensitivity, and suitable for point-of-care testing (POCT) has become an important research direction in this field.

[0003] In recent years, electrochemical sensing technology has shown significant potential in the field of food safety detection due to its advantages such as rapid response, portable equipment, high sensitivity, and easy miniaturization. By designing specific recognition elements and efficient signal amplification strategies, electrochemical sensors can achieve highly selective detection of target substances. Among them, the performance of electrode materials is the core factor determining the sensitivity and stability of the sensor. Porous silicon (PSi) has attracted much attention in the field of biosensing due to its high specific surface area, adjustable pore structure, and good biocompatibility. Its porous structure can effectively load functional nanomaterials or biomolecules, providing an ideal substrate for constructing high-performance composite electrodes. However, the conductivity and catalytic activity of traditional porous silicon-based sensors are still insufficient, limiting their application in the detection of trace substances. Summary of the Invention

[0004] To solve the above technical problems, the purpose of the present invention is to provide an electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material. The preparation method of the sensor includes the following steps:

[0005] (1) Preparing a porous silicon layer on the polished surface of a single-sided polished silicon wafer by a metal-assisted chemical etching method;

[0006] (2) Depositing a metal film on the non-polished surface of the porous silicon material;

[0007] (3) Depositing platinum nanoflowers on the surface of the porous silicon layer by a potentiostatic deposition method to obtain a porous silicon-platinum nanoflower material;

[0008] (4) Modify the surface of the porous silicon-platinum nanoflower material with a thiolated DNA probe rich in T, and block it with 2-mercaptoethanol to obtain an electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material.

[0009] Furthermore, the silicon wafer used in step (1) is a single-sided polished p-type silicon wafer.

[0010] Furthermore, the specific method for preparing silicon porous silicon on the polished surface of the single-sided polished silicon wafer in step (1) is as follows: Immerse the single-sided polished silicon wafer in a mixed solution of concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30% (volume ratio 3:1), soak for 10 minutes, then take it out and put it into a 1-10% hydrofluoric acid solution and soak for 10 minutes, then transfer it to a 1-10% hydrofluoric acid, 1-10 mM silver nitrate aqueous solution, soak for 20-80 seconds to obtain metal nanoparticles on the silicon wafer, and wash with a large amount of water; put the sample into an aqueous solution of 0.1-0.5 M hydrogen peroxide and 5-20% hydrofluoric acid for corrosion for 10-60 minutes, and then soak it in a nitric acid solution with a concentration of 10-30 wt%, that is, a porous silicon layer is obtained on the polished surface of the silicon wafer.

[0011] Furthermore, the thickness of the metal film in step (2) is 10-500 nanometers; the metal material is aluminum, copper, platinum, nickel, titanium, cobalt, palladium, tungsten, etc.; the method for depositing the metal film includes but is not limited to vacuum evaporation, sputtering coating, chemical vapor deposition, etc.

[0012] Furthermore, the specific method of step (3) is: Immerse the polysilicon layer of the silicon wafer in the electrolyte, and the electrolyte is a 5-25 g / L chloroplatinic acid solution containing 0.25 mol / L sulfuric acid. Using 0.3 V (vsAg / AgCl) as the working potential, deposit for 20-100 seconds by the potentiostatic method to obtain a porous silicon-platinum nanoflower material on the polished surface of the silicon wafer.

[0013] Furthermore, the nucleotide sequence of the DNA probe rich in T in step (4) is: 5’-HS-C6H 12 -TTT TTT TTC TTT GTT TTG GTT CTT TCT TTG G-3’;

[0014] The solution of the thiolated DNA probe rich in T is prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH = 7.5), its concentration is 2.5 μM, and its modification amount is 0.065-0.83 mL / cm 2 ;

[0015] The concentration of the 2-mercaptoethanol is 0.05-0.5 mol / L, and the blocking time is 0.5-2 h.

[0016] The electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material obtained by the above preparation method is applied to the electrochemical detection of melamine in the present invention.

[0017] Specifically, using the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material as the working electrode, a three-electrode system is formed with a counter electrode and a reference electrode. The cyclic voltammetry method is used to measure the current response of different concentrations of melamine, and the standard curve method is adopted to calculate the concentration of melamine in the sample to be measured.

[0018] The counter electrode is a platinum wire electrode, and the reference electrode is a silver / silver chloride electrode.

[0019] The parameters of the cyclic voltammetry method are: potential window -0.2 to +0.4 V, scanning speed 50 mV / s.

[0020] When the electrochemical sensor is used, the metal-coated film layer of the modified electrode is connected to the circuit (such as by welding), so that the electrical signal generated by the porous silicon-platinum nanoflower-DNA complex during the test is input into the circuit through the conduction of the silicon wafer.

[0021] When the porous silicon-platinum nanoflower-DNA composite material is immersed in the melamine solution, since melamine can bind to T through three hydrogen bonds, the T-rich DNA modified on the platinum nanoflower can be used as a probe to capture melamine in the solution, covering the electrode surface with melamine, and the steric hindrance effect generated during this process will hinder the electron transfer on the electrode surface. The greater the concentration of melamine in the solution, the more melamine adsorbed on the electrode surface, and the smaller the current signal obtained. Based on this principle, the detection of melamine is realized.

[0022] In addition to the role of the substrate material, the porous silicon in the nano-composite structure also has the effect of enhancing the adsorption effect of melamine. During the process of the nano-composite capturing melamine, due to the large number of hydroxyl groups on the surface of the porous silicon, it is negatively charged in the solution environment used (such as a 0.2 M phosphate buffer solution containing 5 mM KCl and pH 7.2), while melamine is positively charged in this solution environment. Therefore, there is an electrostatic adsorption effect between the two, and the huge surface area of the porous silicon further enhances the adsorption effect of the material surface on melamine, thereby improving the detection sensitivity. The huge surface area of the flower-shaped platinum nano-material is also beneficial to the sensitivity of the sensor. In addition, in the test solution, the negatively charged surface of the porous silicon can have an electrostatic repulsion effect on common coexisting substances and interfering substances that are also negatively charged, such as cyanuric acid, cyanuric acid, and proteins, which makes the sensor have good selectivity.

[0023] The effects and advantages of the present invention:

[0024] The present invention provides an electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material. The DNA rich in T is used as a probe to capture melamine in the solution, and the influence of the steric hindrance effect of this process on the electrode signal is used as the principle to detect melamine. The porous silicon substrate has a huge surface area and has an adsorption function for substrates; the platinum material modified on the electrode with a nanoflower structure also provides a huge surface area for the electrode. These characteristics of the material make the sensor have good sensitivity. In addition, in the test solution, the negatively charged porous silicon surface has an electrostatic repulsion effect on common coexisting substances and interfering substances that are also negatively charged, which makes the sensor have good selectivity. These provide a practical method for the on-site rapid testing of melamine in food inspection technology. Description of the Drawings

[0025] Figure 1 It is a scanning electron microscope photograph of the porous silicon-platinum nanoflower material in Example 1;

[0026] Figure 2 It is the result of detecting melamine by the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material in Example 1;

[0027] Figure 3 It is the result of detecting melamine by the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material in Example 2;

[0028] Figure 4 It is the result of detecting melamine by the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material in Example 3;

[0029] Figure 5 It is the CV test result of the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material in Example 3;

[0030] Figure 6 It is the selectivity test result of the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material in Example 3. Detailed Description of the Invention

[0031] The present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The electrochemical testing method used in the above specific embodiments of the present invention is as follows: At room temperature, the porous silicon-platinum nanoflower-DNA composite modified electrode is immersed in a melamine solution for 30 minutes, then the material is taken out and rinsed with ultrapure water. Then, it is used as the working electrode, and cyclic voltammetry measurement is carried out in a 0.2 M phosphate buffer solution (pH = 7.2, containing 5 mM KCl) using a three-electrode system (a platinum wire as the counter electrode and silver / silver chloride as the reference electrode). The electrode material made of the porous silicon-platinum nanoflower-DNA composite with a melamine capture probe immobilized on its surface in this method is for single use. For each measurement, a new unused electrode needs to be selected and immersed in the test solution, and after immersion, it is cleaned, and then the response of the electrode to melamine in the test solution is studied by cyclic voltammetry in the above phosphate buffer solution.

[0033] The nucleotide sequence of the thiolated DNA probe rich in T used in the following specific embodiments of the present invention is: 5’-HS-C6H 12 -TTT TTT TTC TTT GTT TTG GTT CTT TCT TTG G-3’, which is synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The thiolated DNA probe solution rich in T is prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH = 7.5), and its concentration is 2.5 μM.

[0034] It should be noted that the raw materials or reagents not specifically described in the following embodiments of the present invention are all ordinary commercially available products. The preparation methods or electrochemical detection methods not specifically described are conventional methods in the art.

[0035] Example 1

[0036] An electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material includes the following steps:

[0037] (1) Immerse a single-sided polished P-type 1Ω silicon wafer (size: 1.2 x 1.2 cm) in a mixed solution of concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30% (volume ratio 3:1) for 10 minutes, then take it out and immerse it in a 3 wt% hydrofluoric acid solution for 10 minutes, and then transfer it to a 10 mL aqueous solution of 3% hydrofluoric acid and 5 mM silver nitrate and soak for 45 seconds to obtain metal nanoparticles on the silicon wafer, and wash it with a large amount of water; put the sample into an aqueous solution of 0.15 M hydrogen peroxide and 8 wt% hydrofluoric acid for corrosion for 15 minutes, and then soak it in a 10 wt% nitric acid solution for 5 minutes, that is, a porous silicon layer is obtained on the polished surface of the silicon wafer.

[0038] (2) Use the sputtering coating method to deposit a 300 nm thick aluminum metal on the non-polished surface.

[0039] (3) Then, contact the porous silicon surface of the sample with the chloroplatinic acid electrolyte, and use the potentiostatic deposition method to obtain the porous silicon-platinum nanoflower material. Use a 2.5 g / L chloroplatinic acid solution containing 0.25 mol / L sulfuric acid as the electrolyte, with a working potential of 0.3 V (vs Ag / AgCl), and deposit for 30 seconds by the potentiostatic method to obtain the porous silicon-platinum nanoflower material on the polished surface of the silicon wafer.

[0040] (4) Then, take 0.3 mL of the thiolated DNA probe test solution rich in T and drop-coat it on the surface of the porous silicon-platinum nanoflower material electrode. After drying at room temperature, block it with 2-mercaptoethanol with a concentration of 0.1 mol / L for 0.5 h to obtain the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material.

[0041] Figure 1 This is the scanning electron microscope photograph of the porous silicon-platinum nanoflower material in this example. It can be seen that platinum nanoflowers are loaded on the surface of the porous silicon.

[0042] Use the electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material prepared in Example 1 as the working electrode, and form a three-electrode system with the counter electrode and the reference electrode. Use cyclic voltammetry to measure the current response of different concentrations of melamine, and calculate the concentration of melamine in the test sample by the standard curve method; the counter electrode is a platinum wire electrode, and the reference electrode is a silver / silver chloride electrode; the parameters of the cyclic voltammetry are: potential window -0.2 to +0.4 V; scanning speed 50 mV / s; the electrolyte is 0.2 M phosphate buffer solution (pH 7.2, containing 5 mM KCl).

[0043] Figure 2 This is the graph of the response current of the sensor in this example at +0.20 V plotted against the melamine concentration. The figure shows that within the test range, the peak current value has a good linear relationship with the logarithm of the melamine concentration, and its sensitivity is -3.26×10 -5 μA / nM.

[0044] Example 2

[0045] An electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material, comprising the following steps:

[0046] (1) Immerse a single-side polished P-type 1Ω silicon wafer (size: 1.2 x 1.2 cm) into a mixed solution of concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30% (volume ratio 3:1), soak for 10 minutes, then take it out and put it into a 10wt% hydrofluoric acid solution for 10 minutes, and then transfer it to 10 mL of 8wt% hydrofluoric acid and 10 mM silver nitrate aqueous solution, soak for 75 seconds to obtain metal nanoparticles on the silicon wafer, and wash with a large amount of water; put the sample into an aqueous solution of 0.4M hydrogen peroxide and 15wt% hydrofluoric acid for corrosion for 45 minutes, and then put it into a 10wt% nitric acid solution for 5 minutes, then porous silicon is obtained on the polished surface of the silicon wafer.

[0047] (2) Use the sputtering coating method to modify a 400-nm-thick aluminum metal on the non-polished surface.

[0048] (3) Then contact the porous silicon surface of the sample with chloroplatinic acid electrolyte, and use the potentiostatic deposition method to obtain a porous silicon-platinum nanoflower material. Use an 8.0 g / L chloroplatinic acid solution containing 0.25 mol / L sulfuric acid as the electrolyte, with a working potential of 0.3V (vs Ag / AgCl), and deposit for 60 seconds by the potentiostatic method to obtain a porous silicon-platinum nanoflower material on the polished surface of the silicon wafer.

[0049] (4) The same as Example 1.

[0050] Figure 3 For the sensor in this example, plot the response current at +0.20V against the melamine concentration (the testing method is the same as in Example 1). The figure shows that within the testing range, the peak current value has a good linear relationship with the logarithm of the melamine concentration, and its sensitivity is -6.60×10 -5 μA / nM.

[0051] Example 3

[0052] An electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material, comprising the following steps:

[0053] (1) and (2) are the same as in Example 2.

[0054] (3) Then contact the porous silicon surface of the sample with chloroplatinic acid electrolyte, and use the potentiostatic deposition method to obtain a porous silicon-platinum nanoflower material. Use a 20.0 g / L chloroplatinic acid solution containing 0.25 mol / L sulfuric acid as the electrolyte, with a working potential of 0.3V (vs Ag / AgCl), and deposit for 100 seconds by the potentiostatic method to obtain a porous silicon-platinum nanoflower material on the polished surface of the silicon wafer.

[0055] (4) The same as in Example 2.

[0056] Figure 4The response current of the sensor in this example at +0.20 V was plotted against the melamine concentration (the testing method was the same as in Example 1). The figure shows that within the testing range, the peak current value has a good linear relationship with the logarithm of the melamine concentration, and its sensitivity is -7.72×10 -5 μA / nM.

[0057] Figure 5 This is the CV test result of the sensor for melamine in this example. Figure 6 This is the selectivity test result of the sensor in this example. In a 0.2 M phosphate buffer solution containing 5 mM KCl with a pH of 7.2, the response signal of the sensor to 500 nmol / L melamine is one order of magnitude higher than the response signals of cyanuric acid, isocyanuric acid, urea, and bovine serum albumin at the same concentration. This proves that the sensor has good selectivity. In the solution environment used, the porous silicon surface carries negative charges, which has an electrostatic repulsion effect on common coexisting substances and interfering substances such as isocyanuric acid, cyanuric acid, and proteins that also carry negative charges, making the sensor have good selectivity.

[0058] As can be seen from the above examples, the sensor obtained by using this method has high sensitivity when detecting melamine. Moreover, during the preparation process, the sensitivity of melamine detection can be controlled by changing parameters such as the etching time of silicon, the concentration of chloroplatinic acid in the electrolyte, and the potentiostatic deposition time. For example, in Examples 1 - 3, by changing the etching conditions and electrodeposition conditions of the silicon material, the sensitivity of the porous silicon - platinum nanoflower - DNA composite sensor for detecting melamine was controlled from -3.26×10 -5 μA / nM to -7.72×10 -5 μA / nM. In addition, experiments have proved that this sensor has good selectivity.

[0059] The above is only the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a melamine electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material, characterized in that, It includes the following steps: (1) Prepare a porous silicon layer on the polished surface of the silicon wafer; (2) Deposit a metal film on the other side of the silicon wafer; (3) Deposit platinum nanoflowers on the surface of the porous silicon layer to obtain a porous silicon-platinum nanoflower material layer; (4) Modify a T-rich thiolated DNA probe on the surface of the porous silicon-platinum nanoflower material layer and seal it with 2-mercaptoethanol to obtain an electrochemical sensor based on a porous silicon-platinum nanoflower-DNA composite material.

2. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 1, characterized in that, In step (1), the silicon wafer is a single-sided polished p-type silicon wafer; The specific steps for preparing the porous silicon layer are as follows: Immerse the polished surface of the silicon wafer in a mixed solution of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, then take it out and immerse it in a 1-10% hydrofluoric acid solution for 10 minutes, and then transfer it to a 1-10% hydrofluoric acid and 1-10 mM silver nitrate aqueous solution and soak for 20-80 seconds to obtain metal nanoparticles on the silicon wafer, and wash with water; Put the sample into an aqueous solution of 0.1-0.5 M hydrogen peroxide and 5-20% hydrofluoric acid for corrosion for 10-60 minutes, and then soak it in a nitric acid solution with a concentration of 10-30 wt% to obtain a porous silicon layer.

3. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 1, characterized in that, In step (2), the thickness of the metal film is 10-500 nanometers; the material of the metal includes aluminum, copper, platinum, nickel, titanium, cobalt, palladium, and tungsten.

4. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 1, characterized in that, The specific steps of step (3) are as follows: Immerse the polysilicon layer surface of the silicon wafer in the electrolyte and deposit it by a constant potential method for 20-100 seconds; the electrolyte is a 5-25 g / L chloroplatinic acid solution containing 0.25 mol / L sulfuric acid.

5. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 1, characterized in that The nucleotide sequence of the thiolated DNA probe rich in T described in step (4) is: 5'-HS-C6H 12 -TTT TTT TTC TTT GTT TTG GTT CTT TCT TTG G-3'.

6. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 1, characterized in that, The specific steps of step (4) are as follows: Drop the thiolated DNA probe solution rich in T on the surface of the porous silicon-platinum nanoflower material layer, and after drying, seal it with 2-mercaptoethanol solution; the dropping amount of the thiolated DNA probe solution rich in T is 0.065 - 0.83 mL / cm 2 .

7. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 6, wherein the concentration of the T-rich thiolated DNA probe solution is 2.5 μM.

8. The preparation method of the melamine electrochemical sensor based on the porous silicon-platinum nanoflower-DNA composite material according to claim 6, characterized in that, The concentration of the 2-mercaptoethanol solution is 0.05-0.5 mol / L; the sealing time is 0.5-2 h.

9. A sensor prepared by the method according to any one of claims 1-8.

10. Application of the sensor prepared by the method according to claim 9 in the electrochemical detection of melamine.

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