An aptamer sensor for chronoamperometric detection and its preparation method and application

Abstract

The application belongs to the field of nano-biosensors, and discloses an aptamer sensor for chronoamperometry detection, a preparation method and application thereof. Gold nanoparticles are loaded on conductive glass by using in-situ reduction method, and then DNA aptamer is modified. The DNA aptamer can specifically adsorb target bacteria in a test solution on the surface of the material and generate a current response linearly related to the concentration of the bacteria solution. A PBS buffer solution containing MgCl2 is used as an electrolyte, and the chronoamperometry method can be used to measure the current response of different concentrations of Staphylococcus aureus. The new nano-biosensor provides a practical new method for on-site rapid detection of bacteria, and can be widely used in the fields of food detection and environmental detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical biosensors, specifically relating to an aptamer sensor for chronoamperometry detection, its preparation method and application, and particularly to a chronoamperometry sensor based on ITO conductive glass-gold nanoparticle-DNA composite material for Staphylococcus aureus detection, its preparation method and application. Background Technology

[0002] Microbiological testing in food is crucial for food safety. Currently used methods for detecting foodborne pathogens, such as plate culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), are cumbersome and time-consuming. Electrochemical detection methods have emerged to address this need. Due to their high sensitivity and ease of operation, electrochemical detection is widely used in analytical testing. Common electrochemical detection methods based on three-electrode systems include cyclic voltammetry, linear sweep voltammetry, differential pulse / square wave voltammetry, chronoamperometry, anodic stripping voltammetry, and electrochemical impedance spectroscopy. For example, Chinese patent CN103048369A uses a reduced graphene oxide-gold nanoparticle composite material to modify a thiolized Staphylococcus aureus whole-cell capture probe using a layer-by-layer self-assembly method. The binding of Staphylococcus aureus to the site causes a change in electrochemical impedance value, enabling quantitative detection of Staphylococcus aureus. However, this sensor is time-consuming to test, and the impedance method has a slow response, complex data interpretation, sensitivity to environmental interference, and poor stability. Chinese patent CN113588758A constructs a sandwich-type near-infrared photoelectrochemical sensor for Staphylococcus aureus. Although it also uses chronoamperometry, it requires the coordinated use of an excitation light source and a signal probe to obtain a photocurrent signal related to the concentration of Staphylococcus aureus. This detection system is complex to operate and costly. Similarly, CN112824884A constructs a photoelectrochemical aptamer sensor for chronoamperometry testing, but it requires intermittent illumination conditions, and this relatively complex operation limits its application. Chinese patent CN114088784A constructs an aptamer-catechol-chitosan membrane on a glassy carbon electrode and uses cyclic voltammetry to test Staphylococcus aureus. However, cyclic voltammetry requires continuous scanning within the potential window until the signal stabilizes, which is time-consuming, and the catechol used in the fabrication process is toxic and poses a certain environmental hazard. In summary, there is currently no simple electrochemical sensor for detecting Staphylococcus aureus based on chronoamperometry. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide an aptamer sensor for chronoamperometry detection, its preparation method, and its application.

[0004] The present invention first provides an electrochemical aptamer sensor for chronoamperometry detection, comprising gold nanoparticles sequentially modified on a conductive substrate, a modified electrode constructed from a DNA aptamer, and a PBS buffer solution as an electrolyte; the PBS buffer solution contains MgCl2.

[0005] Furthermore, the PBS buffer solution has a pH of 6.8, a phosphate concentration of 0.2 M, and a MgCl2 concentration of 5 mM.

[0006] Furthermore, the conductive substrate is selected from ITO conductive glass and FTO conductive glass.

[0007] This invention further provides a method for preparing the above-mentioned electrochemical aptamer sensor for chronoamperometry detection, comprising the following steps:

[0008] (1) Immerse ITO conductive glass in chloroauric acid solution, add sodium borohydride and mix thoroughly to react, and ITO conductive glass-gold nanoparticles can be obtained.

[0009] (2) Thiolized DNA aptamers were modified on the surface of ITO conductive glass-gold nanoparticles and blocked with 2-mercaptoethanol to obtain ITO conductive glass-gold nanoparticle-DNA composite material.

[0010] The specific method of step (1) is as follows: immerse the sample in a chloroauric acid solution with a mass fraction of 0.3-2%, add sodium borohydride with a mass fraction of 0.1-1.2%, shake to mix thoroughly, and continue the reaction for 10-60 minutes to obtain ITO conductive glass-gold nanoparticles.

[0011] Among them, the DNA aptamer mentioned in step (2) is the thiolated Staphylococcus aureus aptamer, and the nucleotide sequence of the thiolated Staphylococcus aureus aptamer is: 5'-SH-(CH2)6-GCTT CCAG CTTA TTGA ATAA AGAC GGGGGGGG GGAC CGGC GTAT GAGT GAAG ATGG GGGC GCTG AAGC GCGG AAGC.

[0012] The thiolated Staphylococcus aureus aptamer solution was prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH=7.5) at a concentration of 2.5 μM, and the modification amount was 0.5-10.0 mL.

[0013] The concentration of 2-mercaptoethanol is 0.05-0.5 mol / L, and the sealing time is 0.5-2h.

[0014] This invention also provides the application of the ITO conductive glass-gold nanoparticle-DNA composite material obtained by the above preparation method in the electrochemical detection of Staphylococcus aureus.

[0015] Specifically, using an ITO conductive glass-gold nanoparticle-DNA composite material as the working electrode, a three-electrode system was formed with a counter electrode and a reference electrode. PBS buffer solution containing MgCl2 was used as the electrolyte. The current response of Staphylococcus aureus at different concentrations was determined by chronoamperometry, and the concentration of Staphylococcus aureus in the test sample was calculated using the standard curve method.

[0016] Optionally, the counter electrode is a platinum wire electrode, and the reference electrode is a silver / silver chloride electrode.

[0017] Optionally, the parameters of the chronoamperometry method are: operating potential +0.32 V, scan time 7.5 s.

[0018] In use, the ITO conductive glass-gold nanoparticle-DNA composite material is used as the working electrode and connected to the electrochemical workstation circuit so that the electrical signal generated during the test is introduced into the circuit.

[0019] The electrochemical aptamer sensor for chronoamperometry detection and its preparation method provided by this invention have the following advantages compared with the prior art:

[0020] (1) This invention is based on the chronoamperometry method. It achieves rapid response by recording the change of current signal over time. It eliminates the need for complex sample pretreatment and long-term cultivation process, significantly shortening the detection cycle and providing a rapid and practical on-site detection method for food testing and environmental inspection.

[0021] (2) This invention employs a chemical reduction method to in-situ modify gold nanoparticles, significantly increasing the specific surface area of ​​the electrode, improving the amount of DNA aptamers immobilized, and enhancing electron transfer efficiency. Simultaneously, Mg is introduced into the PBS buffer solution. 2+ It can effectively induce conformational changes in DNA aptamers, enhancing their specific recognition ability for Staphylococcus aureus, thereby obtaining a significantly amplified current response signal and achieving highly sensitive detection of low concentrations of Staphylococcus aureus.

[0022] (3) The present invention uses DNA aptamers that can specifically recognize Staphylococcus aureus, effectively eliminating interference from other microorganisms or matrices in complex samples, and ensuring the accuracy and reliability of the detection results.

[0023] (4) The ITO conductive glass-gold nanoparticle-DNA composite material constructed in this invention has a stable structure, and the gold nanoparticles are firmly bonded to the conductive substrate and DNA aptamer. The modified electrode can maintain good electrochemical activity and bio-recognition performance, and has good repeatability and batch consistency, which is beneficial to the mass production and practical application of the sensor.

[0024] (5) The three-electrode system structure adopted in this invention is simple, the detection process does not require large precision instruments, the operation is simple, and it is easy to achieve miniaturization and portability. The ITO conductive glass used is low in cost and suitable for large-scale screening and use in grassroots units.

[0025] The aptamer sensor and its detection method provided by this invention can be widely used in the fields of pathogen monitoring in the food industry, detection of microbial pollution in environmental water bodies, and rapid screening of clinical samples, and have significant social benefits and good industrialization prospects. Attached Figure Description

[0026] Figure 1 This is a scanning electron microscope image of the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1.

[0027] Figure 2 The chronocurrent response of the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1 to different concentrations of Staphylococcus aureus is shown.

[0028] Figure 3 The results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1 are shown.

[0029] Figure 4 The results of cyclic voltammetry detection of Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1 are shown.

[0030] Figure 5 According to Figure 4 The relationship between the peak current and the bacterial concentration was obtained.

[0031] Figure 6 The results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in Example 2 are shown.

[0032] Figure 7 The results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in Example 3 are shown.

[0033] Figure 8The results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in Comparative Example 1 are shown. Detailed Implementation

[0034] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] The Staphylococcus aureus used in the following embodiments of the present invention was obtained from the American Type Culture Collection (ATCC) and was calibrated using the plate count method.

[0036] The electrochemical testing method used in the above specific embodiments of the present invention is as follows: The ITO conductive glass-gold nanoparticle-DNA composite material was incubated in diluted Staphylococcus aureus bacterial solution at a constant temperature of 37°C for 15 minutes. The material was then removed and rinsed with ultrapure water. It was then used as the working electrode, and a three-electrode system (with a platinum wire as the counter electrode and silver / silver chloride as the reference electrode) was used for chronoamperometry measurement in 0.2 M phosphate buffer solution (pH 6.8, containing 5 mM MgCl2). The electrode material made of the ITO conductive glass-gold nanoparticle-DNA composite material in this method is for single use. For each measurement, a new, unused electrode is selected, incubated in the test bacterial solution, and then washed. The response of the electrode to Staphylococcus aureus in the test bacterial solution is then studied using chronoamperometry in the aforementioned phosphate buffer solution.

[0037] The nucleotide sequence of the thiolized Staphylococcus aureus aptamer used in the following specific embodiments of the present invention is: 5'-SH-(CH2)6-GCTT CCAG CTTA TTGA ATAA AGAC GGGG GGGG GGAC CGGC GTAT GAGTGAAG ATGG GGGC GCTG AAGC GCGG AAGC, synthesized by Shanghai Sangon Biotech Co., Ltd. The thiolized Staphylococcus aureus aptamer test solution was prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH=7.5) at a concentration of 2.5 μM.

[0038] It should be noted that, unless otherwise specified, all raw materials or reagents used in the following embodiments of the present invention are commercially available products. Preparation methods or electrochemical detection methods not specifically described are conventional methods in the art.

[0039] Example 1

[0040] A method for fabricating a chronoamperometry sensor based on ITO conductive glass-gold nanoparticle-DNA composite material includes the following steps:

[0041] (1) Immerse the ITO conductive glass (20×40mm) in 5mL of 0.5% chloroauric acid solution, then add 0.5mL of 0.25% sodium borohydride and shake to mix thoroughly. Continue the reaction for 30 minutes to obtain ITO conductive glass-gold nanoparticles.

[0042] (2) Then, 3.0 mL of thiolated Staphylococcus aureus aptamer test solution was dropped onto the surface of ITO conductive glass-gold nanoparticle material, dried at room temperature, and then blocked with 0.1 mol / L 2-mercaptoethanol for 0.5 h to obtain ITO conductive glass-gold nanoparticle-DNA composite material.

[0043] (3) The electrochemical testing method is as follows: The ITO conductive glass-gold nanoparticle-DNA composite material was incubated in diluted Staphylococcus aureus bacterial solution for 15 minutes at a constant temperature of 37℃. The material was then removed and rinsed with ultrapure water. It was then used as the working electrode, and a three-electrode system (platinum wire as the counter electrode and silver / silver chloride as the reference electrode) was used for chronoamperometry measurement in 0.2 M phosphate buffer solution (pH 6.8, containing 5 mM MgCl2). The parameters for chronoamperometry were: working potential +0.32 V, scan time 7.5 s.

[0044] Figure 1 This is a scanning electron microscope image of the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1.

[0045] Figure 2 The chronocurrent response of the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1 to different concentrations of Staphylococcus aureus is shown.

[0046] Figure 3 The figure shows the results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material from Example 1. The figure also shows the results at 10~10... 9 Within the test range of CFU / mL, the current value showed a good linear relationship with the logarithm of the Staphylococcus aureus concentration (the linear fitting equation was: Y = -0.4008X + 12.7784, R0). 2 =0.998).

[0047] Figure 4 The results of detecting Staphylococcus aureus using cyclic voltammetry with the ITO conductive glass-gold nanoparticle-DNA composite material in Example 1 are shown. The conditions were: potential window -0.1~+0.5V, scan rate 50mV / s. Figure 5 According to Figure 4The relationship between the peak current and bacterial concentration was obtained. It can be seen that compared with the chronoamperometry method, the linear correlation between the signal obtained by the cyclic voltammetry and the concentration is weaker (the linear fitting equation is: Y = -0.41139X + 5.8758, R0). 2 =0.973).

[0048] Example 2

[0049] A method for fabricating a chronoamperometry sensor based on ITO conductive glass-gold nanoparticle-DNA composite material includes the following steps:

[0050] (1) Immerse the ITO conductive glass in 5 mL of 1.0% chloroauric acid solution, then add 0.5 mL of 0.5% sodium borohydride solution and shake to mix thoroughly. Continue the reaction for 30 minutes to obtain ITO conductive glass-gold nanoparticles.

[0051] (2) Then, 5.0 mL of thiolated Staphylococcus aureus aptamer solution was dropped onto the surface of ITO conductive glass-gold nanoparticles. After drying at room temperature, it was blocked with 0.1 mol / L 2-mercaptoethanol for 1.5 h to obtain ITO conductive glass-gold nanoparticle-DNA composite material.

[0052] Figure 6 The figure shows the results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in this embodiment. The figure shows a good linear relationship between the current value and the logarithm of the Staphylococcus aureus concentration within the test range (the linear fitting equation is: Y = -0.30X + 11.8342, R...). 2 =0.997).

[0053] Example 3

[0054] A method for fabricating a chronoamperometry sensor based on ITO conductive glass-gold nanoparticle-DNA composite material includes the following steps:

[0055] (1) Immerse the ITO conductive glass in 5 mL of 1.5% chloroauric acid solution, then add 0.5 mL of 0.75% sodium borohydride and shake to mix thoroughly. Continue the reaction for 45 minutes to obtain ITO conductive glass-gold nanoparticles.

[0056] (2) Then, 0.5 mL of thiolated Staphylococcus aureus aptamer solution was dropped onto the surface of ITO conductive glass-gold nanoparticles. After drying at room temperature, it was blocked with 7.5 mol / L 2-mercaptoethanol for 1.5 h to obtain ITO conductive glass-gold nanoparticle-DNA composite material.

[0057] Figure 7 The figure shows the results of detecting Staphylococcus aureus using the ITO conductive glass-gold nanoparticle-DNA composite material in this embodiment. The figure shows a good linear relationship between the current value and the logarithm of the Staphylococcus aureus concentration within the test range (the linear fitting equation is: Y = -0.25X + 11.0898, R0). 2 =0.996).

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 1 is that the electrolyte used for electrochemical detection does not contain MgCl2. It can be seen that, compared to Example 1, the signal obtained using an electrolyte without MgCl2 shows a weaker linear correlation with concentration (the linear fitting equation is: Y = -0.20X + 11.6659, R0). 2 =0.837).

[0060] As can be seen from the above examples, the ITO conductive glass-gold nanoparticle-DNA composite material obtained by this method can be used as a working electrode to detect Staphylococcus aureus by electrochemical methods.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An electrochemical aptamer sensor for chronoamperometry detection, characterized in that, The modified electrode, constructed by sequentially modifying gold nanoparticles and DNA aptamers onto a conductive substrate, serves as the working electrode, and PBS buffer solution serves as the electrolyte; the PBS buffer solution contains MgCl2.

2. The electrochemical aptamer sensor for chronoamperometry detection according to claim 1, characterized in that, The PBS buffer solution has a pH of 6.8, a phosphate concentration of 0.2 M, and a MgCl2 concentration of 5 mM.

3. A method for preparing an electrochemical aptamer sensor for chronoamperometry detection as described in claim 1, characterized in that, The method for preparing the working electrode includes the following steps: (1) Immerse ITO conductive glass in chloroauric acid solution, add sodium borohydride and mix thoroughly to react, and ITO conductive glass-gold nanoparticles can be obtained. (2) Thiolized DNA aptamers were modified on the surface of ITO conductive glass-gold nanoparticles and blocked with 2-mercaptoethanol to obtain ITO conductive glass-gold nanoparticle-DNA composite material.

4. The method for preparing an electrochemical aptamer sensor for chronoamperometry detection according to claim 3, characterized in that, The specific method of step (1) is as follows: Immerse the ITO conductive glass in a chloroauric acid solution with a mass fraction of 0.3-2%, then add sodium borohydride with a mass fraction of 0.1-1.2%, shake to mix thoroughly, and continue the reaction for 10-60 minutes to obtain ITO conductive glass-gold nanoparticles.

5. The method for preparing an electrochemical aptamer sensor for chronoamperometry detection according to claim 1, characterized in that, The aptamer mentioned in step (2) is a thiolized Staphylococcus aureus aptamer, whose nucleotide sequence is: 5'-SH-(CH2)6-GCTT CCAG CTTA TTGA ATAA AGAC GGGG GGGG GGAC CGGC GTAT GAGT GAAGATGG GGGC GCTG AAGC GCGG AAGC.

6. The method for preparing an electrochemical aptamer sensor for chronoamperometry detection according to claim 1, characterized in that, The concentration of the thiolized DNA aptamer solution is 2.5 μmol / L, and the modification amount is 0.5-10.0 mL.

7. The method for preparing an electrochemical aptamer sensor for chronoamperometry detection according to claim 1, characterized in that, The concentration of the 2-mercaptoethanol is 0.05-0.5 mol / L, and the sealing time is 0.5-2h.

8. The application of the electrochemical aptamer sensor for chronoamperometry detection as described in claim 1 or 2 in the electrochemical detection of Staphylococcus aureus.

9. The application according to claim 8, characterized in that, The working electrode, together with the counter electrode and the reference electrode, forms a three-electrode system. The current response of Staphylococcus aureus at different concentrations is determined by electrochemical chronoamperometry, and the concentration of Staphylococcus aureus in the sample is calculated by the standard curve method.

10. The application according to claim 9, characterized in that, The parameters for the chronoamperometry method are: operating potential +0.32 V, scan time 7.5 s.

Images