An electrochemical aptasensor and its preparation method and application

Abstract

The present invention belongs to the technical field of biosensors, and particularly relates to an electrochemical aptamer sensor, a preparation method and an application thereof, comprising the following steps: mixing a chloroauric acid solution with a carbon quantum dot solution, heating in an oil bath to obtain an Au@CDs suspension, adding the Au@CDs suspension into a chitosan solution and ultrasonically mixing to obtain an Au@CDs / CS composite nanomaterial; using a gold silicon wafer as a working electrode, modifying the surface of the working electrode with the Au@CDs / CS composite nanomaterial, and using glutaraldehyde as a cross-linking agent to self-assemble an amino-modified aptamer on the electrode surface to obtain an electrochemical aptamer sensor. The present invention selects a nanomaterial to modify the working electrode, which can improve the electrochemical activity of the sensing interface, increase the binding sites and binding stability of the recognition element, and is one of the important strategies for improving the sensitivity and stability of the electrochemical sensor.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to an electrochemical aptamer sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Illicit drugs and their metabolites are discharged into municipal sewage with urine, feces, etc. Dynamically monitoring the types, concentrations, and discharge area characteristics of illicit drugs and their metabolites in municipal sewage, based on the principle of sewage epidemiology method, can be used to evaluate the per capita consumption of illicit drugs in a specific area.

[0003] Moreover, real-time monitoring and traceability analysis of the concentration levels of illicit drugs and their metabolites in community sewage pipelines can be used as a drug testing technology.

[0004] In addition, illicit drugs that are not effectively removed by the sewage treatment process will eventually enter the surface water system, and even pollute groundwater and drinking water sources, which are a type of new pollutant. It is reported that the detection rate of illicit drugs in tap water in different regions of the world has reached 4 - 34%. Although the concentration level of illicit drugs in environmental water bodies is relatively low (ng / L), due to their high biological targeting activity, they can have obvious ecological toxic effects on aquatic organisms at trace levels, and can harm terrestrial organisms and human health through bioaccumulation, food chain transmission, drinking water, etc., posing an ecological risk that cannot be ignored.

[0005] However, due to the trace concentration levels (ng / L) of illicit drugs and their metabolites in sewage and the presence of many interfering substances, the sensitivity and specificity requirements for detection methods are relatively high. Currently, the main detection methods are extraction and concentration pretreatment and ultra-high performance liquid chromatography - tandem mass spectrometry (LC-MS / MS). The detection is time-consuming, laborious, costly, and can only be carried out in the laboratory, unable to meet the requirements of on-site real-time rapid monitoring and big data analysis of illicit drugs in sewage. Most of the existing portable detection methods for illicit drugs and their metabolites in sewage are improvements based on fluorescence immunoassay strips and portable spectroscopy methods used for biological samples. Not only does the water sample need to be concentrated and pretreated, but only qualitative and semi-quantitative detection of drugs can be carried out. Compared with spectroscopy, electrochemical methods are not sensitive to light and are not easily interfered by the sample matrix. A few use electrochemical methods for detection, but there are still problems of high detection limits and low sensitivity when applied to water sample detection, and pre-concentration treatment is required. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide an electrochemical aptamer sensor, a preparation method thereof, and an application thereof. Selecting nanomaterials to modify the working electrode can improve the electrochemical activity of the sensing interface, increase the binding sites and binding stability of the recognition element, which is one of the important strategies to improve the sensitivity and stability of electrochemical sensors.

[0007] To solve the above technical problems, the present invention adopts the following technical methods:

[0008] A preparation method of an electrochemical aptasensor, comprising the following steps:

[0009] Using a biomass material as a raw material, adding it to water, carbonizing it by microwave heating to obtain a carbonized solution, centrifuging and filtering the carbonized solution, and then concentrating and drying it by rotary evaporation to obtain dried carbon quantum dots. Then, the dried carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution;

[0010] Adopting an in-situ growth method, using carbon quantum dots as a reducing agent to in-situ reduce Au(Ⅲ) in chloroauric acid to gold nanoparticles (AuNPs). Mix the chloroauric acid solution with the carbon quantum dot solution and heat it in an oil bath until a stable purple-red Au@CDs suspension is formed. To enhance the adhesion of the composite nanomaterial, introduce the natural polymer material chitosan (CS), add the Au@CDs suspension to the chitosan solution and ultrasonically mix to obtain an Au@CDs / CS composite nanomaterial;

[0011] Using a gold silicon wafer as a working electrode, modifying the surface of the working electrode with the Au@CDs / CS composite nanomaterial, using glutaraldehyde as a cross-linking agent, self-assembling the amino-modified aptamer on the electrode surface, and blocking the active sites on the electrode surface with bovine serum albumin (BSA) to obtain an electrochemical aptasensor.

[0012] In a preferred embodiment of the present invention, the volume ratio of the chloroauric acid solution to the carbon quantum dot solution is 0.5 - 5, the concentration of the chloroauric acid solution is 0.5 mg / mL - 2 mg / mL, and the concentration of the carbon quantum dot solution is 3 mg / mL - 8 mg / mL.

[0013] In a preferred embodiment of the present invention, the concentration of the chitosan solution is 0.5 mg / mL - 2.5 mg / mL, and the volume ratio of the chitosan solution to the AuCDs suspension is 1:1.

[0014] In a preferred embodiment of the present invention, the heating temperature in the oil bath is 60°C - 100°C, and the heating time is 60 min - 90 min.

[0015] In a preferred embodiment of the present invention, the output frequency of the microwave heating is 500 W - 700 W, and the heating time is 20 min - 40 min.

[0016] In a preferred embodiment of the present invention, the biomass material is banana peel.

[0017] Another object of the present invention is to provide an electrochemical aptamer sensor prepared by the preparation method described in any one of the above.

[0018] A third object of the present invention is to provide an application of the electrochemical aptamer sensor described above in detecting trace methamphetamine in environmental water bodies.

[0019] In a preferred embodiment of the present invention, the detection range of methamphetamine in water is 1 pg / L to 1 μg / L, the detection limit reaches 0.87 pg / L, and the spiked recovery rate can reach 92.4 - 104.6%.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention uses biomass materials as raw materials, carbonizes them by microwave heating to obtain a carbonized solution, and obtains dry carbon quantum dots after post-treatment of the carbonized solution. Mix the chloroauric acid solution and the carbon quantum dot solution, heat in an oil bath to obtain an Au@CDs suspension, and add the Au@CDs suspension to the chitosan solution and mix by ultrasonic to obtain an Au@CDs / CS composite nanomaterial; use a gold-silicon wafer as a working electrode, modify the surface of the working electrode with the Au@CDs / CS composite nanomaterial, and use glutaraldehyde as a cross-linking agent to self-assemble the amino-modified aptamer on the electrode surface to obtain an electrochemical aptamer sensor. The present invention uses chloroauric acid (HAuCl 4 ) and carbon quantum dots (CDs) as raw materials to prepare Au@CDs composite nanomaterials. Selecting a nano metal-carbon-based composite material as the working electrode modification material, the special composite nanomaterial has the advantages of monomer nanomaterials and unique properties such as high specific surface area, high conductivity, and high stability, and can realize the multiple amplification effect of electrochemical signals, improving the sensitivity and stability of the sensor.

[0022] 2. The present invention uses a gold-silicon wafer as a working electrode and an Au@CDs composite nanomaterial as the working electrode modification material, and uses an aptamer as a biorecognition element to prepare a highly sensitive electrochemical aptamer biosensor for detecting methamphetamine in water bodies. It has good detection performance such as stability / reproducibility and specificity. This method is applicable to the detection of trace methamphetamine in actual water samples such as drinking water, surface water, and sewage. The purpose of quickly detecting trace methamphetamine in water bodies with a micro sample (ten microliters) can be achieved, avoiding the cumbersome water sample preconcentration treatment steps, and laying a foundation for the real-time online monitoring of prohibited drugs in environmental water bodies.

[0023] 3. The present invention selects nanomaterials to modify the working electrode, which can improve the electrochemical activity of the sensing interface, increase the binding sites and binding stability of the recognition element, and is one of the important strategies to improve the sensitivity and stability of electrochemical sensors. Description of the Drawings

[0024] Figure 1 Schematic diagram of the preparation and detection process of the electrochemical aptasensor of the present invention.

[0025] Figure 2 Standard curve of the electrochemical aptasensor prepared in Example 1 of the present invention.

[0026] Figure 3 (a) Reproducibility; (b) Stability; (c) Specificity test results of the electrochemical aptasensor prepared in Example 1 of the present invention.

[0027] Figure 4 Results of the influence of water quality parameters (a) pH, (b) temperature, (c) suspended particulate matter, (d) ionic strength on the performance of the electrochemical aptasensor prepared in Example 1 of the present invention. Detailed implementation mode

[0028] The following combines the embodiments of the present invention and uses preferred embodiments and accompanying drawings for detailed description. The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0029] It should be noted that all the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be obtained through the market or prepared by existing methods.

[0030] Example 1

[0031] A preparation method of an electrochemical aptasensor for detecting methamphetamine in water bodies, comprising the following steps:

[0032] (1) Weigh 5 g of fresh banana peel and add it to 100 mL of deionized water to obtain a mixture. Then transfer the mixture to a microwave oven and heat it to carbonize it. The heating microwave frequency is 600 W and the heating time is 30 min. Centrifuge and filter the carbonized solution to remove large particles. Finally, pre-concentrate the solution by rotary evaporation and freeze-dry it to obtain dry carbon quantum dots. Dissolve the dry carbon quantum dots in water to obtain a carbon quantum dot solution.

[0033] (2) Add the 1 mg / mL HAuCl 4 solution to the 5 mg / mL carbon quantum dot solution in a certain proportion to obtain a mixed solution, where HAuCl 4The volume ratio of the solution to the carbon quantum dot solution is 2. Then, the mixed solution is heated in an oil bath at 90 °C for 80 min until a stable purple-red Au@CDs suspension is formed. To enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan is introduced. The Au@CDs suspension is added to the 2 mg / mL chitosan solution according to a volume ratio of 1:1 and ultrasonically mixed for 15 min to obtain the Au@CDs / CS composite nanomaterial.

[0034] (3) Using a gold silicon wafer (area 20 mm 2 ) as the working electrode, 10 μL of the Au@CDs / CS composite nanomaterial is modified on the electrode surface. Using glutaraldehyde as a cross-linking agent, the electrode is immersed in glutaraldehyde for 1 h. 10 μL of the amino-modified aptamer with a concentration of 1 μM is self-assembled on the electrode surface. After incubation for 8 h, 10 μL of bovine serum albumin (BSA) with a concentration of 1% is used to block the active sites on the electrode surface to prepare the electrochemical aptamer biosensor.

[0035] Example 2

[0036] A preparation method of an electrochemical aptamer sensor for methamphetamine detection in water bodies, comprising the following steps:

[0037] (1) Weigh 5 g of fresh banana peel and add it to 100 mL of deionized water to obtain a mixture. Then, transfer the mixture to a microwave oven and heat it to carbonize. The microwave heating frequency is 500 W and the heating time is 40 min. The carbonized solution is centrifuged and filtered to remove large particles. Finally, the solution is pre-concentrated by rotary evaporation and freeze-dried to obtain dry carbon quantum dots. The dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution.

[0038] (2) Add the 0.5 mg / mL HAuCl 4 solution to the 3 mg / mL carbon quantum dot solution in a certain proportion to obtain a mixed solution, where the volume ratio of the HAuCl 4 solution to the CDs solution is 0.5. Then, the mixed solution is heated in an oil bath at 60 °C for 90 min until a stable purple-red Au@CDs suspension is formed. To enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan is introduced. The Au@CDs suspension is added to the 0.5 mg / mL chitosan solution according to a volume ratio of 1:1 and ultrasonically mixed for 15 min to obtain the Au@CDs / CS composite nanomaterial.

[0039] (3) Using a gold silicon wafer (area 20 mm 2) As the working electrode, 10 μL of the Au@CDs / CS composite nanomaterial was modified on the electrode surface. Using glutaraldehyde as a crosslinking agent, the electrode was immersed in glutaraldehyde for 1 h. Then, 10 μL of the amino-modified aptamer with a concentration of 1 μM was self-assembled on the electrode surface. After incubation for 8 h, 10 μL of bovine serum albumin (BSA) with a concentration of 1% was used to block the active sites on the electrode surface, thus preparing the electrochemical aptamer biosensor.

[0040] Example 3

[0041] A preparation method of an electrochemical aptamer sensor for detecting methamphetamine in water bodies, comprising the following steps:

[0042] (1) Weigh 5 g of fresh banana peel and add it to 100 mL of deionized water to obtain a mixture. Then, transfer the mixture to a microwave oven and heat it to carbonize. The heating microwave frequency is 700 W and the heating time is 20 min. Centrifuge and filter the carbonized solution to remove large particles. Finally, pre-concentrate the solution by rotary evaporation and freeze-dry it to obtain dry carbon quantum dots. Dissolve the dry carbon quantum dots in water to obtain a carbon quantum dot solution.

[0043] (2) Add the 2 mg / mL HAuCl 4 solution to the 8 mg / mL CDs solution in a certain proportion, where the volume ratio of the HAuCl 4 solution to the CDs solution is 5. Then, heat the mixed solution in an oil bath at 100 °C for 60 min until a stable purple-red Au@CDs suspension is formed. To enhance the adhesion of the composite nanomaterial, introduce the natural polymer material chitosan. Add the Au@CDs suspension to the 2.5 mg / mL chitosan solution in a volume ratio of 1:1 and ultrasonically mix for 15 min to obtain the Au@CDs / CS composite nanomaterial.

[0044] (3) Using a gold silicon wafer (area 20 mm 2 ) as the working electrode, 10 μL of the Au@CDs / CS composite nanomaterial was modified on the electrode surface. Using glutaraldehyde as a crosslinking agent, the electrode was immersed in glutaraldehyde for 1 h. Then, 10 μL of the amino-modified aptamer with a concentration of 1 μM was self-assembled on the electrode surface. After incubation for 8 h, 10 μL of bovine serum albumin (BSA) with a concentration of 1% was used to block the active sites on the electrode surface, thus preparing the electrochemical aptamer biosensor.

[0045] Result analysis

[0046] The electrochemical aptamer sensor prepared in Example 1 of the present invention was used to detect methamphetamine. The schematic diagram of its preparation and detection is as Figure 1 shown. Using Ag / AgCl as the reference electrode and a platinum sheet as the counter electrode, 0.1 mol L-1 5 mmol / L in KCl solution -1 [Fe(CN)6] 3- / 4- (1:1) as a redox probe, the detection range of methamphetamine in water is 1 pg / L - 1 μg / L, the detection limit reaches 0.87 pg / L, the spiked recovery rate can reach 92.4 - 104.6%, the standard curve is y = 1.0391log C + 4.52, the correlation coefficient r = 0.997, and the standard curve is as Figure 2 shown.

[0047] Five parallel electrochemical aptasensors were prepared for the detection of methamphetamine at a concentration of 0.1 ng / L. As Figure 3 shown in a, the electrochemical signal changes of methamphetamine detected by five parallel sensors are small, and the relative standard deviation (RSD) is 2.8%, indicating that the prepared electrochemical aptasensors have good reproducibility.

[0048] After the prepared electrochemical aptasensors were stored at 4°C for 1, 3, 5, 7, 10, and 15 days respectively, they were used for the detection of 0.1 ng / L methamphetamine. From Figure 3 b, it can be seen that after the electrochemical aptasensors were stored for 15 days, the electrochemical signal intensity can still maintain 94.4% of the initial value, indicating that the prepared electrochemical aptasensors have good stability.

[0049] Humic acid (HA), sodium alginate (SA), and bovine serum albumin (BSA) were used to simulate typical dissolved organic matter humus, polysaccharides, and protein substances in water respectively. 100 mg / L of HA, SA, BSA, and 0.1 ng / L methamphetamine were incubated on the surface of the electrochemical aptasensor respectively. The electrochemical signal response value (ΔJ) of 0.1 ng / L methamphetamine is 9.8 times, 170 times, and 4.3 times that of 100 mg / L HA, SA, and BSA, significantly higher than the ΔJ of three typical dissolved organic matters, indicating that the prepared electrochemical aptasensor has good specificity for methamphetamine ( Figure 3 c).

[0050] In order to investigate the influence of water quality parameters on the performance of the electrochemical aptasensor, at different pH (5 - 8), temperature (4 - 30°C), suspended particulate matter (0 - 400 mg / L), and ionic strength (0 - 100 mM), the prepared sensor was used to detect 0.1 ng / L methamphetamine. From Figure 4It can be seen that when the pH is in the range of 5 - 6, the electrochemical signal response value ΔJ is relatively low. When the pH is 7 and 8, ΔJ increases significantly, indicating that the prepared electrochemical aptamer sensor is suitable for pH conditions that are slightly alkaline to neutral. The pH ranges of actual drinking water, surface water, and sewage are mostly in the range of 7 - 8. Therefore, the prepared electrochemical aptamer sensor is suitable for the detection of actual water bodies.

[0051] When the temperature is in the range of 4 - 20 °C, ΔJ shows an upward trend, but the change is not significant, and the error is about 3%. When the temperature is 30 °C, ΔJ increases significantly. This indicates that the prepared electrochemical aptamer sensor is suitable for a relatively wide environmental water temperature range (4 - 20 °C). Figure 4 b).

[0052] As Figure 4 shown in c, ΔJ gradually increases with the increase in the concentration of suspended particles, indicating that the suspended particles in the water body have a greater impact on the electrochemical signal of the sensor. During the actual detection process, the water sample can be detected after simple filtration.

[0053] When the ionic strength is in the range of 0 - 10 mM, ΔJ does not change significantly, and the electrochemical signal of the sensor is not affected. When the ionic strength is 10 - 100 mM, ΔJ increases significantly, affecting the electrochemical signal of the sensor. For actual water bodies, the ionic strength ranges of drinking water and surface water are generally 0.5 - 5 mM, and the ionic strength of sewage is usually higher than 30 mM. This indicates that the electrochemical aptamer sensor is not affected by ionic strength when detecting methamphetamine in drinking water and surface water, but may be affected by ionic strength when detecting methamphetamine in sewage.

[0054] Methamphetamine with concentrations of 0.1, 1, and 10 ng / L was added to tap water, river water, lake water, and sewage respectively, and the electrochemical aptamer sensor was used for detection. The detection results are shown in Table 1. As can be seen from the table, the average spiked recovery rate of methamphetamine in the four actual water samples is 92.4 - 104.6%, indicating that the prepared electrochemical aptamer sensor is suitable for the detection of trace methamphetamine in different types of environmental water bodies.

[0055] Table 1 Spiked detection results of actual water samples

[0056]

[0057] In summary, the present invention uses a gold-silicon wafer as a working electrode, an Au@CDs composite nanomaterial as a working electrode modification material, and an aptamer as a biorecognition element to prepare a highly sensitive electrochemical aptasensor for detecting methamphetamine in water. It has good detection performance such as stability / reproducibility and specificity. This method is applicable to the detection of trace methamphetamine in actual water samples such as drinking water, surface water, and sewage. The purpose of quickly detecting trace methamphetamine in water with a micro-sample (ten microliters) is achieved, avoiding the cumbersome water sample preconcentration treatment steps, and laying a foundation for the real-time on-line monitoring of prohibited drugs in environmental water bodies.

[0058] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0059] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Application of an electrochemical aptamer sensor in detecting trace amounts of methamphetamine in environmental water, characterized in that: The detection range of methamphetamine in water is 1 pg / L~1 μg / L, the detection limit is 0.87 pg / L, and the spike recovery rate can reach 85.7%~104.6%; The preparation method of the electrochemical aptamer sensor comprises the following steps: Biomass material is used as a raw material, added into water, and carbonized by microwave heating to obtain a carbonized solution, the carbonized solution is centrifuged, filtered, concentrated, and dried to obtain dry carbon quantum dots, and then the dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution; Mixing the chloroauric acid solution with the carbon quantum dot solution, heating in an oil bath to obtain an Au@CDs suspension, adding the Au@CDs suspension to a chitosan solution for ultrasonic mixing to obtain an Au@CDs / CS composite nanomaterial; The volume ratio of chloroauric acid solution to carbon quantum dot solution is 2, the concentration of chloroauric acid solution is 0.5 mg / mL~2 mg / mL, and the concentration of carbon quantum dot solution is 5 mg / mL; The Au@CDs / CS composite nanomaterial is modified on the working electrode surface with a gold silicon wafer as the working electrode, and the amino-modified aptamer is self-assembled on the electrode surface with glutaraldehyde as a cross-linking agent to obtain an electrochemical aptamer sensor.

2. The use of the electrochemical aptamer sensor according to claim 1 in detecting trace amounts of methamphetamine in environmental water, characterized in that: The concentration of chitosan solution was 0.5 mg / mL~2.5 mg / mL, and the volume ratio of chitosan solution to Au@CDs suspension was 1:

1.

3. The use of the electrochemical aptamer sensor according to claim 1 in detecting trace amounts of methamphetamine in environmental water, characterized in that: The heating temperature in the oil bath is 60°C~100°C, and the heating time is 60 min~90 min.

4. The use of the electrochemical aptamer sensor according to claim 1 in detecting trace amounts of methamphetamine in environmental water, characterized in that: The microwave heating output frequency is 500 W~700 W, and the heating time is 20 min~40 min.

5. The use of the electrochemical aptamer sensor according to claim 1 in detecting trace amounts of methamphetamine in environmental water, characterized in that: The biomass material is banana peel.

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