Electrochemical aptamer sensor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to an electrochemical aptamer sensor and a preparation method and application thereof.The preparation method comprises the following steps that a chloroauric acid solution and a carbon quantum dot solution are mixed and heated in an oil bath, Au-CDs suspension liquid is obtained, the Au-CDs suspension liquid is added into a chitosan solution, ultrasonic mixing is conducted, and an Au-CDs / CS composite nanomaterial is obtained; the electrochemical aptamer sensor is obtained by taking a gold silicon wafer as a working electrode, modifying the Au-coated CDs / CS composite nanomaterial on the surface of the working electrode, and self-assembling an amino-modified aptamer on the surface of the electrode by taking glutaraldehyde as a cross-linking agent. Increasing the binding site and binding stability of the recognition element is one of 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 in particular relates to an electrochemical aptamer sensor and a preparation method and application thereof. Background Art

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

[0003] Not only that, real-time monitoring and traceability analysis of the concentration levels of illegal drugs and their metabolites in community sewage networks can be used as a drug testing technology.

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

[0005] However, since the content of banned drugs and their metabolites in sewage is mostly at trace concentration levels (ng / L) and there are many interfering substances, the sensitivity and specificity of the detection method are required to be high. At present, the detection methods mainly use extraction concentration pretreatment and ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). The detection is time-consuming, labor-intensive, and costly, and can only be performed in the laboratory, which cannot meet the needs of real-time rapid monitoring and big data analysis of banned drugs in sewage. Most of the existing portable detection methods for banned drugs and their metabolites in sewage are improved on the basis of fluorescent immunoassays and portable spectroscopy used in biological samples. Not only do water samples need to be concentrated and pretreated, but only qualitative and semi-quantitative detection of drugs can be performed. Compared with spectroscopy, electrochemical methods are insensitive to light and are not easily interfered by sample matrices. 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] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide an electrochemical adapter sensor and its preparation method and application. The use of 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, and is one of the important strategies to improve the sensitivity and stability of electrochemical sensors.

[0007] In order to solve the above technical problems, the present invention adopts the following technical methods:

[0008] A method for preparing an electrochemical adaptor sensor comprises the following steps:

[0009] 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 and filtered, and then concentrated and dried by rotary evaporation to obtain dry carbon quantum dots, and then the dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution;

[0010] The in-situ growth method is adopted, and Au(III) in chloroauric acid is in-situ reduced to gold nanoparticles (AuNPs) using carbon quantum dots as a reducing agent. The chloroauric acid solution is mixed with the carbon quantum dot solution, and heated in an oil bath until a stable purple-red Au@CDs suspension is generated. In order to enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan (CS) is introduced, and the Au@CDs suspension is added to the chitosan solution for ultrasonic mixing to obtain the Au@CDs / CS composite nanomaterial;

[0011] The Au@CDs / CS composite nanomaterial was modified on the working electrode surface with a gold silicon wafer as the working electrode, glutaraldehyde was used as a cross-linking agent, the amino-modified aptamer was self-assembled on the electrode surface, and bovine serum albumin (BSA) was used to block the active sites on the electrode surface to obtain an electrochemical aptamer sensor.

[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 to 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. to 100° C., and the heating time is 60 min to 90 min.

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

[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 any of the preparation methods described above.

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

[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 is 0.87 pg / L, and the spike recovery rate can reach 92.4% to 104.6%.

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

[0021] 1. The present invention uses biomass materials as raw materials, carbonizes them by microwave heating to obtain a carbonized solution, and then post-processes the carbonized solution to obtain dried carbon quantum dots. A chloroauric acid solution is mixed with a carbon quantum dot solution, and heated in an oil bath to obtain an Au@CDs suspension. The Au@CDs suspension is added to a chitosan solution for ultrasonic mixing to obtain an Au@CDs / CS composite nanomaterial; a gold silicon wafer is used as a working electrode, and the Au@CDs / CS composite nanomaterial is modified on the surface of the working electrode. Glutaraldehyde is used as a cross-linking agent, and an amino-modified aptamer is self-assembled on the electrode surface to obtain an electrochemical aptamer sensor. The present invention uses chloroauric acid (HAuCl4) and carbon quantum dots (CDs) as raw materials to prepare Au@CDs composite nanomaterials, and selects nano-metal-carbon composite materials as working electrode modification materials. The special composite nanomaterial has the advantages of monomer nanomaterials, and has unique properties such as high specific surface area, high conductivity, and high stability, which can achieve multiple amplification effects of electrochemical signals and improve the sensitivity and stability of the sensor.

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

[0023] 3. The present invention uses nanomaterials to modify the working electrode, which can improve the electrochemical activity of the sensing interface and increase the binding sites and binding stability of the recognition element. It is one of the important strategies to improve the sensitivity and stability of electrochemical sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the preparation and detection process of the electrochemical aptamer sensor of the present invention.

[0025] Figure 2 This is the standard curve of the electrochemical aptamer sensor prepared in Example 1 of the present invention.

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

[0027] Figure 4 The results show the effects of water quality parameters (a) pH, (b) temperature, (c) suspended particles, and (d) ionic strength on the performance of the electrochemical aptamer sensor prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The following is a detailed description of the technical solutions in the embodiments of the present invention in combination with the preferred embodiments and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] It should be noted that all professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection 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 purchased from the market or prepared by existing methods.

[0030] Example 1

[0031] A method for preparing an electrochemical aptamer sensor for detecting methamphetamine in water comprises 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, the heating time is 30 min, the carbonized solution is centrifuged to remove large particles, and finally, the solution is pre-concentrated by rotary evaporation and freeze-dried to obtain dry carbon quantum dots, and the dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution.

[0033] (2) 1 mg / mL HAuCl4 solution was added to 5 mg / mL carbon quantum dot solution in a certain proportion to obtain a mixed solution, wherein the volume ratio of HAuCl4 solution to carbon quantum dot solution was 2, and then the mixed solution was heated in a 90°C oil bath for 80 min until a stable purple-red Au@CDs suspension was generated. In order to enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan was introduced, and the Au@CDs suspension was added to 2 mg / mL chitosan solution in a volume ratio of 1:1 and ultrasonically mixed for 15 min to obtain Au@CDs / CS composite nanomaterial.

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

[0035] Example 2

[0036] A method for preparing an electrochemical aptamer sensor for detecting methamphetamine in water comprises 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 it, the heating microwave frequency is 500 W, the heating time is 40 min, the carbonized solution is centrifuged to remove large particles, and finally, the solution is pre-concentrated by rotary evaporation and freeze-dried to obtain dry carbon quantum dots, and the dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution.

[0038] (2) 0.5 mg / mL HAuCl4 solution was added to 3 mg / mL carbon quantum dot solution in a certain proportion to obtain a mixed solution, wherein the volume ratio of HAuCl4 solution to CDs solution was 0.5, and then the mixed solution was heated in a 60°C oil bath for 90 min until a stable purple-red Au@CDs suspension was generated. In order to enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan was introduced, and the Au@CDs suspension was added to the 0.5 mg / mL chitosan solution in a volume ratio of 1:1 and ultrasonically mixed for 15 min to obtain the Au@CDs / CS composite nanomaterial.

[0039] (3) Gold silicon wafer (area 20mm 2) was used as the working electrode, 10 μL of Au@CDs / CS composite nanomaterials were modified on the electrode surface, glutaraldehyde was used as a cross-linking agent, the electrode was immersed in glutaraldehyde for 1 h, 10 μL of 1 μM amino-modified aptamer was self-assembled on the electrode surface, after incubation for 8 h, 10 μL of 1% bovine serum albumin (BSA) was used to block the active sites on the electrode surface to prepare an electrochemical aptamer biosensor.

[0040] Example 3

[0041] A method for preparing an electrochemical aptamer sensor for detecting methamphetamine in water comprises 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 it, the heating microwave frequency is 700 W, the heating time is 20 min, the carbonized solution is centrifuged to remove large particles, and finally, the solution is pre-concentrated by rotary evaporation and freeze-dried to obtain dry carbon quantum dots, and the dry carbon quantum dots are dissolved in water to obtain a carbon quantum dot solution.

[0043] (2) 2 mg / mL HAuCl4 solution was added to 8 mg / mL CDs solution in a certain proportion, where the volume ratio of HAuCl4 solution to CDs solution was 5. The mixed solution was then heated in an oil bath at 100°C for 60 min until a stable purple-red Au@CDs suspension was generated. In order to enhance the adhesion of the composite nanomaterial, the natural polymer material chitosan was introduced. The Au@CDs suspension was added to 2.5 mg / mL chitosan solution in a volume ratio of 1:1 and ultrasonically mixed for 15 min to obtain Au@CDs / CS composite nanomaterials.

[0044] (3) Gold silicon wafer (area 20mm 2 ) was used as the working electrode, 10 μL of Au@CDs / CS composite nanomaterials were modified on the electrode surface, glutaraldehyde was used as a cross-linking agent, the electrode was immersed in glutaraldehyde for 1 h, 10 μL of 1 μM amino-modified aptamer was self-assembled on the electrode surface, after incubation for 8 h, 10 μL of 1% bovine serum albumin (BSA) was used to block the active sites on the electrode surface to prepare an electrochemical aptamer biosensor.

[0045] Results Analysis

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

[0047] Five parallel electrochemical aptasensors were prepared for the detection of methamphetamine at a concentration of 0.1 ng / L. Figure 3 As shown in a, the electrochemical signals of methamphetamine detected by 5 parallel sensors changed slightly, with a relative standard deviation (RSD) of 2.8%, indicating that the prepared electrochemical aptasensor has good reproducibility.

[0048] The prepared electrochemical aptasensor was stored at 4°C for 1, 3, 5, 7, 10 and 15 days, and then used to detect 0.1 ng / L methamphetamine. Figure 3 b It can be seen that after the electrochemical aptamer sensor was stored for 15 days, the electrochemical signal intensity can still maintain 94.4% of the initial value, indicating that the prepared electrochemical aptamer sensor has good stability.

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

[0050] In order to investigate the effect of water quality parameters on the performance of electrochemical aptamer sensors, the prepared sensors were used to detect methamphetamine at a concentration of 0.1 ng / L under different pH (5-8), temperature (4-30°C), suspended particles (0-400 mg / L) and ionic strength (0-100 mM). Figure 4 It can be seen that at pH 5-6, the electrochemical signal response value ΔJ is low, and at pH 7 and 8, ΔJ increases significantly, indicating that the prepared electrochemical aptamer sensor is suitable for neutral to alkaline pH conditions. The pH range of actual drinking water, surface water and sewage is mostly in the range of 7-8, so the prepared electrochemical aptamer sensor is suitable for the detection of actual water bodies.

[0051] At temperatures between 4 and 20°C, ΔJ shows an upward trend, but the change is not large, with an error of about 3%. At 30°C, ΔJ increases significantly. This indicates that the prepared electrochemical aptamer sensor is suitable for a wide range of ambient water temperatures (4 to 20°C). Figure 4 b).

[0052] Depend on Figure 4 c shows that ΔJ increases gradually with the increase of 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. In the actual detection process, the water sample can be detected after simple filtration.

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

[0056]

[0057] In summary, the present invention uses a gold silicon wafer as a working electrode, Au@CDs composite nanomaterials as a working electrode modification material, and an aptamer as a biorecognition element to prepare a highly sensitive electrochemical aptamer sensor to detect methamphetamine in water bodies, with good stability / reproducibility and specificity and other detection performances, and the method is applicable to the detection of trace methamphetamine in water samples such as drinking water, surface water and sewage in actual water bodies. The purpose of realizing the rapid detection of trace methamphetamine in water bodies with a trace amount (ten microliters) of sample is avoided, and the cumbersome water sample pre-concentration treatment steps are avoided, laying the foundation for real-time online monitoring of prohibited drugs in environmental water bodies.

[0058] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 do not necessarily refer 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, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0059] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an electrochemical aptamer sensor, characterized in that: The following steps are involved: 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 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 method for preparing the electrochemical aptamer sensor according to claim 1, characterized in that: 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 3-8 mg / mL.

3. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: The concentration of the chitosan solution was 0.5 mg / mL to 2.5 mg / mL, and the volume ratio of the chitosan solution to the Au@CDs suspension was 1:

1.

4. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: The heating temperature in the oil bath is 60°C to 100°C, and the heating time is 60min to 90min.

5. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: The microwave heating output frequency is 500W to 700W, and the heating time is 20min to 40min.

6. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: The biomass material is banana peel.

7. An electrochemical aptamer sensor prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the electrochemical aptamer sensor according to claim 7 in detecting trace amounts of methamphetamine in environmental water.

9. The use according to claim 8, characterized in that: The detection range of methamphetamine in water is 1pg / L~1μg / L, the detection limit is 0.87pg / L, and the spiked recovery rate can reach 92.4%~104.6%.

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