Portable electrochemical biosensor with aptamer as biological sensitive element and application of portable electrochemical biosensor

Abstract

The invention discloses a portable electrochemical biosensor with a nucleic acid aptamer as a biological sensitive element and application, and relates to the technical field of electrochemical biosensing. Graphene and gold nanoparticles are modified on an electrode in an electrochemical reduction manner, so that electron transfer is promoted, the effective surface area of the electrode is increased, the loading capacity of an aptamer is improved, and a stable and portable aptamer sensor is constructed. The portable electrochemical biosensor provided by the invention adopts the aptamer with strong specificity as a biological sensitive element, and can accurately identify and bind a target substance (such as isocarbophos), so that high-sensitivity and high-selectivity detection is realized, and interference of other substances is reduced. The sensor is suitable for the fields of environment monitoring, food safety, agricultural production and the like, harmful substances such as organophosphorus pesticides and the like can be effectively detected, and guarantee is provided for the ecological environment and public health.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical biosensors, and in particular to a portable electrochemical biosensor using nucleic acid aptamers as biosensing elements and applications thereof. Background Art

[0002] Organophosphorus compounds, such as nerve agents and organophosphorus pesticides, have a wide range of applications and are widely used in agriculture and the military. They pose a serious threat to the environment and human health. Their mechanism of causing human poisoning is through strongly inhibiting the activity of acetylcholinesterase (AChE), resulting in the accumulation of acetylcholine (ACh) in the body, causing acute poisoning with severe disorders of the nervous system. Effective organophosphorus protection or rapid detection research is still very necessary.

[0003] Traditional organophosphorus detection methods are mainly based on chromatography, such as gas chromatography-flame photometric detection (GC-FPD), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), etc. These traditional instrumental analysis methods have the significant advantage of high accuracy and are used by many countries in the world as the preferred detection methods for formulating national standards for pesticide detection. However, these detection methods have the disadvantages of expensive equipment, large and precise instruments, complex sample pretreatment, and strong professional operation, which greatly limits their application in field testing and large-scale screening.

[0004] Therefore, establishing an analytical method with the characteristics of rapid analysis, high sensitivity, simple operation, and portability is an important research direction for organophosphorus detection. Electrochemical biosensors have become an important tool for organophosphorus detection due to their simple structure, rapid response, and high sensitivity. In particular, sensors based on acetylcholinesterase and nucleic acid aptamers have shown great potential in environmental monitoring due to their high selectivity and high sensitivity. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a portable electrochemical biosensor using nucleic acid aptamers as biosensing elements and its application.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: a method for preparing a portable electrochemical biosensor using a nucleic acid aptamer as a biosensor element, comprising the following steps:

[0007] S1. Pretreatment of screen-printed electrodes: Select screen-printed carbon electrodes as substrates and pretreat the electrodes to improve their surface activity and conductivity;

[0008] S2. Electrode modification: Graphene modification: Graphene oxide is dispersed in a suitable solvent and modified on the surface of SPCE by electrochemical reduction. The reduction treatment of graphene oxide can further improve its conductivity.

[0009] Gold nanoparticle modification: A tetrachloroauric acid solution is dropped onto the electrode, and gold nanoparticles are deposited on the electrode surface by electrochemical reduction or chemical reduction to increase the surface conductivity and biocompatibility;

[0010] S3. Fixation of nucleic acid aptamers: The nucleic acid aptamer MB solution was dripped onto the surface of the SPCE working electrode and incubated in a 4°C refrigerator overnight. The aptamer MB solution was self-assembled with gold nanoparticles at its terminal to fix the aptamer on the modified electrode surface, ensuring that the aptamer was firmly bound to the electrode surface and that its biological activity was maintained.

[0011] S4. Assembly and optimization of the sensor: Assemble the modified electrode, reference electrode and counter electrode into a complete three-electrode system; and optimize the performance of the sensor by adjusting parameters such as the loading amount of the aptamer, the modification amount of graphene and gold nanoparticles, and the incubation time of the aptamer and the target to ensure that its sensitivity and selectivity are optimal;

[0012] S5. Sensor testing and verification: Use a portable electrochemical workstation to perform electrochemical testing on the sensor to verify its detection performance for the target; measure parameters such as sensitivity, linear range, and detection limit, and evaluate the repeatability, anti-interference ability, and stability of the sensor;

[0013] S6. Portable integration: Combine the optimized electrochemical biosensor with a portable potentiostat.

[0014] Preferably, in step S3, the preparation of the nucleic acid aptamer MB solution comprises the following steps:

[0015] L1. Storage and preliminary treatment of nucleic acid aptamers: The customized synthetic nucleic acid aptamer MB was stored in a centrifuge tube in the form of dry powder. Before opening the lid for use, it was centrifuged at 4000 rpm for 2 minutes to prevent loss;

[0016] L2. Preparation and storage of mother solution: Add a certain amount of TE buffer (pH=8.0) to a centrifuge tube to prepare a mother solution and store it in a -20°C refrigerator;

[0017] L3. Solution dilution before the experiment: Before each experiment, take a certain amount of mother solution and dilute it with TE buffer (pH=8.0);

[0018] L4. Formation of hairpin structure of aptamer MB: In order to form a good hairpin structure of the aptamer MB fixed on the electrode surface, the aptamer MB solution was placed in a 95° C. water bath and heated for 5 min, and then slowly cooled to room temperature.

[0019] Preferably, in step S4, the optimization of the sensor comprises the following steps:

[0020] S401, modification amount of gold nanoparticles: prepare a series of concentrations of tetrachloroauric acid solution (0.1, 0.3, 0.5, 0.7, 0.9 mM), deposit different amounts of gold nanoparticles on the electrode, and determine the optimal amount of gold nanoparticles by testing the electrochemical response under each modification amount;

[0021] S402, optimization of the aptamer MB loading amount: after determining the optimal amount of gold nanoparticles, prepare a series of aptamer MB solutions (0.8-2.2 μM) and fix them on the surface of the electrode modified with gold nanoparticles and reduced graphene oxide, test the electrochemical response at each concentration by electrochemical method, and select the aptamer MB concentration that can provide the best signal intensity;

[0022] S403. Optimization of the incubation time of nucleic acid aptamer MB and nucleic acid aptamer S: react the sensor and the target at different incubation times to determine the optimal reaction time; determine the incubation time that can produce the maximum signal response by measuring the change of the electrochemical signal to avoid insufficient or excessive reaction.

[0023] Preferably, the concentration of the tetrachloroauric acid solution is 0.5 mM; the concentration of the nucleic acid aptamer MB solution is 2.0 μM; and the incubation time of the nucleic acid aptamer MB and the nucleic acid aptamer S is 100 min.

[0024] The present invention also provides the use of a portable electrochemical biosensor prepared based on the method in the detection of isocarbophos.

[0025] Compared with the prior art, this solution has the following beneficial effects:

[0026] High sensitivity and high selectivity: The sensor uses highly specific nucleic acid aptamers as biosensitive elements, which can accurately identify and bind to target substances (such as isothiocarb), thereby achieving high-sensitivity and high-selectivity detection and reducing interference from other substances.

[0027] Rapid detection: Since electrochemical sensors can instantly capture changes in electrical signals, the detection process is rapid and suitable for on-site real-time detection to meet the needs of rapid response.

[0028] Strong portability: The sensor combines screen-printed electrodes and a small potentiostat. It is small in size, light in weight, easy to carry, and suitable for field applications and mobile detection.

[0029] Highly cost-effective: Using screen-printed electrodes and nanomaterials, the sensor manufacturing process is simple and low-cost, suitable for large-scale production and promotion.

[0030] Good stability and repeatability: By optimizing electrode modification and sensing conditions, the sensor maintains stable performance in long-term use, with good signal repeatability, ensuring the reliability of the detection results.

[0031] Broad application prospects: This sensor is suitable for environmental monitoring, food safety, agricultural production and other fields. It can effectively detect harmful substances such as organophosphorus pesticides and provide protection for the ecological environment and public health. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the construction process of the SPCE / ERGO / AuNPs / MB / MCH sensor in an embodiment of the present invention;

[0033] Figure 2 is a schematic diagram of the SPCE / ERGO / AuNPs / MB / MCH sensor for detecting isocarbophos in an embodiment of the present invention;

[0034] Figure 3 : is a diagram of surface color change during the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor in an embodiment of the present invention (wherein: (A) bare SPCE; (B) SPCE / GO; (C) SPCE / ERGO; (D) SPCE / ERGO / AuNPs; (E) SPCE / ERGO / AuNPs / MB; (F) SPCE / ERGO / AuNPs / MB / MCH);

[0035] Figure 4 is a SEM image of the construction process of the SPCE / ERGO / AuNPs / MB / MCH sensor in an embodiment of the present invention (wherein: (A) bare SPCE; (B) SPCE / GO; (C) SPCE / ERGO; (D) SPCE / ERGO / AuNPs; (E) SPCE / ERGO / AuNPs / MB; (F) SPCE / ERGO / AuNPs / MB / MCH);

[0036] Figure 5 is the EDS image of the SPCE / ERGO / AuNPs / MB / MCH sensor in the embodiment of the present invention;

[0037] Figure 6 is the FTIR image of GO before and after electrochemical reduction in an embodiment of the present invention;

[0038] Figure 7 The electrochemical reduction process of GO and HAuCl4 in the embodiment of the present invention (wherein: (A) CV curve of electrochemical reduction of GO; (B) CV curve of electrochemical reduction of HAuCl4);

[0039] Figure 8 : are the cyclic voltammetry curves during the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor in the embodiment of the present invention (wherein: (a) Bare SPCE; (b) SPCE / GO; (c) SPCE / ERGO; (d) SPCE / ERGO / AuNPs; (e) SPCE / ERGO / AuNPs / MB; (f) SPCE / ERGO / AuNPs / MB / MCH);

[0040] Fig. 9 is the electrochemical impedance curve during the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor in the embodiment of the present invention;

[0041] Fig.10 The CV curves of the electrodes in 1 mM K3Fe(CN)6 (containing 0.1 M KCl) solution at different scan rates (0.01, 0.02, 0.05, 0.1, 0.15, 0.2 V / s) and the linear fitting diagrams of the square roots of different scan rates and the oxidation peak current in the electrode effective surface area test of the embodiments of the present invention (among which: (A) SPCE; (B) SPCE / GO; (C) SPCE / ERGO);

[0042] Fig.11 are SEM images and optical photographs of different electrodes in the embodiments of the present invention (including: (A) SPCE; (B) SPCE / AuNPs; (C) SPCE / ERGO / AuNPs);

[0043] Fig.12 These are the SEM images and optical photographs of gold nanoparticles after SPCE / ERGO electrodeposition in the embodiment of the present invention, wherein the concentration of HAuCl4 deposition solution (0.1, 0.3, 0.5, 0.7, 0.9 mM;

[0044] Fig.13 The effect of HAuCl4 concentration on the ACV response current of the sensor in the embodiment of the present invention;

[0045] Fig.14 It is the optimization of the nucleic acid aptamer MB loading amount in the embodiment of the present invention;

[0046] Fig.15 It is the optimization of the incubation time of nucleic acid aptamer MB and nucleic acid aptamer S in the embodiment of the present invention;

[0047] Fig.16 : is the ACV curve of the SPCE / ERGO / AuNPs / MB / MCH sensor in the embodiment of the present invention after incubation in a mixed solution of isocarbophos and nucleic acid aptamer S at different concentrations (wherein: isocarbophos concentration: 50, 100, 500, 1000, 5000, 10000, 50000 μg / L);

[0048] Fig.17 1 is a calibration curve of the ACV peak current change rate ΔI and the logarithm of the isocarbothion concentration of the SPCE / ERGO / AuNPs / MB / MCH sensor in the embodiment of the present invention. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0050] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0051] This example uses the nucleic acid aptamer SS24-S-35 (referred to as nucleic acid aptamer S) and the hairpin nucleic acid aptamer (referred to as nucleic acid aptamer MB) that can specifically recognize isocarbothion. It is a stable electroactive substance that undergoes oxidation reaction at a certain potential to produce electrochemical signals, and is therefore often used as a marker for electrochemical sensors. These functionalized aptamers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The sequence is as follows (5′-3′):

[0052] Nucleic acid aptamer MB:HS-SH(CH2)6-CTGCA CAAGAATCGCTGCAG -Methylene Blue

[0053] Aptamer S:AGCTTG CTGCAGCGATTCTTG ATCGCCACAGAGCT

[0054] The structural formula of methylene blue is:

[0055] Embodiment 1:

[0056] 1 Preparation of aptamer solution

[0057] All custom-synthesized aptamers (aptamer MB with methylene blue and thiol connected at both ends and aptamer S complementary to it) were stored in a centrifuge tube in the form of dry powder. Before opening the lid for use, centrifuge at 4000 rpm for 2 minutes to prevent loss. Then, a certain amount of TE buffer (pH = 8.0) was added to the centrifuge tube to prepare a mother solution and stored in a -20°C refrigerator. Before each experiment, a certain amount of mother solution was diluted with TE buffer (pH = 8.0). In order to make the aptamer MB fixed on the electrode surface form a good hairpin structure, the aptamer MB solution was placed in a 95°C water bath and heated for 5 minutes, and then slowly cooled to room temperature.

[0058] 2 Construction of nucleic acid aptamer electrochemical sensor

[0059] First, 10 μL of 1 mg / mL graphene oxide dispersion was dripped on the surface of the working electrode of the screen-printed carbon electrode (SPCE). After natural drying at room temperature, the working area of ​​the SPCE was placed in a 0.5 M KCl solution, and cyclic voltammetry (CV) was scanned for 15 cycles to electroreduced graphene oxide (GO) to graphene (ERGO) (potential range -1.5 to 0 V, scan rate 0.05 V / s). After rinsing with deionized water, blow dry with an ear bulb, then place the SPCE working area in a 0.5mM HAuCl4 solution, and perform 10 CV scans to deposit gold nanoparticles on the electrode surface (potential range -0.8 to -0.2V, scan rate 0.01V / s). After rinsing with deionized water, blow dry with an ear bulb, and drop 10μL of nucleic acid aptamer MB solution (2μM) on the surface of the SPCE working electrode. Place it in a 4°C refrigerator and incubate overnight. At this time, the -SS- connected to the 5′ of the nucleic acid aptamer and the gold nanoparticles on the electrode surface form an Au-S bond through self-assembly, so that the nucleic acid aptamer MB is stably fixed on the electrode surface to form a hairpin structure. On the second day, gently rinse the electrode surface with deionized water to remove unbound nucleic acid aptamer MB. Finally, drop 5μL of 1mM 6-mercapto-1-hexanol (MCH) on the surface of the working electrode to block the unoccupied active sites. The preparation process is as follows Figure 1 shown.

[0060] 3. Detection of isothiocarb using aptamer electrochemical sensor

[0061] like Figure 2As shown, after obtaining the initial signal of the sensor, when isocarbothion is not present, the nucleic acid aptamer MB fixed on the electrode binds to the nucleic acid aptamer S, and the probe hairpin structure opens to make the methylene blue away from the electrode surface. At this time, the oxidation current signal of methylene blue decreases more than the initial signal; when isocarbothion is present, isocarbothion competes with the nucleic acid aptamer MB for binding to the nucleic acid aptamer S, and a part of the nucleic acid aptamer S is consumed by isocarbothion. Therefore, the opening amount of the nucleic acid aptamer hairpin structure decreases, making the methylene blue less away from the electrode surface, and the oxidation current signal of methylene blue decreases less than the initial signal.

[0062] The specific experimental operation is as follows: the prepared SPCE / ERGO / AuNPs / MB / MCH sensor is first subjected to ACV scanning in 10mM PBS (pH=8.0) to measure the initial oxidation peak current value I0, and then the modified electrode is placed in a mixed solution of a certain concentration of isothiocarb and nucleic acid aptamer S for 100min, washed with deionized water, and then subjected to ACV scanning in 10mM PBS (pH=8.0) to obtain the oxidation peak current value I1 after binding with the nucleic acid aptamer. The change in the oxidation peak current value is obtained by the following formula: ΔI=I0-I1

[0063] Finally, the calibration curve of the oxidation peak current value change ΔI and different concentrations of isocarbophos was obtained.

[0064] 4. Processing of actual samples

[0065] The cucumbers used as actual samples in this study were purchased from a local vegetable market (Changsha, China). The cucumbers were peeled with a peeler and cut into small pieces of about 2 cm × 2 cm with scissors. 5 g was weighed on a balance and placed in a solution containing 20 mL of water and 20 mL of acetone (1:1) for 10 min and then filtered by ultrasonication for 30 min. The filtrate was concentrated by rotary evaporation at 70 °C and then filtered with a 0.22 μm filter membrane to obtain the supernatant. Different known concentrations of isocarbothion standard solutions were added to prepare spiked samples. Three measurements were performed using different sensors, and the corresponding concentration values ​​were obtained according to the standard curve, which were divided by the actual added concentration to calculate the spiked recovery of the sample. In addition, isocarbothion standard solution was directly added to the tap water sample to prepare the spiked sample, and the test process was consistent with the above.

[0066] 5 Experimental results

[0067] 5.1 Morphology and structure characterization of aptamer electrochemical sensors

[0068] 5.1.1 Optical photos

[0069] like Figure 3As shown in the figure, during the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor, the color of the working electrode surface changed significantly, reflecting the successful progress of each step. The bare SPCE surface was black, turned brown after drop coating of graphene oxide, and turned gray-black after electrochemical reduction. After electrodeposition of HAuCl4, the surface was bright golden yellow, indicating that the gold nanoparticles were successfully loaded. After drop coating of the nucleic acid aptamer MB solution, the electrode surface turned green due to the superposition of the blue color of methylene blue and the golden yellow substrate. After rinsing with deionized water and drop coating of mercaptohexanol, the green part was significantly reduced. This series of color changes verified the successful construction of the sensor.

[0070] 5.1.2 Scanning electron microscopy characterization

[0071] like Figure 4 As shown, Figure 4 (A) shows that the bare SPCE surface is composed of carbon particles and pores. Figure 4 In (B), the SPCE / GO surface is covered with wrinkles of graphene oxide, which increases the effective area of ​​the electrode but reduces the conductivity. Figure 4 (C) shows the reduced SPCE / ERGO, whose clear wrinkled structure indicates a significantly improved conductivity. Figure 4 (D) Gold nanoparticles (particle size <100 nm) are uniformly distributed on the surface of SPCE / ERGO / AuNPs, providing a good matrix for the loading of nucleic acid aptamers. Figure 4 (E) shows the film coverage after SPCE / ERGO / AuNPs modified aptamer MB. Figure 4 (F) SEM image clarity decreases after showing 6-mercapto-1-hexanol blocks the unoccupied active sites, indicating its successful loading. Figure 5 The EDS results confirmed the presence of elements such as C, O, Au, N, P, and S, further verifying the successful loading of nucleic acid aptamer MB and MCH. These results confirmed the successful implementation of each step of the sensor from the perspective of optical appearance, micromorphology, and elemental analysis.

[0072] 5.1.3 Fourier transform infrared spectroscopy characterization

[0073] In order to further illustrate the reduction effect of graphene oxide introduced into the sensor, the Fourier transform infrared spectrum of graphene oxide (GO) before and after electrochemical reduction was tested. Figure 6 GO in 3207cm -1 A broader and stronger absorption band appears at 1597cm -1 The absorption peak at corresponds to the sp 2 The stretching vibration absorption peak of hybrid carbon atom C=C. 1370cm -1 The bending vibration absorption peak corresponding to C-OH is 1232 cm-1 The 1040 cm-1 peak is the stretching vibration absorption peak of CO in ether or lactone, while the 1040 cm-1 peak is the stretching vibration absorption peak of CO in ether or lactone. -1 The absorption peak at comes from the stretching vibration of CO in the epoxy structure. The data show that most of the oxygen-containing functional groups are eliminated in ERGO after electrochemical reduction.

[0074] 6 Electrochemical behavior of nucleic acid aptamer sensors

[0075] 6.1 Electrochemical reduction of GO and electrodeposition of AuNPs by cyclic voltammetry

[0076] The GO reduction process and AuNPs electrodeposition process are as follows Figure 7 shown. Figure 7 (A) shows an obvious reduction peak near -1.35 V, indicating that the oxygen-containing functional groups on the graphene oxide surface begin to be reduced. As the number of scans increases, the reduction peak current decreases sharply and disappears completely after 15 cycles, and the CV curve tends to be stable, indicating that the reduction process is rapid and irreversible. Figure 7 (B) shows the CV curve of electrochemically deposited gold nanoparticles. The deposition curve is smooth, with a reduction peak near -0.4 V. As the number of scans increases, the reduction peak current gradually decreases and tends to stabilize. Compared with the reduction of graphene oxide, the reduction process of gold is smoother and slower.

[0077] 6.2 Cyclic voltammetric response of the construction process of SPCE / ERGO / AuNPs / MB / MCH

[0078] like Figure 8 As shown, the effect of different material loadings on the electrochemical response of the SPCE / ERGO / AuNPs / MB / MCH sensor during its construction was studied in a test solution of 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (1:1) containing 0.1 M KCl. Figure 8 In (a), the CV response of the bare SPCE shows a large redox peak potential difference and a current of about 160 μA, indicating its poor conductivity and electrode reversibility. Figure 8 In (b), after modification of graphene oxide, the current drops sharply to about 10 μA, indicating a significant decrease in conductivity. Graphene oxide needs to be reduced to enhance conductivity. Figure 8 In (c), the redox peak current of the SPCE / ERGO electrode increases significantly, and the potential difference decreases to about 0.2 V, indicating that the conductivity and electrode reversibility are improved. Figure 8 In (d), after the deposition of gold nanoparticles, the current further increases and the potential difference decreases because the gold nanoparticles accelerate the electron transfer. Figure 8 In (e), after loading with nucleic acid aptamer MB, the current decreases because the nucleic acid aptamer hinders electron transfer. Figure 8In (f), the 6-mercapto-1-hexanol at the blocked sites further reduced the current. The above results indicate that the graphene oxide was successfully reduced and all materials were successfully modified to the electrode.

[0079] 6.3 Electrochemical impedance spectroscopy of the SPCE / ERGO / AuNPs / MB / MCH construction process

[0080] Electrochemical impedance spectroscopy (EIS) is used to characterize the properties of electrode interfaces, and its curve includes a semicircle (controlled by charge transfer) and a straight line (controlled by diffusion). Fig. 9 The EIS curve changes during the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor are shown. The charge transfer resistance of bare SPCE is about 3000Ω. Fig. 9 In (A), after modification with graphene oxide, due to the increase in film thickness, the SPCE / GO electrode shows two large arcs, corresponding to [Fe(CN)6] 3- / 4- Diffusion migration and electron transport in graphene oxide. Fig. 9 In (B, C), after graphene oxide is reduced to ERGO, the arc diameter is significantly reduced, and charge transfer becomes easier. After adding gold nanoparticles, the arc almost disappears and approaches a straight line, indicating that the charge transfer resistance is extremely small. After loading the poorly conductive nucleic acid aptamer MB and 6-mercapto-1-hexanol, the arc diameter increases slightly and the charge transfer resistance increases, which is consistent with the CV results.

[0081] In summary, the successful preparation of the layer-by-layer modified SPCE / ERGO / AuNPs / MB / MCH sensor was confirmed by optical photographs and SEM, FTIR, CV, EIS and other methods, and the successful electroreduction of graphene oxide (GO) to ERGO and the successful reduction of HAuCl4 to AuNPs during the modification process were confirmed.

[0082] Embodiment 2:

[0083] This embodiment further optimizes the nucleic acid aptamer sensor based on the embodiment 1.

[0084] Effect of reduced graphene oxide (ERGO) on sensor structure

[0085] The effective surface area of ​​the electrode is the area where the electrochemical reaction actually occurs, and the presence of the modifier will change the effective surface area of ​​the electrode. 3- / 4-The effective surface area of ​​the modified electrode can be calculated by fitting the redox peak current of the electrode surface with the square root of the scan rate. The specific method is as follows: bare SPCE, SPCE / GO electrode, and SPCE / ERGO electrode are subjected to CV scans at different scan rates in 1mM K3Fe(CN)6 (containing 0.1M KCl) solution, and then the oxidation peak current value and the square root of the scan rate are linearly fitted to obtain the results shown in the figure. Fig.10 shown.

[0086] According to the Randles-Savcik equation:

[0087] i p =2.69×10 5 An 3 / 2 D 1 / 2 Cv 1 / 2

[0088] where i p is the current value of the redox peak current, A is the effective surface area of ​​the electrode (cm 2 ), n is the number of electron transfers. For 1 mM K3Fe(CN)6 solution containing 0.1 M KCl, the values ​​of the parameters in the formula are n = 1, D = 7.6 × 10 -6 cm 2 / s, C = 1 × 10 -6 mol / cm 3 , v is the scanning speed.

[0089] According to the formula, i p With v 1 / 2 is proportional to linear relationship, so we can p With v 1 / 2 A linear fitting plot was performed, and the effective surface area A of each electrode was calculated based on the obtained slope as shown in Table 1. The effective surface area of ​​bare SPCE is 0.08363 cm 2 , slightly smaller than its theoretical area of ​​0.1256cm 2 (The working electrode diameter is about 4 mm); the effective surface area of ​​GO modified electrode SPCE / graphene oxide is 0.00757 cm 2 , the effective area decreases rapidly, which indicates that although graphene oxide has many wrinkles, its poor conductivity makes it have great resistance to chemical reactions, and the effective surface area even decreases. The effective surface area of ​​ERGO modified electrode SPCE / ERGO is 0.16714cm 2 , which is about twice the effective surface area of ​​bare SPCE. From the above data, it can be judged that the introduction of ERGO does increase the effective surface area of ​​the electrode.

[0090] Table 1 Square root linear slope of different electrodes and scan rate and effective surface area of ​​electrodes

[0091]

[0092]

[0093] SPCE and SECE / ERGO electrodes were electrodeposited with gold nanoparticles in HAuCl4 solution with the same concentration. Their SEM images and optical photographs are shown in Figure 2. Fig.11 As shown. From the optical photos, we can see that the SPCE / AuNPs electrode is golden yellow, and the black carbon substrate is faintly visible, while the SPCE / ERGO / AuNPs electrode is white gold, which is relatively dense and the substrate is almost invisible. The SEM image can also confirm this. In the SEM image, the gold nanoparticles on the surface of the SPCE / ERGO / AuNPs electrode are much denser than those of the SPCE / AuNPs electrode. Therefore, it is verified that the introduction of ERGO can provide more active sites for the loading of gold nanoparticles, which is conducive to loading more nucleic acid aptamers MB to improve the response signal of the sensor, which is reasonable and beneficial.

[0094] Optimization of HAuCl4 deposition solution concentration

[0095] After verifying the rationality of introducing ERGO into the sensor, the concentration of HAuCl4 deposition solution was optimized to obtain the best gold nanoparticle morphology and distribution to facilitate the loading of nucleic acid aptamer MB. SPCE / ERGO electrodes were electro-deposited in HAuCl4 solutions of different concentrations (0.1, 0.3, 0.5, 0.7, 0.9 mM) and then subjected to SEM testing ( Fig.12 ). The results showed that as the concentration increased, the gold color deepened, indicating an increase in the amount of deposition. SEM images showed that at 0.5 mM, the gold nanoparticles were small and uniform in size, densely distributed, and had no agglomeration, which was most conducive to the loading and stable formation of nucleic acid aptamer MB. ACV test ( Fig.13 )The results showed that the sensor obtained the maximum response current at a concentration of 0.5 mM HAuCl4. Therefore, the optimal concentration of HAuCl4 in subsequent experiments was determined to be 0.5 mM.

[0096] Embodiment 3:

[0097] This embodiment further optimizes the nucleic acid aptamer sensor based on embodiment 2.

[0098] Optimization of nucleic acid aptamer MB loading

[0099] After determining that the optimal concentration of HAuCl4 deposition solution was 0.5 mM, a series of SPCE / ERGO / AuNPs electrodes were prepared in parallel, and different concentrations of nucleic acid aptamer MB solutions (0.8-2.2 μM) were modified to determine the maximum MB loading capacity. The complete SPCE / ERGO / AuNPs / MB / MCH sensor was constructed to measure the ACV response current in 10 mM PBS (pH = 8.0). The results are shown in Figure 2. Fig.14 As shown in the figure, with the increase of aptamer MB concentration, the ACV response current gradually increased, indicating that the aptamer MB bound to the electrode surface through Au-S bonds increased. When the concentration reached 2.0 μM, the response current remained stable, and the aptamer MB loading on the electrode surface reached saturation. Therefore, 2.0 μM was taken as the optimal aptamer MB solution concentration.

[0100] Embodiment 4:

[0101] This embodiment further optimizes the nucleic acid aptamer sensor based on embodiment 3.

[0102] Optimization of incubation time of aptamer MB and aptamer S

[0103] After the construction of the SPCE / ERGO / AuNPs / MB / MCH sensor, the effect of incubation time on the binding degree between aptamer MB and aptamer S was studied. If the incubation time is too short, the two cannot fully pair and bind, resulting in inaccurate experimental results or reduced sensitivity, while if the incubation time is too long, it is not conducive to the demand for rapid detection. Fig.15 As shown in the figure, with the increase of incubation time, the ACV response current gradually decreases. This is because the nucleic acid aptamer MB on the electrode surface binds more nucleic acid aptamer S, so that its "hairpin-type" spatial conformation is opened, making the signal molecule methylene blue away from the electrode surface. However, the ACV response current remains relatively stable after 100 minutes, indicating that the nucleic acid binding process on the sensor is basically completed. Therefore, 100 minutes is the optimal incubation time.

[0104] Embodiment 5:

[0105] In this example, based on Example 4, the detection performance of the nucleic acid aptamer sensor for isocarbophos was studied.

[0106] Under the optimal experimental conditions (HAuCl4 deposition solution concentration of 0.5 mM, aptamer MB solution concentration of 2 μM, aptamer MB and aptamer S incubation time of 100 min), we incubated the SPCE / ERGO / AuNPs / MB / MCH sensor in a solution of aptamer S containing different concentrations of isothiocarb for 100 min, and then tested it in 10 mM PBS (pH = 8.0) to obtain the ACV response current change of the sensor as shown in Figure 2. Fig.16 As shown. It can be seen that as the concentration of isocarbothion increases, the ACV response current value increases. This is because isocarbothion can compete with the nucleic acid aptamer MB to bind to the nucleic acid aptamer S, which reduces the opening of the hairpin structure on the electrode surface, and the ACV response current decreases relative to the initial value. The change in the response current ΔI and the logarithmic value of the isocarbothion concentration are curve-fitted to obtain a linear calibration curve as shown in Fig.17 As shown. The linear equation is ΔI = 7.007-3.106lgC ICP (R 2 =0.991).

[0107] According to the calculation method of detection limit, substitute the data, 3S b1 =0.66, then the minimum analysis signal of the sensor is X L =27.61, (X L is the minimum analysis signal, is the blank mean, S b1 is the standard deviation of the blank value, k is a constant related to the confidence level, generally 3) Substituting the minimum analytical signal into the linear equation, the minimum detection concentration obtained is the detection limit C of the sensing method. L =9.1μg / L, so the linear detection range of the sensor is 50μg / L-50mg / L, and the detection limit is 9.1μg / L (31.46nM). This linear range also includes the Chinese national standard value for the detection of organophosphorus pesticide residues in agricultural products (0.2mg / kg).

[0108] Study on the detection of isocarbophos in actual samples

[0109] Cucumber peel and tap water were selected as the actual sample test objects. A known concentration of isocarbothion was added to the tap water and cucumber peel extract for spike recovery detection to evaluate the actual application ability of the SPCE / ERGO / AuNPs / MB / MCH biosensor. Within the detection range of the sensor, two concentrations of isocarbothion of 100 and 1000 μg / L were selected to be added to the blank samples of cucumber peel and tap water. The detection results of this method were compared with their theoretical values. The recovery results are shown in Table 3. The spike recovery rates of these two types of real samples were between 93.80% and 114.53%, and the detection deviation was small. Therefore, the SPCE / ERGO / AuNPs / MB / MCH sensor showed good accuracy in detecting isocarbothion in actual samples and has great practical application potential in the field of organophosphorus on-site detection.

[0110] Table 3 Recovery results of SPCE / ERGO / AuNPs / MB / MCH sensor in cucumber peel and tap water samples

[0111]

[0112] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing a portable electrochemical biosensor using a nucleic acid aptamer as a biosensor element, characterized in that: The following steps are involved: S1. Pretreatment of screen-printed electrodes: Select screen-printed carbon electrodes as substrates and pretreat the electrodes to improve their surface activity and conductivity; S2. Electrode modification: Graphene modification: Graphene oxide is dispersed in a suitable solvent and modified on the surface of SPCE by electrochemical reduction. The reduction treatment of graphene oxide can further improve its conductivity. Gold nanoparticle modification: A tetrachloroauric acid solution is dropped onto the electrode, and gold nanoparticles are deposited on the electrode surface by electrochemical reduction or chemical reduction to increase the surface conductivity and biocompatibility; S3. Fixation of nucleic acid aptamers: The nucleic acid aptamer MB solution was dripped onto the surface of the SPCE working electrode and incubated in a 4°C refrigerator overnight. The aptamer MB solution was self-assembled with gold nanoparticles at its terminal to fix the aptamer on the modified electrode surface, ensuring that the aptamer was firmly bound to the electrode surface and that its biological activity was maintained. S4. Assembly and optimization of the sensor: Assemble the modified electrode, reference electrode and counter electrode into a complete three-electrode system; and optimize the performance of the sensor by adjusting parameters such as the loading amount of the aptamer, the modification amount of graphene and gold nanoparticles, and the incubation time of the aptamer and the target to ensure that its sensitivity and selectivity are optimal; S5. Sensor testing and verification: Use a portable electrochemical workstation to perform electrochemical testing on the sensor to verify its detection performance for the target; measure parameters such as sensitivity, linear range, and detection limit, and evaluate the repeatability, anti-interference ability, and stability of the sensor; S6. Portable integration: Combine the optimized electrochemical biosensor with a portable potentiostat.

2. A method for preparing a portable electrochemical biosensor using a nucleic acid aptamer as a biosensor element according to claim 1, characterized in that: In step S3, the preparation of the nucleic acid aptamer MB solution comprises the following steps: L1. Storage and preliminary treatment of nucleic acid aptamers: The customized synthetic nucleic acid aptamer MB was stored in a centrifuge tube in the form of dry powder. Before opening the lid for use, it was centrifuged at 4000 rpm for 2 minutes to prevent loss; L2. Preparation and storage of mother solution: Add a certain amount of TE buffer (pH=8.0) to a centrifuge tube to prepare a mother solution and store it in a -20°C refrigerator; L3. Solution dilution before the experiment: Before each experiment, take a certain amount of mother solution and dilute it with TE buffer (pH=8.0); L4. Formation of hairpin structure of aptamer MB: In order to form a good hairpin structure of the aptamer MB fixed on the electrode surface, the aptamer MB solution was placed in a 95° C. water bath and heated for 5 min, and then slowly cooled to room temperature.

3. The method for preparing a portable electrochemical biosensor using a nucleic acid aptamer as a biosensor according to claim 1, characterized in that: In step S4, the optimization of the sensor includes the following steps: S401, modification amount of gold nanoparticles: prepare a series of concentrations of tetrachloroauric acid solution (0.1, 0.3, 0.5, 0.7, 0.9 mM), deposit different amounts of gold nanoparticles on the electrode, and determine the optimal amount of gold nanoparticles by testing the electrochemical response under each modification amount; S402, optimization of the aptamer MB loading amount: after determining the optimal amount of gold nanoparticles, prepare a series of aptamer MB solutions (0.8-2.2 μM) and fix them on the surface of the electrode modified with gold nanoparticles and reduced graphene oxide, test the electrochemical response at each concentration by electrochemical method, and select the aptamer MB concentration that can provide the best signal intensity; S403. Optimization of the incubation time of nucleic acid aptamer MB and nucleic acid aptamer S: react the sensor and the target at different incubation times to determine the optimal reaction time; determine the incubation time that can produce the maximum signal response by measuring the change of the electrochemical signal to avoid insufficient or excessive reaction.

4. The method for preparing a portable electrochemical biosensor using a nucleic acid aptamer as a biosensor according to claim 3, characterized in that: The concentration of the tetrachloroauric acid solution is 0.5 mM; the concentration of the nucleic acid aptamer MB solution is 2.0 μM; and the incubation time of the nucleic acid aptamer MB and the nucleic acid aptamer S is 100 min.

5. Use of a portable electrochemical biosensor prepared by the method according to any one of claims 1 to 4 in on-site detection of organophosphorus.

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