A sandwich structure sensor and its manufacturing method and application

Abstract

The present invention discloses a sandwich structure sensor, comprising a working electrode, a reference electrode and a counter electrode; the working electrode comprises a gold electrode, a conductive molecular imprinting polymer layer and an antibody labeled with a luminescent molecule in sequence, a hole for capturing azithromycin is arranged in the conductive molecular imprinting polymer layer, and the antibody labeled with a luminescent molecule is combined with azithromycin through intermolecular forces. The present invention also discloses a method for preparing a sandwich structure sensor, and the application of the sandwich structure sensor in monitoring trace concentrations of azithromycin in a sewage treatment plant. The present invention utilizes the tertiary amine structure of azithromycin itself to act as a co-reactant of the electrochemiluminescence of terpyridine ruthenium, obtains a higher light response, and realizes the monitoring of azithromycin at a lower concentration, with high detection sensitivity and high precision; the constructed sensor eliminates the pre-enrichment and purification steps in the sample determination process, shortens the detection cycle, and does not require the use of large instruments.

Description

Technical Field

[0001] The invention relates to a detector and a manufacturing method thereof, and in particular to a sandwich structure sensor and a manufacturing method and application thereof. Background Art

[0002] In recent years, the extensive use of macrolide antibiotics has brought great health risks, and they need to be included in the monitoring list. In view of their stable electrochemical properties (no characteristic chemical reactions such as redox, etc.) and the lack of characteristic physicochemical properties (characteristic chromogenic groups, complexation), the current monitoring methods mainly rely on LC-MS. However, as a gold standard, LC-MS has superior accuracy and reliability, but it also has disadvantages such as long detection cycle, cumbersome pretreatment steps, and the need for off-site monitoring. Biosensors are just complementary to them. Among them, electrochemiluminescence, as a high-sensitivity, low-background noise signal conversion and amplification strategy, is widely used in the detection of trace pollutants.

[0003] In existing research, there have been many examples of electrochemiluminescent sandwich immunosensors, but all of these inventions use the traditional double antibody sandwich design. Chinese patent application number 201810745154.1 discloses an electrochemiluminescent immunosensor for the preparation of a severe fever with thrombocytopenia syndrome virus detection kit. The primary antibody is modified with Fe3O4@SiO2 as a carrier, and the secondary antibody is labeled with CdZnTeS quantum dots. The concentration of SFTSV is read out using an electrochemiluminescent signal. This method has good specificity, but it requires multiple monoclonal antibodies corresponding to the detected object, and the double antibody, as a biological macromolecule, has poor conductivity, which greatly hinders the electron transfer in the electrochemiluminescent reaction. On the other hand, this type of invention often uses a primary antibody or a nucleic acid aptamer to capture the target substance in the sample to be tested, which limits the scope of application to a physiological environment (antibodies are easily inactivated) and cannot be used in common sewage systems.

[0004] Molecular imprinting, as a specific recognition technology with simple preparation and good stability, has been used in a variety of electrochemiluminescent sensors. Combining molecular imprinting polymers with a wide tolerance range with biological elements can improve the stability of sandwich immunosensors in actual water sample monitoring. In addition, the incorporation of conductive polymers can simply adjust the conductivity of molecular imprinting membranes and improve the efficiency of electrochemiluminescent reactions.

[0005] At present, reports on sandwich immunity focus on the application of double antibody sandwich, that is, both ends of the sandwich structure are antibodies, which are mainly suitable for target detection under physiological conditions. Fluctuating temperature, pH, ionic strength, etc. in sewage and natural water samples can easily lead to antibody inactivation, affecting sensor performance. On the other hand, the conductivity of antibodies is poor. When the sensor undergoes an electrochemiluminescence reaction, both antibodies hinder the efficiency of electron transfer, affecting its sensitivity. In addition, there are currently no reports of molecular imprinting-antibody sandwich immunosensors based on electrochemiluminescence detection of macrolide antibiotics. Moreover, the accuracy of existing molecular imprinting sensors needs to be further improved. Summary of the invention

[0006] Purpose of the invention: In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a sandwich structure sensor with high electron transfer efficiency and low detection limit. Another purpose of the present invention is to provide a simple and convenient method for preparing a sandwich structure sensor. Another purpose of the present invention is to provide a sandwich structure sensor with a shortened detection cycle for use in monitoring trace concentrations of azithromycin in sewage treatment plants.

[0007] Technical solution: The sandwich structure sensor described in the present invention includes a working electrode, a reference electrode and a counter electrode; the working electrode includes a gold electrode, a conductive molecular imprinting polymer layer and an antibody labeled with a luminescent molecule in sequence, a hole for capturing azithromycin is arranged in the conductive molecular imprinting polymer layer, and the antibody labeled with a luminescent molecule is combined with azithromycin through intermolecular forces.

[0008] Furthermore, the sandwich structure sensor is connected to an electrochemiluminescence analyzer, the reference electrode is a saturated silver / silver chloride electrode, and the counter electrode is a platinum wire.

[0009] The method for preparing the sandwich structure sensor comprises the following steps:

[0010] Step 1: Purify the antibody labeled with luminescent molecule and use Pure protein separation and purification system removes unbound luminescent molecules and impurities;

[0011] Step 2: Take 10-50 μL of acetonitrile solution of functional monomer and auxiliary monomer and drop it on the polished gold electrode surface, let it stand at room temperature for 60-180 minutes, rinse it, complete self-assembly, drop the acetonitrile DMF mixed solution of template molecule on the electrode surface, insert the treated electrode into the acetonitrile solution of cross-linking monomer for electropolymerization, and then stir in acetonitrile acetic acid solution to remove the template molecule;

[0012] Step 3, immersing the electrode obtained in step 2 into the water sample to be tested and stirring at a speed of 200 to 500 rpm for 10 to 60 minutes, then taking it out and gently rinsing it with deionized water to remove unbound impurities;

[0013] Step 4: apply a sealant on the surface of the electrode obtained in step 3, rinse with deionized water or PBS solution, then apply the antibody labeled with the luminescent molecule in step 1 on the surface of the electrode, rinse and stir until the unbound antibody is removed, and obtain a sandwich structure sensor.

[0014] Furthermore, in step 1, the luminescent molecule is di(2,2'-bipyridine)-4,4'-dicarboxybipyridine-ruthenium di(hexafluorophosphate), the antibody is a mouse anti-monoclonal antibody, and the initial molar ratio of the luminescent molecule to the antibody is 5 to 25: 1. The specification of the dialysis bag or separation column used for purification is that the molecular weight is not greater than 1 / 3 of the molecular weight of the labeled antibody.

[0015] Furthermore, in step 2, the gold electrode is polished and ultrasonically cleaned alternately with ethanol and ultrapure water until the CV scan is stable in a 0.5 mM sulfuric acid solution. The functional monomer is p-mercaptophenylboronic acid, the auxiliary monomer is 2-mercaptothiophene or phenylethylmercaptan, the template molecule is azithromycin, and the cross-linking monomer is any one of 3,4-ethylenedioxythiophene, aniline, and pyrrole. The volume percentage of DMF in the acetonitrile DMF mixed solution is 20% to 90%. The molar ratio of the functional monomer to the template molecule is 1:1.

[0016] The auxiliary monomer is preferably 2-mercaptothiophene, and the acetonitrile DMF mixed solution and the acetonitrile solution are both polar solvents, which can ensure the complete dissolution of all functional monomers and auxiliary monomers and good miscibility between solvents. Alcohol solvents should not be selected to prevent them from reacting with boric acid groups and reducing the binding efficiency of functional monomers and template molecules. When the concentration of functional monomers and auxiliary monomers is 1mM, 50vol% DMF is added.

[0017] Furthermore, in step 3, in the acetic acid acetonitrile mixed solution, the volume ratio of acetic acid to acetonitrile is 8 to 9:1. The electrolyte for electropolymerization can be any one of tetrabutylammonium perchlorate, tetrabutylammonium hexafluorophosphate, and lithium perchlorate. The counter electrode used for polymerization is a platinum wire, and the non-aqueous reference electrode is a silver electrode in an acetonitrile solution of 0.1M corresponding electrolyte.

[0018] Preferably, when 2 mM 3,4-ethylenedioxythiophene is used as a cross-linking monomer and 0.1 M tetrabutylammonium perchlorate is used as an electrolyte, the polymerization potential window is selected to be -0.5 to 1.3 V, the number of cyclic voltammetry scans is 5, and the scan rate is 100 mV / s.

[0019] Furthermore, in step 4, the stirring is preferably at 300 rpm for 30 minutes.

[0020] Furthermore, in step five, the blocking agent is any one of 1-2wt% bovine serum albumin, casein, and skimmed milk powder. Using 1wt% methanol to shield unbound boronic acid bonds before blocking is more effective. The pH of the PBS solution is 7.2-7.4. The antibody labeled with the luminescent molecule in step one is preferably 40μg / mL, 20μL. The rotation speed of the elution stirring is 100-300rpm, preferably 300rpm, for 5 minutes.

[0021] The sandwich structure sensor is used in monitoring trace concentration of azithromycin in sewage treatment plants. A three-electrode system is formed in a 10-fold PBS solution containing 0.05-0.2M tri-n-propylamine and 0.5-2wt% Tween 20, and an electrochemiluminescence analyzer is used to monitor and record the current and light signal of +0.9V.

[0022] Further, 0.1 M tri-n-propylamine and 2 wt % Tween 20 are preferred.

[0023] Working principle: The sensor uses molecular imprinting polymer to capture the target molecule (azithromycin) in the water sample to be tested, and then blocks the nonspecific binding sites on the electrode surface. Then, the target antibody labeled with terpyridine ruthenium derivatives specifically binds to the exposed sites of the template molecule on the electrode surface, and finally reads the concentration through the electrochemiluminescence signal of terpyridine ruthenium derivatives. The functional monomers in the molecular imprinting polymer film use the reaction of the boric acid group and the hydroxyl group of the pollutant (BO bond energy 124kcal / mol) to capture the target pollutant. The thiol group irreversibly assembles the functional monomer to the gold electrode surface, so molecules containing both thiol and boronic acid groups are selected as functional monomers; in order to expose as many boronic acid groups as possible for binding to the target pollutant, the thiol and boronic acid groups are modified at the para position of the five-membered ring or six-membered ring of the monomer. In order to avoid too small a spacing between the functional monomers, resulting in too large a steric hindrance during the assembly process, a thiol-containing monomer without a boric acid group is selected for adjustment during the self-assembly process; in order to ensure that the monomers on the electrode surface have strong activity during the electropolymerization process, thiophene or benzene ring-containing monomers with a high electron cloud density are selected. This design uses electropolymerization to complete the construction of molecular imprinting polymers, in which the cross-linking monomers are selected from conductive polymer monomers to promote electron transfer. As the test object, the tertiary amine structure contained in azithromycin can be used as a co-reactant to promote the electrochemiluminescence reaction of terpyridine ruthenium derivatives and enhance the signal intensity. The higher the corresponding azithromycin concentration in the sample, the higher the concentration captured on the electrode surface, the greater the binding amount of the labeled antibody, and the stronger the light signal of the sandwich structure of MIP-azithromycin-labeled antibody in electrochemiluminescence analysis. The concentration can be read according to the light intensity and the calibration curve.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0025] 1. The tertiary amine structure of azithromycin itself is used as a co-reactant for the electrochemiluminescence of terpyridine ruthenium derivatives to obtain a higher light response and achieve the monitoring of azithromycin at a lower concentration, with high detection sensitivity and accuracy;

[0026] 2. The constructed sensor eliminates the pre-enrichment and purification steps in the sample measurement process, shortens the detection cycle, and does not require the use of large instruments. It is expected to be used for in-situ monitoring of trace concentrations of azithromycin in sewage treatment plants. It has strong economic benefits and practical value, and provides a solution for multi-channel and simultaneous monitoring of multiple high-risk pollutants;

[0027] 3. Using molecular imprinted polymers to replace the primary antibody in traditional sandwich immunosensors solves the problem that biorecognition elements are easily affected and inactivated by water quality fluctuations in sewage, greatly improves the stability of the sensor, and broadens the application scenarios;

[0028] 4. Electrochemiluminescence is used as a signal conversion and amplification strategy, with low background noise and greatly improved detection sensitivity compared to the molecular imprinting electrode-SWV electrochemical detection method. Compared with the traditional sandwich immunosensor, a molecular imprinting network is constructed on the electrode surface using conductive polymers to improve electron transfer efficiency and reduce the detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a preparation flow chart of the present invention;

[0030] Figure 2 is a fluorescence spectrum of the labeled antibody of the present invention, wherein A is an emission spectrum and B is an excitation spectrum;

[0031] Figure 3 It is the standard curve for detecting the protein concentration in the antibody by the BCA method of the present invention;

[0032] Figure 4 It is a standard curve for detecting label concentration by the fluorescence method of the present invention;

[0033] Figure 5 is the SDS-PAGE electrophoresis result of the labeled antibody of the present invention;

[0034] Figure 6 The present invention Peak diagram of pure purified antibody;

[0035] Figure 7 is a cyclic voltammogram of the electropolymerized molecular imprinted polymer of the present invention;

[0036] Figure 8 This is a comparison diagram of the SWV signals of the electrode in the potassium ferrocyanide solution before and after polymerization of the present invention;

[0037] Fig. 9It is a schematic diagram of the structure of the sensor of the present invention;

[0038] Fig.10 It is a schematic diagram of the application of the sensor of the present invention;

[0039] Fig.11 is a current-voltage diagram of an electrochemiluminescence test performed by the sensor of the present invention in the presence of a co-reactant;

[0040] Fig.12 is a light intensity-voltage diagram of the electrochemiluminescence test performed by the sensor of the present invention in the presence of a co-reactant;

[0041] Fig.13 3 is a current-voltage diagram of the sensor of the present invention, wherein A represents the absence of the detected object, B represents the absence of the co-reactant, and C represents the absence of the conductive polymer.

[0042] Fig.14 is a light intensity-voltage diagram of the sensor of the present invention, wherein A represents the absence of the detected object, B represents the absence of the co-reactant, and C represents the absence of the conductive polymer;

[0043] Fig.15 is a current / capacity-time diagram of constant potential deposition of gold nanoparticles of the present invention;

[0044] Fig.16 is a scanning electron microscope image of the surface of the sensor of the present invention;

[0045] Fig.17 is an atomic force microscope photograph of the surface of the sensor of the present invention;

[0046] Fig.18 is the calibration curve of the sensor of the present invention. DETAILED DESCRIPTION

[0047] In the following examples, the raw materials used have the following properties:

[0048] Bis(2,2'-bipyridine)-4,4'-dicarboxybipyridine-ruthenium bis(hexafluorophosphate) Ru(bpy)2(dcbpy)(PF6)2, molecular weight MW=947.57 g / mol.

[0049] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, molecular weight MW = 155.24 g / mol, density 0.877 g / cm 3 .

[0050] NHS: N-hydroxysuccinimide, molecular weight MW = 115.09 g / mol, density 1.65 g / cm 3 .

[0051] DMF, density 0.945 g / cm3 .

[0052] The 10x PBS solution was purchased directly, pH=7.2-7.4, and the 1x PBS solution was obtained by diluting it.

[0053] Example 1

[0054] like Figure 1 , a method for preparing a sandwich structure sensor, comprising the following steps:

[0055] Step A, Antibody 7 labeling:

[0056] (1) Dissolve 25 mg of Ru(bpy)2(dcbpy)(PF6)2 in 1 mL of DMF to prepare a 26.4 mmol / LX solution. Mix well and store in a refrigerator at 4°C (keep out moisture).

[0057] (2) Ice making.

[0058] (3) Dissolve 0.31 g EDC and 0.12 g NHS in 4 mL DMF to obtain solution Y (freshly prepared), in which the concentrations of EDC and NHS are 500 mM and 200 mM, respectively, and cool in an ice-water bath.

[0059] (4) Take 190 μL of solution X and 100 μL of solution Y, add 710 μL of DMF to obtain 5 mM [Ru-NHS], place in an ice-water bath, stir at 0°C for 30 minutes, and then leave at room temperature for 5 hours.

[0060] (5) The dialysis bag was placed in 1 mM EDTA2Na (pH=8) and boiled for 10 minutes. The bag was then cooled and placed in a 30% alcohol solution and stored in a refrigerator at 4°C.

[0061] (6) Take Antibody 7 out of the refrigerator and equilibrate it to room temperature. Dilute Antibody 7 to 1 mg / mL with 1x PBS.

[0062] (7) Take 30 μL of 5 mM Ru-NHS solution at room temperature and add it to 1 mL of 1 mg / mL solution of antibody 7. Wrap in tin foil and shake at room temperature for 1 hour.

[0063] (8) Excess Ru-NHS was divided into 100 μL portions and stored at -20°C to prevent contamination from repeated freezing and thawing.

[0064] (9) Add 20 μL of 2 M glycine and incubate for 15 min to terminate the reaction.

[0065] (10) Add the solution to the dialysis bag and rinse the tube wall slightly with buffer solution, then tie it with thread and clamp it with a dialysis clamp. Place it in 1x PBS buffer solution and let it stand for 8 hours, then change the water and let it stand for another 8 hours.

[0066] (11) Take out the spin filter centrifuge tube, add ultrapure water, and make sure that the water completely passes through the membrane. Pre-cool it in an ice bath or refrigerator for a few minutes, and pour out the water. Gently add the remaining liquid in the dialysis bag to the spin filter centrifuge tube, so that the liquid level does not exceed the white line on the top of the tube. Centrifuge the centrifuge tube at 1000×g for 3-5 minutes.

[0067] (12) Use pure protein purification system to further purify the labeled antibody 7. The separation column used was Superdex TM 200Increase 10 / 300GL Prepacked Tricorn TM Columns, the buffer was 1× PBS (pH=7.4), the flow rate was 0.075 mL / min, and the absorbance at 280 nm and 455 nm was detected simultaneously.

[0068] The fluorescent property of the marker Ru(bpy)2(dcbpy)(PF6)2 can be used to determine the concentration of the label. Figure 2 When the excitation wavelength is fixed at 290 nanometers, the molecule has an emission peak at 655 nm, and the emission fluorescence intensity reaches the maximum. The protein concentration of the labeled antibody 7 is detected using a micro BCA kit, and the standard curve is as follows Figure 3 The standard curve of the fluorescent assay label is shown in Figure 4 As shown. Combined Figure 5 The protein electrophoresis results showed that the molecular weight of the labeled antibody 7 was about 190 kDa, from which the labeling rate (label concentration / antibody concentration×100%) could be calculated.

[0069] through The antibody 7 further purified by the pure protein purification system eliminates the interference of impurities such as unreacted luminescent molecules and protein polymers, and has superior performance. Figure 6 , take the outflow from tubes 15, 16, and 17 as the labeled antibody 7 used in the experiment, and freeze it at -20℃ for later use after packaging.

[0070] Step B, pre-assembly of monomers and template molecules:

[0071] (1) Pretreatment of the gold electrode 4: The gold electrode 4 was polished with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al2O3 in sequence, and then ultrasonically cleaned with ethanol and ultrapure water for 3 times, each time for 30 seconds, and finally CV scanning was performed in a 0.5 mM sulfuric acid solution until it was stable.

[0072] (2) Monomer self-assembly: Prepare an assembly solution, the solute of which is 1 mM p-mercaptophenylboronic acid (functional monomer), 1 mM 2-thiophenethiol (auxiliary monomer), and the solvent is acetonitrile + DMF (volume ratio is 1:1), and prepare the assembly solution. Take 20 μL of the assembly solution droplet and apply it on the surface of the gold electrode 4, and let it stand at room temperature for 2 hours.

[0073] (3) The electrode assembled in step (2) was stirred in acetonitrile for 5 minutes to remove excess monomers. Then, 30 μL of azithromycin 8 (template molecule) in acetonitrile / DMF solution (volume ratio of 2:1) was drop-coated on the electrode surface, left to stand at room temperature for 30 minutes, and then inserted into acetonitrile and stirred for 30 minutes to remove unbound template molecules.

[0074] Step C, molecular imprinting membrane preparation:

[0075] (1) A gold electrode 4 after simple elution was used as the working electrode 1, a platinum wire as the counter electrode 3, and a silver electrode filled with 0.1M tetrabutylammonium perchlorate as the reference electrode 2 for cyclic voltammetric electropolymerization. The polymerization solution was an acetonitrile solution containing 0.1M tetrabutylammonium perchlorate and 2mM 3,4-ethylenedioxythiophene (EDOT). The parameters were set to -0.5V to 1.25V, and the scanning speed was 50mV / s. Figure 7 The self-assembled monomers on the electrode surface oxidize to produce free radicals that initiate the polymerization of EDOT in the solution. As the number of scans increases and the assembled monomers are consumed, the current value gradually decreases, and the reaction on the electrode surface decreases. Figure 8 The square wave voltammetry scanning results of the electrode in potassium ferrocyanide solution showed that its conductivity increased slightly, and PEDOT was successfully polymerized. During the preparation process, the water contact angle test was performed on the gold electrode 4, the gold electrode 4 after assembling the monomer, and the gold electrode 4 after polymerizing PEDOT. The results are shown in Table 1 below. It can be seen that the assembled monomer enhances the hydrophobicity of its surface (the contact angle becomes larger), while the hydrophilicity after depositing PEDOT is enhanced (the contact angle becomes smaller), which is stronger than the bare gold electrode 4.

[0076] Table 1 Water contact angle of gold electrode at different stages

[0077] electrode gold Gold electrode after assembling monomers Gold electrode after assembling monomers and polymerizing PEDOT Contact angle / ° 56.3±3.10 59.0±4.12 44.6±5.56

[0078] (2) Gently rinse the PEDOT-polymerized electrode with deionized water and acetonitrile, and then remove the template molecule in 20 mL of 8:2 acetonitrile-acetic acid solution (V / V) at 300 rpm for 30 minutes. After completion, discard the waste liquid and add 20 mL of 8:2 acetonitrile-acetic acid solution (V / V) to ensure that the template is completely removed.

[0079] Step D, selective capture of pollutants in the sample:

[0080] The electrode after the template molecule is removed is immersed in the water sample to be tested and stirred at 200 rpm for 60 minutes, then taken out and the surface is gently rinsed with deionized water to remove unbound impurities.

[0081] Step E, sandwich structure electrode construction:

[0082] The electrode was blown dry with nitrogen, and 20 μL of 1% wt bovine serum albumin was dripped on the surface and incubated at room temperature for 60 minutes. After gentle rinsing with 1x PBS solution (pH = 7.2-7.4), 20 μL of 50 μg / mL labeled antibody 7 was dripped on the electrode surface and incubated at room temperature for 120 minutes. After a simple rinse, the electrode was immersed in 1x PBS and stirred at 100 rpm for 15 minutes to remove unbound antibody 7, and a sandwich structure sensor was prepared.

[0083] like Fig. 9 The sandwich structure sensor comprises, from bottom to top, a gold electrode 4, a conductive molecular imprinting polymer layer 5, and an antibody 7 labeled with terpyridine ruthenium 6. The conductive molecular imprinting polymer layer 5 has a hole 9 for capturing azithromycin 8.

[0084] Application 1

[0085] like Fig.10 , the sandwich structure sensor prepared in Example 1 was subjected to electrochemiluminescence measurement:

[0086] In a 10-fold PBS solution containing 0.2M tri-n-propylamine and 1% Tween 20, the sensor prepared in Example 1 is used as the working electrode 1, the platinum wire is used as the counter electrode 3, and the saturated silver / silver chloride electrode is used as the reference electrode 2 to form a three-electrode system. The solution is placed above the light sampler 11 of the electrochemiluminescence analyzer 10 to obtain the highest photoelectric conversion efficiency. The detection limit is sub-nM level using the electrochemiluminescence reaction determination. Cyclic voltammetry scanning is performed using the Xi'an Ruimai electrochemiluminescence analyzer 10, the photomultiplier tube high voltage is 800V, and the current and light signals are recorded. The results are as follows Figures 11-12 As shown, it can be seen that the optical signal reaches a maximum near 0.9 V, and the reduction peak at 0.5 V corresponds to the reduction of gold.

[0087] In addition, the electrochemiluminescence performance of the sensor under different conditions was compared in the experiment. Figures 13-14 When there is no analyte in the water sample, the light signal is weak (A), and there is almost no light signal when the co-reactant tripropylamine is not present in the scanning solution (B). In the presence of the analyte and the co-reactant but without polymerizing PEDOT, the signal is also weak (C), indicating that the conductive polymer significantly enhances electron transfer.

[0088] Example 2

[0089] A method for preparing a sandwich structure sensor comprises the following steps:

[0090] Step A is the same as in Example 1;

[0091] Step B, pre-assembly of monomers and template molecules:

[0092] (1) Pretreatment of gold electrode 4: The glassy carbon electrode with a diameter of 3 mm was polished with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al2O3, followed by alternating ultrasonic cleaning with ethanol and ultrapure water for 3 times, each time for 30 seconds, and finally CV scanning in 0.5 mM sulfuric acid solution until stable. Square wave voltammetry was used to scan 0.5-0 V in 10 times PBS buffer containing a mixture of 10 mM potassium ferrocyanide / potassium ferrocyanide. The polishing was completed when the signal peak reached 250 μA. The scanning parameters were set to a step size of 2 mV, an amplitude of 25 mV, and a balance time of 60 seconds. The counter electrode 3 used in the scan was a platinum wire, and the reference electrode 2 was a saturated calomel electrode or a saturated silver / silver chloride electrode.

[0093] Insert the polished glassy carbon electrode into 10 times PBS buffer containing 2 mM tetrachloroauric acid, use the glassy carbon electrode as the working electrode 1, the saturated calomel electrode as the reference electrode 2, and the platinum wire as the counter electrode 3, and use the chronocoulometry method to deposit gold on the surface of the glassy carbon electrode. Fig.15 As shown, the control potential is -0.2 V and the deposition charge is 1 mC.

[0094] (2) Monomer self-assembly: Prepare an assembly solution containing 0.5 mM p-mercaptophenylboronic acid (functional monomer) and 1 mM phenylethylmercaptan (auxiliary monomer), and the solvent is acetonitrile + DMF (volume ratio of 1:1). Take 20 μL and drop it on the surface of the gold electrode 4 and let it stand at room temperature for 2 hours.

[0095] (3) After a simple rinse, 20 μL of 0.5 mM mercaptohexanol was used for incubation for 30 minutes to block the unreacted sites. Then, 20 μL of 10 mM azithromycin 8 in acetonitrile / DMF solution (volume ratio of 1:1) was drop-coated on the electrode surface and allowed to stand for 2 hours to allow the functional monomers on the electrode surface to fully capture the template molecules. The electrode was then placed in acetonitrile and stirred at 300 rpm for 5 minutes to remove the unbound template molecules.

[0096] Step C, molecular imprinting membrane preparation:

[0097] (1) The cleaned electrode was inserted into an acetonitrile solution containing 0.1 M tetrabutylammonium perchlorate and 4 mM EDOT, and a +1.4 V constant potential deposition was performed using a non-aqueous anion electrode as a reference, and the polymerization charge was controlled to be 0.1 mC.

[0098] (2) After the polymerization, the electrode was stirred at 300 rpm for 60 minutes in 10 mL of 9:1 acetonitrile-acetic acid solution (V / V) to remove the template. At this point, the molecular imprinted polymer electrode was prepared and can be used for detection and analysis. The surface morphology was characterized using scanning electron microscopy and atomic force microscopy. Figures 16-17 The diameter of the nanoparticles on the electrode surface is about 100 nanometers and is evenly distributed, which increases the specific surface area of ​​the electrode.

[0099] Step D, selective capture of pollutants in the sample:

[0100] Add 10 μM azithromycin 8 stock solution (aqueous solution containing 1 / 100,000 DMF as solubilizer) to deionized water to prepare a series of standard samples. Take 10 mL of the standard sample, immerse the prepared electrode below the liquid surface, incubate at room temperature at 300 rpm for 30 minutes, and then rinse with deionized water.

[0101] Step E, sandwich structure electrode construction:

[0102] 1% wt bovine serum albumin was used for incubation for 30 minutes to block the nonspecific binding sites on the electrode surface. Subsequently, 20 μL of 40 μg / mL labeled antibody 7 was drop-coated on the electrode surface and incubated at 37°C for 60 minutes. After a simple rinse, the electrode was immersed in 1× PBS and stirred at 300 rpm for 5 minutes to remove the unbound antibody 7, and a sandwich structure sensor was prepared.

[0103] Application 2

[0104] The sandwich structure sensor prepared in Example 2 was subjected to electrochemiluminescence measurement:

[0105] The obtained electrode was used as the working electrode 1, the platinum wire as the counter electrode 3, and the saturated silver / silver chloride as the reference electrode 2, and the electrochemiluminescence test was carried out in a 10-fold PBS solution containing 0.1M tri-n-propylamine and 2% Tween 20. The program was set to 15 cycles, each cycle included constant potential pulses of 0V 2s, -0.5V 2s, 0V 5s, and +0.9V 10ms, and the light-electric signal of the last 3ms of the +0.9V pulse in each cycle was recorded, and the average value was the electrochemiluminescence signal of the obtained sensor. After testing, the detection limit of the sensor in deionized water was 0.03nM, the detection limit was 0.07nM, the linear range was 0.1-10nM, and the calibration curve was as shown in Fig.18 As shown in Table 2 below, the sensor has good selectivity, and analogs such as erythromycin, clarithromycin, roxithromycin, carbamazepine, and bisphenol A have little interference in its detection.

[0106] Table 2 Signal response of the obtained sensor to the interferents

[0107] Pollutants Dosage(nM) Detection amount (nM) Recovery rate Azithromycin 10.00 10.48 104.8％ Erythromycin 10.00 1.81 18.1％ Carbamazepine 10.00 0.50 5.0％ Roxithromycin 10.00 0.29 2.9％ Clarithromycin 10.00 0.44 4.4％ Bisphenol A 10.00 0.05 0.5％

[0108] Example 3

[0109] A method for preparing a sandwich structure sensor comprises the following steps:

[0110] Step A, Antibody 7 labeling:

[0111] (1) Dissolve 25 mg of Ru(bpy)2(dcbpy)(PF6)2 in 1 mL of DMF to prepare a 26.4 mmol / LX solution. Mix well and store in a refrigerator at 4°C (keep out moisture).

[0112] (2) Ice making.

[0113] (3) Dissolve 0.31 g EDC and 0.12 g NHS in 4 mL DMF to obtain solution Y (freshly prepared), in which the concentrations of EDC and NHS are 500 mM and 200 mM, respectively, and cool in an ice-water bath.

[0114] (4) Take 190 μL of solution X and 100 μL of solution Y, add 710 μL of DMF to obtain 5 mM [Ru-NHS], place in an ice-water bath, stir at 0°C for 30 minutes, and then leave at room temperature for 5 hours.

[0115] (5) The dialysis bag was placed in 1 mM EDTA2Na (pH=8) and boiled for 10 minutes. The bag was then cooled and placed in a 30% alcohol solution and stored in a refrigerator at 4°C.

[0116] (6) Take Antibody 7 out of the refrigerator and equilibrate it to room temperature. Dilute Antibody 7 to 1 mg / mL with 1x PBS.

[0117] (7) Take 30 μL of 5 mM Ru-NHS solution at room temperature and add it to 1 mL of 1 mg / mL solution of antibody 7. Wrap in tin foil and shake at room temperature for 1 hour.

[0118] (8) Excess Ru-NHS was divided into 100 μL portions and stored at -20°C to prevent contamination from repeated freezing and thawing.

[0119] (9) Add 20 μL of 2 M glycine and incubate for 15 min to terminate the reaction.

[0120] (10) Add the solution to the dialysis bag and rinse the tube wall slightly with buffer solution, then tie it with thread and clamp it with a dialysis clamp. Place it in 1x PBS buffer solution and let it stand for 8 hours, then change the water and let it stand for another 8 hours.

[0121] (11) Take out the spin filter centrifuge tube, add ultrapure water, and make sure that the water completely passes through the membrane. Pre-cool it in an ice bath or refrigerator for a few minutes, and pour out the water. Gently add the remaining liquid in the dialysis bag to the spin filter centrifuge tube, so that the liquid level does not exceed the white line on the top of the tube. Centrifuge the centrifuge tube at 1000×g for 3-5 minutes.

[0122] (12) Use pure protein purification system to further purify the labeled antibody 7. The separation column used was Superdex TM 200Increase 10 / 300GL Prepacked Tricorn TM Columns, the buffer was 1x PBS (pH=7.4), and the flow rate was 0.075 mL / min.

[0123] Step B, pre-assembly of monomers and template molecules:

[0124] (1) Pretreatment of the gold electrode 4: The gold electrode 4 was polished with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al2O3 in sequence, and then ultrasonically cleaned with ethanol and ultrapure water for 3 times, each time for 30 seconds, and finally CV scanning was performed in a 0.5 mM sulfuric acid solution until it was stable.

[0125] (2) Monomer self-assembly: Prepare an assembly solution, whose solute is 1 mM p-mercaptophenylboronic acid (functional monomer), 1 mM 2-thiophenethiol (auxiliary monomer), and the solvent is acetonitrile + DMF (volume ratio is 8:2). Apply 10 μL of the assembly solution droplet on the surface of the gold electrode 4 and let it stand at room temperature for 60 minutes.

[0126] (3) The electrode assembled in step (2) was stirred in acetonitrile for 5 minutes to remove excess monomers. Then, 30 μL of azithromycin 8 (template molecule) in acetonitrile / DMF solution (volume ratio of 8:2) was drop-coated on the electrode surface, left to stand at room temperature for 30 minutes, and then inserted into acetonitrile and stirred for 120 minutes to remove unbound template molecules.

[0127] Step C, molecular imprinting membrane preparation:

[0128] (1) A gold electrode 4 after simple elution was used as the working electrode 1, a platinum wire was used as the counter electrode 3, and a silver electrode filled with 0.1M tetrabutylammonium perchlorate was used as the reference electrode 2 for cyclic voltammetric electropolymerization. The polymerization solution was an acetonitrile solution of 2mM 3,4-ethylenedioxythiophene (EDOT) containing 0.1M tetrabutylammonium perchlorate. The parameters were set to -0.5V to 1.25V, and the scanning speed was 50mV / s.

[0129] (2) Gently rinse the PEDOT-polymerized electrode with deionized water and acetonitrile, and then remove the template molecule in 10 mL of 9:1 acetonitrile-acetic acid solution (V / V) at 300 rpm for 30 minutes. After completion, discard the waste liquid and add 10 mL of 9:1 acetonitrile-acetic acid solution (V / V) to ensure that the template is completely removed.

[0130] Step D, selective capture of pollutants in the sample:

[0131] The electrode after the template molecule is removed is immersed in the water sample to be tested and stirred at a speed of 500 rpm for 10 minutes, then taken out and the surface is gently rinsed with deionized water to remove unbound impurities.

[0132] Step E, sandwich structure electrode construction:

[0133] The electrode was dried with nitrogen, and 20 μL of 2% wt bovine serum albumin was applied on the surface and incubated at room temperature for 60 minutes. After gentle rinsing with 1x PBS solution, 20 μL of 50 μg / mL labeled antibody 7 was applied on the electrode surface and incubated at room temperature for 120 minutes. After a simple rinse, the electrode was immersed in 1x PBS and stirred at 100 rpm for 15 minutes to remove unbound antibody 7, and a sandwich structure sensor was prepared.

[0134] Application 3

[0135] The sandwich structure sensor prepared in Example 3 was subjected to electrochemiluminescence measurement:

[0136] The obtained electrode was used as the working electrode 1, the platinum wire was used as the counter electrode 3, and the saturated silver / silver chloride was used as the reference electrode 2. An electrochemiluminescence test was performed in a 10-fold PBS solution containing 0.05 M tri-n-propylamine and 0.5 wt % Tween 20.

[0137] Example 4

[0138] A method for preparing a sandwich structure sensor comprises the following steps:

[0139] Step A, Antibody 7 labeling:

[0140] (1) Dissolve 25 mg of Ru(bpy)2(dcbpy)(PF6)2 in 1 mL of DMF to prepare a 26.4 mmol / LX solution. Mix well and store in a refrigerator at 4°C (keep out moisture).

[0141] (2) Ice making.

[0142] (3) Dissolve 0.31 g EDC and 0.12 g NHS in 4 mL DMF to obtain solution Y (freshly prepared), in which the concentrations of EDC and NHS are 500 mM and 200 mM, respectively, and cool in an ice-water bath.

[0143] (4) Take 190 μL of solution X and 100 μL of solution Y, add 710 μL of DMF to obtain 5 mM [Ru-NHS], place in an ice-water bath, stir at 0°C for 30 minutes, and then leave at room temperature for 5 hours.

[0144] (5) The dialysis bag was placed in 1 mM EDTA2Na (pH=8) and boiled for 10 minutes. The bag was then cooled and placed in a 30% alcohol solution and stored in a refrigerator at 4°C.

[0145] (6) Take Antibody 7 out of the refrigerator and equilibrate it to room temperature. Dilute Antibody 7 to 1 mg / mL with 1x PBS.

[0146] (7) Take 6.25 μL of 5 mM Ru-NHS solution at room temperature and add it to 1 mL of 1 mg / mL solution of antibody 7. Wrap in tin foil and shake at room temperature for 1 hour.

[0147] (8) Excess Ru-NHS was divided into 100 μL portions and stored at -20°C to prevent contamination from repeated freezing and thawing.

[0148] (9) Add 20 μL of 2 M glycine and incubate for 15 min to terminate the reaction.

[0149] (10) Add the solution to the dialysis bag and rinse the tube wall slightly with buffer solution, then tie it with thread and clamp it with a dialysis clamp. Place it in 1x PBS buffer solution and let it stand for 8 hours, then change the water and let it stand for another 8 hours.

[0150] (11) Take out the spin filter centrifuge tube, add ultrapure water, and make sure that the water completely passes through the membrane. Pre-cool it in an ice bath or refrigerator for a few minutes, and pour out the water. Gently add the remaining liquid in the dialysis bag to the spin filter centrifuge tube, so that the liquid level does not exceed the white line on the top of the tube. Centrifuge the centrifuge tube at 1000×g for 3-5 minutes.

[0151] (12) Use pure protein purification system to further purify the labeled antibody 7. The separation column used was Superdex TM 200Increase 10 / 300GL Prepacked Tricorn TM Columns, the buffer was 1x PBS (pH=7.4), and the flow rate was 0.075 mL / min.

[0152] Step B, pre-assembly of monomers and template molecules:

[0153] (1) Pretreatment of the gold electrode 4: The gold electrode 4 was polished with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al2O3 in sequence, and then ultrasonically cleaned with ethanol and ultrapure water for 3 times, each time for 30 seconds, and finally CV scanning was performed in a 0.5 mM sulfuric acid solution until it was stable.

[0154] (2) Monomer self-assembly: Prepare an assembly solution, the solute of which is 1 mM p-mercaptophenylboronic acid (functional monomer), 1 mM 2-thiophenethiol (auxiliary monomer), and the solvent is acetonitrile + DMF (volume ratio of 1:9). Apply 50 μL of the assembly solution droplet on the surface of the gold electrode 4 and let it stand at room temperature for 120 minutes.

[0155] (3) The electrode assembled in step (2) was stirred in acetonitrile for 5 minutes to remove excess monomers. Then, 30 μL of azithromycin 8 (template molecule) in acetonitrile / DMF solution (volume ratio of 1:9) was drop-coated on the electrode surface, left to stand at room temperature for 75 minutes, and then inserted into acetonitrile and stirred for 30 minutes to remove unbound template molecules.

[0156] Step C, molecular imprinting membrane preparation:

[0157] (1) A gold electrode 4 after simple elution was used as the working electrode 1, a platinum wire was used as the counter electrode 3, and a silver electrode filled with 0.1M tetrabutylammonium perchlorate was used as the reference electrode 2 for cyclic voltammetric electropolymerization. The polymerization solution was an acetonitrile solution of 2mM 3,4-ethylenedioxythiophene (EDOT) containing 0.1M tetrabutylammonium perchlorate. The parameters were set to -0.5V to 1.25V, and the scanning speed was 50mV / s.

[0158] (2) Gently rinse the PEDOT-polymerized electrode with deionized water and acetonitrile, and then remove the template molecule in 20 mL of 8:1 acetonitrile-acetic acid solution (V / V) at 300 rpm for 30 minutes. After completion, discard the waste liquid and add 20 mL of 8:1 acetonitrile-acetic acid solution (V / V) to ensure that the template is completely removed.

[0159] Step D, selective capture of pollutants in the sample:

[0160] The electrode after the template molecule is removed is immersed in the water sample to be tested and stirred at 300 rpm for 30 minutes, then taken out and the surface is gently rinsed with deionized water to remove unbound impurities.

[0161] Step E, sandwich structure electrode construction:

[0162] The electrode was blown dry with nitrogen, and 20 μL of 1.5% wt bovine serum albumin was dripped on the surface and incubated at room temperature for 60 minutes. After gentle rinsing with 1x PBS solution, 20 μL of 50 μg / mL labeled antibody 7 was dripped on the electrode surface and incubated at room temperature for 120 minutes. After a simple rinse, the electrode was immersed in 1x PBS and stirred at 100 rpm for 15 minutes to remove unbound antibody 7, and a sandwich structure sensor was prepared.

[0163] Application 4

[0164] The sandwich structure sensor prepared in Example 4 was subjected to electrochemiluminescence measurement:

[0165] The obtained electrode was used as the working electrode 1, the platinum wire was used as the counter electrode 3, and the saturated silver / silver chloride was used as the reference electrode 2. Electrochemiluminescence test was performed in a 10-fold PBS solution containing 0.2 M tri-n-propylamine and 2 wt % Tween 20.

[0166] Example 5

[0167] A method for preparing a sandwich structure sensor comprises the following steps:

[0168] Step A, Antibody 7 labeling:

[0169] (1) Dissolve 25 mg of Ru(bpy)2(dcbpy)(PF6)2 in 1 mL of DMF to prepare a 26.4 mmol / LX solution. Mix well and store in a refrigerator at 4°C (keep out moisture).

[0170] (2) Ice making.

[0171] (3) Dissolve 0.31 g EDC and 0.12 g NHS in 4 mL DMF to obtain solution Y (freshly prepared), in which the concentrations of EDC and NHS are 500 mM and 200 mM, respectively, and cool in an ice-water bath.

[0172] (4) Take 190 μL of solution X and 100 μL of solution Y, add 710 μL of DMF to obtain 5 mM [Ru-NHS], place in an ice-water bath, stir at 0°C for 30 minutes, and then leave at room temperature for 5 hours.

[0173] (5) The dialysis bag was placed in 1 mM EDTA2Na (pH=8) and boiled for 10 minutes. The bag was then cooled and placed in a 30% alcohol solution and stored in a refrigerator at 4°C.

[0174] (6) Take Antibody 7 out of the refrigerator and equilibrate it to room temperature. Dilute Antibody 7 to 1 mg / mL with 1x PBS.

[0175] (7) Take 30 μL of 5 mM Ru-NHS solution at room temperature and add it to 1 mL of 1 mg / mL solution of antibody 7. Wrap in tin foil and shake at room temperature for 1 hour.

[0176] (8) Excess Ru-NHS was divided into 100 μL portions and stored at -20°C to prevent contamination from repeated freezing and thawing.

[0177] (9) Add 20 μL of 2 M glycine and incubate for 15 min to terminate the reaction.

[0178] (10) Add the solution to the dialysis bag and rinse the tube wall slightly with buffer solution, then tie it with thread and clamp it with a dialysis clamp. Place it in 1x PBS buffer solution and let it stand for 8 hours, then change the water and let it stand for another 8 hours.

[0179] (11) Take out the spin filter centrifuge tube, add ultrapure water, and make sure that the water completely passes through the membrane. Pre-cool it in an ice bath or refrigerator for a few minutes, and pour out the water. Gently add the remaining liquid in the dialysis bag to the spin filter centrifuge tube, so that the liquid level does not exceed the white line on the top of the tube. Centrifuge the centrifuge tube at 1000×g for 3-5 minutes.

[0180] (12) Use pure protein purification system to further purify the labeled antibody 7. The separation column used was Superdex TM 200Increase 10 / 300GL Prepacked Tricorn TM Columns, the buffer was 1x PBS (pH=7.4), and the flow rate was 0.075 mL / min.

[0181] Step B, pre-assembly of monomers and template molecules:

[0182] (1) Pretreatment of the gold electrode 4: The gold electrode 4 was polished with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al2O3 in sequence, and then ultrasonically cleaned with ethanol and ultrapure water for 3 times, each time for 30 seconds, and finally CV scanning was performed in a 0.5 mM sulfuric acid solution until it was stable.

[0183] (2) Monomer self-assembly: Prepare an assembly solution, the solute of which is 1 mM p-mercaptophenylboronic acid (functional monomer), 1 mM 2-thiophenethiol (auxiliary monomer), and the solvent is acetonitrile + DMF (volume ratio is 1:1), and prepare the assembly solution. Take 30 μL of the assembly solution droplet and apply it on the surface of the gold electrode 4, and let it stand at room temperature for 90 minutes.

[0184] (3) The electrode assembled in step (2) was stirred in acetonitrile for 5 minutes to remove excess monomers. Then, 30 μL of azithromycin 8 (template molecule) in acetonitrile / DMF solution (volume ratio of 2:1) was drop-coated on the electrode surface, left to stand at room temperature for 100 minutes, and then inserted into acetonitrile and stirred for 30 minutes to remove unbound template molecules.

[0185] Step C, molecular imprinting membrane preparation:

[0186] (1) A gold electrode 4 after simple elution was used as the working electrode 1, a platinum wire was used as the counter electrode 3, and a silver electrode filled with 0.1M tetrabutylammonium perchlorate was used as the reference electrode 2 for cyclic voltammetric electropolymerization. The polymerization solution was an acetonitrile solution of 2mM 3,4-ethylenedioxythiophene (EDOT) containing 0.1M tetrabutylammonium perchlorate. The parameters were set to -0.5V to 1.25V, and the scanning speed was 50mV / s.

[0187] (2) Gently rinse the polymerized PEDOT electrode with deionized water and acetonitrile, and then remove the template molecule in 20 mL of 8:2 acetonitrile-acetic acid solution (V / V) at 300 rpm for 30 minutes. After completion, discard the waste liquid and add 20 mL of 8:2 acetonitrile-acetic acid solution (V / V) to ensure that the template is completely removed.

[0188] Step D, selectively capture the added pollutants in the effluent of the secondary sedimentation tank of tap water, synthetic sewage and domestic sewage treatment plants:

[0189] The electrode after the template molecule is removed is immersed in the water sample to be tested and stirred at 400 rpm for 40 minutes, then taken out and the surface is gently rinsed with deionized water to remove unbound impurities.

[0190] Step E, sandwich structure electrode construction:

[0191] The electrode was dried with nitrogen, and 20 μL of 1% wt bovine serum albumin was dripped on the surface and incubated at room temperature for 60 minutes. After gentle rinsing with 1x PBS solution, 20 μL of 50 μg / mL labeled antibody 7 was dripped on the electrode surface and incubated at room temperature for 120 minutes. After a simple rinse, the electrode was immersed in 1x PBS and stirred at 100 rpm for 15 minutes to remove unbound antibody 7, and a sandwich structure sensor was prepared.

[0192] Application 5

[0193] The sandwich structure sensor prepared in Example 5 was subjected to electrochemiluminescence measurement:

[0194] The obtained electrode was used as the working electrode 1, the platinum wire was used as the counter electrode 3, and the saturated silver / silver chloride was used as the reference electrode 2. An electrochemiluminescence test was performed in a 10-fold PBS solution containing 0.1 M tri-n-propylamine and 1 wt % Tween 20.

[0195] Table 3 Recovery rate of the obtained sensors in different sewage

[0196]

[0197] Example 6

[0198] This implementation case optimizes the key parameters in the preparation and use of the obtained sensor. Considering that the key group of the functional monomer to capture pollutants is the boric acid bond, it is considered to use small molecules containing hydroxyl groups (methanol, ethanol) or ortho-hydroxyl groups (1,2-propylene glycol) to block them in a targeted manner.

[0199] The remaining steps of this embodiment are the same as those of embodiment 1, with the only difference being that: taking the molecularly imprinted polymer electrode treated in step C, 20 μL of 1% wt methanol, ethanol, and propylene glycol aqueous solutions were drop-coated on the surface, respectively, followed by incubation with antibody 7 and electrochemiluminescence testing.

[0200] The results are shown in Table 3 below. Methanol exhibits the best blocking effect due to the smallest steric hindrance, followed by ethanol, and propylene glycol is the worst. However, due to the presence of a large number of nonspecific binding sites on the electrode surface, its background noise still needs to be further controlled. In contrast, the blocking effect of 1%wt bovine serum albumin is superior, and the sensor blocked with methanol first and then with bovine serum albumin obtains a lower background signal value. Therefore, 20μL 1%wt methanol + 20μL 1%wt bovine serum albumin blocking in sequence is the best solution.

[0201] Table 4 Comparison of the effects of different sealants

[0202] Sealing agent Methanol Ethanol 1,2-Propanediol BSA Methanol + BSA Background signal 3205 3836 4250 382 275

[0203] Example 7

[0204] The concentration of labeled antibody 7 used in the detection process also needs to be optimized. Too low a concentration of antibody 7 will lead to weak signals, insensitive responses, and narrow linear ranges, while too high a concentration of antibody 7 will lead to high background responses, high detection costs, and poor sensitivity, both of which will affect the signal-to-noise ratio of the sensor.

[0205] The remaining steps of this embodiment are the same as those of embodiment 1, with the only difference being that: the molecular imprinted polymer electrodes treated in steps C and D were respectively blocked with 20 μL 1% wt methanol + 20 μL 1% wt bovine serum albumin. Subsequently, 20 μL 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 200 μg / mL of labeled antibody 7 were dripped on the surface, respectively, and electrochemiluminescence test was performed after incubation at 37°C for 60 minutes. Among them, 40 μg / mL of labeled antibody 7 showed the best signal-to-noise ratio and economy, so 40 μg / mL was determined as the optimal antibody 7 concentration.

[0206] Example 8

[0207] The concentration of co-reactants in electrochemiluminescence reactions directly affects the intensity of the signal.

[0208] The remaining steps of this embodiment are the same as those of embodiment 1, except that the molecularly imprinted polymer electrode treated in step E is tested in solutions with different tri-n-propylamine concentrations. The results show that low concentrations of tri-n-propylamine have a poor signal enhancement effect, and 100 mM can significantly enhance the optical signal, while 200 mM only slightly enhances it, so 100 mM is determined to be the optimal co-reaction reagent concentration.

Claims

1. A method for preparing a sandwich structure sensor, characterized in that: The following steps are involved: Step 1, purifying the antibody (7) labeled with the luminescent molecule (6) to remove unbound luminescent molecules and impurities; Step 2: Take the acetonitrile solution of the functional monomer and the auxiliary monomer and apply it to the surface of the polished gold electrode (4), let it stand at room temperature for 60 to 180 minutes, rinse it, complete the self-assembly, and apply the acetonitrile DMF mixed solution of the template molecule to the electrode surface, insert the treated electrode into the acetonitrile solution of the cross-linking monomer for electropolymerization, and then stir it in the acetonitrile acetic acid solution to remove the template molecule; Step 3, immerse the electrode obtained in step 2 into the water sample to be tested and stir at a speed of 200-500 rpm for 10-60 minutes, then take it out and rinse; Step 4: drip a sealing agent on the surface of the electrode obtained in step 3, rinse with deionized water or PBS solution, then drip an antibody (7) labeled with the luminescent molecule (6) in step 1 on the surface of the electrode, rinse and stir until the unbound antibody (7) is removed, and obtain a sandwich structure sensor; In the step 1, the luminescent molecule is di(2,2'-bipyridine)-4,4'-dicarboxylbipyridine-ruthenium di(hexafluorophosphate), the antibody is a mouse anti-monoclonal antibody, and the initial molar ratio of the luminescent molecule to the antibody is 5-25:1; In the step 2, the functional monomer is p-mercaptophenylboronic acid, the auxiliary monomer is 2-mercaptothiophene or phenylethylmercaptan, and the template molecule is azithromycin (8); In the step 2, the cross-linking monomer is any one of 3,4-ethylenedioxythiophene, aniline and pyrrole.

2. The method for preparing a sandwich structure sensor according to claim 1, characterized in that: In the step 2, the gold electrode is polished and ultrasonically cleaned alternately with ethanol and ultrapure water until the CV scan is stable in a sulfuric acid solution.

3. The method for preparing a sandwich structure sensor according to claim 1, characterized in that: In the step 2, in the acetonitrile-acetic acid solution, the volume ratio of acetonitrile to acetic acid is 5-9:

1.

4. The method for preparing a sandwich structure sensor according to claim 1, characterized in that: In the step 4, the blocking agent is 1-2 wt% of any one of bovine serum albumin, casein, and skimmed milk powder.

5. The method for preparing a sandwich structure sensor according to claim 1, characterized in that: The sandwich structure sensor comprises a working electrode (1), a reference electrode (2) and a counter electrode (3); the working electrode (1) comprises a gold electrode (4), a conductive molecular imprinting polymer layer (5) and an antibody (7) labeled with a luminescent molecule (6) in sequence; a hole (9) for capturing azithromycin (8) is arranged in the conductive molecular imprinting polymer layer (5); and the antibody (7) labeled with the luminescent molecule (6) is combined with the azithromycin (8) through intermolecular forces.

6. The method for preparing a sandwich structure sensor according to claim 5, characterized in that: The sandwich structure sensor is connected to an electrochemiluminescence analyzer (10), the reference electrode (2) is a saturated silver / silver chloride electrode, and the counter electrode (3) is a platinum wire.

7. Application of the sandwich structure sensor prepared by the method for preparing a sandwich structure sensor according to claim 5 in monitoring trace concentration of azithromycin in a sewage treatment plant.

Images