CN115184431B - Preparation method and application of molecular imprinting electrochemical sensor with double-signal probe strategy - Google Patents
Preparation method and application of molecular imprinting electrochemical sensor with double-signal probe strategy Download PDFInfo
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- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention belongs to the technical field of analytical chemistry and electrochemistry, and discloses a preparation method and application of a molecular imprinting electrochemical sensor with a double-signal probe strategy. Fixing a first template molecule IgG onto Prussian blue@gold nanoparticles/graphene oxide/screen printing carbon electrode through gold sulfide bonds to obtain the molecular imprinting electrochemical sensor, fixing a second template molecule IgM onto multiwall carbon nanotube-thionine-gold nanoparticles/bovine serum albumin/IgG/Prussian blue@gold nanoparticles/graphene oxide/screen printing carbon electrode through gold sulfide bonds, electrochemically polymerizing pyrrole, eluting the IgG and the IgM by using eluent to form imprinting cavities complementary with the two template molecules in three-dimensional space to obtain the molecular imprinting electrochemical sensor. The molecularly imprinted electrochemical sensor provided by the invention can detect IgG and IgM simultaneously.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and electrochemistry, and particularly relates to a preparation method and application of a molecular imprinting electrochemical sensor with a double-signal probe strategy.
Background
Immunoglobulins (Ig) play an important role in many defense mechanisms of organisms against various potentially damaging objects, such as viruses or bacteria. Five classes of common immunoglobulins have been reported in the human circulatory system, namely immunoglobulins A, G, M, E and D. Excessive Ig levels can cause rheumatoid arthritis, liver disease and infectious diseases, but low Ig levels can also cause humoral immunodeficiency and metabolic diseases. Since 12 months 2019, igG and IgM have become important indicators for assessing early or infectious stages due to outbreaks of new coronal epidemics. Enzyme-linked immunosorbent assay is commonly used for detecting IgG and IgM, but has the defects of expensive instrument, complex operation, time consumption in measurement and the like. The electrochemical detection has the advantages of high sensitivity, low cost, simple operation and the like, thereby having great potential in medical treatment.
The molecular imprinting electrochemical sensor combines a molecular imprinting technology with an electrochemical sensing technology, adopts a Molecular Imprinting Polymer (MIP) as a specific molecular recognition element, and enhances the selective recognition performance of the electrochemical sensor. The molecular imprinting electrochemical sensor has the advantages of high sensitivity, good selectivity, high analysis speed, easy miniaturization and the like, and has been widely applied to detection of multiple targets in serum. However, so far, the study of MIPs has been mainly directed to small molecules. Since proteins are biological macromolecules, which have relatively large molecular weights, complex surface morphologies, and variable molecular conformations, there is a need to find a suitable method for molecular imprinting of proteins.
Disclosure of Invention
In order to detect IgG and IgM simultaneously, the invention provides a preparation method and application of a molecular imprinting electrochemical sensor.
The technical scheme is as follows: in order to solve the technical problems, the invention adopts the following technical scheme:
a molecular imprinting electrochemical sensor with a double-signal probe strategy can detect IgG and IgM simultaneously. A Screen Printing Carbon Electrode (SPCE) is used as a substrate electrode, graphene Oxide (GO) is used for modifying the SPCE, prussian blue@gold nanoparticles (PB@AuNPs) are electrodeposited on the surface of the GO/SPCE in one step, immunoglobulin G (IgG) is used as a first template molecule for modifying the surface of the PB@AuNPs/GO/SPCE, and PB is a medium for indicating the change of an IgG signal. The remaining binding sites were blocked with Bovine Serum Albumin (BSA). Preparing multiwalled carbon nanotubes-thionine-gold nanoparticles (MWCNTs-TH-AuNPs), modifying the multiwalled carbon nanotubes-thionine-gold nanoparticles on the surface of BSA/IgG/PB@AuNPs/GO/SPCE, modifying immunoglobulin M (IgM) as a second template molecule on the surface of MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, wherein TH is a medium for indicating IgM signal change. And then, forming a polymer layer by electropolymerized pyrrole (Py) to further embed the IgG and IgM, and finally eluting the IgG and IgM template molecules by using eluent to form a imprinting cavity, thereby successfully preparing the molecular imprinting electrochemical sensor.
A molecular imprinting electrochemical sensor with a double-signal probe strategy, wherein a first template molecule IgG is fixed on Prussian blue@gold nano particles/graphene oxide/screen printing carbon electrode PB@AuNPs/GO/SPCE through gold sulfide bonds to obtain IgG/PB@AuNPs/GO/SPCE, and a second template molecule IgM is fixed on multi-wall carbon nano tubes-thionine-gold nano particles/bovine serum albumin/IgG/Prussian blue@gold nano particles/graphene oxide/screen printing carbon electrode MWCNTs-TH-AuNPs/BSA/IgG@AuNPs/GO/SPCE to obtain IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE; finally, the pyrrole is electrochemically polymerized to obtain PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, and after the IgG and IgM are eluted by eluent, imprinting cavities complementary with the two template molecules in three-dimensional space are formed, so that the molecular imprinting electrochemical sensor is obtained.
Further, the preparation method of the Prussian blue@gold nanoparticle/graphene oxide/screen-printed carbon electrode PB@AuNPs/GO/SPCE comprises the following steps: dissolving anhydrous ferric trichloride, potassium ferricyanide and potassium chloride in a hydrochloric acid solution, and then adding a chloroauric acid solution into the solution to obtain a PB@AuNPs solution; transferring the GO solution by a liquid transferring gun, dripping the GO solution on the surface of the SPCE, and drying under an infrared lamp to obtain GO/SPCE; scanning in the potential range of 0-1V by cyclic voltammetry, and electrodepositing PB@AuNPs layer on the surface of GO/SPCE to form PB@AuNPs/GO/SPCE.
Further, the preparation method of the multiwall carbon nanotube-thionine-gold nanoparticle/bovine serum albumin/IgG/Prussian blue @ gold nanoparticle/graphene oxide/screen-printed carbon electrode MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE comprises the following steps:
dripping an IgG solution on the surface of PB@AuNPs/GO/SPCE, airing, and then removing BSA as a closed droplet to coat on the surface of the BSA/IgG/PB@AuNPs/GO/SPCE; dissolving MWCNTs in a polyethyleneimine water solution, and performing ultrasonic dispersion; mixing the TH water solution with the solution, vigorously stirring, after stirring is finished, washing the solid substance obtained after centrifugal separation with ultrapure water for a plurality of times, dispersing in water to obtain an MWCNTs-TH solution, adding the MWCNTs-TH solution into the AuNPs solution, stirring uniformly, and centrifuging to obtain a solid which is the MWCNTs-TH-AuNPs composite material; the MWCNTs-TH-AuNPs composite material is dispersed in water in an ultrasonic way, and is coated on the surface of BSA/IgG/PB@AuNPs/GO/SPCE by a pipette, so that the MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE is obtained.
Further, the elution step is as follows: and (3) transferring 10% acetic acid solution containing 10% sodium dodecyl sulfate by mass fraction and having a volume fraction of 10% by using a pipette, dripping the solution on the surface of PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, eluting for 0.5-2.5 h, and removing template molecules IgG and IgM.
Further, when the PB@AuNPs layer is electrodeposited on the surface of the GO/SPCE, the cyclic voltammetry scanning rate is 90-110 mV s –1 The number of scanning turns is 10-30.
Further, the phosphate buffer solution of pyrrole is dripped on the surface of IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, and the cyclic voltammetry scanning rate is 25-125 mV s –1 Preferably 75mV s –1 The number of deposition turns is 3-15, and a PPy polymerization layer is formed.
Further, the IgG concentration is 0.5-2.0. Mu.g mL –1 The method comprises the steps of carrying out a first treatment on the surface of the IgM concentration of 400-600 ng mL –1 。
Further, the concentration of the hydrochloric acid solution was 0.1mM; the concentration of chloroauric acid solution was 0.2mM.
The invention also provides application of the molecularly imprinted electrochemical sensor with the double-signal probe strategy in detecting IgG and IgM.
Further, incubating the molecularly imprinted electrochemical sensor in a solution containing IgG and IgM for 1-6 h, and recording a differential pulse voltammogram within a potential range of-0.5V to realize the simultaneous detection of the IgG and the IgM. The invention also provides a preparation method of the molecular imprinting electrochemical sensor with the double-signal probe strategy, which comprises the following steps:
step a, configuring PB@AuNPs solution: weighing anhydrous ferric trichloride, potassium ferricyanide and potassium chloride, dissolving in a 0.1M hydrochloric acid solution, and then adding a 0.2mM chloroauric acid solution into the solution to obtain a PB@AuNPs solution;
step b, preparing PB@AuNPs/GO/SPCE: transferring 10 mu L of GO solution by a liquid transferring gun, dripping the solution on the surface of the SPCE, and drying under an infrared lamp to obtain GO/SPCE; scanning 15 circles at a certain scanning rate in the potential range of 0-1V by using a cyclic voltammetry, and electrodepositing a PB@AuNPs layer on the surface of GO/SPCE to form PB@AuNPs/GO/SPCE;
step c, preparing BSA/IgG/PB@AuNPs/GO/SPCE: firstly, transferring 10 mu L of IgG solution with a certain concentration to a PB@AuNPs/GO/SPCE surface by using a pipetting gun, airing at 4 ℃, and transferring 10 mu L of BSA as a closed droplet to the electrode surface to obtain BSA/IgG/PB@AuNPs/GO/SPCE;
step d, preparing an MWCNTs-TH-AuNPs composite material: weighing 10mg of MWCNTs, dissolving in a Polyethyleneimine (PEI) aqueous solution, and performing ultrasonic dispersion; mixing TH water solution with a certain concentration with the solution, vigorously stirring, after stirring is finished, washing the solid substance obtained after centrifugal separation with ultrapure water for a plurality of times, dispersing in water to obtain MWCNTs-TH solution, transferring 0.5mL of the MWCNTs-TH solution by a pipetting gun, adding the MWCNTs-TH solution into 2.5mL of AuNPs solution, stirring for a certain time, and centrifuging to obtain the solid, namely the MWCNTs-TH-AuNPs composite material.
Step e, preparing IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE: dispersing the MWCNTs-TH-AuNPs composite material prepared in the step d in 4mL of water by ultrasonic, transferring 10 mu L of the MWCNTs-TH-AuNPs composite material to the surface of the electrode in the step c by using a liquid-transferring gun, naturally airing, transferring 10 mu L of IgM solution with a certain concentration to the surface of the electrode by using the liquid-transferring gun, airing at 4 ℃, and obtaining IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;
step f, preparing PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE: transferring 100 mu L of 0.01M phosphate buffer solution containing 0.1M pyrrole to the surface of the electrode prepared in the step e by using a pipette, and polymerizing for a certain number of circles by using a cyclic voltammetry at a certain scanning rate within a potential range of-0.3-0.8V to obtain PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;
step g, preparing a molecularly imprinted electrochemical sensor: and d, transferring 100 mu L of 10% (v/v) acetic acid solution containing 10% (w%) sodium dodecyl sulfate to the surface of the electrode prepared in the step f by using a pipetting gun, and eluting for a certain time to remove template molecules IgG and IgM, thereby obtaining the molecular imprinting electrochemical sensor.
Further, in the step a, the mass ratio of anhydrous ferric trichloride, potassium ferricyanide and potassium chloride is 0.04-0.05: 0.08 to 0.09: the volume ratio of 0.7-0.8,0.1M hydrochloric acid to 0.2mM chloroauric acid is 10-50: 0.6 to 0.7.
Further, in the step b, the volume of the GO solution removed by the pipette is 8-12 mu L; the cyclic voltammetry scanning rate is 90-110 mV s –1 The electrode is modified with the scanning circle number of 10-30, and a PB@AuNPs layer can be formed on the surface of the GO/SPCE.
Further, in step c, the volume of IgG removed by the pipette is 8-12. Mu.L, and the concentration is 0.5-2.0. Mu.g mL –1 The volume of the BSA blocking solution removed by the pipette is 10-30 mu L, and the incubation time is 0.5-2 h.
In step d, weighing PEI with the mass of 5-15 mg, MWCNTs with the mass of 5-15 mg, the volume of the PEI aqueous solution for dissolving the MWCNTs with the volume of 5-15 mL, and transferring the solution with the volume of 0.5-1.5 mL and the concentration of 1-3 mg mL by a pipette –1 The volume of MWCNTs-TH is 0.25-1.0 mL, the volume of AuNPs solution is 1.5-3.0 mL, and the stirring time is 10-14 h. Wherein the mass ratio of PEI to MWCNTs is 5-15: 5-15, wherein the volume ratio of PEI aqueous solution, TH solution, MWCNTs-TH and AuNPs solution is 5-15: 0.5 to 1.5:0.25 to 1.0:1.5 to 3.0.
Further, in step e, the volume of the MWCNTs-TH-AuNPs solution is 8-12 mu L, and the concentration of the removed IgM is 400-600 ng mL –1 。
Further, in step f, a volume of 50 to 150. Mu.L of a phosphate buffer solution of 0.01M pH 7.4 containing 0.1M pyrrole is removed, and a cyclic voltammetry scan rate is 25 to 125mV s –1 Preferably 75mV s –1 The deposition circle number is 3-15, preferably7 turns were selected to form a PPy polymer layer.
Further, in step g, the pipette is used to remove 100 to 200. Mu.L of 10% acetic acid solution containing 10% sodium dodecyl sulfate by mass fraction, and the elution time is 0.5 to 2.5 hours, preferably 2 hours.
Further, the method comprises the steps of: the molecular imprinting electrochemical sensor is applied to the simultaneous detection of IgG and IgM, the re-incubation time is 1-6 h, preferably 4h, and the differential pulse voltammogram is recorded within the potential range of-0.5V so as to realize the simultaneous detection of the IgG and the IgM.
A molecular imprinting electrochemical sensor with a double-signal probe strategy is prepared by the preparation method.
In a third aspect, the use of a molecularly imprinted electrochemical sensor of the dual signal probe strategy for simultaneous detection of IgG and IgM is provided.
In some embodiments, the application comprises: the molecularly imprinted electrochemical sensor was placed in a solution containing 1 μg mL – 1 IgG and 500ng mL –1 Incubation was performed in solution of IgM.
Further, the application further includes: and recording a cyclic voltammogram of the molecular imprinting sensor recombined with the IgG and the IgM in a potential range of-0.6-0.4V, recording a differential pulse voltammogram of the molecular imprinting sensor recombined with the IgG and the IgM in a potential range of-0.5V, and realizing simultaneous detection of the IgG and the IgM through PB and TH in the change of peak current before and after the recombination of the IgG and the IgM.
Advantageous effects
The surface molecular imprinting is an effective method for synthesizing the Western blotting polymer, which not only can improve the mass transfer process of protein molecules and avoid residues, but also can obtain binding sites with higher affinity in a polymer matrix, thereby improving the recognition efficiency. Electropolymerization is a simple method that can be used to form western-blot substrates, polypyrrole (PPy) is often used as a polymer substrate material for western-blot due to its high stability, biocompatibility and conductivity, and its controllable thickness.
The nano materials such as gold nano particles (AuNPs), multi-wall carbon nano tubes (MWCNTs), graphene Oxide (GO) and the like have the excellent characteristics of large specific surface, strong electric conductivity, high biocompatibility and the like, are often used for modifying electrodes, and can remarkably improve the analysis performance of an electrochemical sensor such as higher sensitivity and faster electron transfer rate. When these nanomaterials are used in molecularly imprinted electrochemical sensors, they can also provide a rich active site for binding to template molecules.
When the electrochemical signal of the object to be detected is not obvious during electrochemical detection, the change of the concentration of the object to be detected is often required to be indicated by means of an electrochemical medium so as to realize the detection of the concentration of the object to be detected. Prussian Blue (PB) and Thionine (TH) are often chosen as electrochemical vehicles because of their good redox reversibility.
According to the preparation method and the application of the molecular imprinting electrochemical sensor with the double-signal probe strategy, after IgG and IgM template molecules in the polypyrrole layer are eluted, imprinting cavities complementary with the IgG and IgM template molecules in the three-dimensional space are reserved, and the imprinting cavities are favorable for transmission of electrons in the redox process of PB and TH. After re-binding the IgG and IgM molecules, the blotting cavity is again occupied, resulting in a decrease in the oxidation peak currents of PB and TH, thereby allowing simultaneous detection of IgG and IgM.
Drawings
FIG. 1 is a differential pulse voltammogram of SPCE, GO/SPCE, PB@AuNPs/GO/SPCE, igG/PB@AuNPs/GO/SPCE, BSA/IgG/PB@AuNPs/GO/SPCE prepared in example one;
FIG. 2 is a differential pulse voltammogram of BSA/IgG/PB@AuNPs/GO/SPCE, MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, igM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE prepared in example one;
FIG. 3 is a cyclic voltammogram of the molecularly imprinted electrochemical sensor prepared in example one, after polypyrrole has been polymerized, eluted, and after incubation in IgG and IgM solutions;
FIG. 4 is a differential pulse voltammogram of a molecularly imprinted electrochemical sensor prepared in example one for detecting solutions containing different concentrations of IgG and IgM;
FIG. 5 is a molecular imprinting electrochemical sensor prepared in example oneWhen IgG and IgM were detected simultaneously, the reduction in TH peak current (ΔI TH ) A linear graph (A) of IgG concentration vs. value (lgC) and a reduction in PB peak current (. DELTA.I) PB ) A linear graph (B) of IgM concentration vs. value (lgC);
FIG. 6 is a UV-visible absorption spectrum of MWCNTs-TH-AuNPs prepared in example one.
FIG. 7 shows the specificity of the molecularly imprinted electrochemical sensor prepared in example one.
Fig. 8 is a cyclic voltammogram of a non-imprinted electrochemical sensor prepared in comparative example one after polypyrrole, elution, and incubation in IgG and IgM solutions.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following examples.
Embodiment one:
a preparation method of a molecular imprinting electrochemical sensor with a double-signal probe strategy comprises the following steps:
(1) 0.0405g of anhydrous ferric trichloride, 0.0823g of potassium ferricyanide and 0.7455g of potassium chloride are weighed and dissolved in 100mL of 0.1M hydrochloric acid solution, and then 0.689mL of 29mM chloroauric acid solution is added to the solution, so that PB@AuNPs solution can be obtained.
(2) 2mg of GO powder is weighed, dissolved in 1mL of ultrapure water, uniformly dispersed by ultrasonic, dripped on the surface of SPCE, and dried under an infrared lamp, thus obtaining the GO/SPCE. As in fig. 1, go/SPCE has no oxidation peak; transferring 10 μL PB@AuNPs solution onto GO/SPCE with a pipette, and applying cyclic voltammetry to a potential range of 0-1V for 100mV s –1 And electrodepositing a PB@AuNPs layer on the surface of the GO/SPCE to form PB@AuNPs/GO/SPCE. As shown in FIG. 1, PB@AuNPs/SPCE shows an oxidation peak of PB at 0.1V, which indicates that PB is an excellent electrochemical vehicle. PB is used as a first electrochemical probe to indicate the change of the signal of IgG.
(3) mu.L of 1.0. Mu.g mL was removed first with a pipette –1 The IgG solution was used to modify the surface of the electrode obtained in step (2), and incubated at 4 ℃. As shown in FIG. 1, compared with PB@AuNPs/SPCE, igG/PB@AuNPs/GO/SPCE oxygenThe peak current is obviously reduced, because IgG is a biological macromolecule with poor conductivity, and electron transfer on the surface of the electrode is inhibited after the thiol is combined with AuNPs; and then 10 mu L of BSA is taken as a blocking solution to incubate the electrode for 1h at 4 ℃ to obtain BSA/IgG/PB@AuNPs/GO/SPCE, wherein the current is continuously reduced as shown in figure 1 due to poor conductivity of the BSA.
(4) And (3) transferring 10 mu L of MWCNTs-TH-AuNPs solution to be coated on the surface of the electrode in the step (3), and drying at room temperature to obtain the MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE. As shown in FIG. 2, the MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE showed an oxidation peak of TH at-0.2V, indicating that TH is also an excellent electrochemical vehicle, while the oxidation peak position of PB is almost unchanged. TH as a second electrochemical probe, indicating a change in IgM signal; mu.L of 500ng mL was removed with a pipette –1 And incubating the electrode at 4 ℃ to obtain the IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE. As shown in FIG. 2, the oxidation peak of TH is reduced, and electron transport is hindered due to the binding of the bio-macromolecule IgM with poor conductivity to AuNPs in the MWCNTs-TH-AuNPs through thiol.
(5) Removing 100 mu L of 0.01M phosphate buffer solution containing 0.1M pyrrole, dripping the solution onto the surface of the electrode prepared in the step (4), and using a cyclic voltammetry to perform a potential range of-0.3-0.8V for 75mV s –1 And polymerizing for 7 circles to obtain the PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE. As shown in fig. 3, after electropolymerization of pyrrole, the peaks of PB and TH disappeared, since the polypyrrole layer was formed on the electrode surface, blocking the redox processes of PB and TH.
(6) And (3) transferring 100 mu L of 10% acetic acid eluting solution containing 10% sodium dodecyl sulfate to the surface of the electrode prepared in the step (5) for eluting template molecules, and after eluting for 2 hours, alternately flushing the electrode with ultrapure water and phosphate buffer solution for multiple times to remove residual IgG and IgM on the surface, thereby obtaining the molecular imprinting electrochemical sensor. As shown in fig. 3, redox peaks of PB and TH occur, and IgG and IgM can be eluted from the polymeric layer due to the ability of the acidic eluent to break the protein structure, thereby forming a blotting cavity, which is advantageous for the redox reaction of PB and TH. Remove 10. Mu.L of the solution containing 1.0. Mu.g mL –1 IgG and 500ng mL – 1 Solutions of IgM were incubated at 4 ℃ for 4h in a molecularly imprinted electrochemical sensor. As shown in fig. 3, peak currents for PB and TH were significantly reduced because IgG and IgM re-occupy the print cavity, thereby inhibiting the redox processes for PB and TH.
(7) Prepared molecularly imprinted electrochemical sensor in the presence of IgG (0.05 ng mL –1 ～100ng mL –1 ) And IgM (0.05 ng mL) –1 ～100ng mL –1 ) Is incubated for 4h. As shown in FIG. 4, the oxidation peak of TH appears at about-0.23V, which is useful for indicating a change in IgM concentration, and the oxidation peak of PB appears at about 0.11V, which is useful for indicating a change in IgG concentration. As IgG and IgM concentrations increase, peak currents of PB and TH decrease gradually due to IgG and IgM occupying the corresponding print cavities, thereby inhibiting the transport of electrons generated during PB and TH redox.
(8) When the prepared molecularly imprinted electrochemical sensor detects IgG and IgM simultaneously, the reduction value of peak current and the concentration logarithmic value are in linear relation. As shown in FIG. 5A, the linear equation of IgM is ΔI TH (μA)＝1.662lgC+4.813(R 2 =0.9962), linear range of 0.05ng mL –1 ～100ng mL –1 As shown in FIG. 5B, the linear equation of IgG is ΔI PB (μA)＝2.130lgC+9.802(R 2 =0.9945), linear range of 0.05ng mL –1 ～100ng mL –1 . According to the triple signal-to-noise ratio, the detection limit of IgM is 28.61pg mL –1 The detection limit of IgG is 23.93pg mL –1 . The above results demonstrate that the molecularly imprinted electrochemical sensor is capable of detecting IgG and IgM simultaneously.
(9) And transferring 0.5mL of MWCNTs-TH solution, adding the MWCNTs-TH solution into 2.5mL of AuNPs solution, stirring for 12h, and centrifuging to obtain solid which is dispersed in water, thus obtaining the solution of the MWCNTs-TH-AuNPs composite material. As shown in fig. 6, curve a is the uv-vis spectrum of AuNPs, the characteristic absorption band of AuNPs appears at 519nm, indicating that AuNPs is successfully prepared, curve b is the uv-vis spectrum of MWCNT-TH, two characteristic absorption bands appear at 285 and 601nm, respectively belonging to pi-pi transition of TH aromatic ring and N-pi transition of c=n bond, indicating that TH molecule is non-covalently attached to MWCNTs surface by pi-pi stacking effect, curve C is the uv-vis spectrum of MWCNT-TH-AuNPs, similar to the spectrum of MWCNT-TH-AuNPs (curve b), but the absorption band at 601nm is blue shifted to 597nm, probably due to coordination of N atom of TH with Au 519, and disappearance of absorption band at nm is attributed to aggregation of AuNP on MWCNT-TH. The above results indicate that the MWCNT-TH-AuNPs composites have been successfully synthesized.
(10) By detecting 100ng mL –1 IgG and IgM, and 10-fold concentration (1. Mu.g mL) –1 ) Bovine Serum Albumin (BSA), hemoglobin (HGB), L-tryptophan (L-Trp) and D-tyrosine (D-Tyr) were used to investigate the specificity of the molecularly imprinted electrochemical sensor (FIG. 7). The test result shows that after the sensor is incubated in BSA, HGB, L-Trp and D-Tyr solution with 10 times concentration for 4 hours, the sensor has delta I PB And DeltaI TH The value is obviously lower than that of the detection of IgG and IgM, which indicates that the molecular imprinting electrochemical sensor has good specificity.
Comparative example one:
to further demonstrate the successful preparation of the molecularly imprinted electrochemical sensor, a non-imprinted electrochemical sensor was additionally prepared, which was operated in the same manner as in example one, steps (1) to (6), except that no IgG and IgM template molecules were added. As shown in fig. 8, no redox peaks of PB and TH occur due to the polypyrrole layer blocking the redox process of PB and TH. Since no IgG and IgM exist, when the electrode is eluted by the acid eluent, a blotting cavity which is favorable for electron transfer cannot be formed, and therefore the eluted electrode still has no oxidation-reduction peaks of PB and TH. Remove 10. Mu.L of the solution containing 1.0. Mu.g mL –1 IgG and 500ng mL –1 Solutions of IgM were incubated at 4 ℃ for 4h on the molecularly imprinted electrochemical sensor, as in fig. 8, with almost unchanged current signal, indicating the absence of imprinted cavities. The comparison of the molecular imprinting electrochemical sensor and the non-imprinting electrochemical sensor further proves that the molecular imprinting electrochemical sensor can detect IgG and IgM simultaneously.
Claims (10)
1. A molecular imprinting electrochemical sensor with a double-signal probe strategy is characterized in that a first template molecule IgG is fixed on Prussian blue@gold nanoparticles/graphene oxide/screen printing carbon electrodes PB@AuNPs/GO/SPCE through gold sulfide bonds to obtain IgG/PB@AuNPs/GO/SPCE, and a second template molecule IgM is fixed on multi-wall carbon nano tubes-thionine-gold nanoparticles/bovine serum albumin/IgG/Prussian blue@gold nanoparticles/graphene oxide/screen printing carbon electrodes MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE through gold sulfide bonds to obtain IgM/MWCNTs-TH-AuNPs/BSA/PB@AuNPs/GO/SPCE; finally, the pyrrole is electrochemically polymerized to obtain PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, and after the IgG and IgM are eluted by eluent, imprinting cavities complementary with the two template molecules in three-dimensional space are formed, so that the molecular imprinting electrochemical sensor is obtained.
2. The preparation method of the molecular imprinting electrochemical sensor with the dual-signal probe strategy as claimed in claim 1, wherein the preparation method of Prussian blue@gold nanoparticles/graphene oxide/screen-printed carbon electrode PB@AuNPs/GO/SPCE is as follows: dissolving anhydrous ferric trichloride, potassium ferricyanide and potassium chloride in a hydrochloric acid solution, and then adding a chloroauric acid solution into the solution to obtain a PB@AuNPs solution; transferring the GO solution by a liquid transferring gun, dripping the GO solution on the surface of the SPCE, and drying under an infrared lamp to obtain GO/SPCE; scanning in the potential range of 0-1V by cyclic voltammetry, and electrodepositing PB@AuNPs layer on the surface of GO/SPCE to form PB@AuNPs/GO/SPCE.
3. The preparation method of the molecular imprinting electrochemical sensor of the dual-signal probe strategy according to claim 2, wherein the preparation method of the multi-wall carbon nanotube-thionine-gold nanoparticle/bovine serum albumin/IgG/Prussian blue @ gold nanoparticle/graphene oxide/screen-printed carbon electrode MWCNTs-TH-AuNPs/BSA/IgG/pb @ AuNPs/GO/SPCE comprises the following steps:
dripping an IgG solution on the surface of PB@AuNPs/GO/SPCE, airing, and then removing BSA as a closed droplet to coat on the surface of the BSA/IgG/PB@AuNPs/GO/SPCE; dissolving MWCNTs in a polyethyleneimine water solution, and performing ultrasonic dispersion; mixing the TH water solution with the solution, vigorously stirring, after stirring is finished, washing the solid substance obtained after centrifugal separation with ultrapure water for a plurality of times, dispersing in water to obtain an MWCNTs-TH solution, adding the MWCNTs-TH solution into the AuNPs solution, stirring uniformly, and centrifuging to obtain a solid which is the MWCNTs-TH-AuNPs composite material; the MWCNTs-TH-AuNPs composite material is dispersed in water in an ultrasonic way, and is coated on the surface of BSA/IgG/PB@AuNPs/GO/SPCE by a pipette, so that the MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE is obtained.
4. The method for preparing a molecularly imprinted electrochemical sensor according to the dual signal probe strategy of claim 2, wherein the eluting step is: and (3) transferring 10% acetic acid solution containing 10% sodium dodecyl sulfate by mass fraction and having a volume fraction of 10% by using a pipette, dripping the solution on the surface of PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, eluting for 0.5-2.5 h, and removing template molecules IgG and IgM.
5. The method for preparing a molecular imprinting electrochemical sensor with a dual-signal probe strategy according to claim 2, wherein the cyclic voltammetry scanning rate is 90-110 mV s when the PB@AuNPs layer is electrodeposited on the surface of GO/SPCE –1 The number of scanning turns is 10-30; dripping pyrrole phosphate buffer solution on the surface of IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE, and scanning at cyclic voltammetry scanning rate of 25-125 mV s –1 The number of deposition turns is 3-15, and a PPy polymerization layer is formed.
6. The method for preparing a molecularly imprinted electrochemical sensor with a dual-signal probe strategy according to claim 2, wherein the concentration of IgG is 0.5-2.0 μg mL –1 The method comprises the steps of carrying out a first treatment on the surface of the IgM concentration of 400-600 ng mL –1 。
7. The preparation method of the double-signal probe strategy molecular imprinting electrochemical sensor according to claim 2, wherein the mass ratio of anhydrous ferric trichloride to potassium ferricyanide to potassium chloride is 0.04-0.05: 0.08-0.09: 0.7-0.8; the mass ratio of PEI to MWCNTs is 5-15: 5 to 15; the volume ratio of PEI aqueous solution, TH solution, MWCNTs-TH and AuNPs solution is 5-15: 0.5 to 1.5:0.25 to 1.0:1.5 to 3.0.
8. The method for preparing a molecularly imprinted electrochemical sensor of a dual signal probe strategy according to claim 2, wherein the concentration of the hydrochloric acid solution is 0.1mM; the concentration of chloroauric acid solution was 0.2mM; the volume ratio of the hydrochloric acid to the chloroauric acid is 10-50: 0.6 to 0.7.
9. Use of a molecularly imprinted electrochemical sensor of the dual signaling probe strategy of claim 1 for detecting IgG and IgM.
10. The use according to claim 9, wherein the molecularly imprinted electrochemical sensor is incubated in a solution containing IgG and IgM for 1-6 h, and a differential pulse voltammogram is recorded in a potential range of-0.5V, to achieve simultaneous detection of IgG and IgM.
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