CN114705739B - Capacitive immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy - Google Patents
Capacitive immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- 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
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
Abstract
The invention relates to capacitance sensing, and discloses a capacitive immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy. The invention firstly extracts silk fibroin and synthesizes CdSeQDs, and loads Ab on CdSeQDs-PDA 2 And preparing an RGO-Ag-ZnO-PPy composite film, and then preparing the capacitive immunosensor for detecting the silk fibroin through a layer-by-layer self-assembly process. The capacitive biosensor of the present invention can adjust the change in capacitance by the affinity interaction between the biological recognition surface and the target analyte, thereby producing high sensitivity and selectivity.
Description
Technical Field
The invention relates to the field of capacitance sensing, in particular to a capacitance immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy.
Background
The Chinese has been the major country of textiles since ancient times, and the produced textiles are rich in variety, exquisite in technology and comfortable and breathable. The most popular textile is silk in China, so China is also called "silk country". The silk relics not only have values in various aspects such as science, technology, culture, art and the like, but also are historic witnessed persons with alternating societies and humane blending. The main component of silk in silk cultural relics is mulberry silk which mainly comprises silk fibroin and sericin, wherein the silk fibroin is the main component of silk and accounts for about 70% of the total weight. However, the mulberry silk in the silk cultural relics is used as an organic polymer material, and is easily degraded by the influence of light, heat, acid and alkali, microorganisms and the like in an underground tomb environment all the year round, so that the crystallinity, molecular weight and other structures and performances are changed, on the other hand, the silk cultural relics are often accompanied with a plurality of impurities when being unearthed, and the real effective components are very few. The conventional silk fibroin detection method has low sensitivity, is greatly influenced by impurity interference, is not suitable for detecting silk relics, and therefore has important significance in developing a method for detecting ancient silk fabrics, which has good sensitivity and strong specificity.
The analysis methods reported at home and abroad for textile residues mainly comprise a chemical degradation method, a biological mass spectrometry method and the like. However, ancient textiles have complex components, and tiny component changes can cause larger errors in mass spectrometry, and the whole experimental process also has to be subjected to experimental steps such as residue extraction, enzyme digestion, mass spectrometry, result analysis and the like, which are relatively complicated. Therefore, it is important to find a method for identifying textile residues with extremely high sensitivity, extremely high specificity, and high speed and efficiency.
Supercapacitors have attracted considerable attention due to their high energy density, excellent reversibility, and longer cycle life compared to conventional capacitors and batteries. Supercapacitors are classified into two types, electric double layer capacitors and pseudocapacitance capacitors, according to their energy storage mechanism. Hybrid supercapacitors are advantageous for their numerous attractive advantages, including low maintenance costs, high energy and power density, rapid charge and discharge rates, good research interests, and long cycle life. If capacitance calculation is combined with biological immunity detection, interference of background signals can be greatly reduced. In addition, the advantages of simple equipment, low cost and high sensitivity are attracting attention.
Disclosure of Invention
In order to solve the technical problems, the invention provides a capacitive immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy. The invention firstly extracts silk fibroin and synthesizes CdSeQDs, and loads Ab on CdSeQDs-PDA 2 Preparing an RGO-Ag-ZnO-PPy composite film, and then preparing the capacitive immunosensor for detecting the silk fibroin through a layer-by-layer self-assembly process; the RGO electrode material with self-supporting characteristic is prepared, so that the preparation process can be simplified, the proportion of the pseudo-capacitance material can be further improved, and the interaction between materials can be enhanced; conductive polymer PPy is widely used in various applications such as organic electronics, supercapacitors and energy converters due to its low cost processing technology and good conductivity. The presence of functional moieties in the polymer backbone provides a platform for better immobilization of different biomolecules such as antibodies, DNA, enzymes, aptamers, etc. Capacitive biosensors may be formed from a biological recognition surface and a target analyteThe affinity interactions regulate the change in capacitance, resulting in high sensitivity and selectivity.
The specific technical scheme of the invention is as follows: a capacitive immunosensor for detecting silk fibroin based on RGO-Ag-ZnO-PPy comprises the following steps:
Step 1: extraction of silk fibroin: the silkworm cocoons are firstly put in Na 2 CO 3 Boiling in water solution, and washing to remove sericin; dissolving the obtained silk fibroin fibers in a calcium chloride mixed solution after drying; after dialysis, centrifugation, freeze-drying and grinding, silk fibroin is obtained.
Step 2: chemical vapor deposition of ZnO nanowires: placing the ZnO powder and carbon powder mixture in a central reaction area of a quartz tube, and placing a silicon wafer coated with an Au catalyst in front of the reaction area; heating for reaction, and introducing a mixed gas of argon and oxygen as carrier gas during the reaction; after the reaction is completed, the ZnO nanowires are separated, added into isopropanol and dispersed by ultrasonic waves.
Step 3: preparation of graphene oxide: adding graphite nano-sheets into chromic acid washing liquid for ultrasonic dispersion; mechanically stirring at 40-50deg.C, pouring water, and suction filtering; washing, baking, cooling to room temperature, and preparing into graphene oxide slurry.
Step 4: preparation of RGO: heating the uniform mixture of the graphene oxide slurry and the hydroiodic acid for reaction; and (5) refrigerating for standby after washing.
The graphene structure prepared by the method has ideal self-supporting characteristics.
Step 5: functional layer RGO-Ag-ZnO membrane electrode assembly: tiling a polyvinylidene fluoride porous filter membrane; coating the isopropanol solution of RGO and Ag nanowires obtained in the step 4 and the ZnO nanowire dispersion liquid obtained in the step 2 on the surface of the film in sequence; vacuum drying; stripping the RGO-Ag-ZnO film, and cleaning to remove possible iodine; and (5) drying and cutting to obtain the RGO-Ag-ZnO film electrode of the functional layer.
The present invention contemplates that the binding sites of biological probes are generally limited, and that direct binding of a single nanomaterial to a biological probe results in little change in the current signal. The RGO-Ag-ZnO film serving as a functional layer is a high-efficiency load material, and can overcome the limitation of the self-properties of a single nano material.
The invention considers that the polyvinylidene fluoride (PVDF) microporous filter membrane is a porous membrane, similar to paper, and has good flexibility and high mechanical strength.
Step 6: pretreatment of an ITO conductive glass current collector: and 5, taking an ITO conductive glass electrode, carrying out ultrasonic cleaning and drying, and adhering the RGO-Ag-ZnO film electrode of the functional layer obtained in the step 5 to the surface of the ITO conductive glass electrode.
Considering that most of the traditional high-pseudocapacitance substances are powdery raw materials, the working electrode is required to be prepared by coating acetylene black and a binder on a substrate, and the electrode material has high internal resistance and poor electrochemical performance. The electrode material with self-supporting characteristic can simplify the preparation process, further improve the proportion of the pseudo-capacitance material and enhance the interaction between the pseudo-capacitance material and the matrix.
Step 7: electrochemical deposition of polypyrrole (PPy): preparing a mixed solution of pyrrole monomer and KCl as electrolyte; and (3) using an electrochemical workstation CHI660E, using a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, and using a constant current method to electrochemically deposit polypyrrole on the electrode obtained in the step (6) to prepare the RGO-Ag-ZnO-PPy composite material.
Polypyrrole is widely used in various applications such as organic electronics, supercapacitors and energy converters due to its low cost processing technology and good electrical conductivity. The presence of functional moieties in the polymer backbone provides a platform for better immobilization of different biomolecules such as antibodies, DNA, enzymes, aptamers, etc.
Step 8: core-shell structure CdSeQDs-PDA@Ab 2 Is prepared from the following steps: mixing thioglycollic acid and water uniformly, adding cadmium chloride aqueous solution, stirring uniformly, regulating pH to 9.5-10.5, introducing nitrogen to remove oxygen in the solution, continuously adding NaHSe solution, stirring uniformly, heating the obtained reaction precursor liquid under the protection of nitrogen for reaction, finally cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifugally cleaning, adding polygalamine, fully combining at 30-35 ℃, flushing, and adding into rabbit anti-mouse anti-silk fibroin antibody Ab 2 Incubating and washing the solution to obtain CdSeQDs-PDA@Ab 2 And (5) storing for standby.
The CdSeQDs of the invention is used as a narrow bandgap semiconductor, and is combined with PDA and secondary antibody to increase the steric hindrance so as to increase the response of a current signal.
Step 9: activating an ITO-RGO-Ag-ZnO-PPy membrane electrode: and (3) dropwise adding an aqueous solution of MPA on the surface of the RGO-Ag-ZnO-PPy composite material obtained in the step (7) after drying by adopting a dropwise coating method, incubating to form a saturated MPA monolayer, washing with a PBS buffer solution, soaking the obtained electrode modified with the MPA in an MES buffer solution for incubation, converting the terminal carboxyl of the MPA into active NHS ester, and washing with the PBS buffer solution to obtain the activated ITO-RGO-Ag-ZnO-PPy membrane electrode.
Step 10: layer-by-layer self-assembled capacitive immunosensor: dropwise adding CB solution of the silk fibroin obtained in the step 1 to the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode to enable terminal amino groups of the silk fibroin to be combined with activated carboxyl groups, thoroughly cleaning by using PBS buffer solution to remove unbound antigens, then blocking the electrode by using BSA solution to block non-specific binding sites possibly existing on the surface of the electrode, taking out, cleaning by using PBS buffer solution, and continuously dropwise adding mouse anti-silk fibroin antibody Ab 1 Washing the solution with PBS buffer solution at 25-35deg.C for 50-70 min to wash the non-immobilized murine anti-silk fibroin antibody Ab 1 And finally, dripping the CdSeQDs-PDA@Ab obtained in the step 8 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 25-35deg.C for 50-70 min 2 Thus obtaining the capacitive immunosensor for detecting the silk fibroin.
Preferably, the step 1 specifically includes: adding 1-3-g silkworm cocoons into 100-120 ml Na 0.3-0.7wt% 2 CO 3 Boiling in water solution for 30-40 min, and washing with distilled water for 3-5 times to completely remove sericin; drying silk fibroin fiber at 50-60deg.C for 24-30 h; dissolving the dried silk fibroin fiber in 80-120 ml calcium chloride mixed solution at 96-100deg.C for 1.5-2 h; dialyzing the dissolved mixed solution for 10-15 times by using a cellulose dialysis bag with the molecular weight cutoff of 8000-10000, and replacing distilled water every 3-4 h; centrifuging at 6000-8000 r/min, lyophilizing supernatant for 2-3 days, and grinding Grinding to obtain silk fibroin; the molar ratio of the calcium chloride to the ethanol to the distilled water in the calcium chloride mixed solution is 1 (1.5-2.5) (7-8).
Preferably, step 2 specifically includes: placing a ZnO powder and carbon powder mixture with the molar ratio of 0.8-1.2:1 in a central reaction area of a quartz tube, and placing a silicon wafer coated with an Au catalyst of 5-10nm in front of the reaction area; setting the reaction temperature to 850-950 ℃ and the reaction time to 15-30 min, and introducing argon with the constant flow rate of 80-120sccm and oxygen with the constant flow rate of 3-7sccm as carrier gases during the reaction; after the reaction is completed, znO nano-wire is separated, and the solid-to-liquid ratio is 2-3 mg ml -1 Adding into isopropanol, and ultrasonic treating for 10-15 min to disperse uniformly, and storing.
Preferably, the step 3 specifically includes: adding 100-120mg of graphite nano-sheets with the sheet diameter of 500-800 mu m into 560-600 ml chromic acid washing liquid, and performing ultrasonic dispersion for 30-40 min; mechanically stirring at 40-50deg.C for 10-20 min, pouring 1.5-2L water, and suction filtering; washing the filtrate with water and ethanol for 3-5 times, baking at 110-120deg.C for 3-3.5 hr, cooling to room temperature, and making into 20-30mg ml -1 And (3) oxidizing the graphene slurry for later use.
Preferably, the step 4 specifically includes: heating the uniform mixture of the graphene oxide slurry and 40-50. 50 wt% of hydroiodic acid according to the volume ratio of 8-12:1 at the temperature of 85-90 ℃ for 2-2.5 h; washing with water and ethanol for 3-5 times, and refrigerating at 4deg.C.
Preferably, the step 5 specifically includes: taking 40-50 ml and 2-3 mg ml of ZnO nanowire dispersion liquid obtained in the step 2 -1 Isopropyl alcohol solution of Ag nanowire 40-50-ml, respectively and ultrasonically dispersing uniformly; a vacuum pump of a vacuum suction filtration system is started, and a polyvinylidene fluoride porous filter membrane is paved at the top end of a conical flask and is soaked; coating the isopropanol solution of RGO and Ag nanowires obtained in the step 4 and the ZnO nanowire dispersion liquid obtained in the step 2 on the surface of the film in sequence; vacuum drying at 30-40deg.C 18-24 h; stripping the RGO-Ag-ZnO film, washing with water and ethanol for 2-3 times, and removing possible iodine; and (5) drying and cutting to obtain the RGO-Ag-ZnO film electrode of the functional layer.
Preferably, step 6 specifically includes: and 5, taking an ITO conductive glass electrode, respectively ultrasonically cleaning the ITO conductive glass electrode by using acetone, ethanol and deionized water for 10-20 min, drying the ITO conductive glass electrode at 50-60 ℃, and adhering the RGO-Ag-ZnO film electrode of the functional layer obtained in the step 5 to the surface of the ITO conductive glass electrode.
Preferably, the step 7 specifically includes: preparing 50-60 ml of mixed solution of 0.03-0.07 mmol/L pyrrole monomer and 0.03-0.07 mmol/L KCl as electrolyte; and (3) using an electrochemical workstation CHI660E, using a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, and using a constant current method to electrochemically deposit polypyrrole on the electrode obtained in the step (6), wherein the current is 8-12 mA, and the deposition time is 300-500 s, so as to prepare the RGO-Ag-ZnO-PPy composite material.
Preferably, the step 8 specifically includes: mixing 0.80-0.88 mL thioglycollic acid and 55-65 mL water uniformly, adding 45-55 mL 0.1M cadmium chloride aqueous solution, stirring uniformly, adjusting pH to 9.5-10.5, then introducing nitrogen gas to remove oxygen in the solution, continuing to add 11-13 mL 0.2M NaHSe solution, stirring uniformly, reacting the obtained reaction precursor solution at 75-85 ℃ under nitrogen protection for 3-5 h, finally cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifuging and cleaning, adding 10-20 mL polygalamine, fully combining 1-3 h at 30-35 ℃, flushing for 3-5 min, and adding 8-12 ul mL -1 Rabbit anti-murine anti-silk fibroin antibody Ab 2 Incubation at 30-35 ℃ for 0.5-1.5. 1.5 h, flushing for 3-5 min, and preserving at 1-5 ℃ for standby.
Preferably, step 9 specifically includes: and (3) dropwise adding 10-20 ul of 0.05M MPA aqueous solution on the surface of the RGO-Ag-ZnO-PPy composite material obtained in the step (7) after drying by adopting a dropwise coating method, incubating for 50-70min at 50-60 ℃ to form a saturated MPA monolayer, washing with PBS buffer, soaking the obtained electrode modified with MPA in MES buffer, incubating for 50-70min at 55-65 ℃, converting the terminal carboxyl of the MPA into active NHS ester, and washing with PBS buffer to obtain the activated ITO-RGO-Ag-ZnO-PPy membrane electrode.
Preferably, step 10 specifically includes: dropwise adding 10-20 ul1ul/ml CB solution of silk fibroin obtained in step 1 onto the surface of activated ITO-RGO-Ag-ZnO-PPy membrane electrode to combine amino terminal with activated carboxyl, thoroughly washing with PBS buffer solution to remove unbound antigen, and dissolving with 10-20 ul of 0.8-1.2wt% BSABlocking the electrode for 25-35 min, blocking 0.5-1.5-h with 0.8-1.2wt% BSA solution, taking out, washing with PBS buffer solution, and dripping 10-20 ul1ul/ml mouse anti-silk fibroin antibody Ab 1 Washing the solution with PBS buffer solution at 25-35deg.C for 50-70 min to wash the non-immobilized murine anti-silk fibroin antibody Ab 1 Finally, dropwise adding the CdSeQDs-PDA@Ab obtained in the step 8 of 10-20 and 20 ul 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 25-35deg.C for 50-70 min 2 Thus obtaining the capacitive immunosensor for detecting the silk fibroin.
Steric hindrance is an important signal amplification strategy because most biomolecules, such as protein molecules, are less conductive. Therefore, when target molecules (mainly protein molecules) are modified on the surface of the electrode, a steric hindrance effect is generated on the surface of the electrode, so that transfer and transmission of electrons are hindered, and electrochemical response is further affected. The invention constructs an indirect immunosensor, which is different from a conventional sandwich immunosensor.
The capacitive biosensor of the present invention can adjust the change of capacitance by the affinity interaction between the biological recognition surface and the target analyte, thereby producing high sensitivity and selectivity, and the capacitive sensor is easy to manufacture and can be manufactured in a miniaturized manner.
Compared with the prior art, the invention has the following technical effects:
(1) The graphene structure prepared by the method has ideal self-supporting characteristics.
(2) The present invention contemplates that the binding sites of biological probes are generally limited, and that direct binding of a single nanomaterial to a biological probe results in little change in the current signal. The RGO-Ag-ZnO film serving as a functional layer is a high-efficiency load material, and can overcome the limitation of the self-properties of a single nano material.
(3) The invention considers that the polyvinylidene fluoride (PVDF) microporous filter membrane is a porous membrane, similar to paper, and has good flexibility and high mechanical strength.
(4) Considering that most of high pseudo-capacitance materials are powdery raw materials under the common condition, the working electrode is required to be prepared by coating acetylene black and a binder on a substrate, the electrode material has large internal resistance and poor electrochemical performance, and the electrode material with self-supporting characteristics is prepared by the method, so that the preparation process can be simplified, the proportion of the pseudo-capacitance material is further improved, and the interaction between the electrode material and a substrate can be enhanced.
(5) The conductive polymer of the present invention is widely used in various applications such as organic electronics, supercapacitors, and energy converters due to its low cost processing technology and good electrical conductivity. The presence of functional moieties in the polymer backbone provides a platform for better immobilization of different biomolecules such as antibodies, DNA, enzymes, aptamers, etc.
(6) The CdSeQDs of the invention is used as a narrow bandgap semiconductor, and is combined with PDA and secondary antibody to increase the steric hindrance so as to increase the response of a current signal.
(7) The steric hindrance effect is an important signal amplification strategy, because most biomolecules such as protein molecules have poor conductivity, so when target molecules (mainly protein molecules) are modified on the surface of an electrode, the steric hindrance effect is generated on the surface of the electrode, thereby blocking the transfer and transmission of electrons and further affecting the electrochemical response. The invention constructs an indirect immunosensor, which is different from a conventional sandwich immunosensor.
(8) The capacitive biosensor of the present invention can adjust the change of capacitance by the affinity interaction between the biological recognition surface and the target analyte, thereby producing high sensitivity and selectivity, and the capacitive sensor is easy to manufacture and can be manufactured in a miniaturized manner.
Detailed Description
The invention is further described below with reference to examples.
Example 1
Step 1: extraction of silk fibroin: 1g of domestic silkworm cocoons are put in 100 ml of 0.5 percent Na 2 CO 3 Boiling in water solution for 30min, and washing with distilled water for 3 times to completely remove sericin; drying the degummed silk fiber in a drying oven at 50 ℃ for 24 h; the dried silk fibroin fiber is mixed with 100 ml calcium chloride mixed solution at 98 DEG C(molar ratio of calcium chloride, ethanol and distilled water is 1:2:8) for 1.5h; the dissolved mixed solution is dialyzed for 10 times by using a dialysis bag (MWCO: 8000), and distilled water is replaced every 3 h; purifying the obtained solution using a centrifuge (6000 r/min); finally, taking supernatant, freeze-drying and grinding to obtain silk fibroin;
step 2: chemical vapor deposition of ZnO nanowires: placing a ZnO powder and carbon powder mixture (the molar ratio is 1:1) in a central reaction area of a quartz tube, and placing a silicon wafer coated with a 5nm Au catalyst in front of the reaction area; setting the reaction temperature to 850 ℃ and the reaction time to 15min, and introducing argon gas with the constant flow rate of 100sccm and oxygen gas mixture with the constant flow rate of 5sccm as carrier gas during the reaction; after the reaction was completed, the ZnO nanowires were separated from the substrate and dissolved in isopropanol (2 mg ml -1 ) Uniformly dispersing the materials by ultrasonic treatment for 10 min, and preserving the materials for later use;
step 3: preparation of graphene oxide: adding 100 mg graphite nano-sheets (sheet diameter 500 μm) into 560 ml chromic acid washing liquid, and performing ultrasonic dispersion for 30 min; then mechanically stirring for 10 min at 40 ℃, pouring 1.5L of deionized water, and carrying out suction filtration; sequentially washing the filtrate with water and ethanol for 3 times, baking at 110deg.C for 3 hr, cooling to room temperature, and preparing into 20 mg ml -1 Is reserved for use;
step 4: preparation of RGO: a homogeneous mixture of the above graphene oxide slurry and hydroiodic acid (HI) (40 wt.%) (volume ratio: graphene oxide slurry: hi=10:1) was reacted in an oven at 85 ℃ for 2 h; washing with water and ethanol for 3 times, and refrigerating at 4deg.C;
step 5: functional layer RGO-Ag-ZnO membrane electrode assembly: 40 ml of ZnO nanowire dispersion in the step 2 was taken, and isopropyl alcohol solution (2 mg ml) of Ag nanowire was taken -1 ) 40, ml, uniformly dispersing by ultrasonic wave; a vacuum pump of a vacuum suction filtration system is opened, and a polyvinylidene fluoride (PVDF) porous filter membrane is paved at the top end of a conical flask and is soaked; coating RGO of the step 4, silver nanowires of the step 5 and ZnO nanowires of the step 2 on the surface of the solution in sequence; placing the device in a vacuum oven 18 h at 30 ℃; cutting a small incision on the edge of PVDF filter membrane with a blade, peeling RGO-Ag-ZnO membrane from the substrate, using Washing with water and ethanol for 2 times to remove possible iodine; drying, cutting into 1cm multiplied by 2cm to obtain a functional layer RGO-Ag-ZnO membrane electrode for later use;
step 6: pretreatment of an ITO conductive glass current collector: taking an ITO conductive glass electrode with the area of 1cm multiplied by 3 cm, respectively ultrasonically cleaning the ITO conductive glass electrode with acetone, ethanol and deionized water for 10 min, drying the ITO conductive glass electrode in a baking oven at 50 ℃, and adhering the RGO-Ag-ZnO film electrode of the functional layer in the step 5 on the surface of the ITO conductive glass for later use;
step 7: electrochemical deposition of polypyrrole (PPy): preparing a 50 ml mixed solution of 0.05 mmol/L pyrrole monomer and 0.5 mmol/L KCl as electrolyte; electrochemical workstation CHI660E is used, a platinum wire electrode is a counter electrode, a silver/silver chloride electrode is used as a reference electrode, polypyrrole is electrochemically deposited on the electrode prepared in the step 6 by using a constant current method (10 mA electrodeposition 300 s), and the electrode area immersed in electrolyte is 1 multiplied by 1cm 2 Preparing RGO-Ag-ZnO-PPy composite material for later use;
step 8: core-shell structure CdSeQDs-PDA@Ab 2 Is prepared from the following steps: mixing thioglycollic acid of 0.8 mL and deionized water of 55 mL uniformly, adding 0.1M cadmium chloride aqueous solution of 45 mL, stirring uniformly, adjusting the pH of the solution to 9.5 by NaOH solution, introducing high-purity nitrogen to remove oxygen in the solution, continuously adding 11 mL of 0.2M NaHSe solution, stirring uniformly, reacting the obtained reaction precursor solution at 75 ℃ under the protection of high-purity nitrogen for 3 h, finally cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifuging, cleaning to constant volume, adding 10 mL poly-dopa polyamine, fully combining 1 h at 30 ℃, washing with deionized water for 3 min, adding 10 ul mL of 10 mL -1 Rabbit anti-murine anti-silk fibroin antibody Ab 2 Incubating at 30 ℃ for 0.5. 0.5 h, and washing with deionized water for 3 min to obtain CdSeQDs-PDA@Ab 2 And storing at 5 ℃ for standby;
step 9: activating an ITO-RGO-Ag-ZnO-PPy membrane electrode: dropwise adding 10 ul of 0.05M aqueous solution of MPA on the surface of the electrode obtained in the step 7 after drying by adopting a dropwise coating method, incubating for 50 min at 50 ℃ to form a saturated MPA monolayer, washing with PBS buffer, soaking the electrode modified with MPA in MES buffer (containing 0.05M EDC and 0.03M NHS), incubating for 50 min at 55 ℃, converting the terminal carboxyl of the MPA into active NHS ester, washing with PBS buffer, and obtaining an activated ITO-RGO-Ag-ZnO-PPy membrane electrode;
step 10: layer-by-layer self-assembled capacitive immunosensor: dropwise adding 10 ul1ul/ml of CB solution of the silk fibroin obtained in the step 1 onto the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode, enabling terminal amino groups of the CB solution to be combined with activated carboxyl groups, thoroughly cleaning by using PBS buffer solution to remove unbound antigens, and then sealing the electrode by using 10 ul 0.8% BSA solution at 35 ℃ for 25 min; subsequently, 0.5. 0.5 h was blocked with 0.8% BSA solution to block non-specific binding sites possibly present on the electrode surface, and after removal, washed with PBS buffer, and further dropwise added with 10 ul1ul/ml murine anti-silk fibroin antibody (Ab) 1 ) Washing the solution with PBS buffer solution at 25deg.C for 50 min 1 And (3) dripping 10 g/ul of the antibody, wherein the CdSeQDs-PDA@Ab is obtained in the step (8) 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 25deg.C for 50 min 2 Obtaining the capacitive immunosensor for detecting the silk fibroin;
step 11: electrochemical measurement: all electrochemical measurements were performed in a standard three-electrode system with a platinum electrode as the counter electrode and silver/silver chloride as the reference electrode; electrochemical performance is characterized by adopting a CHI660B electrochemical workstation, a Cyclic Voltammetry (CV) voltage window is between 0 and 1V, and the scanning speed is 10mV/s; under different currents of 20 mu A, measuring an electrostatic rectification charge-discharge (GCD) test within a potential range of 0-1V; EIS measurements were performed at a frequency range of 0.01Hz-100kHz and an open circuit potential, with an alternating current disturbance of 5mV; according to experimental data, calculating mass specific capacitance, wherein the mass specific capacitance calculation formula of the supercapacitor electrode is C m =i×Δt/(m×Δv), wherein: i is constant current; Δt is the discharge time; m is the total mass added to the electrode; deltaV is the potential difference during discharge.
Examples
Step 1: extraction of silk fibroin: the 2 g cocoons were treated with 110 ml of 0.5% Na 2 CO 3 Boiling in water solution for 35 min, and washing with distilled water for 4 times to completely remove sericin; degummed silk fiberDrying 27. 27 h in a 55 ℃ oven; dissolving the dried silk fibroin fibers in a 100 ml calcium chloride mixed solution (molar ratio of calcium chloride, ethanol and distilled water is 1:2:8) at 98 ℃ to obtain a solution of 1.5 h; the dissolved mixed solution is dialyzed for 12 times by using a dialysis bag (MWCO: 8000), and distilled water is replaced every 3.5 h; the obtained solution was purified using a centrifuge (7000 r/min); finally, taking supernatant, freeze-drying and grinding to obtain silk fibroin;
step 2: chemical vapor deposition of ZnO nanowires: placing a ZnO powder and carbon powder mixture (the molar ratio is 1:1) in a central reaction area of a quartz tube, and placing a silicon wafer coated with 7 nm Au catalyst in front of the reaction area; setting the reaction temperature to 900 ℃ and the reaction time to 20min, and introducing argon gas with the constant flow rate of 100sccm and oxygen gas mixture with the constant flow rate of 5sccm as carrier gas during the reaction; after the reaction was completed, the ZnO nanowires were separated from the substrate and dissolved in isopropanol (2.5 mg ml -1 ) Uniformly dispersing the materials by ultrasonic treatment for 10 min, and preserving the materials for later use;
step 3: preparation of graphene oxide: adding 110 mg graphite nano-sheets (with the sheet diameter of 700 mu m) into 580 ml chromic acid washing liquid, and performing ultrasonic dispersion for 35 min; then mechanically stirring for 15 min at 45 ℃, pouring 1.8L of deionized water, and carrying out suction filtration; the filter media is washed with water and ethanol for 4 times, baked for 3.3 hours at 115 ℃, cooled to room temperature and prepared into 25 mg ml -1 Is reserved for use;
step 4: preparation of RGO: a homogeneous mixture of the above graphene oxide slurry and hydroiodic acid (HI) (45 wt.%) (volume ratio: graphene oxide slurry: hi=10:1) was reacted in an oven at 85 ℃ for 2 h; washing with water and ethanol for 4 times in sequence, and refrigerating at 4 ℃ for standby;
step 5: functional layer RGO-Ag-ZnO membrane electrode assembly: 45ml of ZnO nanowire dispersion in the step 2 was taken, and an isopropanol solution (2.5 mg ml of Ag nanowire was taken -1 ) 45ml, and uniformly dispersing by ultrasonic waves; a vacuum pump of a vacuum suction filtration system is opened, and a polyvinylidene fluoride (PVDF) porous filter membrane is paved at the top end of a conical flask and is soaked; coating RGO of the step 4, silver nanowires of the step 5 and ZnO nanowires of the step 2 on the surface of the solution in sequence; placing the device at 35deg.CIs 20h; cutting a small incision on the edge of the PVDF filter membrane by a blade, peeling the RGO-Ag-ZnO membrane from the substrate, and washing with water and ethanol for 3 times to remove possible iodine; drying, cutting into 1cm multiplied by 2cm to obtain a functional layer RGO-Ag-ZnO membrane electrode for later use;
step 6: pretreatment of an ITO conductive glass current collector: taking an ITO conductive glass electrode with the area of 1cm multiplied by 3 cm, respectively ultrasonically cleaning the ITO conductive glass electrode with acetone, ethanol and deionized water for 15 min, drying the ITO conductive glass electrode in a baking oven at 55 ℃, and adhering the RGO-Ag-ZnO film electrode of the functional layer in the step 5 on the surface of the ITO conductive glass for later use;
Step 7: electrochemical deposition of polypyrrole (PPy): preparing 55 ml mixed solution of 0.05 mmol/L pyrrole monomer and 0.5 mmol/L KCl as electrolyte; electrochemical workstation CHI660E is used, a platinum wire electrode is a counter electrode, a silver/silver chloride electrode is used as a reference electrode, polypyrrole is electrochemically deposited on the electrode prepared in the step 6 by using a constant current method (10 mA electrodeposition 400 s), and the electrode area immersed in electrolyte is 1 multiplied by 1cm 2 Preparing RGO-Ag-ZnO-PPy composite material for later use;
step 8: core-shell structure CdSeQDs-PDA@Ab 2 Is prepared from the following steps: mixing thioglycollic acid of 0.84 mL and deionized water of 60 mL uniformly, adding cadmium chloride aqueous solution of 0.1M into the mixture, stirring uniformly, regulating the pH of the solution to 10 by NaOH solution, introducing high-purity nitrogen to remove oxygen in the solution, continuously adding NaHSe solution of 0.2M of 12 mL and stirring uniformly, reacting the obtained reaction precursor solution at 80 ℃ under the protection of high-purity nitrogen for 4 h, finally cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifugally cleaning to constant volume, adding polydopamine of 15 mL, fully combining 2 h at 35 ℃, washing with deionized water for 4 min, and adding 15 ul mL of 15 mL -1 Rabbit anti-murine anti-silk fibroin antibody Ab 2 Incubating 1h at 30 ℃ and washing with deionized water for 4 min to obtain CdSeQDs-PDA@Ab 2 And storing at 1 ℃ for standby;
step 9: activating an ITO-RGO-Ag-ZnO-PPy membrane electrode: dripping 15 ul of 0.05M aqueous solution of MPA on the surface of the electrode obtained in the step 7 after drying by adopting a dripping method, incubating for 60min at 55 ℃ to form a saturated MPA monolayer, washing by using PBS buffer, soaking the electrode modified with MPA in MES buffer (containing 0.05M EDC and 0.03M NHS), incubating for 60min at 60 ℃, converting the terminal carboxyl of the MPA into active NHS ester, washing by using PBS buffer, and obtaining an activated ITO-RGO-Ag-ZnO-PPy membrane electrode;
step 10: layer-by-layer self-assembled capacitive immunosensor: dropwise adding 15 ul of 1ul/ml of CB solution of the silk fibroin obtained in the step 1 to the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode, enabling terminal amino groups of the CB solution to be combined with activated carboxyl groups, thoroughly cleaning the surface of the activated ITO-RGO-Ag-ZnO-PPy membrane electrode by using PBS buffer solution to remove unbound antigens, and then sealing the electrode by using a 1% BSA solution of 15 ul at 37 ℃ for 30 min; subsequently, blocking with 1% BSA solution for 1h to block non-specific binding sites possibly present on the electrode surface, taking out, washing with PBS buffer, and further dripping 15 ul1ul/ml of murine anti-silk fibroin antibody (Ab) 1 ) Washing the solution with PBS buffer solution at 30deg.C for 60min 1 The antibody is finally dripped with 15 ul CdSeQDs-PDA@Ab obtained in the step 8 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 30deg.C for 60min 2 Obtaining the capacitive immunosensor for detecting the silk fibroin;
step 11: electrochemical measurement: all electrochemical measurements were performed in a standard three-electrode system with a platinum electrode as the counter electrode and silver/silver chloride as the reference electrode; electrochemical performance is characterized by adopting a CHI660B electrochemical workstation, a Cyclic Voltammetry (CV) voltage window is between 0 and 1V, and the scanning rate is 500mV/s; under different currents of 50 mu A, measuring an electrostatic rectification charge-discharge (GCD) test within a potential range of 0-1V; EIS measurements were performed at a frequency range of 0.01Hz-100kHz and an open circuit potential, with an alternating current disturbance of 5mV; according to experimental data, calculating mass specific capacitance, wherein the mass specific capacitance calculation formula of the supercapacitor electrode is C m =i×Δt/(m×Δv), wherein: i is constant current; Δt is the discharge time; m is the total mass added to the electrode; deltaV is the potential difference during discharge.
Examples
Step 1: extraction of silk fibroin: 3g of domestic silkworm cocoons were treated with 120 ml of 0.5% Na 2 CO 3 Boiling in water solution for 40 min, and washing with distilled water for 5 times to completely remove sericin; drying the degummed silk fiber in a drying oven at 60 ℃ for 30 h; dissolving the dried silk fibroin fibers in a 100 ml calcium chloride mixed solution (the molar ratio of calcium chloride, ethanol and distilled water is 1:2:8) at 98 ℃ for 2 h; using a dialysis bag (MWCO: 8000) to carry out 15 times of dialysis on the dissolved mixed solution, and replacing distilled water every 4 hours; purifying the obtained solution using a centrifuge (8000 r/min); finally, taking supernatant, freeze-drying and grinding to obtain silk fibroin;
step 2: chemical vapor deposition of ZnO nanowires: placing a ZnO powder and carbon powder mixture (the molar ratio is 1:1) in a central reaction area of a quartz tube, and placing a silicon wafer coated with a 10nm Au catalyst in front of the reaction area; setting the reaction temperature to 950 ℃ and the reaction time to 30min, and introducing argon gas with the constant flow rate of 100sccm and oxygen gas mixture with the constant flow rate of 5sccm as carrier gas during the reaction; after the reaction was completed, the ZnO nanowires were separated from the substrate and dissolved in isopropanol (3 mg ml -1 ) Uniformly dispersing the materials by ultrasonic treatment for 15 min, and preserving the materials for later use;
step 3: preparation of graphene oxide: adding 120 mg graphite nano-sheets (with the sheet diameter of 800 mu m) into 600 ml chromic acid washing liquid, and performing ultrasonic dispersion for 40 min; mechanically stirring at 50deg.C for 20 min, pouring 2L deionized water, and suction filtering; sequentially washing the filtrate with water and ethanol for 5 times, baking at 120deg.C for 3.5 hr, cooling to room temperature, and preparing into 30 mg ml -1 Is reserved for use;
step 4: preparation of RGO: a homogeneous mixture of the above graphene oxide slurry and hydroiodic acid (HI) (50 wt%) was reacted in an oven at 90 ℃ for 2.5h (volume ratio: graphene oxide slurry: hi=10:1); washing with water and ethanol for 5 times, and refrigerating at 4deg.C;
step 5: functional layer RGO-Ag-ZnO membrane electrode assembly: 50 ml of the ZnO nanowire dispersion in step 2 was taken, and an isopropanol solution (3 mg ml) of Ag nanowires was taken -1 ) 50, ml, uniformly dispersing by ultrasonic waves; a vacuum pump of a vacuum suction filtration system is opened, and a polyvinylidene fluoride (PVDF) porous filter membrane is paved at the top end of a conical flask and is soaked; sequentially applying the solutions to a surfaceCoating RGO of the step 4, silver nanowires of the step 5 and ZnO nanowires of the step 2 in sequence; placing the device in a vacuum oven 24 h at 40 ℃; cutting a small incision on the edge of the PVDF filter membrane by a blade, peeling the RGO-Ag-ZnO membrane from the substrate, and washing with water and ethanol for 3 times to remove possible iodine; drying, cutting into 1cm multiplied by 2cm to obtain a functional layer RGO-Ag-ZnO membrane electrode for later use;
step 6: pretreatment of an ITO conductive glass current collector: taking an ITO conductive glass electrode with the area of 1cm multiplied by 3 cm, respectively ultrasonically cleaning the ITO conductive glass electrode with acetone, ethanol and deionized water for 20 min, drying the ITO conductive glass electrode in a baking oven at 60 ℃, and adhering the RGO-Ag-ZnO film electrode of the functional layer in the step 5 on the surface of the ITO conductive glass for later use;
Step 7: electrochemical deposition of polypyrrole (PPy): preparing a 60 ml mixed solution of 0.05 mmol/L pyrrole monomer and 0.5 mmol/L KCl as electrolyte; electrochemical workstation CHI660E is used, a platinum wire electrode is a counter electrode, a silver/silver chloride electrode is used as a reference electrode, polypyrrole is electrochemically deposited on the electrode prepared in the step 6 by using a constant current method (10 mA electrodeposition 400 s), and the electrode area immersed in electrolyte is 1 multiplied by 1cm 2 Preparing RGO-Ag-ZnO-PPy composite material for later use;
step 8: core-shell structure CdSeQDs-PDA@Ab 2 Is prepared from the following steps: mixing thioglycollic acid of 0.88 and mL with deionized water of 65 mL, adding cadmium chloride aqueous solution of 0.1M of 55 mL and stirring uniformly, regulating pH to 10.5 with NaOH solution, introducing high-purity nitrogen gas to remove oxygen in the solution, continuously adding NaHSe solution of 0.2M of 13 mL and stirring uniformly, reacting the obtained reaction precursor solution at 85 ℃ under the protection of high-purity nitrogen gas for 5 h, cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifuging, cleaning to constant volume, adding 20 mL poly-dopa polyamine, fully combining with 3 h at 35 ℃, washing with deionized water for 5 min, adding 10 ul mL of 20 mL -1 Rabbit anti-murine anti-silk fibroin antibody Ab 2 Incubating 1.5. 1.5 h at 35 ℃ and washing with deionized water for 5 min to obtain CdSeQDs-PDA@Ab 2 And storing at 5 ℃ for standby;
step 9: activating an ITO-RGO-Ag-ZnO-PPy membrane electrode: dropwise adding 20 ul of 0.05M aqueous solution of MPA on the surface of the electrode obtained in the step 7 after drying by adopting a dropwise coating method, incubating for 70min at 60 ℃ to form a saturated MPA monolayer, washing with PBS buffer, soaking the electrode modified with MPA in MES buffer (containing 0.05M EDC and 0.03M NHS), incubating for 70min at 65 ℃, converting the terminal carboxyl of the MPA into active NHS ester, washing with the PBS buffer, and obtaining an activated ITO-RGO-Ag-ZnO-PPy membrane electrode;
step 10: layer-by-layer self-assembled capacitive immunosensor: dropwise adding 20 ul of 1ul/ml of CB solution of the silk fibroin obtained in the step 1 onto the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode, enabling terminal amino groups of the CB solution to be combined with activated carboxyl groups, thoroughly cleaning by using PBS buffer solution to remove unbound antigens, and then sealing the electrode by using 20 ul of 1.2% BSA solution at 40 ℃ for 35 min; subsequently, 1.5. 1.5 h was blocked with 1.2% BSA solution to block non-specific binding sites possibly present on the electrode surface, and after removal, washed with PBS buffer, followed by dropwise addition of 20 ul1ul/ml murine anti-silk fibroin antibody (Ab) 1 ) Washing the solution with PBS buffer solution at 35deg.C for 70min 1 And (3) dripping 20 g ul of the antibody, wherein the CdSeQDs-PDA@Ab is obtained in the step (8) 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 35℃for 70min 2 Obtaining the capacitive immunosensor for detecting the silk fibroin;
step 11: electrochemical measurement: all electrochemical measurements were performed in a standard three-electrode system with a platinum electrode as the counter electrode and silver/silver chloride as the reference electrode; electrochemical performance is characterized by adopting a CHI660B electrochemical workstation, a Cyclic Voltammetry (CV) voltage window is between 0 and 1V, and the scanning rate is 1000mV/s; under different currents of 80 mu A, measuring an electrostatic rectification charge-discharge (GCD) test within a potential range of 0-1V; EIS measurements were performed at a frequency range of 0.01Hz-100kHz and an open circuit potential, with an alternating current disturbance of 5mV; according to experimental data, calculating mass specific capacitance, wherein the mass specific capacitance calculation formula of the supercapacitor electrode is C m =i×Δt/(m×Δv), wherein: i is constant current; Δt is the discharge time; m is the total mass added to the electrode; deltaV is the potential difference during discharge.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. The capacitive immunosensor for detecting the silk fibroin based on RGO-Ag-ZnO-PPy is characterized by comprising the following steps of:
step 1: extraction of silk fibroin: the silkworm cocoons are firstly put in Na 2 CO 3 Boiling in water solution, and washing to remove sericin; dissolving the obtained silk fibroin fibers in a calcium chloride mixed solution after drying; obtaining silk fibroin after dialysis, centrifugation, freeze drying and grinding;
step 2: chemical vapor deposition of ZnO nanowires: placing the ZnO powder and carbon powder mixture in a central reaction area of a quartz tube, and placing a silicon wafer coated with an Au catalyst in front of the reaction area; heating for reaction, and introducing a mixed gas of argon and oxygen as carrier gas during the reaction; after the reaction is completed, znO nanowires are separated and added into isopropanol for ultrasonic dispersion;
step 3: preparation of graphene oxide: adding graphite nano-sheets into chromic acid washing liquid for ultrasonic dispersion; mechanically stirring at 40-50deg.C, pouring water, and suction filtering; washing, baking, cooling to room temperature, and preparing graphene oxide slurry;
Step 4: preparation of RGO: heating the uniform mixture of the graphene oxide slurry and the hydroiodic acid for reaction; refrigerating for standby after washing;
step 5: functional layer RGO-Ag-ZnO membrane electrode assembly: tiling a polyvinylidene fluoride porous filter membrane; coating the isopropanol solution of RGO and Ag nanowires obtained in the step 4 and the ZnO nanowire dispersion liquid obtained in the step 2 on the surface of the film in sequence; vacuum drying; stripping the RGO-Ag-ZnO film; drying and cutting to obtain a functional layer RGO-Ag-ZnO membrane electrode;
step 6: pretreatment of an ITO conductive glass current collector: taking an ITO conductive glass electrode, carrying out ultrasonic cleaning and drying, and adhering the RGO-Ag-ZnO film electrode of the functional layer obtained in the step 5 to the surface of the ITO conductive glass electrode;
step 7: electrochemical deposition of polypyrrole: preparing a mixed solution of pyrrole monomer and KCl as electrolyte; using an electrochemical workstation CHI660E, using a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, and using a constant current method to electrochemically deposit polypyrrole on the electrode obtained in the step 6 to prepare an RGO-Ag-ZnO-PPy composite material;
step 8: core-shell structure CdSeQDs-PDA@Ab 2 Is prepared from the following steps: mixing thioglycollic acid and water uniformly, adding cadmium chloride aqueous solution, stirring uniformly, regulating pH to 9.5-10.5, introducing nitrogen to remove oxygen in the solution, continuously adding NaHSe solution, stirring uniformly, heating the obtained reaction precursor liquid under the protection of nitrogen for reaction, finally cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifugally cleaning, adding polygalamine, fully combining at 30-35 ℃, flushing, and adding into rabbit anti-mouse anti-silk fibroin antibody Ab 2 Incubating and washing the solution to obtain CdSeQDs-PDA@Ab 2 Storing for later use;
step 9: activating an ITO-RGO-Ag-ZnO-PPy membrane electrode: dropwise adding an aqueous solution of MPA on the surface of the RGO-Ag-ZnO-PPy composite material obtained in the step 7 after drying by adopting a dropwise coating method, incubating to form a saturated MPA monolayer, washing with a PBS buffer solution, soaking the obtained electrode modified with MPA in an MES buffer solution for incubation, converting the terminal carboxyl of the MPA into active NHS ester, and washing with the PBS buffer solution to obtain an activated ITO-RGO-Ag-ZnO-PPy membrane electrode;
step 10: layer-by-layer self-assembled capacitive immunosensor: dropwise adding CB solution of the silk fibroin obtained in the step 1 to the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode to enable terminal amino groups of the silk fibroin to be combined with activated carboxyl groups, thoroughly cleaning by using PBS buffer solution to remove unbound antigens, then blocking the electrode by using BSA solution to block non-specific binding sites possibly existing on the surface of the electrode, taking out, cleaning by using PBS buffer solution, and continuously dropwise adding mouse anti-silk fibroin antibody Ab 1 Washing the solution with PBS buffer solution at 25-35deg.C for 50-70 min to wash the non-immobilized murine anti-silk fibroin antibody Ab 1 And finally, dripping the CdSeQDs-PDA@Ab obtained in the step 8 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 25-35deg.C for 50-70 min 2 Thus obtaining the capacitive immunosensor for detecting the silk fibroin.
2. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 2 specifically comprises the following steps: placing a ZnO powder and carbon powder mixture with the molar ratio of 0.8-1.2:1 in a central reaction area of a quartz tube, and placing a silicon wafer coated with an Au catalyst of 5-10nm in front of the reaction area; setting the reaction temperature to 850-950 ℃ and the reaction time to 15-30 min, and introducing argon with the constant flow rate of 80-120sccm and oxygen with the constant flow rate of 3-7sccm as carrier gases during the reaction; after the reaction is completed, znO nano-wire is separated, and the solid-to-liquid ratio is 2-3 mg ml -1 Adding into isopropanol, and ultrasonic treating for 10-15 min to disperse uniformly, and storing.
3. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 3 specifically comprises the following steps: adding 100-120mg of graphite nano-sheets with the sheet diameter of 500-800 mu m into 560-600 ml chromic acid washing liquid, and performing ultrasonic dispersion for 30-40 min; mechanically stirring at 40-50deg.C for 10-20 min, pouring 1.5-2L water, and suction filtering; washing the filtrate with water and ethanol for 3-5 times, baking at 110-120deg.C for 3-3.5 hr, cooling to room temperature, and making into 20-30mg ml -1 And (3) oxidizing the graphene slurry for later use.
4. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 4 specifically comprises the following steps: heating the uniform mixture of the graphene oxide slurry and 40-50. 50 wt% of hydroiodic acid according to the volume ratio of 8-12:1 at the temperature of 85-90 ℃ for 2-2.5 h; washing with water and ethanol for 3-5 times, and refrigerating at 4deg.C.
5. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, which comprisesIs characterized in that: the step 5 specifically comprises the following steps: taking 40-50 ml and 2-3 mg ml of ZnO nanowire dispersion liquid obtained in the step 2 -1 Isopropyl alcohol solution of Ag nanowire 40-50-ml, respectively and ultrasonically dispersing uniformly; a vacuum pump of a vacuum suction filtration system is started, and a polyvinylidene fluoride porous filter membrane is paved at the top end of a conical flask and is soaked; coating the isopropanol solution of RGO and Ag nanowires obtained in the step 4 and the ZnO nanowire dispersion liquid obtained in the step 2 on the surface of the film in sequence; vacuum drying at 30-40deg.C 18-24 h; stripping the RGO-Ag-ZnO film, washing with water and ethanol for 2-3 times, and removing possible iodine; and (5) drying and cutting to obtain the RGO-Ag-ZnO film electrode of the functional layer.
6. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 6 specifically comprises the following steps: and 5, taking an ITO conductive glass electrode, respectively ultrasonically cleaning the ITO conductive glass electrode by using acetone, ethanol and deionized water for 10-20 min, drying the ITO conductive glass electrode at 50-60 ℃, and adhering the RGO-Ag-ZnO film electrode of the functional layer obtained in the step 5 to the surface of the ITO conductive glass electrode.
7. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 7 specifically comprises the following steps: preparing 50-60 ml of mixed solution of 0.03-0.07 mmol/L pyrrole monomer and 0.03-0.07 mmol/L KCl as electrolyte; and (3) using an electrochemical workstation CHI660E, using a platinum wire electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, and using a constant current method to electrochemically deposit polypyrrole on the electrode obtained in the step (6), wherein the current is 8-12 mA, and the deposition time is 300-500 s, so as to prepare the RGO-Ag-ZnO-PPy composite material.
8. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 8 specifically comprises the following steps: mixing 0.80-0.88 mL thioglycollic acid and 55-65 mL water uniformly, adding 45-55 mL 0.1M cadmium chloride aqueous solution, stirring uniformly, adjusting pH to 9.5-10.5, introducing nitrogen to remove oxygen in the solution, continuously adding 11-13 mL 0.2M NaHSe solution, stirring uniformly, reacting the obtained reaction precursor solution at 75-85deg.C under nitrogen protection for 3-5 h, and finally obtaining bright yellow CCooling dSeQDs solution to room temperature, centrifuging, cleaning, adding 10-20 ml polyDOPA polyamine, fully binding 1-3 h at 30-35deg.C, washing for 3-5 min, adding into solution containing 8-12 ul ml -1 Rabbit anti-murine anti-silk fibroin antibody Ab 2 Incubation at 30-35 ℃ for 0.5-1.5. 1.5 h, flushing for 3-5 min, and preserving at 1-5 ℃ for standby.
9. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 9 specifically comprises the following steps: and (3) dropwise adding 10-20 ul of 0.05M MPA aqueous solution on the surface of the RGO-Ag-ZnO-PPy composite material obtained in the step (7) after drying by adopting a dropwise coating method, incubating for 50-70min at 50-60 ℃ to form a saturated MPA monolayer, washing with PBS buffer, soaking the obtained electrode modified with MPA in MES buffer, incubating for 50-70min at 55-65 ℃, converting the terminal carboxyl of the MPA into active NHS ester, and washing with PBS buffer to obtain the activated ITO-RGO-Ag-ZnO-PPy membrane electrode.
10. The capacitive immunosensor for silk fibroin detection as claimed in claim 1, wherein: the step 10 specifically comprises the following steps: dropwise adding 10-20 ul1ul/ml CB solution of silk fibroin obtained in the step 1 onto the surface of an activated ITO-RGO-Ag-ZnO-PPy membrane electrode, enabling terminal amino groups of the CB solution to be combined with activated carboxyl groups, thoroughly washing the surface of the activated ITO-RGO-Ag-ZnO-PPy membrane electrode by using PBS buffer solution to remove unbound antigens, blocking the electrode by using 0.8-1.2wt% BSA solution of 10-20 ul for 25-35 min, blocking 0.5-1.5 h by using 0.8-1.2wt% BSA solution, taking out the electrode, washing the electrode by using PBS buffer solution, continuously dropwise adding 10-20 ul1ul/ml murine anti-silk fibroin antibody Ab 1 Washing the solution with PBS buffer solution at 25-35deg.C for 50-70 min to wash the non-immobilized murine anti-silk fibroin antibody Ab 1 Finally, dropwise adding the CdSeQDs-PDA@Ab obtained in the step 8 of 10-20 and 20 ul 2 Washing the unfixed CdSeQDs-PDA@Ab with PBS buffer solution at 25-35deg.C for 50-70 min 2 Thus obtaining the capacitive immunosensor for detecting the silk fibroin.
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| CN112834591A (en) * | 2021-01-06 | 2021-05-25 | 浙江理工大学 | Based on CdSeQDs-TiO2Preparation method of photoelectrochemical immunosensor for silk fibroin detection of @ Au |
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