阅读说明：本技术 一种用于检测角蛋白的光电化学免疫传感器的制备方法 (Preparation method of photoelectrochemical immunosensor for detecting keratin ) 是由 王秉 周晴晴 李青青 王钟元 胡智文 万军民 于 2021-01-06 设计创作，主要内容包括：本发明涉及光电化学传感领域,公开了一种用于检测角蛋白的光电化学免疫传感器的制备方法,本发明首先提取丝素蛋白的提取并合成AuNPs和聚多巴胺,并使CdSeQDs-PDA负载Ab-2,然后逐层自组装制备间接型传感器。AuNPs由于局域表面等离子体共振效应来增强光催化活性。MoS-2-GO构成了2D-2D异质结,通过能带的匹配,来增大光电流信号的响应。如PDA膜上的醌官能团可以通过迈克尔加成反应,与胺封端的抗体共价偶联。CdSeQDs发射峰与Au NPs等离子体带之间有重叠,由于竞争吸收光激子能量转移(EET)的作用,可以减小的光电流信号。(The invention relates to the field of photoelectrochemical sensing, and discloses a preparation method of a photoelectrochemical immunosensor for detecting keratin 2 And then self-assembling layer by layer to prepare the indirect sensor. AuNPs enhance photocatalytic activity due to localized surface plasmon resonance effects. MoS 2 GO forms a 2D-2D heterojunction, and the response of the photocurrent signal is increased through the matching of energy bands. Quinone functional groups on films such as PDA can be reacted by Michael additionIt should be covalently coupled to an amine-terminated antibody. The CdSeQDs emission peak and Au NPs plasma band are overlapped, and the light current signal can be reduced due to the action of competitive absorption Exciton Energy Transfer (EET).)

1. A method for preparing a photoelectrochemical immunosensor for the detection of keratin, comprising the steps of:

step 1: extraction of wool keratin: adding wool into ethanol, stirring for 1-3h, washing with deionized water, and drying; adding 4-6g of dried wool into CB buffer solution according to a bath ratio of 1:18-20, adding 1-1.4g of sodium sulfite, reacting at 55-65 ℃ for 25-35min, washing with deionized water and drying, soaking 1.8-2.2g of the treated wool into 18-22ml of 1-ethyl-3-methylimidazol diethyl phosphate ionic liquid, adding 20-25ml of dimethyl sulfoxide, and carrying out ultrasonic treatment; adding 18-22ml of 1-allyl-3-methylimidazolium acetate ionic liquid into the obtained solution, adding 20-25ml of dimethyl sulfoxide, performing ultrasonic centrifugation, taking supernatant, dialyzing in a dialysis bag, freeze-drying, and grinding to obtain wool keratin;

step 2: preparation of AuNPs: 30-34ml of 0.01% HAuCl was taken₄Heating the solution, adding 38.8mM sodium citrate solution, reacting for 15-20min, cooling to room temperature, and refrigerating;

and step 3: MoS₂Hydrothermal preparation of GO nanocomposites: under the action of ultrasonic wave, dispersing 45-55mg of GO powder into 60ml of distilled water, and then adding 0.5-0.6g of NaMoO₄And 0.7-0.75g of thiourea are added into the obtained suspension for mixing, the obtained mixed solution is transferred into an autoclave for reaction at the temperature of 230-240 ℃ for 20-30h, the mixture is cooled to the room temperature, and black MoS is collected by centrifugation₂Washing GO with distilled water and absolute ethyl alcohol, and finally drying at 50-60 ℃ for later use;

and 4, step 4: preparation of polydopamine: adding 8-10ml of absolute ethyl alcohol and 18-20ml of deionized water into a container, and then adding 550-; stirring the obtained mixed solution for 25-30min, and slowly adding 2ml of 45-55mg/ml dopamine solution; then stirring and reacting for 25-30h at normal temperature, and centrifuging the obtained solution for 3-7min at 8000-;

and 5: core-shell structure CdSeQDs-PDA @ Ab₂The preparation of (1): 0.80-0.88mL of thioglycolic acid and 55-65mL of deionized water are uniformly mixed, then 45-55mL of 0.1M aqueous solution of cadmium chloride is added into the mixture and uniformly stirred, the pH value of the solution is adjusted to 9.5-10.5 by NaOH solution, then high-purity nitrogen is introduced to remove oxygen in the solution, then 11-13mL of 0.2M NaHSe solution is continuously added into the solution and uniformly stirred, then the obtained reaction precursor solution reacts for 3-5 hours at 75-85 ℃ under the protection of the high-purity nitrogen, finally the obtained bright yellow CdSeQDs solution is cooled to room temperature, centrifugally cleaned and fixed, 10-15mL of polyaminoamine prepared in the step 4 is added, the mixture is fully combined for 1-3 hours at the temperature of 30-35 ℃, the deionized water is used for washing for 3-5 minutes, and the mixture is added into 15-20mL of Ab containing 10ul/mL₂Incubating at 30-35 deg.C for 0.5-1.5h in BSA solution, washing with deionized water for 3-5min, and storing at 1-5 deg.C;

step 6: activated MoS₂GO/AuNPs/ITO electrodes: 180-200ul mercaptopropionic acid was added to 1ml of MoS obtained in step 3₂Continuously performing ultrasonic treatment for 20-30h in a mixed suspension of GO and 1mg AuNPs obtained in the step 2, and centrifugally collecting carboxyl modified MoS₂GO/AuNPs, MoS carboxylated finally₂Dispersing GO/AuNPs into 2ml of 0.05wt% CS solution, dripping 15-20ul of the solution on an ITO electrode, standing at room temperature overnight, and drying at 70-80 ℃ for 0.5-1.5h, finally soaking the electrode in 1ml of 0.05M MES buffer solution, incubating at 50-60 ℃ for 50-70min, converting the terminal carboxyl of mercaptopropionic acid into active NHS ester, and washing with PBS buffer solution;

and 7: pretreating an ITO conductive glass electrode: taking the area of 0.35-0.45cm²The ITO conductive glass electrode is ultrasonically cleaned for 10-20min by acetone, ethanol and deionized water respectively, and dried at 50-60 ℃ overnight;

and 8: constructing an indirect type immunoprobe: dropwise adding 5-10 ul of 1ul/ml of the CB solution of the keratin obtained in the step 1 to combine the terminal amino group with the activated carboxyl, thoroughly washing with PBS buffer solution to remove unbound antigen, and sealing the electrode with 10-15 ul of 1% BSA at 35-40 ℃ for 20-40 min; blocking with 1% BSA for 0.5-1.5 hr to block non-specific binding sites on the electrode surface, washing with PBS buffer, and adding 3-7ul 1ul/ml solution of rabbit anti-keratin antibody (Ab)₁Placing the antibody solution at 25-35 deg.C for 50-70min, washing the unfixed Ab with PBS buffer₁Finally, 8-12ul of the CdSeQDs-PDA @ Ab obtained in the step 5 is dripped₂Placing at 25-35 deg.C for 50-70min, washing unfixed CdSeQDs-PDA @ Ab with PBS buffer solution₂And obtaining the photoelectrochemistry immunosensor.

2. The method of claim 1, wherein in step 1: the centrifugation rate is 7000-8000r/min, and the centrifugation time is 9-15 min; dialyzing the obtained solution in deionized water for 2-3 days by using a cellulose dialysis bag with the molecular weight cutoff of 7000-10000, replacing water every 4-5h, and carrying out vacuum freeze drying on the keratin solution for 2-3 days.

3. The method of claim 1, wherein in steps 1 and 8: the CB buffer pH = 9.6.

4. The method of claim 1, wherein in step 2: heating to a temperature of 95-105 deg.C, adding sodium citrate solution, stirring at constant speed during reaction, and observing color changing from blue to red.

5. The method according to claim 1, wherein the concentrated aqueous ammonia has a concentration of 25 to 28wt% in step 4.

6. The method of claim 1, wherein in step 5, Ab is₂Is polyclonal antibody of goat anti-rabbit immunoglobulin.

7. The method according to claim 1, wherein in step 6, MoS in the suspension is present₂-GO concentration of 3-7mg.ml^-1The concentration of the AuNPs is 0.34-0.39 ug/ml.

8. The method of claim 1, wherein in step 6, the MES buffer comprises 0.05M EDC and 0.03M NHS.

9. The method of claim 1, wherein the PBS buffer has a pH =7.4 in steps 6 and 8.

Technical Field

The invention relates to the field of photoelectrochemical sensing, in particular to a preparation method of a photoelectrochemical immunosensor for detecting keratin.

Background

Since ancient China, the fabric is a big textile, and the produced fabric is rich in variety, exquisite in process, comfortable and breathable. The production of wool fabric is far ahead, and wool fabric cultural relics are historical visitors of social alternation and human-character interaction. The main component of wool is wool, and the protein contained in wool is mainly keratin. However, as an organic polymer material, wool in the wool fabric cultural relic is susceptible to degradation caused by light, heat, acid, alkali, microorganisms and the like in an underground burial environment all the year round, so that the changes of structures and performances such as crystallinity and molecular weight are caused; on the other hand, when the cultural relics are unearthed, a plurality of impurities are often accompanied, and the real effective components are few. Therefore, the conventional detection method has low sensitivity, is greatly influenced by impurity interference and is not suitable for detecting cultural relics, so that the method for detecting the ancient wool fabric, which has good sensitivity and strong specificity, has important significance.

The analytical methods for textile residues reported at home and abroad mainly comprise a chemical degradation method, a biological mass spectrometry method and the like. However, the ancient textiles have complex components, the mass spectrometry can be carried out with large errors due to small component changes, and the whole experimental process is complicated because the experimental steps of residue extraction, enzyme digestion, mass spectrometry, result analysis and the like are required. Therefore, it is very important to find a method with extremely high sensitivity, extremely high specificity, rapidness and high efficiency for identifying textile residues.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a photoelectrochemical immunosensor for detecting keratin₂Then preparing the indirect MoS by a layer-by-layer self-assembly process₂GO/Au NPs sensors, and finally the sensor performance was tested with a self-assembled PEC system.

The specific technical scheme of the invention is as follows: a method for preparing a photoelectrochemical immunosensor for detection of keratin, comprising the steps of:

step 1: extraction of wool keratin: adding wool into ethanol, stirring for 1-3h, washing with deionized water, and drying; adding 4-6g of dried wool into CB buffer solution according to a bath ratio of 1:18-20, adding 1-1.4g of sodium sulfite, reacting at 55-65 ℃ for 25-35min, washing with deionized water and drying, soaking 1.8-2.2g of the treated wool into 18-22ml of 1-ethyl-3-methylimidazol diethyl phosphate ionic liquid, adding 20-25ml of dimethyl sulfoxide, and carrying out ultrasonic treatment; adding 18-22ml of 1-allyl-3-methylimidazolium acetate ionic liquid into the obtained solution, adding 20-25ml of dimethyl sulfoxide, performing ultrasonic centrifugation, taking supernatant, dialyzing in a dialysis bag, freeze-drying, and grinding to obtain the wool keratin.

Step 2: preparation of AuNPs: 30-34ml of 0.01% HAuCl was taken₄Heating the solution, adding 38.8mM sodium citrate solution, continuing to react for 15-20min, cooling to room temperature, and refrigerating for later use.

The fluorescence resonance between the semiconductor nanocrystal and AuNPs nanoparticles can transfer energy, and the AuNPs enhance the photocatalytic activity due to the local surface plasmon resonance effect. Because the emission of the semiconductor material can excite surface plasmon resonance in the metal nanomaterial within a short working distance.

And step 3: MoS₂Hydrothermal preparation of GO nanocomposites: under the action of ultrasonic wave, dispersing 45-55mg of GO powder into 60ml of distilled water, and then adding 0.5-0.6g of NaMoO₄And 0.7-0.75g of thiourea are added into the obtained suspension for mixing, the obtained mixed solution is transferred into an autoclave for reaction at the temperature of 230-240 ℃ for 20-30h, the mixture is cooled to the room temperature, and black MoS is collected by centrifugation₂and/GO is washed by distilled water and absolute ethyl alcohol and finally dried at 50-60 ℃ for later use.

MoS in the invention₂GO forms a 2D-2D heterojunction, the response of a photocurrent signal is increased through the matching of energy bands, and the two-dimensional (2D) nano material has the advantages of large specific surface area, excellent optical and electrical properties and the like. MoS₂Is a layered structure that is acted upon by weak van der waals forces. The structure has the advantages of narrow band gap, high carrier mobility and the like.

And 4, step 4: preparation of polydopamine: adding 8-10ml of absolute ethyl alcohol and 18-20ml of deionized water into a container, and then adding 550-; stirring the obtained mixed solution for 25-30min, and slowly adding 2ml of 45-55mg/ml dopamine solution; then, the reaction is stirred for 25-30h at normal temperature, and the obtained solution is centrifuged for 3-7min at 8000-10000rpm for later use.

The present invention takes into account that the binding sites of biological probes are generally limited by combining nanomaterials with polydopamine membrane materials having a large surface area. The bionic material Polydopamine (PDA) can spontaneously form uniform ultrathin coatings on the surfaces of different types of inorganic matters and organic matters, has high biocompatibility, has strong absorption in the whole ultraviolet-visible (UV-vis) and Near Infrared (NIR) regions, can compete to absorb photons and excitons by combining with secondary antibodies, and improves the quenching property of system photocurrent. And PDA has abundant functional groups, such as quinone functional groups on PDA membranes, which can be covalently coupled to amine terminated antibodies via michael addition reaction, thus loading more biocomposites.

And 5: core-shell structure CdSeQDs-PDA @ Ab₂The preparation of (1): mixing 0.80-0.88mL of thioglycolic acid and 55-65mL of deionized water uniformly, adding 45-55mL of 0.1M aqueous solution of cadmium chloride, stirring uniformly, adjusting the pH of the solution to 9.5-10.5 by using NaOH solution, introducing high-purity nitrogen to remove oxygen in the solution, and continuing to remove oxygen in the solutionAdding 11-13ml of 0.2M NaHSe solution, stirring uniformly, reacting the obtained reaction precursor solution at 75-85 ℃ for 3-5h under the protection of high-purity nitrogen, cooling the obtained bright yellow CdSeQDs solution to room temperature, centrifugally cleaning to constant volume, adding 10-15ml of polyamine prepared in the step 4, fully bonding for 1-3h at 30-35 ℃, washing with deionized water for 3-5min, adding 15-20ml of Ab containing 10ul/ml₂Incubating at 30-35 deg.C for 0.5-1.5h in BSA solution, washing with deionized water for 3-5min, and storing at 1-5 deg.C.

The CdSeQDs as a narrow-bandgap semiconductor are combined with PDA and a secondary antibody, can compete to absorb photons and excitons, increase steric hindrance and reduce the response of photocurrent signals.

Step 6: activated MoS₂GO/AuNPs/ITO electrodes: 180-200ul mercaptopropionic acid was added to 1ml of MoS obtained in step 3₂Continuously performing ultrasonic treatment for 20-30h in a mixed suspension of GO and 1mg AuNPs obtained in the step 2, and centrifugally collecting carboxyl modified MoS₂GO/AuNPs, MoS carboxylated finally₂Dispersing GO/AuNPs into 2ml of 0.05wt% CS solution, dropwise adding 15-20ul of ITO electrode, standing overnight at room temperature, drying at 70-80 ℃ for 0.5-1.5h, finally soaking the electrode in 1ml of 0.05M MES buffer solution, placing in 50-60 ℃ for incubation for 50-70min, converting the terminal carboxyl of mercaptopropionic acid into active NHS ester, and washing with PBS buffer solution.

The GO surface of the invention has rich carboxyl active sites, and can be further covalently coupled with biomolecules, and meanwhile, the dispersibility of the solution is improved.

And 7: pretreating an ITO conductive glass electrode: taking the area of 0.35-0.45cm²The ITO conductive glass electrode is ultrasonically cleaned by acetone, ethanol and deionized water for 10-20min and dried at 50-60 ℃ overnight.

And 8: constructing an indirect type immunoprobe: dropwise adding 5-10 ul of 1ul/ml of the CB solution of the keratin obtained in the step 1 to combine the terminal amino group with the activated carboxyl, thoroughly washing with PBS buffer solution to remove unbound antigen, and sealing the electrode with 10-15 ul of 1% BSA at 35-40 ℃ for 20-40 min; subsequently, blocking with 1% BSA for 0.5-1.5h to block the possible presence of electrode surfaceThe non-specific binding sites were removed and washed with PBS buffer, and 3-7ul of 1ul/ml rabbit anti-keratin antibody solution (Ab) was added dropwise₁Placing the antibody solution at 25-35 deg.C for 50-70min, washing the unfixed Ab with PBS buffer₁Finally, 8-12ul of the CdSeQDs-PDA @ Ab obtained in the step 5 is dripped₂Placing at 25-35 deg.C for 50-70min, washing unfixed CdSeQDs-PDA @ Ab with PBS buffer solution₂And obtaining the electrochemical immunosensor.

Preferably, in step 1: the centrifugation rate is 7000-8000r/min, and the centrifugation time is 9-15 min; dialyzing the obtained solution in deionized water for 2-3 days by using a cellulose dialysis bag with the molecular weight cutoff of 7000-10000, replacing water every 4-5h, and carrying out vacuum freeze drying on the keratin solution for 2-3 days.

Preferably, in steps 1 and 8: the CB buffer pH = 9.6.

Preferably, in step 2: heating to a temperature of 95-105 deg.C, adding sodium citrate solution, stirring at constant speed, and observing color changing from blue to red.

Preferably, in step 4, the concentration of the concentrated ammonia water is 25-28 wt%.

Preferably, in step 5, Ab is₂Is polyclonal antibody of goat anti-rabbit immunoglobulin.

Preferably, in step 6, MoS in the suspension is₂-GO concentration of 3-7mg.ml^-1The concentration of the AuNPs is 0.34-0.39 ug/ml.

Preferably, in step 6, the MES buffer contains 0.05M EDC and 0.03M NHS.

Preferably, the PBS buffer has a pH =7.4 in steps 6 and 8.

Compared with the prior art, the invention has the beneficial effects that:

(1) the fluorescence resonance between the semiconductor nanocrystal and AuNPs nanoparticles can transfer energy, and the AuNPs enhance the photocatalytic activity due to the local surface plasmon resonance effect. Because the emission of the semiconductor material can excite surface plasmon resonance in the metal nanomaterial within a short working distance.

(2) MoS of the invention₂GO forms a 2D-2D heterojunction, the response of a photocurrent signal is increased through the matching of energy bands, and the two-dimensional (2D) nano material has the advantages of large specific surface area, excellent optical and electrical properties and the like. MoS₂Is a layered structure that is acted upon by weak van der waals forces. The structure has the advantages of narrow band gap, high carrier mobility and the like.

(3) The present invention takes into account that the binding sites of biological probes are generally limited by combining nanomaterials with polydopamine membrane materials having a large surface area. The bionic material Polydopamine (PDA) can spontaneously form uniform ultrathin coatings on the surfaces of different types of inorganic matters and organic matters, has high biocompatibility, has strong absorption in the whole ultraviolet-visible (UV-vis) and Near Infrared (NIR) regions, can compete to absorb photons and excitons by combining with secondary antibodies, and improves the quenching property of system photocurrent. And PDA has abundant functional groups, such as quinone functional groups on PDA membranes, which can be covalently coupled to amine terminated antibodies via michael addition reaction, thus loading more biocomposites.

(4) The CdSeQDs as a narrow-bandgap semiconductor are combined with PDA and a secondary antibody, can compete to absorb photons and excitons, increase steric hindrance and reduce the response of photocurrent signals.

(5) In the PEC immunoassay of the invention, besides the steric hindrance effect which is an important signal amplification strategy, the CdSeQDs emission peak and the Au NPs plasma band are overlapped, and the light current signal can be reduced due to the action of competitive absorption light Exciton Energy Transfer (EET). The invention constructs an indirect immunosensor, which is different from the conventional sandwich photoelectrochemical immunosensor. The detection base solution was phosphate buffered saline (PBS, 0.01mol/L, pH =7.4) containing 0.1M Ascorbic Acid (AA) as an electron donor for the photoanode current.

Drawings

FIG. 1 is a high resolution TEM image of the CdSeQDs obtained in example 1;

FIG. 2 shows the MoS obtained in example 1₂-scanning electron microscopy (left) and transmission electron microscopy (right) of GO;

FIG. 3 shows MoS obtained in example 1₂Scanning electron micrograph (c).

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

Step 1: extraction of wool keratin: adding wool into ethanol, stirring for 1h, washing with deionized water, and drying; taking 4g of dried wool, mixing the dried wool with a mixture of 1: adding 18 parts of bath ratio into CB 9.6 buffer solution, adding 1g of sodium sulfite, reacting for 25min at 55 ℃, washing with deionized water for 3 times, drying, taking 1.8g of treated wool, immersing into 18ml of 1-ethyl-3-methylimidazolium diethyl phosphate ionic liquid, adding 20ml of dimethyl sulfoxide, and carrying out ultrasonic treatment; adding 18ml of 1-allyl-3-methylimidazole acetate ionic liquid into the obtained solution, adding 20ml of dimethyl sulfoxide, centrifuging at the speed of 7000r/min for 9min, dialyzing the obtained solution in deionized water for 2 days by using a cellulose dialysis bag with the molecular weight cutoff of 7000, changing water every 4h, carrying out vacuum freeze drying on the keratin solution for 2 days, and grinding to obtain wool keratin;

step 2: preparation of AuNPs: 30ml of 0.01% HAuCl were taken₄Heating the solution in a 100ml round-bottom flask to a temperature stable at 95 ℃, adding 38.8mM sodium citrate solution, continuing to react for 15min, continuously stirring at a constant speed during the reaction, observing the color, turning blue and then red, cooling to room temperature, and storing in a refrigerator at 4 ℃ for later use;

and step 3: MoS₂Hydrothermal preparation of GO nanocomposites: dispersing 45mg GO powder into 60ml distilled water under the action of ultrasonic waves, and then adding 0.5g NaMoO₄And 0.7g of thiourea were added to the above suspension and mixed, the homogeneous mixture was transferred to an autoclave and reacted at 230 ℃ for 24 hours, cooled to room temperature, centrifuged to collect black MoS₂GO, washing 5 times with distilled water and absolute ethanol, and finally drying overnight at 50 ℃ for later use;

and 4, step 4: preparation of polydopamine: add 8ml absolute ethanol and 18ml deionized water to a clean beaker followed by 550ul of concentrated ammonia (25 wt%); the resulting mixed solution was stirred for 25min on a magnetic stirrer, and then 2ml of dopamine solution (50 mg/ml) was slowly added, followed by reaction for 25h under magnetic stirring at room temperature. Centrifuging the obtained solution at 8000rpm for 5 min;

and 5: core-shell structure CdSeQDs-PDA @ Ab₂The preparation of (1): 0.80ml of thioglycolic acid and 55ml of deionized water are uniformly mixed, 45ml of 0.1M cadmium chloride aqueous solution is added into the mixture and uniformly stirred, the pH value of the solution is adjusted to 9.5 by 0.1M NaOH solution, high-purity nitrogen is introduced to remove oxygen in the solution, 11ml of 0.2M NaHSe solution is continuously added into the solution and uniformly stirred, the reaction precursor solution is reacted for 4 hours at 75 ℃ under the protection of the high-purity nitrogen, finally, the bright yellow CdSeQDs solution is cooled to room temperature, centrifugal cleaning and constant volume are carried out, 10ml of PDA prepared in the step 4 is added, the mixture is fully combined for 2 hours at 30 ℃, the deionized water is used for washing for 3 minutes, and 15ml of PDA containing 10ul/ml Ab is added₂Incubating (goat anti-rabbit immunoglobulin polyclonal antibody) in BSA solution at 30 deg.C for 1h, washing with deionized water for 3min, and storing at 4 deg.C;

step 6: activated MoS₂GO/AuNPs/ITO electrodes: 180ul of mercaptopropionic acid was added to step 31 ml of MoS₂-GO（5mg.ml^-1) And step 21 ml of AuNPs (0.35 ug/ml) suspension, continuously carrying out ultrasonic treatment for 24h, and centrifugally collecting carboxyl modified MoS₂GO/AuNPs, MoS carboxylated finally₂Dispersing GO/AuNPs in 2ml CS (0.05 wt%) solution, adding 15ul ITO electrode dropwise, standing overnight at room temperature, drying at 70 deg.C for 1h, soaking the electrode in 1ml 0.05M EDC/0.03M NHS MES buffer solution, incubating in 50 deg.C oven for 50min, converting terminal carboxyl group of mercaptopropionic acid into active NHS ester, and incubating with 0.01 mol.L^-1Washing with PBS buffer (pH 7.4);

and 7: pretreating an ITO conductive glass electrode: taking the area of 0.40cm²The ITO conductive glass electrode is ultrasonically cleaned for 10min by acetone, ethanol and deionized water respectively, and dried in an oven at 50 ℃ overnight;

and 8: constructing an indirect type immunoprobe: dripping deviceAdding 5ul of 1ul/ml of the solution of keratin CB obtained in the step 1 to bind the terminal amino group to the activated carboxyl group, washing thoroughly with PBS (pH =7.4) to remove the unbound antigen, blocking the electrode with 10ul of 1% BSA at 37 ℃ for 30min, then blocking the electrode with 1% BSA for 1h to block any non-specific binding sites on the electrode surface, removing the electrode, and blocking the electrode with 0.01 mol. L^-1Washing with PBS buffer (pH 7.4), and further dropwise adding keratin antibody (Ab) 5ul and 1ul/ml₁) Placing the solution in an oven at 30 deg.C for 50min, and adding 0.01mol/L^-1Non-immobilized Ab was washed with Phosphate Buffered Saline (PBS) (pH 7.4)₁(rabbit anti-silk fibroin antibody solution), and finally 10ul of step 5 CdSeQDs-PDA @ Ab is dripped₂Placing in an oven at 30 deg.C for 50min, and heating with 0.01 mol. L^-1Washing of the unfixed CdSeQDs-PDA @ Ab with Phosphate Buffered Saline (PBS) (pH 7.4)₂And placing the electrode in a detection base solution which is phosphate buffer solution (PBS, 0.01mol/L, pH7.4) containing 0.1M Ascorbic Acid (AA), and completing the assembly of the electrochemical immunosensor.

FIG. 1 is a high resolution TEM image of the CdSeQDs obtained in example 1; FIG. 2 shows the MoS obtained in example 1₂-scanning electron microscopy (left) and transmission electron microscopy (right) of GO; FIG. 3 shows MoS obtained in example 1₂Scanning electron micrograph (c).

Example 2

Step 1: extraction of wool keratin: adding wool into ethanol, stirring for 2h, washing with deionized water, and drying; taking 5g of dried wool, mixing the dried wool with a mixture of 1: adding a bath ratio of 19 into a CB 9.6 buffer solution, adding 1.2g of sodium sulfite, reacting for 30min at 60 ℃, washing with deionized water for 4 times, drying, soaking 2g of the treated wool into 20ml of 1-ethyl-3-methylimidazolium diethyl phosphate ionic liquid, adding 23ml of dimethyl sulfoxide, and carrying out ultrasonic treatment; adding 20ml of 1-allyl-3-methylimidazole acetate ionic liquid into the obtained solution, adding 23ml of dimethyl sulfoxide, centrifuging at the speed of 7500r/min for 11min, dialyzing the obtained solution in deionized water for 2 days by using a cellulose dialysis bag with the molecular weight cutoff of 9000, changing water every 4h, carrying out vacuum freeze drying on the keratin solution for 2 days, and grinding to obtain wool keratin;

step 2: preparation of AuNPs: 32ml of 0.01% HAuCl were taken₄Heating the solution in a 100ml round-bottom flask to a temperature stable at 100 deg.C, adding 38.8mM sodium citrate solution, reacting for 18min, stirring at constant speed, observing color, turning blue and then red, cooling to room temperature, and storing in a refrigerator at 4 deg.C;

and step 3: MoS₂Hydrothermal preparation of GO nanocomposites: dispersing 50mg GO powder into 60ml distilled water under the action of ultrasonic waves, and then adding 0.55g NaMoO₄And 0.73g of thiourea were added to the above suspension and mixed, the homogeneous mixture was transferred to an autoclave and reacted at 235 ℃ for 24 hours, cooled to room temperature, centrifuged to collect black MoS₂GO, washing 6 times with distilled water and absolute ethyl alcohol, and finally drying overnight at 55 ℃ for later use;

and 4, step 4: preparation of polydopamine: 9ml of absolute ethanol and 19ml of deionized water were added to a clean beaker followed by 580ul of concentrated aqueous ammonia (26 wt%); the resulting mixed solution was stirred on a magnetic stirrer for 27min, and then 2ml of dopamine solution (50 mg/ml) was slowly added, followed by reaction for 27h with magnetic stirring at room temperature. Centrifuging the obtained solution at 9000rpm for 5 min;

and 5: core-shell structure CdSeQDs-PDA @ Ab₂The preparation of (1): 0.84ml of thioglycolic acid and 55ml of deionized water are uniformly mixed, then 50ml of 0.1M cadmium chloride aqueous solution is added into the mixture and uniformly stirred, the pH value of the solution is adjusted to 10 by 0.1M NaOH solution, then high-purity nitrogen is introduced to remove oxygen in the solution, then 12ml of 0.2M NaHSe solution is continuously added into the solution and uniformly stirred, then the reaction precursor solution reacts for 4 hours at 80 ℃ under the protection of high-purity nitrogen, finally the bright yellow CdSeQDs solution is cooled to room temperature, centrifugal cleaning and volume fixing are carried out, 12ml of PDA prepared in the step 4 is added, the mixture is fully combined for 2 hours at 33 ℃, deionized water is used for washing for 4 minutes, and 18ml of PDA containing 10ul/ml Ab is added₂Incubating (goat anti-rabbit immunoglobulin polyclonal antibody) in BSA solution at 32 deg.C for 1h, washing with deionized water for 4min, and storing at 4 deg.C;

step 6: activated MoS₂GO/AuNPs/ITO electrodes: 190ul of mercapto groupPropionic acid was added to step 31 ml MoS₂-GO（5mg.ml^-1) And step 21 ml of AuNPs (0.35 ug/ml) suspension, continuously carrying out ultrasonic treatment for 24h, and centrifugally collecting carboxyl modified MoS₂GO/AuNPs, MoS carboxylated finally₂Dispersing GO/AuNPs in 2ml CS (0.05 wt%) solution, adding 18ul ITO electrode dropwise, standing overnight at room temperature, drying at 70-80 deg.C for 1h, soaking the electrode in 1ml MES buffer solution of 0.05M EDC/0.03M NHS, incubating at 55 deg.C for 60min, converting terminal carboxyl group of mercaptopropionic acid into active NHS ester, and incubating with 0.01 mol.L^-1Washing with PBS buffer (pH 7.4);

and 7: pretreating an ITO conductive glass electrode: taking the area of 0.40cm²The ITO conductive glass electrode is ultrasonically cleaned for 15min by acetone, ethanol and deionized water respectively, and dried in an oven at 55 ℃ overnight;

and 8: constructing an indirect type immunoprobe: dropping 8ul of 1ul/ml of the solution of keratin CB of step 1 to allow the terminal amino group to bind to the activated carboxyl group, washing thoroughly with PBS (pH =7.4) to remove unbound antigen, blocking the electrode with 13 ul of 1% BSA at 37 ℃ for 30min, then blocking 1h with 1% BSA to block non-specific binding sites that may be present on the electrode surface, taking out, and blocking with 0.01 mol. L^-1Washing with PBS buffer (pH 7.4), and further dropwise adding keratin antibody (Ab) 5ul and 1ul/ml₁) Placing the solution in an oven at 30 deg.C for 60min, and adding 0.01mol/L^-1Non-immobilized Ab was washed with Phosphate Buffered Saline (PBS) (pH 7.4)₁(rabbit anti-silk fibroin antibody solution), and finally 10ul of step 5 CdSeQDs-PDA @ Ab is dripped₂Placing in an oven at 30 deg.C for 60min, and heating with 0.01 mol. L^-1Washing of the unfixed CdSeQDs-PDA @ Ab with Phosphate Buffered Saline (PBS) (pH 7.4)₂And placing the electrode in a detection base solution which is phosphate buffer solution (PBS, 0.01mol/L, pH7.4) containing 0.1M Ascorbic Acid (AA), and completing the assembly of the electrochemical immunosensor.

Example 3

Step 1: extraction of wool keratin: adding wool into ethanol, stirring for 3h, washing with deionized water, and drying; taking 6g of dried wool, mixing the dried wool with a mixture of 1: adding 20 parts of bath ratio into CB 9.6 buffer solution, adding 1.4g of sodium sulfite, reacting for 35min at 65 ℃, washing with deionized water for 5 times, drying, soaking 2.2g of treated wool into 22ml of 1-ethyl-3-methylimidazole diethyl phosphate ionic liquid, adding 25ml of dimethyl sulfoxide, and carrying out ultrasonic treatment; adding 22ml of 1-allyl-3-methylimidazole acetate ionic liquid into the obtained solution, adding 25ml of dimethyl sulfoxide, centrifuging at the speed of 8000r/min for 15min, dialyzing the obtained solution in deionized water for 3 days by using a cellulose dialysis bag with the molecular weight cutoff of 10000, changing water every 5h, carrying out vacuum freeze drying on the keratin solution for 3 days, and grinding to obtain wool keratin;

step 2: preparation of AuNPs: 34ml of 0.01% HAuCl was taken₄Heating the solution in a 100ml round-bottom flask to a temperature stable at 105 deg.C, adding 38.8mM sodium citrate solution, reacting for 20min, stirring at constant speed, observing color, turning blue and then red, cooling to room temperature, and storing in a refrigerator at 4 deg.C;

and step 3: MoS₂Hydrothermal preparation of GO nanocomposites: dispersing 55mg GO powder into 60ml distilled water under the action of ultrasonic waves, and then adding 0.6g NaMoO₄And 0.75g of thiourea were added to the above suspension and mixed, the homogeneous mixture was transferred to an autoclave and reacted at 240 ℃ for 24 hours, cooled to room temperature, centrifuged to collect black MoS₂GO, washing 7 times with distilled water and absolute ethyl alcohol, and finally drying overnight at 60 ℃ for later use;

and 4, step 4: preparation of polydopamine: 10ml of absolute ethanol and 20ml of deionized water were added to a clean beaker, followed by 600ul of concentrated ammonia (28 wt%); the resulting mixed solution was stirred for 30min on a magnetic stirrer, then 2ml of dopamine solution (50 mg/ml) was slowly added, followed by reaction for 30h under magnetic stirring at room temperature. Centrifuging the obtained solution at 10000rpm for 5min for later use;

and 5: core-shell structure CdSeQDs-PDA @ Ab₂The preparation of (1): 0.88ml of thioglycolic acid and 65ml of deionized water are taken and mixed evenly, then 55ml of 0.1M cadmium chloride aqueous solution is added into the mixture and stirred evenly, the pH value of the solution is adjusted to 10.5 by 0.1M NaOH solution,introducing high-purity nitrogen to remove oxygen in the solution, continuously adding 13ml of 0.2M NaHSe solution, uniformly stirring, reacting the reaction precursor solution at 85 ℃ under the protection of the high-purity nitrogen for 4h, cooling the bright yellow CdSeQDs solution to room temperature, centrifugally cleaning to constant volume, adding 15ml of PDA prepared in the step 4, fully combining for 2h at 30-35 ℃, washing for 5min by deionized water, adding 20ml of the PDA containing 10ul/ml Ab₂Incubating (goat anti-rabbit immunoglobulin polyclonal antibody) in BSA solution at 35 deg.C for 1h, washing with deionized water for 5min, and storing at 4 deg.C;

step 6: activated MoS₂GO/AuNPs/ITO electrodes: 200ul mercaptopropionic acid was added to step 31 ml of MoS₂-GO（5mg.ml^-1) And step 21 ml of AuNPs (0.35 ug/ml) suspension, continuously carrying out ultrasonic treatment for 24h, and centrifugally collecting carboxyl modified MoS₂GO/AuNPs, MoS carboxylated finally₂Dispersing GO/AuNPs in 2ml CS (0.05 wt%) solution, dropping 20ul ITO electrode, standing overnight at room temperature, drying at 80 deg.C for 1h, soaking the electrode in 1ml 0.05M EDC/0.03M NHS MES buffer, incubating in 60 deg.C oven for 70min, converting the terminal carboxyl group of mercaptopropionic acid into active NHS ester, and incubating with 0.01 mol.L^-1Washing with PBS buffer (pH 7.4);

and 7: pretreating an ITO conductive glass electrode: taking the area of 0.40cm²The ITO conductive glass electrode is ultrasonically cleaned for 20min by acetone, ethanol and deionized water respectively, and dried in an oven at 50-60 ℃ overnight;

and 8: constructing an indirect type immunoprobe: dropping 10ul of 1ul/ml of the solution of keratin CB of step 1 to allow the terminal amino group to bind to the activated carboxyl group, washing thoroughly with PBS (pH =7.4) to remove unbound antigen, blocking the electrode with 15ul of 1% BSA at 37 ℃ for 30min, then blocking 1h with 1% BSA to block non-specific binding sites that may be present on the electrode surface, taking out, and blocking with 0.01 mol. L^-1Washing with PBS buffer (pH 7.4), and further dropwise adding keratin antibody (Ab) 5ul and 1ul/ml₁) Placing the solution in an oven at 30 deg.C for 70min, and adding 0.01mol/L^-1Non-immobilized Ab was washed with Phosphate Buffered Saline (PBS) (pH 7.4)₁(rabbit anti-silk fibroin antibody solution), and finally 10ul of step 5 CdSeQDs-PDA @ Ab is dripped₂Placing in an oven at 30 deg.C for 50-70min, and heating with 0.01 mol. L^-1Washing of the unfixed CdSeQDs-PDA @ Ab with Phosphate Buffered Saline (PBS) (pH 7.4)₂And placing the electrode in a detection base solution which is phosphate buffer solution (PBS, 0.01mol/L, pH7.4) containing 0.1M Ascorbic Acid (AA), and completing the assembly of the electrochemical immunosensor.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.