阅读说明：本技术 一种用于检测胶原蛋白的光电化学免疫传感器的制备方法 (Preparation method of photoelectrochemical immunosensor for detecting collagen ) 是由 王秉 周晴晴 傅悦悦 彭志勤 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胶原蛋白的光电化学免疫传感器的制备方法,本发明首先提取胶原蛋白并合成g-C-3N-4-MoS-2纳米复合半导体材料,接着进行锰卟啉的制备,并使IMAs负载Ab-2,然后逐层自组装制备间接型传感器。卟啉纳米管具有较大的比表面积,可增强电信号,提高催化能力。g-C-3N-4-MoS-2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,可以有效的抑制光生电子空穴对的重组并且增强其光捕获能力。含有聚苯乙烯微球为核心MPS100/羧基珠粒,其高分子聚合物表面带有丰富的羧基,可以与活性氨基共价偶联。(The invention relates to the field of photoelectrochemical sensing, and discloses a preparation method of a photoelectrochemical immunosensor for detecting collagen 3 N 4 ‑MoS 2 Nano composite semiconductor material, preparing manganese porphyrin and loading IMAs with Ab 2 And then self-assembling layer by layer to prepare the indirect sensor. The porphyrin nanotube has large specific surface area, and can enhance electric signals and improve catalytic capability. g-C 3 N 4 ‑MoS 2 A 2D-2D heterojunction is formed,by matching energy bands, the response of a photocurrent signal is increased, the recombination of photo-generated electron-hole pairs can be effectively inhibited, and the light capture capability of the photo-generated electron-hole pairs can be enhanced. The MPS 100/carboxyl bead with polystyrene microsphere as core has rich carboxyl on the surface of polymer and can be covalently coupled with active amino.)

1. A preparation method of a photoelectrochemical immunosensor for detecting collagen is characterized by comprising the following steps:

step 1: extracting collagen: cutting 15-20g fresh cow leather into 0.5-1cm pieces, cleaning with clear water, adding 8-12ml sodium carbonate solution with mass fraction of 5-10% as degreasing solution, adding 40-50ml warm water of 35-45 deg.C, stirring for 10-15min, filtering, and repeating the above operations; placing in 20-25ml 3% acetic acid for 40-50h, centrifuging for 1-3h at a centrifugation rate of 7000-8000r/min, sucking supernatant, adding 10-15ml 25-35% concentrated saline, salting out for 25-30min, dissolving precipitate in 15-20ml 0.1% acetic acid, placing in a dialysis bag, dialyzing in 20-25ml 1mol/L HCl solution for 2-3 days by using a cellulose dialysis bag with a cut-off molecular weight of 7000-13000, and changing water every 4-5h for 4-6 times; adding chloroform into the product obtained by dialysis, opening the container mouth, wrapping with sterile gauze, placing on a sterile workbench to volatilize chloroform to obtain collagen solution, and freeze-drying under vacuum for use;

step 2: preparing a manganese porphyrin nano material: introducing nitrogen into a reaction container for degassing, adding 120ml of 100-120 ml of N, N-diethylformamide after 8-10 min, heating to slightly boil and refluxing, then adding 80-90mg of tetrasulfonic acid phenyl porphyrin, stirring for 8-10 min until the mixture is clear and transparent, then adding 90-100 mg of manganese chloride, mechanically stirring at the temperature of 100 ℃ and under the nitrogen atmosphere, cooling a reaction product to room temperature after 6-10 h, pouring into 130 ml of 120-130 ml of deionized water, standing for 3-5h, filtering, and sequentially washing a filter cake with water and ethanol respectively; collecting a first powder color band by using reagent-grade silica gel as an adsorbent and a chloroform/methanol solution as an eluting agent, performing vacuum rotary evaporation to obtain a manganoporphyrin nanotube, and putting the manganoporphyrin nanotube into a dryer for storage for later use;

and step 3: g-C₃N₄-MoS₂Preparing a nano composite material: placing the crucible with the cover containing 4.8-5.0g of white melamine powder into a muffle furnace, calcining at 500-550 ℃ for 3-5h, naturally cooling to room temperature, and adding yellow g-C₃N₄Grinding the product to a powder and then milling 1g g-C₃Adding N4 powder into 80-120ml of 5M HNO₃Refluxing the solution at the temperature of 120-125 ℃ for 20-30h, cooling, centrifuging, and washing with deionized water until the pH value is 6.8-7.2; finally drying in a vacuum oven at 30-35 ℃ for 101-5h to obtain carboxylated g-C₃N₄(ii) a MoS is prepared by dissolving sodium molybdate and thiourea in a molar ratio of 1:4-6 in 40-50ml of ultrapure water₂Taking carboxylated g-C₃N₄Adding MoS₂In the solution, carrying out hydrothermal reaction; finally, collecting the cooled sample, washing with ultrapure water, and drying under the protection of nitrogen for later use;

and 4, step 4: immunomagnetic beads and Ab₂The core-shell structure of (a): taking 1-1.2 mg of carboxyl magnetic beads in a centrifuge tube, washing once with 200 mul of deionized water with 180 pieces of organic solvent, and removing the supernatant; then washed with 150-Supernatant fluid; add MES to resuspend and add diluted Ab₂Oscillating for reaction; adding EDC solution, reacting at 800-1000 rpm/min for 1.5-2.5h, removing supernatant to obtain loaded Ab₂The immunomagnetic beads of (a); sequentially washing by PBST and PBS, adding 400-fold L TT buff, carrying out shake reaction for 20-40 min, removing supernatant, washing by PBS, and then blocking by BSA solution for 4-6 h; adding 0.8-1.2 ml of a mixture containing 0.01% Tween 20 and 0.02% NaN₃Uniformly mixing the PBS solution by shaking to obtain immunomagnetic bead mixed solution, and refrigerating for later use;

and 5: pre-assembling an ITO conductive glass electrode: ultrasonic cleaning with acetone, ethanol and deionized water for 10-20min, blowing with nitrogen, and wrapping ITO electrode with fluorinated sealing tape to make its effective area fixed at 0.14-0.18cm²Dropwise adding the manganoporphyrin obtained in the step 2 and the g-C obtained in the step 3 onto the electrode₃N₄-MoS₂With a CS acetic acid solution; then the electrode is put into a silica gel drying box to be dried at room temperature and dried to obtain g-C₃N₄-MoS₂ITO electrode dipped and washed with ultrapure water to remove manganese porphyrin and g-C which are not bonded on the surface₃N₄ -MoS₂And CS, followed by dropping 10-15ul of 1% GA solution on the electrode and keeping at room temperature for 0.5-1.5h, after which excess GA is washed off with deionized water; dripping 8-12ul of 10ul/ml of the CB solution of the collagen obtained in the step 1 onto the surface of an electrode to enable the terminal amino group of the CB solution to be combined with the aldehyde group of GA;

step 6: constructing an indirect type immunoprobe: thoroughly washing with PBS buffer to remove the antigen not bound to the electrode surface in step 5, blocking the electrode with 10-15ul of 1% BSA at 35-40 deg.C for 20-40 min, blocking with 1% BSA for 0.5-1.5h to block non-specific binding sites possibly existing on the electrode surface, taking out, washing with PBS buffer, and continuously dropwise adding 5-10ul of 1ul/ml rabbit anti-collagen antibody solution, i.e. Ab₁The solution is placed at 25-35 ℃ for 50-70min, and the unfixed Ab is washed with PBS buffer₁Finally, 8-12ul of the Ab load obtained in the step 4 is dripped₂The immune magnetic beads are placed at the temperature of 30-35 ℃ for 50-70min, the unfixed immune magnetic beads are washed by PBS buffer solution, and the electrodes are placed in detection base solution, thus completing the assembly of the photoelectrochemical immunosensor.

2. The method according to claim 1, wherein in step 1, 0.05 to 0.1ml of chloroform is added to the dialyzed product, the mouth of the container is opened, the container is covered with sterile gauze, the chloroform is volatilized on a sterile table for 40 to 50 hours to obtain a collagen solution, and the collagen solution is freeze-dried under vacuum for 2 to 3 days for use.

3. The preparation method as claimed in claim 1, wherein in step 2, the filter cake is washed twice with water and ethanol in sequence, 150-250 mesh reagent grade silica gel is used as adsorbent, and chloroform/methanol solution with volume ratio of (4-6): 2 is used as eluent.

4. The process according to claim 1, wherein 0.8 to 1.2g of carboxylated g-C is taken in step 3₃N₄3ml of MoS was added₂In the solution, and carrying out hydrothermal reaction for 20-30h at the temperature of 180-190 ℃.

5. The method of claim 1, wherein in step 4, 35-40 μ l 100 mM MES is added for resuspension, and Ab diluted 1000 times is added₂Oscillating for reaction for 20-40 min; adding 40-50 mul 10 mg/ml EDC solution, reacting at 800-1000 rpm/min for 1.5-2.5h, removing the supernatant to obtain the loaded Ab₂The immunomagnetic beads of (a); the Ab₂Is goat anti-rabbit anti-1 type collagen.

6. The method according to claim 1, wherein in step 5, the manganoporphyrin obtained in step 2 and the g-C obtained in step 3 are added dropwise to the electrode in a ratio of 1:1:2.5-3.5₃N₄-MoS₂Mixing with 0.05wt% CS acetic acid solution 10-15 ul; drying the electrode in a silica gel drying box at room temperature for 10-15 hr, and drying at 70-80 deg.C for 1.5-2.5 hr to obtain g-C₃N₄-MoS₂an/ITO electrode.

7. The method of claim 1, wherein the CB solution has a pH =9.6 and the PBS buffer has a pH = 7.4.

8. The method of claim 1, wherein in step 5, the assay base solution is PBS buffer containing 0.1M ascorbic acid.

Technical Field

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

Background

Abnormal changes in collagen have been implicated in a number of fibrotic diseases, and in the development and metastasis of tumors, the distribution and type of collagen has also changed so that each type is extracted separately at times during extraction. Collagen has great influence on the normal function and wound healing of cells, tissues and organs as a medical biomaterial, and has more superiority than the metal, ceramic or chemical materials which are used in the past, so that the collagen has been widely used clinically for nearly 20 years.

The ancient adhesive as an organic polymer material usually contains a large amount of collagen, the type I collagen is the most main component in connective tissue interstitium, and the general products mostly use the type I collagen as the material. Collagen detection is carried out on the adhesive, and cultural relic development can be traced. However, since the old buried environment is susceptible to degradation by light, heat, acid, alkali, microorganisms and the like, the structure and performance of the buried environment are changed, such as crystallinity, molecular weight and the like, and on the other hand, the cultural relics are often accompanied by a plurality of impurities when coming out of the earth, and the real effective components are few. Therefore, the conventional detection method has low sensitivity and is greatly influenced by impurity interference, and is not suitable for detecting cultural relics.

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 cause 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. The combination of the photoexcitation process with electrochemical detection allows the PEC sensor to greatly reduce the interference of background signals, and in addition, the device is simple, low cost and high sensitivity.

Porphyrin is a compound with a large pi structure and bionic properties of a plurality of important enzyme activity points, is also an active center of a plurality of proteins and enzymes in the nature, and is widely researched in various fields due to unique structure and superior properties. When the collagen cultural relics and metal utensils such as copper and iron are buried underground, metal ions can interact with collagen to form mineralized textiles, but the traditional detection technology cannot effectively solve the problem of the material quality of the mineralized textiles. The IMB can detect collagen from the mineralized sample. The functional group of the immune ligand can be hydroxyl (-OH), amino (-NH)₂) Carboxyl (-COOH), aldehyde (-CHO), so that the carrier microsphere has different physical properties of hydrophobicity-hydrophilicity, non-polarity, positive charge-negative charge and the like.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a photoelectrochemical immunosensor for detecting collagen₃N₄-MoS₂Preparing a nano composite material and immunomagnetic beads, and finally preparing the indirect IMB @ Ab by a layer-by-layer self-assembly process₂/ Ab₁/collagen 1/g-C₃N₄-MoS₂ITO photoelectrochemical immunosensor.

The specific technical scheme of the invention is as follows:

a preparation method of a photoelectrochemical immunosensor for detecting collagen comprises the following steps:

step 1: extracting collagen: cutting 15-20g fresh cow leather into 0.5-1cm pieces, cleaning with clear water, adding 8-12ml sodium carbonate solution with mass fraction of 5-10% as degreasing solution, adding 40-50ml warm water of 35-45 deg.C, stirring for 10-15min, filtering, and repeating the above operations; placing in 20-25ml 3% acetic acid for 40-50h, centrifuging for 1-3h at a centrifugation rate of 7000-8000r/min, sucking supernatant, adding 10-15ml 25-35% concentrated saline, salting out for 25-30min, dissolving precipitate in 15-20ml 0.1% acetic acid, placing in a dialysis bag, dialyzing in 20-25ml 1mol/l HCl solution for 2-3 days by using a cellulose dialysis bag with a cut-off molecular weight of 7000-13000, and changing water every 4-5h for 4-6 times; adding chloroform into the product obtained by dialysis, opening the container mouth, wrapping with sterile gauze, placing on a sterile workbench to volatilize chloroform to obtain collagen solution, and freeze-drying under vacuum for use.

The collagen mainly takes type I as a main component, the molecular weight is between 80kDa and 130kDa, the filtration and the repeated operation are carried out for two times, the purity of the collagen is favorably improved, the extraction frequency is not too small, otherwise, the purity is not enough, and the collagen denaturation is easy to occur when the extraction frequency is too large, so the extraction frequency is 3 to 4 times.

Step 2: preparing a manganese porphyrin nano material: introducing nitrogen into a reaction container for degassing, adding 120ml of 100-120 ml of N, N-diethylformamide after 8-10 min, heating to slightly boil and refluxing, then adding 80-90mg of tetrasulfonic acid phenyl porphyrin, stirring for 8-10 min until the mixture is clear and transparent, then adding 90-100 mg of manganese chloride, mechanically stirring at the temperature of 100 ℃ and under the nitrogen atmosphere, cooling a reaction product to room temperature after 6-10 h, pouring into 130 ml of 120-130 ml of deionized water, standing for 3-5h, filtering, and sequentially washing a filter cake with water and ethanol respectively; and collecting the first powder color band of the crude product by using reagent-grade silica gel as an adsorbent and a chloroform/methanol solution as an eluting agent, performing vacuum rotary evaporation to obtain a manganoporphyrin nanotube, and putting the manganoporphyrin nanotube into a dryer for storage and standby.

The porphyrin nanotube has large specific surface area, and can enhance electric signals and improve catalytic capability. Porphyrin nanotubes and g-C₃N₄-MoS₂Excellent properties of heterojunctions, bothThe compound of (2) is beneficial to reducing oxidation or reduction overpotential, improving the sensitivity of the material to light, increasing the mobility of electrons due to the huge specific surface area of the material, and being beneficial to converting optical signals into electric signals.

And step 3: g-C₃N₄-MoS₂Preparing a nano composite material: placing the crucible with the cover containing 4.8-5.0g of white melamine powder into a muffle furnace, calcining at 500-550 ℃ for 3-5h, naturally cooling to room temperature, and adding yellow g-C₃N₄Grinding the product to a powder and then milling 1g g-C₃Adding N4 powder into 80-120ml of 5M HNO₃Refluxing the solution at the temperature of 120-125 ℃ for 20-30h, cooling, centrifuging, and washing with deionized water until the pH value is 6.8-7.2; finally drying in a vacuum oven at 30-35 ℃ for 101-5h to obtain carboxylated g-C₃N₄(ii) a MoS is prepared by dissolving sodium molybdate and thiourea in a molar ratio of 1:4-6 in 40-50ml of ultrapure water₂Taking carboxylated g-C₃N₄Adding MoS₂In the solution, carrying out hydrothermal reaction; finally, the cooled sample was collected, washed with ultrapure water, and dried under nitrogen for future use.

g-C as a non-metallic semiconductor photoactive material₃N₄The photoelectric properties of (2) have been receiving wide attention, such as low energy gap (2.69eV), high chemical stability and thermal stability, good biocompatibility, environmental friendliness, and abundant raw materials. Although g-C₃N₄Has the advantages, but has very limited application in photoelectrochemistry because of high recombination rate of photogenerated electron-hole pairs, and must be applied to g-C₃N₄And (5) carrying out modification treatment. 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. Despite MoS₂Nanomaterials have relatively low forbidden band widths, but also behave as g-C in themselves₃N₄As well as having a number of disadvantages. G to C₃N₄And MoS₂Combined nano-materials, g-C₃N₄-MoS₂Form a 2D-2D heterojunction, and the 2D/2D heterojunction is compared with the OD/2D heterojunctionAnd 1D/2D heterojunction, the diffusion distance of electrons is relatively short, and through the matching of energy bands, the photoelectric conversion efficiency is high, the photocurrent is relatively stable, the recombination of a photo-generated electron hole pair can be effectively inhibited, and the light capture capability of the photo-generated electron hole pair is enhanced.

And 4, step 4: immunomagnetic beads and Ab₂The core-shell structure of (a): taking 1-1.2 mg of carboxyl magnetic beads in a centrifuge tube, washing once with 200 mul of deionized water with 180 pieces of organic solvent, and removing the supernatant; then washing the mixture for 1-2 times by using 150-plus 250 mul 100 mM MES, and removing the supernatant; add MES to resuspend and add diluted Ab₂Oscillating for reaction; adding EDC solution, reacting at 800-1000 rpm/min for 1.5-2.5h, removing supernatant to obtain loaded Ab₂The immunomagnetic beads of (a); sequentially washing by PBST and PBS, adding 400-fold L TT buff, carrying out shake reaction for 20-40 min, removing supernatant, washing by PBS, and then blocking by BSA solution for 4-6 h; adding 0.8-1.2 ml of a mixture containing 0.01% Tween 20 and 0.02% NaN₃And vibrating and uniformly mixing the PBS solution to obtain immunomagnetic bead mixed solution, and refrigerating for later use.

MPS 100/carboxyl bead with polystyrene microsphere as core has rich carboxyl on the surface of high molecular polymer, and can be covalently coupled with active amino. An antibody is a protein with free reactive amino groups that can bind to carboxyl groups to form stable acyl ammonium bonds. The immunomagnetic beads can also increase steric hindrance to reduce the response of the photocurrent signal.

And 5: pre-assembling an ITO conductive glass electrode: ultrasonic cleaning with acetone, ethanol and deionized water for 10-20min, blowing with nitrogen, and wrapping ITO electrode with fluorinated sealing tape to make its effective area fixed at 0.14-0.18cm²Dropwise adding the manganoporphyrin obtained in the step 2 and the g-C obtained in the step 3 onto the electrode₃N₄-MoS₂With a CS acetic acid solution; then the electrode is put into a silica gel drying box to be dried at room temperature and dried to obtain g-C₃N₄-MoS₂ITO electrode dipped and washed with ultrapure water to remove manganese porphyrin and g-C which are not bonded on the surface₃N₄ -MoS₂And CS, followed by dropping 10-15ul of 1% GA solution on the electrode and keeping at room temperature for 0.5-1.5h, after which excess GA is washed off with deionized water; mixing 8-12ul of collagen obtained in step 1 at a concentration of 10ul/mlAnd (3) dropwise adding the CB solution to the surface of the electrode to enable the terminal amino group to be combined with the aldehyde group of GA.

Step 6: constructing an indirect type immunoprobe: thoroughly washing with PBS buffer to remove the antigen not bound to the electrode surface in step 5, blocking the electrode with 10-15ul of 1% BSA at 35-40 deg.C for 20-40 min, blocking with 1% BSA for 0.5-1.5h to block non-specific binding sites possibly existing on the electrode surface, taking out, washing with PBS buffer, and continuously dropwise adding 5-10ul of 1ul/ml rabbit anti-collagen antibody solution, i.e. Ab₁The solution is placed at 25-35 ℃ for 50-70min, and the unfixed Ab is washed with PBS buffer₁Finally, 8-12ul of the Ab load obtained in the step 4 is dripped₂The immune magnetic beads are placed at the temperature of 30-35 ℃ for 50-70min, the unfixed immune magnetic beads are washed by PBS buffer solution, and the electrodes are placed in detection base solution, thus completing the assembly of the photoelectrochemical immunosensor.

Preferably, in step 1, 0.05-0.1ml of chloroform is added to the product obtained by dialysis, the container mouth is opened, the container is wrapped by sterile gauze, the sterile gauze is placed on a sterile workbench for 40-50h to volatilize the chloroform, and the collagen solution is obtained and is frozen and dried for 2-3 days in vacuum for standby.

Preferably, in the step 2, the filter cake is washed twice by water and ethanol in sequence, 150-250-mesh reagent grade silica gel is used as an adsorbent, and a chloroform/methanol solution with a volume ratio of (4-6): 2 is used as an eluent.

Preferably, in step 3, 0.8 to 1.2g of carboxylated g-C are taken₃N₄3ml of MoS was added₂In the solution, and carrying out hydrothermal reaction for 20-30h at the temperature of 180-190 ℃.

Preferably, in step 4, 35-40 μ l 100 mM MES resuspension is added, and Ab diluted 1000 times is added₂Oscillating for reaction for 20-40 min; adding 40-50 mul 10 mg/ml EDC solution, reacting at 800-1000 rpm/min for 1.5-2.5h, removing the supernatant to obtain the loaded Ab₂The immunomagnetic beads of (a); the Ab₂Is goat anti-rabbit anti-1 type collagen.

Preferably, in step 5, the manganoporphyrin obtained in step 2 and the g-C obtained in step 3 are dropped onto the electrode in a ratio of 1:1:2.5-3.5₃N₄-MoS₂With 0.05% by weight of CS acetic acid solution10-15 ul; drying the electrode in a silica gel drying box at room temperature for 10-15 hr, and drying at 70-80 deg.C for 1.5-2.5 hr to obtain g-C₃N₄-MoS₂an/ITO electrode.

Preferably, the CB solution has a pH =9.6 and the PBS buffer has a pH = 7.4.

Preferably, in step 5, the detection base solution of the present invention is a phosphate buffered saline (PBS, 0.01mol/L, pH =7.4) containing 0.1M Ascorbic Acid (AA) as an electron donor for the photoanode current.

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

(1) the collagen mainly takes type I as a main component, the molecular weight is between 80kDa and 130kDa, the filtration and the repeated operation are carried out for two times, the purity of the collagen is favorably improved, the extraction frequency is not too small, otherwise, the purity is not enough, and the collagen denaturation is easy to occur when the extraction frequency is too large, so the extraction frequency is 3 to 4 times.

(2) The porphyrin nanotube has large specific surface area, and can enhance electric signals and improve catalytic capability. Porphyrin nanotubes and g-C₃N₄-MoS₂The heterojunction has excellent properties, the combination of the two is favorable for reducing oxidation or reduction overpotential and improving the sensitivity of the material to light, the huge specific surface area of the material can increase the mobility of electrons and is favorable for converting optical signals into electric signals.

(3) g-C as a non-metallic semiconductor photoactive material₃N₄The photoelectric properties of (2) have been receiving wide attention, such as low energy gap (2.69eV), high chemical stability and thermal stability, good biocompatibility, environmental friendliness, and abundant raw materials. Although g-C₃N₄Has the advantages, but has very limited application in photoelectrochemistry because of high recombination rate of photogenerated electron-hole pairs, and must be applied to g-C₃N₄And (5) carrying out modification treatment. 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. Despite MoS₂Nanomaterials have relatively low forbidden band widths, but they also act as suchSame g-C₃N₄As well as having a number of disadvantages. G to C₃N₄And MoS₂Combined nano-materials, g-C₃N₄-MoS₂Compared with OD/2D and 1D/2D heterojunctions, the 2D/2D heterojunctions have the advantages that the diffusion distance of electrons is relatively short, the photoelectric conversion efficiency is high through energy band matching, the photocurrent is relatively stable, the recombination of photo-generated electron hole pairs can be effectively inhibited, and the light capture capability of the photo-generated electron hole pairs is enhanced.

(4) MPS 100/carboxyl bead with polystyrene microsphere as core has rich carboxyl on the surface of high molecular polymer, and can be covalently coupled with active amino. An antibody is a protein with free reactive amino groups that can bind to carboxyl groups to form stable acyl ammonium bonds. The immunomagnetic beads can also increase steric hindrance to reduce the response of the photocurrent signal.

(5) The detection base solution of the invention is phosphate buffer solution (PBS, 0.01mol/L, pH =7.4) containing 0.1M Ascorbic Acid (AA) as an electron donor for the photoanode current.

Drawings

FIG. 1 shows the MoS obtained in example 3₂High resolution SEM images of different magnifications of (1);

FIG. 2 is a graph of the self-assembled impedance of example 3: a is a bare ITO electrode; b is manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; c is antigen/manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; d is Ab₁antigen/manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; e is IMAs @ Ab₂/Ab₁antigen/manganoporphyrin/g-C₃N₄-MoS₂an/ITO electrode.

Detailed Description

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

Example 1

Step 1: extracting collagen: cutting 15g of fresh cow leather into small pieces of 0.5cm, cleaning with clear water, adding 10ml of a sodium carbonate solution with the mass fraction of 5% as a degreasing solution, continuously adding 40ml of warm water with the temperature of 35 ℃, magnetically stirring for 10min, filtering, repeating the operations again, putting the mixture into 20ml of 3% acetic acid for 48h, centrifuging for 2h at the speed of 7000r/min, sucking the supernatant, adding 10ml of 30% concentrated saline, salting out for 25min, dissolving the precipitate into 15ml of 0.1% acetic acid, placing the precipitate into a dialysis bag, dialyzing for 2 days in 20ml of 1mol/l HCl by using a cellulose dialysis bag with the intercepted molecular weight of 7000, changing water every 4h for 5 times, adding 0.05ml of chloroform, opening the bottle mouth, wrapping the solution with sterile gauze, placing the sterile workbench for 48h to volatilize the chloroform, obtaining a collagen solution, and carrying out vacuum freeze drying for 2 days for later use;

step 2: preparing a manganese porphyrin nano material: introducing nitrogen into a reaction bottle for degassing, adding 100ml of N, N-diethylformamide after 8min, heating to slightly boil and reflux, then adding 80 mg of phenyl porphyrin tetrasulfonate, stirring for 8min until the mixture is clear and transparent, then adding 90mg of manganese chloride, mechanically stirring at 100 ℃ under the nitrogen atmosphere, cooling the reaction product to room temperature after 8h, pouring the reaction product into 120ml of deionized water, standing for 4h, filtering, and sequentially washing a filter cake twice by using water and ethanol. The crude product obtained is adsorbed by 200 mesh reagent grade silica gel, chloroform/methanol (V)₁:V₂=5: 2) collecting the first powder color band as eluent, performing vacuum rotary evaporation to obtain manganese porphyrin nanotube, placing the manganese porphyrin nanotube in a dryer, and storing for later use;

and step 3: g-C₃N₄-MoS₂Preparing a nano composite material: placing the crucible with the cover containing 4.8g of white melamine powder into a muffle furnace, calcining at 500 ℃ for 4h, naturally cooling to room temperature, and adding yellow g-C₃N₄The product was ground to a powder and then 1g g-C3N4 powder was placed in 100ml HNO3(5M), cooled at 120 ℃ under reflux for 24h, centrifuged and washed with deionized water to pH 7.0. Finally, the product is dried in a vacuum oven at 30 ℃ for 12 hours to prepare carboxylated g-C₃N₄MoS was prepared by dissolving sodium molybdate and thiourea (1:5 molar ratio) in 40ml of ultrapure water₂1.0g of carboxylated g-C₃N₄3ml of MoS2 solution were added and the hydrothermal reaction was carried out at 180 ℃ for 24 h. Finally, the cooled sample was collectedWashed with ultrapure water and in N₂Drying under protection for later use;

and 4, step 4: preparing immunomagnetic beads: taking 1mg of carboxyl Magnetic Beads (MB) in a centrifuge tube, washing the centrifuge tube once by using 180 microliter of deionized water, and removing a supernatant; then washing with 200 mul 100 mM MES for 1-2 times, and removing the supernatant; add 35 μ l 100 mM MES for resuspension, add Ab diluted 1000 times₂(goat anti-rabbit anti-1 type collagen), and shaking for 30 min; adding 40 mul of 10 mg/ml EDC solution (used as prepared) for reaction at 900rpm/min for 2h, and removing the supernatant; obtain the supported Ab₂The immunomagnetic beads are washed by 1ml of PBST for 2-4 times, washed by 1ml of PBS for 3 times, added with 500 muL of TT buff to shake for reaction for 30min, removed of supernatant, washed by 100 muL of PBS for 3 times, and then sealed by 1ml of BSA solution for about 5 h. 1ml of PBS (containing 0.01% Tween 20 and 0.02% NaN) was added₃) The solution is shaken and mixed evenly to obtain 1 mg/ml immunomagnetic bead mixed solution, and the mixed solution is stored at 4 ℃ for standby.

And 5: pre-assembling an ITO conductive glass electrode: ultrasonic cleaning with acetone, ethanol and deionized water for 10min, blowing with nitrogen, and wrapping ITO electrode with fluorinated sealing tape to fix effective area at 0.16cm²Dropwise adding manganese porphyrin obtained in the step 2 and steps 3 g-C onto the electrode₃N₄-MoS₂To CS (0.05wt% in acetic acid solution) ratio 1:1: 3, then placing the electrode into a silica gel drying box, drying for 12 hours at room temperature, and drying for 2 hours at 70-80 ℃ to obtain g-C₃N₄-MoS₂ITO electrode, dipped and washed 3 times with ultrapure water to remove manganese porphyrin and g-C which are not bonded on the surface₃N₄ -MoS₂And CS, followed by dropping 10ul of 1% GA solution on the electrode and holding at room temperature for 1h, after which excess GA was washed off with deionized water; dripping 10ul of collagen CB obtained in the step 1 into the surface of the electrode in a volume of 10ul/ml to enable terminal amino groups of the collagen CB to be bonded with aldehyde groups of GA;

step 6: constructing an indirect type immunoprobe: after thoroughly washing with PBS (pH =7.4) to remove the antigen not bound to the electrode surface in step 5, the electrode was blocked with 10ul of 1% BSA at 37 ℃ for 30min, and then blocked with 1% BSA for 1h to block non-specific binding sites that may be present on the electrode surface, and after removal, the electrode was washed with 0.01mol · L^-1PBS buffer (pH 7.4) and then 5ul of collagen antibody (Ab) at 1ul/ml was added dropwise thereto₁) 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-collagen antibody solution), and finally 10ul of Ab loaded in step 4 is dripped₂The immunomagnetic beads are placed in an oven at 30 ℃ for 50min, and 0.01 mol.L is added^-1The non-immobilized immunomagnetic beads were washed with Phosphate Buffered Saline (PBS) (pH 7.4), and the electrodes were placed in a detection base solution containing 0.1M Ascorbic Acid (AA) (PBS, 0.01mol/L, pH =7.4), thereby completing the assembly of the photoelectrochemical immunosensor.

FIG. 1 shows the MoS obtained in example 3₂High resolution SEM images of different magnifications of (1); FIG. 2 is a graph of the self-assembled impedance of example 3. Wherein: a is a bare ITO electrode; b is manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; c is antigen/manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; d is Ab₁antigen/manganoporphyrin/g-C₃N₄-MoS₂An ITO electrode; e is IMAs @ Ab₂/Ab₁antigen/manganoporphyrin/g-C₃N₄-MoS₂an/ITO electrode.

Example 2

Step 1: extracting collagen: cutting 18g of fresh cow leather into small pieces of 0.7cm, cleaning with clear water, adding 10ml of a sodium carbonate solution with the mass fraction of 5-10% as a degreasing solution, continuously adding 45ml of warm water at 40 ℃, magnetically stirring for 12min, filtering, repeating the operations again, putting into 23ml of 3% acetic acid for 48h, centrifuging for 2h at the speed of 7500r/min, sucking the supernatant, adding 13ml of 30% concentrated saline, salting out for 28min, dissolving the precipitate into 17ml of 0.1% acetic acid, placing into a dialysis bag, dialyzing for 2 days in 23ml of 1mol/l HCl by using a cellulose dialysis bag with the molecular weight cutoff of 10000, changing water for 5 times every 4h, adding 0.07ml of chloroform, opening the bottle mouth, wrapping with sterile gauze, placing a sterile workbench for 48h to volatilize the chloroform to obtain a collagen solution, and carrying out vacuum freeze drying for 3 days for later use;

step 2: preparing a manganese porphyrin nano material: introducing nitrogen into the reaction bottleDegassing with gas, adding 110ml of N, N-diethylformamide after 8-10 min, heating to slightly boil and refluxing, then adding 85mg of phenyl porphyrin tetrasulfonate, stirring for 9min until the mixture is clear and transparent, then adding 95 mg of manganese chloride, mechanically stirring at 105 ℃ under nitrogen atmosphere, cooling the reaction product to room temperature after 8h, pouring into 125 ml of deionized water, standing for 4h, filtering, and washing the filter cake twice with water and ethanol in sequence. The crude product obtained is adsorbed by 200 mesh reagent grade silica gel, chloroform/methanol (V)₁:V₂=5: 2) collecting the first powder color band as eluent, performing vacuum rotary evaporation to obtain manganese porphyrin nanotube, placing the manganese porphyrin nanotube in a dryer, and storing for later use;

and step 3: g-C₃N₄-MoS₂Preparing a nano composite material: placing the crucible with the cover containing 4.9g of white melamine powder into a muffle furnace, calcining at 525 ℃ for 4h, naturally cooling to room temperature, and adding yellow g-C₃N₄The product was ground to a powder and then 1g g-C3N4 powder was placed in 100ml HNO3(5M), cooled at 123 ℃ under reflux for 24h, centrifuged and washed with deionized water to pH 7.0. Finally, the product is dried in a vacuum oven at 33 ℃ for 12 hours to prepare carboxylated g-C₃N₄MoS was prepared by dissolving sodium molybdate and thiourea (1:5 molar ratio) in 40-50ml of ultrapure water₂1.0g of carboxylated g-C₃N₄3ml of MoS2 solution were added and the hydrothermal reaction was carried out at 185 ℃ for 24 h. Finally, the cooled sample was collected, washed with ultrapure water, and washed in N₂Drying under protection for later use;

and 4, step 4: preparing immunomagnetic beads: 1.1 mg of carboxyl Magnetic Beads (MB) are taken out of a centrifuge tube, and washed once by 190 mul of deionized water, so that the supernatant is removed; then washing with 200 mul 100 mM MES for 1-2 times, and removing the supernatant; add 38 μ l 100 mM MES for resuspension, add Ab diluted 1000 times₂(goat anti-rabbit anti-1 type collagen), and shaking for 30 min; adding 45 mul of 10 mg/ml EDC solution (used as prepared) for reaction at 900rpm/min for 2h, and removing the supernatant; obtain the supported Ab₂The immunomagnetic beads are washed 2-4 times by 1ml PBST, washed 3 times by 1ml PBS, added with 500 mu L TT buff to shake for reaction for 30min, removed of supernatant, washed 3 times by 100 mu L PBS, and then dissolved by 1ml BSASealing the solution for about 5 h. 1ml of PBS (containing 0.01% Tween 20 and 0.02% NaN) was added₃) The solution is shaken and mixed evenly to obtain 1 mg/ml immunomagnetic bead mixed solution, and the mixed solution is stored at 4 ℃ for standby.

And 5: pre-assembling an ITO conductive glass electrode: ultrasonic cleaning with acetone, ethanol and deionized water for 15min, blowing with nitrogen, and wrapping ITO electrode with fluorinated sealing tape to fix effective area at 0.16cm²Dropwise adding manganese porphyrin obtained in the step 2 and steps 3 g-C onto the electrode₃N₄-MoS₂To CS (0.05wt% in acetic acid solution) ratio 1:1: 3, then placing the electrode into a silica gel drying box, drying for 12 hours at room temperature, and drying for 2 hours at 75 ℃ to obtain g-C₃N₄-MoS₂ITO electrode, dipped and washed 3 times with ultrapure water to remove manganese porphyrin and g-C which are not bonded on the surface₃N₄ -MoS₂And CS, followed by dropping 12ul of 1% GA solution on the electrode and holding at room temperature for 1h, after which excess GA was washed off with deionized water; dripping 10ul of collagen CB obtained in the step 1 into the surface of the electrode in a volume of 10ul/ml to enable terminal amino groups of the collagen CB to be bonded with aldehyde groups of GA;

step 6: constructing an indirect type immunoprobe: after thoroughly washing with PBS (pH =7.4) to remove the antigen not bound to the electrode surface in step 5, the electrode was blocked with 12ul of 1% BSA at 37 ℃ for 30min, and then blocked with 1% BSA for 1h to block non-specific binding sites that may be present on the electrode surface, and after removal, the electrode was washed with 0.01mol · L^-1Washing with PBS buffer (pH 7.4), and further dropwise adding collagen antibody (Ab) 5-10ul 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-collagen antibody solution), and finally 10ul of Ab loaded in step 4 is dripped₂The immunomagnetic beads are placed in an oven with the temperature of 30-35 ℃ for 60min, and 0.01 mol.L is used^-1The non-immobilized immunomagnetic beads were washed with Phosphate Buffered Saline (PBS) (pH 7.4), and the electrodes were placed in a detection base solution containing 0.1M Ascorbic Acid (AA) (PBS, 0.01mol/L, pH =7.4), thereby completing the assembly of the photoelectrochemical immunosensor.

Example 3

Step 1: extracting collagen: cutting 20g of fresh cow leather into small blocks of 1cm, cleaning with clear water, adding 10ml of sodium carbonate solution with the mass fraction of 10% as degreasing fluid, continuously adding 50ml of warm water at 45 ℃, magnetically stirring for 15min, filtering, repeating the operation once more, putting the mixture into 25ml of 3% acetic acid for 48h, centrifuging for 2h at the speed of 8000r/min, sucking supernatant, adding 30% of concentrated saline 15ml, salting out for 30min, dissolving precipitate into 20ml of 0.1% acetic acid, placing the precipitate into a dialysis bag, dialyzing for 3 days in 25ml of 1mol/l HCl by using a cellulose dialysis bag with the intercepted molecular weight of 13000, changing water every 5h, adding 0.1ml of chloroform, opening the bottle mouth, wrapping with sterile gauze, placing a sterile workbench for 48h to volatilize the chloroform, obtaining collagen solution, and carrying out vacuum freeze drying for 3 days for later use;

step 2: preparing a manganese porphyrin nano material: introducing nitrogen into a reaction bottle for degassing, adding 120ml of N, N-diethylformamide after 10min, heating to slightly boil and refluxing, then adding 90mg of phenyl porphyrin tetrasulfonate, stirring for 10min until the mixture is clear and transparent, then adding 100 mg of manganese chloride, mechanically stirring at 110 ℃ under the nitrogen atmosphere, cooling the reaction product to room temperature after 8h, pouring the reaction product into 130 ml of deionized water, standing for 4h, filtering, and sequentially washing a filter cake twice by using water and ethanol. The crude product obtained is adsorbed by 200 mesh reagent grade silica gel, chloroform/methanol (V)₁:V₂=5: 2) collecting the first powder color band as eluent, performing vacuum rotary evaporation to obtain manganese porphyrin nanotube, placing the manganese porphyrin nanotube in a dryer, and storing for later use;

and step 3: g-C₃N₄-MoS₂Preparing a nano composite material: placing the crucible with the cover containing 5.0g of white melamine powder into a muffle furnace, calcining at 550 ℃ for 4h, naturally cooling to room temperature, and adding yellow g-C₃N₄The product was ground to a powder and then 1g g-C3N4 powder was placed in 100ml HNO3(5M), cooled at 125 ℃ under reflux for 24h, centrifuged and washed with deionized water to pH 7.0. Finally, the product is dried in a vacuum oven at 35 ℃ for 12 hours to prepare carboxylated g-C₃N₄MoS was prepared by dissolving sodium molybdate and thiourea (1:5 molar ratio) in 50ml of ultrapure water₂1.0g of carboxylated g-C₃N₄3ml of MoS2 solution were added and the hydrothermal reaction was carried out at 190 ℃ for 24 h. Finally, the cooled sample was collected, washed with ultrapure water, and washed in N₂Drying under protection for later use;

and 4, step 4: preparing immunomagnetic beads: taking 1.2 mg of carboxyl Magnetic Beads (MB) in a centrifuge tube, washing once with 200 mul of deionized water, and removing the supernatant; then washed with 200 μ l 100 mM MES for 2 times, and the supernatant was removed; 40 μ l 100 mM MES was added for resuspension, and Ab diluted 1000 times was added₂(goat anti-rabbit anti-1 type collagen), and shaking for 30 min; adding 50 mul of 10 mg/ml EDC solution (used as prepared) for reaction at 900rpm/min for 2h, and removing the supernatant; obtain the supported Ab₂The immunomagnetic beads are washed by 1ml of PBST for 2-4 times, washed by 1ml of PBS for 3 times, added with 500 muL of TT buff to shake for reaction for 30min, removed of supernatant, washed by 100 muL of PBS for 3 times, and then sealed by 1ml of BSA solution for about 5 h. 1ml of PBS (containing 0.01% Tween 20 and 0.02% NaN) was added₃) The solution is shaken and mixed evenly to obtain 1 mg/ml immunomagnetic bead mixed solution, and the mixed solution is stored at 4 ℃ for standby.

And 5: pre-assembling an ITO conductive glass electrode: ultrasonic cleaning with acetone, ethanol and deionized water for 20min, blowing with nitrogen, and wrapping ITO electrode with fluorinated sealing tape to fix effective area at 0.16cm²Dropwise adding manganese porphyrin obtained in the step 2 and steps 3 g-C onto the electrode₃N₄-MoS₂To CS (0.05wt% in acetic acid solution) ratio 1:1: 3, then placing the electrode into a silica gel drying box, drying for 12 hours at room temperature, and drying for 2 hours at 80 ℃ to obtain g-C₃N₄-MoS₂ITO electrode, dipped and washed 3 times with ultrapure water to remove manganese porphyrin and g-C which are not bonded on the surface₃N₄ -MoS₂And CS, followed by dropping 15ul of 1% GA solution onto the electrode and holding at room temperature for 1h, after which excess GA was washed off with deionized water; dripping 10ul of collagen CB obtained in the step 1 into the surface of the electrode in a volume of 10ul/ml to enable terminal amino groups of the collagen CB to be bonded with aldehyde groups of GA;

step 6: constructing an indirect type immunoprobe: after thoroughly washing with PBS (pH =7.4) to remove the antigen not bound to the electrode surface in step 5, 15ul of 1% BSA was added at 37 ℃Blocking the electrode for 30min, then blocking with 1% BSA for 1h to block non-specific binding sites that may be present on the electrode surface, and removing with 0.01 mol. L^-1After washing with PBS buffer (pH 7.4), 10ul of 1ul/ml collagen antibody (Ab) was added dropwise thereto₁) 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-collagen antibody solution), and finally 10ul of Ab loaded in step 4 is dripped₂The immunomagnetic beads are placed in an oven at the temperature of 30-35 ℃ for 70min, and 0.01 mol.L is used^-1The non-immobilized immunomagnetic beads were washed with Phosphate Buffered Saline (PBS) (pH 7.4), and the electrodes were placed in a detection base solution containing 0.1M Ascorbic Acid (AA) (PBS, 0.01mol/L, pH =7.4), thereby completing the assembly of the photoelectrochemical 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.