CN111717909B - Preparation method of sandwich type photoelectrochemical sensor for detecting procalcitonin by using fullerene-stannic oxide - Google Patents
Preparation method of sandwich type photoelectrochemical sensor for detecting procalcitonin by using fullerene-stannic oxide Download PDFInfo
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Abstract
The invention relates to a preparation method of a sandwich type photoelectrochemical sensor for detecting procalcitonin based on fullerene-stannic oxide. According to the invention, the composite material of tin tetraoxide sensitized by fullerene quantum dots and cadmium selenide together is used as the substrate photosensitive material, fullerene has excellent efficiency of transferring electrons, and can effectively enhance photocurrent response of the tin oxide material, and secondly, carboxyl functionalized cadmium selenide can effectively connect with biomolecules, and simultaneously, the composite material is used as a sensitizer to further promote basic photocurrent response, and the stability of the sensor is improved. The iron disulfide has good light absorption performance, is used as a marker for marking procalcitonin secondary antibody to construct a sandwich type sensor, can effectively improve the sensitivity of the sensor, and realizes the ultra-sensitive detection of procalcitonin. The detection limit is 3.5 fg/mL.
Description
Technical Field
The invention relates to a preparation method of a sandwich type photoelectrochemical sensor for detecting procalcitonin based on fullerene-stannic oxide. Specifically, the sandwich type photoelectrochemical sensor for detecting procalcitonin is prepared by adopting common sensitization of fullerene quantum dots and cadmium selenide to prepare a substrate photosensitive material and adopting iron disulfide as a marker to mark a secondary antibody, and belongs to the technical field of novel functional materials and biosensing detection.
Background
Procalcitonin (PCT) is a protein whose levels in plasma are elevated when severe bacterial, fungal and parasitic infections and sepsis and multi-organ failure, bacterial endotoxins play a crucial role in the induction process, and localized limited bacterial infections, mild infections and chronic inflammation do not lead to an elevation, nor does PCT rise in autoimmune, allergic and viral infections, and thus PCT is a specific indicator of severe bacterial and fungal inflammation and also a reliable indicator of sepsis and multi-organ failure associated with inflammatory activity, reflecting the activity of systemic inflammatory reactions. In severe bacterial infectious diseases such as sepsis and MODS, the degree of PCT elevation is a reflection of inflammatory activity. PCT is not only an acute indicator for differential diagnosis, but also a parameter for monitoring inflammatory activity. Its advantage over other inflammatory markers is that severe infections cause significant increases in PCT concentrations (> 10 ug/L), whereas less severe infections or less severe sepsis cause only moderate increases in PCT. Further, PCT can also be used as a post-cure monitoring indicator of severe inflammation. PCT returned to the normal reference range within a few days if the inflammatory stimulus was no longer present. Therefore, it is necessary to establish an analysis method for rapidly and accurately detecting procalcitonin. There are many existing methods for detecting procalcitonin antigen, such as the detection by time-resolved fluorescence immunochromatography (Huang De Zhi, Yihao, Liufei, etc.. the establishment and performance evaluation of procalcitonin time-resolved fluorescence immunochromatography detection method [ J ]. third Jun Med. academic newspaper, 2019, 41(06): 581-); enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay for procalcitonin, CN 201610401787.1); chemiluminescence immune methods (procalcitonin chemiluminescence detection reagent and detection method based on nano-antibody, CN201810235448. X) and the like, but enzyme-linked immunoassay is complicated in operation and expensive in price; the controllability of fluorescence analysis is poor, and the toxicity is high; long detection time of electrochemical luminescence analysis, and the like. The invention designs a novel sandwich-type photoelectrochemical sensor which has the advantages of high analysis speed, simple operation, good stability and low detection limit, and the detection limit of the sandwich-type photoelectrochemical sensor designed by the invention on procalcitonin antigen reaches 3.5 fg/mL.
The stannic oxide is a semiconductor material containing a multi-valence metal element, has excellent photosensitive activity and photocatalytic performance, is different from single-valence tin dioxide or stannous oxide, has excellent photostability, and is more favorable for generation and transfer of photo-generated electrons. However, after the pure tin tetraoxide material is excited by light, in the process of rapidly forming photo-generated electrons, partial electrons and holes are also compounded, so that the excellent performance of the material is not obvious. The carbon material with the fullerene structure has excellent electron transfer performance, can be used as an electron acceptor by itself, receives electrons and transfers the electrons rapidly. And due to the good conductivity of the material with the carbon nano structure, the fullerene quantum dot is used for sensitizing the stannic oxide material in the invention, so that the transfer process of photo-generated electrons can be more effectively realized, and higher photocurrent response can be obtained. The stannic oxide is in a flower-shaped structure, provides more active sites for the loading of fullerene quantum dots, but is not beneficial to the connection of subsequent biomolecules because the fullerene is difficult to perform radical functionalization, and the carboxyl functionalized cadmium selenide material is applied to connect the biomolecules. In addition, cadmium selenide is used as an excellent sensitizer, so that the photoelectric response of the substrate is further improved, and the stability of the sensor is improved. In the subsequent process of biomolecule modification, the photoelectric current is gradually reduced, the iron disulfide nanoparticles with the function of absorbing light energy are used as a marker to mark a second antibody, so that when the sensor is modified to the last layer, the photoelectric current is greatly reduced, the change value of the photoelectric current is improved, the content of a detected object is judged by utilizing the relation between the change value and the concentration of the detected object, the introduction of iron disulfide improves the sensitivity of the sensor, the ultra-sensitive detection of procalcitonin is realized, and the detection limit reaches 3.5 fg/mL.
The photoelectrochemical detection method has the characteristics of simple operation, high sensitivity, low background signal and the like, has been developed into an analysis method with great application potential, and has wide application prospects in the fields of food safety, environmental sanitation, medicine and the like. The application of the tin tetraoxide material in the aspect of the photoelectrochemical sensor is not reported. The sandwich type photoelectrochemical sensor for detecting procalcitonin under visible light is successfully constructed on the basis of the fullerene quantum dot sensitized patterned stannic oxide material. The sensor takes flower-shaped stannic oxide which is co-sensitized by fullerene quantum dots and cadmium selenide nano particles as a substrate photosensitive material, the efficient electron transfer function of fullerene and the excellent sensitization of carboxylated cadmium selenide improve the photoelectric response of the material and the stability of the sensor, and meanwhile, the functionalized cadmium selenide is easily connected with biomolecules, so that the establishment of a sensing biological platform is guaranteed. The iron disulfide is used as a marker and a second antibody is marked, so that the sensitivity of photocurrent signal change when the concentration of a target substance changes is realized, and the ultrasensitive detection of procalcitonin is realized. The photoelectrochemical sensor prepared by the invention has the advantages of low cost, high sensitivity, good specificity, quick detection, easy preparation and the like, realizes quick and ultrasensitive detection of procalcitonin in a visible light region, and effectively overcomes the defects of the existing procalcitonin detection method.
Disclosure of Invention
One of the objects of the present invention is to use a flower-like tri-tin tetroxide material as a photosensitive material, and a semiconductor material containing a polyvalent metal has excellent photoelectric activity and photostability.
The other purpose of the invention is to utilize the fullerene quantum dots to sensitize the tin tetraoxide material, the fullerene has the function of rapidly transferring electrons, the conductivity and the photoelectric response can be improved, and in addition, the flower-shaped tin oxide provides a large specific surface area to load a large amount of fullerene.
The third purpose of the invention is to further sensitize the substrate material by carboxyl functionalized cadmium selenide, on one hand, the photocurrent is further improved, and the stability of the sensor is improved, and on the other hand, the carboxyl is easy to be connected with the biomolecule, so that the subsequent modification of the sensor is guaranteed.
The fourth purpose of the invention is to use iron disulfide as a marker for marking procalcitonin secondary antibody, during the process of modifying antigen and antibody layer by layer, the sensor can block electron transfer and reduce photocurrent, and the iron disulfide can absorb light energy, so that the photocurrent is further reduced, the change of photocurrent signal is increased, and the detection sensitivity of a target object is improved.
The sandwich-type photoelectrochemical sensor with high sensitivity, good stability and high detection speed is prepared by using stannic oxide sensitized by fullerene quantum dots and cadmium selenide together as a substrate photosensitive material and using iron disulfide as a marker to mark a procalcitonin secondary antibody, so that the purpose of ultrasensitive detection of procalcitonin under the condition of visible light is realized.
The technical scheme of the invention is as follows:
1. the preparation method of the sandwich-type photoelectrochemical sensor for detecting procalcitonin based on fullerene-stannic oxide is characterized by comprising the following steps:
(1) preparation of flower-like stannic oxide material
Dissolving 1.0-1.5 g of stannous chloride dihydrate and 3.0-3.5 g of trisodium citrate dihydrate into 10-15 mL of ultrapure water, and stirring and mixing the solution at room temperature to obtain a solution A; dissolving 0.25-0.35 g of sodium hydroxide in 10-15 mL of ultrapure water, and uniformly stirring at room temperature to obtain a solution B; mixing the solution A and the solution B, stirring at room temperature for 18-24 h, transferring the mixed solution into a high-pressure reaction kettle, carrying out hydrothermal reaction at 150-200 ℃ for 10-16 h, naturally cooling after the reaction is finished, washing the product for 5 times by using ultrapure water, and drying at 30-60 ℃ for 10-14 h to obtain a flower-shaped stannic oxide material;
(2) preparation of fullerene quantum dots
Dissolving 1-5 mg of fullerene powder in 1-5 mL of anhydrous toluene solvent, uniformly stirring at room temperature to obtain a purple red solution, adding 1-5 mL of ultrapure water into the solution, carrying out ultrasonic treatment for 16-24 h until toluene is completely removed, and storing the obtained faint yellow fullerene quantum dot solution in a 4 ℃ refrigerator for later use;
(3) preparation of cadmium selenide nanoparticles
Dissolving 0.02-0.04 g of selenium powder and 0.02-0.04 g of sodium borohydride in 1-5 mL of ultrapure water, and vigorously stirring at room temperature until the solution becomes clear, wherein the solution is used as a solution A; dissolving 0.02-0.04 g of cadmium chloride dihydrate in 50-100 mL of ultrapure water, adding 100-300 muL of thioglycollic acid solution under stirring, and then adjusting the pH of the solution to 9 by using 0.5-1M of sodium hydroxide aqueous solution to obtain a solution B; pouring the solution A into the solution B, stirring for 2-6 h at room temperature under the mixed solution, washing the obtained product for 5 times by using ultrapure water, and drying for 10-14 h at 30-60 ℃ to obtain carboxyl functionalized cadmium selenide nanoparticles;
(4) preparation of iron disulfide nanoparticles
Dissolving 0.5-0.8 g of trisodium citrate dihydrate and 0.1-0.5 g of polyvinylpyrrolidone K-30 into 50-100 mL of ultrapure water under stirring, adding 1.0-1.5 g of ferrous sulfate heptahydrate into the solution, continuously stirring for 30 min, dropwise adding 10-20 mL of a 1.0-2.0M sodium hydroxide aqueous solution into the mixed solution, then adding 0.2-0.5 g of sulfur powder into the solution, continuously and vigorously stirring to obtain a uniform mixed solution, transferring the mixed solution into a high-pressure reaction kettle, reacting for 16-24 h at 150-200 ℃, washing the product with ultrapure water and anhydrous ethanol for 3 times respectively after the reaction is finished, and drying for 10-14 h at 30-60 ℃ to obtain iron disulfide nanoparticles;
(5) preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15 mol/L, and taking the aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask, and preparing an aqueous solution with the concentration of 1/15 mol/L as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 5.0-8.0;
(6) preparation of iron disulfide-labeled procalcitonin secondary antibody
Taking 1.0-5.0 mg of prepared iron disulfide nanoparticle solution into 1.0 mL of PBS buffer solution with pH of 7.4, then adding 10-30 μ L of cross-linking agent, wherein the cross-linking agent is a mixed solution composed of 5 mg/mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide, activating at 37 ℃ for 10-30 min, then adding 100-500 μ L of procalcitonin secondary antibody with the concentration of 1 μ g/mL into the solution, activating at 37 ℃ for 5-10 h, centrifugally washing with the PBS buffer solution, dispersing the obtained product in 1 mL of PBS buffer solution, and storing in a refrigerator at 4 ℃ for later use;
(7) preparation of photoelectrochemical sensor
1) Ultrasonically cleaning conductive glass by using liquid detergent, acetone, ethanol and ultrapure water in sequence, and drying the conductive glass in a nitrogen atmosphere;
2) dripping 10 mu L of 1-5 mg/mL of tin tetraoxide aqueous solution onto a conductive surface of the ITO conductive glass, and naturally drying at room temperature;
3) dripping 10 muL of prepared fullerene quantum dot solution on the surface of the modified electrode, and naturally airing at room temperature;
4) continuously dropwise adding 10 mu L of 1-5 mg/mL cadmium selenide aqueous solution on the surface of the modified electrode, and naturally airing at room temperature;
5) dropwise adding 4 mu L of mixed solution of 5-10 mg/mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-5 mg/mL of N-hydroxysuccinimide in a volume ratio of 1:1 on the surface of the modified electrode, and naturally airing at room temperature to a wet film state;
6) dropwise adding 6 muL of procalcitonin first antibody of 1-5 mug/mL, naturally airing at room temperature to a wet film state, and then washing the electrode with PBS buffer solution;
7) dropwise adding 1-3% bovine serum albumin solution prepared by PBS buffer solution with pH of 7.4 on the surface of the modified electrode, airing in a refrigerator at 4 ℃, and then washing the electrode by the PBS buffer solution;
8) dropwise adding procalcitonin antigen of 6 muL and 1-5 mug/mL, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution;
9) dropwise adding 6 mu L of the prepared procalcitonin secondary antibody with the iron disulfide mark, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution; and (3) preparing the sandwich type photoelectric chemical sensor for detecting procalcitonin.
2. The method for detecting a photoelectrochemical sensor manufactured by the manufacturing method according to claim 1, comprising the steps of:
(1) testing by using an electrochemical workstation and a three-electrode system, taking a saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and taking the prepared ITO modified sensor as a working electrode, wherein the testing is carried out in 10 mL of PBS (phosphate buffer solution) with the pH value of 5.0-8.0 and 0.01-0.5 mol/L of ascorbic acid buffer solution;
(2) detecting the procalcitonin antigen by a time-current method, setting the voltage to be-0.1V, the running time to be 120 s, and the wavelength of a light source to be 400-450 nm;
(3) after the electrodes are placed, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve;
(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen standard solution for detection.
The linear range of the sensor for detecting procalcitonin is 0.01 pg/mL-10 ng/mL, and the detection limit is 3.5 fg/mL.
The chemicals required for material synthesis were all purchased from local reagent stores and were not reprocessed.
Advantageous effects of the invention
(1) The invention successfully prepares the patterned material of the stannic oxide containing the polyvalent metal, the material contains the metal elements with various valence states, the visible light is used for exciting the material, the transmission is more facilitated after the formation of the photo-generated electrons, and the visible light stability and the sensitivity are realized.
(2) The patterned tin oxide has a large specific surface area, can load a large amount of fullerene, and the fullerene has an excellent electron transfer effect, can efficiently promote electron transfer and improve photocurrent.
(3) The carboxyl functionalized cadmium selenide is used for further sensitizing the substrate material to overcome the defect that fullerene is not easy to functionalize so as to realize the connection with biomolecules, and meanwhile, the cadmium selenide is used as an excellent sensitizing agent, so that the photocurrent is further improved, and the stability of the sensor is improved.
(4) The iron disulfide capable of absorbing visible light energy is used as a marker for marking the procalcitonin secondary antibody, and the change value of the current is increased when the sensor is modified layer by layer in the biological molecules through the marking of the iron disulfide, so that the change value of the photocurrent is increased when the concentration of a detected object is changed, and the detection sensitivity of the sensor is improved.
(5) The sandwich-type photoelectrochemical sensor prepared by the invention is used for detecting procalcitonin, has short response time, wide linear range, low detection limit, good stability and reproducibility, and can realize simple, quick, high-sensitivity and specific detection. The linear range of the procalcitonin detection is 0.01 pg/mL-10 ng/mL, and the detection limit is 3.5 fg/mL.
Detailed description of the preferred embodiments
EXAMPLE 1 preparation of photoelectrochemical sensor
(1) Preparation of flower-like stannic oxide material
Dissolving 1.0 g of stannous chloride dihydrate and 3.0 g of trisodium citrate dihydrate into 10 mL of ultrapure water, and stirring and uniformly mixing the solution at room temperature to obtain solution A; dissolving 0.25 g of sodium hydroxide in 10 mL of ultrapure water, and uniformly stirring at room temperature to obtain a solution B; mixing the solution A and the solution B, stirring at room temperature for 18 h, transferring the mixed solution into a high-pressure reaction kettle, carrying out hydrothermal reaction at 150 ℃ for 10 h, naturally cooling after the reaction is finished, washing the product for 5 times by using ultrapure water, and drying at 30 ℃ for 10 h to obtain a flower-shaped stannic oxide material;
(2) preparation of fullerene quantum dots
Dissolving 1 mg of fullerene powder in 1 mL of anhydrous toluene solvent, uniformly stirring at room temperature to obtain a mauve solution, adding 1 mL of ultrapure water into the solution, carrying out ultrasonic treatment for 16 h until toluene is completely removed, and storing the obtained light yellow fullerene quantum dot solution in a refrigerator at 4 ℃ for later use;
(3) preparation of cadmium selenide nanoparticles
Dissolving 0.02 g of selenium powder and 0.02 g of sodium borohydride in 1 mL of ultrapure water together, and violently stirring at room temperature until the solution becomes clear, wherein the solution is used as a solution A; dissolving 0.02 g of cadmium chloride dihydrate in 50 mL of ultrapure water, adding 100 muL of thioglycollic acid solution under stirring, and then adjusting the pH of the solution to 9 by using 0.5M of sodium hydroxide aqueous solution to obtain solution B; pouring the solution A into the solution B, stirring for 2 hours at room temperature under the mixed solution, washing the obtained product for 5 times by using ultrapure water, and drying for 10 hours at 30 ℃ to prepare carboxyl functionalized cadmium selenide nano-particles;
(4) preparation of iron disulfide nanoparticles
Dissolving 0.5 g of trisodium citrate dihydrate and 0.1 g of polyvinylpyrrolidone K-30 in 50 mL of ultrapure water under stirring, adding 1.0 g of ferrous sulfate heptahydrate into the solution, continuously stirring for 30 min, dropwise adding 10 mL of 1.0M sodium hydroxide aqueous solution into the mixed solution, then adding 0.2 g of sulfur powder into the solution, continuously and violently stirring to obtain a uniform mixed solution, transferring the mixed solution into a high-pressure reaction kettle, reacting for 16 h at 150 ℃, washing the product for 3 times by using ultrapure water and absolute ethyl alcohol after the reaction is finished, and drying for 10 h at 30 ℃ to obtain iron disulfide nanoparticles;
(5) preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and putting the disodium hydrogen phosphate dodecahydrate into a 500 mL volumetric flask to prepare 1/15 mol/L aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask, and preparing aqueous solution with the concentration of 1/15 mol/L to be used as solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 5.0-8.0;
(6) preparation of iron disulfide-labeled procalcitonin secondary antibody
Taking 1.0 mg of the prepared iron disulfide nanoparticle solution into 1.0 mL of PBS buffer solution with pH of 7.4, then adding 10 muL of a cross-linking agent, wherein the cross-linking agent is a mixed solution consisting of 5 mg/mL of 1-ethyl- (3-dimethylamino-C grade) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide, activating for 10 min at 37 ℃, then adding 100 muL of procalcitonin second antibody with the concentration of 1 mug/mL into the solution, activating for 5 h at 37 ℃, then centrifugally washing by using PBS buffer solution, dispersing the obtained product in 1 mL of PBS buffer solution, and storing for later use in a 4 ℃ refrigerator;
(7) preparation of photoelectrochemical sensor
1) Ultrasonically cleaning conductive glass by using a detergent, acetone, ethanol and ultrapure water in sequence, and drying the conductive glass in a nitrogen atmosphere;
2) dropwise adding 10 mu L of 1 mg/mL of tin tetraoxide aqueous solution to a conductive surface of the ITO conductive glass, and naturally drying at room temperature;
3) dripping 10 muL of prepared fullerene quantum dot solution on the surface of the modified electrode, and naturally airing at room temperature;
4) continuously dropwise adding 10 muL of 1 mg/mL cadmium selenide aqueous solution on the surface of the modified electrode, and naturally airing at room temperature;
5) dropwise adding 4 mu L of mixed solution of 5 mg/mL of 1-ethyl- (3-dimethylamino-propyl) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide in a volume ratio of 1:1 on the surface of the modified electrode, and naturally airing the mixture to a wet film state at room temperature;
6) dropwise adding 6 muL of procalcitonin first antibody of 1 mug/mL, naturally airing to a wet film state at room temperature, and then washing the electrode with PBS buffer solution;
7) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1% prepared by PBS buffer solution with the pH of 7.4 on the surface of the modified electrode, airing in a refrigerator at 4 ℃, and then washing the electrode by the PBS buffer solution;
8) dropwise adding procalcitonin antigen of 6 muL and 1 mug/mL, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution;
9) dropwise adding 6 mu L of the prepared procalcitonin secondary antibody with the iron disulfide mark, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution; the sandwich type photoelectrochemical sensor for detecting procalcitonin is prepared.
Example 2 preparation of a photoelectrochemical sensor
(1) Preparation of flower-like stannic oxide material
Dissolving 1.3 g of stannous chloride dihydrate and 3.2 g of trisodium citrate dihydrate in 14 mL of ultrapure water, and stirring and uniformly mixing the solution at room temperature to obtain solution A; dissolving 0.28 g of sodium hydroxide in 14 mL of ultrapure water, and uniformly stirring at room temperature to obtain a solution B; mixing the solution A and the solution B, stirring at room temperature for 20 h, transferring the mixed solution into a high-pressure reaction kettle, carrying out hydrothermal reaction at 160 ℃ for 12 h, naturally cooling after the reaction is finished, washing the product for 5 times by using ultrapure water, and drying at 40 ℃ for 12 h to obtain a flower-shaped stannic oxide material;
(2) preparation of fullerene quantum dots
Dissolving 3 mg of fullerene powder in 3 mL of anhydrous toluene solvent, uniformly stirring at room temperature to obtain a mauve solution, adding 3 mL of ultrapure water into the solution, carrying out ultrasonic treatment for 16 h until toluene is completely removed, and storing the obtained faint yellow fullerene quantum dot solution in a refrigerator at 4 ℃ for later use;
(3) preparation of cadmium selenide nanoparticles
Dissolving 0.03 g of selenium powder and 0.03 g of sodium borohydride in 3 mL of ultrapure water together, and violently stirring at room temperature until the solution becomes clear, wherein the solution is used as a solution A; dissolving 0.03 g of cadmium chloride dihydrate in 80 mL of ultrapure water, adding 200 muL of thioglycollic acid solution under stirring, and then adjusting the pH of the solution to 9 by using 0.5M of sodium hydroxide aqueous solution to obtain solution B; pouring the solution A into the solution B, stirring for 3 h at room temperature under the mixed solution, washing the obtained product for 5 times by using ultrapure water, and drying for 12 h at 40 ℃ to obtain carboxyl functionalized cadmium selenide nano-particles;
(4) preparation of iron disulfide nanoparticles
Dissolving 0.6 g of trisodium citrate dihydrate and 0.3 g of polyvinylpyrrolidone K-30 in 80 mL of ultrapure water under stirring, adding 1.3 g of ferrous sulfate heptahydrate into the solution, continuously stirring for 30 min, dropwise adding 15 mL of 1.5M sodium hydroxide aqueous solution into the mixed solution, then adding 0.4 g of sulfur powder into the solution, continuously and violently stirring to obtain a uniform mixed solution, transferring the mixed solution into a high-pressure reaction kettle, reacting for 18 h at 180 ℃, washing the product for 3 times by using ultrapure water and absolute ethyl alcohol after the reaction is finished, and drying for 14 h at 60 ℃ to obtain iron disulfide nanoparticles;
(5) preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15 mol/L, and taking the aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask, and preparing an aqueous solution with the concentration of 1/15 mol/L as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 5.0-8.0;
(6) preparation of iron disulfide-labeled procalcitonin secondary antibody
Taking 3.0 mg of the prepared iron disulfide nanoparticle solution into 1.0 mL of PBS buffer solution with pH of 7.4, then adding 20 μ L of a cross-linking agent, wherein the cross-linking agent is a mixed solution consisting of 5 mg/mL of 1-ethyl- (3-dimethylamino-C grade) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide, activating at 37 ℃ for 20 min, then adding 300 μ L of procalcitonin second antibody with the concentration of 1 μ g/mL into the solution, activating at 37 ℃ for 10 h, then centrifugally washing with the PBS buffer solution, dispersing the obtained product in 1 mL of PBS buffer solution, and storing in a refrigerator at 4 ℃ for later use;
(7) preparation of photoelectrochemical sensor
1) Ultrasonically cleaning conductive glass by using a detergent, acetone, ethanol and ultrapure water in sequence, and drying the conductive glass in a nitrogen atmosphere;
2) dripping 10 mu L of 4 mg/mL of tin tetraoxide aqueous solution onto a conductive surface of the ITO conductive glass, and naturally airing at room temperature;
3) dropwise adding 10 muL of prepared fullerene quantum dot solution on the surface of the modified electrode, and naturally airing at room temperature;
4) continuously dropwise adding 10 muL of 4 mg/mL cadmium selenide aqueous solution on the surface of the modified electrode, and naturally airing at room temperature;
5) dripping 4 mu L of mixed solution of 8 mg/mL 1-ethyl- (3-dimethylamino-propyl) carbodiimide hydrochloride and 3 mg/mL N-hydroxysuccinimide with the volume ratio of 1:1 on the surface of the modified electrode, and naturally airing the mixture to a wet film state at room temperature;
6) dropwise adding 6 muL of procalcitonin first antibody of 5 mug/mL, naturally airing to a wet film state at room temperature, and then washing the electrode with PBS buffer solution;
7) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 2% prepared by PBS buffer solution with the pH of 7.4 on the surface of the modified electrode, airing in a refrigerator at 4 ℃, and then washing the electrode by the PBS buffer solution;
8) dropwise adding procalcitonin antigen of 6 mu L and 4 mu g/mL, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution;
9) dropwise adding 6 mu L of the prepared procalcitonin secondary antibody with the iron disulfide mark, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution; the sandwich type photoelectrochemical sensor for detecting procalcitonin is prepared.
EXAMPLE 3 preparation of photoelectrochemical sensor
(1) Preparation of flower-like stannic oxide material
Dissolving 1.5 g of stannous chloride dihydrate and 3.5 g of trisodium citrate dihydrate in 15 mL of ultrapure water, and stirring and uniformly mixing the solution at room temperature to obtain solution A; dissolving 0.35 g of sodium hydroxide in 15 mL of ultrapure water, and uniformly stirring at room temperature to obtain a solution B; mixing the solution A and the solution B, stirring at room temperature for 24 h, transferring the mixed solution into a high-pressure reaction kettle, carrying out hydrothermal reaction at 200 ℃ for 16 h, naturally cooling after the reaction is finished, washing the product for 5 times by using ultrapure water, and drying at 60 ℃ for 14 h to obtain a flower-shaped stannic oxide material;
(2) preparation of fullerene quantum dots
Dissolving 5 mg of fullerene powder in 5 mL of anhydrous toluene solvent, uniformly stirring at room temperature to obtain a mauve solution, adding 5 mL of ultrapure water into the solution, carrying out ultrasonic treatment for 24 hours until toluene is completely removed, and storing the obtained light yellow fullerene quantum dot solution in a refrigerator at 4 ℃ for later use;
(3) preparation of cadmium selenide nanoparticles
Dissolving 0.04 g of selenium powder and 0.04 g of sodium borohydride in 5 mL of ultrapure water together, and vigorously stirring at room temperature until the solution becomes clear, wherein the solution is used as a solution A; dissolving 0.04 g of cadmium chloride dihydrate in 100 mL of ultrapure water, adding 300 muL of thioglycollic acid solution under stirring, and then adjusting the pH of the solution to 9 by using 1M of sodium hydroxide aqueous solution to obtain solution B; pouring the solution A into the solution B, stirring for 6 hours at room temperature under the mixed solution, washing the obtained product for 5 times by using ultrapure water, and drying for 14 hours at the temperature of 60 ℃ to prepare carboxyl functionalized cadmium selenide nano-particles;
(4) preparation of iron disulfide nanoparticles
Dissolving 0.8 g of trisodium citrate dihydrate and 0.5 g of polyvinylpyrrolidone K-30 in 100 mL of ultrapure water under stirring, adding 1.5 g of ferrous sulfate heptahydrate into the solution, continuously stirring for 30 min, dropwise adding 20 mL of 2.0M sodium hydroxide aqueous solution into the mixed solution, then adding 0.5 g of sulfur powder into the solution, continuously and violently stirring to obtain a uniform mixed solution, transferring the mixed solution into a high-pressure reaction kettle, reacting for 24 h at 200 ℃, washing the product for 3 times by using ultrapure water and absolute ethyl alcohol after the reaction is finished, and drying for 14 h at 60 ℃ to obtain iron disulfide nanoparticles;
(5) preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15 mol/L, and taking the aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask, and preparing aqueous solution with the concentration of 1/15 mol/L to be used as solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 5.0-8.0;
(6) preparation of iron disulfide-labeled procalcitonin secondary antibody
Taking 5.0 mg of the prepared iron disulfide nanoparticle solution into 1.0 mL of PBS buffer solution with pH of 7.4, then adding 30 μ L of a cross-linking agent, wherein the cross-linking agent is a mixed solution consisting of 5 mg/mL of 1-ethyl- (3-dimethylamino-C) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide, activating at 37 ℃ for 30 min, then adding 500 μ L of procalcitonin second antibody with the concentration of 1 μ g/mL into the solution, activating at 37 ℃ for 10 h, then centrifugally washing with the PBS buffer solution, dispersing the obtained product in 1 mL of PBS buffer solution, and storing in a refrigerator at 4 ℃ for later use;
(7) preparation of photoelectrochemical sensor
1) Ultrasonically cleaning conductive glass by using a detergent, acetone, ethanol and ultrapure water in sequence, and drying the conductive glass in a nitrogen atmosphere;
2) dripping 10 mu L of 5 mg/mL of tin tetraoxide aqueous solution onto a conductive surface of the ITO conductive glass, and naturally airing at room temperature;
3) dropwise adding 10 muL of prepared fullerene quantum dot solution on the surface of the modified electrode, and naturally airing at room temperature;
4) continuously dropwise adding 10 muL and 5 mg/mL of cadmium selenide aqueous solution on the surface of the modified electrode, and naturally airing at room temperature;
5) dripping 10 mg/mL of mixed solution of 1-ethyl- (3-dimethylamino-propyl) carbodiimide hydrochloride and 5 mg/mL of N-hydroxysuccinimide with the volume ratio of 1:1, namely 4 mu L, on the surface of the modified electrode, and naturally airing the mixture to a wet film state at room temperature;
6) dropwise adding 6 muL of procalcitonin first antibody of 5 mug/mL, naturally airing to a wet film state at room temperature, and then washing the electrode with PBS buffer solution;
7) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 3% prepared by PBS buffer solution with the pH of 7.4 on the surface of the modified electrode, airing in a refrigerator at 4 ℃, and then washing the electrode by the PBS buffer solution;
8) dripping 6 muL of procalcitonin antigen and 5 mug/mL of procalcitonin antigen, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution;
9) dropwise adding 6 mu L of the prepared procalcitonin secondary antibody with the iron disulfide mark, naturally airing in a refrigerator at 4 ℃, and then washing the electrode with PBS buffer solution; the sandwich type photoelectrochemical sensor for detecting procalcitonin is prepared.
Example 4 detection of Procalcitonin
(1) An electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared ITO modified sensor is used as a working electrode, and the testing is carried out in 10 mL of PBS with the pH value of 5.0 and 0.01 mol/L of ascorbic acid buffer solution;
(2) detecting procalcitonin antigen by a time-current method, setting the voltage to be-0.1V, the running time to be 120 s and the light source wavelength to be 400 nm;
(3) after the electrodes are placed, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve;
(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen sample solution for detection.
Example 5 detection of Procalcitonin
(1) An electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared ITO modified sensor is used as a working electrode, and the testing is carried out in 10 mL of PBS with the pH value of 7.0 and 0.1 mol/L of ascorbic acid buffer solution;
(2) detecting procalcitonin antigen by a time-current method, setting the voltage to be 0V, the running time to be 120 s and the light source wavelength to be 430 nm;
(3) after the electrodes are placed, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve;
(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen standard solution for detection.
Example 6 detection of Procalcitonin
(1) An electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared ITO modified sensor is used as a working electrode, and the testing is carried out in 10 mL of PBS (phosphate buffer solution) with the pH value of 8.0 and 0.5 mol/L of ascorbic acid buffer solution;
(2) detecting procalcitonin antigen by a time-current method, setting the voltage to be 0.1V, the running time to be 120 s and the light source wavelength to be 450 nm;
(3) after the electrodes are placed, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve;
(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen standard solution for detection.
Claims (2)
1. The preparation method of the sandwich-type photoelectrochemical sensor for detecting procalcitonin based on fullerene-stannic oxide is characterized by comprising the following steps:
(1) preparation of flower-like stannic oxide material
Dissolving 1.0-1.5 g of stannous chloride dihydrate and 3.0-3.5 g of trisodium citrate dihydrate into 10-15 mL of ultrapure water, and stirring and mixing the solution at room temperature to obtain a solution A; dissolving 0.25-0.35 g of sodium hydroxide in 10-15 mL of ultrapure water, and uniformly stirring at room temperature to obtain a solution B; mixing the solution A and the solution B, stirring at room temperature for 18-24 h, transferring the mixed solution into a high-pressure reaction kettle, carrying out hydrothermal reaction at 150-200 ℃ for 10-16 h, naturally cooling after the reaction is finished, washing the product for 5 times by using ultrapure water, and drying at 30-60 ℃ for 10-14 h to obtain a flower-shaped stannic oxide material;
(2) preparation of fullerene quantum dots
Dissolving 1-5 mg of fullerene powder in 1-5 mL of anhydrous toluene solvent, uniformly stirring at room temperature to obtain a mauve solution, adding 1-5 mL of ultrapure water into the solution, carrying out ultrasonic treatment for 16-24 h until toluene is completely removed, and storing the obtained faint yellow fullerene quantum dot solution in a 4 ℃ refrigerator for later use;
(3) preparation of cadmium selenide nanoparticles
Dissolving 0.02-0.04 g of selenium powder and 0.02-0.04 g of sodium borohydride in 1-5 mL of ultrapure water, and vigorously stirring at room temperature until the solution becomes clear, wherein the solution is used as a solution A; dissolving 0.02-0.04 g of cadmium chloride dihydrate in 50-100 mL of ultrapure water, adding 100-300 muL of thioglycollic acid solution under stirring, and then adjusting the pH of the solution to 9 by using 0.5-1M of sodium hydroxide aqueous solution to obtain a solution B; pouring the solution A into the solution B, stirring for 2-6 h at room temperature under the mixed solution, washing the obtained product for 5 times by using ultrapure water, and drying for 10-14 h at 30-60 ℃ to obtain carboxyl functionalized cadmium selenide nanoparticles;
(4) preparation of iron disulfide nanoparticles
Dissolving 0.5-0.8 g of trisodium citrate dihydrate and 0.1-0.5 g of polyvinylpyrrolidone K-30 in 50-100 mL of ultrapure water under stirring, adding 1.0-1.5 g of ferrous sulfate heptahydrate into the solution, continuously stirring for 30 min, dropwise adding 10-20 mL of 1.0-2.0M sodium hydroxide aqueous solution into the mixed solution, then adding 0.2-0.5 g of sulfur powder into the solution, continuously and violently stirring to obtain a uniform mixed solution, transferring the mixed solution into a high-pressure reaction kettle, reacting for 16-24 h at 150-200 ℃, washing the product for 3 times by using ultrapure water and anhydrous ethanol after the reaction is finished, and drying for 10-14 h at 30-60 ℃ to obtain iron disulfide nanoparticles;
(5) preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15 mol/L, and taking the aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask, and preparing aqueous solution with the concentration of 1/15 mol/L to be used as solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 5.0-8.0;
(6) preparation of iron disulfide-labeled procalcitonin secondary antibody
Taking 1.0-5.0 mg of prepared iron disulfide nanoparticle solution into 1.0 mL of PBS buffer solution with pH of 7.4, then adding 10-30 μ L of cross-linking agent, wherein the cross-linking agent is a mixed solution consisting of 5 mg/mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1 mg/mL of N-hydroxysuccinimide, activating at 37 ℃ for 10-30 min, then adding 100-500 μ L of procalcitonin secondary antibody with the concentration of 1 μ g/mL into the solution, activating at 37 ℃ for 5-10 h, then centrifugally washing with the PBS buffer solution, dispersing the obtained product in 1 mL of PBS buffer solution, and storing in a refrigerator at 4 ℃ for later use;
(7) preparation of photoelectrochemical sensor
1) Ultrasonically cleaning conductive glass by using liquid detergent, acetone, ethanol and ultrapure water in sequence, and drying the conductive glass in a nitrogen atmosphere;
2) dripping 10 mu L of 1-5 mg/mL of tin tetraoxide aqueous solution onto a conductive surface of the ITO conductive glass, and naturally drying at room temperature;
3) dripping 10 muL of prepared fullerene quantum dot solution on the surface of the modified electrode, and naturally airing at room temperature;
4) continuously dropwise adding 10 muL of 1-5 mg/mL of cadmium selenide aqueous solution on the surface of the modified electrode, and naturally airing at room temperature;
5) dropwise adding 4 mu L of mixed solution of 5-10 mg/mL 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-5 mg/mL N-hydroxysuccinimide in a volume ratio of 1:1 on the surface of the modified electrode, and naturally airing at room temperature to a wet film state;
6) dropwise adding 6 muL of procalcitonin first antibody of 1-5 mug/mL, naturally airing at room temperature to a wet film state, and then washing the electrode with PBS buffer solution;
7) dropwise adding 1-3% bovine serum albumin solution prepared by PBS buffer solution with pH of 7.4 on the surface of the modified electrode, airing in a refrigerator at 4 ℃, and then washing the electrode by the PBS buffer solution;
8) dripping 6 muL of procalcitonin antigen and 1-5 mug/mL of procalcitonin antigen, naturally airing in a refrigerator at 4 ℃, and then washing an electrode by using PBS buffer solution;
9) dropwise adding 6 mu L of prepared procalcitonin secondary antibody with an iron disulfide mark, naturally airing in a refrigerator at 4 ℃, and then washing an electrode by using PBS buffer solution; and (3) preparing the sandwich type photoelectric chemical sensor for detecting procalcitonin.
2. The method for detecting a photoelectrochemical sensor manufactured by the manufacturing method according to claim 1, comprising the steps of:
(1) testing by using an electrochemical workstation and a three-electrode system, taking a saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and taking the prepared ITO modified sensor as a working electrode, wherein the testing is carried out in 10 mL of PBS (phosphate buffer solution) with the pH value of 5.0-8.0 and 0.01-0.5 mol/L of ascorbic acid buffer solution;
(2) detecting the procalcitonin antigen by a time-current method, setting the voltage to be-0.1V, the running time to be 120 s, and the wavelength of a light source to be 400-450 nm;
(3) after the electrodes are placed, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve;
(4) and replacing the procalcitonin antigen sample solution to be detected with the procalcitonin antigen standard solution for detection.
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