CN109932407B - Sandwich type prostate specific antigen photoelectrochemical detection method based on in-situ signal amplification - Google Patents
Sandwich type prostate specific antigen photoelectrochemical detection method based on in-situ signal amplification Download PDFInfo
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Abstract
The invention discloses a sandwich type prostate specific antigen photoelectrochemical detection method based on in-situ signal amplification. The method uses Au NCs as a substrate material, promotes the transfer of photogenerated electrons due to good conductivity, utilizes the sulfydryl of the mercaptophenylboronic acid to form a bond with a gold cone, and the bond of the boronic acid and the amino of an antibody form a cyclic bond, so that a prostate specific antigen antibody (Ab) is formed1) Is secured to the electrode interface. Chitosan (CS) has enzyme-like effect, and can be used for preparing TiO2‑B@CS‑Ag+Complex and Secondary antibody (Ab)2) And combining to form the photoelectrochemical probe. Through immune reaction, Ag is generated in situ under the action of mimic enzyme2S; due to Ag2S energy sensitizing TiO2The photoelectric signal of-B is increased along with the increase of the antigen concentration and the increase of the marker concentration. The method of the invention can realize the concentration of the prostate specific antigen to be 1 multiplied by 10‑6Detection in the range of ng/mL to 50 ng/mL.
Description
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing detection, and particularly relates to sandwich type prostate specific antigen photoelectrochemical detection based on in-situ signal amplification.
Background
Prostate cancer is one of the high-grade cancers in men, and no effective drug for treating prostate cancer exists so far. Prostate Specific Antigen (PSA), an effective biomarker, can detect not only early stage Prostate cancer but also the effect of Prostate cancer treatment, and therefore it is important to detect PSA content sensitively and accurately. At present, common methods for detecting PSA mainly include fluorescence, electrochemiluminescence, surface enhanced Raman scanning, electrochemistry, enzyme-linked immunosorbent assay and the like. However, although these methods can detect the PSA content to some extent, they have limitations in some aspects, such as the need for large-scale detection equipment and long detection time. Therefore, the discovery of a simple and sensitive method for detecting the PSA content is a problem to be solved by current scientific research workers.
Compared with other detection methods, the photoelectrochemistry detection uses light as an excitation signal and detects an electric signal, and different forms of energy are adopted as the excitation signal and the detection signal, so that the excitation signal and the detection signal are not interfered with each other, the background signal is lower, and higher sensitivity can be obtained. However, in order to achieve sensitive detection better, how to amplify the detection signal and reduce the background signal also becomes a problem to be solved. According to the signal amplification strategies reported in the literature, target cyclic amplification with enzyme participation, signal amplification based on nano materials, chain hybridization reaction signal amplification, rotary rolling amplification, dendritic immobilized signal amplification and the like are planned. TiO 22The B nano material has relatively more gaps and continuous channels due to the perovskite-like structure, so that the B nano material has higher electron transfer capability and higher photoelectric effect. It has been reported in the literature that chitosan has properties that mimic enzymes, as shown in H2O2Where present, catalytic reactions may occur.
In the present invention, we refer to TiO2the-B is compounded with chitosan and Ag + to form a compound TiO2-B@CS-Ag+Binding with secondary antibody to form labeled secondary antibody, and then H2O2If present with S2O3 2-Reacting to generate Ag in situ2And S. Due to Ag2Forbidden band width ratio of S to TiO2small-B, excited by light when it strikes the electrode surface, Ag2The electrons in the S valence band are transferred to the conduction band to form electron-hole pairs, Ag2Enrichment of electrons in S conduction band and transfer to TiO2The conduction band of-B, so that the photogenerated electron-hole recombination is slower and the photoelectric signal is enhanced. As the concentration of the analyte increases, the concentration of the labeled secondary antibody immobilized on the electrode surface increases due to the immunoreaction, and H2O2In the presence of (A), S2O3 2-Reduction reaction to produce S2-With Ag+Combined to form Ag2And S, increasing photocurrent, realizing sensitive detection of the prostate antigen according to the phenomenon, and providing a platform for clinical detection.
Disclosure of Invention
The invention aims to provide a method for in-situ generation of Ag2S-sensitized TiO2A sandwich type photoelectrochemical sensor of-B mesoscopic crystals, a preparation method and a detection application thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
(1) pretreatment of GCE: firstly, mechanically grinding and polishing a chamois leather paved with alumina powder by GCE, washing residual powder on the surface by secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally completely washing the chamois leather by ethanol, dilute acid and water in sequence;
（2）Au/Ab1preparation of PSA modified electrode: dripping 3 μ L of gold cone solution on clean glassy carbon electrode surface, oven drying under infrared lamp, cooling to room temperature, dripping 3 μ L of mercaptophenylboronic acid solution on modified electrode surface, incubating at 4 deg.C for 50min, washing with secondary waterRemoving the excess solution; the modified electrode was exposed to 5. mu.L of prostate specific antigen antibody Ab at a concentration of 1 mg/mL1Incubation at 4 ℃ for 50min in solution, followed by removal of excess prostate specific antigen antibody Ab using phosphate buffered pH 7.41Then, the electrode is immersed into 20 mu L BSA with the concentration of 1.0 wt.% for 1 h, the non-specific active sites on the electrode surface are blocked, and the surface residual liquid is flushed by secondary water, thus obtaining Ab1a/Au modified electrode; immersing the electrodes in 5 μ Ι _ of different prostate cancer cell antigen (PSA) standard solutions and incubating at 4 ℃ for 50 min; washing the surface of the electrode with a phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain Au/Ab1a/PSA modified electrode;
（3） Au/Ab1/PSA/Ab2/Ag2preparing an S modified electrode: weighing a certain amount of TiO2The solid powder of the B is dispersed evenly by secondary water to prepare suspension with the concentration of 3 mg/mL, and TiO is added2Mixing the solution B and the chitosan solution according to the volume ratio of 1:2, shaking for 30min, centrifuging the reacted solution at the rotating speed of 5000 rpm for 10min, removing the redundant chitosan solution, and then adding a certain amount of secondary water for dispersing to form TiO2-B @ CS solution. According to AgNO3Solution with TiO2-B @ CS in a volume ratio of 1:1, adding a certain concentration of AgNO3Solution addition to TiO2Shaking the solution to react for 30min to make Ag+Is fully adsorbed to TiO2-B surface, removing excess Ag by centrifugation+Dispersing the solid with secondary water to form treated TiO2-B solution. Mixing the prostate specific antigen secondary antibody and the treated TiO according to the volume ratio of 1:32Mixing the solutions B, reacting at 4 deg.C for 2h, centrifuging to remove unadsorbed secondary antibody, dispersing with phosphate buffer solution with pH of 7.4, adding a certain amount of BSA solution to block inactive sites to obtain Ab2-TiO2-B@CS-Ag+And placing the glass in a refrigerator for standby. Draw up a defined amount of Ab with a pipette2-TiO2-B@CS-Ag+Solution, drop-coated onto Au/Ab1Ag modified electrode, incubating at 4 deg.C for 1 h, and cleaning with secondary waterWashing to remove excessive physical adsorption to obtain Au/Ab1/PSA/Ab2An electrode; 0.26M of H at room temperature2O2With 0.18M of S2O3 2-The solution was applied dropwise to the modified Au/Ab1/PSA/Ab2Electrode surface, reaction for 15 min, at H2O2With TiO2In-situ generation of Ag under the catalytic action of-B @ CS2S, obtaining Au/Ab1/PSA/Ab2/Ag2And an S electrode.
The above TiO compound2-B preparation of nanorod material:
TiO2the preparation of the-B nano rod is synthesized by a solvothermal method by taking a titanium nano wire as a precursor. 1 g TiO2Dispersed in 75 mL 15M aqueous KOH. After shaking for 10 minutes, the resulting suspension was transferred to a 100 mL reaction kettle, which was placed at 170 ℃ for 72 h and then cooled to room temperature. The resulting deposit was washed with acetic acid solution until the pH was 7.0. The final product was collected by centrifugation and dried in a forced air oven at 60 ℃ for 12 h. TiO 22Synthesis of-B, 300 mg of the TiO prepared are first reacted2The nanowire precursor is dispersed in 50mL of 8M acetic acid solution, and then the solution is transferred to a reaction kettle and heated to 180 ℃ for reaction for 24 hours. The resulting product was collected by centrifugation, washed with water twice and ethanol, and dried overnight at 60 ℃.
Preparation of the above gold cones (Au NCs):
prior to spin coating the polystyrene, the Teflon film was first cut into 1.5 cm x 1.5 cm squares and rinsed with ethanol and a second rinse, then plasma water for 3 minutes. 1mL of a 2.5 w/v% polystyrene solution was aspirated, centrifuged, and transferred to a mixed solution of ethanol and methanol at a volume ratio of 2: 1. A surfactant (TX100) was added to the solution at a volume ratio of 0.2%, followed by adjustment of the polystyrene concentration to about 5 w/v%. The polystyrene was then spin coated on clean Teflon film and left at room temperature for several minutes to allow the solvent to dry. With O2The polystyrene/Teflon surface was plasma etched for a certain time. Finally, the surface is coated with 50 nm gold by thermal evaporation to obtain the gold cone.
The invention relates to a TiO-based material2The prostate specific antigen photoelectrochemical sensor of the-B mesoscopic crystal comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts Au/Ab1/PSA/Ab2/Ag2An S-modified electrode prepared by the method of 1) polishing a glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence; 2) Au/Ab1/PSA/Ab2/Ag2Preparing an S modified electrode: dripping 3 mu L of gold cone solution on the surface of a clean glassy carbon electrode, drying under an infrared lamp, cooling to room temperature, dripping 3 mu L of mercapto-phenylboronic acid solution on the surface of the modified electrode, incubating for 50min at 4 ℃, washing with secondary water, and removing redundant solution; the modified electrode was exposed to 5. mu.L of prostate specific antigen antibody Ab at a concentration of 1 mg/mL1Incubation at 4 ℃ for 50min in solution, followed by removal of excess prostate specific antigen antibody Ab using phosphate buffered pH 7.41Then, the electrode is immersed into 20 mu L of BSA with the concentration of 1.0 wt.% for 1 h, the non-specific active sites on the surface of the electrode are blocked, and the residual liquid on the surface is washed away by a phosphate buffer solution with the pH of 7.4, thus obtaining Au/Ab1Modifying the electrode; immersing the electrodes in 5 μ Ι _ of different prostate cancer cell antigen (PSA) standard solutions and incubating at 4 ℃ for 50 min; washing the surface of the electrode with a phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain Au/Ab1a/PSA modified electrode; aspirate 5. mu.L of labeled Secondary antibody Ab2-TiO2-B@CS-Ag+Drop-coating onto Au/Ab1PSA modified electrode surface, incubation at 4 deg.C for 1 h, washing off unreacted excess secondary antibody with phosphate buffer solution of pH 7.4 to obtain Au/Ab1/PSA/Ab2Modifying the electrode; finally, 5. mu.L of 0.26M H was aspirated2O2With 0.18M of S2O3 2-The solution was applied dropwise to the modified Au/Ab1/PSA/Ab2The surface of the electrode is coated with a metal,reaction for 15 min at H2O2With TiO2In-situ generation of Ag under the catalytic action of-B @ CS2S, obtaining Au/Ab1/PSA/Ab2/Ag2And an S electrode.
The invention relates to a TiO-based material2-B mesoscopic crystal photoelectrochemical sensor for use in a method for detection of prostate specific antigens, characterised by the steps of: 1) the assay was performed using a three-electrode system with Au/Ab1/PSA/Ab2/Ag2The S modified electrode is a working electrode, the Ag/AgCl is a reference electrode, the platinum wire electrode is a counter electrode, a photoelectrochemical workstation is used for detecting, the voltage is set to be-0.1V, the lamp is switched on and switched off every 10S, and a monochromatic light excitation light source emitted by a xenon lamp is filtered by a monochromator before being used; 2) detection by photoelectrochemical station in PBS buffer pH 7.4 at 1X 10-6ng/mL-50 ng/mL of a series of prostate specific antigen standard solutions with different solubilities, and drawing a working curve by recording different current signals generated before and after turning on and off a lamp; the sample solution to be detected replaces the prostate specific antigen standard solution for detection, and the detection result can be obtained through a working curve.
The invention has the following remarkable advantages:
(1) gold is used as a noble metal, has good conductivity, and the gold cone is used as a substrate material, can effectively promote the transfer of photogenerated electrons, enhances the photoelectric response, and is TiO2-B @ CS has a catalytic effect in H2O2In the presence of TiO2-B @ CS catalysis of S2O3 2-Generation of S2-，S2-With Ag+Reaction to form Ag2S, enhancement of photocurrent.
(2) The PSA to be detected and the corresponding labeled secondary antibody have immunoreaction, the concentration of the labeled secondary antibody is increased along with the increase of the concentration of the substance to be detected, the photoelectric signal is gradually enhanced, and the sensitive detection of the PSA is realized through the phenomenon.
Drawings
FIG. 1 shows a TiO-based composition according to the invention2Schematic diagram of construction process of sandwich type biosensor for amplifying in-situ signal。
FIG. 2A shows different concentrations of 1X 10-6ng/mL-50 ng/mL (a-i) prostate specific antigen standard solution, photocurrent response graph of sensing electrode.
FIG. 2B is a linear curve corresponding to standard solutions of different concentrations.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
Based on TiO2The preparation method of the photoelectrochemical sensor of the mesoscopic crystal (as shown in figure 1) comprises the following steps:
(1) pretreating a glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence;
（2） Au/Ab1preparation of PSA modified electrode: dripping 3 mu L of gold cone (Au NCs) solution with the concentration of 2 mg/mL on the surface of a clean glassy carbon electrode, drying under an infrared lamp, and cooling to room temperature; then, 3. mu.L of 5 mM mercaptophenylboronic acid solution was applied dropwise to the modified electrode surface, incubated at 4 ℃ for 50min, washed with water twice, the excess solution removed, and the modified electrode was incubated with 5. mu.L of 1 mg/mL prostate-specific antigen antibody Ab1Incubation at 4 ℃ for 50min in solution, followed by removal of excess prostate specific antigen antibody Ab using phosphate buffered pH 7.41Then, the electrode is immersed into 20 mu L of Bovine Serum Albumin (BSA) solution with the concentration of 1.0 wt.% for 1 h, the non-specific active sites on the surface of the electrode are blocked, and the residual liquid on the surface is washed away by phosphoric acid buffer solution with the pH of 7.4, thus obtaining Au/Ab1Modifying the electrode, immersing the electrode into 5 mu L of prostate cancer cell antigen (PSA) standard solutions with different concentrations, incubating for 50min at 4 ℃, washing the surface of the electrode with a phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain Au/Ab1a/PSA modified electrode;
（3） Au/Ab1/PSA/Ab2/Ag2preparing an S modified electrode: weighing a certain amount of TiO2The solid powder of the B is dispersed evenly by secondary water to prepare suspension with the concentration of 3 mg/mL, and TiO is added2Mixing the solution B and 0.5 wt.% of chitosan solution according to the volume ratio of 1:2, shaking for 30min, centrifuging the reacted solution at the rotation speed of 5000 rpm for 10min, removing the redundant chitosan solution, and adding a certain amount of secondary water for dispersing to form TiO2-B @ CS solution; according to AgNO3Solution with TiO2-B @ CS volume ratio of 1:1, AgNO concentration of 0.1 mol/L3Solution addition to TiO2Shaking the solution to react for 30min to make Ag+Is fully adsorbed to TiO2-B surface, removing excess Ag by centrifugation+Dispersing the solid with secondary water to form treated TiO2-a solution B; the prostate specific antigen secondary antibody (Ab) is added according to the volume ratio of 1:32) With treated TiO2Mixing the solutions B, reacting at 4 deg.C for 2h, centrifuging to remove unadsorbed secondary antibody, dispersing with phosphate buffer solution with pH of 7.4, adding a certain amount of BSA solution to block inactive sites to obtain Ab2-TiO2-B@CS-Ag+Placing the solution in a refrigerator for later use; draw up a defined amount of Ab with a pipette2-TiO2-B@CS-Ag+Solution, drop-coated onto Au/Ab1Incubating on PSA modified electrode at 4 deg.C for 1 h, washing with secondary water, removing excessive physical adsorption to obtain Au/Ab1/PSA/Ab2An electrode; 0.26M of H at room temperature2O2With 0.2M AgNO3The solution was applied dropwise to the modified Au/Ab1/PSA/Ab2Electrode surface, reaction for 15 min, at H2O2With TiO2In-situ generation of Ag under the catalytic action of-B @ CS2S, obtaining Au/Ab1/PSA/Ab2/Ag2And an S electrode.
Example 2
TiO used in example 12-preparation of material B:
TiO2the preparation of-B nano-rod uses titanium nano-wire asThe precursor is synthesized by a solvothermal method. 1 g TiO2Dispersed in 75 mL of 15M aqueous KOH. After shaking for 10 minutes, the resulting suspension was transferred to a 100 mL reaction kettle, which was placed at 170 ℃ for 72 h and then cooled to room temperature. The resulting deposit was washed with acetic acid solution until the pH was 7.0. Collecting the final product by centrifugation, and drying in a forced air oven at 60 deg.C for 12 h to obtain TiO2And (3) a nanowire precursor. TiO 22Synthesis of-B, 300 mg of the TiO prepared are first reacted2The nanowire precursor is dispersed in 50mL of 8M acetic acid solution, and then the solution is transferred to a reaction kettle and heated to 180 ℃ for reaction for 24 hours. The resulting product was collected by centrifugation, washed with water twice and ethanol, and dried overnight at 60 ℃.
Preparation of gold cones (Au NCs) used in example 1:
prior to spin coating the polystyrene, the Teflon film was first cut into 1.5 cm x 1.5 cm squares and rinsed with ethanol and a second rinse, then plasma water for 3 minutes. 1mL of a 2.5 w/v% polystyrene solution was aspirated, centrifuged, and transferred to a mixed solution of ethanol and methanol at a volume ratio of 2: 1. A surfactant (TX100) was added to the solution at a volume ratio of 0.2%, followed by adjustment of the polystyrene concentration to about 5 w/v%. The polystyrene was then spin coated on clean Teflon film and left at room temperature for several minutes to allow the solvent to dry. With O2The polystyrene/Teflon surface was plasma etched for a certain time. Finally, the surface is coated with 50 nm gold by thermal evaporation to obtain the gold cone.
Example 3
Based on TiO2-B crystal photoelectrochemical sensor for prostate specific antigen detection method, comprising the steps of:
(1) the assay was performed using a three-electrode system, with Au/Ab prepared in example 11/PSA/Ab2/Ag2The S modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is a counter electrode, a photoelectrochemical workstation is used for detection, the voltage is set to be-0.1V, the lamps are switched on and off every 10S, and a monochromatic light excitation light source emitted by a xenon lamp is used before being usedFiltering by a color instrument;
(2) detection by photoelectrochemical station in PBS buffer pH 7.4 at 1X 10-6ng/mL-50 ng/mL of a series of prostate specific antigen standard solutions with different concentrations, and drawing a working curve by recording different current signals generated before and after the lamp is turned on and off. FIG. 2A shows different concentrations of 1X 10-6ng/mL-50 ng/mL (a-i) prostate specific antigen standard solution, sensing the photocurrent response of the electrode. Fig. 2B is a graph of the photocurrent response of the sensing electrode versus the concentration of the prostate specific antigen standard solution.
And (3) replacing the prostate specific antigen standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
Claims (4)
1. A sandwich type prostate specific antigen photoelectrochemical detection method based on in-situ signal amplification is characterized by comprising the following steps:
(1) pretreatment of the glassy carbon electrode GCE: firstly, mechanically grinding and polishing a chamois leather paved with alumina powder by GCE, washing residual powder on the surface by secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally completely washing the chamois leather by ethanol, dilute acid and water in sequence;
（2） Au/Ab1preparation of PSA modified electrode: dripping 3 μ L of Au NCs solution on clean glassy carbon electrode surface, drying under infrared lamp, cooling to room temperature, dripping 3 μ L of mercaptophenylboronic acid solution on modified electrode surface, incubating at 4 deg.C for 50min, washing with secondary water, removing excess solution, and adding 5 μ L of antibody Ab of prostate specific antigen with concentration of 1 mg/mL1Incubation at 4 ℃ for 50min in solution, followed by removal of excess prostate specific antigen antibody Ab using phosphate buffered pH 7.41Then, the electrode is immersed into 20 mu L of bovine serum albumin BSA solution with the concentration of 1.0 wt.% for 1 h, the non-specific active sites on the surface of the electrode are blocked, and the residual liquid on the surface is washed away by phosphoric acid buffer solution with the pH of 7.4, thus obtaining Au/Ab1Modifying the electrode by immersing the electrode in 5 μ L of prostate cancer cells of different concentrationsIncubating the antigen PSA standard solution at 4 ℃ for 50min, washing the surface of the electrode with a phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain Au/Ab1a/PSA modified electrode;
（3） Au/Ab1/PSA/Ab2/Ag2preparing an S modified electrode: weighing a certain amount of TiO2The solid powder of the B is dispersed evenly by secondary water to prepare suspension with the concentration of 3 mg/mL, and TiO is added2Mixing the solution B and the chitosan solution according to the volume ratio of 1:2, shaking for 30min, centrifuging the reacted solution at the rotating speed of 5000 rpm for 10min, removing the redundant chitosan solution, and then adding a certain amount of secondary water for dispersing to form TiO2-B @ CS solution; according to AgNO3Solution with TiO2-B @ CS in a volume ratio of 1:1, adding a certain concentration of AgNO3Solution addition to TiO2Shaking the solution to react for 30min to make Ag+Is fully adsorbed to TiO2-B surface, removing excess Ag by centrifugation+Dispersing the solid with secondary water to form treated TiO2-a solution B; the prostate specific antigen secondary Ab is added according to the volume ratio of 1:32With treated TiO2Mixing the solutions B, reacting at 4 deg.C for 2h, centrifuging to remove unadsorbed secondary antibody, dispersing with phosphate buffer solution with pH of 7.4, adding a certain amount of BSA solution to block inactive sites to obtain Ab2-TiO2-B@CS-Ag+Placing the solution in a refrigerator for later use; draw up a defined amount of Ab with a pipette2-TiO2-B@CS-Ag+Solution, drop-coated onto Au/Ab1Incubating on PSA modified electrode at 4 deg.C for 1 h, washing with secondary water, removing excessive physical adsorption to obtain Au/Ab1/PSA/Ab2An electrode; 0.26M of H at room temperature2O2With 0.18M of S2O3 2-The solution was applied dropwise to the modified Au/Ab1/PSA/Ab2Electrode surface, reaction for 15 min, at H2O2With TiO2In-situ generation of Ag under the catalytic action of-B @ CS2S, obtaining Au/Ab1/PSA/Ab2/Ag2An S electrode;
(4) detection of prostate specific antigen: the assay was performed using a three-electrode system with Au/Ab1/PSA/Ab2/Ag2The S modified electrode is a working electrode, the Ag/AgCl is a reference electrode, the platinum wire electrode is a counter electrode, a photoelectrochemical workstation is used for detecting, the voltage is set to be-0.1V, the lamp is switched on and switched off every 10S, and a monochromatic light excitation light source emitted by a xenon lamp is filtered by a monochromator before being used; detection by photoelectrochemical station in PBS buffer pH 7.4 at 1X 10-6ng/mL-50 ng/mL of a series of prostate specific antigen standard solutions with different concentrations, and drawing a working curve by recording different current signals generated before and after turning on and off a lamp; the sample solution to be detected replaces the prostate specific antigen standard solution for detection, and the detection result can be obtained by checking a working curve;
the TiO is2-B material is prepared by the following method: TiO 22The preparation of the nano-rod is synthesized by taking the titanium nano-wire as a precursor through a solvothermal method, and 1 g of TiO2Dispersing into 75 mL of 15M KOH aqueous solution, shaking for 10 minutes, transferring the obtained suspension into a 100 mL reaction kettle, placing the reaction kettle at 170 ℃ for reaction for 72 hours, cooling to room temperature, washing the obtained deposit with an acetic acid solution until the pH value is 7.0, collecting the final product by centrifugation, and drying in a 60 ℃ air-blast oven for 12 hours to obtain TiO2Nano wire, then taking 300 mg of prepared TiO2Dispersing the nanowire precursor in 50mL of 8M acetic acid solution, transferring the solution to a reaction kettle, heating to 180 ℃, reacting for 24 hours, centrifugally collecting the obtained product, washing with secondary water and ethanol, and drying at 60 ℃ overnight to obtain TiO2-B。
2. The method of claim 1, wherein the Au NCs is prepared by the following method:
before spin-coating polystyrene, the Teflon film was first cut into 1.5 cm x 1.5 cm squares and rinsed with ethanol and secondary water, then with plasma water for 3 minutes; 1mL of a 2.5 w/v% polystyrene solution was aspirated, centrifuged, and then transferredTransferring the mixture into a mixed solution of ethanol and methanol in a volume ratio of 2: 1; adding 0.2% by volume of surfactant TX100 to the solution, and then adjusting the concentration of polystyrene to about 5 w/v%; the polystyrene was then spin-coated onto a clean Teflon film and left to stand at room temperature for several minutes to allow the solvent to dry; with O2Etching the surface of the polystyrene/Teflon by plasma for a certain time; finally, the surface is coated with 50 nm gold by thermal evaporation to obtain the gold cone.
3. Based on TiO2The photoelectrochemical biosensor for prostate specific antigen of-B comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts Au/Ab1/PSA/Ab2/Ag2An S-modified electrode prepared by the method of 1) polishing a glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence; 2) Au/Ab1/PSA/Ab2/Ag2Preparing an S modified electrode: dripping 3 mu L of gold cone solution on the surface of a clean glassy carbon electrode, drying under an infrared lamp, cooling to room temperature, dripping 3 mu L of mercapto-phenylboronic acid solution on the surface of the modified electrode, incubating for 50min at 4 ℃, washing with secondary water, and removing redundant solution; the modified electrode was exposed to 5. mu.L of prostate specific antigen antibody Ab at a concentration of 1 mg/mL1Incubation at 4 ℃ for 50min in solution, followed by removal of excess prostate specific antigen antibody Ab using phosphate buffered pH 7.41Then, the electrode is immersed into 20 mu L BSA with the concentration of 1.0 wt.% for 1 h, the non-specific active sites on the electrode surface are blocked, and the surface residual liquid is flushed by secondary water, thus obtaining Ab1a/Au modified electrode; immersing the electrodes in 5 mu L of different prostate cancer cell antigen PSA standard solutions, and incubating for 50min at 4 ℃; washing the surface of the electrode with a phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain Au/Ab1a/PSA modified electrode; aspirate 5. mu.L of labeled secondary antibodyAb2-TiO2-B@CS-Ag+Drop-coating onto Au/Ab1PSA modified electrode surface, incubation at 4 deg.C for 1 h, washing off unreacted excess secondary antibody with phosphate buffer solution of pH 7.4 to obtain Au/Ab1/PSA/Ab2Modifying the electrode; finally, 5. mu.L of 0.26M H was aspirated2O2With 0.18M of S2O3 2-The solution was applied dropwise to the modified Au/Ab1/PSA/Ab2Electrode surface, reaction for 15 min, at H2O2With TiO2In-situ generation of Ag under the catalytic action of-B @ CS2S, obtaining Au/Ab1/PSA/Ab2/Ag2An S electrode;
TiO2-B material is prepared by the following method: TiO 22The preparation of the nano-rod is synthesized by taking the titanium nano-wire as a precursor through a solvothermal method, and 1 g of TiO2Dispersing into 75 mL of 15M KOH aqueous solution, shaking for 10 minutes, transferring the obtained suspension into a 100 mL reaction kettle, placing the reaction kettle at 170 ℃ for reaction for 72 hours, cooling to room temperature, washing the obtained deposit with an acetic acid solution until the pH value is 7.0, collecting the final product by centrifugation, and drying in a 60 ℃ air-blast oven for 12 hours to obtain TiO2Nano wire, then taking 300 mg of prepared TiO2Dispersing the nanowire precursor in 50mL of 8M acetic acid solution, transferring the solution to a reaction kettle, heating to 180 ℃, reacting for 24 hours, centrifugally collecting the obtained product, washing with secondary water and ethanol, and drying at 60 ℃ overnight to obtain TiO2-B。
4. A TiO-based material prepared by the process of any one of claims 1-22-B mesoscopic crystal photoelectrochemical sensor for use in a method for detection of prostate specific antigens, characterised by the steps of: 1) the assay was performed using a three-electrode system with Au/Ab1/PSA/Ab2/Ag2The S modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is a counter electrode, a photoelectrochemical workstation is used for detection, the set voltage is 0.1V, the lamps are switched on and off every 10S, and a monochromatic light excitation light source emitted by the xenon lamp is filtered by a monochromator before being used(ii) a 2) Detection by photoelectrochemical station in PBS buffer pH 7.4 at 1X 10-6 ng/mL-50 ng/mL of a series of prostate specific antigen standard solutions with different solubilities, and drawing a working curve by recording different current signals generated before and after turning on and off a lamp; the sample solution to be detected replaces the prostate specific antigen standard solution for detection, and the detection result can be obtained through a working curve.
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| CN110596364B (en) * | 2019-09-12 | 2020-06-23 | 江南大学 | Construction method and application of ratio electrochemical sensor for prostate specific antigen detection |
| CN110687182A (en) * | 2019-10-30 | 2020-01-14 | 云南大学 | Preparation method of electrochemical immunosensor for detecting prostate specific antigen |
| CN111273014B (en) * | 2020-03-06 | 2024-01-30 | 安徽大学 | Photoelectrochemical immunosensor for detecting prostate specific antigen and preparation method thereof |
| CN111624338B (en) * | 2020-06-06 | 2022-12-16 | 青岛科技大学 | Preparation method of photoelectrochemical immunosensor for detecting prostate specific antigen |
| CN112129940B (en) * | 2020-09-14 | 2023-01-10 | 青岛科技大学 | Cathode photoelectrochemical immunosensor for amplifying detection signal by using bilirubin oxidase and preparation method and application thereof |
| CN114199956B (en) * | 2021-11-02 | 2022-12-09 | 东莞理工学院 | Photoelectric sensor for detecting human prostate specific antigen by immune reaction |
| CN114113051B (en) * | 2021-12-16 | 2023-06-16 | 南京信息工程大学 | Preparation method and application of PSMA electrochemiluminescence sensor |
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