Based on WO3The building of the photoelectric sensor of/BiOI in conjunction with enzymatic precipitated phase
阅读说明：本技术 基于WO3/BiOI与酶催化沉淀相结合的光电传感器的构建 (Based on WO3The building of the photoelectric sensor of/BiOI in conjunction with enzymatic precipitated phase ) 是由 颜梅 苗培 张晶 李增军 赵悦英 于京华 于 2019-08-29 设计创作，主要内容包括：本发明公开了一种基于WO3/BiOI与酶催化沉淀相结合的光电传感器的构建方法,首先合成了具有良好电化学信号的花状WO3结构,该结构有大的比表面积,能够在负载更多的检测物；又在花状WO3上负载了BiOI,进一步增加电化学信号；另外在二抗上面修饰了HRP,它可以催化4-CN与H2O2迅速反应生成沉淀物附着在电极表面,极大的阻碍了电极表面的电荷传输,导致光电信号的减小；同时,Ab2-HRP生物偶联物通过特异性结合在抗原上,增大了传感器的空间位阻,进一步的引起光电信号的减小；这样通过层层修饰构建成了检测前列腺抗原的电化学传感器。(The invention discloses a kind of construction method of photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase, the flower-shaped WO3 structure with good electrochemical signals has been synthesized first, which has big specific surface area, can load more detectable substances；BiOI has been loaded on flower-shaped WO3 again, has further increased electrochemical signals；In addition HRP has been modified on secondary antibody, it can be catalyzed 4-CN with H2O2 rapidly react generate sediment be attached to electrode surface, greatly hinder electrode surface charge transmission, lead to the reduction of photosignal；Meanwhile Ab2-HRP bioconjugate increases the steric hindrance of sensor, further causes the reduction of photosignal by specifically binding on antigen；The electrochemical sensor of detection prostate antigen has been built by modification layer by layer in this way.)
1. being based on WO3The building of the photoelectric sensor of/BiOI in conjunction with enzymatic precipitated phase, feature the following steps are included:
(1) WO is synthesized3；
(2) WO is synthesized3/ BiOI hetero-junctions；
(3) ITO/WO is constructed3/ BiOI electrode；
(4) two anti-horseradish peroxidase (Ab2-HRP) compounds are synthesized；
(5) building of photoelectric sensor (PEC)；
(6) Electrochemical Detection of photoelectric sensor.
2. being based on WO according to claim 13The building of the photoelectric sensor of/BiOI in conjunction with enzymatic precipitated phase, synthesis WO3, it is characterized in that: by the WCl of 1.0 g6It is dissolved in the dehydrated alcohol of 50 mL, 4 h is heated in the water-bath of 160 oC；Will After the product cooled to room temperature arrived, respectively three times with dehydrated alcohol and milli-Q water, dry 12 h at 60 oC.
3. the building of the photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase according to claim 1, synthesis WO3/ BiOI hetero-junctions, it is characterized in that: the 0.3 g WO that step (1) is synthesized3It is dissolved in 60 mL ultrapure waters, while agitating The KI of 1.0 mmol is added；Continue after stirring 40 min, by 1.0 mmol Bi (NO3)3·5H2O is added in above-mentioned mixed solution, Then to reaction mixture carry out ultrasonic treatment 15 min, 3 h are then stirred at room temperature, finally under 8000 rmp revolving speeds from The heart handles 2 min, is washed 3 times with ultrapure water and dehydrated alcohol, is dried respectively at 60 oC.
4. the building of the photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase according to claim 1, building ITO/WO3/ BiOI electrode, it is characterized in that: electro-conductive glass is indium tin oxide glass (ITO), electro-conductive glass is cut into 4.0 × 0.5 cm strip is successively cleaned by ultrasonic 5 min with acetone soln, secondary distilled water and dehydrated alcohol, then dries under a nitrogen It is spare；The WO for being 2.0 mg/mL by the concentration that ito glass is placed on step (2) synthesis320 are ultrasonically treated in/BiOI mixed solution Min obtains ITO/WO after then calcining 2 h at 200 oC3/ BiOI electrode.
5. the building of the photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase according to claim 1, synthesis Two anti-horseradish peroxidase (Ab2-HRP) compounds, it is characterized in that: being 20 μ g mL by 50 μ L concentration−11- (3- diformazan Aminopropyl) -3- ethyl-carbodiimide hydrochloride and 50 μ L concentration are 10 mg mL−1N- hydroxysuccinimide solution add Enter to 800 mL concentration be 50 mg/mL Ab2 solution in, then by the concentration of 200 mL be 1.0 mg/mL HRP with it is above-mentioned And then solution mixing, 3 h of incubated under agitation at 30 oC are incubated for 12 h at 4 oC；Finally, in 6000 rpm centrifugal rotational speeds Lower 15 min of centrifugal treating obtains Ab2-HRP compound, and is diluted to 800 mL using the phosphate buffer of pH 7.4.
6. the building of the photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase according to claim 1, photoelectricity The building of sensor (PEC), it is characterized in that: being then 10 μ g 6 μ L concentration with ultrapure water ITO/WO3/BiOI electrode mL−1Primary antibody, that is, Ab1 16 h are incubated at 4 oC, with phosphate buffer cleaning down 3 times of pH 7.4；Continue 20 μ of drop coating The bovine serum albumin(BSA) of L 3% blocks nonspecific binding site, with phosphate buffer cleaning down 3 times of pH 7.4, by 20 μ L various concentration prostate antigen is added dropwise to electrode surface, after being incubated for 30 min at room temperature, with the phosphate buffer of pH 7.4 Washing 3 times；Continue the Ab2-HRP that the synthesis of 20 μ L steps (4) is added dropwise；It is 1.0 mg that above-mentioned electrode, which is then placed in concentration, mL-14-CN and concentration be 1.5 mg mL-1H2O2Mixed solution in 10 min, finally use ultrapure water electrode 3 It is secondary.
7. the building of the photoelectric sensor based on WO3/BiOI in conjunction with enzymatic precipitated phase according to claim 1, photoelectricity The Electrochemical Detection of sensor, it is characterized in that: the modified electrode that step (5) is handled well is as working electrode, it is platinum to electrode Silk electrode, reference electrode are Ag/AgCl electrodes, and bias numerical value is 0 V, and for xenon lamp as source stimulating, electrolytic cell is pH's 7.4 Phosphate-buffered liquid system measures electric current I-T curve to carry out the detection of photoelectric properties.
Technical field
The present invention relates to the quantitative detection fields of prostate-specific antigen, are in particular based on WO3/BiOI and enzyme The method for being catalyzed the building for the photoelectric sensor that precipitated phase combines.
Background technique
Tumor markers be tumour cell synthesized by gene expression, secrete or body because to tumour generate reaction And the substance generated extremely, it plays an important role in early diagnosis of tumor.The early detection demand of cancer is to its tumour mark Show the method for highly selective, highly sensitive, the quick analysis detection of object.Therefore, it is good pernicious to establish simple and quick sensitive and selectivity Knubble biological marker new detecting method, early detection to malignant tumour and and treatment effectiveness evaluation there is highly important meaning Justice.
Its working principle of optical electro-chemistry sensed immunological sensor mainly utilizes the specific recognition function of Ag-Ab, institute The inductive signal of generation is converted into exportable electrochemical signals by signal adapter, the concentration of the signal and target analytes Certain proportionate relationship is presented, so as to realize the quantitative analysis to target substance.In recent years, optical electro-chemistry immunosensor By the concern of many researchers because its immune response high specific, shirtsleeve operation, high sensitivity, it is highly selective with And the advantages that speed is fast is analyzed, it is obtained extensively in clinical medicine, environmental pollution analyte detection, food analysis, biotechnology field Using.
Summary of the invention
It is an object of the invention to construct a kind of Photoelectrochemistrbiosensor biosensor for prostate-specific antigen detection.
In order to solve the above-mentioned technical problem, the present invention is realized by following measures: one kind based on WO3/BiOI with Enzymatic precipitated phase combine photoelectric sensor building, feature the following steps are included:
(1) by the WCl of 1.0 g6It is dissolved in the dehydrated alcohol of 50 mL, 4 h is heated in the water-bath of 160 oC；By what is obtained After product cooled to room temperature, respectively three times with dehydrated alcohol and milli-Q water, dry 12 h at 60 oC；
(2) the 0.3 g WO for synthesizing step (1)3It is dissolved in 60 mL ultrapure waters, is added 1.0 mmol's while agitating KI；Continue after stirring 40 min, by 1.0 mmol Bi (NO3)3·5H2O is added in above-mentioned mixed solution, then mixes to reaction Object carries out 15 min of ultrasonic treatment, 3 h is then stirred at room temperature, finally 2 min of centrifugal treating under 8000 rmp revolving speeds, uses Ultrapure water and dehydrated alcohol wash 3 times respectively, dry at 60 oC；
(3) electro-conductive glass is indium tin oxide glass (ITO), electro-conductive glass is cut into 4.0 × 0.5 cm strips, successively with third Ketone solution, secondary distilled water and dehydrated alcohol are cleaned by ultrasonic 5 min, then drying for standby under a nitrogen；Ito glass is placed on step Suddenly the WO that the concentration of (2) synthesis is 2.0 mg/mL320 min are ultrasonically treated in/BiOI mixed solution, are then forged at 200 oC After burning 2 h, ITO/WO is obtained3/ BiOI electrode；
It (4) is 20 μ g mL by 50 μ L concentration−11- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 50 μ L concentration is 10 mg mL−1N- hydroxysuccinimide solution be added 800 mL concentration be 50 mg/mL Ab2 solution in, so The HRP that the concentration of 200 mL is 1.0 mg/mL is mixed with above-mentioned solution afterwards, 3 h of incubated under agitation at 30 oC, and then 4 12 h are incubated under oC；Finally, 15 min of centrifugal treating obtains Ab2-HRP compound under 6000 rpm centrifugal rotational speeds, and use The phosphate buffer of pH 7.4 is diluted to 800 mL；
(5) ultrapure water ITO/WO3/BiOI electrode is used, is then 10 μ g mL 6 μ L concentration−1Primary antibody, that is, Ab1 4 16 h are incubated under oC, with phosphate buffer cleaning down 3 times of pH 7.4；Continue the bovine serum albumin(BSA) of 20 μ L 3% of drop coating Nonspecific binding site is blocked, with phosphate buffer cleaning down 3 times of pH 7.4, by 20 μ L various concentration prostates Antigen is added dropwise to electrode surface, after being incubated for 30 min at room temperature, is washed 3 times with the phosphate buffer of pH 7.4；Continue to be added dropwise The Ab2-HRP of 20 μ L steps (4) synthesis；It is 1.0 mg mL that above-mentioned electrode, which is then placed in concentration,-14-CN and concentration be 1.5 mg mL-1H2O2Mixed solution in 10 min, finally use ultrapure water electrode 3 times；
(6) modified electrode for handling step (5) well is platinum electrode to electrode as working electrode, and reference electrode is Ag/ AgCl electrode, bias numerical value are 0 V, and for xenon lamp as source stimulating, electrolytic cell is the phosphate-buffered liquid system of pH 7.4, measurement Electric current I-T curve carries out the detections of photoelectric properties.
Beneficial effects of the present invention:
(1) present invention is low in cost, experimental implementation is simple, easy control of reaction conditions.
(2) Ab2 marker HRP can be catalyzed 4-CN and H2O2Reaction generates sediment and is attached to electrode surface rapidly, greatly Hinder electrode surface charge transmission, lead to the reduction of photosignal.
(3) Ab2-HRP bioconjugate increases the steric hindrance of sensor through specific binding on antigen, into The reduction for causing photosignal of one step.
Specific embodiment
For a further understanding of the present invention, implemented in conjunction with the embodiments according to technical solution of the present invention, is provided specific Embodiment:
(1) by the WCl of 1.0 g6It is dissolved in the dehydrated alcohol of 50 mL, 4 h is heated in the water-bath of 160 oC；By what is obtained After product cooled to room temperature, respectively three times with dehydrated alcohol and milli-Q water, dry 12 h at 60 oC；
(2) the 0.3 g WO for synthesizing step (1)3It is dissolved in 60 mL ultrapure waters, is added 1.0 mmol's while agitating KI；Continue after stirring 40 min, by 1.0 mmol Bi (NO3)3·5H2O is added in above-mentioned mixed solution, then mixes to reaction Object carries out 15 min of ultrasonic treatment, 3 h is then stirred at room temperature, finally 2 min of centrifugal treating under 8000 rmp revolving speeds, uses Ultrapure water and dehydrated alcohol wash 3 times respectively, dry at 60 oC；
(3) electro-conductive glass is indium tin oxide glass (ITO), electro-conductive glass is cut into 4.0 × 0.5 cm strips, successively with third Ketone solution, secondary distilled water and dehydrated alcohol are cleaned by ultrasonic 5 min, then drying for standby under a nitrogen；Ito glass is placed on step Suddenly the WO that the concentration of (2) synthesis is 2.0 mg/mL320 min are ultrasonically treated in/BiOI mixed solution, are then forged at 200 oC After burning 2 h, ITO/WO is obtained3/ BiOI electrode；
It (4) is 20 μ g mL by 50 μ L concentration−11- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 50 μ L concentration is 10 mg mL−1N- hydroxysuccinimide solution be added 800 mL concentration be 50 mg/mL Ab2 solution in, so The HRP that the concentration of 200 mL is 1.0 mg/mL is mixed with above-mentioned solution afterwards, 3 h of incubated under agitation at 30 oC, and then 4 12 h are incubated under oC；Finally, 15 min of centrifugal treating obtains Ab2-HRP compound under 6000 rpm centrifugal rotational speeds, and use The phosphate buffer of pH 7.4 is diluted to 800 mL；
(5) ultrapure water ITO/WO3/BiOI electrode is used, is then 10 μ g mL 6 μ L concentration−1Primary antibody, that is, Ab1 4 16 h are incubated under oC, with phosphate buffer cleaning down 3 times of pH 7.4；Continue the bovine serum albumin(BSA) of 20 μ L 3% of drop coating Nonspecific binding site is blocked, with phosphate buffer cleaning down 3 times of pH 7.4, by 20 μ L various concentration prostates Antigen is added dropwise to electrode surface, after being incubated for 30 min at room temperature, is washed 3 times with the phosphate buffer of pH 7.4；Continue to be added dropwise The Ab2-HRP of 20 μ L steps (4) synthesis；It is 1.0 mg mL that above-mentioned electrode, which is then placed in concentration,-14-CN and concentration be 1.5 mg mL-1H2O2Mixed solution in 10 min, finally use ultrapure water electrode 3 times；
(6) modified electrode for handling step (5) well is platinum electrode to electrode as working electrode, and reference electrode is Ag/ AgCl electrode, bias numerical value are 0 V, and xenon lamp is as source stimulating, and electrolytic cell is that the phosphate that pH is 7.4 rushes solution, before change The concentration of column gland specific antigen, in 1.0 pg mL−1To 10 ng mL−1In range, the changing value and concentration of photocurrent response Logarithm good linear relationship, and linear equation is presented are as follows:I=161.25 12.53logc, related coefficient 0.997, Detection is limited to 0.2 pg/mL, and therefore, this work realizes high sensitivity, detects prostate-specific antigen to high stability.