CN114674893B - Construction of sensor based on two-dimensional heterojunction and nano enzyme combination - Google Patents
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Abstract
The invention discloses a construction method of a sensor based on two-dimensional heterojunction and nano enzyme combination, which firstly synthesizes Ti with good photoelectrochemical signals 3 C 2 /MgIn 2 S 4 The material is a two-dimensional flower-like structure with large specific surface area, can effectively increase the antibody growth area, and simultaneously contains Ti 3 C 2 Is a novel two-dimensional material with metal-like property, which is characterized in MgIn 2 S 4 A Schottky barrier is formed between the two electrodes, so that recombination of electrons and empty is effectively avoided, and a stable photocurrent signal can be provided for construction of the sensor; in addition, znMOF enzyme is modified on the secondary antibody as a signal amplification carrier, and the steric hindrance effect brought by the introduction of the secondary antibody marker ZnMOF enzyme can effectively block electron transfer and reduce photocurrent, and simultaneously ZnMOF nano enzyme can also be used as a simulated peroxidase to effectively catalyze 4-chloro-1-naphthol to generate corresponding precipitate, so that the photocurrent is further reduced, and the sensitivity of the sensor is improved.
Description
Technical Field
The invention relates to the field of quantitative detection of prostate specific antigen, in particular to construction of a sensor based on two-dimensional heterojunction and nano-enzyme combination.
Background
It is counted that the number of new cancer patients in China is about 160 ten thousand per year, and the number of cancer deaths per year is about 130 ten thousand. In many death reasons of residents in large and medium cities in China, cancer is the first death cause, and the existing modes such as radiotherapy and chemotherapy can cause serious damage to normal cells when treating the cancer, so that the early detection and treatment of the cancer have very important significance for diagnosing malignant tumors, and a novel method for detecting malignant tumor biomarkers is established, and has very important value for early detection and treatment effect evaluation of the malignant tumors.
Photoelectrochemical immunosensors are widely used in the fields of disease diagnosis, food safety detection, environmental protection, and the like due to their high sensitivity. Photoelectrochemical immunosensor outputs a signal expressed by a sensitive biological material as an electric signal by immobilizing the sensitive biological material such as an active substance of enzyme, antigen, antibody, DNA, etc. as a recognition element. The specificity recognition of the biological material enables the photoelectrochemical immunosensor to have good specificity and sensitivity to the diagnosis of tumors.
Disclosure of Invention
The invention aims to construct a photoelectrochemical sensor for detecting prostate specific antigen based on combination of a two-dimensional heterojunction and nano enzyme.
In order to solve the technical problems, the invention is realized by the following measures: the construction of the sensor based on the combination of the two-dimensional heterojunction and the nano enzyme is characterized by comprising the following steps:
(1) Synthesis of Ti 3 C 2 : 0.8. 0.8g LiF was dissolved in 9mol/L of 10mL HCl and stirred at room temperature for 5min, then 0.5g Ti was slowly added 3 Al 2 To prevent boiling and continuously stir 24h at 25 ℃ and then washing with deionized water multiple times until the pH is around 6; finally, adding 100mL deionized water, and shaking the mixture evenly by hand until the mixture is separated; and (3) obtaining a final black solution through multiple centrifugation, and finally placing the black solution in a refrigerator at a temperature of 4 ℃ for preservation.
(2) Synthesis of Ti 3 C 2 /MgIn 2 S 4 Heterojunction: mgCl of 0.1016 and 0.1016g 2 InCl of 0.2932g 3 And 0.3005g thioacetamide are dissolved in 70mL ethylene glycol in this order, and 1.5g of Ti synthesized in the step (1) is then dissolved in 3 C 2 Adding into the mixed solution; thereafter the homogeneous solution was sealed in an autoclave and reacted at 180 ℃ 12h; after cooling the resulting product to room temperature, it was collected by centrifugation with deionized water and ethanol multiple times and dried overnight at 60 ℃.
(3) Construction of ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode: the conductive glass is indium tin oxide glass (ITO), the conductive glass is cut into 4.0x0.5 cm strips, sequentially washed by acetone solution, secondary distilled water and absolute ethyl alcohol for 5min in an ultrasonic manner, and then dried under nitrogen for standby; ti synthesized in the step (2) with the concentration of 2.0mg/mL 3 C 2 /MgIn 2 S 4 Dripping the mixture on ITO glass, drying at 60 ℃, and then calcining at 200 ℃ for 2h to obtain ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode.
(4) Synthesizing ZnMOF nano enzyme: 0.34. 0.34g of terephthalic acid and 1.2g of Zn (NO 3 ) 2 ·6H 2 O was dispersed in N, N-dimethylformamide of 40mL and heated at 90℃to 21 h. The synthesized crystalline and colorless ZnMOF was collected by centrifugation and washed several times with deionized water; finally, drying in air atmosphere to obtain ZnMOF nano enzyme.
(5) Synthesis of SA-MOF: dissolving the ZnMOF synthesized in the step (4) of 4mg in an ethanol solution of 4mL, adding 200 mu L of 3-aminopropyl triethoxysilane and carrying out ultrasonic treatment of 1h to obtain an aminated ZnMOF material, centrifuging the obtained material, dispersing the material in PBS of 2mL, adding glutaraldehyde solution of 0.2mL, incubating 1h at room temperature, adding 2mg/mL of Streptavidin (SA) of 0.2mL and incubating 1h at 4 degrees C, washing and centrifuging, and dissolving the obtained precipitate in PBS of 2mL to obtain SA-ZnMOF.
(6) Synthesis of SA-Ab2-ZnMOF: adding the secondary antibody, namely Ab2, with the concentration of 1mL of 10 mug/mL into the synthesized product in the step (5), incubating for 2h at 4 ℃, and washing 3 times by using phosphate buffer with the pH of 7.4 to remove the Ab2 without complexing, thus obtaining the Sa-Ab2-ZnMOF.
(7) Construction of a Photosensor (PEC): rinsing ITO/Ti with ultra pure water 3 C 2 /MgIn 2 S 4 The electrode was then incubated 16h with 6 μl of primary antibody, ab1, at a concentration of 10 μg/mL at 4 ℃ and thoroughly rinsed 3 times with phosphate buffer pH 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing 3 times with phosphate buffer solution with pH of 7.4, and concentrating 20 mu L of the non-specific binding sitesDripping the degree prostate antigen on the surface of the electrode, incubating for 30min at room temperature, and washing 3 times by using phosphate buffer solution with pH of 7.4; and (3) continuing to dropwise add 20 mu L of SA-Ab2-ZnMOF synthesized in the step (6), and incubating for 4h at room temperature.
(8) Electrochemical detection by a photoelectric sensor: and (3) taking the modified electrode treated in the step (7) as a working electrode, taking a platinum wire electrode as a counter electrode, taking an Ag/AgCl electrode as a reference electrode, taking a bias voltage value of 0V, taking a xenon lamp as a light source for stimulation, taking a phosphate buffer solution system (1 mol/L ascorbic acid) with pH of 7.4 as an electrolytic cell, and measuring a current I-T curve to detect photoelectric performance.
The invention has the beneficial effects that:
(1) The invention has low cost, simple experimental operation and easy control of reaction conditions.
(2) Synthetic Ti 3 C 2 /MgIn 2 S 4 Is of a two-dimensional flower-like structure, has larger specific surface area, can effectively increase the antibody growth area, and simultaneously has Ti 3 C 2 Is a novel two-dimensional material with metal-like property, which is characterized in MgIn 2 S 4 And a Schottky barrier is formed between the two electrodes, so that recombination of electrons and empty is effectively avoided, and a stable photocurrent signal can be provided for construction of the sensor.
(3) The steric hindrance effect brought by the introduction of the secondary antibody marker ZnMOF enzyme can effectively prevent electron transfer and reduce photocurrent, and simultaneously the ZnMOF nano enzyme can also be used as a simulated peroxidase to effectively catalyze 4-chloro-1-naphthol to generate corresponding precipitate, so that the photocurrent is further reduced and the sensitivity of the sensor is improved.
Detailed Description
For further understanding of the present invention, embodiments are provided by combining the technical solutions of the present invention with examples:
(1) Synthesis of Ti 3 C 2 : 0.8. 0.8g LiF was dissolved in 9mol/L of 10mL HCl and stirred at room temperature for 5min, then 0.5g Ti was slowly added 3 Al 2 To prevent boiling and continuously stir 24h at 25 ℃ and then washing with deionized water multiple times until the pH is around 6; finallyAdding 100mL deionized water, and shaking by hand until separation; and (3) obtaining a final black solution through multiple centrifugation, and finally placing the black solution in a refrigerator at a temperature of 4 ℃ for preservation.
(2) Synthesis of Ti 3 C 2 /MgIn 2 S 4 Heterojunction: mgCl of 0.1016 and 0.1016g 2 InCl of 0.2932g 3 And 0.3005g thioacetamide are dissolved in 70mL ethylene glycol in this order, and 1.5g of Ti synthesized in the step (1) is then dissolved in 3 C 2 Adding into the mixed solution; thereafter the homogeneous solution was sealed in an autoclave and reacted at 180 ℃ 12h; after cooling the resulting product to room temperature, it was collected by centrifugation with deionized water and ethanol multiple times and dried overnight at 60 ℃.
(3) Construction of ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode: the conductive glass is indium tin oxide glass (ITO), the conductive glass is cut into 4.0x0.5 cm strips, sequentially washed by acetone solution, secondary distilled water and absolute ethyl alcohol for 5min in an ultrasonic manner, and then dried under nitrogen for standby; ti synthesized in the step (2) with the concentration of 2.0mg/mL 3 C 2 /MgIn 2 S 4 Dripping the mixture on ITO glass, drying at 60 ℃, and then calcining at 200 ℃ for 2h to obtain ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode.
(4) Synthesizing ZnMOF nano enzyme: 0.34. 0.34g of terephthalic acid and 1.2g of Zn (NO 3 ) 2 ·6H 2 O was dispersed in N, N-dimethylformamide of 40mL and heated at 90℃to 21 h. The synthesized crystalline and colorless ZnMOF was collected by centrifugation and washed several times with deionized water; finally, drying in air atmosphere to obtain ZnMOF nano enzyme.
(5) Synthesis of SA-MOF: dissolving the ZnMOF synthesized in the step (4) of 4mg in an ethanol solution of 4mL, adding 200 mu L of 3-aminopropyl triethoxysilane, and carrying out ultrasonic treatment on the mixture for 1h to obtain an aminated ZnMOF material, centrifuging the obtained material, dispersing the material in PBS of 2mL, adding glutaraldehyde solution of 0.2mL, incubating the material at room temperature for 1h, adding Streptavidin (SA) of 2mg/mL of 0.2mL, incubating the material at 4 ℃ for 1h, washing and centrifuging the material, and dissolving the obtained precipitate in PBS of 2mL to obtain the SA-ZnMOF.
(6) Synthesis of SA-Ab2-ZnMOF: adding the secondary antibody, namely Ab2, with the concentration of 1mL of 10 mug/mL into the synthesized product in the step (5), incubating for 2h at 4 ℃, and washing 3 times by using phosphate buffer with the pH of 7.4 to remove the Ab2 without complexing, thus obtaining the Sa-Ab2-ZnMOF.
(7) Construction of a Photosensor (PEC): rinsing ITO/Ti with ultra pure water 3 C 2 /MgIn 2 S 4 The electrode was then incubated 16h with 6 μl of primary antibody, ab1, at a concentration of 10 μg/mL at 4 ℃ and thoroughly rinsed 3 times with phosphate buffer pH 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding site, thoroughly flushing 3 times by using phosphate buffer solution with the pH of 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of an electrode, incubating for 30min at room temperature, and washing 3 times by using the phosphate buffer solution with the pH of 7.4; and (3) continuing to dropwise add 20 mu L of SA-Ab2-ZnMOF synthesized in the step (6), and incubating for 4h at room temperature.
(8) Electrochemical detection by a photoelectric sensor: the modified electrode treated in the step (7) is used as a working electrode, a counter electrode is a platinum wire electrode, a reference electrode is an Ag/AgCl electrode, the bias value is 0V, a xenon lamp is used as a light source for stimulation, an electrolytic cell is a phosphate buffer system (1 mol/L ascorbic acid) with pH of 7.4, and the photoelectric performance is detected by measuring a current I-T curve, so that a linear equation I= -2.08log (c) -14.44, the correlation coefficient is 0.994, the detection limit is 0.04pg/mL, and the high-sensitivity detection of the prostate specific antigen is realized.
Claims (6)
1. The construction of the sensor based on the combination of the two-dimensional heterojunction and the nano enzyme is characterized by comprising the following steps:
(1) Synthesis of Ti 3 C 2 ；
(2) Synthesis of Ti 3 C 2 /MgIn 2 S 4 A heterojunction;
(3) Construction of ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode;
(4) Synthesizing ZnMOF nano enzyme;
(5) Synthesizing SA-ZnMOF;
(6) Synthesizing SA-Ab2-ZnMOF;
(7) Construction of a photoelectric sensor (PEC);
(8) Electrochemical detection of a photoelectric sensor;
wherein Ti is synthesized 3 C 2 Is characterized in that: 0.8g of LiF was dissolved in 9mol/L of 10mL of HCl and stirred at room temperature for 5min, followed by slow addition of 0.5g of Ti 3 AlC 2 To prevent boiling and continuously stirring at 25 ℃ for 24 hours, and then washing with deionized water for a plurality of times until the pH value is about 6; finally, adding 100mL of deionized water, and shaking the mixture evenly by hand until separation; obtaining a final black solution through multiple centrifugation, and finally placing the black solution in a refrigerator at 4 ℃ for preservation;
wherein Ti is synthesized 3 C 2 /MgIn 2 S 4 The heterojunction is characterized in that: 0.1016g of MgCl 2 0.2932g of InCl 3 And 0.3005g of thioacetamide are dissolved in 70mL of ethylene glycol in sequence, and then 1.5g of Ti synthesized in the step (1) is dissolved 3 C 2 Adding into the mixed solution; thereafter, the homogeneous solution was sealed in an autoclave and reacted at 180℃for 12 hours; cooling the obtained product to room temperature, centrifugally collecting the product with deionized water and ethanol for multiple times, and drying the product at 60 ℃ overnight;
wherein the construction of the Photosensor (PEC) is characterized by: rinsing ITO/Ti with ultra pure water 3 C 2 /MgIn 2 S 4 The electrode was then incubated with 6. Mu.L of primary antibody Ab1 at a concentration of 10. Mu.g/mL for 16h at 4℃and thoroughly rinsed 3 times with phosphate buffer at pH 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding site, thoroughly flushing 3 times by using phosphate buffer solution with the pH of 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of an electrode, incubating for 30min at room temperature, and washing 3 times by using the phosphate buffer solution with the pH of 7.4; continuing to dropwise add 20 mu L of SA-Ab2-ZnMOF synthesized in the step (6), incubating for 4 hours at room temperature, and adding the modified electrode in the presence of 1mM H 2 O 2 Is incubated for 20min with 10mM 4-chloro-1-naphthol solution.
2. The construction of the two-dimensional heterojunction and nano-enzyme binding sensor according to claim 1, and the construction of ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode, characterized by: the conductive glass is indium tin oxide glass (ITO), the conductive glass is cut into strips of 4.0 multiplied by 0.5cm, sequentially washed by acetone solution, secondary distilled water and absolute ethyl alcohol for 5min in an ultrasonic manner, and then dried under nitrogen for standby; ti synthesized in the step (2) with the concentration of 2.0mg/mL 3 C 2 /MgIn 2 S 4 Dripping on ITO glass, drying at 60deg.C, calcining at 200deg.C for 2 hr to obtain ITO/Ti 3 C 2 /MgIn 2 S 4 An electrode.
3. The construction of the two-dimensional heterojunction and nano-enzyme binding sensor, according to claim 1, for synthesizing ZnMOF nano-enzyme, which is characterized in that: 0.34g of terephthalic acid and 1.2g of Zn (NO) 3 ) 2 ·6H 2 O was dispersed in 40mL of N, N-dimethylformamide and heated at 90℃for 21h, and the synthesized crystalline and colorless ZnMOF was collected by centrifugation and washed several times with deionized water; finally, drying in air atmosphere to obtain ZnMOF nano enzyme.
4. The construction of the two-dimensional heterojunction and nano-enzyme binding sensor according to claim 1, wherein the SA-MOF is synthesized by the following steps: dissolving 4mg of ZnMOF synthesized in the step (4) in 4mL of ethanol solution, adding 200 mu L of 3-aminopropyl triethoxysilane, carrying out ultrasonic treatment for 1h to obtain an aminated ZnMOF material, centrifuging the obtained material, dispersing the material in 2mL of PBS, adding 0.2mL of glutaraldehyde solution, incubating for 1h at room temperature, adding 0.2mL of 2mg/mL of Streptavidin (SA), incubating for 1h at 4 ℃, and finally washing and centrifuging to obtain a precipitate, and dissolving the precipitate in 2mL of PBS to obtain the SA-ZnMOF.
5. The construction of the sensor based on two-dimensional heterojunction and nano-enzyme combination according to claim 1, synthesizing SA-Ab2-ZnMOF, which is characterized in that: adding 1mL of secondary antibody with the concentration of 10 mug/mL, namely Ab2, into the synthesized product of the step (5), incubating for 2 hours at the temperature of 4 ℃, and washing 3 times by using phosphate buffer solution with the pH of 7.4 to remove the Ab2 without complexing, thus obtaining the Sa-Ab2-ZnMOF.
6. The construction of the sensor based on the combination of the two-dimensional heterojunction and the nano-enzyme according to claim 1, wherein the electrochemical detection of the photoelectric sensor is characterized in that: the modified electrode treated in the step (7) is used as a working electrode, a counter electrode is a platinum wire electrode, a reference electrode is an Ag/AgCl electrode, the bias value is 0V, a xenon lamp is used as a light source for stimulation, and a current I-T curve is measured in an electrolytic cell of a phosphate buffer system with pH 7.4 containing 1mol/L ascorbic acid to detect photoelectric performance.
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| CN110243887A (en) * | 2019-06-18 | 2019-09-17 | 济南大学 | Construction method based on ZnO/CdS Yu CdTe quantum dual signal photoelectric sensor |
| CN113138213A (en) * | 2021-04-20 | 2021-07-20 | 济南大学 | Preparation of signal amplification sensor based on enzyme-like MOF |
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| CN113138213A (en) * | 2021-04-20 | 2021-07-20 | 济南大学 | Preparation of signal amplification sensor based on enzyme-like MOF |
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