CN109187379B - Construction and application of natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor - Google Patents
Construction and application of natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor Download PDFInfo
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Abstract
The invention discloses construction and application of a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor, which comprises the following steps: soaking the prepared zinc oxide array in the extracted natural chlorophyll solution, and covalently bonding the natural chlorophyll and the ZnO surface; adding an alpha-fetoprotein antibody (Ab), fixing the Ab on the surface of an electrode, and blocking the Ab with Bovine Serum Albumin (BSA); finally, the alpha fetoprotein with different concentrations is introduced, and the purpose of measuring the alpha fetoprotein is realized by detecting the change of a photocurrent signal caused by antigen-antibody specific reaction. The preparation method of the natural chlorophyll sensitizing agent obtained by the invention is green and environment-friendly, the operation is simple and rapid, and the prepared natural chlorophyll sensitizing zinc oxide-based photoelectrochemical immunosensor has good biocompatibility, low cost, energy conservation and environmental protection. Therefore, the method has better application prospect in the photoelectric biosensing technology and the biomedical field.
Description
Technical Field
The invention relates to a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor, which is characterized in that a sample containing alpha fetoprotein antigen is fixed on a prepared BSA/Ab/CHL-ZnO electrode, the photocurrent change value of the sample is detected, and the photocurrent change value is brought into an obtained equation, so that the purpose of detecting the alpha fetoprotein concentration is achieved, and the natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor belongs to the field of analytical chemistry and nano materials.
Background
The photoelectrochemical immunosensor is a new technology for analyzing by using the concentration change of a substance to be detected related to photogenerated current or photogenerated voltage. The zinc oxide is an important inorganic functional material, has the advantages of good photoelectric property, no toxicity, chemical stability, good biocompatibility and the like, and can be widely applied to the photoelectrochemistry biosensing technology. Pure zinc oxide can only absorb ultraviolet light due to large forbidden band width, and natural pigment has good absorption to visible light, good biocompatibility and easy extraction.
In recent years, the research of nano materials in the field of photoelectrochemical biosensors is also increasing. The nano material has excellent physical, chemical, electro-catalysis and other properties, and the photoelectric chemical biosensor based on the nano material has the excellent properties of smaller volume, higher speed, higher detection sensitivity and the like. As an important semiconductor ceramic material, zinc oxide has many useful properties such as piezoelectricity, light absorption and emission, high voltage-current nonlinearity, sensitivity to gases and chemicals, and good catalytic activity. Pure zinc oxide can only absorb ultraviolet rays, is insensitive to visible light, and needs a sensitizer to enhance the sensitivity. At present, inorganic substances (such as noble metals and quantum dots) and organic substances (macrocyclic compounds) are mainly adopted to modify the surface of ZnO, so that the absorption of visible light is enhanced. But has the disadvantages of high cost, complex process, poor biocompatibility and the like, and especially the synthesis process of the organic matters can cause environmental pollution. The natural pigment has rich sources, good biocompatibility, easy extraction and environmental protection. By using the sensitization of natural pigment, ZnO can be irradiated by visible light to generate photocurrent, and the semiconductor electrode itself can be prevented from being excited, so that the consumption caused by corrosion and dissolution can be avoided.
Disclosure of Invention
The invention aims to provide a photoelectric chemical immunosensor based on natural chlorophyll sensitized zinc oxide and a method for detecting alpha fetoprotein.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for constructing a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor comprises the following steps:
1) cleaning ITO glass, then adopting a spin-coating method to dissolve zinc acetate in ethanol to form a solution, flatly paving the zinc acetate ethanol solution on the dried ITO glass, and forming a seed layer on the surface of the ITO after calcination; 2) preparing an aqueous solution consisting of zinc nitrate hexahydrate, hexamethylenetetramine and polyethyleneimine, adding ammonia water, and preparing a ZnO nano array structure growth solution by using purified water; 3) soaking the ITO glass loaded with the ZnO seed layer on the surface, prepared in the step 1), into the ZnO nano array growth solution prepared in the step 2), reacting for 4 hours at 85 ℃, calcining at 450 ℃ to prepare a ZnO nano material, and scraping redundant ZnO on the ZnO nano material to obtain a ZnO electrode; 4) picking and cleaning the leaves of Pueraria lobata Ohwi, removing veins, air-drying, cutting, adding silicon dioxide, grinding to prevent chlorophyll from being damaged, adding ethanol for soaking, filtering to obtain a chlorophyll extracting solution, concentrating chlorophyll to obtain a chlorophyll concentrated solution, placing the ZnO electrode prepared in the step 3) into a beaker filled with the chlorophyll concentrated solution for soaking, taking out after soaking, cleaning with ethanol to remove adhesion substances, drying to obtain a natural chlorophyll sensitization ZnO nano material (CHL-ZnO), and scraping off excessive ZnO on the natural chlorophyll sensitization ZnO nano material to obtain a CHL-ZnO electrode; 5) introducing an alpha-fetoprotein antibody (Ab) onto the CHL-ZnO electrode obtained in step 4): dropwise adding 10 muL 100 mug/mL alpha fetoprotein antibody (Ab) onto the CHL-ZnO electrode, incubating at 37 ℃, and rinsing with purified water for several times to obtain the Ab/CHL-ZnO electrode; 6) and (3) dropwise adding 15 mu L of 1 wt% Bovine Serum Albumin (BSA) blocking solution onto the Ab/CHL-ZnO electrode prepared in the step 5), incubating at 37 ℃ to block the non-specific binding site, and rinsing with purified water to obtain the electrode (BSA/Ab/CHL-ZnO) serving as the photoelectrochemical immunosensor.
The invention discloses an application of a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor, which comprises the following steps: 1) soaking the prepared zinc oxide array in the extracted natural chlorophyll solution, and covalently bonding the natural chlorophyll and the ZnO surface; 2) adding an alpha-fetoprotein antibody (Ab), fixing the Ab on the surface of an electrode, and blocking the Ab with Bovine Serum Albumin (BSA); finally, the alpha fetoprotein with different concentrations is introduced, and the purpose of measuring the alpha fetoprotein is realized by detecting the change of a photocurrent signal caused by antigen-antibody specific reaction.
The method for detecting the alpha fetoprotein antigen of the natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor comprises the following steps: fixing alpha fetoprotein antigen (Ag) with different concentrations of 0.005 ng/mL-50 ng/mL on a BSA/Ab/CHL-ZnO electrode, detecting the change value of the photocurrent, wherein the photocurrent signal is weaker along with the increase of the concentration of the alpha fetoprotein antigen, and the photocurrent signal and the logarithm of the concentration of the alpha fetoprotein antigen (Ag) are in a linear relation within the concentration range of 0.005 ng/mL-50 ng/mL.
Specifically, the invention adopts the following technical scheme: the natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor is characterized in that natural chlorophyll is used as a sensitizing agent, the photocurrent response of zinc oxide under visible light irradiation is enhanced, and a natural chlorophyll sensitized zinc oxide (CHL-ZnO) electrode introduced with an alpha fetoprotein antigen antibody is used as a working electrode to carry out photocurrent detection.
The CHL-ZnO material is prepared by the following method: cleaning an electric conduction glass (ITO) electrode, spin-coating 5mM zinc acetate growth solution on an electric conduction surface, calcining at 300 ℃ for 20 min, putting the electric conduction surface in an aqueous solution containing 15 mM hexamethylenetetramine, 30 mM zinc nitrate hexahydrate, 10 mM polyethyleneimine and 0.5M ammonia water at 85 ℃ for 4 h in a water bath, taking out the electric conduction glass, drying, and calcining at 450 ℃ for 30 min to obtain a ZnO nano material; and scraping 1 x 0.5cm of ZnO on the zinc oxide nano material to obtain the ZnO electrode. Soaking the green yellow kudzuvine leaves in 200 mL of ethanol for 4 hours to obtain a natural pigment extracting solution, filtering and concentrating by 4 times, placing the dried ZnO nano material in the extracting solution for soaking for 16 hours, washing off pigments not attached to the surface by using the ethanol, taking out and drying to obtain a natural chlorophyll sensitized ZnO nano material; and scraping 1 x 0.5cm of ZnO on the natural chlorophyll sensitization ZnO nano material to obtain the CHL-ZnO electrode.
A method for detecting alpha fetoprotein based on a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor comprises the following steps: 1) putting the ITO electrode into a cleaning agent, deionized water, acetone, ethanol and the like, ultrasonically cleaning, flatly paving a zinc acetate growth solution on the ITO electrode, and calcining and carrying out water bath to obtain a ZnO nano material; and scraping 1 x 0.5cm of ZnO on the ZnO nano material to obtain the ZnO electrode. 2) Extracting natural Chlorophyll (CHL) from Pueraria chlorotolucrata leaf, soaking ZnO nanometer material in natural chlorophyll solution to obtain natural chlorophyll-sensitized ZnO nanometer material (CHL-ZnO); and scraping 1 x 0.5cm of ZnO on the natural chlorophyll sensitization ZnO nano material to obtain the CHL-ZnO electrode. And an alpha fetoprotein antibody is introduced on the surface of the zinc oxide base material to construct a zinc oxide base photoelectrochemical biosensor; 3) A three-electrode system is adopted for testing, a CHL-ZnO electrode is taken as a working electrode, a platinum wire electrode is taken as a counter electrode, saturated Ag/AgCl is taken as a reference electrode, the electrodes are inserted into 0.1M phosphate buffer solution, a xenon lamp light source is used for simulating visible light irradiation, and a photoinduced current signal is detected; 4) and (3) plotting the photoinduced current signal to the alpha fetoprotein concentration to obtain a standard curve, wherein the photocurrent signal and the logarithm of the alpha fetoprotein antigen concentration are in a linear relation within the range of 0.005 ng/mL-50 ng/mL.
The buffer solution is phosphate buffer solution, the electrolyte added in the buffer solution is NaCl, and the electrolyte concentration is 0.1M.
The invention adopts the following specific technical scheme:
preparation of natural chlorophyll sensitized ZnO
The ITO glass is subjected to ultrasonic cleaning by sequentially adopting a cleaning agent, deionized water, acetone, ethanol and the like, then 5 nM zinc acetate solution is spread on dry ITO by adopting a spin-coating method, and the ITO glass is calcined at 300 ℃ for 20 min to form a seed layer on the surface of the ITO. Reformulating with 30 mM Zn (NO)3)215 mM hexamethylenetetramine, 10 mM polyethyleneimine and 0.5M NH3·H2ZnO growth liquid of O. Soaking the ITO loaded with the ZnO seed layer into a growth solution, reacting for 4 h at 85 ℃, and calcining for 30 min at 450 ℃ to prepare a ZnO nano material; and scraping 1 x 0.5cm of ZnO on the ZnO nano material to obtain the ZnO electrode.
Picking and cleaning the leaves of Pueraria lobata Ohwi, removing veins, air-drying, weighing 11 g, cutting, adding silicon dioxide, grinding to prevent chlorophyll from being damaged, adding 200 mL of ethanol, soaking for 4 h, filtering and purifying to obtain chlorophyll extract, soaking the prepared ZnO in a beaker for 16 h, cleaning with ethanol to remove physical adhesion, taking out and drying to obtain a natural chlorophyll sensitization ZnO electrode (CHL-ZnO electrode); and scraping 1 x 0.5cm of ZnO on the natural chlorophyll sensitization ZnO nano material to obtain the CHL-ZnO electrode.
(II) preparation of immunosensor
First, an alpha-fetoprotein antibody was introduced onto the obtained CHL-ZnO electrode. And (3) dripping 10 muL 100 mug/mL alpha fetoprotein antibody on the CHL-ZnO compound, incubating at 37 ℃ for 2 h, and rinsing with purified water for several times to obtain an Ab/CHL-ZnO electrode. Then, 15 μ L of 1 wt% BSA blocking solution was added dropwise to the Ab/CHL-ZnO electrode and incubated at 37 ℃ for 30 min to block non-specific binding sites. After rinsing with purified water, the resulting electrode (designated BSA/Ab/CHL-ZnO) was used as a photoelectrochemical immunosensor. And (3) dropwise adding 10 mu L of alpha fetoprotein antigen solution with a certain concentration (0.005 ng/mL-50 ng/mL) to the prepared photoelectrochemical immunosensor, and incubating for 1 h at 37 ℃. The resulting electrode (designated as Ag/BSA/Ab/CHL-ZnO) was rinsed several times with purified water before photocurrent detection.
(III) generation and detection of photocurrent signals of natural chlorophyll sensitization sensor
The experiment was performed at room temperature using a conventional three-electrode system, in which a platinum wire was used as the counter electrode, a saturated silver chloride electrode was used as the reference electrode, and CHL-ZnO was used as the working electrode. And (3) placing the modified electrode in 0.1M phosphate buffer solution, applying a voltage of 0V, and carrying out photoelectrochemical detection under simulated natural light.
(IV) determination of alpha-fetoprotein
And (5) introducing the alpha fetoprotein antibody and the antigens with different concentrations onto the CHL-ZnO electrode, obtaining the photocurrent intensity corresponding to the concentration according to the method in the step (three) after incubation is finished, and drawing a working curve.
The invention has the advantages of
(1) The invention takes natural chlorophyll as a sensitizer, and utilizes the good photoelectric response performance of the sensitizer, so that the constructed zinc oxide-based photoelectrochemical immunosensor has high sensitivity and good reproducibility;
(2) the preparation method of the natural chlorophyll sensitizing agent obtained by the invention is green and environment-friendly, the operation is simple and rapid, and the prepared natural chlorophyll sensitizing zinc oxide-based photoelectrochemical immunosensor has good biocompatibility, low cost, energy conservation and environmental protection. Therefore, the method has better application prospect in the photoelectric biosensing technology and the biomedical field.
Drawings
FIG. 1 is a schematic diagram of the principle of the photoelectrochemical immunosensor of the present invention.
FIG. 2 is a UV-visible absorption spectrum of ZnO and CHL-ZnO.
FIG. 3 is an infrared spectrum of ZnO and CHL-ZnO.
FIG. 4 is the electrochemical impedance spectrum of different electrodes, wherein a is ZnO electrode, b is CHL-ZnO electrode, c is Ab/CHL-ZnO electrode, d is BSA/Ab/CHL-ZnO electrode, and e is 5 ng/mL Ag/BSA/Ab/CHL-ZnO electrode.
FIG. 5 is a graph showing the change in photocurrent at the alpha-fetoprotein antigen concentration (a-e = 0.005 ng/mL-50 ng/mL).
FIG. 6 is a linear plot of alpha fetoprotein antigen photocurrent signal versus concentration.
Detailed Description
The invention is described in detail below with reference to the following figures and examples:
the invention principle introduces:
the principle of the natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor is shown in fig. 1, which shows the modification process of the ZnO electrode (a in fig. 1) and the basic principle of photoelectrochemical detection (b in fig. 1). As shown in a of fig. 1, firstly, the prepared zinc oxide array is placed in the extracted natural chlorophyll solution to be soaked for 16 h, and the natural chlorophyll is covalently bonded with the surface of ZnO. Secondly, adding an alpha-fetoprotein antibody (Ab), fixing the Ab on the surface of an electrode, and blocking the Ab with Bovine Serum Albumin (BSA); finally, the alpha fetoprotein with different concentrations is introduced, and the purpose of measuring the alpha fetoprotein is realized by detecting the change of a photocurrent signal caused by antigen-antibody specific reaction. In b of fig. 1, the natural chlorophyll not only serves as a carrier to provide a biocompatible environment for the alpha fetoprotein antibody in the immunoassay, but also serves as a photosensitive substance to improve photoelectrochemical properties and enhance photocurrent under the irradiation of visible light. Specifically, under simulated sunlight, the natural chlorophyll absorbs solar energy, generates photo-generated electrons, and is directly transferred to the conduction band of ZnO by the natural chlorophyll, thereby enhancing photocurrent. More importantly, the excellent conductivity of the natural chlorophyll improves the electron transfer at the interface, and the photocurrent efficiency is remarkably improved.
The method specifically comprises the following steps:
preparation of natural chlorophyll sensitized ZnO compound
1) The ITO glass (purchased from Guangdong Fushan crystal source responsibility Co., Ltd.) is subjected to ultrasonic cleaning by sequentially adopting a cleaning agent, deionized water, acetone, ethanol and the like, then 0.0278 g of zinc acetate is dissolved in 25 mL of ethanol (purchased from national medicine group chemical reagent Co., Ltd.) solution and is flatly laid on the dry ITO by adopting a spin coating method, and the ITO glass is calcined at 300 ℃ for 20 min to form a seed layer on the surface of the ITO. 2) 0.9177 g of zinc nitrate hexahydrate (purchased from national pharmaceutical products chemical Co., Ltd.), 0.1774 g of hexamethylenetetramine (HMTA, purchased from national pharmaceutical products chemical Co., Ltd.), 0.3001 g of polyethyleneimine (PEI, purchased from national pharmaceutical products chemical Co., Ltd.) were dissolved in 50 mL of purified water, and 2.548 mL of aqueous ammonia (NH)3·H2O, purchased from national chemical group, ltd), and then 50 mL of purified water is added to prepare the growth liquid of the ZnO nano-array structure. 3) And (2) soaking the ITO loaded with the ZnO seed layer on the surface, prepared in the step 1), into the ZnO nano-array growth solution prepared in the step 2), reacting for 4 h at 85 ℃, and calcining for 30 min at 450 ℃ to prepare the ZnO nano-material. And scraping 1 x 0.5cm of ZnO on the ZnO nano material to obtain the ZnO electrode.
4) Picking and cleaning the leaves of the Pueraria lobata Ohwi, removing veins, air-drying, weighing 11 g, cutting, adding silicon dioxide (purchased from national drug group chemical reagent Co., Ltd.) to grind to prevent chlorophyll from being damaged, adding 200 mL of ethanol to soak for 4 h, filtering to obtain chlorophyll extract, concentrating chlorophyll by 4 times, putting the ZnO electrode prepared in the step 3) into a beaker to soak for 16 h, cleaning with ethanol to remove physical adhesion, taking out and drying to obtain the natural chlorophyll sensitization ZnO nano material (CHL-ZnO). And scraping 1 x 0.5cm of ZnO on the natural chlorophyll sensitization ZnO nano material to obtain the CHL-ZnO electrode.
(II) preparation of immunosensor
1) First, an alpha-fetoprotein antibody (Ab, available from tokyo biotechnology responsibility ltd) was introduced onto the CHL-ZnO electrode obtained in step (i): and (3) dropwise adding 10 muL 100 mug/mL alpha fetoprotein antibody (Ab) onto the CHL-ZnO electrode, incubating at 37 ℃ for 2 h, and rinsing with purified water for several times to obtain the Ab/CHL-ZnO electrode. 2) Then, 15 μ L of 1 wt% bovine serum albumin (BSA, purchased from Biotechnology engineering (Shanghai) Ltd.) blocking solution was added dropwise to the Ab/CHL-ZnO electrode prepared in step 1), and incubated at 37 ℃ for 30 min to block the nonspecific binding site. After rinsing with purified water, the resulting electrode (designated BSA/Ab/CHL-ZnO) was used as a photoelectrochemical immunosensor. 3) Adding 10 mu L of alpha-fetoprotein antigen (Ag, purchased from Nanjing Sanchen Biotechnology responsibility Co., Ltd.) solution with a certain concentration (0.005 ng/mL-50 ng/mL) into the photoelectrochemical immunosensor (BSA/Ab/CHL-ZnO) prepared in the step 2), incubating for 1 h at 37 ℃ to obtain an electrode (Ag/BSA/Ab/CHL-ZnO), and rinsing the finally obtained electrode (Ag/BSA/Ab/CHL-ZnO) with purified water for several times for later use.
(III) generation and detection of photocurrent signals of natural chlorophyll sensitization sensor
And (2) carrying out an experiment at room temperature by adopting a traditional three-electrode system, wherein a platinum wire is used as a counter electrode, a saturated silver chloride electrode is used as a reference electrode, and the CHL-ZnO electrode obtained in the step (I) is used as a working electrode. The CHL-ZnO electrode is placed in 0.1M phosphate buffer solution, voltage of 0V is applied, and photoelectrochemical detection is carried out under simulated natural light.
(IV) determination of alpha-fetoprotein
And (5) introducing the alpha fetoprotein antibody (Ab) and the alpha fetoprotein antigen (Ag) with different concentrations onto the CHL-ZnO electrode, obtaining the photocurrent intensity corresponding to the concentration according to the method in the step (three) after incubation is finished, and drawing a working curve.
The technology for measuring alpha fetoprotein based on the natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor comprises the following specific detection methods: and dripping a sample containing the alpha-fetoprotein antigen onto the immunosensor, and detecting the change value of the photoelectric signal.
(V) measuring the ultraviolet-visible absorption spectrum
And (2) scraping ZnO (ZnO) on the ZnO electrode obtained in the step (I) and ZnO (CHL-ZnO) on the natural chlorophyll sensitization ZnO electrode from conductive glass, dispersing the ZnO (ZnO) and the ZnO (CHL-ZnO) in 1 mL of water, and quickly measuring an ultraviolet visible absorption spectrogram after uniformly mixing, wherein the spectrogram shows that natural chlorophyll can effectively enhance the absorption of ZnO to visible light.
(VI) measuring the infrared spectrum
And (3) scraping ZnO (ZnO) on the ZnO electrode obtained in the step (I) and ZnO (CHL-ZnO) on the natural chlorophyll sensitization ZnO electrode from conductive glass, adding a proper amount of sample into ground KBr, grinding and tabletting, and analyzing. The infrared spectrum of fig. 3 can be obviously characterized, chlorophyll and zinc oxide surface form covalent binding, and the construction of the natural chlorophyll sensitization sensor is successful.
(VII) measuring electrochemical AC impedance spectrum
Measuring the sample impedance by AC impedance method on CHI660D electrochemical workstation, using saturated Ag/AgCl electrode as reference electrode, platinum wire electrode as counter electrode, ZnO electrode obtained in step (I), CHL-ZnO electrode obtained in step (II), Ab/CHL-ZnO electrode obtained in step (II), BSA/Ab/CHL-ZnO electrode and Ag/BSA/Ab/CHL-ZnO electrode 5 ng/mL as working electrodes, and K of 5mM3[Fe(CN)6]／K4[Fe(CN)6]The solution is used as an electrolytic cell solution, and the impedance of the sample is measured. The electrochemical impedance spectrum of fig. 4 is an effective means for explaining impedance change information and electrode characteristics in the modification process, and the impedance value gradually increases with the addition of chlorophyll, antibody, bovine serum albumin and antigen, which can also indicate that the construction of the conduction interface of the natural chlorophyll sensitization sensor is completed.
According to the invention, a photocurrent detection technology is used, alpha fetoprotein (Ag) antigens with different concentrations (0.005 ng/mL-50 ng/mL) are fixed on a BSA/Ab/CHL-ZnO electrode, and the photocurrent change value is detected. The experimental results are shown in the attached fig. 5 and fig. 6, and it can be seen from the graphs that the photocurrent signal is weaker as the alpha-fetoprotein antigen concentration is increased within a certain range. Within the concentration range of 0.005 ng/mL-50 ng/mL (a-e), the photocurrent signal and the logarithm of the concentration of alpha-fetoprotein antigen (Ag) form a linear relation.
Claims (1)
1. A method for constructing a natural chlorophyll sensitized zinc oxide based photoelectrochemical immunosensor comprises the following steps:
1) cleaning ITO glass, then adopting a spin-coating method to dissolve zinc acetate in ethanol to form a solution, flatly paving the zinc acetate ethanol solution on the dried ITO glass, and forming a seed layer on the surface of the ITO after calcination; 2) preparing an aqueous solution consisting of zinc nitrate hexahydrate, hexamethylenetetramine and polyethyleneimine, adding ammonia water, and preparing a ZnO nano array structure growth solution by using purified water; 3) soaking the ITO glass loaded with the seed layer on the surface, prepared in the step 1), into the growth liquid of the ZnO nano array structure, prepared in the step 2), reacting for 4 hours at 85 ℃, calcining at 450 ℃ to prepare a ZnO nano material, and scraping redundant ZnO on the ZnO nano material to obtain a ZnO electrode; 4) picking and cleaning the leaves of Pueraria lobata Ohwi, removing veins, air-drying, cutting, adding silicon dioxide, grinding to prevent chlorophyll from being damaged, adding ethanol for soaking, filtering to obtain a chlorophyll extracting solution, concentrating chlorophyll to obtain a chlorophyll concentrated solution, placing the ZnO electrode prepared in the step 3) into a beaker filled with the chlorophyll concentrated solution for soaking, taking out after soaking, cleaning with ethanol to remove adhesion substances, drying to obtain a natural chlorophyll sensitization ZnO nano material CHL-ZnO, and scraping off excessive ZnO on the natural chlorophyll sensitization ZnO nano material to obtain a CHL-ZnO electrode; 5) introducing an alpha-fetoprotein antibody Ab onto the CHL-ZnO electrode obtained in step 4): dripping 10 muL 100 mug/mL alpha fetoprotein antibody Ab onto a CHL-ZnO electrode, incubating at 37 ℃, and rinsing with purified water for several times to obtain an Ab/CHL-ZnO electrode; 6) and (3) dropwise adding 15 mu L of 1 wt% bovine serum albumin BSA blocking solution onto the Ab/CHL-ZnO electrode prepared in the step 5), incubating at 37 ℃ to block the non-specific binding site, and rinsing with purified water to obtain the electrode BSA/Ab/CHL-ZnO serving as the photoelectrochemical immunosensor.
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| CN105241937A (en) * | 2015-09-03 | 2016-01-13 | 福建医科大学 | Preparation of ZnO-based photo-electro-chemistry biosensor for detecting DNA |
| CN107064509A (en) * | 2017-04-21 | 2017-08-18 | 济南大学 | Detect the preparation and application of the optical electro-chemistry immunosensor of carcinomebryonic antigen |
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| CN105241937A (en) * | 2015-09-03 | 2016-01-13 | 福建医科大学 | Preparation of ZnO-based photo-electro-chemistry biosensor for detecting DNA |
| CN107064509A (en) * | 2017-04-21 | 2017-08-18 | 济南大学 | Detect the preparation and application of the optical electro-chemistry immunosensor of carcinomebryonic antigen |
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