WO2021180132A1 - 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法 - Google Patents

基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法 Download PDF

Info

Publication number
WO2021180132A1
WO2021180132A1 PCT/CN2021/080030 CN2021080030W WO2021180132A1 WO 2021180132 A1 WO2021180132 A1 WO 2021180132A1 CN 2021080030 W CN2021080030 W CN 2021080030W WO 2021180132 A1 WO2021180132 A1 WO 2021180132A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
carcinoembryonic antigen
modified
conductive glass
detection method
Prior art date
Application number
PCT/CN2021/080030
Other languages
English (en)
French (fr)
Inventor
张敏
谢永泽
程发良
宾倩韵
谢世磊
吕维忠
Original Assignee
东莞理工学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东莞理工学院 filed Critical 东莞理工学院
Publication of WO2021180132A1 publication Critical patent/WO2021180132A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
    • G01N21/69—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence specially adapted for fluids, e.g. molten metal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to the field of carcinoembryonic antigen detection, in particular to a carcinoembryonic antigen detection method based on a Fe 2 O 3 material monolayer photoelectrochemical sensor.
  • the traditional methods commonly used for the detection of tumor markers include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (ECL), fluorescence immunoassay (IFA), mass spectrometry and other immunoassay methods.
  • ELISA enzyme-linked immunosorbent assay
  • ECL electrochemiluminescence
  • IFA fluorescence immunoassay
  • mass spectrometry and other immunoassay methods.
  • the defects of the above detection methods include: 1. The excitation source and detection signal are the same, both of which are electrical (optical) signals. 2. The input of a larger sample volume is required. 3. Need to resort to large and complex equipment. 4. Long time period.
  • Carcinoembryonic antigen is an acidic glycoprotein with the characteristics of human embryonic antigen, which exists on the surface of cancer cells differentiated from endoderm cells. CEA is used as a specific marker for early diagnosis of colon cancer and rectal cancer. Breast cancer, lung cancer and other malignant tumors will also increase its concentration. Therefore, carcinoembryonic antigen is an important broad-spectrum tumor marker. Its low concentration detection is of great significance, and it is of great help to the early detection and treatment of cancer.
  • Photoelectrochemical detection is a rapidly developing detection technology in recent years.
  • the excitation source (light) and detection signal (current) are completely separated, which greatly reduces background noise and makes it more sensitive than traditional fluorescence and electrochemical technologies; the time period is very short ,
  • the test can be completed in about 15 to 30 seconds; the input sample amount is less, only micrograms and micro-upgrading of other sample amounts are required.
  • the prior art photoelectrochemical methods for detecting the concentration of cancer blank antigens mostly use double-layer photoelectric immunosensors, which have wide light absorption band gaps, low photoelectric absorption efficiency, and high detection limits, and cannot detect low concentrations of cancer blank antigens. , Is not conducive to the early diagnosis and condition monitoring of malignant tumors.
  • the carcinoembryonic antigen detection method based on Fe 2 O 3 material single-layer photoelectrochemical sensor includes the following steps: a. Preparation of modified ⁇ Fe 2 O 3 optoelectronic materials; b. Assembly of modified ⁇ Fe 2 O 3 optoelectronic materials in step a The detection electrode system, c. The assembled electrode system in step b is used for quantitative detection of carcinoembryonic antigen.
  • the process of preparing modified ⁇ Fe 2 O 3 is to take FTO conductive glass, clean it with ethanol, acetone, and water, and then dry it with nitrogen for later use; configure a mixture of FeCl 3 and NaNO 3 Solution: Put the FTO conductive glass into the reactor with the conductive surface facing down, and then add the configured solution to hydrothermally heat 1400°C-1600°C for 4 hours to obtain Fe2O3; the FTO conductive glass after the water heat is naturally cooled, cleaned, and put into the horse Calcined in a furnace at 7500°C-8500°C for 30 minutes to obtain modified ⁇ Fe 2 O 3 uniformly grown on conductive glass.
  • the assembled electrode system is a three-electrode system, wherein the electrode based on the modified ⁇ Fe 2 O 3 FTO conductive glass is the working electrode, the calomel electrode is the reference electrode, and the platinum wire The electrode is the counter electrode.
  • step b the preparation process of the modified ⁇ Fe 2 O 3 FTO conductive glass working electrode is assembling by layer-by-layer incubation, and the FTO conductive glass in step a is assembled with ethanol, acetone, and After washing with ionized water, drip EDS and NHS, and let stand at room temperature to activate the carboxyl groups on the electrode surface; then drip antibody and incubate at low temperature for 10h-14h to make the antibody and the electrode surface produce a stable link; then drip bovine serum albumin , To block the non-specific binding site on the antibody and retain the specific site that binds to the carcinoembryonic antigen; then drip the carcinoembryonic antigen and incubate for several hours at room temperature, so that the carcinoembryonic antigen binds to the electrode surface through the antibody to form a complete electrode system.
  • step c during the quantitative detection of carcinoembryonic antigen, the pH is 7-8, the photovoltage is 0.1v-0.3v, and the electron donor is 0.5 mol L-1-1.5 mol L-1 ascorbic acid. Phosphate buffer solution.
  • the present invention synthesizes ⁇ Fe 2 O 3 with good photoelectric absorption efficiency and photoelectric conversion efficiency as a photoelectric material combination, and constructs a photoelectric immunosensor based on photoelectric materials.
  • the light absorption band gap is wide, and the photoelectric efficiency is high.
  • the quantitative detection of low-concentration carcinoembryonic antigen is achieved, and the detection limit is lower than that of photoelectric materials in the prior art.
  • the present invention improves the sensitivity of the sensor by loading modified semiconductor optoelectronic materials (modified Fe 2 O 3 ) with high photoelectric conversion efficiency (modified Fe 2 O 3) and introducing antibody markers, so that the detection accuracy of cancer blank antigens is higher.
  • the present invention adopts the practical application of photoelectric immunosensor in the qualitative and quantitative analysis of tumor markers in human serum.
  • This detection method has high selectivity and low detection limit, which is beneficial to early diagnosis and condition monitoring of malignant tumors.
  • the detection method of the present invention has a simple process and low detection cost.
  • step a the process of preparing modified ⁇ Fe 2 O 3 is to take FTO conductive glass, clean it with ethanol, acetone, and water, and then dry it with nitrogen for use; configure a mixed solution of 1M FeCl 3 and 0.5M NaNO 3; Put the FTO conductive glass into the reactor with the conductive surface facing down, and then add the prepared solution to hydrothermally heat at 1500°C for 4 hours to obtain Fe 2 O 3 ; the FTO conductive glass after the water heat is naturally cooled, cleaned, and placed in the muffle furnace It is calcined at 8000°C for 30 minutes to obtain modified ⁇ Fe 2 O 3 uniformly grown on conductive glass.
  • alpha iron hydrothermal method 2 O 3 obtained alpha iron 2 O 3 can be grown uniformly FTO conductive glass surface, such that subsequent detection, alpha iron uniform and stable 2 O 3 For photoelectric conversion, the detection result of cancer blank antigen is more accurate.
  • the assembled electrode system is a three-electrode system, wherein the electrode based on modified ⁇ Fe 2 O 3 FTO conductive glass is the working electrode, the calomel electrode is the reference electrode, and the platinum wire electrode is the counter electrode.
  • the preparation cost of the detection material is low, and the detection accuracy is high.
  • step b the preparation process of the modified ⁇ Fe 2 O 3 FTO conductive glass working electrode is assembling by layer-by-layer incubation, and the FTO conductive glass in step a is washed with ethanol, acetone, and deionized water.
  • Figure 1 is a schematic diagram of the electron flow in the detection process of the present invention.
  • Figure 2 shows the unmodified Fe 2 O 3 scanning electron microscope image and the modified Fe 2 O 3 scanning electron microscope image.
  • Figure 3 is a schematic diagram of the assembly process of the electrode system.
  • Figure 4 is a schematic diagram of the photocurrent intensity of the electrode under different modification degrees.
  • Figure 5 is a schematic diagram of photocurrent intensity under different detection conditions.
  • Figure 6 is a schematic diagram of photocurrent intensity under different CEA concentrations.
  • the carcinoembryonic antigen detection method based on Fe 2 O 3 material single-layer photoelectrochemical sensor includes the following steps: a. Preparation of modified ⁇ Fe 2 O 3 optoelectronic materials; b. Assembly of modified ⁇ Fe 2 O 3 optoelectronic materials in step a The detection electrode system, c. The assembled electrode system in step b is used for quantitative detection of carcinoembryonic antigen.
  • FIG. 1 it is a schematic diagram of the electron flow in the detection process of the present invention.
  • a combination of optoelectronic materials with good photoelectric absorption efficiency and photoelectric conversion efficiency is screened and synthesized; methods such as covalent bonding, covalent cross-linking, affinity adsorption and other methods are used to identify and bind antigen and antibody specifically , Assemble conductive glass, photoelectric materials, and objects to be detected into complete electrodes, and use the specific binding of antigen and antibody to assemble complete photoelectric sensors; use hydrothermal, electrodeposition, and chemical immersion deposition methods to prepare photoelectric materials ; By optimizing synthesis and detection conditions, a photoelectric immunosensor based on photoelectric materials is constructed, and linear analysis is adopted to realize the application in the detection of tumor markers.
  • step a the process of preparing modified ⁇ Fe 2 O 3 is to take FTO conductive glass, clean it with ethanol, acetone, and water, and then dry it with nitrogen for later use; configure a mixed solution of 1M FeCl 3 and 0.5M NaNO 3;
  • the conductive glass is placed in the reactor with the conductive surface facing down, and then the prepared solution is added and heated at 1500°C for 4 hours to obtain Fe 2 O 3 ;
  • the FTO conductive glass after the water heating is naturally cooled, cleaned, and placed in the muffle furnace at 8000°C It is calcined for 30 minutes to obtain modified ⁇ Fe 2 O 3 uniformly grown on the conductive glass.
  • the unmodified Fe 2 O 3 SEM image and the modified Fe 2 O 3 SEM image As shown in Figure 2, the unmodified Fe 2 O 3 SEM image and the modified Fe 2 O 3 SEM image. It can be seen from Figure 2 that the surface of the modified Fe 2 O 3 scanning electron microscope image is relatively uniform, which facilitates the subsequent combination of photoelectric materials and antigens, and makes the final detection result more accurate.
  • FIG. 3 it is a schematic diagram of the assembly process of the detection electrode system of the present invention.
  • the assembled electrode system is a three-electrode system, wherein the electrode based on modified ⁇ Fe 2 O 3 FTO conductive glass is the working electrode, the calomel electrode is the reference electrode, and the platinum wire electrode is the counter electrode.
  • step b the preparation process of the modified ⁇ Fe 2 O 3 FTO conductive glass working electrode is assembling by layer-by-layer incubation, and the FTO conductive glass in step a is washed with ethanol, acetone, and deionized water, and then dropped Apply EDS and NHS, and let stand at room temperature to activate the carboxyl groups on the electrode surface; then drip the antibody and incubate at low temperature for 10h-14h to make the antibody and the electrode surface have a stable link; then drip the bovine serum albumin to block the non-specificity on the antibody
  • the binding site retains the specific site that binds to the carcinoembryonic antigen; then the carcinoembryonic antigen is dripped and incubated for several hours at room temperature, so that the carcinoembryonic antigen binds to the electrode surface through the antibody to form a complete electrode system.
  • FIG. 4 it is a schematic diagram of the photocurrent intensity of the electrode under different modification degrees.
  • a is a blank conductive glass
  • b is an electrode modified with ⁇ Fe 2 O 3
  • c is an electrode modified with ⁇ Fe 2 O 3 and an antibody
  • d is an electrode that captures carcinoembryonic antigen.
  • FIG. 5 it is a schematic diagram of photocurrent intensity under different detection conditions, where A is a schematic diagram of photocurrent intensity under different pH values, B is a schematic diagram of photocurrent intensity under different photovoltage conditions, and C is a schematic diagram of photocurrent intensity under different photovoltage conditions. Schematic diagram of photocurrent intensity at body concentration. It can be seen from Figure 5 that when the carcinoembryonic antigen is quantitatively detected, the pH is 7.4, the photovoltage is 0.2v, and the electron donor is a phosphate buffer solution of 1 mol L-1 ascorbic acid, the photocurrent intensity is the highest at this time.
  • FIG. 6 it is a schematic diagram of photocurrent intensity under different CEA concentrations. It can be seen from Figure 6 that when the cancer blank antigen is detected, the photocurrent intensity changes linearly with the concentration. When the cancer blank antigen concentration is low, the photocurrent intensity is higher, indicating the detection limit of the detection method of the present invention. Low, high detection accuracy, which is conducive to early diagnosis and condition monitoring of malignant tumors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Plasma & Fusion (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Electrochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，包括如下步骤，a、制备改性αFe 2O 3光电材料，b、取步骤a中的改性αFe 2O 3光电材料组装检测电极系统，c、采用步骤b中的组装电极系统进行癌胚抗原的定量检测。检测方法灵敏度高、选择性高、检测限低、有利于恶性肿瘤的早期诊断和病情监控。

Description

基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法
相关申请的交叉引用。
本申请要求于2020年3月12日提交中国专利局，申请号为202010168567.5，发明名称为“基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉癌胚抗原检测领域，特别是涉及一种基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法。
背景技术
目前，对肿瘤标志物的检测常用的传统方法有，酶联免疫吸附法（ELISA）、电化学发光法（ECL）、荧光免疫法（IFA）、质谱分析法等免疫分析方法。以上检测方法存在的缺陷包括：1、激发源和检测信号相同，均是电（光）信号。2、需要较大样品量的输入。3、需要借助大型复杂的仪器设备。4、时间周期较长。
癌胚抗原（carcinoembryonic antigen，CEA），是一种具有人类胚胎抗原特性的酸性糖蛋白，存在于内胚层细胞分化而来的癌症细胞表面。把CEA作为早期诊断结肠癌和直肠癌的特异性标志物，乳腺癌、肺癌及其他恶性肿瘤也会是其浓度升高，因此，癌胚抗原是一种重要的广谱肿瘤标志物。对其的低浓度检测有重要意义，对癌症的早期检查与治疗有很大帮助。
光电化学检测是近年来快速发展的检测技术，激发源（光）与检测信号（电流）完全分离，大幅降低了背景噪声，使其比传统的荧光和电化学技术灵敏度更高；时间周期非常短，约15至30秒即可完成检测；输入样品量较少，只需微克、微升级别的样品量。
 技术问题
但现有技术的光电化学用于检测癌坯抗原浓度的方法，多采用双层光电免疫传感器，光吸收带隙宽、光电吸收效率低，检测限高，不能对低浓度的癌坯抗原进行测定，不利于恶性肿瘤的早期诊断和病情监控。
 技术解决方案
根据本申请的各种实施例，提供一种灵敏度高、选择性高、检测限低、有利于恶性肿瘤的早期诊断和病情监控的基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法。
基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，包括如下步骤，a、制备改性αFe 2O 3光电材料，b、取步骤a中的改性αFe 2O 3光电材料组装检测电极系统，c、采用步骤b中的组装电极系统进行癌胚抗原的定量检测。
在其中一个实施例中，步骤a中，制备改性αFe 2O 3的过程为，取FTO导电玻璃，以乙醇、丙酮、水分别清洗超声后氮气吹干备用；配置FeCl 3与NaNO 3的混合溶液；将FTO导电玻璃导电面朝下放入反应釜，再加入配置好的溶液进行水热1400℃-1600℃4小时，得到Fe2O3；水热后的FTO导电玻璃自然冷却、清洗，放入马弗炉7500℃-8500℃煅烧30分钟，得到均匀生长在导电玻璃上的改性αFe 2O 3。
在其中一个实施例中，步骤b中，组装的电极系统为三电极系统，其中基于改性αFe 2O 3的FTO导电玻璃为基底的电极为工作电极，甘汞电极为参比电极，铂丝电极为对电极。
在其中一个实施例中，步骤b中，改性αFe 2O 3的FTO导电玻璃工作电极的制备过程为，采用逐层孵育法进行组装，将步骤a中的FTO导电玻璃用乙醇、丙酮、去离子水洗净后，滴涂EDS与NHS，室温下静置，使电极表面羧基活化；然后滴涂抗体，低温孵育10h-14h，使抗体与电极表面产生稳定的链接；然后滴涂牛血清蛋白，封闭抗体上的非特异性结合位点，保留与癌胚抗原结合的特异性位点；然后滴涂癌胚抗原，常温孵育数小时，使癌胚抗原通过抗体与电极表面结合，形成完整的电极体系。
在其中一个实施例中，步骤c中，癌胚抗原定量检测时，PH为7-8，光电压为0.1v-0.3v，电子供体为0.5 mol L-1-1.5 mol L-1抗坏血酸的磷酸盐缓冲溶液。
 有益效果
1、一方面，本发明合成了具有较好光电吸收效率和光电转换效率的αFe 2O 3作为光电材料组合，构建了基于光电材料的光电免疫传感器，光吸收带隙宽，光电效率高，实现了低浓度癌胚抗原的定量检测，检测限低于现有技术中的光电材料。第二方面，本发明通过负载高光电转换效率的改性半导体光电材料（改性Fe 2O 3）和引入抗体标志物的途径，提高了传感器的灵敏度，使得癌坯抗原的检测精度更高。第三方面，本发明采用光电免疫传感器在人血清中肿瘤标志物的定性和定量分析的实际应用，此种检测方法选择性高，检测限低，有利于恶性肿瘤的早期诊断和病情监控。第四方面，本发明的检测方法，流程简单，检测成本低。
2、步骤a中，制备改性αFe 2O 3的过程为，取FTO导电玻璃，以乙醇、丙酮、水分别清洗超声后氮气吹干备用；配置1M FeCl 3与0.5M NaNO 3的混合溶液；将FTO导电玻璃导电面朝下放入反应釜，再加入配置好的溶液进行水热1500℃ 4小时，得到Fe 2O 3；水热后的FTO导电玻璃自然冷却、清洗，放入马弗炉8000℃煅烧30分钟，得到均匀生长在导电玻璃上的改性αFe 2O 3。在改性αFe 2O 3光电材料的制备过程中，采用水热法制备αFe 2O 3，得到的αFe 2O 3能均匀生长在FTO导电玻璃表面，使得后续检测时，αFe 2O 3均匀稳定的进行光电转化，从而对癌坯抗原的检测结果更为精确。
3、步骤b中，组装的电极系统为三电极系统，其中基于改性αFe 2O 3的FTO导电玻璃为基底的电极为工作电极，甘汞电极为参比电极，铂丝电极为对电极。检测材料制备成本低，且检测精度高。
4、步骤b中，改性αFe 2O 3的FTO导电玻璃工作电极的制备过程为，采用逐层孵育法进行组装，将步骤a中的FTO导电玻璃用乙醇、丙酮、去离子水洗净后，滴涂EDS与NHS，室温下静置，使电极表面羧基活化；然后滴涂抗体，低温孵育10h-14h，使抗体与电极表面产生稳定的链接；然后滴涂牛血清蛋白，封闭抗体上的非特异性结合位点，保留与癌胚抗原结合的特异性位点；然后滴涂癌胚抗原，常温孵育数小时，使癌胚抗原通过抗体与电极表面结合，形成完整的电极体系。采用此种方法制定工作电极，电极表面羧基活化程度高，由于抗体上除了癌坯抗原结合的特异性位点之外的所有非特异性结合位点均已封闭，在检测过程中，抗体和癌坯抗原的结合更为精确，检测结果精确度高，可靠性高，且检测限低。
5、在检测时，由于pH对生物材料的活性和光电材料都有影响，因此不同环境pH对检测效果有着一定影响，在pH为7-8，特别是PH为7.4时，此时整个体系的光电流最大。同时，不同光电材料的光电压不同，αFe 2O 3的最优光电压为0.2V。在检测过程中，电子供体向整个体系提供了绝大多数的自由电子，它的浓度对检测结果有影响，在抗坏血酸浓度达到0.1M后，继续增大抗坏血酸的浓度也不会使光电流有明显增加，因此，电子供体选用0.5 mol L-1-1.5 mol L-1抗坏血酸的磷酸盐缓冲溶液，最优为选用1 mol L-1抗坏血酸。
附图说明
为了更好地描述和说明这里公开的那些发明的实施例和/或示例，可以参考一幅或多幅附图。用于描述附图的附加细节或示例不应当被认为是对所公开的发明、目前描述的实施例和/或示例以及目前理解的这些发明的最佳模式中的任何一者的范围的限制。
图1 为本发明的检测过程中的电子流动原理图。
图2为未改性的Fe 2O 3扫描电镜图像和改性的Fe2O3扫描电镜图像。
图3为电极体系组装过程示意图。
图4为不同修饰程度下电极的光电流强度示意图。
图5为不同的检测条件下的光电流强度示意图。
图6为不同CEA浓度下的光电流强度示意图。
 本发明的实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，包括如下步骤，a、制备改性αFe 2O 3光电材料，b、取步骤a中的改性αFe 2O 3光电材料组装检测电极系统，c、采用步骤b中的组装电极系统进行癌胚抗原的定量检测。
如图1所示，为本发明的检测过程中的电子流动原理图。
从图1可看出，在单层的无标光电免疫传感器中，由于受空间位阻效应的影响，电子从溶液中的电子供体到光电材料（或电子从光电材料到电子受体）的转移过程受到阻碍，导致了光生电子-空穴对的复合，改变了光电流的输出，最后通过光电流的变化来确定样品中肿瘤标志物（CEA）的含量。
本发明中，筛选、合成了具有良好光电吸收效率和光电转换效率的光电材料组合；利用共价键合法、共价交联法、亲和吸附法等方法和抗原抗体之间的特异识别与结合，将导电玻璃、光电材料、待检测物组装为完整的电极，利用抗原抗体的特异性结合，组装完整的光电传感器；采用水热法、电沉积法和化学浸渍沉积法等方法，制备光电材料；通过优化合成与检测条件，构建基于光电材料的光电免疫传感器，采用线性分析，实现在肿瘤标记物检测中的应用。
步骤a中，制备改性αFe 2O 3的过程为，取FTO导电玻璃，以乙醇、丙酮、水分别清洗超声后氮气吹干备用；配置1M FeCl 3与0.5M NaNO 3的混合溶液；将FTO导电玻璃导电面朝下放入反应釜，再加入配置好的溶液进行水热1500℃4小时，得到Fe 2O 3；水热后的FTO导电玻璃自然冷却、清洗，放入马弗炉8000℃煅烧30分钟，得到均匀生长在导电玻璃上的改性αFe 2O 3。
如图2所示，为未改性的Fe 2O 3扫描电镜图像和改性的Fe2O3扫描电镜图像。从图2可看出，经改性的Fe 2O 3扫描电镜图像表面比较均匀，便于后续光电材料与抗原的结合，使得最后的检测结果更为精确。
如图3所示，为本发明的检测电极体系组装的过程示意图。
步骤b中，组装的电极系统为三电极系统，其中基于改性αFe 2O 3的FTO导电玻璃为基底的电极为工作电极，甘汞电极为参比电极，铂丝电极为对电极。
步骤b中，改性αFe 2O 3的FTO导电玻璃工作电极的制备过程为，采用逐层孵育法进行组装，将步骤a中的FTO导电玻璃用乙醇、丙酮、去离子水洗净后，滴涂EDS与NHS，室温下静置，使电极表面羧基活化；然后滴涂抗体，低温孵育10h-14h，使抗体与电极表面产生稳定的链接；然后滴涂牛血清蛋白，封闭抗体上的非特异性结合位点，保留与癌胚抗原结合的特异性位点；然后滴涂癌胚抗原，常温孵育数小时，使癌胚抗原通过抗体与电极表面结合，形成完整的电极体系。
如图4所示，为不同修饰程度下电极的光电流强度示意图。其中，a为空白导电玻璃，b为修饰了αFe 2O 3的电极，c为修饰了αFe 2O 3和抗体的电极，d为捕捉了癌胚抗原的电极。
从图4可看出，空白导电玻璃几乎没有光电流响应。修饰了αFe 2O 3后，光电流响应有了极大的提高。依次修饰抗体牛血清蛋白、癌胚抗原后，光电流响应逐渐下降，证明电极的组装是成功的，各个材料均进入了相应位置。
如图5所示，为不同检测条件下的光电流强度示意图，其中，A为不同pH值的条件下光电流强度示意图，B为不同光电压条件下的光电流强度示意图，C为不同电子供体浓度下的光电流强度示意图。由图5可看出，癌胚抗原定量检测时，PH为7.4，光电压为0.2v，电子供体为1 mol L-1抗坏血酸的磷酸盐缓冲溶液时，此时光电流强度最大。
如图6所示，为不同CEA浓度下的光电流强度示意图。从图6可看出，在进行癌坯抗原检测时，光电流强度随浓度的变化呈线性变化，当癌坯抗原浓度较低时，光电流强度较高，说明本发明的检测方法的检测限低，检测精度高，有利于恶性肿瘤的早期诊断和病情监控。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (1)

1、基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，其特征在于：包括如下步骤，a、制备改性αFe 2O 3光电材料，b、取步骤a中的改性αFe 2O 3光电材料组装检测电极系统，c、采用步骤b中的组装电极系统进行癌胚抗原的定量检测。
2、根据权利要求1所述的基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，其特征在于：步骤a中，制备改性αFe 2O 3的过程为，取FTO导电玻璃，以乙醇、丙酮、水分别清洗超声后氮气吹干备用；配置FeCl 3与NaNO 3的混合溶液；将FTO导电玻璃导电面朝下放入反应釜，再加入配置好的溶液进行水热1400℃-1600℃4小时，得到Fe 2O 3；水热后的FTO导电玻璃自然冷却、清洗，放入马弗炉7500℃-8500℃煅烧30分钟，得到均匀生长在导电玻璃上的改性αFe2O3。
3、根据权利要求2所述的基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，其特征在于：步骤b中，组装的电极系统为三电极系统，其中基于改性αFe 2O 3的FTO导电玻璃为基底的电极为工作电极，甘汞电极为参比电极，铂丝电极为对电极。
4、根据权利要求3所述的基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，其特征在于：步骤b中，改性αFe 2O 3的FTO导电玻璃工作电极的制备过程为，采用逐层孵育法进行组装，将步骤a中的FTO导电玻璃用乙醇、丙酮、去离子水洗净后，滴涂EDS与NHS，室温下静置，使电极表面羧基活化；然后滴涂抗体，低温孵育10h-14h，使抗体与电极表面产生稳定的链接；然后滴涂牛血清蛋白，封闭抗体上的非特异性结合位点，保留与癌胚抗原结合的特异性位点；然后滴涂癌胚抗原，常温孵育数小时，使癌胚抗原通过抗体与电极表面结合，形成完整的电极体系。
5、根据权利要求4所述的基于Fe 2O 3材料单层光电化学传感器的癌胚抗原检测方法，其特征在于：步骤c中，癌胚抗原定量检测时，PH为7-8，光电压为0.1v-0.3v，电子供体为0.5 mol L-1-1.5 mol L-1抗坏血酸的磷酸盐缓冲溶液。
PCT/CN2021/080030 2020-03-12 2021-03-10 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法 WO2021180132A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010168567.5A CN111307902B (zh) 2020-03-12 2020-03-12 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法
CN202010168567.5 2020-03-12

Publications (1)

Publication Number Publication Date
WO2021180132A1 true WO2021180132A1 (zh) 2021-09-16

Family

ID=71145626

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/080030 WO2021180132A1 (zh) 2020-03-12 2021-03-10 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法

Country Status (2)

Country Link
CN (1) CN111307902B (zh)
WO (1) WO2021180132A1 (zh)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114113262A (zh) * 2021-11-29 2022-03-01 山西大学 一种Ti-C3N4/ZnO纳米棒复合材料的制备方法及其应用
CN114280123A (zh) * 2021-12-20 2022-04-05 济南大学 一种用于四环素检测的光电化学传感器制备方法
CN114384131A (zh) * 2021-10-13 2022-04-22 河海大学 一种全固态z型异质结的制备方法及应用
CN114609218A (zh) * 2022-05-01 2022-06-10 济南大学 一种用于检测神经元特异性烯醇化酶的光电化学免疫传感器的制备方法
CN114674896A (zh) * 2022-03-28 2022-06-28 济南大学 一种基于控制释放zif-8屏蔽壳层的光电化学nse传感器的制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111307902B (zh) * 2020-03-12 2024-01-12 东莞理工学院 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103308560A (zh) * 2013-06-04 2013-09-18 中国科学院微电子研究所 一种室温检测nh3的气体传感器的制作方法
CN104122393A (zh) * 2014-07-31 2014-10-29 济南大学 一种光电化学三维纸芯片的制备及其在肿瘤检测中的应用
CN106645350A (zh) * 2016-12-29 2017-05-10 西南大学 聚多巴胺修饰n型半导体材料在构建光电免疫传感器中的应用
CN110779976A (zh) * 2019-11-15 2020-02-11 东莞理工学院 基于UiO-66(NH2)-硫化银复合材料的癌坯抗原检测方法
CN110823972A (zh) * 2019-11-21 2020-02-21 湖北工程学院 基于Fe2O3电极的无酶葡萄糖光电化学传感器及其制备方法与检测方法
CN111307902A (zh) * 2020-03-12 2020-06-19 东莞理工学院 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106855533A (zh) * 2017-01-17 2017-06-16 济南大学 一种基于rGO/CdSe:Ca的前列腺特异性抗原夹心型光电化学传感器的制备方法
CN107045010B (zh) * 2017-01-19 2019-03-15 济南大学 基于二硫化锡-介孔氮化碳的光电化学传感器的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103308560A (zh) * 2013-06-04 2013-09-18 中国科学院微电子研究所 一种室温检测nh3的气体传感器的制作方法
CN104122393A (zh) * 2014-07-31 2014-10-29 济南大学 一种光电化学三维纸芯片的制备及其在肿瘤检测中的应用
CN106645350A (zh) * 2016-12-29 2017-05-10 西南大学 聚多巴胺修饰n型半导体材料在构建光电免疫传感器中的应用
CN110779976A (zh) * 2019-11-15 2020-02-11 东莞理工学院 基于UiO-66(NH2)-硫化银复合材料的癌坯抗原检测方法
CN110823972A (zh) * 2019-11-21 2020-02-21 湖北工程学院 基于Fe2O3电极的无酶葡萄糖光电化学传感器及其制备方法与检测方法
CN111307902A (zh) * 2020-03-12 2020-06-19 东莞理工学院 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN, YAN ET AL.: "Detection of CEA in human serum using surface-enhanced Raman spectroscopy coupled with antibody-modified Au and γ-Fe2O3@Au nanoparticles", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 121, 15 January 2016 (2016-01-15), XP029421013, ISSN: 0731-7085, DOI: 10.1016/j.jpba.2016.01.027 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114384131A (zh) * 2021-10-13 2022-04-22 河海大学 一种全固态z型异质结的制备方法及应用
CN114113262A (zh) * 2021-11-29 2022-03-01 山西大学 一种Ti-C3N4/ZnO纳米棒复合材料的制备方法及其应用
CN114280123A (zh) * 2021-12-20 2022-04-05 济南大学 一种用于四环素检测的光电化学传感器制备方法
CN114280123B (zh) * 2021-12-20 2023-06-30 济南大学 一种用于四环素检测的光电化学传感器制备方法
CN114674896A (zh) * 2022-03-28 2022-06-28 济南大学 一种基于控制释放zif-8屏蔽壳层的光电化学nse传感器的制备方法
CN114674896B (zh) * 2022-03-28 2023-07-04 济南大学 一种基于控制释放zif-8屏蔽壳层的光电化学nse传感器的制备方法
CN114609218A (zh) * 2022-05-01 2022-06-10 济南大学 一种用于检测神经元特异性烯醇化酶的光电化学免疫传感器的制备方法

Also Published As

Publication number Publication date
CN111307902B (zh) 2024-01-12
CN111307902A (zh) 2020-06-19

Similar Documents

Publication Publication Date Title
WO2021180132A1 (zh) 基于Fe2O3材料单层光电化学传感器的癌胚抗原检测方法
CN110376380B (zh) 一种电化学酶联免疫传感器及其制备与检测抗原的应用
Zheng et al. Electrochemiluminescence and photoelectrochemistry dual-signal immunosensor based on Ru (bpy) 32+-functionalized MOF for prostate-specific antigen sensitive detection
CN110927238A (zh) 一种检测前列腺特异性抗原的夹心型光电化学传感器的制备方法及应用
CN112505120B (zh) 一种双电极光电化学免疫传感器及其制备方法
CN109613244B (zh) 一种Ag@Pt-CuS标记的免疫传感器的制备方法及应用
Sun et al. Chemiluminescence excited paper-based photoelectrochemical competitive immunosensing based on porous ZnO spheres and CdS nanorods
Zhong et al. Dual-wavelength responsive photoelectrochemical aptasensor based on ionic liquid functionalized Zn-MOFs and noble metal nanoparticles for the simultaneous detection of multiple tumor markers
CN113092452A (zh) 一种生物化学传感器的制备方法及应用
CN110441535B (zh) 一种基于Pd NCs功能化CuInOS检测降钙素原的电化学免疫传感器的制备方法
CN114923968A (zh) 一种检测新冠病毒核壳蛋白的光电化学生物传感器的制备方法及应用
CN113588758A (zh) 一种基于AgBiS2的光电化学传感器检测金黄色葡萄球菌的方法
Li et al. A novel photoelectrochemical sensor based on SiNWs@ PDA for efficient detection of myocardial infarction
CN112526135A (zh) 一种检测前列腺特异性抗原的光电化学生物传感器的制备方法及应用
Sun et al. Silver-functionalized gC 3 N 4 nanohybrids as signal-transduction tags for electrochemical immunoassay of human carbohydrate antigen 19-9
CN109856076B (zh) 检测细胞的组合物及检测方法
CN109060904B (zh) 一种基于介孔花状氧化锡复合材料的光电化学n端前脑钠肽传感器的制备方法及应用
CN112649607B (zh) 一种夹心免疫试剂盒及其应用
CN111766281B (zh) 一种基于尖晶石型锰铁氧体的夹心型光电化学免疫传感器及其制备方法
CN115290630A (zh) 一种基于聚多巴胺修饰氧化钴猝灭磷掺杂石墨氮化碳电化学发光的免疫传感器的制备方法
CN112098648B (zh) 一种检测肝癌患者血清生物标志物的方法
CN113281389A (zh) 一种基于2,4,6-三氨基嘧啶调控的类石墨相氮化碳的电化学免疫传感器的制备方法
Lin et al. Surface plasmon resonance‐enhanced photoelectrochemical immunoassay of prostate‐specific antigen based on BiOCl‐Au heterojunction
CN111766282B (zh) 一种基于尖晶石型锌铁氧体的竞争型光电化学免疫传感器的制备方法及应用
CN109884316A (zh) 一种用于检测肿瘤m2型丙酮酸激酶的试剂盒及制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21768962

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 03/01/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21768962

Country of ref document: EP

Kind code of ref document: A1