A digital single-molecule electrochemical detection method for alkaline phosphatase
A technology of molecular electrochemistry and phosphatase, which is applied in the field of analysis and detection, can solve the problems that hinder the application and lack of SEM, and achieve the effects of single-molecule electrochemical detection, improved reliability and accuracy, and enhanced signal-to-noise ratio
Inactive Publication Date: 2018-10-12
WUHAN UNIV
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However, the lack of reliable and accurate quantitative methods hinders the application of SEM in complex systems
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Embodiment 1
[0036] 1. Preparation of microelectrode array
[0037] We fabricated microelectrode arrays with a microfabrication method combined with soft lithography. Use ITO glass slides as substrates, place them on a homogenizer, spin-coat AZ4620 photoresist (forward rotation 600rpm, 15s; back rotation 2000rpm, 30s), put the ITO slides on a baking table at 75°C and bake for 3min , and then placed on a baking table at 105°C for 5 minutes. After the ITO glass slide is cooled, a film film with a micro-electrode array pattern designed with n micro-electrode strips is used as a mask, at 17.4mw / cm 2 Expose for 40s under the UV exposure intensity, then use developer (AZ400K:H 2 O=1:3) Develop the ITO glass slide until the photoresist in the exposed area is just removed and rinsed with ultrapure water. Then soak the ITO glass slide in concentrated hydrochloric acid, wash off the ITO film on the other parts of the ITO glass slide except the photoresist, and obtain a microelectrode array contai...
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Abstract
The invention discloses a method for detecting alkaline phosphatase through digital single-molecule electrochemistry. The method comprises the steps that single alkaline phosphatase (ALP) catalyzes a substrate p-amino phenol phosphate (a-APP) of single alkaline phosphatase (ALP) to generate reductive p-aminophenol (p-AP), p-AP can immediately reduce Ag<+> into Ag<0> to be deposited on the surface of microelectrode, and an electrical signal is obtained through metal stripping voltammetry finally. According to the method, independent nanometer micro-electrode arrays are constructed by means of soft lithography and used for a digital analyzing high-throughput parallel experiment. By diluting enzyme concentration and controlling the volume reaction liquid, it is guaranteed that each microelectrode at most contains a single enzyme molecule, and therefore single molecule detection of alkaline phosphatase is achieved. By combining digital analysis, the detection limit of ALP is reduced to 1 aM from 50 aM, the sensitivity and reliability of the method are effectively improved, and the method is successfully used for ALP detection of a complex system of hepatoma carcinoma cells. The method has a wide application prospect in exploration of the physicochemical property of molecules, single biomolecule detection and single cell analysis.
Description
technical field [0001] The invention relates to a method for digital single-molecule electrochemical detection of alkaline phosphatase, which belongs to the field of analysis and detection. Background technique [0002] With the development of science and technology, people's exploration of life and nature has gone deep into the level of single cell and single molecule, which has brought great challenges to conventional analysis methods. Traditional experiments are aimed at molecular aggregates, and what is obtained is the average behavior of substances, which often conceals a lot of important information. Accurate and sensitive detection of single molecules, especially biomolecules such as DNA and proteins, can lead to new breakthroughs in biology and clinical medicine. In addition, single-molecule detection can reveal molecular heterogeneity, intermolecular interactions, and physicochemical properties of individual molecules. Microscopy, optics, and electronics are the t...
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IPC IPC(8): G01N27/42G01N27/48
CPCG01N27/42G01N27/48
Inventor 张志凌伍珍庞代文
Owner WUHAN UNIV