Intracellular purine electrochemical-detection method based on enzyme catalysis
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A detection method and electrochemical technology, applied in the field of detection of purine bases in cells, can solve problems such as the inability to obtain purine elemental content, and achieve the effect of improving accuracy
Inactive Publication Date: 2014-07-23
JIAMUSI UNIVERSITY
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[0005] The purpose of the present invention is to solve the problem that the content of purine in the process of intracellular nucleotide metabolism cannot be obtained by using the prior art, and provides an electrochemical detection method for intracellular purine based on enzyme catalysis
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specific Embodiment approach 1
[0024] Embodiment 1: In this embodiment, an enzyme-catalyzed intracellular purine electrochemical detection method is implemented according to the following steps:
[0025] 1. Drawing of standard curve
[0026] Configure the standard to a concentration of 5×10 -6 mol / L~15×10 -6 mol / L solution to obtain a standard solution, use an electrochemical detector to detect the electrochemical signal peak of the standard solution, obtain the peak area of the standard through analysis, and draw the standard curve;
[0027] The standard substance described in step 1 is guanine, hypoxanthine, adenine, xanthine or xanthine oxidase;
[0028] The standard curve described in step 1 takes the electrochemical signal peak area of the standard as the ordinate, and the concentration of the standard as the abscissa;
[0029] Second, the secondary electrochemical detection of the lysate
[0030] ①, to contain 3 × 10 5 pcs / dish~5×10 5 Add 500 μL of PBS buffer solution to a 60 mm culture dish...
specific Embodiment approach 2
[0039] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the electrochemical detector described in step 1 is an electrochemical detector with a three-electrode system. Other parameters are the same as in the first embodiment.
specific Embodiment approach 3
[0040] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that the three-electrode system described in step 1 is that the ionic liquid / multi-walled carbon modified electrode is used as the working electrode, and silver / silver chloride is used as the reference electrode. The ratio electrode and platinum wire are the opposite poles. Other parameters are the same as those in Embodiment 1 or Embodiment 2.
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Abstract
The invention discloses an intracellular purine electrochemical-detection method based on enzyme catalysis, which relates to an intracellular purine base detection method and aims at solving the problem in the prior art that the content of simple substances of purine in a metabolism process of intracellular nucleotide cannot be obtained. The electrochemical-detection method comprises the following steps: I. drawing standard curves, i.e., obtaining electrochemical signals by an electrochemical detector, calculating peak areas and drawing the standard curve of each standard substance; II. electrochemically detecting lysate twice, i.e., performing primary electrochemical detection on the lysate of a to-be-detected cell to obtain a xanthine-guanine mixed signal peak and an adenine-hypoxanthine mixed signal peak; adding a xanthine oxidase solution and performing second time of electrochemical detection to obtain a guanine signal peak and an adenine signal peak; III. calculating the content of single purine, i.e., by combining the standard curves with the result obtained by subtracting the peak area obtained before adding xanthine oxidase from the peak area obtained after adding the xanthine oxidase, calculating the content of each purine base. The electrochemical-detection method can be used in the field of cell detection.
Description
technical field [0001] The invention relates to a method for detecting purine bases in cells, in particular to electrochemical detection of purines in cells catalyzed by xanthine oxidase. Background technique [0002] Cell electrochemistry, which has emerged in the past ten years, is a new frontier field based on the combination of electrochemical principles, experimental methods and cell molecular biology techniques. It detects the electrochemical signals of electroactive substances in cells to reflect their content. Variety. At present, although the theory and method are not perfect, it has become an important means of cell analysis by virtue of its simplicity, rapidity, sensitivity, non-toxicity, and low cost. [0003] As a new research field, the electrochemical response mechanism of cells is still unclear. In 2005, Ju Xing first proposed that the voltammetric response of the complete cell may be generated by the guanine in the cytoplasm quickly passing through the cell...
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