Electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility and analysis application of electrochemical fast sweep voltammetry method

An analysis application and repeatability technology, which is applied in the field of electrochemical analysis technology and biosensing, can solve the problems of poor repeatability and reproducibility, cumbersome material synthesis, complex chemical reactions, etc., and achieve high reproducibility and short analysis time , the effect of high scan rate

Pending Publication Date: 2021-06-29
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods still have some limitations, such as complex chemical reactions, insufficient sensitivity, cumbersome material synthesis, etc., resulting in poor repeatability and reproducibility

Method used

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  • Electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility and analysis application of electrochemical fast sweep voltammetry method
  • Electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility and analysis application of electrochemical fast sweep voltammetry method
  • Electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility and analysis application of electrochemical fast sweep voltammetry method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 electrochemical biosensor comprises the following steps:

[0033] Bare electrode: use 1μm, 0.3μm, 0.05μm Al on the gold electrode with a diameter of 2mm 2 o 3 The powder was polished to a mirror surface, then ultrasonically washed with water and absolute ethanol for 5 min, and dried with nitrogen, the gold electrode was placed in 0.5M H 2 SO 4 The solution was scanned by cyclic voltammetry (CV) until the signal was stable. Finally, dry it again with nitrogen gas for later use.

[0034] Electrode 1: Add 5 μL of thiol DNA with a concentration of 10 μM dropwise onto the surface of the treated gold electrode, and place it in a refrigerator at 4°C overnight. After the incubation, gently rinse the surface of the electrode to remove unbound thiol DNA, and then immerse the electrode in 2-mercaptoethanol (MCH) at a concentration of 2 mM for 1 h to block the active sites that are not bound to thiol DNA .

[0035] Electrode 2: Prepare TdT reac...

Embodiment 2

[0037] Embodiment 2 feasibility analysis comprises the following steps:

[0038] According to the preparation steps of the electrochemical biosensor in Example 1 above, the TdT mixed reaction solution added dropwise to electrode 2 includes 0.2 μL of TdT solution with a concentration of 0 and 100 U / mL, 1 μL of a dTTP solution with a concentration of 10 mM, and 2 μL of 5×TdTbuffer and 2 μL of double distilled water. Other steps and experimental conditions are the same as the electrode preparation steps, and are used for the feasibility detection of the invention.

[0039] Using FSCV detection, the prepared electrode 3 is used as the working electrode, the platinum electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode, and placed in the PBS electrolyte solution of pH=7, the initial potential is set to 0V, and the termination potential is 1V. The scanning speed was set to 400V / s. The result is as figure 1 (C TdT =100U / mL), the...

Embodiment 3

[0043] Embodiment 3 TdT enzyme activity and the detection of inhibitor

[0044] According to the preparation steps of the above-mentioned Example 1 and the electrochemical response of different TdT / PP in Example 2, by changing the final concentration of TdT (0.1-190U / mL) or PP (0-9mM) in the mixed reaction solution, using FSCV To test the sensor, set the initial potential to 0V, the end potential to 1V, and the scan speed to 400V / s. like image 3 A. The voltage response of the sensor to TdT has a good linear relationship with the concentration. The linear correlation equation between the voltage response of the sensor and the concentration of TdT is ①y=0.0009x+0.050, R 2 =0.984, ②y=-0.001x-0.110, R 2 =0.991, ③y=0.0009x+0.048, R 2 =0.986 and ④y=-0.001x-0.115, R 2 = 0.994. The linear equations of the two selected periodic data are similar. Within the allowable range of error, the detection linear range is 0.5-150U / mL, and the detection limits are ①0.067U / mL, ②0.1U / mL, ③0.06...

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Abstract

The invention discloses an electrochemical fast sweep voltammetry method with high-order repeatability and reproducibility and analysis application thereof. A data form presented by the method has the characteristics of obvious high-order repeatability and repeatability, and data results in hundreds of cycles are highly consistent. The final concentration of TdT (0.1-190 U / mL) or PP (0-9 mM) in a mixed reaction solution is changed, an FSCV is adopted to test a sensor, the initial potential is set to be 0 V, the termination potential is set to be 1 V, the scanning speed is set to be 400 V / s, and on the basis, the fast sweep method is applied to the analysis and detection of TdT and a small-molecule inhibitor of TdT. The method has the advantages that the sensitivity is high, the specificity is strong, the analysis time is short, the response speed is high, the preparation is simple, the electrochemical signals with high-order repeatability and reproducibility can be output, and the low-concentration TdT activity analysis can be well realized; the detection limits are (1) 0.067 U / mL, (2) 0.1 U / mL, (3) 0.067 U / mL and (4) 0.1 U / mL respectively, the inhibitor detection IC50 values are 1.7 mM, 1.6 mM, 1.5 mM and 1.7 mM respectively, and the application prospect is good.

Description

technical field [0001] The present invention relates to two fields: electrochemical analysis technology and biosensing, especially relates to an electrochemical fast-sweep voltammetry method with high-order repeatability, and is applied to terminal deoxynucleotidyl transferase and its small molecule inhibition Agent biosensing analysis. Background technique [0002] Terminal deoxynucleotidyl transferase (TdT) differs from other DNA polymerases in its special ability to incorporate nucleotides into the 3′-OH terminus of single-stranded DNA in a template-independent manner. Expression of the TdT gene is normally suppressed in most human cells but is abnormally high in acute lymphoblastic leukemia cells and some other leukemia-associated cells. It is worth noting that acute lymphoblastic leukemia accounts for about 25% of the most common cancers in childhood, so the detection of TdT is crucial for the development of cancer-related drugs and basic biochemical research. Despite...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3275G01N27/48
Inventor 王琦郭智勇胡宇芳郝婷婷王邃
Owner NINGBO UNIV
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