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Method for detecting ATP (adenosine triphosphate) based on resolution type nucleic acid aptamer and thioflavin T

A nucleic acid aptamer, thioflavin technology, applied in the field of biosensing and analysis, can solve the problems of limited use, low specificity, false positive signals, etc., and achieve the effect of rapid method and improved sensitivity

Active Publication Date: 2021-07-13
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the optical detection of biomolecules, the optical method of "turn-off" mode often produces false positive signals due to many factors causing fluorescence quenching, resulting in low detection specificity. limited use

Method used

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  • Method for detecting ATP (adenosine triphosphate) based on resolution type nucleic acid aptamer and thioflavin T
  • Method for detecting ATP (adenosine triphosphate) based on resolution type nucleic acid aptamer and thioflavin T
  • Method for detecting ATP (adenosine triphosphate) based on resolution type nucleic acid aptamer and thioflavin T

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Feasibility analysis of ATP detection

[0035] 1) DNA pretreatment: In order to remove the secondary structure of the ATP aptamer, the aptamer was pretreated before use. First, heat Apt-1 and Apt-2 with a volume of 4 µL and a concentration of 10 µM and 42 µL of buffer solution II at 90-95°C for 5-10 minutes, and then incubate on ice for 10-15 minutes.

[0036] 2) Add 4 µL of 100 µM ThT solution to the pretreated solutions containing Apt-1 and Apt-2, and react at 36~38°C for 50~60 minutes. One group was used as a blank control, and the other group was added with 4 µL of 100 mM ATP as a positive control.

[0037] Such as figure 2 As shown, the blank group has weak fluorescence, and the positive control group has obvious fluorescence enhancement signal, which is about 2 times stronger, which fully demonstrates the feasibility of the scheme.

Embodiment 2

[0038] Embodiment 2: The specificity analysis that ATP detects

[0039] Add 4 µL of ATP, GTP, CTP, UTP and dNTPs at a concentration of 100 mM to the pretreated solutions containing Apt-1 and Apt-2, and then add 4 µL of a ThT solution at a concentration of 100 µM, and finally add the buffer solution Ⅱ After the total volume of the solution reaches 100 µL, react at 36-38°C for 50-60 minutes. Samples containing only Apt-1, Apt-2 and ThT served as blank controls.

[0040] Such as image 3 As shown, the fluorescence intensities of the blank, GTP, CTP and UTP systems are relatively weak. Similar to the system with the addition of ATP, the fluorescence intensity of the system after the addition of dNTPs is also significantly enhanced. This is because the dNTPs also contain the same concentration of ATP in addition to the analogs containing ATP, so they have a significant fluorescence response. The results show that this method has Good specificity.

Embodiment 3

[0041] Embodiment 3: the sensitivity analysis of ATP detection

[0042] Add different volumes (0, 0.2, 0.4, 0.5, 2, 3, 4 µL, 100 mM) of ATP solution to the pretreated solutions containing Apt-1 and Apt-2, and then add 4 µL of 100 µM ThT solution, finally add buffer solution II until the total volume of the solution is 100 µL, then react at 36~38°C for 50~60 minutes.

[0043] Such as Figure 4 As shown, as the concentration of ATP gradually increased in the range of 0-1000 μM, the fluorescence signal intensity also gradually increased. Moreover, within the range of ATP concentration of 0-500 µM, there is a good linear relationship between the fluorescence signal intensity and the ATP concentration, and its linear regression equation is y=0.66x+364.38 (R 2 =0.96398), where y represents the fluorescence intensity value, and x represents the concentration of ATP in the system (µM). According to the 3σ rule, the limit of detection (LOD) of ATP in the buffer solution system was c...

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Abstract

The invention discloses a method for detecting ATP (adenosine triphosphate) based on a resolution type nucleic acid aptamer and thioflavin T, and belongs to the field of biosensing and analysis. The method comprises the following steps: respectively adding split aptamers Apt-1 and Apt-2 into a Tris-HCl buffer solution I, wherein the sequences of the split aptamers Apt-1 and Apt-2 are respectively Apt-1: SEQ ID NO.1 and Apt-2: SEQ ID NO.2; adding Apt-1 and Apt-2 into a Tris-HCl buffer solution II according to a molar ratio of 1:1 for pretreatment; and adding a to-be-detected solution containing ATP into a Tris-HCl buffer solution II containing ThT and the pretreated resolution type aptamer, performing fluorescence detection with 440 nm as the excitation wavelength, and when a fluorescence signal is enhanced, increasing the concentration of ATP in the to-be-detected solution, wherein the molar ratio of the ThT to the pretreated resolution type aptamer is 10:1. The method is simple, convenient, rapid and low in cost, has high selectivity and practicability and shows better detection performance in complex biological serum, and the sensitivity is improved by two orders of magnitude.

Description

technical field [0001] The invention belongs to the field of biological sensing and analysis, and in particular relates to a method for detecting adenosine triphosphate (ATP) based on a split-type nucleic acid aptamer and thioflavin T. Background technique [0002] Aptamer is a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) sequence, which is usually obtained from a nucleic acid molecule library by using the exponential enrichment ligand system evolution technique (Systematic evolution of ligands by exponentialenrichment, SELEX). filtered out. According to reports, nucleic acid aptamers can bind to a variety of target substances with high specificity and high selectivity, and when nucleic acid aptamers bind to target substances, their own configuration will change accordingly. So far, nucleic acid aptamers for various target substances have been reported, including small organic molecules (ATP, tetracycline), metal ions (Hg + 、K + , Pb 2+ 、Sr 2+ ), proteins (tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428Y02A50/30
Inventor 刘兴奋孙棋黄艳琴范曲立
Owner NANJING UNIV OF POSTS & TELECOMM
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