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Biosensor for detecting ATP activity and preparation method and application thereof

A biosensor and active technology, applied in the field of biosensors, can solve the problems of photobleaching by fluorescence method and poor sensitivity of colorimetric method, and achieve the effects of high sensitivity, improving signal-to-background ratio, and avoiding fluorescent photobleaching.

Inactive Publication Date: 2019-03-22
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the above-mentioned problems of detecting ATP activity in traditional methods, the present invention provides a method that uses nucleic acid chains and gold nanoparticles as substrates to achieve sensitive and accurate detection of ATP activity based on Surface-Enhanced Raman Scattering (SERS) technology, overcoming Solved the problem of photobleaching of fluorescence method and the problem of poor sensitivity of colorimetric method

Method used

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  • Biosensor for detecting ATP activity and preparation method and application thereof
  • Biosensor for detecting ATP activity and preparation method and application thereof
  • Biosensor for detecting ATP activity and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Pure ATP Concentration Detection

[0038] 1. Modification of Raman dyes and nucleic acid strands to the surface of gold nanoparticles:

[0039] a. Take 1 mL nano-gold solution in a centrifuge tube, centrifuge for 10 min, and centrifuge the two tubes at the same time for later use. Centrifuge until the supernatant is colorless and transparent, remove the supernatant, and add 300 μL of sterile water to concentrate the nano-gold solution to 3 nM. Transfer to a 1 mL glass vial, seal it with tinfoil, and add 12 ul of Raman dye (4-NTP) with a concentration of 0.25 nM.

[0040] b. After standing at room temperature for 30 minutes, add 150 μL (the concentration ratio of Walker Chain / Protect Chain hybridization solution to Track Chain is 20:1) 30 μM nucleic acid chain modified with -SH, mix well, and place at 4 °C for 24 h.

[0041] c. Slowly add 50 μL of PBS buffer several times, add magnets (soaked in aqua regia the day before) and stir for 10 min, then continue t...

Embodiment 2

[0046] Embodiment 2 The present invention detects the sensitivity of ATP method

[0047] 1. Modification of Raman dyes and nucleic acid strands to the surface of gold nanoparticles:

[0048] Same as in Example 1, the reaction substrate is still the above-mentioned gold nanoparticle solution labeled with nucleic acid.

[0049] 2. Sensitive detection in homogeneous solution

[0050] Add 2 uL of (ATP / ADP / GTP / CTP) solution containing 2 uL of exonuclease III (2 U / mL), 2 uL of 10×Buffer (10 mM Tris-HCl, 50 μM NaCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9), 10 uL of gold nanoparticles solution modified with nucleic acid strands and Raman dye (add water to make up to 20 uL reaction system), another blank control sample (blank); shake for 30 s, put Into a 37 °C water bath for 60 min.

[0051] After 60 min, the mixed solution was taken out from the water bath, the color change was observed, and detected by a Raman spectrometer to obtain the SERS spectrum of each group of samples. From the r...

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Abstract

The invention relates to the technical field of biosensors, in particular to a biosensor for detecting ATP (triphosadenine) activity based on surface-enhancement Raman scattering generated by nanogoldparticle aggregation. By means of the characteristic that ATP can be specifically combined with a nucleic acid aptamer of the ATP, Walker Chains of the surfaces of the nanogold particles are released, so that shorter Track Chains on the surfaces of nanogold are catalytically hydrolyzed by means of nuclease, and therefore, the nanogold particles lose nucleic acid protection and are aggregated in ahigh salt buffer solution to generate a surface plasma resonance effect, thereby enhancing the electromagnetic field intensity of the surfaces of the nanogold particles greatly. A Raman dye marked onthe surfaces of the nanogold particles generates a surface-enhancement Raman scattering (SERS) effect and Raman spectra are shown in special positions. When a reaction solution does not contain ATP,Protect Chains cannot be replaced, so that a follow-up nanogold aggregation reaction cannot be carried out, and therefore, no Raman scattering spectra are generated. The biosensor detects the ATP activity quickly, sensitively and safely by taking nucleic acid chains modified on the surfaces of the nanogold particles.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a biosensor for detecting ATP (adenosine triphosphate) activity based on the surface-enhanced Raman scattering generated by the agglomeration of gold nanoparticles and its preparation method and application. Background technique [0002] Adenosine Triphosphate (ATP) is composed of adenine (A) and three phosphate groups, the chemical formula is C 10 h 16 N 5 o 13 P 3 , the molecular weight is 507.184. It exists in all organisms from microorganisms to higher animals and plants, and is the direct source of energy for various life activities. However, the content of ATP itself in the body is not high, and its molar concentration in cells is usually 1-10 mM. The main function of ATP in cells is to provide energy, participate in the metabolism of fat, protein, sugar and nucleic acid, and play an irreplaceable role in maintaining the normal function of organisms. ATP is an im...

Claims

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Application Information

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IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 王玉王海旺黄加栋刘素王敬锋宋晓蕾张雪李莎莎赵一菡瞿晓南张儒峰
Owner UNIV OF JINAN
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