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Split aptamer sensor based on nanogold as well as preparation method and application of sensor

An aptamer sensor and nano-gold technology, applied in the field of sensors, can solve the problems of high detection limit, high operation difficulty, weakened affinity, etc., and achieve the effects of low detection limit, simple operation and good specificity.

Pending Publication Date: 2021-04-09
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The analysis of the above studies found that the detection limit of split-type nucleic acid aptamers for ATP detection is generally relatively high, which is not conducive to the detection of low-concentration ATP. This is because after the nucleic acid aptamer splits, the specificity is enhanced, but its affinity with the target molecule is relatively weakened. , when the concentration is too low, it is difficult to capture a small amount of ATP molecules in the solution due to steric hindrance
Some researchers enhanced the reaction process through various molecular modifications (ATP detection limit 2.4 nmol / L) and chemical process changes (ATP detection limit 0.02 nmol / L), which effectively reduced the detection limit of split-type aptamer sensors. However, the preparation process of the sensor is complicated, the operation is difficult and the relative cost is high.

Method used

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  • Split aptamer sensor based on nanogold as well as preparation method and application of sensor
  • Split aptamer sensor based on nanogold as well as preparation method and application of sensor
  • Split aptamer sensor based on nanogold as well as preparation method and application of sensor

Examples

Experimental program
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Effect test

Embodiment 1

[0041]The preparation method based on nano-gold split body sensor, the steps are as follows:

[0042]5 μmol / L modified sulfhydryl groups were activated at 10 mmol / L, pH = 7 TRIS-HCl reaction 2 h. 20 μL, 5 μmol / L of P2 solution was added, and 8 μL, 1 nmol / L of AUNPs were added. After 20 min, the temperature was added to 20 min, 1 μL, 500 mmol / L, pH = 5 citrate solution was added and oscillated at 37 ° C. 3 h was cultured, and the body P2 and the nano-gold were bonded through the Au-S key. The extra P2 chain was removed at 14,000 R / min to remove excess P2 chains, and the AUNPS @ P2 coupling can be obtained. 20 μL, 5 μmol / L of modified fluorescent group FAM was added to the AUNPS @ P2 coupled, and the medium temperature culture solution can be obtained to obtain a sensor working solution at 4 ° C.

[0043]ATP was detected: 6 of the above working solutions were added, and 20 μl of concentrations were added to an ATP of 1 nmol / L, 15 nmol / L, 65 nmol / L, 100 nmol / L, 200 nmol...

Embodiment 2

[0046]The preparation method based on nano-gold split body sensor, the steps are as follows:

[0047]The preparation step of the present embodiment is the same as in Example 1, and only 0.5 μL, 1 μL, 1.5 μl, 2 μl of, 2.5 μL, 3 μl of 500 mmol / L, pH = 5, respectively. Sodium citrate solution, prepared a working solution different from the initial fluorescence intensity, such asimage 3 . With the increase of sodium citrate content, the initial fluorescence intensity of the working solution is presented and decreased before enhancement, such asimage 3 (B). This is because the amount of sodium citrate is too small, the suitable low pH solution environment cannot be formed, and the Au-S button is less in a short time, resulting in a naked state of the part of the AUNPS surface. Single-strand base P1 is easily adapt to the nano-gold surface, the distance between the FAM and AUNPS, thereby generating energy transfer from the FAM to the AUNPS surface, resulting in fluorescence quenching. As t...

Embodiment 3

[0049]The preparation method based on nano-gold split body sensor, the steps are as follows:

[0050]The preparation step of the present embodiment is the same as that of Example 1, maintaining the P2 concentration constant, only the volume of the AUNPS increases from 1 μL to 15 μL, and the AUNPS and P2 ratios have been prepared, and the initial fluorescence spectrum such asFigure 5 (A), peak changesFigure 5 (B). As can be seen from the figure, as the AUNPS concentration increases, the working solution is highly reduced to decrease. When the AUNPS volume is 8 μL, the fluorescent signal of the working solution is the strongest. It is understood that when the AUNPS concentration is less than 8 μl, the fluorescence intensity of the working solution increases with the concentration of AUNPS, which is due to the auNPS concentration is too low, a large number of P2 can be modified on a single AUNPS, and the AUNPS surface is almost P2 chain. Enclosed; at this time, the fluorescent signal in t...

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Abstract

The invention belongs to the field of sensors, and relates to a split aptamer sensor, in particular to a split aptamer sensor based on nanogold as well as a preparation method and application of the split aptamer sensor. Based on an energy transfer principle, nanogold is used for quenching an FAM fluorophore; and in combination with a split type aptamer recognition mechanism, a high-sensitivity split type aptamer sensor based on nanogold is constructed. The preparation conditions of the sensor are subjected to experimental optimization, such as the use amount of sodium citrate in the combination process of AuNPs and P2, the ratio of AuNPs to P2, the stabilization time of a working solution and the like. When the optimized working solution is used for detecting ATP, a result shows that the detection range of the sensor is 30pmol / L to 15nmol / L, the high-sensitivity linear detection range is 30pmol / L to 3330pmol / L, the detection limit is 30pmol / L, and the sensor has high specificity and has great application potential.

Description

Technical field[0001]The present invention belongs to the field of sensors, and involves splitting aptamer sensors, particularly refers to nano-gold-based split-sensitive sensors and preparation methods and applications thereof.Background technique[0002]Triphosadenine (ATP) is a direct source of various life activity energy in the biology, which plays a key role in biochemical reactions and drug analysis. Common cardiopathy, Parkinson syndrome and Alzheimer are closely related to the concentration of ATP, and the detection of ATP concentrations is of great significance in pathology and life science research. The traditional method of detecting ATP mainly depends on expensive instrumentation, such as high performance liquid chromatography, isotope trace method, etc., although the sensitivity is high, the sample production requirements are high, and the experimental operation is cumbersome, and the instrument is expensive to limit its promotion. .[0003]Aptamer refers to an oligonucleo...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/573G01N21/64
CPCG01N33/587G01N33/582G01N33/5735G01N21/6428G01N2021/6432
Inventor 姜利英任林娇秦自瑞张培孟晓龙徐晓萍张吉涛陈青华
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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