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A surface plasmon resonance sensor and its preparation and application

A surface-immobilized, miRNA-21 technology, applied in the field of biosensors, can solve the problems of immature research and application of SPR sensors

Active Publication Date: 2017-02-22
重庆医科大学国际体外诊断研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research and application of SPR sensors for miRNA in biological samples is still immature

Method used

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  • A surface plasmon resonance sensor and its preparation and application
  • A surface plasmon resonance sensor and its preparation and application
  • A surface plasmon resonance sensor and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Construction of a surface plasmon resonance sensor for detecting MicroRNA-21

[0102] 1. Materials and methods

[0103] 1.1 Materials

[0104] miRNA-21 was purchased from Dalian Takara Company, auxiliary DNA probe 1 and auxiliary DNA probe 2 were synthesized by Shanghai Sangon Bioengineering Co., Ltd., streptavidin was purchased from Sigma-Aldrich, KH 2 PO 4 , NaCl, Na 3 PO 4 .12H 2 O, EDTA-2Na and other reagents were purchased from Chongqing Maoye Chemical Reagent Co., Ltd. The composition and components of the hybridization solution were: 450mMNaCl, 30mM NaCl 3 PO 4 .12H 2 O, 3mM EDTA-2Na, 0.25% Triton×100, pH7.4.

[0105] 1.2 Testing instruments

[0106] The Biocore X-type surface plasmon resonance instrument is a product of Biocore AB, Sweden.

[0107] 1.3 Detection principle

[0108] The working chip is a bare gold chip, and the sulfhydryl-labeled capture probe is firmly immobilized on the bare gold chip through a gold-sulfur bond, and then the...

Embodiment 2

[0123] Embodiment 2 is used to detect the surface plasmon resonance sensor characterization and inspection of MicroRNA-21

[0124] 1. Characterization of Immobilized Capture Probes on a Working Chip

[0125] Such as figure 1 It was shown that the sulfhydryl-labeled capture probes could be well immobilized on the bare gold chip.

[0126] 2. The characterization of adding miRNA-21, auxiliary DNA probes 1 and 2, streptavidin and regeneration solution NaOH respectively is as follows figure 2 Shown are the SPR response signals of adding miRNA-21, auxiliary DNA probes 1 and 2, streptavidin and regeneration solution NaOH at each step:

[0127] △RU1 is the SPR response signal level of miRNA-21;

[0128] △RU2 is the SPR response signal level of the DNA concatemer formed by auxiliary DNA probes 1 and 2;

[0129] △RU3 is the SPR response signal level generated by the combination of streptavidin and auxiliary DNA probe 1-labeled biotin;

[0130] And it can be seen that the regenerat...

Embodiment 3

[0133] Example 3 is used to detect the surface plasmon resonance sensor of MicroRNA-21 and the optimization of its use conditions

[0134] We also have several important conditions in the experimental process, namely capture probes of different sequences, auxiliary DNA probes 1 and 2 of different sequences, ratio of auxiliary DNA probes 1 and 2, concentration of auxiliary DNA probes 1 and 2, The concentration of streptavidin for these three assay conditions was further optimized. For each condition, five points were selected from low concentration to high concentration to carry out a series of experiments.

[0135] 1. Investigate the influence of different capture probes on the surface plasmon resonance sensor:

[0136] The capture probes CP1, CP2, and CP3 were immobilized on the same sensor chip and sealed with MCH to prepare the sensor chip.

[0137] Add 100nM miRNA-2140μL, the flow rate is 5μL.min -1 , the obtained sensing signal is as Figure 4 , it can be seen that the ...

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Abstract

The invention provides a surface plasma resonance sensor for detecting miRNA-21. The surface plasma resonance sensor comprises a working chip, wherein the working chip is formed by fixing a capture probe aiming at miRNA-21 on a bare gold chip. The miRNA-21 is detected by using the sensor disclosed by the invention; a calibration curve is subjected to four-parameter fitting; a regression equation refers to Y=(A1-A2) / (1+(X / X0)P)+A2, wherein A1 is equal to -69.95+ / -7.34, A2 is equal to 2890.50+ / -18.50, X0 is equal to 5.27+ / -0.16, and P is equal to 0.47+ / -0.01; the detection range is between 0.01nM and 1000nM; the correlation coefficient is 0.9978; and the lowest detection limit is 0.009nM. Compared with the prior art, the non-enzyme and label-free sensor can realize real-time monitoring, is high in sensitivity, high in specificity, low in cost, easy and convenient to operate and short in detection period, is suitable for measuring actual samples and the like and is expected to become a sensor with actual application value.

Description

technical field [0001] The invention relates to the field of biosensors, in particular to a surface plasmon resonance sensor for detecting MiRNA-21 and its preparation and application. Background technique [0002] MicroRNA (miRNA) is a kind of highly conserved short-chain non-coding small molecule (18-25nt) single-stranded RNA discovered in recent years. Studies have found that it plays a variety of regulatory roles in the process of cell growth and development, and has become a tumor biological agent. hot areas of scientific research. miRNA is very stable in plasma and serum, can resist the degradation of RNA cleavage enzymes (RNases), and its content can still remain relatively stable under harsh environments. Therefore, miRNA has some characteristics required as an ideal biomarker, which makes it possible for it to be used as a biomarker for clinical diagnosis. [0003] Traditional miRNA detection methods mainly include Northern blotting, microarray hybridization, RT-P...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/552
Inventor 丁世家丁小娟颜玉蓉程伟袁泰先李胜强张晔李丹丹
Owner 重庆医科大学国际体外诊断研究院
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