Hydrogel-Based Method for Fluorometric Analysis of HCR and Cation Exchange Reactions

A cation exchange and fluorescence analysis technology, applied in the field of molecular detection, can solve the problems of limited trace analyte detection and complicated operation, and achieve the effect of increasing probe loading, improving reaction efficiency and improving amplification efficiency.

Active Publication Date: 2021-09-28
NANJING UNIV
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Problems solved by technology

However, most of the detection is carried out in a homogeneous solution, using magnetic beads for separation, the operation is relatively cumbersome, and the large detection solution volume also limits the detection of trace analytes

Method used

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  • Hydrogel-Based Method for Fluorometric Analysis of HCR and Cation Exchange Reactions
  • Hydrogel-Based Method for Fluorometric Analysis of HCR and Cation Exchange Reactions
  • Hydrogel-Based Method for Fluorometric Analysis of HCR and Cation Exchange Reactions

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: in combination with figure 1 , Silanization of glass sheets and preparation of hydrogel arrays

[0037] (1) Preparation of silanized substrates: Ultrasonic cleaning of ordinary glass sheets with acetone, ethanol and deionized water for 2 minutes respectively, blowing dry with nitrogen gas, and then treating them with a plasma cleaner to activate surface hydroxyl groups, and then putting them into 0.05% (v / v) silylating agent (3-acryloyloxypropyltrichlorosilane) in anhydrous toluene for 1 hour in a nitrogen-filled glove bag. Afterwards, the glass slides were cleaned with toluene, ethanol and deionized water, blown dry with nitrogen gas, and cured in an oven at 100°C for 2 hours, and stored in a dry box for future use.

[0038] (2) Preparation of hydrogel array: first prepare the prepolymer solution, 7.5% (v / v) PEG-DA (MW700), 22.5% (v / v) 20mM PEG-DA aqueous solution (MW 3400), 40% (v / v) PEG (MW 200), 15% (v / v) 20mM Acryl-PEG-COOH in water, and 10% (v / v) 6...

Embodiment 2

[0039] Embodiment 2: in combination with figure 2 , taking miRNA-21 as an example to illustrate that the fluorescence analysis method based on hydrogel HCR and cation exchange reaction is used for the detection of microRNA

[0040] (1) Probe modification: Activate the carboxyl group of the hydrogel array with 0.4M EDC / 0.1M NHS MES buffer (25mM, pH5.5) at room temperature for 40 minutes, add 10μL of 0.5μM capture solution to each array spot after washing Probe CP, placed in a humid chamber and reacted overnight at room temperature.

[0041] (2) Hybridization chain reaction: first, drop 10 μL containing different concentrations of standard miRNA or samples to be tested at each array point, incubate at 37°C for 1 hour, then add 10 μL of an equal volume mixed solution of 1 μM hybridization chain hairpin H1 and H2 dropwise after washing, Incubate at 37°C for 2 hours, the DNA single strand of the capture probe triggers the hybridization chain reaction between H1 and H2, and assemb...

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Abstract

The invention relates to a fluorescence analysis method based on hydrogel-based HCR and cation exchange reaction. The C-C double bond is modified on the surface of the substrate through the silanization reaction, and the radical polymerization reaction induced by ultraviolet light is used on the substrate with the help of a photomask. On-chip construction of PEG hydrogel arrays. The target molecule opens the hairpin structure of the capture probe modified on the hydrogel, triggers the hybridization chain reaction between biotinylated hairpins H1 and H2, and forms a long string of double-stranded DNA at the sensing site. Biotinylated quantum dots CdS were immobilized to the sensing sites through a biotin-streptavidin-specific binding reaction. by Ag + Initiate CdS to undergo cation exchange reaction, Cd 2+ The fluorescence of Rhod‑5N is greatly enhanced. The fluorescence of array spots is quantified by a gene chip scanner, so as to achieve highly sensitive and specific analysis of target molecules. The method of the invention has the advantages of simple operation, low cost, high sensitivity, good specificity and high universality.

Description

technical field [0001] The invention specifically relates to a fluorescence analysis method for microRNA based on hydrogel HCR and cation exchange reaction, and belongs to the technical field of molecular detection. Background technique [0002] In the field of biosensing, fluorescence analysis has many advantages over other technologies, such as simplicity, sensitivity, the availability of organic dyes with different optical properties, and the rapid development of optical imaging. Fluorescence analysis has been widely used in biological systems, Disease control and diagnosis, biological defense, environmental monitoring and other fields. The most common method of fluorescence analysis is to label the reporter molecules with fluorescent dyes, so as to quantitatively analyze the target according to the fluorescence intensity. However, further improvements in the sensitivity of fluorescence-based bioassays have been hampered by the low luminescence intensity of organic dyes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12Q1/6813
CPCC12Q1/6813G01N21/6428G01N2021/6439C12Q2525/301
Inventor 刘颖鞠熀先吴利娜
Owner NANJING UNIV
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