Quantitative biomolecule detection method

Abstract

The invention discloses a quantitative biomolecule detection method comprising the following steps of: firstly, designing fluorescently-labeled H1* and H2* probes according to a target biomolecule sequence; secondly, heating solution with set concentration of fluorophore-labeled stem-loop-structure nucleic acid probes H1* and H2* on PCR (Polymerase Chain Reaction) instrument for 2 minutes at 95 DEG C, and then placing the solution away from light for 1 hour to 2 hours at a room temperature so as to allow a stem-loop structure to close fully; thirdly, preparing solution with set specification concentration from target biomolecules, respectively adding the biomolecule solution with different specification concentration and the H1* and H2* solution in a plurality of centrifuge tubes, and performing HCR (Hybridization Chain Reaction) on the biomolecule solution and the H1* and H2* solution which are mixed uniformly at the room temperature; after the HCR is finished, adding a proper amount of carbon materials in the mixed solution according to the concentration of the H1* and H2* in the second step, reacting the solution for 30 minutes at a room temperature, and detecting and recording fluorescent signals of the system; and finally, performing quantitative analysis on the target biomolecules by virtue of the fluorescent signals. According to the quantitative biomolecule detection method, the cost is low, the sensitivity is high, and the operation is simple.

Description

technical field [0001] The invention relates to a quantitative detection method of biomolecules, in particular to a quantitative detection method of biomolecules based on nucleic acid hybridization chain reaction. Background technique [0002] High sensitivity and high selectivity detection of important living substances such as nucleic acids and proteins is of great significance for in-depth revealing of their biological functions and disease diagnosis and other research fields. Taking nucleic acid analysis as an example, the content of some important nucleic acid fragments is often extremely low in the human body. To achieve high sensitivity and specificity detection, amplification technology is usually used. Nucleic acid amplification techniques can be mainly divided into two categories: temperature cycling amplification and constant temperature amplification. Polymerase chain reaction (PCR) is currently the most widely used temperature cycle nucleic acid amplification t...

AI Technical Summary

Problems solved by technology

However, there is still a lack of sensitive and effective means for how to analyze the HCR products and then give the quantitative information of the initial target molecules.

Currently, the reported methods for detecting HCR products mainly include gel electrophoresis and fluorescent labeling methods. The gel electrophoresis detection method has low sensitivity and cannot meet the analysis requirements of trace biomolecules; Tan et al. (Angew.Chem.Int.Ed. , 2011, 50, 401-404) were labeled with Pyrene fluorescent molecules at the two ends of the H1 and H2 probes, and Pyrene dimers would be formed after the HCR reaction, whose fluorescence emission wavelength and intensity were compared with those of H1 or H2 monomers Significant changes allow quantification of the initial priming molecule via fluorescence information from the dimer

However, this technology requires fluorescent molecular labels at both ends of each nucleic acid probe, which is cumbersome and expensive

However, if fluorescent dimer technology is not used, and conventional single fluorescent molecular labeling is performed on each H1 or H2 monomer, there will be no difference in the fluorescent signal of the system before and after the HCR reaction, and it is impossible to distinguish the HCR product

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