A method for the detection of biomacromolecules based on nanohomogeneous time-resolved fluorescence immunoassay and droplet microfluidics

Abstract

The invention belongs to the field of micro-fluidic chips, and relates to a biological macromolecule detection method. An alpha-LISA technology and a droplet-based microfluidics technology are combined and optimized to prepare a micro-fluidic chip liquid. A novel biological macromolecule detection method with a high sensitivity is provided. A three-phase mixing technology is adopted. A passive mixing structure is used to fully and evenly mix the reaction liquid. A cross focusing technology is used to form droplets. The generated droplets have the advantage of smaller volume, and thus the reactions become faster. Moreover, the requirements on the samples are lower, the reactions are specific, sensitive, fast, and full; the washing does not need to be carried out after reactions; complicated complexes such as complete proteins, enzyme complex, bacteriophage, and the like, can be detected; the technical bottleneck of conventional commercial liquid chips at present can be broken through; the provided method can be applied to fast clinical biological macromolecule detection; the detection sensitivity and specificity are both improved, and the required sample amount is reduced.

Description

Technical field [0001] The invention belongs to the field of biomedical research and clinical application, and relates to a detection method of biological macromolecules; in particular, it relates to a microfluidic chip based on nano homogeneous time-resolved fluorescence immunity and droplet microfluidic technology and a method for detecting biological macromolecules . Background technique [0002] The prior art discloses that the liquid-phase chip technology is a modern biochip technology developed under the background of post-genomics with functional genomics and proteomics as the research object. This technology uses antigen-antibody high-specific recognition ability and high-strength binding ability to capture specific targets for further qualitative and quantitative research, such as detection of protein markers, capture of tumor cells, etc. Compared with the traditional solid-phase chip based on the ELISA principle, the liquid-phase chip has the advantages of large flux, ...

AI Technical Summary

Problems solved by technology

However, the traditional solid-liquid two-phase chip based on ELISA technology has the following disadvantages: 1. The antibody needs to be fixed on the chip plate, which will lead to an increase in the antibody fixation step for chip production. 2. Since the reaction is a solid-liquid two-phase reaction, Therefore, the reaction is only carried out on the bottom surface of the liquid chamber where the antibody is fixed, and the reaction efficiency is greatly reduced; 3. After several reactions, the antibody is exhausted, which is not conducive to the reuse of the chip; 4. During the application of the solid-liquid chip, at least 6-step washing, the process is cumbersome;

[0003] As a representative product of the liquid phase chip, Flexmap 3D is a device for biomolecular detection based on xMAP technology (a new generation of liquid phase chip detection platform developed by Luminex), which is based on xMAP technology. Compared with the solid-phase chip clinical application methods (such as ELISA method), although there are advantages such as high specificity, high throughput, high sensitivity, good reproducibility, and less amount of detection serum, it also has the following disadvantages at the same time: 1. After the antigen-antibody reaction, the Flexmap 3D liquid phase chip needs to wash the unspecific antibodies, and the washing step will not only increase the test time, but also the process is cumbersome. The fixed antigen is washed away, resulting in false negatives, which seriously affects the authenticity of the test results; 2. Due to the large size of the entire detection reaction system, a large volume of antigen and antibody is required to participate in the reaction, which increases the additional consumption of detection reagents and is not conducive to For the detection of rare samples, at the same time, the large reaction system also forces the antibody to be incubated for a long time to ensure that the reaction is fully carried out, resulting in a prolonged overall reaction time, which is not conducive to the application of this equipment to the screening of large-scale clinical samples

[0004] The liquid phase chip based on the prior art has defects such as low specificity, cumbersome detection steps, large detection error, slow reaction rate, large consumption of reagents and samples, etc. Phase-time-resolved fluorescence immunoassay and droplet microfluidics microfluidic chip and method for detecting biomacromolecules

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