CN-121995050-A - Complete virus particle detection and quantification system and method based on droplet microfluidic

CN121995050ACN 121995050 ACN121995050 ACN 121995050ACN-121995050-A

Abstract

The invention discloses a complete virus particle detection quantification system and method based on droplet microfluidics, which belong to the technical field of micro-nano, and comprise single virus adsorption microspheres and two subsystems, wherein the single virus adsorption microspheres are coupled with antibodies through streptavidin chemical bonds, single viruses are adsorbed by means of antigen-antibody specific binding, the single virus nucleic acid detection subsystem wraps microsphere droplets with viruses, microsphere-virus sample aqueous phase liquid and fluorescent probe reagents are contained, single virus nucleic acids are detected in the droplets through RT-PCR amplification, and fluorescent probes are used for calibrating and comparing fluorescence intensity. The high-flux droplet screening subsystem analyzes droplet size and fluorescence signal intensity, identifies single-virus-containing droplets, accurately counts the fluorescence droplets, and realizes quantitative analysis of single-virus layers. The invention adopts a method combining nucleic acid and protein detection to identify complete virus particles, and realizes high-sensitivity detection and absolute quantitative analysis of a single virus layer by a droplet microfluidic technology.

Inventors

NAN LANG
WEI XUEYONG
CEN HAOZHANG
XIA HE
ZHANG CHUANYU
GAO WEIZHUO

Assignees

西安交通大学
香港城市大学

Dates

Publication Date: 20260508
Application Date: 20251215
Priority Date: 20250321

Claims (10)

1. A droplet microfluidic-based whole viral particle detection quantification system, comprising: The single virus adsorption microsphere is used for coupling with an antibody through a chemical bond of streptavidin, and realizing adsorption of the microsphere to single virus through specific combination of antigen and antibody; The single virus nucleic acid detection subsystem is used for coating the single virus adsorption microsphere with viruses to form a liquid drop coated with microsphere-virus sample aqueous phase liquid and fluorescent probe reagent, carrying out RT-PCR amplification treatment on the liquid drop, carrying out single virus layer nucleic acid molecule detection in the liquid drop, carrying out specific calibration on virus nucleic acid by using a fluorescent probe, and comparing the fluorescence intensity generated after the virus nucleic acid amplification; The high-flux droplet screening subsystem is used for analyzing the droplet size and the fluorescence signal intensity, identifying the droplets wrapped with the single viruses, and accurately counting the fluorescence droplets to realize quantitative analysis of the single virus layer.
2. The droplet microfluidic-based complete virus particle detection and quantification system according to claim 1, wherein the particle size of the single virus adsorption microsphere is 0.5-5 μm, streptavidin is modified on the surface of the single virus adsorption microsphere, and a streptavidin-biotin binding bond is adopted as a chemical bond of the streptavidin.
3. The complete virus particle detection and quantification system based on droplet micro-fluidic control according to claim 1, wherein the adsorption of the microsphere to the single virus is realized through the specific binding of antigen and antibody, and the method specifically comprises the steps that the antigen is protein on the surface of the virus, and the antibody is a monoclonal antibody capable of specifically capturing the target virus.
4. The droplet microfluidic-based complete viral particle detection and quantification system according to claim 1, wherein the fluorescent probe is an oligonucleotide fluorescent probe for specifically calibrating viral nucleic acid.
5. The droplet microfluidic-based complete viral particle detection and quantification system according to claim 1, wherein the droplet encapsulation of single virus adsorption microspheres with viruses, in particular, comprises: The liquid drop wrapping process uses a microfluidic technology, water phase liquid with microsphere-virus samples and a reagent containing fluorescent probes are introduced into a micro-channel, the water phase liquid with microsphere-virus samples and the reagent containing fluorescent probes in the micro-channel are sheared by oil phase liquid to form liquid drops wrapped with single microspheres, and the single virus adsorption microspheres in the liquid drops are adsorbed with empty virus shells, free proteins or complete virus particles.
6. The droplet microfluidic based complete viral particle detection and quantification system according to claim 1, wherein the high throughput droplet screening subsystem comprises a semiconductor pumped solid state laser, cylindrical lens, reflective mirror, objective lens, dichroic beamsplitter, beam splitting cube, high speed camera, and photomultiplier; The semiconductor pump solid laser is used for exciting fluorescent signals to obtain laser beams, the laser beams are converged through a cylindrical lens to form a slit, the slit is focused on a plane where a micro-channel is located through an objective lens after being reflected by a reflecting lens, the fluorescent signals are excited and then transmitted back along an original light path through the objective lens, the fluorescent signals are transmitted into a beam splitting cube after being reflected by a dichroic spectroscope, the beam splitting cube divides the fluorescent signals into two paths of beam splitting signals, one path of beam splitting signals are reflected into a high-speed camera to be imaged, and the other path of beam splitting signals are transmitted into a photomultiplier after being filtered by an optical filter to convert optical signals into electrical signals.
7. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 6, wherein the high-throughput droplet screening subsystem further comprises an analysis device, the analysis device is used for collecting electric signals detected by a photomultiplier tube, and the number of fluorescent droplets wrapped with single viruses is counted by analyzing and identifying the fluorescent droplets wrapped with the single viruses through a LabVIEW program.
8. A droplet microfluidic-based complete viral particle detection and quantization method, implemented based on the droplet microfluidic-based complete viral particle detection and quantization system according to any one of claims 1 to 7, comprising: S1, coupling with an antibody through a chemical bond of streptavidin to prepare a single virus adsorption microsphere, adsorbing viruses by using the single virus adsorption microsphere, and performing mixed incubation in an aqueous phase solution to form aqueous phase liquid of a microsphere-virus sample; S2, coating liquid drops in a micro-channel by utilizing aqueous phase liquid of a microsphere-virus sample and a reagent containing a fluorescent probe to form liquid drops coating single microspheres, and carrying out RT-PCR amplification treatment on the liquid drops; S3, injecting the amplified liquid drops into a micro-channel, then placing the liquid drops on an objective table for accurate alignment, scanning fluorescent signals of the liquid drops one by utilizing a liquid drop screening subsystem, and utilizing a preset threshold value and the detected fluorescent signals to realize quantitative analysis of a single virus layer.
9. The method for detecting and quantifying complete virus particles based on droplet microfluidic according to claim 7, wherein virus is adsorbed by using single virus adsorption microspheres and mixed incubation is performed in aqueous phase solution to form aqueous phase liquid of microsphere-virus sample, and specifically comprising: And (3) mixing and incubating the virus sample and the single virus adsorption microsphere in an aqueous phase solution, adjusting the number ratio of the single virus adsorption microsphere to the virus to be more than 10:1, adsorbing the single virus on the surface of the single virus adsorption microsphere, and performing centrifugation and washing operation when the virus is adsorbed on the single virus adsorption microsphere to remove free nucleic acid molecules.
10. The method for detecting and quantifying complete virus particles based on droplet microfluidic according to claim 7, wherein the method for detecting and quantifying complete virus particles based on droplet microfluidic according to claim 7 is characterized in that droplets are packed in a microchannel by using an aqueous phase liquid of a microsphere-virus sample and a reagent containing a fluorescent probe to form droplets for packing individual microspheres, and the method for carrying out RT-PCR amplification treatment on the droplets specifically comprises: introducing aqueous phase liquid with microsphere-virus sample and reagent containing fluorescent probe into the micro-channel, and then mixing the aqueous phase liquid with the oil phase solution in the micro-channel at the intersection of the micro-channel; Adjusting the flow rate to generate uniform-size liquid drops which are wrapped with single microspheres through the action of shearing force; The generated droplets are put into a nucleic acid amplification instrument for temperature circulation, polymerase chain reaction is carried out, amplification of single viruses is completed, and fluorescent signals are amplified.

Description

Complete virus particle detection and quantification system and method based on droplet microfluidic Technical Field The invention belongs to the technical field of micro-nano, and particularly relates to a complete virus particle detection and quantification system and method based on droplet micro-fluidic. Background The known viral diseases in the world are about four hundred kinds, which brings serious threat to human health and life. Taking the example of a new coronavirus that continues to be popular in recent years, the global accumulation of established cases has been up to 6 hundred million and 4000 thousands in a short four year period, and causes death in excess of 660 thousands. Virus detection plays a vital role in the monitoring and treatment of epidemic diseases. By detecting the viruses with lower concentration in the body fluid, the early diagnosis of infectious diseases can be realized, so that effective isolation and treatment measures can be timely taken, healthy people without diseases can be screened early, and inconvenience caused by long-term isolation can be avoided. The quantitative analysis of the virus can evaluate the severity of the disease, and then a proper and effective treatment scheme is adopted. For example, the quantitative detection of the EB virus can accurately reflect the growth condition of the virus in the body in the treatment process of the nasopharyngeal carcinoma, so that the quantitative detection can be used as a detection index of the metastasis and recurrence of the nasopharyngeal carcinoma. Currently, for the clinical detection of viruses, the nucleic acid or antigen protein of the virus is mainly determined by Polymerase Chain Reaction (PCR) or immunoadsorption reaction. However, both methods are performed on products after virus lysis, and cannot distinguish between infectious intact virus particles and infectious virus fragments (e.g., free nucleic acids, proteins, empty capsids, etc.), thereby reducing the accuracy of the detection and limiting the evaluation of the infectivity. Meanwhile, the quantitative analysis of viruses mostly adopts a plaque experiment, and the content of the viruses is quantified by measuring the number of plaques formed after the viruses infect cells. This method, while highly reliable, requires a long (4-10 days) incubation to form a countable plaque, greatly limiting its testing efficiency. In view of the problems existing in the current virus detection and quantitative analysis, it is an urgent need to find a detection and quantization method capable of accurately identifying complete virus particles and having high sensitivity, high precision and high throughput. Disclosure of Invention In order to overcome the defects of the prior art, the invention aims to provide a complete virus particle detection and quantification system and method based on droplet microfluidic, so as to solve the technical problem that the detection means is single in the virus detection method. In order to achieve the above purpose, the invention is realized by adopting the following technical scheme: The invention provides a complete virus particle detection and quantification system based on droplet microfluidic, which comprises the following components: The single virus adsorption microsphere is used for coupling with an antibody through a chemical bond of streptavidin, and realizing adsorption of the microsphere to single virus through specific combination of antigen and antibody; The single virus nucleic acid detection subsystem is used for coating the single virus adsorption microsphere with viruses to form a liquid drop coated with microsphere-virus sample aqueous phase liquid and fluorescent probe reagent, carrying out RT-PCR amplification treatment on the liquid drop, carrying out single virus layer nucleic acid molecule detection in the liquid drop, carrying out specific calibration on virus nucleic acid by using a fluorescent probe, and comparing the fluorescence intensity generated after the virus nucleic acid amplification; The high-flux droplet screening subsystem is used for analyzing the droplet size and the fluorescence signal intensity, identifying the droplets wrapped with the single viruses, and accurately counting the fluorescence droplets to realize quantitative analysis of the single virus layer. As a further improvement of the invention, the particle size of the single virus adsorption microsphere is 0.5-5 mu m, and streptavidin is modified on the surface of the single virus adsorption microsphere, and the chemical bond of the streptavidin adopts a streptavidin-biotin bonding bond. As a further improvement of the invention, the adsorption of the microsphere to the single virus is realized through the specific combination of antigen and antibody, and the method specifically comprises the steps that the antigen is protein on the surface of the virus, and the antibody is a monoclonal antibody capable of specifically capturing t