Complete virus particle detection and quantification system and method based on droplet microfluidics

Abstract

The invention discloses a complete virus particle detection quantification system and method based on droplet microfluidics, and belongs to the field of micro-nano technology, the system comprises a single virus adsorption microsphere and two subsystems; the single virus adsorption microspheres are coupled with an antibody through a streptavidin chemical bond, and single viruses are adsorbed by virtue of antigen-antibody specific binding; the single virus nucleic acid detection subsystem wraps microsphere liquid drops with viruses, water-phase liquid containing microsphere-virus samples and a fluorescent probe reagent are subjected to RT-PCR amplification, single virus nucleic acid is detected in the liquid drops, and fluorescent probes are used for calibrating and comparing fluorescence intensity. And the high-throughput liquid drop screening subsystem analyzes the size of the liquid drop and the intensity of the fluorescence signal, identifies the liquid drop containing the single virus, and accurately counts the fluorescence liquid drops, so that quantitative analysis on the single virus level is realized. Complete virus particles are identified by adopting a method of combining nucleic acid detection and protein detection, and high-sensitivity detection and absolute quantitative analysis on a single virus level are realized by adopting a droplet microfluidic technology.

Description

Technical Field

[0001] This invention belongs to the field of micro-nano technology, specifically relating to a complete virus particle detection and quantification system and method based on droplet microfluidics. Background Technology

[0002] Currently, there are over 400 known viral diseases worldwide, posing a serious threat to human health and life. Taking the COVID-19 pandemic as an example, in just four years, the global cumulative number of confirmed cases has reached 640 million, resulting in over 6.6 million deaths. Viral testing plays a crucial role in the monitoring and treatment of infectious diseases. Detecting low concentrations of the virus in bodily fluids allows for early diagnosis of infectious diseases, enabling timely and effective isolation and treatment measures. It also allows for early identification of healthy individuals, avoiding the inconvenience of prolonged isolation. Quantitative analysis of the virus can assess the severity of the disease, leading to appropriate and effective treatment plans. For example, quantitative detection of Epstein-Barr virus (EBV) can accurately reflect the rise and fall of the virus in the body during nasopharyngeal carcinoma treatment, thus serving as an indicator for detecting metastasis and recurrence of nasopharyngeal carcinoma.

[0003] Currently, clinical detection of viruses primarily relies on polymerase chain reaction (PCR) or immunosorbent assay (IRISA) to measure viral nucleic acids or antigen proteins. However, both methods detect products of viral lysis, failing to distinguish between infectious intact viral particles and non-infectious viral fragments (such as free nucleic acids, proteins, and empty capsids). This reduces detection accuracy and limits their ability to assess infectivity. Meanwhile, quantitative viral analysis often employs plaque assays, quantifying viral load by measuring the number of plaques formed after viral infection of cells. While this method offers high reliability, it requires a long incubation period (4-10 days) to form countable plaques, significantly limiting its testing efficiency.

[0004] Given the current problems in virus detection and quantitative analysis, it is urgent to find a detection and quantification method that can accurately identify intact virus particles and has high sensitivity, high precision and high throughput. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a complete virus particle detection and quantification system and method based on droplet microfluidics, so as to solve the technical problem of the single detection means in virus detection methods.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a complete virus particle detection and quantification system based on droplet microfluidics, comprising: Single-virus adsorption microspheres: The single-virus adsorption microspheres are used to couple with antibodies via chemical bonds of streptavidin, and the adsorption of a single virus by the microspheres is achieved through the specific binding of antigen and antibody. Single-virus nucleic acid detection subsystem: used to encapsulate single-virus adsorbed microspheres containing viruses into droplets, forming droplets containing an aqueous liquid containing microsphere-virus samples and fluorescent probe reagents; RT-PCR amplification is performed on the droplets, nucleic acid molecules at the single-virus level are detected within the droplets, the viral nucleic acid is specifically labeled using fluorescent probes, and the fluorescence intensity generated after viral nucleic acid amplification is compared. High-throughput droplet screening subsystem: Analyzes droplet size and fluorescence signal intensity to identify droplets containing single viruses; accurately counts fluorescent droplets to achieve quantitative analysis at the single virus level.

[0007] As a further improvement of the present invention, the single virus adsorption microspheres have a particle size of 0.5~5μm, and the surface of the single virus adsorption microspheres is modified with streptavidin, wherein the chemical bond of streptavidin adopts a streptavidin-biotin binding bond.

[0008] As a further improvement of the present invention, the adsorption of a single virus by microspheres through the specific binding of antigen and antibody specifically includes: the antigen being a protein on the surface of the virus, and the antibody being a monoclonal antibody capable of specifically capturing the target virus.

[0009] As a further improvement of the present invention, the fluorescent probe is an oligonucleotide fluorescent probe that specifically labels viral nucleic acid.

[0010] As a further improvement of the present invention, the single virus-adsorbed microspheres carrying the virus are encapsulated in droplets, specifically including: The droplet encapsulation process utilizes microfluidics, where an aqueous liquid containing a microsphere-virus sample and a reagent containing a fluorescent probe are introduced into a microchannel. Within the microchannel, the aqueous liquid containing the microsphere-virus sample and the reagent containing the fluorescent probe are sheared by an oil-phase liquid, forming droplets encapsulating individual microspheres. The single-virus adsorbed microspheres within the droplets adsorb empty viral capsids, free proteins, or intact viral particles.

[0011] As a further improvement of the present invention, the high-throughput droplet screening subsystem includes a semiconductor-pumped solid-state laser, a cylindrical lens, a reflecting mirror, an objective lens, a dichroic beam splitter, a beam splitter cube, a high-speed camera, and a photomultiplier tube. The semiconductor-pumped solid-state laser is used to excite the fluorescence signal to obtain a laser beam. After the laser beam is focused by a cylindrical lens, it forms a narrow slit. After being reflected by a reflecting mirror, it is focused by an objective lens onto the plane where the microchannel is located. After the fluorescence signal is excited, it is transmitted back through the objective lens along the original optical path. After being reflected by a dichroic beam splitter, it is transmitted into a beam splitter cube. The beam splitter cube splits the fluorescence signal into two beam split signals. One beam split signal is reflected into a high-speed camera for imaging, while the other beam split signal is filtered by a filter and transmitted into a photomultiplier tube to convert the optical signal into an electrical signal.

[0012] As a further improvement of the present invention, the high-throughput droplet screening subsystem also includes an analysis device, which is used to collect the electrical signal detected by the photomultiplier tube, and analyze and identify fluorescent droplets containing single viruses through a LabVIEW program, thereby realizing the counting of the number of fluorescent droplets containing single viruses.

[0013] Secondly, the present invention also provides a complete viral particle detection and quantification method based on droplet microfluidics, implemented based on the above-mentioned complete viral particle detection and quantification system based on droplet microfluidics, comprising: S1. Single-virus adsorbed microspheres were prepared by coupling streptavidin with antibodies via chemical bonds; the single-virus adsorbed microspheres were used to adsorb viruses and were mixed and incubated in an aqueous solution to form an aqueous liquid of microsphere-virus sample. S2. Using the aqueous phase of the microsphere-virus sample and reagents containing fluorescent probes, droplets are encapsulated in microchannels to form droplets encapsulating individual microspheres. The droplets are then subjected to RT-PCR amplification. S3. Inject the amplified droplets into the microchannel, then place them on the stage for precise alignment. Use the droplet screening subsystem to scan the fluorescence signals of each droplet. Utilize preset thresholds and the detected fluorescence signals to achieve quantitative analysis at the single-virus level.

[0014] As a further improvement of the present invention, viruses are adsorbed using single-virus adsorption microspheres and then mixed and incubated in an aqueous solution to form an aqueous liquid containing microspheres and virus samples, specifically including: Virus samples and single-virus adsorbed microspheres were mixed and incubated in an aqueous solution. The ratio of single-virus adsorbed microspheres to viruses was adjusted to more than 10:1, so that individual viruses were adsorbed onto the surface of the single-virus adsorbed microspheres. When the viruses were adsorbed onto the single-virus adsorbed microspheres, centrifugation and washing were performed to remove free nucleic acid molecules.

[0015] As a further improvement of the present invention, droplets are encapsulated in a microfluidic channel using an aqueous liquid phase containing a microsphere-virus sample and a reagent containing a fluorescent probe to form droplets encapsulating individual microspheres. The droplets are then subjected to RT-PCR amplification, specifically including: An aqueous phase liquid containing microsphere-virus samples and a reagent containing fluorescent probes are introduced into a microchannel, and then the liquid merges with the oil phase solution in the microchannel at the intersection of the microchannels. Adjusting the flow rate generates uniformly sized droplets containing individual microspheres through shear force. The generated droplets are placed in a nucleic acid amplification instrument for temperature cycling, where a polymerase chain reaction occurs to amplify a single virus and amplify the fluorescence signal.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention aims to eliminate interference from viral fragments by combining antigen adsorption and nucleic acid labeling, enabling the identification of intact viral particles and achieving single-virus level detection within microdroplets, thus achieving highly sensitive and high-throughput quantitative viral analysis. Microspheres conjugated with specific antibodies adsorb single viruses by binding to surface antigens, and are then encapsulated in droplets as single microspheres. Reverse transcription polymerase chain reaction (RT-PCR) is used to specifically fluorescently label the thermally lysed viral nucleic acid molecules. Free nucleic acid molecules cannot be adsorbed by the microspheres and are washed away, while free proteins or empty viral capsids cannot be fluorescently labeled, thus allowing for the identification of intact viral particles. Based on a high-throughput droplet screening system, droplets are scanned one by one to identify droplets encapsulating single viral particles, achieving ultra-high sensitivity detection and accurate viral quantity counting. This method combines the advantages of precise identification, high sensitivity, high accuracy, and absolute quantification, and is expected to provide a theoretical and practical foundation for accurate and efficient viral detection and disease diagnosis.

[0017] (1) This method uses microspheres coupled with specific antibodies to bind and adsorb antigen proteins on the surface of the virus, thereby washing away free nucleic acid molecules to eliminate their interference. At the same time, it differs from traditional virus lysis detection methods, ensuring the integrity of virus particles to the greatest extent, laying the foundation for subsequent nucleic acid detection.

[0018] (2) This method utilizes droplet microfluidics to confine a single virus and its contained single nucleic acid molecules within a picoliter-level reaction system, thereby increasing the concentration of the analyte by hundreds of thousands of times, ensuring efficient PCR amplification and fluorescence labeling detection. The effective combination of nucleic acid detection and previous antigen detection can accurately identify complete viral particles.

[0019] (3) This method relies on an automated droplet detection platform to scan and analyze droplet samples one by one, which can effectively identify extremely low concentrations of virus particles and perform absolute quantitative analysis of their quantity. It has the advantages of high sensitivity, high precision, high throughput, and absolute quantification, and is expected to provide new ideas for the quantitative analysis of viruses and the accurate diagnosis of diseases.

[0020] (4) The automated droplet detection system can effectively excite the fluorescent signal of the droplet, and through the automated program, it can digitally detect and calibrate the photoelectric signal. At the same time, it can accurately identify the single droplet level, and the detection frequency reaches the kHz level.

[0021] Furthermore, streptavidin is attached to the surface of the microspheres, and biotin is attached to the crystallizable (Fc) region of the specific antibody. The combination of the two can couple the antibody to the surface of the microspheres.

[0022] Furthermore, by coupling microspheres with specific antibodies, the antigen proteins on the surface of the virus are adsorbed, thereby washing away free nucleic acid molecules and eliminating their interference.

[0023] Furthermore, the ratio of microspheres to viruses can be adjusted to achieve specific adsorption of individual viruses by the microspheres.

[0024] Furthermore, specific fluorescent labeling of viral nucleic acids can be achieved through RT-PCR amplification. Free proteins and empty viral capsids cannot produce fluorescent signals, thus eliminating their interference.

[0025] Furthermore, the automated droplet detection system can scan and analyze droplets one by one, achieving highly sensitive detection and quantification of single virus particles.

[0026] Furthermore, by combining antigen detection and nucleic acid detection, complete influenza virus particles can be accurately identified, and absolute quantitative analysis of the virus can be completed through high-throughput droplet screening.

[0027] In summary, this invention combines antigen detection and nucleic acid detection to identify intact viral particles, offering advantages such as high sensitivity, high precision, high throughput, and absolute quantification. This provides a new approach for quantitative viral analysis and accurate disease diagnosis. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the specific adsorption of virus particles by the microspheres of the present invention. Figure 2 This is a schematic diagram illustrating the process and principle of single-virus nucleic acid detection within droplets according to the present invention. Figure 3 This is a fluorescence image of a single virus droplet from the present invention; Figure 4 This is a schematic diagram of the high-throughput droplet screening system of the present invention; Figure 5 This is a schematic diagram of the fluorescence detection platform of the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] The present invention will now be described in further detail with reference to the accompanying drawings: See Figures 1 to 5 This embodiment provides a complete virus particle detection and quantification system based on droplet microfluidics. It includes a laser that emits a specific wavelength of laser light to excite a specific fluorescence signal for detection. The laser beam is converged by a cylindrical lens to form a slit, and then reflected by a series of lenses before being focused onto the plane of the microchannel by an objective lens. Droplets flowing through the channel are excited to fluoresce, and the fluorescence is transmitted back along the original optical path through the objective lens. The emitted light is reflected by a dichroic beam splitter and then into a beam-splitting cube, where it is reflected into a high-speed camera for imaging and into a photomultiplier tube, converting the optical signal into an electrical signal. The detected electrical signal is transmitted to a computer via a data acquisition card. An automated program analyzes and identifies fluorescent droplets encapsulating single viruses, and high-throughput screening is used to accurately count their numbers. The specific implementation is as follows.

[0031] The system provided in this embodiment includes: Single-virus adsorbed microspheres: The single-virus adsorbed microspheres are used to couple with antibodies via chemical bonds of streptavidin, and the adsorption of a single virus by the microspheres is achieved through the specific binding of antigen and antibody.

[0032] Preferably, in this embodiment, the single virus adsorption microspheres have a particle size of 0.5~5μm, and the surface of the single virus adsorption microspheres is modified with streptavidin, and the chemical bond of streptavidin adopts a streptavidin-biotin binding bond.

[0033] The antigen is a protein on the surface of the virus, and the antibody is a monoclonal antibody that can specifically capture the target virus.

[0034] In this embodiment, the ratio of single-virus adsorption microspheres to viruses is greater than 10:1, ensuring that only a single virus is adsorbed onto the surface of the microspheres. This embodiment uses a single-virus adsorption microsphere to virus ratio of 10:1.

[0035] Single-virus nucleic acid detection subsystem: The single-virus nucleic acid detection subsystem includes droplet encapsulation of single-virus adsorbed microspheres, nucleic acid amplification, and fluorescence labeling. Specifically, it is used to encapsulate virus-containing single-virus adsorbed microspheres into droplets, forming droplets containing an aqueous liquid containing a microsphere-virus sample and a fluorescent probe reagent; the droplets are subjected to RT-PCR amplification, nucleic acid molecules at the single-virus level are detected within the droplets, the viral nucleic acid is specifically labeled using a fluorescent probe, and the fluorescence intensity generated after viral nucleic acid amplification is compared.

[0036] Encapsulating virus-laden single-virus adsorbed microspheres into droplets, specifically including: The droplet encapsulation process utilizes microfluidics. An aqueous liquid containing a microsphere-virus sample and a reagent containing a fluorescent probe are introduced into a microchannel. The microchannel employs a focusing structure. Within the microchannel, the aqueous liquid containing the microsphere-virus sample and the reagent containing the fluorescent probe are sheared by the oil phase liquid, forming droplets encapsulating individual microspheres. The single-virus adsorbed microspheres within the droplets adsorb empty viral capsids, free proteins, or intact viral particles.

[0037] After encapsulating microspheres in droplets, viral lysis and nucleic acid amplification were performed. When the microspheres in the droplets adsorbed empty viral capsids or free proteins after viral lysis, they did not contain viral nucleic acid and could not be fluorescently labeled. Therefore, there was no fluorescent signal in the droplets after nucleic acid amplification. When the microspheres in the droplets adsorbed intact viral particles, viral nucleic acid was present after lysis. The fluorescence signal intensity was significantly enhanced after nucleic acid amplification, forming a clear contrast with the other droplets.

[0038] Fluorescent probes are fluorescent probes that can specifically label viral nucleic acids.

[0039] High-throughput droplet screening subsystem: Analyzes droplet size and fluorescence signal intensity to identify droplets containing single viruses; accurately counts fluorescent droplets to achieve quantitative analysis at the single virus level.

[0040] The high-throughput droplet screening subsystem includes two functions: droplet identification and virus identification. It calculates the total number of droplets and viruses by setting different fluorescence signal intensity thresholds. Specifically, the high-throughput droplet screening subsystem includes a semiconductor-pumped solid-state laser, cylindrical lenses, reflecting mirrors, objective lenses, a dichroic beam splitter, a beam-splitting cube, a high-speed camera, and a photomultiplier tube. The semiconductor-pumped solid-state laser is used to excite the fluorescence signal to obtain a laser beam. After the laser beam is focused by a cylindrical lens, it forms a narrow slit. After being reflected by a reflecting mirror, it is focused by an objective lens onto the plane where the microchannel is located. After the fluorescence signal is excited, it is transmitted back through the objective lens along the original optical path. After being reflected by a dichroic beam splitter, it is transmitted into a beam splitter cube. The beam splitter cube splits the fluorescence signal into two beam split signals. One beam split signal is reflected into a high-speed camera for imaging, while the other beam split signal is filtered by a filter and transmitted into a photomultiplier tube to convert the optical signal into an electrical signal.

[0041] In addition, the high-throughput droplet screening subsystem also includes an analysis device, which is used to collect electrical signals detected by photomultiplier tubes, analyze and identify fluorescent droplets containing single viruses through LabVIEW program, and realize the counting of fluorescent droplets containing single viruses.

[0042] In summary, by coupling microspheres with specific antibodies to bind and adsorb antigen proteins on the surface of the virus, free nucleic acid molecules can be washed away to eliminate their interference. Furthermore, unlike traditional virus lysis detection methods, this approach maximizes the integrity of the virus particles.

[0043] By utilizing droplet microfluidics, individual viruses and their contained nucleic acid molecules are confined within a picoliter-level reaction system, thereby increasing the concentration of the analyte by hundreds of thousands of times. This ensures efficient PCR amplification and fluorescence labeling detection. Furthermore, the effective combination of nucleic acid detection and previous antigen detection allows for the accurate identification of intact viral particles.

[0044] Example 2 A complete viral particle detection and quantification method based on droplet microfluidics includes the following steps: S1. A monoclonal antibody conjugated with biotin targeting viral surface antigen proteins and polystyrene microspheres with streptavidin attached to their surface are mixed and incubated in an aqueous solution to form specific capture microspheres that can adsorb viruses.

[0045] S2. The virus sample and the capture microspheres are mixed and incubated in an aqueous solution. The ratio of microspheres to viruses is adjusted to more than 10:1 so that individual viruses are adsorbed onto the surface of the microspheres. Then, centrifugation and washing are performed to remove free nucleic acid molecules. S3. The aqueous phase liquid containing the microsphere-virus sample and the reagent containing the fluorescent probe are introduced into the microfluidic channel in parallel flow, and then converge with the oil phase solution at the intersection. Adjusting the flow rate generates uniformly sized droplets encapsulating individual microspheres through shear force. The generated droplets are placed in a nucleic acid amplification instrument for temperature cycling, where polymerase chain reaction occurs, effectively amplifying the single virus and amplifying the fluorescence signal. S4. Inject the amplified droplets into the microchannel, then place them precisely on the stage and align them. Use a droplet screening system to scan the fluorescence signals of each droplet. Droplets containing intact virus particles will produce fluorescence signals. By setting appropriate thresholds and analyzing detection signals, single viruses can be accurately identified and their quantities can be quantified.

[0046] Example 3 A complete viral particle detection and quantification system and method based on droplet microfluidics, including a single-virus nucleic acid detection system and a high-throughput droplet screening system.

[0047] like Figure 1 As shown, a monoclonal antibody conjugated with biotin targeting viral surface antigen proteins and polystyrene microspheres with streptavidin attached to their surface are mixed and incubated in an aqueous solution to form specific capture microspheres that can adsorb viruses. Then, centrifugation and washing are performed to remove free nucleic acid molecules.

[0048] like Figure 2 As shown, droplet generation was performed using a microfluidic system. The aqueous phase contained microspheres containing a virus sample and fluorescent probes for subsequent RT-PCR labeling. The aqueous and oil phases met at the interface, and by adjusting the flow rates of both phases, uniformly sized droplets encapsulating individual microspheres were generated through shear force. The generated droplets contained microspheres adsorbed with empty viral capsids, free proteins, or intact viral particles. The generated droplets underwent viral lysis and were then placed in a nucleic acid amplification instrument for temperature cycling, resulting in polymerase chain reaction (PCR) for efficient amplification of the single virus and effective amplification of the fluorescence signal. Only droplets with microspheres adsorbing intact viral particles exhibited viral nucleic acid molecules after lysis, resulting in a significantly enhanced fluorescence signal intensity after nucleic acid amplification, forming a clear contrast with the other droplets. Figure 3 As shown.

[0049] like Figure 4 As shown, the amplified droplets are injected into the microchannel, using methods such as... Figure 5 The fluorescence detection platform shown scans the fluorescence signals of droplets one by one. Droplets containing intact virus particles will produce fluorescence signals. By setting appropriate virus and droplet detection thresholds and combining the detection signal analysis, single viruses can be accurately identified and their numbers can be quantitatively counted.

[0050] like Figure 5The fluorescence detection platform shown is mounted on an optical vibration damping table to maintain its level and prevent external disturbances, while a curtain is placed around it to prevent interference from visible light. A semiconductor-pumped solid-state laser emits a laser of a specific wavelength to excite fluorescence signals for detection. This laser beam is focused by a cylindrical lens to form a slit, then reflected by a series of lenses before being focused onto the plane of the microchannel by an objective lens. Droplets flowing through the channel are excited to fluoresce, and the fluorescence is transmitted back along the original optical path through the objective lens. The emitted light is reflected by a dichroic beam splitter and enters a beam-splitting cube. A portion of the light is reflected into a high-speed camera for imaging, while the remaining light is filtered and transmitted to a photomultiplier tube, converting the optical signal into an electrical signal. The detected electrical signal is transmitted to a computer via a data acquisition card, where LabVIEW software analyzes and identifies fluorescent droplets encapsulating single viruses, and performs high-throughput screening to accurately count their numbers.

[0051] This invention discloses a complete virus particle detection and quantification system and method based on droplet microfluidics. It combines antigen detection and nucleic acid detection, eliminating interference from free nucleic acid molecules through the specific binding of antigen and antibody. By encapsulating the virus in droplets and amplifying it with RT-PCR, a clear contrast is formed between the fluorescence signals of complete virus particles and virus fragments. A high-throughput droplet screening system is used to monitor and digitally read the fluorescence intensity in real time, ultimately achieving high-sensitivity detection and absolute quantitative analysis at the single-virus level.

[0052] Specifically, it includes the following steps: S1. A monoclonal antibody conjugated with biotin targeting viral surface antigen proteins and polystyrene microspheres with streptavidin attached to their surface are mixed and incubated in an aqueous solution to form specific capture microspheres that can adsorb viruses.

[0053] S2. The virus sample and the capture microspheres are mixed and incubated in an aqueous solution. The ratio of microspheres to viruses is adjusted to more than 10:1 so that individual viruses are adsorbed onto the surface of the microspheres. Then, centrifugation and washing are performed to remove free nucleic acid molecules. S3. The aqueous phase liquid containing the microsphere-virus sample and the reagent containing the fluorescent probe are introduced into the microchannel, and then meet with the oil phase solution at the intersection. Adjusting the flow rate generates uniformly sized droplets encapsulating individual microspheres through shear force. The generated droplets are placed in a nucleic acid amplification instrument for temperature cycling, where polymerase chain reaction occurs, completing the effective amplification of a single virus and effectively amplifying the fluorescence signal. S4. Inject the amplified droplets into the microchannel, then place them precisely on the stage and align them. Use a droplet screening system to scan the fluorescence signals of each droplet. Droplets containing intact virus particles will produce fluorescence signals. By setting appropriate thresholds and analyzing detection signals, single viruses can be accurately identified and their quantities can be quantified.

[0054] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A complete virus particle detection and quantification system based on droplet microfluidics, characterized in that, include: Single-virus adsorption microspheres: The single-virus adsorption microspheres are used to couple with antibodies via chemical bonds of streptavidin, and the adsorption of a single virus by the microspheres is achieved through the specific binding of antigen and antibody. Single-virus nucleic acid detection subsystem: used to encapsulate single-virus adsorbed microspheres containing viruses into droplets, forming droplets containing an aqueous liquid containing microsphere-virus samples and fluorescent probe reagents; RT-PCR amplification is performed on the droplets, nucleic acid molecules at the single-virus level are detected within the droplets, the viral nucleic acid is specifically labeled using fluorescent probes, and the fluorescence intensity generated after viral nucleic acid amplification is compared. High-throughput droplet screening subsystem: Analyzes droplet size and fluorescence signal intensity to identify droplets containing single viruses; accurately counts fluorescent droplets to achieve quantitative analysis at the single virus level.

2. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 1, characterized in that, The single-virus adsorption microspheres have a particle size of 0.5~5μm, and the surface of the single-virus adsorption microspheres is modified with streptavidin. The chemical bond of the streptavidin adopts a streptavidin-biotin binding bond.

3. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 1, characterized in that, The adsorption of a single virus by microspheres through the specific binding of antigen and antibody specifically includes: the antigen being a protein on the surface of the virus, and the antibody being a monoclonal antibody that can specifically capture the target virus.

4. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 1, characterized in that, The fluorescent probes described are oligonucleotide fluorescent probes that specifically label viral nucleic acids.

5. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 1, characterized in that, Encapsulating virus-laden single-virus adsorbed microspheres into droplets, specifically including: The droplet encapsulation process utilizes microfluidics, where an aqueous liquid containing a microsphere-virus sample and a reagent containing a fluorescent probe are introduced into a microchannel. Within the microchannel, the aqueous liquid containing the microsphere-virus sample and the reagent containing the fluorescent probe are sheared by an oil-phase liquid, forming droplets encapsulating individual microspheres. The single-virus adsorbed microspheres within the droplets adsorb empty viral capsids, free proteins, or intact viral particles.

6. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 1, characterized in that, The high-throughput droplet screening subsystem includes a semiconductor-pumped solid-state laser, cylindrical lenses, reflecting mirrors, objectives, a dichroic beam splitter, a beam splitter cube, a high-speed camera, and a photomultiplier tube; The semiconductor-pumped solid-state laser is used to excite the fluorescence signal to obtain a laser beam. After the laser beam is focused by a cylindrical lens, it forms a narrow slit. After being reflected by a reflecting mirror, it is focused by an objective lens onto the plane where the microchannel is located. After the fluorescence signal is excited, it is transmitted back through the objective lens along the original optical path. After being reflected by a dichroic beam splitter, it is transmitted into a beam splitter cube. The beam splitter cube splits the fluorescence signal into two beam split signals. One beam split signal is reflected into a high-speed camera for imaging, while the other beam split signal is filtered by a filter and transmitted into a photomultiplier tube to convert the optical signal into an electrical signal.

7. The complete virus particle detection and quantification system based on droplet microfluidics according to claim 6, characterized in that, The high-throughput droplet screening subsystem also includes an analysis device, which is used to collect electrical signals detected by photomultiplier tubes, analyze and identify fluorescent droplets containing single viruses through a LabVIEW program, and count the number of fluorescent droplets containing single viruses.

8. A complete viral particle detection and quantification method based on droplet microfluidics, implemented based on the complete viral particle detection and quantification system based on droplet microfluidics as described in any one of claims 1 to 7, characterized in that, include: S1. Single-virus adsorbed microspheres were prepared by coupling streptavidin with antibodies via chemical bonds; the single-virus adsorbed microspheres were used to adsorb viruses and were mixed and incubated in an aqueous solution to form an aqueous liquid of microsphere-virus sample. S2. Using the aqueous phase of the microsphere-virus sample and reagents containing fluorescent probes, droplets are encapsulated in microchannels to form droplets encapsulating individual microspheres. The droplets are then subjected to RT-PCR amplification. S3. Inject the amplified droplets into the microchannel, then place them on the stage for precise alignment. Use the droplet screening subsystem to scan the fluorescence signals of each droplet. Utilize preset thresholds and the detected fluorescence signals to achieve quantitative analysis at the single-virus level.

9. The complete viral particle detection and quantification method based on droplet microfluidics according to claim 7, characterized in that, Viruses are adsorbed using single-virus adsorption microspheres and then mixed and incubated in an aqueous solution to form an aqueous liquid microsphere-virus sample, specifically including: Virus samples and single-virus adsorbed microspheres were mixed and incubated in an aqueous solution. The ratio of single-virus adsorbed microspheres to viruses was adjusted to more than 10:1, so that individual viruses were adsorbed onto the surface of the single-virus adsorbed microspheres. When the viruses were adsorbed onto the single-virus adsorbed microspheres, centrifugation and washing were performed to remove free nucleic acid molecules.

10. The complete viral particle detection and quantification method based on droplet microfluidics according to claim 7, characterized in that, The method involves encapsulating microspheres in an aqueous liquid phase containing a virus sample and reagents containing fluorescent probes within a microfluidic channel, forming droplets that encapsulate individual microspheres. These droplets are then subjected to RT-PCR amplification, specifically including: An aqueous phase liquid containing microsphere-virus samples and a reagent containing fluorescent probes are introduced into a microchannel, and then the liquid merges with the oil phase solution in the microchannel at the intersection of the microchannels. Adjusting the flow rate generates uniformly sized droplets containing individual microspheres through shear force. The generated droplets are placed in a nucleic acid amplification instrument for temperature cycling, where a polymerase chain reaction occurs to amplify a single virus and amplify the fluorescence signal.

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