Single-cell protein high-resolution quantitative detection system and method based on microdroplet compression channel

Abstract

The application provides a single-cell protein high-resolution quantitative detection system and method based on a micro-droplet compression channel, and the system composition comprises: a micro-droplet lysis and generation module, which is used for realizing the falling of a single-cell dyeing antibody in a droplet; a micro-droplet quartz compression channel detection module, which is used for realizing the collection and detection of a single-cell protein fluorescence signal in a micro-droplet; a micro-droplet fluorescence signal collection and processing module, which is used for processing and analyzing the collected fluorescence signal; and a micro-droplet driving module, which is used for realizing the flow rate control of the micro-droplet in a micro-channel. The application reduces optical noise by using quartz instead of a common base material, reduces electrical noise by using a sine wave to control a laser source to modulate fluorescence, and has a significant two-order-of-magnitude improvement in detection resolution compared with existing methods; and the application realizes high reliability and accuracy of detection results by optimizing the composition of a cell lysis solution compared with existing methods.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection, specifically relating to a high-resolution quantitative detection system and method for single-cell proteins based on microdroplet compression channels. Background Technology

[0002] High-resolution quantitative detection of single-cell proteins refers to the quantitative detection of one or more protein molecules in a large number of single cells in a short period of time for certain proteins that are present in small amounts. The results of this protein quantitative analysis provide key parameters for cellular heterogeneity and are helpful for the study of tumor mechanisms and clinical diagnosis and treatment.

[0003] Currently, single-cell protein quantification typically employs fluorescence flow cytometry or mass flow cytometry. Fluorescence flow cytometry uses fluorescently labeled antibodies to stain cells, which are then washed through a capillary tube, with fluorescence intensity quantified by a photomultiplier tube. Subsequently, based on chemically modified calibration beads, fluorescence flow cytometry can accurately quantify single-cell surface proteins. However, due to the lack of corresponding intracellular protein calibration beads, this method cannot accurately quantify the number of intracellular protein molecules in a single cell. Mass flow cytometry uses rare-earth metal-labeled antibodies to stain cells, which are then ionized and analyzed using a time-of-flight mass spectrometer, thereby quantifying protein expression in individual cells. Although this method can simultaneously detect intracellular and cell surface proteins at the single-cell level, the lack of calibration methods prevents it from obtaining the accurate molecular count of single-cell proteins.

[0004] Because microfluidics is well-suited to the size of biological cells (tens to hundreds of micrometers), it has become an important tool for single-cell analysis. For single-cell protein quantification based on microfluidics, there are two main methods: micro-engraving and barcode chip methods. Both methods primarily work by confining single cells within micropores or cavities, followed by cell lysis, protein capture, and quantification. Although these microfluidic methods are based on large array analysis, they cannot continuously characterize individual cells, thus limiting throughput and resulting in low detection rates.

[0005] In recent years, a microfluidic platform based on compressed channels has been developed for high-throughput quantification of specific intracellular proteins in single cells. In this microfluidic cytometry technique, compressed microchannels serve as calibration structures. Cells stained with fluorescently labeled antibodies are forced through the compressed channels, and the raw fluorescence signal is collected. Simultaneously, a fluorescent antibody solution of known concentration is passed through the compressed channels to generate a calibration curve, thereby converting the raw fluorescence signal into the number of specific intracellular proteins. Although this method can obtain the number of intracellular proteins in a single cell, it frequently suffers from clogging due to the small size of the cells compared to the cross-sectional area of ​​the compressed channels, thus affecting detection efficiency.

[0006] Recently, we have been considering using a microdroplet-based method to lyse cells within droplets and then pass the droplets through a compression channel instead of the cells. Since droplets lack the supporting structure of cells, they can pass smoothly through the compression channel under pressure without causing blockage. However, existing methods for quantitative detection of single-cell proteins based on microdroplet compression channels are limited by the dilution effect of microdroplets on cells and significant background noise, resulting in low detection resolution and making them difficult to use effectively for studying tumor heterogeneity.

[0007] Therefore, developing a high-resolution quantitative detection system and method for single-cell proteins based on microdroplet compression channels can solve the cell blockage problem while improving the method resolution, thereby enabling the detection of more protein types. This is of great scientific significance for the study of tumor heterogeneity. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] To develop a high-resolution quantitative detection system and method for single-cell proteins based on microdroplet compression channels, addressing the low resolution issue of existing microdroplet detection methods, and thus meeting the clinical needs for detecting proteins with even lower concentrations.

[0010] (II) Technical Solution

[0011] To address the aforementioned problems, this invention provides a high-resolution quantitative detection system for single-cell proteins based on a microdroplet compression channel. The system includes: a microdroplet lysis and generation module, comprising immunofluorescence-stained cells, cell lysis buffer, fluorinated oil, and a microdroplet generation microchannel. The cell lysis buffer is used to facilitate the shedding of single-cell immunostaining antibodies within the droplets, thereby ensuring uniform fluorescence distribution within the microdroplets. By controlling the flow rates and flow ratio of the two aqueous phases and the oil phase, stable single-cell microdroplet encapsulation is achieved. Uniformly fluorescently distributed microdroplets are generated in the microdroplet generation channel. The two aqueous phases are immunofluorescence-stained cells and cell lysis buffer, and the oil phase is fluorinated oil. The system also includes a microdroplet quartz compression channel detection module, comprising a quartz compression microchannel, a quartz substrate, a chromium window, a photomultiplier tube, and a sinusoidally controlled laser light source. (The quartz substrate material is superior to existing substrate materials such as polydimethylsiloxane (PDMS) and polymethyl methacrylate.) PMMA (methacrylate), with its low autofluorescence, is used as a substitute for PDMS in existing methods as a fluorescence detection substrate. This substrate is then used to create a quartz compressed microchannel for fluorescence detection. Fluorescence excitation is achieved by exciting microdroplet proteins containing single cells of the target cell within the microchannel. A photomultiplier tube converts the detected fluorescence signal into a voltage signal, which is then modulated by a sinusoidally controlled laser light source. This laser light source modulates the fluorescence signal of the single-cell protein in the droplet to a high frequency, thus removing low-frequency electrical noise. The microdroplet fluorescence signal is then received... The collection and processing module includes a signal generator, a lock-in amplifier, a data acquisition card, and a signal acquisition system. The signal generator provides the reference signal required by the lock-in amplifier, namely the AC sine wave signal of the modulated laser source. The high-frequency fluorescence voltage signal modulated by the signal generator is demodulated into a low-frequency voltage signal by the lock-in amplifier. The low-frequency voltage signal is then input to the data acquisition card and converted into a digital signal. The signal acquisition system performs data processing and analysis, and determines the number of protein molecules in a single cell by combining the relationship curve between the number of protein molecules and the voltage signal.

[0012] The uniformly fluorescently distributed microdroplets are single-cell microdroplets stained with fluorescently labeled proteins.

[0013] Furthermore, the lock-in amplifier is used to modulate and demodulate the detected fluorescence voltage signal to eliminate electrical noise in the detection; the data acquisition card is used to acquire the voltage signal and transmit it to the central controller, and the number of protein molecules in a single cell to be detected is determined by the fluorescence voltage signal based on the signal acquisition system.

[0014] Optionally, the system further includes a microdroplet driving module for controlling the flow rate of the test droplets entering the quartz compression microchannel, so that a single droplet can pass through the excitation detection area in the quartz compression microchannel at a uniform speed without droplet breakage or fusion.

[0015] Furthermore, the microdroplet fragmentation and generation module is mainly composed of a microdroplet generation chip.

[0016] The microdroplet generation chip is located on the microscope stage of the microdroplet quartz compression channel detection module. First, microdroplets are formed by the microdroplet generation chip. Then, the microdroplets containing lysis buffer are co-incubated with the target single cell to achieve uniform distribution of fluorescent antibody in the microdroplets.

[0017] Furthermore, the microdroplet quartz compression channel detection module includes a microdroplet quartz compression channel detection chip.

[0018] Furthermore, the microdroplet quartz compression channel detection chip is located on the stage of the microscope of the microdroplet quartz compression channel detection module, and the collection and processing of microdroplet fluorescence signals are realized through the microdroplet fluorescence signal collection and processing module.

[0019] Furthermore, the microdroplet generation chip is provided with a microdroplet generation microchannel. The microdroplet generation microchannel is a cross-shaped channel; the cross-shaped channel has a first end and a second end opposite to each other, as well as a third end and a fourth end opposite to each other; the first end of the cross-shaped channel is the input end for immunofluorescence-stained cells and cell lysis buffer; the second end of the cross-shaped channel is the outflow end for uniformly fluorescently distributed microdroplets; the third end of the cross-shaped channel is the input end for fluorinated oil; the fourth end of the cross-shaped channel contains fluorinated oil; single-cell microdroplets stained with fluorescently labeled proteins are formed within the microdroplet generation microchannel. Both the immunofluorescence-stained cells and the cell lysis buffer are aqueous phases; the fluorinated oil is an oil phase. By optimizing the cell lysis buffer, the immunostaining antibodies within the droplets are removed, thereby ensuring uniform fluorescence distribution within the microdroplets. By adjusting the flow rate ratio and flow rate of the two aqueous and oil phases, stable single-cell microdroplet encapsulation is achieved.

[0020] Furthermore, the microdroplet driving module includes a manual pressure pump and a microtube. First, a layer of perforated polydimethylsiloxane material is bonded to the microdroplet quartz compression channel detection chip. The manual pressure pump is connected to this perforated polydimethylsiloxane material through the microtube to control the flow rate of the microdroplets in the microdroplet driving module.

[0021] Preferably, the microdroplet driving module includes a manual pressure pump and a microtube. One end of the microtube is directly connected to the manual pressure pump, and the other end is directly connected to a layer of perforated polydimethylsiloxane material bonded to the microdroplet quartz compression channel detection chip. By controlling the magnitude of the negative pressure, it is ensured that the generated microdroplets can pass through the quartz compression microchannel at a uniform speed instead of breaking or merging.

[0022] Furthermore, the microdroplet quartz compression channel detection chip includes a quartz compression microchannel, a quartz substrate, a chromium window, a photomultiplier tube, and a sinusoidally controlled laser source. The quartz compression microchannel penetrates the quartz substrate. The chromium window is disposed on the sidewall of the quartz compression microchannel. The photomultiplier tube and the sinusoidally controlled laser source are disposed outside the quartz substrate, facing the chromium window. The microdroplet protein containing a single cell to be detected is fluorescently excited by the microdroplet entering the quartz compression microchannel, and the fluorescence signal is received by the photomultiplier tube and converted into a voltage signal, which is then modulated by the sinusoidally controlled laser source.

[0023] Furthermore, the microdroplet fluorescence signal collection and processing module, including a signal generator, a lock-in amplifier, a data acquisition card, and a signal acquisition system, demodulates the high-frequency fluorescence voltage signal modulated by the signal generator back to its original low-frequency voltage signal using the lock-in amplifier. This signal is then input to the data acquisition card for conversion into a digital signal. The signal acquisition system performs data processing and analysis, and by combining the relationship curve between the number of protein molecules and the voltage signal, determines the number of protein molecules in a single cell to be detected.

[0024] Furthermore, the cell lysis buffer in the microdroplet lysis and generation module includes proteinase K, guanidine hydrochloride, and urea; the concentration of proteinase K in the cell lysis buffer is 1 mg / mL, the concentration of guanidine hydrochloride is 3 mol / L, and the concentration of urea is 3 mol / L.

[0025] Furthermore, the cross-sectional dimensions of the microdroplet generation chip (located on the microscope stage of the microdroplet quartz compression channel detection module, used for microdroplet generation, and a core component of the microdroplet lysis and generation module), and the flow rates and flow ratios of immunofluorescence stained cells, cell lysate, and oil phase, are specified. The cross-sectional dimensions of the microdroplet generation chip are 20 × 20 μm. 2 The flow rates of the aqueous phase of immunofluorescence staining cells and the aqueous phase of cell lysis buffer were controlled at 2 μL / min using an injection pump, while the flow rate of the fluorinated oil phase was 4 μL / min, with a water-to-oil ratio of 1:2. The final microdroplet volume reached 8 pL.

[0026] Furthermore, in the microdroplet fragmentation and generation module, the volume of the microdroplet generated by the channel of the microdroplet generation microchannel, and the corresponding stretching length of the microdroplet, are expressed by the following formula:

[0027]

[0028] Among them, L p w represents the stretching length of the microdroplet within the microchannel where the microdroplet is generated. c Q is the channel width. d and Q c η represents the volumetric flow rates of the aqueous phase and the oil phase, respectively. c γ is the viscosity of the oil phase. dc ρ represents the surface tension of the oil phase, and h represents the height of the microchannels formed by the microdroplets.

[0029] Furthermore, the relevant parameters of the sine wave controlled laser source and lock-in amplifier are as follows: the frequency of the corresponding sine wave is 20kHz, the amplitude is 1V, and the DC bias is 0.5V; while the filter order of the lock-in amplifier is selected as 15th order, and the time constant is selected as 0.5ms.

[0030] Furthermore, the signal acquisition and data processing module includes a preset curve showing the relationship between the number of protein molecules and the voltage signal.

[0031] Optionally, the droplet volume in the excitation detection region (located between the quartz compression microchannel and the quartz substrate in the microdroplet quartz compression channel detection module) is expressed by the following formula:

[0032]

[0033] Among them, V d H is the volume of the droplet. c For the high efficiency of quartz compressed microchannels, W c To compress the width of the microchannel in quartz, W g T represents the width of the chrome window. r T is the time it takes for the droplet head to enter the chromium window. s T represents the time it takes for the droplet to completely enter the chromium window. d This represents the time it takes for the droplet to gradually detach from the chrome window.

[0034] Optionally, the microdroplet fluorescence signal collection and processing module includes a preset curve showing the relationship between protein concentration and voltage signal.

[0035] Another aspect of the present invention provides a method for quantitative detection of single-cell proteins using the single-cell protein detection system based on microdroplet compression channels described above, the method comprising:

[0036] S1 generates stable, uniform fluorescent microdroplets that encapsulate single cells;

[0037] S2, the uniform fluorescent microdroplet is introduced into a quartz compressed microchannel, a sine wave controlled laser source is used to modulate the fluorescence of the uniform fluorescent microdroplet, and then a lock-in amplifier is used to demodulate the noise-reduced fluorescence voltage signal.

[0038] S3, multiple sets of fluorescently labeled antibody solutions with known protein concentrations are introduced into the quartz compression microchannel. A sine wave controlled laser source modulates the fluorescence generated by the equivalent antibody solution. A lock-in amplifier is used to demodulate and obtain the calibration curve of protein concentration and fluorescence voltage signal.

[0039] S4. Fluorescence compensation is performed based on the protein molecule number versus voltage signal curve to obtain the corrected protein molecule number versus voltage signal curve.

[0040] S5, acquire the fluorescence voltage signal of the single cell obtained by the modulation and demodulation detection, and obtain the number of protein molecules in the single cell based on the voltage signal and the modified curve of the relationship between the number of protein molecules and the voltage signal.

[0041] (III) Beneficial Effects

[0042] The present invention has at least the following beneficial effects:

[0043] (1) This invention reduces the volume of microdroplet generation by 10 times by adjusting the flow rate ratio and velocity of the aqueous and oil phases using an injection pump and by redesigning the cross-sectional dimensions of the microdroplet generation chip. This reduces the dilution of the cells by the droplets and successfully improves the sensitivity of the detection method. Furthermore, by generating unsupported droplets to replace cells in the compression channel, the problem of cell blockage in the compression channel is solved.

[0044] (2) By using a quartz substrate instead of the traditional PDMS substrate material to make quartz compressed microchannels, the noise of the background material is reduced; by using a light source modulation method to reduce the electrical noise generated during fluorescence detection, the detection accuracy of the detection method is successfully improved and the detection signal-to-noise ratio is significantly increased; finally, the detection limit (the smallest number of proteins that can be detected in a single cell) is two orders of magnitude higher than that of the existing quantitative detection method based on microdroplet compressed channels, thereby solving the problem of limited resolution in the existing method and realizing the detection needs of more single-cell protein types.

[0045] (3) This invention utilizes effective cell lysis buffer components to achieve stable droplet generation while ensuring effective cell lysis, and successfully achieves effective lysis of 6 proteins (i.e., lysis efficiency of nearly 100% - uniform distribution of multiple protein fluorescent antibodies in the droplet). This solves the problem of inaccurate fluorescence quantitative detection caused by uneven excitation light during laser detection, and improves the accuracy and reliability of the entire fluorescence quantitative detection based on microdroplet compression channel. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the structure of the single-cell protein high-resolution quantitative detection system based on microdroplet compression channels described in an embodiment of the present invention;

[0047] Figure 2 This is a schematic diagram of the microdroplet lysis and generation module in the single-cell protein high-resolution quantitative detection system based on microdroplet compression channels described in this embodiment of the invention.

[0048] Figure 3 This is a process flow diagram of the quartz compression microchannel described in an embodiment of the present invention;

[0049] Figure 4 This is a flowchart of the high-resolution quantitative detection method for single-cell proteins based on microdroplet compression channels according to an embodiment of the present invention;

[0050] Figure 5 The curves showing the change over time in the lysis efficiency of different combinations of lysing agents against cytokines in tumor cells, as described in the embodiments of the present invention.

[0051] Figure 6 This is a simulation diagram of the phase interface motion process of the droplets as described in the embodiments of the present invention;

[0052] Figure 7 This is a simulation of the phase interface motion process of the droplet as described in the embodiments of the present invention, including the cross-shaped channel image during droplet generation and the generated droplet. Detailed Implementation

[0053] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0054] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0055] The first embodiment of this invention provides a high-resolution quantitative detection system for single-cell proteins based on microdroplet compression channels, see [link to relevant documentation]. Figure 1The detection system comprises four modules: a microdroplet lysis and generation module 1, a microdroplet driving module 2, a microdroplet quartz compression channel detection module 3, and a microdroplet fluorescence signal collection and processing module 4. The microdroplet lysis and generation module 1 mainly consists of a microdroplet generation chip, located on the microscope stage of the microdroplet quartz compression channel detection module 3. Microdroplets are first formed using the chip, and then the microdroplets, containing lysis buffer, are co-incubated with target single cells to achieve uniform distribution of fluorescent antibodies within the microdroplets. The microdroplet quartz compression channel detection module 3, also including a microdroplet quartz compression channel detection chip, is also located on the microscope stage. The microdroplet fluorescence signal collection and processing module 4 collects and processes the fluorescence signal from the microdroplets. The microdroplet driving module 2 mainly consists of a manual pressure pump. A layer of perforated polydimethylsiloxane material is bonded to the microdroplet quartz compression channel detection chip, and this perforated material is connected via a microtube to control the flow rate of the microdroplets within the microdroplet quartz compression channel detection module 3.

[0056] See Figure 1A high-resolution quantitative detection system for single-cell proteins based on a microdroplet compression channel is disclosed. The system mainly includes a microdroplet lysis and generation module 1, a microdroplet driving module 2, a microdroplet quartz compression channel detection module 3, and a microdroplet fluorescence signal collection and processing module 4. Specifically, the microdroplet lysis and generation module 1 includes immunofluorescently stained cells 101, cell lysis buffer 102, fluorinated oil 103, and a microdroplet generation microchannel. The microdroplet generation microchannel is disposed on the microdroplet generation chip. The microdroplet generation microchannel is a cross-shaped channel. The first end of the cross-shaped channel is the input end of the immunofluorescently stained cells 101 and cell lysis buffer 102. The second end of the cross-shaped channel is opposite to the first end. The second end of the cross-shaped channel is the outflow end of the single-cell microdroplets stained with fluorescently labeled proteins. The third end of the cross-shaped channel is opposite to the fourth end. The third end of the cross-shaped channel is the input end of the fluorinated oil 103. The fourth end of the cross-shaped channel is filled with fluorinated oil 103. Both immunofluorescence staining cells 101 and cell lysis buffer 102 are aqueous phases; fluorinated oil 103 is the oil phase. The optimized cell lysis buffer 102 allows for the shedding of immunostaining antibodies within the droplets, ensuring uniform fluorescence distribution within the microdroplets. Adjusting the flow rate ratio and velocity of the two aqueous phases (immunofluorescence staining cells 101 and cell lysis buffer 102 and the oil phase (fluorinated oil 103)) achieves stable single-cell microdroplet encapsulation, ensuring high reliability and accuracy of the detection method. Optimizing cell lysis buffer 102 allows for the shedding of immunostaining antibodies within the droplets, ensuring uniform fluorescence distribution within the microdroplets. Adjusting the flow rate ratio and velocity of the two aqueous and oil phases creates microchannels within the microdroplets, resulting in stable single-cell microdroplet encapsulation and uniformly fluorescently distributed microdroplets. These uniformly fluorescently distributed microdroplets are single-cell microdroplets stained with fluorescently labeled proteins.

[0057] In the microdroplet fragmentation and generation module 1, the volume of the microdroplet generated by the cross-shaped channel of the microdroplet generation microchannel, and the corresponding stretching length of the microdroplet, are expressed by the following formula:

[0058]

[0059] Among them, L p w represents the stretching length of the microdroplet within the microchannel where the microdroplet is generated. c To generate microchannel width for microdroplets, Q d and Q c η represents the volumetric flow rates of the aqueous phase and the oil phase, respectively. c γ is the viscosity of the oil phase. dc ρ represents the surface tension of the oil phase, and h represents the height of the microchannels formed by the microdroplets.

[0060] The microdroplet driving module 2 mainly consists of a manual pressure pump 201 and microtubes. The manual pressure pump is connected to the upper through-hole of the microdroplet quartz compression channel via the microtubes, forming a uniform pressure in the microchannel to ensure that the generated microdroplets can pass through the quartz compression microchannel at a uniform speed without breaking or merging, further ensuring the reliability of the detection method. First, a layer of perforated polydimethylsiloxane material is bonded to the microdroplet quartz compression channel detection chip. The manual pressure pump is connected to this perforated polydimethylsiloxane material through the series of microtubes, controlling the flow rate of the microdroplets in the microdroplet quartz compression channel detection module 3. By controlling the negative pressure, it is ensured that the generated microdroplets can pass through the quartz compression microchannel 301 at a uniform speed without breaking or merging.

[0061] The microdroplet quartz compression channel detection module 3 mainly consists of a quartz compression microchannel 301, a quartz substrate 302, a chromium window 303, a photomultiplier tube 304, and a sinusoidally controlled laser source 305. The laser 305, passing through the chromium window 303, excites the fluorescence of microdroplet proteins containing a single cell to be detected within the quartz compression microchannel 301. The photomultiplier tube 304 receives the fluorescence signal and converts it into a voltage signal, which is then modulated by the sinusoidally controlled laser source 305. The quartz compression microchannel 301 penetrates the quartz substrate 302; the chromium window 303 is located on the sidewall of the quartz compression microchannel 301; the photomultiplier tube 304 and the sinusoidally controlled laser source 305 are located outside the quartz substrate 302, facing the chromium window. The quartz compression microchannel 301, the quartz substrate 302, and the chromium window 303 are disposed on the microdroplet quartz compression channel detection chip.

[0062] The microdroplet fluorescence signal collection and processing module 4 mainly consists of a signal generator 401, a lock-in amplifier 402, a data acquisition card 403, and a signal acquisition system 404. When using a laser light source for modulation, the signal generator 401 controls the laser light source 305 to output a sine wave as the carrier wave (which is input as a reference signal to the reference signal terminal of the lock-in amplifier 402). The excited fluorescent droplet pulse signal is used as the signal to be measured and input to the signal to be measured terminal of the lock-in amplifier. The lock-in amplifier 402 is used for noise reduction and demodulation, and then directly connected to the data acquisition card 403 to be converted into a digital signal. The signal acquisition system 404 then analyzes the signal. Stray noise is removed in one step by using a 40-point median filter. The baseline of the fluorescence pulse peak is obtained by using a kernel density estimation method. By selecting a typical trapezoidal pulse peak as a template, the model is trained, the characteristic values ​​of the droplet fluorescence pulse peak are extracted, and the volume and fluorescence intensity of each droplet are calculated. Then, combined with the relationship curve between the number of protein molecules and the voltage signal, the number of protein molecules in a single cell to be detected is determined.

[0063] Optionally, the cell lysis buffer 102 in the microdroplet lysis and generation module 1 includes proteinase K, guanidine hydrochloride and urea; the concentration of proteinase K in the cell lysis buffer 102 is 1 mg / mL, the concentration of guanidine hydrochloride is 3 mol / L and the concentration of urea is 3 mol / L.

[0064] Optionally, the cross-sectional size of the microdroplet generation chip and the flow rate and ratio of immunofluorescence stained cells 101, cell lysis buffer 102, and oil phase 103 are such that the cross-sectional size of the microdroplet generation chip is 20×20µm. 2 The flow rates of the aqueous phase of immunofluorescence staining cells 101 and the aqueous phase of cell lysis buffer 102 were controlled at 2 μL / min by an injection pump, while the flow rate of the fluorinated oil phase was 4 μL / min, with a water-to-oil ratio of 1:2. The final microdroplet volume reached 8 pL.

[0065] Therefore, this invention utilizes effective cell lysis buffer components to achieve stable droplet generation while ensuring effective cell lysis. Compared to existing methods that can only achieve effective lysis of three proteins, this invention successfully extends to the effective lysis of six proteins, increasing the original protein lysis efficiency from less than 50% to nearly 100%. This enables uniform distribution of multiple protein fluorescent antibodies in the droplets, solving the problem of inaccurate fluorescence quantitative detection caused by uneven excitation light in laser detection, and improving the accuracy and reliability of the entire microdroplet compression channel-based fluorescence quantitative detection. Furthermore, this invention reduces the volume of microdroplets generated by adjusting the flow rate ratio and velocity of the aqueous and oil phases using an injection pump and redesigning the cross-sectional dimensions of the droplet generation chip, thereby reducing the dilution of the cells by the droplets. It also reduces background noise by using a quartz substrate instead of the traditional polydimethylsiloxane substrate to create the quartz compressed microchannel. Finally, it employs a light source modulation method to reduce electrical noise generated during fluorescence detection. Ultimately, this invention achieves a two-order-of-magnitude improvement in detection resolution compared to existing microdroplet compressed channel-based quantitative detection methods, enabling the detection of proteins present in small amounts in single cells, such as signaling pathway proteins, thus further meeting the clinical needs for single-cell protein detection.

[0066] It should be noted that the uniform fluorescent microdroplets mentioned above refer to the fluorescent antibodies that specifically bind to the target protein of a single cell under the action of cell lysis buffer, breaking the binding bond between the fluorescent antibody and the target protein. The corresponding fluorescent antibody is uniformly distributed in the droplet. The generation of uniform fluorescent droplets can solve the problem of accurate quantification of proteins at different distribution locations in a single cell.

[0067] See Figure 1The microdroplet quartz compression channel detection module 3 allows uniformly fluorescent microdroplets to pass through a quartz compression microchannel under pressure. A laser source (excitation detection area) controlled by a sine wave generated by a signal generator modulates the fluorescence of the uniformly fluorescent microdroplets. As shown in the microdroplet fluorescence signal collection and processing module 4, the modulated fluorescence signal is received by a photomultiplier tube, input to a lock-in amplifier, and then demodulated using a reference signal of the same frequency generated by the signal generator to obtain a noise-reduced fluorescence voltage signal. This signal is then input to a data acquisition card and converted into a digital signal. By combining the relationship curve between protein concentration and fluorescence voltage, the number of protein molecules in a single test cell is determined.

[0068] The volume of the microdroplets in the excitation detection region described above is expressed by the following formula:

[0069]

[0070] Among them, V d H is the volume of the droplet. c For the high efficiency of quartz compressed microchannels, W c To compress the width of the microchannel in quartz, W g T represents the width of the chrome window. r T is the time it takes for the droplet head to enter the chromium window. s T represents the time it takes for the droplet to completely enter the chromium window. d This represents the time it takes for the droplet to gradually detach from the chrome window.

[0071] In one feasible approach, the microdroplet fluorescence signal collection and processing module 4 has a preset curve showing the relationship between protein concentration and voltage signal.

[0072] See Figure 2 The microdroplet lysis and generation module 1 controls the flow rate of the two aqueous phases to be consistent via an injection pump, ensuring that the immunofluorescence stained cell suspension 101 and cell lysis buffer 102 are mixed in equal volumes. Under the shear force of fluorinated oil 103, stable droplets encapsulating single cells are generated. The effective components of cell lysis buffer 102 (proteinase K 102a, guanidine hydrochloride 102b, urea 102c) ensure uniform distribution of the fluorescent antibody corresponding to the target protein within the droplets. By adjusting the flow rates and ratios of the two aqueous and oil phases, as well as the cross-sectional size of the microdroplet generation channel, the droplet volume is significantly reduced, thereby greatly minimizing the dilution of cells by the droplets.

[0073] The stretching length of the microdroplets in the microdroplet generation chip (located on the microscope stage of the microdroplet quartz compression channel detection module 3, used for microdroplet generation, and a core component of the microdroplet fragmentation and generation module 1) described above is expressed by the following formula:

[0074]

[0075] Among them, L p w represents the stretching length of the microdroplet in the microchannel. c Q is the channel width. d and Q c η represents the volumetric flow rates of the aqueous phase and the oil phase, respectively. c γ is the viscosity of the oil phase. dc ρ is the surface tension of the oil phase, and h is the height of the channel.

[0076] See Figure 3 The present invention also provides a method for fabricating a microfluidic chip (as a quartz detection chip (i.e., a microdroplet quartz compression channel detection chip) in a detection system, which has the characteristic of low optical noise compared to existing PDMS chips), comprising:

[0077] The chip fabrication method is based on two smooth quartz wafers, each 25mm x 75mm in length and width and 1mm thick. One wafer is used to fabricate the channel layer. First, a layer of AZ4903 positive resist is coated onto the clean quartz wafer, and then the channel pattern is exposed (see attached image). Figure 3 (I) The channel pattern was patterned using 0.6% NaOH solution (see Appendix). Figure 3 (II) Based on the selective etching of AZ4903 and quartz using dry etching, a channel layer with a depth of 20 μm was etched (see Appendix). Figure 3 (III) Finally, the inlet and outlet holes of the microchannel are cut using laser processing equipment (see Appendix). Figure 3 (IV)); The other substrate, used as the chip, is fabricated in the same way as before, first by depositing a 150nm chromium thin film on a clean quartz wafer (see Appendix). Figure 3 (V)), then apply another layer of AZ1500 positive resist, expose and develop to pattern the photoresist (see appendix). Figure 3 (VI and VII) Finally, the chromium thin film with photoresist as a mask is etched to complete the chromium patterning (see appendix). Figure 3 (VIII)). After the two-layer structure is completed, they are bonded together using a quartz bonding process to obtain the final quartz chip (see appendix). Figure 3 (IX)).

[0078] Another embodiment of the present invention provides a method for high-resolution quantitative detection of single-cell proteins using the microdroplet compression channel-based single-cell protein detection device described above. See [link to relevant documentation]. Figure 4 , combined Figure 1 The method includes the following steps S1-S5:

[0079] S1 generates stable, uniform fluorescent microdroplets that encapsulate single cells;

[0080] It should be noted that the generation of stable, uniformly fluorescent microdroplets encapsulating single cells can refer to microdroplets encapsulating single cells containing only one type of protein antibody, or it can refer to microdroplets encapsulating single cells containing multiple types of proteins. Uniform fluorescence refers to the breaking of the binding bond between the fluorescent antibody specifically binding to the target protein on the single cell and the target protein under the action of cell lysis buffer, resulting in a uniform distribution of the corresponding fluorescent antibody within the droplet. Therefore, the method of this application embodiment can not only achieve quantitative detection of single cells containing only one type of protein, but also achieve quantitative detection of single cells containing multiple types of proteins. Because the fluorescent antibody is uniformly distributed within the droplet, the fluorescent antibody corresponding to unevenly distributed proteins in the single cell can be uniformly distributed within the droplet using lysis buffer, enabling quantitative detection of cases with uneven protein distribution in single cells.

[0081] S2, the uniform fluorescent microdroplet is introduced into a quartz compressed microchannel, a sine wave controlled laser source is used to modulate the fluorescence of the uniform fluorescent microdroplet, and then a lock-in amplifier is used to demodulate the noise-reduced fluorescence voltage signal.

[0082] Uniformly fluorescent microdroplets are introduced into a quartz compressed microchannel. When a single microdroplet encapsulating a single cell passes sequentially through the excitation region of a sinusoidally controlled laser light source, the single-cell microdroplet stained with fluorescently labeled protein is excited and modulated by the sinusoidally controlled excitation light source, generating correspondingly modulated emission light. This emission light is collected by a photomultiplier tube and sent to the signal terminal of a lock-in amplifier. At the same time, the same frequency signal controlling the excitation light source is sent to the reference terminal of the lock-in amplifier. The core of the lock-in amplifier is the phase-sensitive detection section. The amplitude of the demodulated cell fluorescence signal is output to a signal acquisition card, and the data is input into a computer for display and storage.

[0083] S3, multiple sets of fluorescently labeled antibody solutions with known protein concentrations are introduced into the quartz compression microchannel. A sine wave controlled laser source modulates the fluorescence generated by the equivalent antibody solution. A lock-in amplifier is used to demodulate and obtain the calibration curve of protein concentration and fluorescence voltage signal.

[0084] S4. Fluorescence compensation is performed based on the protein molecule number versus voltage signal curve to obtain the corrected protein molecule number versus voltage signal curve.

[0085] Specifically, multiple sets of fluorescently labeled protein antibody solutions with known protein concentrations are passed through identical quartz compression microchannels. When the antibody solution passes through the sine wave-controlled laser region (i.e., the excitation detection region) in the quartz microchannel, the fluorescently labeled protein antibody solution is modulated by the sine wave-controlled laser, generating correspondingly modulated emission light. This light is then collected by a photomultiplier tube and sent to the test signal terminal of a lock-in amplifier. Simultaneously, a reference signal is generated by a signal generator and input to the reference terminal of the lock-in amplifier. After demodulation by the lock-in amplifier, a voltage signal is obtained, thus yielding a calibration curve of protein concentration versus detection voltage. This calibration curve represents the relationship between protein concentration and voltage signal. Then, fluorescence compensation is performed on this calibration curve using a fluorescence compensation method to obtain a corrected curve showing the relationship between the number of protein molecules and the voltage signal.

[0086] S5, acquire the fluorescence voltage signal of the single cell obtained by the modulation and demodulation detection, and obtain the number of protein molecules in the single cell based on the voltage signal and the modified curve of the relationship between the number of protein molecules and the voltage signal.

[0087] This concludes the detailed description of this embodiment in conjunction with the accompanying drawings. Based on the above description, those skilled in the art should have a clear understanding of the single-cell protein high-resolution quantitative detection system and method based on microdroplet compression channels of the present invention.

[0088] In this embodiment, the material of the support is quartz. Those skilled in the art will understand that, in addition to PDMS, PMMA, SU-8, silicon wafers, and other materials can also be used to form the above-mentioned support.

[0089] In this embodiment of the invention, the cross-section of the quartz compression microchannel is square, but it can be replaced with a circular or rectangular shape as needed for detection.

[0090] In this invention, negative pressure is used to drive the droplets through the channel, but other methods can also be used, such as applying positive pressure at the end of the droplet solution injection channel.

[0091] The second embodiment of the present invention provides a method for reducing the volume of microdroplets. First, the microdroplet generation simulation is redesigned and simulated using the finite element method. According to formula (1.1), the cross-sectional area of ​​the droplet generation is redesigned to be 20×20 μm. 2 Different methods were designed to simulate and generate droplet volumes, and the flow rate ratio and flow rate size that can stably generate droplets, as well as the droplet generation cross-sectional size that can meet the target droplet volume, were investigated.

[0092]

[0093] Among them, L p w represents the stretching length of the microdroplet within the droplet generation microchannel. cQ is the width of the microchannels used for droplet generation. d and Q c η represents the volumetric flow rates of the aqueous phase and the oil phase, respectively. c γ is the viscosity of the oil phase. dc Here, represents the surface tension of the oil phase, and h represents the height of the channel. This invention, based on COMSOL Multiphysics software, simulates and analyzes droplet size in a cross-channel under different flow rate ratios. First, a physical model of microdroplet formation is constructed, simplifying it into a two-phase flow model. The separated multiphase flow module within the microfluidics module of COMSOL Multiphysics software is used to simulate the phase interface motion process of the droplets. The cross-sectional area of ​​the cross-channel is set to 20 × 20 μm. 2 According to formula (1), droplets with a diameter in the range of 10-30 μm are expected to be generated. Fluorinated oil and pure water are selected as the material properties for the aqueous and oil phases, respectively, and the fluid viscosity and density are set to 1.10 × 10⁻³ Pa·s and 1.6 × 10³ kg / m³, respectively. 3 7.70×10⁻³ Pa·s and 1.0×10³ kg / m 3 The simulation results are shown below: (See attached image) Figure 6 (AD) represent the water-oil flow rate ratios (1:10, 1:5, 1:2 and 1:1), respectively.

[0094] Based on theoretical analysis and simulation design, this invention designs and fabricates a material with a cross-sectional area of ​​20×20µm based on a flow focusing method. 2 The droplet formation involved cross-shaped channels. Droplet volume generation verification experiments were conducted based on different flow rate ratios simulated in the simulation, and the results are shown in the table below. The results show that the deviation between the actual generated droplet elongation length and the simulated droplet elongation length is controlled to be approximately 5%. Figure 7 In (A), the flow rate is 0.2-0.2-4, that is, the flow rate of immunofluorescent staining cell 101 is 0.2 μL / min, the flow rate of cell lysis buffer 102 is 0.2 μL / min, and the flow rate of fluorinated oil 103 is 4 μL / min; in (B), the flow rate is 1-1-4, that is, the flow rate of immunofluorescent staining cell 101 is 1 μL / min, the flow rate of cell lysis buffer 102 is 1 μL / min, and the flow rate of fluorinated oil 103 is 4 μL / min; in (C), the flow rate is 2-2-4, that is, the flow rate of immunofluorescent staining cell 101 is 2 μL / min, the flow rate of cell lysis buffer 102 is 2 μL / min, and the flow rate of fluorinated oil 103 is 4 μL / min.

[0095]

[0096] This demonstrates the accuracy and effectiveness of the simulation modeling and formula (1). Considering the stability of droplet formation over a long period and the limitation that the aqueous phase flow rate cannot be too low during actual operation, the oil-water ratio of 1, the aqueous phase flow rate of 2 μL / min, and the oil phase flow rate of 4 μL / min were ultimately selected in this invention to generate droplets. The cross-shaped channel image during droplet formation and the image of the generated droplets are attached. Figure 7 As shown, at a set flow rate, droplets can be stably generated, while the coefficient of variation in droplet size is controlled within 5%. Compared to existing microdroplet generation methods based on compression channels, which produce droplets with a diameter of approximately 50 μm and a final target droplet volume of approximately 80 pL, the droplet volume generated using the above method, while achieving stable droplet generation, corresponds to a droplet diameter of approximately 25 μm, and a final target droplet volume of approximately 8 pL, representing a reduction in droplet volume by approximately 10 times.

[0097] The third embodiment of this invention provides a method for high-resolution quantitative detection of single-cell proteins based on a microdroplet compression channel. By using a quartz compression microchannel 301 instead of polydimethylsiloxane, optical noise is reduced. A sinusoidal AC signal 305 with an AC amplitude of 1Vpp and a DC bias of 0.5V controls the existing solid-state laser for fluorescence excitation. The frequency of this laser is set to 20kHz, approximately 10 times higher than the frequency at which the droplet passes through the compression channel. This effectively and accurately modulates the fluorescence signal of the moving single droplet to a higher frequency domain. The modulated fluorescence signal is then amplified by a single photomultiplier tube 304. Optimal low-frequency noise removal is achieved by setting the filter order of the lock-in amplifier 402 to 15th order and the corresponding time constant to 0.5ms. Finally, the signal is demodulated into a trapezoidal fluorescence pulse, which is sampled by the data acquisition card 403 at an 80kHz sampling rate. Specific values ​​are shown in the table below.

[0098]

[0099] As shown in the table, after three noise reduction methods, the existing method has a detection resolution that is two orders of magnitude higher than that of the PDMS-based microdroplet quantitative detection method.

[0100] The fourth embodiment of this invention provides a highly reliable and stable method for achieving uniform distribution of fluorescent antibodies in droplets. Traditional methods, such as proteinase K (102a)-based lysis methods, only achieve about 50% lysis efficiency for cytokines such as ras, c-Myc, and p-53 in tumor cells. This leads to the inability to achieve single-cell protein quantification based on compressed microchannels. To achieve quantification of more types of proteins, the optimal composition of the cell lysis buffer was determined, and the lysis efficiency of different combinations of lysis agents for cytokines in tumor cells was experimentally investigated. The experimental results are attached. Figure 5As shown (where K represents the abbreviation for proteinase K, G represents the abbreviation for guanidine hydrochloride, and U represents the abbreviation for urea).

[0101] According to existing literature, higher concentrations of these three lysis buffers result in better protein lysis. Therefore, when experimenting with different combinations of lysis buffers, the concentrations of these three lysis buffers were chosen based on their maximum water solubility: proteinase K at 1 mg / mL, guanidine hydrochloride at 3 mol / L, and urea at 3 mol / L. These three lysis buffers were then used to incubate single cells in 96-well plates, and their lysis effects were observed.

[0102] The results showed that, compared to guanidine hydrochloride (G) and urea (U), proteinase K (K) alone had a relatively high cleavage efficiency of about 50% among single protein denaturants. Among the combinations of proteinase K and guanidine hydrochloride, the combination of proteinase K and guanidine hydrochloride had the highest cleavage efficiency, reaching about 70% after 12 hours of cell incubation, but complete cleavage was still not achieved. However, when these three protein denaturants were used together targeting four proteins, most of them were completely cleaved after 12 hours of incubation, and a relatively uniform distribution of fluorescence was achieved. Based on this background, different combinations of protein denaturants were encapsulated in droplets, and microdroplet fluorescence detection was performed using a microdroplet quartz compression channel detection module 3. The pulse peak values ​​of different types of fluorescent droplets were obtained, and the cleavage effect of the cleavage agent combination was determined by statistically analyzing the pulse proportion of uniformly fluorescent droplets.

[0103] The specific statistical values ​​are shown in the table below.

[0104]

[0105] Finally, this invention proposes a method based on the combined use of proteinase K (102a), guanidine hydrochloride (102b), and urea (102c) to achieve a lysis efficiency of nearly 100% for cytokines such as ras, c-Myc, and mutant-p-53 in tumor cells, solving the problem of single-cell protein quantification based on compressed microchannels and achieving complete lysis and quantification of six proteins.

[0106] It should be noted that implementations not shown or described in the accompanying drawings or the main text of the specification are all forms known to those skilled in the art and are not described in detail. Furthermore, the definitions of the various elements above are not limited to the specific structures, shapes, or methods mentioned in the embodiments; those skilled in the art can easily modify or substitute them, for example:

[0107] (1) The directional terms mentioned in the embodiments, such as “up”, “down”, “front”, “back”, “left”, “right”, etc., are only for reference to the directions in the accompanying drawings and are not intended to limit the scope of protection of this disclosure.

[0108] (2) The above embodiments can be used in combination with each other or with other embodiments based on design and reliability considerations. That is, the technical features of different embodiments can be freely combined to form more embodiments.

[0109] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-resolution quantitative detection system for single-cell proteins based on microdroplet compression channels, characterized in that, The system includes: The microdroplet lysis and generation module (1) includes immunofluorescent stained cells (101), cell lysis buffer (102), fluorinated oil (103), and a microdroplet generation microchannel. The cell lysis buffer (102) is used to remove single-cell immunostaining antibodies from the droplets, thereby ensuring uniform fluorescence distribution within the microdroplets. By adjusting the flow rate ratio and flow rate of the two aqueous and oil phases, stable single-cell microdroplet encapsulation is achieved, and microdroplets with uniform fluorescence distribution are generated in the microdroplet generation channel. The aqueous phase consists of immunofluorescent stained cells (101) and cell lysis buffer (102), and the oil phase is fluorinated oil (103). The microdroplet driving module (2) includes a manual pressure pump (201) and a microtube; the manual pressure pump is connected to the upper through hole of the microdroplet quartz compression channel through the microtube, forming a uniform pressure in the microchannel to ensure that the microdroplets can pass through the quartz compression microchannel at a uniform speed instead of breaking or merging. The microdroplet quartz compression channel detection module (3) includes a quartz compression microchannel (301), a quartz substrate (302), a chromium window (303), a photomultiplier tube (304), and a sine wave controlled laser source (305). By exciting fluorescence on microdroplet proteins containing a single cell to be detected within the quartz compression microchannel (301), and using the photomultiplier tube (304) to receive the fluorescence signal and convert it into a voltage signal, the sine wave controlled laser source (305) modulates the fluorescence signal, thereby modulating the single-cell protein fluorescence signal in the droplet to a high frequency to remove low-frequency electrical noise. The microdroplet fluorescence signal collection and processing module (4) includes a signal generator (401). The system comprises a lock-in amplifier (402), a data acquisition card (403), and a signal acquisition system (404). The signal generator (401) modulates the laser source (305) to generate an AC sine wave signal as a reference signal input to the reference signal terminal of the lock-in amplifier (402). The high-frequency fluorescence voltage signal after modulation is input to the test signal terminal of the lock-in amplifier (402). The lock-in amplifier (402) demodulates and reduces noise to a low-frequency voltage signal. The low-frequency voltage signal is input to the data acquisition card (403) to be converted into a digital signal. The signal acquisition system (404) performs data processing and analysis. Combined with the relationship curve between the number of protein molecules and the voltage signal, the number of protein molecules in a single cell to be tested is determined.

2. The system according to claim 1, characterized in that, The microchannel for generating microdroplets is a cross-shaped channel; the cross-shaped channel has a first end and a second end that are opposite to each other, as well as a third end and a fourth end that are opposite to each other; the first end of the cross-shaped channel is the input end for immunofluorescent stained cells (101) and cell lysis buffer (102); the second end of the cross-shaped channel is the outflow end for microdroplets with uniform fluorescence distribution; the third end of the cross-shaped channel is the input end for fluorinated oil (103); and the fourth end of the cross-shaped channel contains fluorinated oil (103).

3. The system according to claim 1, characterized in that, First, a layer of perforated polydimethylsiloxane material is bonded to the microdroplet quartz compression channel detection module (3). A manual pressure pump is connected to this perforated polydimethylsiloxane material through a microtube to control the flow rate of the microdroplets in the microdroplet quartz compression channel detection module (3).

4. The system according to claim 1, characterized in that, The (1) mainly consists of a microdroplet generation chip, and a microdroplet generation microchannel is arranged on the microdroplet generation chip; the cross-sectional size of the microdroplet generation chip is 20*20 um 2 ; The flow rates of the aqueous phase of immunofluorescence stained cells (101) and the aqueous phase of cell lysis buffer (102) were controlled at 2 μL / min by an injection pump, and the flow rate of the oil phase of fluorinated oil (103) was controlled at 4 μL / min, so that the final microdroplet generation volume reached 8 pL.

5. The system according to claim 1, characterized in that, In the microdroplet fragmentation and generation module (1), the volume of the microdroplet generated by the channel of the microdroplet generation microchannel, and the corresponding stretching length of the microdroplet, are expressed by the following formula: （1.1） in, This represents the stretching length of the microdroplet within the microchannels where it is generated. To create the width of the microchannels for microdroplets, and These are the volumetric flow rates of the aqueous phase and the oil phase, respectively. The viscosity of the oil phase. The surface tension of the oil phase, The height of the microchannels generated by the microdroplets.

6. The system according to claim 1, characterized in that, The volume of the droplet in the quartz compression microchannel (301) during microdroplet detection is expressed by the following formula: （1.2） in, Let be the volume of the droplet. For the high density of quartz compressed microchannels, To compress the width of the microchannel in quartz, For the width of the chrome window, The time it takes for the droplet head to enter the chromium window. This represents the time it takes for the droplet to completely enter the chromium window. This represents the time it takes for the droplet to gradually detach from the chrome window.

7. The system according to claim 1, characterized in that... The relevant parameters of the sine wave controlled laser source (305) and lock-in amplifier (402) are as follows: the frequency of the corresponding sine wave is 20kHz, the AC amplitude is 1V, and the DC bias is 0.5V; while the filter order of the lock-in amplifier (402) is selected as 15th order and the time constant is selected as 0.5ms.

8. The system according to claim 1, characterized in that, The signal acquisition and data processing module (4) has a preset curve showing the relationship between the number of protein molecules and the voltage signal.

9. The system according to claim 1, characterized in that, A quartz compression microchannel (301) penetrates a quartz substrate (302); a chromium window (303) is disposed on the sidewall of the quartz compression microchannel (301); a photomultiplier tube (304) and a sine wave controlled laser source (305) are disposed outside the quartz substrate (302), with the photomultiplier tube (304) and the sine wave controlled laser source (305) facing the chromium window (303); by fluorescently exciting the microdroplet protein containing a single cell to be detected in the quartz compression microchannel (301), the photomultiplier tube (304) receives the fluorescence signal and converts it into a voltage signal, and then the sine wave controlled laser source (305) modulates the fluorescence.

10. A method for quantitative detection of single-cell proteins using the system according to any one of claims 1-9, characterized in that, The method includes: S1 generates stable, uniform fluorescent microdroplets that encapsulate single cells; S2, the uniform fluorescent microdroplet is introduced into the quartz compressed microchannel (301), and the uniform fluorescent microdroplet is fluorescently modulated by a sine wave controlled laser light source (305), and then demodulated by a lock-in amplifier (402) to obtain the noise-reduced fluorescent voltage signal; S3, multiple sets of fluorescently labeled antibody solutions with known protein concentrations are introduced into the quartz compression microchannel. A sine wave controlled laser light source modulates the fluorescence generated by the equivalent antibody solution. A lock-in amplifier is used to demodulate and obtain the calibration curve of protein concentration and fluorescence voltage signal. S4. Fluorescence compensation is performed based on the protein molecule number versus voltage signal curve to obtain the corrected protein molecule number versus voltage signal curve. S5, acquire the fluorescence voltage signal of the single cell obtained by the modulation and demodulation detection, and obtain the number of protein molecules in the single cell based on the voltage signal and the modified curve of the relationship between the number of protein molecules and the voltage signal.

Images