Single-cell protein quantification system and method based on diffractive optical elements

Abstract

The application discloses a single-cell protein quantitative detection system and method based on a diffractive optical element, and belongs to the technical field of micro-fluids. The system comprises a fluid control module, a micro-fluid channel module, a light beam shaping module and a data acquisition and processing module. The application improves the single-cell protein quantitative detection resolution. Compared with the uniform light field obtained by light beam interception, the uniform light field formed by the diffractive optical element in the micro-channel detection area has higher intensity, so that higher fluorescence detection signals can be obtained, thereby improving the resolution of the single-cell protein quantitative detection. The application improves the accuracy of the single-cell protein quantitative detection. Due to the existence of the chromium window on the micro-channel substrate, the fluorescence emitted by the fluorescence molecules at different heights in the channel will be blocked by the chromium window to different degrees, resulting in different signals measured by the photoelectric sensor. The diffractive optical element does not need the chromium window, thereby improving the accuracy of the single-cell protein quantitative detection.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics technology, specifically relating to a single-cell protein quantitative detection system and method based on diffractive optical elements. Background Technology

[0002] The cell is the basic unit of morphology, structure, and physiology of living organisms, as well as the basic unit of life function. Current cell research primarily focuses on cell morphology, structure, and components, typically requiring the analysis of a substantial number of cells to obtain average results on cell characteristics. However, cellular heterogeneity is particularly prominent in areas such as microbial infection and immunity (leukocytes), and tumor diagnosis and treatment (tumor cells). Studies at the cell population level often mask underlying molecular mechanisms; single-cell research, on the other hand, better reflects the true state of life events and possesses significant academic value.

[0003] Single-cell protein detection refers to the assessment of the types and quantities of proteins expressed at the single-cell level. Quantitative results of single-cell proteins are crucial parameters for analyzing cellular heterogeneity, making single-cell protein detection a key analytical tool in cell biology research. Currently, single-cell protein quantification typically employs fluorescence flow cytometry or mass flow cytometry. Fluorescence flow cytometry uses cells stained with fluorescently labeled antibodies, based on laser excitation and fluorescence detection, to detect intracellular antigen expression. Due to limitations of fluorescently labeled microspheres, this method can only achieve quantitative detection of membrane proteins. Mass flow cytometry uses cells stained with rare-earth metal-labeled antibodies, which are ionized and analyzed using a time-of-flight mass spectrometer to quantify antigen expression in individual cells. However, due to the lack of calibration methods, this method still cannot obtain the absolute quantity of corresponding proteins in a single cell.

[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. There are two main methods for single-cell protein quantification based on microfluidics: micro-engraving and barcode microchip methods. Both methods 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 and are limited by throughput, resulting in low detection throughput that cannot meet the needs of clinical diagnostics.

[0005] To achieve quantitative detection of proteins at any location on a single cell and improve throughput, cited patent CN111323403A proposes a single-cell protein quantitative detection system based on a three-dimensional uniform light field. The microchannel cross-sectional size of this system is larger than that of the cell, enabling high-throughput cell detection. Simultaneously, it utilizes three-dimensional uniform excitation light to achieve fluorescence detection of proteins at any location on a single cell. However, this method has low laser energy utilization efficiency, affecting the resolution of the protein quantitative detection results. Furthermore, due to the obstruction of the chromium window, the generated fluorescence signal is affected by the height of the cell in the channel, thus impacting the detection accuracy.

[0006] There are two main problems with the existing technology: (1) Low energy utilization efficiency of excitation light. In order to ensure the uniformity of the excitation light field, the current three-dimensional uniform excitation light based on beam interception only takes a small part of the slowly changing intermediate core of the Gaussian beam, and most of the excitation light energy is wasted, which reduces the resolution of the single-cell protein quantitative detection results. (2) The fluorescence detection signal is highly cell-dependent. In order to obtain three-dimensional uniform excitation light, the existing technology sputters and etches chromium windows on the substrate of the microfluidic chip. However, since the chromium window is located at the bottom of the microchannel, the fluorescence emitted by fluorescent molecules at different heights of the microchannel will be blocked to different degrees, resulting in different proportions of fluorescence that can be recovered by the microscope objective, which will reduce the accuracy of single-cell protein quantitative detection. Summary of the Invention

[0007] To address the technical problems mentioned in the background above, this invention utilizes diffractive optical elements to develop a single-cell protein quantitative detection system and method based on diffractive optical elements. This invention can improve the efficiency of excitation light utilization and eliminate the cell-dependent nature of the signal.

[0008] This invention is achieved through the following technical solution:

[0009] A single-cell protein quantitative detection system based on diffractive optical elements includes a fluid control module, a microfluidic channel module, a beam shaping module, and a data acquisition and processing module.

[0010] The fluid control module is used to control the flow rate of the sample flow and the sheath flow channel. It includes a fluid injection pump, a liquid guiding tube, a disposable syringe, and a connector. The disposable syringe is connected to the fluid injection pump, and the fluid injection pump is connected to the microfluidic chip through the liquid guiding tube and the connector.

[0011] The microfluidic channel module is used to focus the sample flow and the cells in it, confining the cells flowing through the detection area within a uniform light field. The microfluidic channel module includes a microfluidic chip, which is provided with a sample inlet channel, a sheath flow channel and a mixed liquid outlet.

[0012] The beam shaping module is used to shape the spot of a high-quality single-mode Gaussian beam. The beam shaping module includes a laser source, a collimator, a beam expander, a diffractive optical element, and a microscope objective. The laser source, collimator, beam expander, diffractive optical element, and microscope objective are arranged sequentially along the emission direction of the source. The laser source is used to provide a high-quality single-wavelength Gaussian beam. The collimator is used to convert the Gaussian beam generated by the laser source into collimated parallel light. The beam expander is used to adjust the size of the collimated Gaussian beam. The diffractive optical element is used for phase modulation. The microfluidic chip is disposed on the focal plane of the microscope objective.

[0013] The data acquisition and processing module includes a photoelectric sensor, a data acquisition card, and a central controller. The photoelectric sensor, the data acquisition card, and the central controller are connected in sequence for communication. The photoelectric sensor is used to convert the fluorescence emission light signal generated by the laser excitation of a single cell into an electrical signal. The data acquisition card is used to transmit the electrical signal to the central controller. The central processor is used for data processing.

[0014] In the above technical solution, the capacity of the disposable syringe is 1 mL.

[0015] In the above technical solution, the flow rate of the fluid injection pump is 0.0033 µL / min-205.30 mL / h.

[0016] In the above technical solution, the microfluidic chip is a quartz microfluidic chip fabricated using reactive ion etching process.

[0017] In the above technical solution, the cross-sectional area of ​​the channel of the microfluidic chip is larger than the cross-sectional area of ​​a single cell to be detected.

[0018] In the above technical solution, the detection channel of the microfluidic chip has a width of 100μm and a height of 50μm.

[0019] In the above technical solution, the central processing unit is a device such as a computer terminal that has data processing and storage functions.

[0020] A method for quantitative detection of proteins in single cells based on diffractive optical elements includes the following steps:

[0021] Step 1: Prepare single-cell solutions for staining with fluorescently labeled proteins;

[0022] Step 2: Pass the single-cell solution described in Step 1 into the microfluidic chip channel, and use the uniform light field generated by the diffraction optical element to perform spatial three-dimensional fluorescence detection on the single cell.

[0023] Step 3: Obtain the calibration curve of protein molecule number versus voltage signal;

[0024] Step 4: Obtain the fluorescence compensation coefficient;

[0025] Step 5: Obtain the single-cell solution voltage signal obtained from the three-dimensional fluorescence detection, correct it according to the fluorescence compensation coefficient to obtain the compensated voltage value, and combine it with the protein molecule number and voltage signal relationship curve to obtain the absolute quantitative value of the single-cell protein molecule number.

[0026] In the above technical solution, in step 1, the single cell contains one type of protein or multiple types of proteins, and the protein distribution in the single cell is uniform or non-uniform.

[0027] In the above technical solution, in step 2, a single-cell solution stained with fluorescently labeled protein is introduced into the microchannel of the microfluidic chip. When a single cell passes through the three-dimensional spatial region (i.e., the excitation detection region) with uniform intensity of a focused laser beam in the microchannel, the single cell stained with fluorescently labeled protein is excited by excitation light of a specific wavelength and generates emission light of the corresponding wavelength. The emission light is collected by a photoelectric sensor, and the detected fluorescence signal is converted into a voltage signal.

[0028] In the above technical solution, the method for obtaining the calibration curve in step 3 is as follows: Multiple sets of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are introduced into the microchannels of the microfluidic chip. A uniform light field modulated by a diffractive optical element is used to perform three-dimensional fluorescence detection on the equivalent antibody solutions. The protein antibody solutions are excited by excitation light of a specific wavelength, generating emission light of the corresponding wavelength. A photodetector then collects the voltage signal of the corresponding wavelength channel, obtaining the relationship curve between the number of protein molecules and the voltage signal. At this time, the fluorescent molecules of the multiple sets of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are uniformly distributed in the excitation detection region. Since the intensity of the excitation light field is consistent in three-dimensional space, the voltage signals obtained from different distributions of the same number of protein molecules in the excitation detection region are the same.

[0029] In the above technical solution, in step 4, the method for obtaining the fluorescence compensation coefficient is as follows: multiple sets of fluorescently labeled protein antibody solutions with known protein molecule numbers are introduced into the microchannel of the same microfluidic chip. When a set of antibody solutions passes through the three-dimensional region (i.e., the excitation detection region) of the laser focusing space in the microchannel, the fluorescence voltage signals of multiple wavelength channels are collected simultaneously by the photoelectric sensor, thereby obtaining multiple curves showing the relationship between the number of protein molecules and the voltage signal. The fluorescence compensation coefficient can be obtained through these curves.

[0030] Beneficial effects:

[0031] (1) This invention improves the resolution of single-cell protein quantitative detection. Compared with the uniform light field obtained by beam interception, the uniform light field formed in the detection area of ​​the microchannel using diffractive optical elements has a higher intensity, thus obtaining a higher fluorescence detection signal, thereby improving the resolution of single-cell protein quantitative detection.

[0032] (2) This invention improves the accuracy of quantitative detection of single-cell proteins. Due to the presence of chromium windows on the microchannel substrate, the fluorescence emitted by fluorescent molecules at different heights within the channel is blocked to varying degrees by the chromium windows, resulting in different signals measured by the photoelectric sensor. Using diffractive optical elements eliminates the need for chromium windows, thereby improving the accuracy of quantitative detection of single-cell proteins. Attached Figure Description

[0033] Figure 1 This is a diagram of the system components and modules.

[0034] Figure 2 This describes the experimental steps for quantitative detection of proteins in single cells.

[0035] Among them, 1 is a fluid injection pump, 2 is a liquid delivery hose, 3 is a sample injection channel, 4 is a sheath flow channel, 5 is a mixed liquid outlet, 6 is a laser source, 7 is a collimator, 8 is a beam expander, 9 is a diffractive optical element, 10 is a microscope objective, 11 is a photoelectric sensor, 12 is a data acquisition card, and 13 is a central controller. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0037] This embodiment mainly consists of hardware devices and experimental procedures.

[0038] The hardware required for this embodiment is as follows: Figure 1 As shown, it mainly includes a fluid control module, a microfluidic channel module, a beam shaping module, and a data acquisition and processing module.

[0039] The fluid control module, used to control the flow rate of the sample flow and sheath flow channels, includes a fluid injection pump 1, a liquid delivery tubing 2, a disposable syringe, and a connector. The fluid injection pump 1, using a 1 mL disposable syringe, can manually adjust the output flow rate to the desired level. It provides an adjustable flow rate within the range of 0.0033 µL / min to 205.30 mL / h. Connected to the microfluidic chip via the liquid delivery tubing and connector, it provides the microfluidic channel module with an adjustable sample flow and sheath flow.

[0040] The microfluidic channel module is used to focus the sample flow and the cells within it, confining the cells flowing through the detection area within a uniform light field. This module is a quartz microfluidic chip fabricated using reactive ion etching (RIE) technology, and mainly consists of a sample inlet channel 3, a sheath flow channel 4, and a mixed liquid outlet 5. The cross-sectional area of ​​the microchannel is larger than the cross-sectional area of ​​a single cell to be detected, and the detection channel width is 100 μm. The height is 50 With the design of the sample flow and sheath flow rates, a single cell to be detected can pass through the detection area where the uniform light field is located, avoiding cell blockage and achieving the high throughput requirement.

[0041] The beam shaping module is used to shape the spot of a high-quality single-mode Gaussian beam. This module includes a laser source 6, a collimator 7, a beam expander 8, a diffractive optical element 9, and a microscope objective 10. The laser source provides a high-quality single-wavelength Gaussian beam. The collimator 7 converts the Gaussian beam generated by the laser (laser source 6) into collimated parallel light. The beam expander 8 adjusts the size of the collimated Gaussian beam to meet the beam size required by the diffractive optical element 9. Then, after phase modulation by the diffractive optical element 9, the beam can be shaped within a 50° radius before and after the focal plane of the microscope objective 10. 50 within the range The square light spot means that a three-dimensional, uniform light field region for excitation light can be obtained within the microchannel.

[0042] The data acquisition and processing module consists of a photoelectric sensor 11, a data acquisition card 12, and a central controller 13. The photoelectric sensor 11 converts the fluorescence emission signal generated by the single cell when excited by laser into an electrical signal, which is then acquired by the data acquisition card 12 and transmitted to the central controller 13 for data processing. The central processor 13 can be a computer terminal or other device with data processing and storage functions.

[0043] This embodiment provides a method for quantitative detection of single-cell proteins using the above-described detection system, combined with the attached... Figure 1 See attached document Figure 2 The method includes the following steps:

[0044] Step 1: Prepare a single-cell solution stained with fluorescently labeled proteins. It should be noted that the single-cell solution prepared here can refer to a single cell containing only one type of protein, or a single cell containing multiple types of proteins; it can refer to a single cell with uniform protein distribution, or a single cell with uneven protein distribution. Therefore, the method of this embodiment can not only quantitatively detect single cells containing only one type of protein, but also single cells containing multiple types of proteins; it can not only quantitatively detect single cells with uniform protein distribution, but also single cells with uneven protein distribution. When quantitatively detecting single cells containing multiple types of proteins, the absolute quantitative values ​​of the number of each protein molecule can be obtained simultaneously.

[0045] Step 2: The single-cell solution is introduced into the microfluidic chip channel, and the single cells are subjected to spatial three-dimensional fluorescence detection using a uniform light field modulated by a diffractive optical element. A single-cell solution stained with fluorescently labeled proteins is introduced into the microchannel of the microfluidic chip. When a single cell sequentially passes through a three-dimensional spatial region (i.e., the excitation detection region) within the microchannel where a focused laser beam has uniform intensity, the fluorescently labeled single cell is excited by excitation light of a specific wavelength, generating emission light of the corresponding wavelength. This emission light is collected by a photoelectric sensor, and the detected fluorescence signal is converted into a voltage signal.

[0046] Step 3: Obtain the calibration curve of protein molecule number versus voltage signal. Specifically, multiple sets of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are introduced into the microchannels of the microfluidic chip. A uniform light field modulated by diffractive optical elements is used to perform three-dimensional fluorescence detection on the equivalent antibody solutions. The protein antibody solutions are excited by excitation light of a specific wavelength, generating emission light of the corresponding wavelength. A photodetector then collects the voltage signal of the corresponding wavelength channel, obtaining the relationship curve between protein molecule number and voltage signal. At this point, the fluorescent molecules of the multiple sets of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are uniformly distributed in the excitation detection area. Since the intensity of the excitation light field is consistent in three-dimensional space, the voltage signal obtained from different distributions of the same number of protein molecules in the excitation detection area is the same. By detecting equivalent antibody solutions of different concentrations and obtaining the fluorescence intensity of the corresponding detection channels, several logarithmic relationships between the equivalent protein molecule number and fluorescence intensity can be obtained. The calibration curve can be obtained by linear fitting with the number of equivalent protein molecules as the x-axis and fluorescence intensity as the y-axis:

[0047] (1)

[0048] in, Indicates fluorescence intensity, Indicates the number of equivalent protein molecules. This indicates the slope of the calibration curve.

[0049] Step 4: Obtain the fluorescence compensation coefficient. Specifically, multiple fluorescently labeled protein antibody solutions with known protein molecule numbers are introduced into the microchannels of the same microfluidic chip. When an antibody solution passes through the three-dimensional region (i.e., the excitation detection region) of the laser focusing space within the microchannel, the fluorescence voltage signals of multiple wavelength channels are simultaneously acquired by a photoelectric sensor. This allows the fluorescence intensity values ​​of the fluorescently labeled antibody solution in all channels to be obtained. The fluorescence compensation coefficient can then be obtained by analyzing the fluorescence intensity values ​​of all types of fluorescently labeled antibody solutions at various channels. (Leakage value) Defined as the ratio of the fluorescence voltage value of fluorescent label n in the overflow channel m to the fluorescence voltage value in its detection channel n, in a system with N types of fluorescent labels and N detection channels, there are a total of There is an overflow value, and naturally there is By collecting all the overflow values, we can obtain the overflow matrix. Assuming This represents the fluorescence intensity vector detected in each channel. Let be the true fluorescence intensity vector of each fluorophore in its main channel, then we have:

[0050] (2)

[0051] in, This represents the fluorescence intensity vector of each detection channel. Represents the overflow matrix. This represents the true fluorescence intensity vector of each fluorophore in its main channel. Therefore, the inverse of the overflow matrix is ​​the fluorescence compensation matrix, from which the specific compensation coefficients can be obtained.

[0052] Step 5: Obtain the single-cell solution voltage signal obtained from the three-dimensional fluorescence detection, correct it according to the fluorescence compensation coefficient to obtain the compensated voltage value, and combine it with the protein molecule number versus voltage signal relationship curve to obtain the absolute quantitative value of the single-cell protein molecule number. That is, the formula for calculating the protein molecule number is:

[0053] (3)

[0054] Represents the protein molecule number vector. This represents a block matrix composed of the slopes of the calibration curves, where the diagonal elements are the slopes of the respective calibration curves, and the remaining elements are 0. Represents the overflow matrix. This represents the fluorescence intensity vector of each detection channel.

[0055] In this embodiment, the diffractive optical element is a phase element, which utilizes the continuous relief or stepped structure etched on the substrate based on the theory of optical wave diffraction to achieve phase modulation of the laser beam. The material used for the diffractive optical element in this embodiment is fused silica, but other optical materials such as ZnSe quartz glass, zinc selenide, or other organic materials can also be used. There are also multiple fabrication processes for diffractive optical elements, such as photolithography, thin film deposition, direct writing, grayscale masking, etc.

[0056] In this embodiment, the collimator and beam expander are used to obtain a collimated Gaussian beam of a certain size. The beam expander has different structures: Keplerian structure and Galilean structure. It can also be matched with lens groups to achieve the effect of beam expansion (or beam contraction). The microscope objective can also be replaced with other focusing lenses with corresponding focal lengths without affecting the spot effect.

[0057] In this embodiment, a transparent material is used as the substrate material for the microfluidic chip. The shape and size of the interface between the sample flow channel inlet, sheath flow channel inlet, and mixed fluid outlet and the fluid control module do not affect the basic functionality. In this invention, the microchannel cross-section is rectangular, but it can also be replaced with a circular or semi-circular shape without affecting the basic functionality.

[0058] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 single-cell protein quantitative detection system based on diffractive optical elements, characterized in that: It includes a fluid control module, a microfluidic channel module, a beam shaping module, and a data acquisition and processing module; The fluid control module is used to control the flow rate of the sample flow and the sheath flow channel. It includes a fluid injection pump, a liquid guiding tube, a disposable syringe, and a connector. The disposable syringe is connected to the fluid injection pump, and the fluid injection pump is connected to the microfluidic chip through the liquid guiding tube and the connector. The microfluidic channel module is used to focus the sample flow and the cells in it, confining the cells flowing through the detection area within a uniform light field. The microfluidic channel module includes a microfluidic chip, which is provided with a sample inlet channel, a sheath flow channel and a mixed liquid outlet. The beam shaping module is used to shape the spot of a high-quality single-mode Gaussian beam. The beam shaping module includes a laser source, a collimator, a beam expander, a diffractive optical element, and a microscope objective. The laser source, collimator, beam expander, diffractive optical element, and microscope objective are arranged sequentially along the emission direction of the source. The laser source is used to provide a high-quality single-wavelength Gaussian beam. The collimator is used to convert the Gaussian beam generated by the laser source into collimated parallel light. The beam expander is used to adjust the size of the collimated Gaussian beam. The diffractive optical element is used for phase modulation. The microfluidic chip is disposed on the focal plane of the microscope objective. The cross-sectional area of ​​the microchannel is larger than that of a single cell to be detected. The detection channel is 100 μm wide and 50 μm high, which allows a single cell to be detected to pass through the detection area where the uniform light field is located. The Gaussian beam, after being phase-modulated by the diffractive optical element, forms a 50μm square spot within a 50μm range before and after the focal plane of the microscope objective, thus obtaining a three-dimensional uniform excitation light field region in the microchannel. The data acquisition and processing module includes a photoelectric sensor, a data acquisition card, and a central controller. The photoelectric sensor, the data acquisition card, and the central controller are connected in sequence for communication. The photoelectric sensor is used to convert the fluorescence emission light signal generated by the laser excitation of a single cell into an electrical signal. The data acquisition card is used to transmit the electrical signal to the central controller, and the central controller is used for data processing.

2. The single-cell protein quantitative detection system based on diffractive optical elements according to claim 1, characterized in that: The capacity of the disposable syringe is 1 mL.

3. The single-cell protein quantitative detection system based on diffractive optical elements according to claim 1, characterized in that: The flow rate of the fluid injection pump is 0.0033 µL / min - 205.30 mL / h.

4. The single-cell protein quantitative detection system based on diffractive optical elements according to claim 1, characterized in that: The microfluidic chip is a quartz microfluidic chip fabricated using reactive ion etching (RIE) technology.

5. The single-cell protein quantitative detection system based on diffractive optical elements according to claim 1, characterized in that: The detection channel of the microfluidic chip has a width of 100μm and a height of 50μm.

6. A method for quantitative detection of proteins in single cells based on diffractive optical elements, characterized in that, Includes the following steps: Step 1: Prepare single-cell solutions for staining with fluorescently labeled proteins; Step 2: Pass the single-cell solution described in Step 1 into the microfluidic chip channel, and use the uniform light field generated by the diffraction optical element to perform spatial three-dimensional fluorescence detection on the single cell. Step 3: Obtain the calibration curve of protein molecule number versus voltage signal; Step 4: Obtain the fluorescence compensation coefficient; Step 5: Obtain the single-cell solution voltage signal obtained from the three-dimensional fluorescence detection, correct it according to the fluorescence compensation coefficient to obtain the compensated voltage value, and combine it with the relationship curve between the number of protein molecules and the voltage signal to obtain the absolute quantitative value of the number of protein molecules in the single cell. In step 1, the single cell contains one or more types of proteins, and the proteins in the single cell may be uniformly or unevenly distributed. In step 2, a single-cell solution stained with fluorescently labeled protein is introduced into the microchannel of the microfluidic chip. When a single cell passes through a three-dimensional spatial region with uniform intensity of a focused laser beam in the microchannel, the single cell stained with fluorescently labeled protein is excited by excitation light of a specific wavelength and generates emission light of the corresponding wavelength. The emission light is collected by a photoelectric sensor, and the detected fluorescence signal is converted into a voltage signal. In step 3, the method for obtaining the calibration curve is as follows: multiple sets of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are introduced into the microchannel of the microfluidic chip. The equivalent antibody solutions are subjected to spatial three-dimensional fluorescence detection using a uniform light field modulated by a diffractive optical element. The protein antibody solutions are excited by excitation light of a specific wavelength, generating emission light of the corresponding wavelength. The voltage signal of the corresponding wavelength channel is then collected by a photodetector to obtain the relationship curve between the number of protein molecules and the voltage signal. In step 4, the method for obtaining the fluorescence compensation coefficient is as follows: multiple sets of fluorescently labeled protein antibody solutions with known protein molecule numbers are introduced into the microchannels of the same microfluidic chip. When a set of antibody solutions passes through the three-dimensional region of the laser focusing space in the microchannel, the fluorescence voltage signals of multiple wavelength channels are simultaneously collected by the photoelectric sensor, thereby obtaining multiple relationship curves between the number of protein molecules and the voltage signal. The fluorescence compensation coefficient is obtained through the relationship curves.

Images