Single-cell protein quantitative detection system and method based on diffractive optical element

Abstract

The invention discloses a single-cell protein quantitative detection system and method based on a diffractive optical element, and belongs to the technical field of microfluidics, the system comprises a fluid control module, a microfluidic channel module, a beam shaping module and a data acquisition and processing module; according to the invention, the single-cell protein quantitative detection resolution is improved. Compared with a uniform light field obtained by light beam interception, the uniform light field formed in the detection area in the micro-channel by using the diffraction optical element has higher intensity, so that a higher fluorescence detection signal can be obtained, and the resolution ratio of single-cell protein quantitative detection is improved; the accuracy of quantitative detection of the single-cell protein is improved. Due to the existence of a chromium window on a micro-channel substrate, fluorescence emitted by fluorescent molecules at different heights in a channel can be blocked by the chromium window to different degrees, so that signals measured by a photoelectric sensor are different. A chromium window is not needed by utilizing the diffractive optical element, so that the accuracy of quantitative detection of the single-cell protein can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and in particular relates to a single-cell protein quantitative detection system and method based on a diffraction optical element. Background Art

[0002] Cells are the basic units of morphology, structure, and physiology of living organisms, and are also the basic units of life functions. Currently, the objects of cell research mainly include cell morphology, structure, and components. It is usually necessary to detect a considerable number of cells for statistical analysis of data to obtain the average results of cell characteristics. However, cell heterogeneity is particularly prominent in the fields of microbial infection and immunity (leukocytes), tumor diagnosis and treatment (tumor cells), etc. In the face of research at the cell population level, there is often the problem of concealing the underlying molecular mechanisms. Single-cell research can better reflect the true situation of life events and has important academic value.

[0003] Single-cell protein detection refers to the evaluation of the types and quantities of protein expression at the single-cell level, and the quantitative results of single-cell proteins are important parameters for analyzing cell heterogeneity. Therefore, single-cell protein detection is a key analytical tool in cell biology research. Currently, single-cell protein quantification is usually performed using fluorescence flow cytometry or mass spectrometry. Fluorescence flow cytometry uses cells stained with fluorescently labeled antibodies to detect antigen expression in cells based on laser excitation and fluorescence detection. Due to the limitations of fluorescently labeled microspheres, this method can only achieve quantitative detection of membrane proteins. Mass spectrometry flow cytometry uses cells stained with rare earth metal-labeled antibodies to be ionized and analyzed using a time-of-flight mass spectrometer to quantify antigen expression in individual cells, but due to the lack of calibration methods, this method still cannot obtain the absolute number of corresponding proteins in single cells.

[0004] Since microfluidics matches the size of biological cells (tens to hundreds of microns), 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. The principle of both methods is to confine single cells in micropores or microcavities, and then perform cell lysis, protein capture and quantification. Although these microfluidic methods are based on large array analysis, they cannot continuously characterize single cells and are limited by throughput, so the detection throughput is low and cannot meet the needs of clinical diagnosis.

[0005] In order to achieve quantitative detection of proteins at any position of a single cell and improve the detection throughput, the cited patent CN111323403A proposes a single-cell protein quantitative detection system based on a three-dimensional uniform light field. The cross-sectional size of the microchannel of this system is larger than that of the cell, which can achieve high-throughput detection of cells. At the same time, three-dimensional uniform excitation light is used to achieve fluorescence detection of proteins at any position of a single cell. However, the laser energy utilization efficiency of this method is low, which affects the resolution of the protein quantitative detection results. At the same time, due to the obstruction of the chrome window, the generated fluorescence signal will be affected by the height of the cell in the channel, thereby affecting the detection accuracy.

[0006] There are two problems with the existing technology: (1) The efficiency of excitation light energy utilization is low. In order to ensure the uniformity of the excitation light field, the three-dimensional uniform excitation light based on beam interception only takes a small part of the middle core with slow intensity change in 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 dependent on the cell. In order to obtain three-dimensional uniform excitation light, the existing technology sputters and etches a chromium window on the substrate of the microfluidic chip. However, since the chromium window is located at the bottom of the microchannel, the fluorescence emitted by the fluorescent molecules at different heights of the microchannel will be blocked to varying degrees, resulting in different proportions of fluorescence that can be recovered by the microscope objective, thereby reducing the accuracy of single-cell protein quantitative detection. Summary of the invention

[0007] In response to the technical problems mentioned in the above background, the present invention uses diffractive optical elements to invent a single-cell protein quantitative detection system and method based on diffractive optical elements. The present invention can improve the utilization efficiency of excitation light and eliminate the cell-height dependence of the signal.

[0008] The present invention is achieved through the following technical solutions:

[0009] A single-cell protein quantitative detection system based on diffractive optical elements, including 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 sheath flow channels, and includes a fluid injection pump, a liquid guide 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 guide tube and the connector.

[0011] The microfluidic channel module is used to achieve focusing of the sample flow and the cells therein, and to limit the cells flowing through the detection area within the range of the uniform light field. The microfluidic channel module includes a microfluidic chip, and the microfluidic chip is provided with a sample injection channel, a sheath flow channel and a mixed liquid outlet;

[0012] The beam shaping module is used to perform spot shaping on a high-quality single-mode Gaussian beam. The beam shaping module includes a laser light source, a collimator, a beam expander, a diffractive optical element and a microscope objective. The laser light source, the collimator, the beam expander, the diffractive optical element and the microscope objective are sequentially arranged along the emission direction of the light source. The laser light source is used to provide a single-wavelength high-quality Gaussian beam. The collimator is used to convert the Gaussian beam generated by the laser light 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 arranged 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, which are communicatively connected in sequence. The photoelectric sensor is used to convert the fluorescent emission light signal generated by a single cell excited by a laser into an electrical signal, the data acquisition card is used to transmit the electrical signal to the central controller, and the central processor is used to perform 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 manufactured by 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 100 μm and a height of 50 μm.

[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] The single cell protein quantitative detection method based on diffractive optical elements includes the following steps:

[0021] Step 1, preparing a single cell solution stained with fluorescently labeled proteins;

[0022] Step 2, passing the single cell solution in step 1 into the channel of the microfluidic chip, and performing spatial three-dimensional fluorescence detection on the single cell using a uniform intensity light field modulated by a diffractive optical element;

[0023] Step 3, obtaining a calibration curve of the number of protein molecules and the voltage signal;

[0024] Step 4, obtaining the fluorescence compensation coefficient;

[0025] Step 5: Obtain the single-cell solution voltage signal obtained according to the spatial 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 single-cell protein molecules.

[0026] In the above technical solution, in step 1, the single cell contains one type of protein or multiple types of proteins, and the proteins in the single cell are evenly distributed or unevenly distributed.

[0027] In the above technical solution, in step 2, a single cell solution stained with a fluorescent marker protein is introduced into a microchannel of a microfluidic chip. When single cells sequentially pass through a spatial three-dimensional region (i.e., an excitation detection region) in which a focused laser beam has uniform intensity in the microchannel, the single cells stained with the fluorescent marker protein are excited by excitation light of a specific wavelength and generate emission light of a 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, in step 3, the method for obtaining the calibration curve is as follows: multiple groups of fluorescently labeled and stained equivalent antibody solutions with known numbers of protein molecules are introduced into the microchannel of the microfluidic chip, and the equivalent antibody solution is subjected to spatial three-dimensional fluorescence detection using a uniform light field generated by modulation of a diffractive optical element, the protein antibody solution is excited by excitation light of a specific wavelength, and an emission light of a corresponding wavelength is generated, and then the voltage signal of the corresponding wavelength channel is collected by a photoelectric detector to obtain a curve of the relationship between the number of protein molecules and the voltage signal. At this time, the fluorescent molecules of multiple groups of fluorescently labeled and stained equivalent antibody solutions with known numbers of protein molecules are uniformly distributed in the excitation detection area. Since the intensity of the excitation light field is consistent in three-dimensional space, the voltage signals obtained by different distributions of the same number of protein molecules in the excitation detection area are the same.

[0029] In the above technical solution, in step 4, the method for obtaining the fluorescence compensation coefficient is as follows: multiple groups of fluorescently labeled protein antibody solutions with known protein molecule numbers are introduced into the microchannel of the same microfluidic chip. When one group of antibody solutions passes through the three-dimensional area of ​​the laser focusing space in the microchannel (i.e., the excitation detection area), 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, and the fluorescence compensation coefficient can be obtained through these curves.

[0030] Beneficial effects:

[0031] (1) The present invention improves the resolution of single-cell protein quantitative detection. Compared with the uniform light field obtained by intercepting the light beam, the uniform light field formed in the detection area in the microchannel using the diffractive optical element has a higher intensity, so a higher fluorescence detection signal can be obtained, thereby improving the resolution of single-cell protein quantitative detection.

[0032] (2) The present invention improves the accuracy of single-cell protein quantitative detection. Due to the presence of the chromium window on the microchannel substrate, the fluorescence emitted by the fluorescent molecules at different heights in the channel will be blocked to varying degrees by the chromium window, resulting in different signals measured by the photoelectric sensor. The use of diffractive optical elements does not require a chromium window, thereby improving the accuracy of single-cell protein quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 This is an experimental procedure for single cell protein quantification.

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

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

[0037] This embodiment is mainly divided into hardware devices and experimental procedures.

[0038] The hardware devices required for this embodiment are 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 is used to control the flow rate of the sample flow and sheath flow channels, and includes a fluid injection pump 1, a liquid guide tube 2, a disposable syringe and a connector. The fluid injection pump 1 can output the required flow rate by manual adjustment. Using a disposable syringe with a capacity of 1 mL, the fluid injection pump can provide an adjustable flow rate within the range of 0.0033 µL / min-205.30 mL / h, and is connected to the microfluidic chip through the liquid guide tube and the connector to provide the microfluidic channel module with adjustable sample flow and sheath flow.

[0040] The microfluidic channel module is used to achieve the focusing of the sample flow and the cells therein, and to limit the cells flowing through the detection area within the range of the uniform light field. The module is a quartz microfluidic chip made by reactive ion etching process, mainly composed of a sample injection 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 is 100 wide. , height 50 , combined with the design of sample flow and sheath flow rate, can enable a single cell to be detected to pass through the detection area where the uniform intensity light field is located, avoiding cell clogging and achieving high throughput requirements.

[0041] The beam shaping module is used to shape the spot of a high-quality single-mode Gaussian beam. The module includes a laser light source 6, a collimator 7, a beam expander 8, a diffractive optical element 9, and a microscope objective 10. The laser light source provides a single-wavelength high-quality Gaussian beam. The collimator 7 converts the Gaussian beam generated by the laser (laser light source 6) into collimated parallel light. The beam expander 8 adjusts the size of the collimated Gaussian beam to reach the beam size required by the diffractive optical element 9. After the phase modulation of the diffractive optical element 9, the beam can be 50 degrees in front and behind the focal plane of the microscope objective 10. Formed within 50 A square light spot is formed, that is, a three-dimensional uniform intensity light field area of ​​the excitation light can be obtained in the microchannel.

[0042] The data acquisition and processing module is composed of a photoelectric sensor 11, a data acquisition card 12 and a central controller 13. The photoelectric sensor 11 converts the fluorescence emission light signal generated by the laser excitation of the single cell into an electrical signal, and the data acquisition card 12 collects the electrical signal and transmits it 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 single cell protein quantitative detection using the above detection system, combined with the attached Figure 1 , refer to the attached Figure 2 , the method comprises 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 stained with fluorescently labeled proteins prepared here may refer to a single cell containing only one type of protein, or a single cell containing multiple types of proteins, and may refer to a single cell having a uniform distribution of proteins, or a single cell having an uneven distribution of proteins. Thus, through the method of the embodiment of the present invention, not only can quantitative detection of single cells containing only one type of protein be achieved, but also quantitative detection of single cells containing multiple types of proteins can be achieved; not only can quantitative detection of the uniform distribution of proteins in single cells be performed, but also quantitative detection of the uneven distribution of proteins in single cells can be performed. Among them, when quantitative detection of single cells containing multiple types of proteins is performed, the absolute quantitative values ​​of the number of these protein molecules can be obtained at the same time.

[0045] Step 2, passing the single cell solution into the microfluidic chip channel, and performing spatial three-dimensional fluorescence detection on the single cell using the uniform intensity light field modulated by the diffraction optical element. A single cell solution stained with fluorescent marker protein is passed into the microchannel of the microfluidic chip. When the single cell passes through the spatial three-dimensional region (i.e., the excitation detection region) in which the focused laser beam in the microchannel has uniform intensity, the single cell stained with fluorescent marker protein is excited by the excitation light of a specific wavelength, and generates emission light of the corresponding wavelength. The emission light is collected by the photoelectric sensor, and the detected fluorescence signal is converted into a voltage signal.

[0046] Step 3, obtain a calibration curve of the number of protein molecules and the voltage signal. Specifically, multiple groups of fluorescently labeled and stained equivalent antibody solutions with known numbers of protein molecules are introduced into the microchannel of the microfluidic chip, and the equivalent antibody solutions are subjected to spatial three-dimensional fluorescence detection using a uniform intensity light field modulated by a diffraction optical element. The protein antibody solution is excited by an excitation light of a specific wavelength to generate emission light of a corresponding wavelength, and then the voltage signal of the corresponding wavelength channel is collected by a photodetector to obtain a relationship curve between the number of protein molecules and the voltage signal. At this point, the fluorescent molecules of multiple groups of fluorescently labeled and stained equivalent antibody solutions with known numbers of protein molecules are uniformly distributed in the excitation detection area. Since the intensity of the excitation light field is consistent in three-dimensional space, the voltage signals obtained when the same number of protein molecules is distributed differently in the excitation detection area are the same. By detecting equivalent antibody solutions of different concentrations and obtaining the fluorescence intensity of the corresponding detection channel, several pairs of equivalent protein molecules and fluorescence intensity can be obtained. The calibration curve can be obtained by linear fitting with the equivalent protein molecules as the horizontal axis and the fluorescence intensity as the vertical axis:

[0047] (1)

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

[0049] Step 4, obtain the fluorescence compensation coefficient. Specifically, a variety of fluorescently labeled protein antibody solutions with known numbers of protein molecules are introduced into the microchannels of the same microfluidic chip. When an antibody solution passes through the three-dimensional area of ​​the laser focusing space in the microchannel (i.e., the excitation detection area), the photoelectric sensor simultaneously collects the fluorescence voltage signals of multiple wavelength channels, thereby obtaining the fluorescence intensity values ​​of the fluorescently labeled antibody solution in all channels. The fluorescence compensation coefficient can be obtained by the fluorescence intensity values ​​of all types of fluorescently labeled antibody solutions in each channel. Overflow value It is defined as the ratio of the fluorescence voltage value of fluorescent marker n in overflow channel m to the fluorescence voltage value of its detection channel n. In a system with N fluorescent markers and N detection channels, there are a total of overflow value, and naturally there is , we can get the leakage matrix by combining all the leakage values , assuming is the fluorescence intensity vector detected by each channel, is the true fluorescence intensity vector of each fluorophore in its main channel, then:

[0050] (2)

[0051] in, represents the fluorescence intensity vector of each detection channel, represents the leakage matrix, 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, and the specific compensation coefficient can be obtained.

[0052] Step 5, obtain the voltage signal of the single cell solution obtained according to the spatial 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 single cell protein molecules. That is, the calculation formula of the number of protein molecules is:

[0053] (3)

[0054] represents the vector of protein molecules, It represents a square matrix composed of the slopes of the calibration curves, whose diagonal elements are the slopes of each calibration curve, and the remaining elements are 0. represents the leakage matrix, Represents the fluorescence intensity vector of each detection channel.

[0055] In this embodiment, the diffractive optical element is a phase element, which is based on the light wave diffraction theory and uses a continuous relief or step-type structure etched on a substrate to achieve phase modulation of the laser beam. In this embodiment, the material used for the diffractive optical element is fused quartz, and other optical materials can also be used, such as ZnSe quartz glass, zinc selenide or other organic materials. There is more than one processing technology for diffractive optical elements, such as photolithography, thin film deposition, direct writing, grayscale masking, etc.

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

[0057] In this embodiment, the substrate material of the microfluidic chip is transparent. The shape and size of the sample flow channel inlet, the sheath flow channel inlet, the mixed fluid outlet and the interface of the fluid control module do not affect the realization of the basic functions. The cross-section of the microchannel in the present invention is rectangular, and can also be replaced with a circular or semicircular shape without affecting the realization of the basic functions.

[0058] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Single cell protein quantitative detection system based on diffractive optical elements, characterized by: 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 sheath flow channels, and includes a fluid injection pump, a liquid guide 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 guide tube and the connector. The microfluidic channel module is used to achieve focusing of the sample flow and the cells therein, and to limit the cells flowing through the detection area within the range of the uniform light field. The microfluidic channel module includes a microfluidic chip, and the microfluidic chip is provided with a sample injection channel, a sheath flow channel and a mixed liquid outlet; The beam shaping module is used to perform spot shaping on a high-quality single-mode Gaussian beam. The beam shaping module includes a laser light source, a collimator, a beam expander, a diffractive optical element and a microscope objective. The laser light source, the collimator, the beam expander, the diffractive optical element and the microscope objective are sequentially arranged along the emission direction of the light source. The laser light source is used to provide a single-wavelength high-quality Gaussian beam. The collimator is used to convert the Gaussian beam generated by the laser light 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 arranged on the focal plane of the microscope objective. The data acquisition and processing module includes a photoelectric sensor, a data acquisition card and a central controller, which are communicatively connected in sequence. The photoelectric sensor is used to convert the fluorescent emission light signal generated by a single cell excited by a laser into an electrical signal, the data acquisition card is used to transmit the electrical signal to the central controller, and the central processor is used to perform 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 manufactured by reactive ion etching process.

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 100 μm and a height of 50 μm.

6. A single cell protein quantitative detection method based on a diffractive optical element, characterized in that: The steps include: Step 1, preparing a single cell solution stained with fluorescently labeled proteins; Step 2, passing the single cell solution in step 1 into the channel of the microfluidic chip, and performing spatial three-dimensional fluorescence detection on the single cell using a uniform intensity light field modulated by a diffractive optical element; Step 3, obtaining a calibration curve of the number of protein molecules and the voltage signal; Step 4, obtaining the fluorescence compensation coefficient; Step 5: Obtain the single-cell solution voltage signal obtained according to the spatial 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 single-cell protein molecules.

7. The single cell protein quantitative detection method based on diffractive optical elements according to claim 6, characterized in that: In step 1, the single cell contains one type of protein or multiple types of proteins, and the proteins in the single cell are evenly or unevenly distributed.

8. The single cell protein quantitative detection method based on diffractive optical elements according to claim 6, characterized in that: In step 2, a single cell solution stained with a fluorescent marker protein is passed through a microchannel of a microfluidic chip. When single cells sequentially pass through a spatial three-dimensional region in the microchannel where a focused laser beam has uniform intensity, the single cells stained with the fluorescent marker protein are excited by excitation light of a specific wavelength and generate emission light of a corresponding wavelength. The emission light is collected by a photoelectric sensor, and the detected fluorescence signal is converted into a voltage signal.

9. The single cell protein quantitative detection method based on diffractive optical elements according to claim 6, characterized in that: In step 3, the method for obtaining the calibration curve is as follows: multiple groups of fluorescently labeled and stained equivalent antibody solutions with known protein molecule numbers are introduced into the microchannel of the microfluidic chip, and the equivalent antibody solutions are subjected to spatial three-dimensional fluorescence detection using a uniform intensity light field modulated by a diffractive optical element. The protein antibody solution is excited by excitation light of a specific wavelength to generate emission light of a corresponding wavelength, and then a voltage signal of the corresponding wavelength channel is collected by a photoelectric detector to obtain a relationship curve between the number of protein molecules and the voltage signal.

10. The single cell protein quantitative detection method based on diffractive optical elements according to claim 6, characterized in that: In step 4, the method for obtaining the fluorescence compensation coefficient is as follows: multiple groups of fluorescently labeled protein antibody solutions with known protein molecule numbers are introduced into the microchannel of the same microfluidic chip. When one group of antibody solutions passes through the three-dimensional area 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, and the fluorescence compensation coefficient is obtained through the relationship curves.

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