Single-cell nine-protein high-throughput quantitative detection device and method

Abstract

The invention discloses a single-cell nine-protein high-throughput quantitative detection device and method, and belongs to the technical field of single-cell analysis. According to the method, three independent adjustable light spots are generated through a multi-laser coupling light path, the sizes and positions of the light spots are adjusted by using a beam expander, a reflector and a scanning lens, and a three-dimensional uniform light field is constructed by combining parallel beam interception and a long-working-distance objective lens, so that uniform excitation and detection of proteins at different distribution positions of a single cell are realized. Through the volume equivalent calibration principle, the fluorescence signal intensity is associated with the number of antibody molecules, and the influence of the protein distribution position on detection is eliminated. The device comprises a signal modulation module, a light spot positioning and uniform light field module and a signal acquisition and processing module, the excitation light intensity variation coefficient of the uniform light field is lower than 1.3%, the detection light variation coefficient is 1.9%, and the detection limit of nine fluorescence channels reaches 10-10000 molecules. The problem of multi-laser crosstalk is solved, synchronous quantification of nine single-cell proteins is supported, and the high-throughput detection requirement of clinical leukemia typing is met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of protein detection, and particularly relates to a single-cell nine-protein high-throughput quantitative detection device and method. BACKGROUND

[0002] Proteins are a large class of organic matter in nature. According to different biological functions, proteins have functions such as life composition, material transportation, reaction catalysis and signal transmission, and dominate or assist in completing almost all life activities in living organisms. Protein analysis at the single-cell level can provide information on cell heterogeneity, and plays an important role in disease diagnosis and biological research.

[0003] Leukemia is a malignant disease originating from hematopoietic stem cells or precursor cells, which has a major threat to people's life and health. There are about 474,000 new cases of leukemia and 310,000 deaths worldwide each year. Leukemia can be divided into two major categories: acute and chronic, and according to the type of cells, it can be divided into lymphocytic and myelocytic. Accurate typing is the premise of effective treatment and prognosis evaluation. The main method for typing leukemia in clinical practice is immunophenotyping by flow cytometry. The principle is to use fluorescently conjugated antibodies to label the proteins to be tested to achieve single-cell multi-parameter and rapid quantitative analysis. The main problem of flow cytometry is that the calibration is based on calibration microspheres with known number of fluorescent molecules on the surface. Due to the lack of internal fluorescent molecule calibration microspheres that can be accurately controlled, the intracellular proteins to be tested cannot be quantitatively determined, which restricts the accuracy of clinical leukemia typing diagnosis.

[0004] Patent CN111323403A proposes a single-cell protein quantitative detection method based on three-dimensional uniform focusing laser, which realizes quantitative detection of multiple distributed proteins in single cells. However, it is limited by the traditional optical path of the fluorescence microscope, and can only produce a single spot, which cannot solve the crosstalk problem of multiple wavelengths of laser. Therefore, a single wavelength laser is used, which can only quantitatively detect three proteins at the same time, and the detection parameters are few. For example, in the clinical typing of leukemia, at least eight to nine proteins need to be detected, and if this method is used, at least three samples are needed, which is not only cumbersome to operate, but also affects the accuracy of the typing results due to the lack of quantitative results of nine proteins in a single cell. Therefore, this method cannot meet the clinical needs.

[0005] In summary, in order to meet the needs of clinical application, it is necessary to realize high-throughput quantitative detection of nine or more proteins with different distribution positions, so it is very necessary to develop a single-cell nine-protein high-throughput quantitative detection method and device based on spot positioning and uniform light field. SUMMARY

[0006] To solve the technical problems proposed in the background art, the application discloses a single-cell nine-protein high-throughput quantitative detection device and method. The application designs and realizes three uniform fluorescence detection areas with adjustable size and position, calibrates the fluorescence signal of single-cell protein labeling, and realizes rapid quantitative detection of nine proteins in multiple distribution positions of single cells by combining the geometric size of the detection area. The main problem of the prior art is that the detection parameters are few. The current method is limited to the fact that the laser can only form a single spot on the sample through the objective lens, and only three proteins can be quantified at the same time. The application solves the crosstalk problem of different wavelength lasers by constructing three adjustable size and position spots through a multi-laser coupling light path, and can simultaneously quantify multiple distribution positions of nine proteins, greatly improving the number of detection parameters.

[0007] The technical solution of the application is as follows:

[0008] A single-cell nine-protein high-throughput quantitative detection device comprises:

[0009] A signal modulation module is composed of a signal generator and a laser, which is used to generate a sinusoidal carrier signal and amplitude modulate the laser;

[0010] A spot positioning and uniform light field module comprises three wavelength laser sources, a beam expander, a mirror, a scanning lens, a beam combiner, a microscope objective and a detection chip, wherein:

[0011] The beam expander is used to adjust the diameter of the laser beam, the mirror adjusts the beam angle, the scanning lens corrects the spot distortion, and the beam combiner couples the three laser beams to the microscope excitation light path;

[0012] The detection chip is provided with a microfluidic channel and a light transmission slit, and the working distance of the objective lens and the height difference of the detection area are greater than 10 times to realize a uniform detection light field;

[0013] A signal acquisition and processing module comprises a photomultiplier tube array, a data acquisition card and a computer, which is used to capture the fluorescence signal and demodulate the single-cell protein quantity.

[0014] In the above technical solution, in the uniform light field module: the excitation light path covers the detection area by intercepting the middle uniform part of the parallel light beam, and the spot size is more than 3 times the size of the detection area; the detection light path uses the long working distance of the objective lens and the microscale difference of the detection area to make the fluorescence molecules of different heights be captured with the same aperture angle.

[0015] In the above technical solution, the microfluidic channel of the detection chip adopts three-dimensional sheath flow focusing technology, the channel material is fused quartz, the light transmission slit width is matched with the spot size, and the error is less than 5 μm.

[0016] In the technical scheme, the expansion ratio of the beam expander is adjustable, the axial position of the mirror is adjustable, and the focal length of the scanning lens ranges from 10 mm to 100 mm.

[0017] In the technical scheme, the laser output wavelength is 405 nm, 488 nm or 640 nm, the modulation frequency is 10-200 kHz, and the amplitude error of the sinusoidal carrier signal is less than 1%.

[0018] In the technical scheme, the excitation light intensity of the uniform light field in the height direction of the detection area has a variation coefficient of less than 0.02%, the light intensity variation coefficients in the length and width directions are 1.3%, and the detection light field variation coefficient is 1.9%.

[0019] In the technical scheme, the width of the light-transmitting slit is 10-200 μm, the slit edge roughness is less than 0.1 μm, and the alignment error with the center of the light spot is less than 2 μm.

[0020] A single-cell nine-protein high-throughput quantitative detection method based on the above device, comprising the following steps:

[0021] (a) Inject the single-cell suspension labeled with fluorescent antibodies into the microfluidic channel, and form a single-cell flow by sheath flow focusing;

[0022] (b) Three independently modulated lasers excite the cells through the uniform light field to generate amplitude-modulated fluorescent signals;

[0023] (c) Photomultiplier tubes capture nine fluorescent channel signals, which are transmitted to a computer through a data acquisition card;

[0024] (d) Demodulate the fluorescent pulse signals, extract the amplitude values in the stable region, and calculate the cell diameter;

[0025] (e) Inject a gradient concentration of fluorescent antibody solution, and establish a fluorescent intensity-molecule number calibration curve based on the volume equivalence principle;

[0026] (f) Convert the single-cell fluorescent amplitude to the number of target proteins to realize synchronous quantification of nine proteins.

[0027] In the technical scheme, the calibration curve in step (e) is established based on the linear relationship between the number of fluorescent molecules in the detection area and the solution concentration, and the calibration error is less than 5%. The concentration gradient of the calibration solution includes 10, 100, 1000, and 10000 molecules / μL.

[0028] In the technical scheme, the fluorescent pulse signal demodulation in step (d) uses lock-in amplification technology, and the noise suppression ratio is greater than 60 dB. The stable region amplitude value calculation is based on the ratio of pulse peak value to baseline variance, and the error tolerance is ±3%.

[0029] Beneficial effects:

[0030] (1) The present application realizes simultaneous quantitative detection of nine proteins in different distribution positions. Compared with the prior art, three independent adjustable light spots are realized by designing excitation and detection light paths, reducing crosstalk, thereby realizing quantitative detection of nine parameters, meeting the clinical leukemia typing detection requirements.

[0031] (2) The present application improves the detection efficiency and reduces the required sample amount. Compared with the prior art, the number of single-tube sample detection markers is increased from three to nine by improving the detection parameters, and the efficiency is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a single cell nine protein high-throughput quantitative detection device structure diagram of the present application;

[0033] Figure 2 It is a detection flow chart;

[0034] Figure 3 It is a detection principle diagram;

[0035] Figure 4 It is a light spot positioning-uniform light field uniformity characterization;

[0036] Figure 5 It is a fluorescence calibration curve of a single cell nine protein high-throughput quantitative detection method of the present application. DETAILED DESCRIPTION

[0037] The present application will be described in detail below in combination with the drawings and specific embodiments. However, the following examples are only used to explain the present application, and the protection scope of the present application should include the entire content of the claims, and through the description of the following examples, those skilled in the art can fully realize the entire content of the claims of the present application.

[0038] EMBODIMENT

[0039] The present embodiment provides a single cell nine protein quantitative detection device and method.

[0040] Device composition: signal modulation module, light spot positioning and uniform light field module, signal acquisition and processing module. As shown in the accompanying Figure 1As shown, the signal modulation module includes a signal generator and a laser, and the sinusoidal carrier signal generated by the signal generator is used to amplitude modulate the laser; the spot positioning and uniform light field module mainly includes an adjustable laser shaping light path, a microscope excitation light path and a detection chip with microchannels and a light transmission slit, which are used to form three independent uniform light field detection areas that do not interfere with each other, and the fluorescent molecules at different positions in the areas have uniform signal strength, so as to ensure the accurate quantification of proteins at different distribution positions; the signal acquisition and processing module includes a photomultiplier tube, a data acquisition card and a computer, the photomultiplier tube is used to convert the received fluorescent signal into an electric signal, the data acquisition card is used to collect the converted voltage signal and transmit it to the computer, and the computer is used for data saving and analysis work.

[0041] In the present application, three laser excitations without crosstalk are realized based on spot positioning, and absolute quantitative detection of proteins at any distribution position of single cells is realized based on uniform light field. Figure 2 As shown. Cells are first combined with antibodies labeled with specific fluorescent probes to realize cell staining (Fig. Figure 2 (a) in the appendix), and then they pass through the quartz channel manufactured by microfabrication technology in the form of single cell flow through three-dimensional sheath flow focusing, and the single cells passing through the detection area are excited by the laser source controlled by the sinusoidal voltage signal (Fig. Figure 2 (b) in the appendix), the excited fluorescent signal is a modulated signal with amplitude changing with fluorescent intensity, which is captured by nine PMTs (Fig. Figure 2 (c) in the appendix), the demodulated single cell fluorescent pulse is divided into rising region, stable region and falling region (Fig. Figure 2 (d) in the appendix), combined with the geometric size of the detection area, they are further converted into the diameter of the cell. By injecting a gradient concentration of antibody solution with fluorescent probe into the quartz microchannel (Fig. Figure 2 (e) in the appendix), based on the principle of equal volume in cell suspension and calibration solution, the correlation between antibody concentration and fluorescent intensity is obtained (Fig. Figure 2 (f) in the appendix), according to the calibration principle of uniform light field, the fluorescent intensity is only related to the number of fluorescent molecules and is not affected by the distribution position, so the correlation between the number of antibodies and the fluorescent intensity is obtained, and the fluorescent amplitude of the fluorescent pulse is converted into the diameter of the cell and the number of single cell proteins (Fig. Figure 2 (g) in the appendix).

[0042] In order to construct a uniform light field, the present invention designs uniform excitation light and uniform detection light. The goal of constructing the excitation light path is to enable the laser to be uniformly irradiated on any position of a single cell, so that the fluorescent pigments labeled on proteins at any distribution position on the cell can be excited at the same time. The goal of constructing the detection light path is to uniformly collect the fluorescence emitted by the fluorescent pigments labeled on proteins at different distribution positions on a single cell. In terms of achieving uniform excitation light, the main challenge is the non-uniformity of the laser beam. In the direction parallel to the emission of the laser beam, the collimated laser is focused on the back focal plane of the microscope objective lens, so that the focused laser beam is emitted from the lens in the form of parallel light. In the direction perpendicular to the emission of the laser, in order to obtain a portion of the laser beam with approximately uniform light intensity in the middle, the size of the light spot must be much larger than the size of the excited detection area. In terms of achieving uniform detection light, the main challenge is that the proportion of fluorescent molecules at different heights captured by the objective lens is different, and the fluorescent molecules at higher heights are affected by the shading area more than the fluorescent molecules at the bottom of the channel, resulting in the fluorescent molecules in the detection area not being uniformly detected. However, it can be found that the greater the difference between the working distance of the objective lens and the height of the detection area, the smaller the impact of different heights in the detection area and the light transmission slit. This is because at a sufficiently long working distance, the tiny height differences of the fluorescent molecules in the detection area can be ignored, and the fluorescence emitted by each fluorescent molecule is almost captured by the objective lens in equal proportion with the same aperture angle. At the same time, the light shielding effect of the light transmission slit on the fluorescent molecules at the top of the detection area can also be ignored. Therefore, the difference between the long working distance of the optical lens and the microscale of the detection area can achieve uniform collection of fluorescence signals. (Appendix Figure 3 Middle (a)

[0043] The present invention designs a method for light spot positioning, the main goal of which is to make the sizes and positions of the three laser spots independently adjustable to cooperate with the light-transmitting slits on the detection channel to form three independent uniform light fields, thereby avoiding crosstalk caused by simultaneous excitation of multiple lasers and increasing the number of detection parameters. The main challenge faced is that the three lasers need to share the excitation light path of the microscope, and the solution is to shape them separately and then combine them to input the microscope. In order to achieve adjustable sizes and positions of the three laser spots, a beam expander with adjustable magnification is used to control the beam diameter, an axially adjustable reflector is used to control the beam angle, and a scanning lens is used to make the light spot image plane flat to reduce light spot distortion. As shown in the attached figure Figure 3 As shown in (b), the three wavelengths of laser light are output from the optical fiber and pass through the collimator. They are then shaped by the beam expander, reflector, and scanning lens. They then enter the excitation light path of the microscope through the beam combiner. After being reflected by the dichroic mirror, they pass through the objective lens and form three independent light spots on the detection channel.

[0044] The present invention combines spot positioning with uniform intensity light field to form three independent detection areas, as shown in the attached Figure 4As shown, the light intensity fluctuation in the three detection regions is less than 10%.

[0045] The fluorescence calibration curve of the single-cell nine-protein high-throughput quantitative detection method based on the embodiment is as shown in the following table: Figure 5 As shown, the calibration curves of the nine fluorescence channels (such as FITC, PE, APC, etc.) are shown in the figure, and the linear slopes of each channel are close, indicating that the detection sensitivity of different fluorescence probes is consistent, meeting the synchronous quantitative requirement of multiple proteins. The calibration curve is generated by injecting a solution of known concentration of fluorescent antibody, and the volume of the solution in the detection region is equivalent to the volume of a single cell, ensuring that the calibration result can be directly used for single-cell protein calculation. The curve still remains linear at the low concentration end, proving that the system has high sensitivity (the detection limit reaches the single molecule level); there is no signal saturation phenomenon at the high concentration end, indicating that the uniform light field avoids the problem of overexposure, and the dynamic range is wide. Figure 5 Through the high linearity and low error calibration curve, the effectiveness of the uniform light field design and the volume equivalent calibration method of the application is verified, which provides a reliable basis for the synchronous absolute quantification of nine proteins in a single cell, and meets the requirement of high-precision detection of multiple parameters for clinical leukemia typing.

[0046] In the application, the detection region based on the uniform light field is not limited to the instrument for controlling the light source, the type of light source, the wavelength of excitation light, or the construction method of the detection light path, as long as a suitable uniform light field region is formed, the detection requirement can be met.

[0047] In the application, the microfluidic channel is not limited to the shape, size or material of the microfluidic channel, as long as the cell can pass through the microchannel without blocking and meet the calibration model, the detection requirement can be met.

[0048] In the application, a positive pressure is applied at the end of the cell solution injection channel, but other methods can also be used, such as using negative pressure to drive the cell solution through the channel.

[0049] The above is only a specific embodiment of the application, which enables those skilled in the art to understand or implement the application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application. Therefore, the application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features described in the application.

Claims

1. A single cell nine protein high-throughput quantitative detection device, characterized in that: include: The signal modulation module consists of a signal generator and a laser, and is used to generate a sinusoidal carrier signal and perform amplitude modulation on the laser; The spot positioning and uniform intensity light field module includes three wavelength laser sources, a beam expander, a reflector, a scanning lens, a beam combiner, a microscope objective lens, and a detection chip, including: The beam expander is used to adjust the laser beam diameter, the reflector adjusts the beam angle, the scanning lens corrects the spot distortion, and the beam combiner couples the three laser beams to the microscope excitation light path; The detection chip is provided with a microfluidic channel and a light-transmitting slit, and the height difference between the objective lens working distance and the detection area is greater than 10 times to achieve a uniform detection light field; The signal acquisition and processing module, including a photomultiplier tube array, a data acquisition card, and a computer, is used to capture the fluorescence signal and demodulate it into the number of single-cell proteins.

2. A single cell nine protein high-throughput quantitative detection device according to claim 1, characterized in that: In the uniform intensity light field module: the excitation light path intercepts the middle uniform part of the parallel light beam to cover the detection area, and the spot size is more than 3 times that of the detection area; the detection light path utilizes the long working distance of the objective lens and the microscale difference of the detection area to capture fluorescent molecules of different heights with the same aperture angle.

3. The high-throughput quantitative detection device for nine proteins in a single cell according to claim 1, characterized in that: The microfluidic channel of the detection chip adopts three-dimensional sheath flow focusing technology, the channel material is fused quartz, the width of the light transmission slit matches the light spot size, and the error is less than 5μm.

4. The high-throughput quantitative detection device for nine proteins in a single cell according to claim 1, characterized in that: The magnification of the beam expander is adjustable, the axial position of the reflector is adjustable, and the focal length range of the scanning lens is 10-100 mm.

5. The high-throughput quantitative detection device for nine proteins in a single cell according to claim 1, characterized in that: The laser output wavelengths are 405nm, 488nm, and 640nm, the modulation frequency is 10-200kHz, and the amplitude error of the sinusoidal carrier signal is less than 1%.

6. The high-throughput quantitative detection device for nine proteins in a single cell according to claim 1, characterized in that: The coefficient of variation of the excitation light intensity of the uniform light field in the height direction of the detection area is less than 0.02%, the coefficient of variation of the light intensity in the length and width directions is 1.3%, and the coefficient of variation of the detection light field is 1.9%.

7. The high-throughput quantitative detection device for nine proteins in a single cell according to claim 3, characterized in that: The width of the light-transmitting slit is 10-200 μm, the roughness of the slit edge is less than 0.1 μm, and the alignment error with the center of the light spot is less than 2 μm.

8. A method for high-throughput quantitative detection of nine proteins in a single cell, based on the device according to any one of claims 1 to 7, comprising the following steps: (a) A single-cell suspension labeled with fluorescent antibodies is injected into a microfluidic channel and focused by sheath flow to form a single-cell stream. (b) Three independently modulated laser beams excite cells through a uniform intensity light field, generating an amplitude-modulated fluorescence signal; (c) Photomultiplier tubes capture nine fluorescence channel signals and transmit them to a computer via a data acquisition card; (d) Demodulate the fluorescence pulse signal, extract the amplitude of the stable region and calculate the cell diameter; (e) Injecting gradient concentration fluorescent antibody solutions and establishing a fluorescence intensity-molecular number calibration curve based on the volume equivalence principle; (f) Converting single-cell fluorescence amplitudes into target protein quantities enables simultaneous quantification of nine proteins.

9. A single cell nine protein high-throughput quantitative detection method according to claim 8, characterized in that: In step (e), the calibration curve is established by the linear relationship between the number of fluorescent molecules in the detection area and the solution concentration, the calibration error is less than 5%, and the concentration gradient of the calibration solution includes 10, 100, 1000, and 10000 molecules / μL.

10. A single cell nine protein high-throughput quantitative detection method according to claim 8, characterized in that: In step (d), the fluorescence pulse signal demodulation adopts phase-locked amplification technology with a noise suppression ratio greater than 60 dB, and the stable region amplitude is calculated based on the ratio of the pulse peak value to the baseline variance with an error tolerance of ±3%.

Images