A high-throughput Raman single-cell sorting device and method

Abstract

The present invention provides a high-throughput single-cell sorting device and method. It includes: a microfluidic chip for placing single-cell samples; a PMT (photomultiplier tube) narrow-band Raman single-cell pre-screening module for pre-screening single-cell samples to obtain narrow-band Raman signals and narrow-band background signals corresponding to single-cell samples; a linear array Raman single-cell identification module for obtaining full-spectrum information of single-cell samples. The chip includes a narrow-band Raman detection pre-screening part, a linear array Raman detection part, a droplet encapsulation forming part, and a single-cell sorting part connected in sequence; the present invention can realize high-throughput detection, analysis, and sorting of cell samples.

Description

Technical Field

[0001] The present invention relates to the field of microscopic Raman single cell sorting, and in particular to a high-throughput Raman single cell sorting device and method. Background Art

[0002] Raman spectroscopy is a highly efficient information identification technology. By analyzing the inelastic scattering spectra of specific incident light on compounds, microscopic Raman spectroscopy can directly detect the vibration or rotation energy levels of compound molecules. By analyzing the Raman characteristic spectra, the molecular composition and structural information of the compound can be obtained.

[0003] However, the existing Raman microscopy technology has defects when used for sample analysis. For example, the single-cell Raman spectrum signal intensity of a single cell is weak, especially when the cell is suspended in a liquid, usually only 10 6-8 One in ten photons passes through Raman scattering, resulting in a long spectral scanning time to obtain a complete and reliable Raman spectral signal. When analyzing a large number of samples, full spectrum identification of each cell will greatly increase the analysis time of the sample, resulting in a lower acquisition throughput.

[0004] The linear array Raman detection products currently on the market are generally used to analyze solid samples or stem cell samples. They can achieve rapid analysis of single-cell samples, but this is all they can do. They are unable to separate and sort single cells.

[0005] The Chinese patent with patent number CN107462566A discloses a Raman spectrometer for detecting a specific narrowband wavenumber range, which can realize rapid and high-sensitivity detection of Raman spectra in a specific narrow wavenumber range. However, when detecting on-site or clinical samples, the specificity of single cells can be determined. However, the patent cannot obtain complete Raman spectral information, and cannot realize high-throughput and rapid single-cell type identification, and then separate and sort single cells for subsequent processing (such as single-cell culture, amplification, etc.). Summary of the invention

[0006] In view of the above defects, the present invention provides a high-throughput Raman single-cell sorting device and method.

[0007] In one aspect, the present invention provides a high-throughput Raman single-cell sorting device, comprising:

[0008] -PMT (photomultiplier tube) narrow-band Raman single-cell pre-screening module, used to perform pre-screening detection on single-cell samples to obtain narrow-band Raman signals and narrow-band background signals corresponding to single-cell samples;

[0009] -Linear Raman single-cell identification module, used to obtain full spectrum information of single-cell samples;

[0010] -Microfluidic chip for placing single cell samples.

[0011] In another preferred example, the narrow-band Raman single-cell pre-screening module includes a laser light source and a photomultiplier tube.

[0012] In another preferred example, the linear array Raman single cell identification module includes a laser light source, a linear array light generator, and a spectrometer.

[0013] In another preferred embodiment, the PMT (photomultiplier tube) narrow-band Raman single-cell pre-screening module and the linear array Raman single-cell identification module have the same laser light source.

[0014] In another preferred embodiment, the microfluidic chip is placed on a three-dimensional moving platform.

[0015] In another preferred embodiment, the microfluidic chip includes a narrow-band Raman detection pre-screening part, a linear array Raman detection part, a droplet package forming part, and a single cell sorting part connected in sequence; the narrow-band Raman detection pre-screening part includes an injection port, a first liquid reservoir, and a first channel connected in sequence, the first channel is provided with a waste liquid tank, and a first sorting electrode is provided on the outside of the first channel; the linear array Raman detection part includes a second liquid reservoir and a second channel in sequence, the second channel is provided with a buffer inlet, and a capture electrode is provided on the outside of the second channel; the droplet package forming part includes an oil reservoir and a droplet package forming port in sequence; the single cell sorting part includes a third channel and at least two separation ports in sequence, and a second sorting electrode is provided on the outside of the third channel.

[0016] In another preferred embodiment, the narrow-band Raman detection pre-screening part quickly and highly sensitively screens out single cells with specificity, and separates single cells with no specificity to a waste liquid outlet for removal by adding electrodes.

[0017] In another preferred embodiment, the linear array Raman detection and identification part can realize high-throughput collection of Raman signals of single cells, obtain full spectrum information of single cells, and identify the type of single cells through database comparison.

[0018] In another preferred embodiment, the droplet encapsulation forming part forms droplets to encapsulate single cells.

[0019] In another preferred embodiment, the single cell sorting part guides the single cells wrapped in the droplets out from different separation channels by loading sorting electrodes.

[0020] In another preferred embodiment, the electrode is connected to an external power source.

[0021] In another preferred embodiment, the width of the first channel is 10-50 um, which is the narrowest dimension.

[0022] In another preferred embodiment, the width of the first liquid storage tank is 80-100 um, which is the widest dimension.

[0023] The single cell samples include bacteria, fungi, microorganisms and the like.

[0024] Another aspect of the present invention provides a high-throughput Raman single-cell sorting method, comprising the following steps:

[0025] S1: single cell sample is injected into the microfluidic chip;

[0026] S2: When the single cell sample is located in the first channel, a PMT narrow-band micro-Raman single cell pre-screening module is used to perform pre-screening detection on the single cell sample, obtain a narrow-band Raman signal and a narrow-band background signal corresponding to the single cell sample, and identify a single cell sample with specificity;

[0027] S3: applying a voltage to the first sorting electrode to allow the single cell sample with specificity to enter the second liquid reservoir and then the second channel;

[0028] S4: applying voltage to the capture electrode, and the linear array Raman single-cell identification module detects the single-cell sample, obtains the Raman spectrum of one or more single-cell samples, and identifies the single-cell sample;

[0029] S5: injecting oil phase into the oil reservoir, so that the single cell sample generates droplets at the droplet encapsulation formation port, and the generated droplets enter the third channel;

[0030] S6: Apply voltage to the second sorting electrode to make the droplets enter different separation ports for separation.

[0031] In another preferred embodiment, the step S2 of identifying a specific single-cell sample includes performing data processing on the obtained narrow-band Raman signal and narrow-band background signal to obtain a signal-to-noise ratio value, and comparing the values ​​according to a preset value to determine whether the single-cell sample is specific.

[0032] In another preferred embodiment, identifying the desired single cell in step S4 includes comparing the Raman spectra of the one or more single cell samples with standard Raman spectral signals or reference Raman spectral signals in the constructed single cell phenotype database to determine the type of the cell.

[0033] In another preferred example, the step S6 applies a voltage to the second sorting electrode to make the droplets enter different separation ports for sorting, including numbering the droplets that enter the third channel in sequence, identifying the single cell samples according to step S4, and applying different voltages in different numbering sequences, so that the droplets enter different separation ports.

[0034] In another preferred embodiment, the single-cell high-throughput detection method includes culturing or amplifying the sorted single-cell samples.

[0035] Beneficial effects of the present invention:

[0036] The present invention describes a high-throughput parallel Raman single-cell sorting device based on the combination of PMT and linear array detection technology. First, a PMT detector is used to perform preliminary screening of cells with specific needs, thereby greatly improving the screening throughput. Then, the screened cells with specificity are controlled by a microfluidic device to perform parallel Raman full-spectrum acquisition. This strategy can avoid full-spectrum analysis of all samples, greatly reducing the sample acquisition time. At the same time, linear array Raman is used to acquire the spectra of specific cells for cell type identification. Finally, the tested specific cells can be sorted out through a microfluidic chip, providing the possibility for further analysis of subsequent cells (such as single-cell culture, single-cell sequencing, etc.), thereby achieving high-throughput detection, analysis and sorting of cell samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the optical path of the high-throughput Raman single-cell sorting device and method combining PMT and linear array Raman of the present invention.

[0038] Figure 2 Schematic diagram of the microfluidic chip.

[0039] The reference numerals are as follows:

[0040] 1. Laser 2. Beam expander collimator 3. Laser beam splitter 41. First reflector 42. Second reflector 43. Third reflector 44. Fourth reflector 45. Fifth reflector 51. First high-pass filter 52. Second high-pass filter 61. First dichroic mirror 62. Second dichroic mirror 71. First microscope group 72. Second microscope group 8. Microfluidic chip 9. Three-dimensional mobile platform 101. First lens 102. Second lens 103. Third lens 11. Pinhole 121. First narrow-band filter 122. Second narrow-band filter 13. Beam splitter prism 141. First PMT 142. Second PMT 151. First visible light beam splitter 152. Second visible light beam splitter 161. First image collector 162. Second image collector 171. First LED light source 172. Second LED light source 18. Cylindrical mirror 19. Slit 20. Spectrometer 21. Linear array light generator 22. Inlet 23. First liquid reservoir 24. Narrow-band Raman detection light 25. First sorting electrode 26. First channel 27. Waste liquid reservoir 28. Second liquid reservoir 29. Buffer inlet 30. Capture electrode 31. Linear array Raman detection light 32. Second channel 33. Oil reservoir 34. Droplet encapsulation formation port 35. Second sorting electrode 36. Third channel 37. First separation port 38. Second separation port 39. Third separation port DETAILED DESCRIPTION

[0041] To facilitate understanding of the embodiments of the present invention, the following will be further explained with reference to specific embodiments in conjunction with the accompanying drawings, and each embodiment does not constitute a limitation on the embodiments of the present invention. In addition, the accompanying drawings are schematic diagrams, so the device and apparatus of the present invention are not limited by the size or proportion of the schematic diagrams.

[0042] It should be noted that in the claims and description of this patent, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including one" do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0043] Embodiment 1

[0044] like Figure 1 The shown includes a PMT narrow-band Raman single-cell pre-screening optical path and a linear array Raman single-cell identification optical path. The PMT narrow-band microscopic Raman single-cell pre-screening optical path and the linear array microscopic Raman single-cell identification optical path are focused in parallel on the microfluidic chip, and a single-cell sample is placed in the microfluidic chip.

[0045] The PMT narrow-band micro-Raman single-cell pre-screening optical path and the linear array micro-Raman single-cell identification optical path form a high-throughput Raman single-cell sorting device and method combined with PMT and linear array Raman. The single-cell Raman spectrometer includes a laser 1, a beam expander collimator 2, a laser beam splitter 3, mirrors 41, 42, 43, 44, 45, high-pass filters 51, 52, dichroic mirrors 61, 62, microscope objective lens groups 71, 72, microfluidic chip 8, three-dimensional displacement platform 9, lenses 101, 102, 103, pinhole 11, narrow-band filters 121, 122, beam splitter prism 13, PMT 141, 142, visible light beam splitters 151, 152, image collectors 161, 162, LED light sources 171, 172, cylindrical mirror 18, slit 19, spectrometer 20, linear array light generator 21, etc. Among them:

[0046] The laser beam emitted by the laser 1 passes through the beam expander collimator 2 to generate parallel light, and the parallel light beam passes through the laser beam splitter 3, the first reflector 41, the second reflector 42, the first high-pass filter 51, the first dichroic mirror 61, and the first microscope objective lens group 71 in sequence to be focused on the single cell sample in the microfluidic chip 8 to generate a Raman signal, and the Raman signal passes through the first microscope objective lens group 71, the first dichroic mirror 61, the first high-pass filter 51, the first lens 101, the pinhole 11, and the beam splitter prism 13 in reverse order, and is evenly divided into the first narrow-band filter 121 and the second narrow-band filter 122 to enter the first PMT 141 and the second PMT 142 respectively.

[0047] The first LED light source 171 passes through the first visible light beam splitter 151, the first dichroic mirror 61, and the first microscope objective lens group 71 in sequence and is focused on the microfluidic chip 8 to obtain single-cell sample image information. The image information passes through the first microscope objective lens 71, the first dichroic film 61, the first visible light beam splitter 151, and the second lens 102 in reverse order to enter the first image collector 161.

[0048] The laser beam emitted by the laser 1 passes through the beam expander collimator 2 to generate parallel light, and the parallel light beam passes through the laser beam splitter 3, the third reflector 43, the fourth reflector 44, the linear array light generator 21, the fifth reflector 45, the second high-pass filter 52, the second dichroic mirror 62, and the second microscope objective lens group 72 in sequence to be focused on the single cell sample in the microfluidic chip 8 to generate a Raman signal. The Raman signal passes through the second microscope objective lens group 72, the second dichroic mirror 62, the second high-pass filter 52, the cylindrical mirror 18, and the slit 19 in reverse order to enter the spectrometer 20.

[0049] The second LED light source 172 passes through the second visible light beam splitter 152, the second dichroic mirror 62, and the second microscope objective lens group 72 in sequence and is focused on the microfluidic chip 8 to obtain single-cell sample image information. The image information passes through the second microscope objective lens 72, the second dichroic film 62, the second visible light beam splitter 152, and the third lens 103 in reverse order to enter the second image collector 162.

[0050] The PMT narrow-band micro-Raman single-cell pre-screening optical path and the linear array micro-Raman single-cell identification optical path are focused on the microfluidic chip in parallel to detect single-cell samples in sequence.

[0051] The three-dimensional displacement platform 9 is placed with a microfluidic chip 8 , and the movement of the three-dimensional displacement platform 9 drives the movement of the microfluidic chip 8 .

[0052] Embodiment 2

[0053] like Figure 2 The above is a schematic diagram of a microfluidic chip, which specifically includes:

[0054] Inlet 22, first liquid reservoir 23, first sorting electrode 25, first channel 26, waste liquid pool 27; the sample enters the first liquid reservoir 23 through the inlet 22 to wait for detection, and as the sample continues to flow into the single cell, it enters the first channel 26 from the first liquid reservoir 23 in turn, and reaches the focusing position of the narrow-band Raman detection light 24. The narrow-band Raman detection can quickly and highly sensitively detect the narrow-band spectral signal of the single cell, and judge whether the single cell has specificity based on the detected narrow-band spectral signal. It is particularly pointed out that the single cell specificity is uncertain, and it is necessary to set the specificity according to the actual experimental needs. Single cells with specificity do not have electrodes applied, and directly enter the detection of the linear array Raman detection part. Single cells without specificity are applied with electrodes and enter the waste liquid port for removal.

[0055] The linear array Raman detection part includes a second liquid reservoir 28, a buffer inlet 29, a capture electrode 20, a linear array Raman detection light 31, and a second channel 32 in sequence; a single cell with specificity enters the second channel 32 from the second liquid reservoir 28, and a buffer is injected into the buffer inlet 29. Under the action of the buffer, the single cells are arranged in a linear shape, and the capture electrode 30 stabilizes the linearly arranged single cells at the focusing position of the linear array Raman detection light beam 31. The focused light beam is irradiated on the single cell, and the single cell generates a full-spectrum Raman signal. The Raman spectrum is collected by a spectrometer, and the computer compares the Raman spectrum with a database to identify the type of the single cell. After the collection is completed, the capture electrode 30 is discharged, and the single cell enters the droplet encapsulation formation part.

[0056] The droplet encapsulation forming part includes an oil reservoir 33 and a droplet encapsulation forming port in sequence; an oil phase is injected 33 into the oil reservoir, and the oil phase can enable single cells to generate droplets at the droplet encapsulation forming port 34, and the generated droplets reach the third channel 36 in sequence.

[0057] The single cell sorting part includes a second sorting electrode 35, a third channel 36, a first separation port 37, a second separation port 38, and a third separation port 39; the droplets entering the third channel in sequence are calculated and numbered according to the identified types, and different electrodes are applied in different numbering sequences, so that the droplets enter different separation ports: the first separation port 37, the second separation port 38, and the third separation port 39.

[0058] Embodiment 3

[0059] The present invention provides a single cell high-throughput Raman single cell sorting device based on a combination of PMT and linear array Raman, and provides a single cell high-throughput detection method, comprising the following steps:

[0060] 1. Prepare single cell samples;

[0061] 2. The single cell sample is injected into the microfluidic chip 8, and the microfluidic chip is placed on the three-dimensional displacement platform 9;

[0062] 3. Single-cell narrow-band Raman signal detection: PMT narrow-band micro-Raman single-cell pre-screening optical path, pre-screening detection of single-cell samples, to obtain narrow-band Raman signals and narrow-band background signals corresponding to single-cell samples;

[0063] Furthermore, step 3 also includes the steps of: performing data processing on the obtained narrow-band Raman signal and narrow-band background signal to obtain a signal-to-noise ratio value, and comparing the values ​​according to a preset value to determine whether the single-cell sample is specific.

[0064] 4. Linear Raman signal detection: The single-cell sample with specificity enters the linear array micro-Raman single-cell identification optical path through the microfluidic chip channel, detects the single-cell sample located in the linear array detection laser beam, and obtains the Raman spectrum of one or more single cells;

[0065] Furthermore, step 4 also includes the steps of: standardizing the Raman spectral signal (baseline subtraction, normalization, stray peak filtering, etc.) and comparing it with the standard Raman spectral signal or the reference Raman spectral signal in the constructed single-cell phenotype database, and using deep learning, database mining and other algorithms to quickly determine the type (type) of the cell.

[0066] 5. Inject oil phase into the oil reservoir to generate droplets of the single-cell sample at the droplet encapsulation formation port, and the generated droplets enter the third channel.

[0067] 6. Apply different electrodes to the identified single-cell droplets, and the single-cell samples enter different separation ports, and the sorted single cells are subsequently processed (such as culture, amplification, etc.).

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A high-throughput Raman single-cell sorting device, characterized in that: include: PMT narrow-band Raman single-cell pre-screening module is used to pre-screen single-cell samples, obtain narrow-band Raman signals and narrow-band background signals corresponding to single-cell samples, and identify single-cell samples with specificity; Linear Raman single-cell identification module, used to obtain full-spectrum information of single-cell samples; Microfluidic chips, used to hold single-cell samples; The microfluidic chip comprises a narrow-band Raman detection pre-screening part, a linear array Raman detection part, a droplet package forming part, and a single cell sorting part connected in sequence; the narrow-band Raman detection pre-screening part comprises an injection port, a first liquid reservoir, and a first channel connected in sequence, the first channel is provided with a waste liquid tank, and a first sorting electrode is provided on the outside of the first channel; the linear array Raman detection part comprises a second liquid reservoir and a second channel in sequence, the second channel is provided with a buffer inlet, and a capture electrode is provided on the outside of the second channel; the droplet package forming part comprises an oil reservoir and a droplet package forming port in sequence; the single cell sorting part comprises a third channel and at least two separation ports in sequence, and a second sorting electrode is provided on the outside of the third channel; The PMT narrow-band Raman single-cell pre-screening module and the linear array Raman single-cell identification module have the same laser light source; The high-throughput Raman single-cell sorting device also includes a PMT narrow-band Raman single-cell pre-screening optical path and a linear array Raman single-cell identification optical path. The PMT narrow-band microscopic Raman single-cell pre-screening optical path and the linear array microscopic Raman single-cell identification optical path are focused on the microfluidic chip in parallel.

2. The high-throughput Raman single-cell sorting device according to claim 1, characterized in that: The PMT narrow-band Raman single-cell pre-screening module includes a laser light source and a photomultiplier tube.

3. The high-throughput Raman single-cell sorting device according to claim 1, characterized in that: The linear array Raman single cell identification module comprises a laser light source, a linear array light generator and a spectrometer.

4. The high-throughput Raman single-cell sorting device according to claim 1, characterized in that: The electrodes are externally connected to a power source.

5. The high-throughput Raman single-cell sorting device according to claim 1, characterized in that: The microfluidic chip is placed on a three-dimensional moving platform.

6. A high-throughput Raman single-cell sorting method, characterized in that: A high-throughput Raman single-cell sorting device according to any one of claims 2 to 5 is used, and comprises the following steps: S1: single cell sample is injected into the microfluidic chip; S2: When the single cell sample is located in the first channel, a PMT narrow-band micro-Raman single cell pre-screening module is used to perform pre-screening detection on the single cell sample, obtain a narrow-band Raman signal and a narrow-band background signal corresponding to the single cell sample, and identify a single cell sample with specificity; S3: applying a voltage to the first sorting electrode to allow the single cell sample with specificity to enter the second liquid reservoir and then the second channel; S4: applying voltage to the capture electrode, and the linear array Raman single-cell identification module detects the single-cell sample, obtains the Raman spectrum of one or more single-cell samples, and identifies the single-cell sample; S5: injecting oil phase into the oil reservoir, so that the single cell sample generates droplets at the droplet encapsulation formation port, and the generated droplets enter the third channel; S6: Apply voltage to the second sorting electrode to make the droplets enter different separation ports for sorting.

7. The high-throughput Raman single-cell sorting method according to claim 6, characterized in that: The step S2 of identifying a single cell sample with specificity includes performing data processing on the obtained narrow-band Raman signal and narrow-band background signal to obtain a signal-to-noise ratio value, and performing comparison according to a preset value to determine whether the single cell sample is specific.

8. The high-throughput Raman single-cell sorting method according to claim 6, characterized in that: Identifying the desired single cell in step S4 includes comparing the Raman spectra of the one or more single cell samples with standard Raman spectral signals or reference Raman spectral signals in the constructed single cell phenotype database to determine the type of the cell.

9. The high-throughput Raman single-cell sorting method according to claim 6, characterized in that: The step S6 of applying voltage to the second sorting electrode to make the droplets enter different separation ports for sorting includes numbering the droplets that enter the third channel in sequence, identifying the single cell samples according to step S4, and applying different voltages in different numbering sequences to make the droplets enter different separation ports.

Images