CN215115894U - A high-throughput Raman single cell sorting device - Google Patents
A high-throughput Raman single cell sorting device Download PDFInfo
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- CN215115894U CN215115894U CN202022136745.4U CN202022136745U CN215115894U CN 215115894 U CN215115894 U CN 215115894U CN 202022136745 U CN202022136745 U CN 202022136745U CN 215115894 U CN215115894 U CN 215115894U
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Abstract
The utility model provides a high flux raman unicellular sorting unit. The method comprises the following steps: the microfluidic chip is used for placing a single cell sample; the PMT (photomultiplier tube) narrow-band Raman single-cell pre-screening module is used for performing pre-screening detection on the single-cell sample to obtain a narrow-band Raman signal and a narrow-band background signal corresponding to the single-cell sample; and the linear array Raman single cell identification module is used for acquiring full spectrum information of the single cell sample. The chip comprises a narrow-band Raman detection pre-screening part, a linear array Raman detection part, a liquid drop package forming part and a single cell sorting part which are connected in sequence; the utility model discloses can realize cell sample's high flux detection, analysis and sorting.
Description
Technical Field
The utility model relates to a micro-Raman unicellular sorting field especially relates to a high flux Raman unicellular sorting unit.
Background
Raman spectrum is a high-efficiency information identification technology, through inelastic scattering spectral line analysis of a compound by specific incident light, micro Raman spectrum can directly detect the molecular vibration or rotation energy level of the compound, and through Raman characteristic spectral line analysis, compound molecular composition and structure information can be obtained.
However, the existing raman microscopy technology has defects when used for sample analysis, and takes the measurement of single cells of microorganisms as an example: single cell Raman spectra have weak signal intensity, especially when the cells are suspended in liquid, usually only 106-8One-half of the photons are scattered by Raman scattering, so that the spectrum scanning time for obtaining a complete and reliable Raman spectrum signal is longer, and when a large number of samples are analyzed, the analysis time of the samples can be greatly increased by performing full spectrum identification on each cell, so that the acquisition flux is lower.
At present, linear array Raman detection products on the market generally analyze solid samples or dry cell samples, can realize rapid analysis of single cell samples, but cannot separate and sort single cells.
Chinese patent No. CN107462566A discloses a raman spectrometer for detecting a specific narrow band wave number range, which can realize fast and highly sensitive detection of a raman spectrum in a specific narrow wave number range, but can determine the specificity of a single cell in field or clinical sample detection, but this patent cannot obtain complete raman spectrum information, cannot realize high-throughput and fast single cell type identification, and further separates and sorts the single cell for subsequent processing (such as single cell culture, amplification, etc.).
SUMMERY OF THE UTILITY MODEL
To above defect, the utility model provides a high flux raman unicellular sorting unit.
The utility model discloses an aspect provides high flux raman unicellular sorting unit, include:
a PMT (photomultiplier tube) narrow-band raman single-cell prescreening module for prescreening detection of the single-cell sample to obtain a narrow-band raman signal and a narrow-band background signal corresponding to the single-cell sample;
-a linear array raman single cell identification module for acquiring full spectral information of the single cell sample;
-a microfluidic chip for placing a single cell sample;
and the light path of the PMT narrow-band Raman single cell pre-screening module and the light path of the linear array Raman single cell identification module are focused on the microfluidic chip in parallel.
In another preferred example, the narrow-band raman single-cell pre-screening module comprises a laser light source and a photomultiplier.
In another preferred embodiment, the linear array raman single cell identification module comprises a laser light source, a linear array light generator and a spectrometer.
In another preferred example, the PMT (photomultiplier tube) narrow-band raman single cell prescreening module and the linear array raman single cell identification module have the same laser light source.
In another preferred example, the microfluidic chip is placed on a three-dimensional moving platform.
In another preferred embodiment, 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 which are connected in sequence; the narrow-band Raman detection pre-screening part comprises a sample inlet, a first liquid storage tank, a first channel and a waste liquid tank, wherein the sample inlet, the first liquid storage tank and the first channel are sequentially connected, a first sorting electrode is arranged on the outer side of the first channel; the linear array Raman detection part sequentially comprises a second liquid storage tank and a second channel, the second channel is provided with a buffer liquid inlet, and the outer side of the second channel is provided with a capture electrode; the liquid drop package forming part sequentially comprises an oil storage pool and a liquid drop package forming opening; the single-cell sorting part sequentially comprises a third channel and at least two separation ports, and a second sorting electrode is arranged outside the third channel.
In another preferred example, the narrow-band raman detection pre-screening part rapidly and highly sensitively screens out specific single cells, and separates the non-specific single cells into a waste liquid port by adding an electrode for removing.
In another preferred embodiment, the linear array raman detection and identification part can realize high-flux single cell raman signal acquisition to obtain full spectrum information of single cells, and single cell types are identified through database comparison.
In another preferred embodiment, the droplet-packing formation section forms a droplet to pack a single cell.
In another preferred example, the single-cell sorting part leads out the droplet-wrapped single cells from different separation channels by loading a sorting electrode.
In another preferred example, the electrode is externally connected with a power supply.
In another preferred embodiment, the width of the first channel is 10-50um, the narrowest dimension.
In another preferred example, the width of the first liquid storage tank is 80-100um, which is the widest dimension.
In another preferred embodiment, the single cell sample comprises bacteria, fungi, microorganisms, and the like.
In another preferred embodiment, the single-cell high-throughput detection method comprises culturing or amplifying the sorted single-cell sample.
The utility model has the advantages that:
the utility model discloses a high flux parallel Raman single cell sorting device based on the combination of PMT and linear array detection technology, firstly, the PMT detector is adopted to primarily screen cells with specific requirements, and the screening flux is greatly improved; and then, the screened specific cells are controlled by a microfluidic device to carry out Raman full-spectrum parallel collection, the strategy can avoid carrying out full-spectrum analysis on all samples, greatly reduce the collection time of the samples, simultaneously adopt linear array Raman to collect the spectrum of the specific cells to carry out cell type identification, finally, the tested specific cells can be sorted out by a microfluidic chip, the possibility (such as single cell culture, single cell sequencing and the like) is provided for further analysis of subsequent cells, and the high-throughput detection, analysis and sorting of the cell samples are realized.
Drawings
Fig. 1 is a schematic diagram of the light path of the high-throughput raman single-cell sorting device and method combining PMT and linear array raman.
Fig. 2 is a schematic diagram of a microfluidic chip.
The reference numbers are as follows:
1. laser 2, beam expanding 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 moving 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 141, first PMT142, 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, linear array spectrometer 21, sample light generator 22 and sample light generator 22 The port 23, the first reservoir 24, the narrow band Raman detection light 25, the first sorting electrode 26, the first channel 27, the waste liquid pool 28, the second reservoir 29, the buffer inlet 30, the capture electrode 31, the linear array Raman detection light 32, the second channel 33, the reservoir 34, the liquid drop wrapping port 35, the second sorting electrode 36, the third channel 37, the first separation port 38, the second separation port 39, and the third separation port
Detailed Description
In order to facilitate understanding of the embodiments of the present invention, the following description will be made in terms of specific embodiments with reference to the accompanying drawings, and the embodiments are not intended to limit the embodiments of the present invention. Furthermore, the drawings are schematic and, thus, the present devices and apparatus are not limited by the size or scale of the schematic.
It is to be noted that in the claims and the description of the present patent, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, the use of the verb "comprise a" to define an element does not exclude the presence of another, same element in a process, method, article, or apparatus that comprises the element.
Example one
As shown in fig. 1, the micro-fluidic chip comprises a PMT narrow-band Raman single cell pre-screening optical path and a linear array Raman single cell identification optical path, wherein the PMT narrow-band Raman single cell pre-screening optical path and the linear array Raman single cell identification optical path are focused on the micro-fluidic chip in parallel, and a single cell sample is contained in the micro-fluidic chip.
The single-cell Raman spectrometer comprises a laser 1, a beam expansion collimator 2, a laser beam splitter 3, reflectors 41, 42, 43, 44 and 45, high- pass filters 51 and 52, dichroic mirrors 61 and 62, a microscope objective group 71 and 72, a microfluidic chip 8, a three-dimensional displacement platform 9, lenses 101, 102 and 103, a pinhole 11, narrow- band filters 121 and 122, a beam splitter prism 13, a PMT141 and 142, visible light beam splitters 151 and 152, image collectors 161 and 162, LED light sources 171 and 172, a cylindrical lens 18, a slit 19, a spectrometer 20, a linear array light generator 21 and the like. Wherein:
the laser beam emitted by the laser 1 passes through the beam expanding collimator 2 to generate parallel light, the parallel light sequentially passes through the laser beam splitter 3, the first reflecting mirror 41, the second reflecting mirror 42, the first high-pass filter 51, the first dichroic mirror 61 and the first microscope objective group 71 to be focused on the single-cell sample in the microfluidic chip 8 to generate a Raman signal, and the Raman signal sequentially reversely passes through the first microscope objective 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, and uniformly enters the first narrow-band filter 121 and the second narrow-band filter 122 to respectively enter the first PMT141 and the second PMT 142.
The first LED light source 171 sequentially passes through the first visible light beam splitter 151, the first dichroic mirror 61, and the first microscope objective group 71 and is focused on the microfluidic chip 8 to obtain image information of the single-cell sample, and the image information sequentially and reversely passes through the first microscope objective 71, the first dichroic mirror 61, the first visible light beam splitter 151, and the second lens 102 and enters the first image collector 161.
The laser beam emitted by the laser 1 passes through the beam expanding collimator 2 to generate parallel light, the parallel light sequentially 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 optical filter 52, the second dichroic mirror 62 and the second microscopic objective group 72 to be focused on the single cell sample in the microfluidic chip 8 to generate a raman signal, and the raman signal sequentially reversely passes through the second microscopic objective group 72, the second dichroic mirror 62, the second high-pass optical filter 52, the cylindrical mirror 18 and the slit to enter the spectrometer 19.
The second LED light source 172 sequentially passes through the second visible light beam splitter 152, the second dichroic mirror 62, and the second microscope objective group 72 to be focused on the microfluidic chip 8, so as to obtain image information of the single-cell sample, and the image information sequentially reversely passes through the second microscope objective 72, the second dichroic mirror 62, the second visible light beam splitter 152, and the third lens 103 and enters the second image collector 162.
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 micro-fluidic chip in parallel to detect the single-cell samples in sequence.
The three-dimensional displacement platform 9 is provided with the micro-fluidic chip 8, and the movement of the three-dimensional displacement platform 9 drives the micro-fluidic chip 8 to move.
Example two
Fig. 2 is a schematic diagram of a microfluidic chip, which specifically includes:
a sample inlet 22, a first liquid storage tank 23, a first sorting electrode 25, a first channel 26 and a waste liquid tank 27; the sample enters the first liquid storage tank 23 through the sample inlet 22 to wait for detection, the single cell continuously flows into the first channel 26 from the first liquid storage tank 23, 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 the single cell is judged whether to have specificity according to the detected narrow-band spectral signal.
The linear array Raman detection part sequentially comprises a second liquid storage tank 28, a buffer liquid inlet 29, a capture electrode 20, a linear array Raman detection light 31 and a second channel 32; the single cells with specificity enter the second channel 32 from the second liquid storage tank 28, buffer solution is injected into the buffer solution inlet 29, the single cells are arranged in a linear shape under the action of the buffer solution, the single cells arranged in the linear shape are stabilized at the focusing position of the linear array Raman detection light beam 31 by the capture electrode 30, the focused light beam irradiates the single cells, the single cells generate full spectrum Raman signals, Raman spectra are collected by a spectrometer, the types of the single cells are identified by comparing the Raman spectra with a database by a computer, the collection is completed, the capture electrode 30 discharges, and the single cells enter a liquid drop package forming part.
The liquid drop package forming part sequentially comprises an oil storage pool 33 and a liquid drop package forming opening; and injecting 33 an oil phase into the oil storage tank, wherein the oil phase can realize that single cells generate liquid drops at the liquid drop package forming port 34, and the generated liquid drops sequentially reach the third channel 36.
The single-cell sorting section includes a second sorting electrode 35, a third channel 36, a first separation port 37, a second separation port 38, a third separation port 39; and (3) counting the liquid drops entering the third channel in sequence according to the identified types, and applying different electrodes according to different numbering sequences so as to enable the liquid drops to enter different separation ports: a first separation port 37, a second separation port 38, and a third separation port 39.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and additions can be made without departing from the method of the present invention, and these improvements and additions should also be regarded as the protection scope of the present invention.
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| CN114252425A (en) * | 2020-09-25 | 2022-03-29 | 中国科学院青岛生物能源与过程研究所 | A high-throughput Raman single cell sorting device and method |
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| CN114252425A (en) * | 2020-09-25 | 2022-03-29 | 中国科学院青岛生物能源与过程研究所 | A high-throughput Raman single cell sorting device and method |
| CN114252425B (en) * | 2020-09-25 | 2025-01-24 | 中国科学院青岛生物能源与过程研究所 | A high-throughput Raman single-cell sorting device and method |
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