CN114252425B - A high-throughput Raman single-cell sorting device and method - Google Patents
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Abstract
本发明提供了一种高通量单细胞分选装置与方法。包括：微流控芯片，用于放置单细胞样品；PMT(光电倍增管)窄带拉曼单细胞预筛选模块，用于对单细胞样品进行预筛选检测，获得对应于单细胞样品的窄带拉曼信号和窄带背景信号；线阵拉曼单细胞识别模块，用于获取单细胞样品的全谱信息。所述芯片包括依次连接窄带拉曼检测预筛选部分、线阵拉曼检测部分、液滴包裹形成部分、单细胞分选部分；本发明可实现细胞样品的高通量检测、分析和分选。
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
The invention relates to the field of microscopic Raman single-cell sorting, in particular to a high-flux Raman single-cell sorting device and method.
Background
The Raman spectrum is a high-efficiency information identification technology, the vibration or rotation energy level of a compound molecule can be directly detected by microscopic Raman spectrum through analyzing inelastic scattering spectral lines of specific incident light rays on the compound, and the composition and structure information of the compound molecule can be obtained through analyzing Raman characteristic spectral lines.
However, the existing raman microscopy technology has defects when being used for sample analysis, taking single-cell measurement of microorganisms as an example, single-cell raman spectrum signal intensity is weaker, particularly when cells are suspended in liquid, only 10 6-8 times of photons usually pass through raman scattering, so that the spectrum scanning time for obtaining complete and reliable raman spectrum signals is longer, and when a large number of samples are analyzed, the analysis time of the samples is greatly increased by carrying out full spectrum identification on each cell, so that the collection flux is lower.
The existing linear array Raman detection products on the market generally analyze solid samples or dry cell samples, so that the single cell samples can be rapidly analyzed, but single cell separation and sorting cannot be performed only in the linear array Raman detection products.
The Chinese patent with the patent number of CN107462566A discloses a Raman spectrometer for detecting a specific narrow-band wave number range, which can realize rapid and high-sensitivity detection of a Raman spectrum in the specific narrow-band wave number range, but can judge the specificity of single cells during on-site or clinical sample detection, but the patent can not obtain finished Raman spectrum information, can not realize high-flux and rapid single cell type identification, and further separate and sort single cells for subsequent treatment (such as single cell culture, amplification and the like).
Disclosure of Invention
Aiming at the defects, the invention provides a high-flux Raman single-cell sorting device and a high-flux Raman single-cell sorting method.
In one aspect, the invention provides a high throughput raman single cell sorting apparatus comprising:
-a PMT (photomultiplier) narrowband raman single-cell pre-screening module for pre-screening the single-cell sample to obtain a narrowband raman signal and a narrowband background signal corresponding to the single-cell sample;
-a linear array raman single cell identification module for obtaining full spectrum information of a single cell sample;
-a microfluidic chip for placing single cell samples.
In another preferred embodiment, 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 embodiment, the PMT (photomultiplier tube) narrowband raman single cell pre-screening module and the linear array raman single cell identification module have the same laser light source.
In another preferred embodiment, the microfluidic chip is placed on a three-dimensional mobile platform.
In another preferred example, the microfluidic 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 sequentially connected, wherein 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 which are sequentially connected, a first sorting electrode is arranged outside the first channel, the linear array Raman detection part sequentially comprises a second liquid storage tank and a second channel, a buffer liquid inlet is arranged in the second channel, a capturing electrode is arranged outside the second channel, the liquid drop package forming part sequentially comprises an oil storage tank and a liquid drop package forming port, the single cell sorting part sequentially comprises a third channel and at least two separating ports, and a second sorting electrode is arranged outside the third channel.
In another preferred embodiment, the pre-screening part of the narrow-band Raman detection screens out single cells with specificity rapidly and with high sensitivity, and separates single cells without specificity to a waste liquid port for removal by means of an electrode.
In another preferred example, the linear array raman detection identification part can acquire the raman signal of single cells in a high flux manner, obtain the full spectrum information of the single cells, and identify the single cell types through database comparison.
In another preferred embodiment, the droplet encapsulation forming portion forms droplets to encapsulate single cells.
In another preferred embodiment, the single cell sorting section directs the droplet-packed single cells out of different separation channels by loading sorting electrodes.
In another preferred embodiment, the electrode is externally connected to a power source.
In another preferred embodiment, the width of the first channel is 10-50um, the narrowest dimension.
In another preferred embodiment, the width of the first reservoir is 80-100um, the widest dimension.
The single cell sample includes bacteria, fungi, microorganisms, and the like.
The invention also provides a high-throughput Raman single-cell sorting method, which comprises the following steps of:
S1, injecting a single-cell sample into a microfluidic chip;
s2, when the single-cell sample is positioned in the first channel, a PMT narrow-band micro-Raman single-cell pre-screening module is used for pre-screening and detecting the single-cell sample, so that a narrow-band Raman signal and a narrow-band background signal corresponding to the single-cell sample are obtained, and the single-cell sample with specificity is identified;
S3, applying voltage to the first sorting electrode to enable the single-cell sample with specificity to enter the second liquid storage tank and enter the second channel;
s4, applying voltage to the capturing electrode, detecting the single-cell sample by the linear array Raman single-cell recognition module, obtaining Raman spectra of one or more single-cell samples, and recognizing the single-cell sample;
S5, injecting an oil phase into the oil storage pool, enabling the single-cell sample to generate liquid drops at a liquid drop wrapping forming port, and enabling the generated liquid drops to enter a third channel;
and S6, applying voltage to the second separation electrode to enable the liquid drops to enter different separation ports for separation.
In another preferred embodiment, the step S2 of identifying the single cell sample with specificity includes performing data processing on the obtained narrowband raman signal and narrowband background signal to obtain a signal to noise ratio value, and comparing the signal to noise ratio value according to a preset value, so as to distinguish whether the single cell sample is specific.
In another preferred embodiment, the identifying of the desired single cell in step S4 comprises comparing the raman spectrum obtained for one or more single cell samples with standard raman spectrum signals or reference raman spectrum signals in a constructed single cell phenotype database to obtain a determined cell type.
In another preferred embodiment, the step S6 of applying a voltage to the second sorting electrode to cause the droplets to enter the different separation openings for sorting includes numbering the droplets sequentially entering the third channel, identifying the single-cell sample according to the step S4, and applying different voltages in different numbering sequences to cause the droplets to enter the different separation openings.
In another preferred embodiment, the single cell high throughput detection method comprises culturing or amplifying the sorted single cell sample.
The invention has the beneficial effects that:
The invention discloses a high-flux parallel Raman single-cell sorting device based on the combination of a PMT and a linear array detection technology, which comprises the steps of firstly adopting a PMT detector to perform primary screening on cells with specific requirements, greatly improving screening flux, then controlling the screened cells with specificity through a microfluidic device to perform Raman full-spectrum parallel collection, avoiding full-spectrum analysis on all samples by the strategy, greatly reducing sample collection time, simultaneously adopting a linear array Raman to collect spectra of the specific cells to perform type identification on the cells, and finally sorting the tested specific cells through a microfluidic chip, thereby providing possibility (such as single-cell culture, single-cell sequencing and the like) for further analysis of subsequent cells and realizing high-flux detection, analysis and sorting of cell samples.
Drawings
FIG. 1 is a schematic diagram of the optical path of the PMT-linear array Raman combined high-throughput Raman single-cell sorting device and method.
Fig. 2 is a schematic diagram of a microfluidic chip.
The reference numerals are as follows:
1. Laser 2, expanded beam collimator 3, laser beam splitter 41, first mirror 42, second mirror 43, third mirror 44, fourth mirror 45, fifth mirror 52, first high pass filter 61, first dichroic mirror 62, second dichroic mirror 71, first microscope set 72, second microscope set 8, microfluidic chip 9, three-dimensional moving stage 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 beam splitter 152, third lens 102, third lens 11, pinhole 121, second narrow band filter 13, second PMT 142, second PMT 151, and third visible beam splitter 152 two visible light beam splitters 161, a first image collector 162, a second image collector 171, a first LED light source 172, a second LED light source 18, a cylindrical mirror 19, a slit 20, a spectrometer 21, a linear array light generator 22, a sample inlet 23, a first reservoir 24, narrow-band Raman detection light 25, a first sorting electrode 26, a first channel 27, a waste liquid reservoir 28, a second reservoir 29, a buffer inlet 30, a trapping electrode 31, linear array Raman detection light 32, a second channel 33, an oil reservoir 34, a droplet packing formation port 35, a second sorting electrode 36, a third channel 37, a first separation port 38, a second separation port 39, a third separation port
Detailed Description
For the purpose of facilitating an understanding of the embodiments of the present invention, reference will now be made to the detailed description of embodiments, taken in conjunction with the accompanying drawings, and the various embodiments are not intended to limit the embodiments of the invention. Furthermore, the drawings are schematic representations, and thus the apparatus and devices of the present invention are not limited by the dimensions or proportions of the schematic representations.
It should be noted that in the claims and the description of this 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. Moreover, 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, an element defined by the phrase "comprising" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Example 1
The device comprises a PMT narrow-band Raman single-cell pre-screening light path and a linear array Raman single-cell identification light path, wherein the PMT narrow-band microscopic Raman single-cell pre-screening light path and the linear array microscopic Raman single-cell identification light path are focused on the microfluidic chip in parallel, and single-cell samples are contained in the microfluidic chip.
The PMT narrow-band micro-Raman single-cell pre-screening light path and the linear array micro-Raman single-cell identification light path form a high-throughput Raman single-cell sorting device and method combining a PMT and a linear array Raman, and the single-cell Raman spectrometer comprises a laser 1, a beam expanding collimator 2, a laser beam splitter 3, reflecting mirrors 41, 42, 43, 44, 45, high-pass filters 51, 52, dichroic mirrors 61, 62, microscope objective groups 71, 72, a microfluidic chip 8, a three-dimensional displacement platform 9, lenses 101, 102, 103, pinholes 11, narrow-band filters 121, 122, a beam splitting prism 13, PMT141, 142, visible beam splitters 151, 152, image collectors 161, 162, LED light sources 171, 172, a cylindrical mirror 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 micro objective lens 71 to be focused on a single cell sample in the microfluidic chip 8 to generate a raman signal, and the raman signal sequentially passes through the first micro objective lens 71, the first dichroic mirror 61, the first high-pass filter 51, the first lens 101, the pinhole 11 and the splitting prism 13 in a reverse direction and uniformly enters the first narrow-band filter 121 and the second narrow-band filter 122 to enter the first PMT141 and the second PMT142 respectively.
The first LED light source 171 sequentially passes through the first visible light beam splitter 151, the first dichroic mirror 61, and the first micro objective lens group 71 to focus on the microfluidic chip 8, so as to obtain image information of the single cell sample, and the image information sequentially passes through the first micro objective lens 71, the first dichroic plate 61, the first visible light beam splitter 151, and the second lens 102 reversely to enter 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 passes through the laser beam splitter 3, the third reflecting mirror 43, the fourth reflecting mirror 44, the linear array light generator 21, the fifth reflecting mirror 45, the second high-pass filter 52, the second dichroic mirror 62 and the second micro objective lens group 72 in sequence to be focused on the single cell sample in the micro fluidic chip 8, a raman signal is generated, and the raman signal passes through the second micro objective lens group 72, the second dichroic mirror 62, the second high-pass filter 52, the cylindrical mirror 18 and the slit in sequence in reverse direction to enter the 19 spectrometer 20.
The second LED light source 172 sequentially passes through the second visible light beam splitter 152, the second dichroic mirror 62, and the second micro objective lens group 72 to focus on the microfluidic chip 8, so as to obtain image information of the single cell sample, and the image information sequentially passes through the second micro objective lens 72, the second dichroic plate 62, the second visible light beam splitter 152, and the third lens 103 reversely to enter the second image collector 162.
And the PMT narrow-band microscopic Raman single-cell pre-screening light path and the linear array microscopic Raman single-cell identification light path are focused on the microfluidic chip in parallel, and single-cell samples are detected sequentially.
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:
The sample enters the first liquid storage tank 23 through the sample inlet 22 to wait for detection, and continuously flows into single cells from the first liquid storage tank 23 to enter the first channel 26 in sequence along with the continuous inflow of the sample to reach the focusing position of the narrow-band Raman detection light 24, the narrow-band Raman detection can rapidly and highly sensitively detect the narrow-band spectrum signal of the single cells, and whether the single cells have specificity or not is judged according to the detected narrow-band spectrum signal.
The linear array Raman detection part sequentially comprises a second liquid storage tank 28, a buffer solution inlet 29, a capture electrode 20, linear array Raman detection light 31 and a second channel 32, single cells with specificity enter the second channel 32 from the second liquid storage tank 28, the buffer solution inlet 29 is filled with buffer solution, the single cells are linearly arranged under the action of the buffer solution, the capture electrode 30 stabilizes the single cells which are linearly arranged on the focusing position of the linear array Raman detection light beam 31, the focusing light beam irradiates the single cells, the single cells generate full-spectrum Raman signals, a Raman spectrum is acquired through a spectrometer, the computer compares the Raman spectrum through a database, the types of the single cells are identified, the acquisition is completed, the capture electrode 30 discharges, and the single cells enter the liquid drop package forming part.
The liquid drop package forming part sequentially comprises an oil storage tank 33 and a liquid drop package forming opening, and oil phase is injected 33 into the oil storage tank, so that single cells can generate liquid drops at the liquid drop package forming opening 34, and the generated liquid drops sequentially reach a third channel 36.
The single cell sorting part comprises a second sorting electrode 35, a third channel 36, a first separating opening 37, a second separating opening 38 and a third separating opening 39, and for the liquid drops entering the third channel in sequence, the number according to the identified type is calculated, and different electrodes are applied according to different number sequences, so that the liquid drops enter different separating openings, namely the first separating opening 37, the second separating opening 38 and the third separating opening 39.
Example III
The invention provides a single-cell high-flux detection method based on a high-flux Raman single-cell sorting device combining PMT and linear array Raman, which comprises the following steps:
1. Preparing a single cell sample;
2. Single-cell samples are injected into a microfluidic chip 8, and the microfluidic chip is placed on a three-dimensional displacement platform 9;
3. The single-cell narrow-band Raman signal detection comprises a PMT narrow-band microscopic Raman single-cell pre-screening light path, and pre-screening detection is carried out on a single-cell sample to obtain a narrow-band Raman signal and a narrow-band background signal corresponding to the single-cell sample;
further, the step3 further comprises the step of performing data processing on the obtained narrow-band Raman signal and the narrow-band background signal to obtain a signal-to-noise ratio value, and comparing according to a preset value to determine whether the single cell sample is specific.
4. The linear array Raman signal detection comprises the steps that a single-cell sample with specificity enters a linear array microscopic Raman single-cell identification light path through a microfluidic chip channel, and single-cell samples positioned on a linear array detection laser beam are detected to obtain one or more Raman spectra of single cells;
further, step 4 includes the step of comparing the raman spectrum signal after normalization (baseline subtraction, normalization, spurious peak filtering, etc.) with standard raman spectrum signals or reference raman spectrum signals in the constructed single cell phenotype database, and simultaneously adopting algorithms such as deep learning, database mining, etc. to rapidly determine the type (kind) of the cell.
5. Injecting an oil phase into the oil storage pool, so that single-cell samples form liquid drops at liquid drop wrapping forming openings, and the generated liquid drops enter a third channel.
6. The identified single cell liquid drops are applied with different electrodes, single cell samples enter different separation ports, and the separated single cells are subjected to subsequent treatment (such as culture, amplification and the like).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (9)
1. A high throughput raman single cell sorting apparatus, comprising:
the PMT narrowband Raman single-cell pre-screening module is used for pre-screening and detecting single-cell samples, obtaining narrowband Raman signals and narrowband background signals corresponding to the single-cell samples, and identifying the single-cell samples with specificity;
The linear array Raman single-cell identification module is used for acquiring full spectrum information of a single-cell sample;
The microfluidic chip is used for placing single-cell samples;
The microfluidic 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 sequentially connected, wherein 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 which 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, a buffer liquid inlet is arranged on the second channel, a capturing electrode is arranged on the outer side of the second channel, the liquid drop package forming part sequentially comprises an oil storage tank and a liquid drop package forming opening, the single cell sorting part sequentially comprises a third channel and at least two separating openings, and a second sorting electrode is arranged on the outer side of the third channel;
the PMT narrow-band Raman single-cell pre-screening module and the linear array Raman single-cell identification module are provided with the same laser light source;
The high-flux Raman single-cell sorting device further comprises a PMT narrow-band Raman single-cell pre-screening light path and a linear array Raman single-cell identification light path, and the PMT narrow-band microscopic Raman single-cell pre-screening light path and the linear array microscopic Raman single-cell identification light path are focused on the microfluidic chip in parallel.
2. The high throughput raman single cell sorting apparatus according to claim 1, wherein said PMT narrowband raman single cell pre-screening module comprises a laser light source and a photomultiplier tube.
3. The high throughput raman single cell sorting apparatus of claim 1, wherein 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 apparatus of claim 1, wherein the electrode is externally connected to a power source.
5. The high throughput raman single cell sorting apparatus according to claim 1, wherein the microfluidic chip is placed on a three-dimensional moving platform.
6. A high throughput raman single cell sorting method, characterized by using the high throughput raman single cell sorting apparatus according to any one of claims 2 to 5, and comprising the steps of:
S1, injecting a single-cell sample into a microfluidic chip;
s2, when the single-cell sample is positioned in the first channel, a PMT narrow-band micro-Raman single-cell pre-screening module is used for pre-screening and detecting the single-cell sample, so that a narrow-band Raman signal and a narrow-band background signal corresponding to the single-cell sample are obtained, and the single-cell sample with specificity is identified;
S3, applying voltage to the first sorting electrode to enable the single-cell sample with specificity to enter the second liquid storage tank and enter the second channel;
s4, applying voltage to the capturing electrode, detecting the single-cell sample by the linear array Raman single-cell recognition module, obtaining Raman spectra of one or more single-cell samples, and recognizing the single-cell sample;
S5, injecting an oil phase into the oil storage pool, enabling the single-cell sample to generate liquid drops at a liquid drop wrapping forming port, and enabling the generated liquid drops to enter a third channel;
And S6, applying voltage to the second separation electrode to enable the liquid drops to enter different separation ports for separation.
7. The high throughput raman single cell sorting method according to claim 6, wherein the step S2 of identifying single cell samples having specificity comprises performing data processing on the obtained narrowband raman signal and narrowband background signal to obtain signal to noise ratio values, and comparing according to preset values to thereby identify whether single cell samples have specificity.
8. The high throughput raman single cell sorting method according to claim 6, wherein said identifying a desired single cell in step S4 comprises comparing said obtaining raman spectra of one or more single cell samples with standard raman spectrum signals or reference raman spectrum signals in a constructed single cell phenotype database to obtain a determined cell type.
9. The high throughput raman single cell sorting method according to claim 6, wherein step S6 of applying a voltage to the second sorting electrode to sort the droplets into different separation openings comprises numbering the droplets sequentially entering the third channel, identifying single cell samples according to step S4, and applying different voltages in different numbering orders to cause the droplets to enter different separation openings.
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| US7057721B2 (en) * | 2002-01-10 | 2006-06-06 | Chemimage Corporation | Wide field method for detecting pathogenic microorganisms |
| CN102019277B (en) * | 2010-10-29 | 2013-05-22 | 北京惟馨雨生物科技有限公司 | Sorter and sorting method for separating cells and particles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN215115894U (en) * | 2020-09-25 | 2021-12-10 | 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 |
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