WO2019080905A1 - Raman activated droplet sorting system and method - Google Patents

Abstract

Provided by the present invention are a Raman activated droplet sorting system and a sorting method, which may achieve high-speed, accurate and lossless droplet sorting. The Raman activated droplet sorting system of the present invention comprises the following units in order from first to last in the liquid flow direction: a liquid flow trapping unit, a signal detecting unit, a droplet generating unit and a sorting control unit, and the Raman activated droplet sorting system further comprises a liquid flow driving unit, a droplet recovering unit, and a liquid flow channel.

Description

Raman activated droplet sorting system and method Technical field

The invention belongs to the field of biotechnology and instrument science, in particular to a Raman-activated droplet sorting system and method, which can realize high-speed, accurate and non-destructive droplet sorting, and is particularly suitable for single cell sorting.

Background technique

Living single cells are the basic unit of life activity and the basic unit of evolution. It is of great significance to study cell biological processes from a single cell level. Rapid separation and acquisition of single cells has become a key technology for single cell research and analysis. Ideal single-cell analysis tools are expected to be processed in a non-invasive, label-free, and high-throughput manner in the natural state of the cell. Raman-activated cell sorting is based on the principle that Raman spectroscopy is the intrinsic biochemical curve of single cells and the "chemical image", and has the ability to single-cell sorting without labeling, non-invasive, and simultaneous analysis of multiple features or phenotypes. Make it an ideal method for single cell research.

There are a series of techniques in the prior art for Raman-activated cell sorting, such as optical forceps, laser jet coupling, etc., which are static versions of Raman-activated cell sorting techniques. Although these systems are simple and practical, their flux is too low, hindering the application of Raman spectroscopy in high-throughput sorting. In order to improve the single-cell sorting flux, Raman-activated sorting flow cytometry based on microfluidic technology has also appeared. In such a technical scheme, cells are fixed by optical forceps and dragged to collect cells, but The throughput of such programs is still not high (~3min/cell), and it is not yet fully met.

Summary of the invention

As mentioned above, there is currently no Raman-activated droplet sorting system and sorting method that can meet the following requirements:

a) high flux requirements for sorting;

b) high accuracy requirements for sorting;

c) No damage or only minor damage to the contents of the sorted droplets, such as single cells.

In addition, the droplet sorting system and sorting method based on Raman spectroscopy also have the following two technical problems: 1 The lens effect produced by the convex/concave shape of the droplet surface will cause the focus to be deformed and the spatial resolution is reduced; The Raman spectrum of the oil phase used for the droplets is too strong, causing significant interference. Both of these problems can make it difficult to accurately obtain a Raman signal when detecting droplets and/or their contents.

The present invention is intended to provide a Raman-activated droplet sorting system and a Raman-activated droplet sorting method that solve the above problems, thereby realizing high-speed, accurate, and non-destructive droplet sorting based on Raman spectroscopy.

In a first aspect of the invention, a Raman-activated droplet sorting system is provided, characterized in that the sorting system comprises the following units in order from the first to the last in the direction of flow:

1) a liquid flow trapping unit for regulating the flow rate of the cell/particle suspension and the buffer, and concentrating the cells/particles to be sorted in the middle of the liquid flow channel to form a stable single cell/particle flow;

2) a signal detecting unit for generating Raman scattered photons, performing Raman laser signal detection on the single cell/particle flow formed by the liquid flow trapping unit, collecting the detection signals and transmitting them to the sorting control unit;

3) a droplet generating unit for introducing an oil phase into the single cell/particle flow detected by the signal detecting unit to form a water-in-oil type droplet;

4) a sorting control unit for analyzing the detection signal from the signal detecting unit, and sorting the droplets generated by the droplet generating unit according to the signal;

The sorting system further includes a liquid flow driving unit and a liquid flow channel for generating a driving force required for liquid flow in the sorting system; the liquid flow channel is a liquid flow in the system Carrier and liquid inlet and outlet.

As a preferred form of the invention, the flow aggregating unit comprises at least one flow channel for the inflow of the cell/particle suspension and at least two flow channels for the influent buffer.

As a preferred form of the invention, the flow agglomeration unit is used to condition the cell/particle suspension and buffer flow rate to form a single cell/particle flow suitable for detection by the signal detection unit.

As a preferred form of the present invention, the signal detecting unit includes a microscope, a stage, a Raman laser source, a signal collecting element, and a high speed CCD.

As a preferred form of the invention, the Raman laser signal alignment used in the signal detecting unit is selected from the combination of one or more of the following: single peak contrast, multi peak contrast, full spectrum alignment.

As a more preferred form of the invention, the signal collecting element is a charge coupled device.

As a more preferred form of the present invention, the signal detecting unit includes a dielectric capturing unit for capturing cells/particles by dielectric means to extend the Raman laser signal acquisition time and increase the acquired signal intensity.

In a preferred form of the invention, the droplet generating unit comprises at least one liquid flow path for flowing into the cell/particle suspension detected by the signal detecting unit, and at least one lateral flow inlet for introducing the oil phase .

As a more preferred form of the present invention, the water-in-oil droplets generated by the droplet generating unit contain an average of no more than one single cell/particle per droplet.

As a more preferable form of the present invention, the water-in-oil droplets generated by the droplet generating unit have an average of 0.3 single cells/particles per droplet.

In one embodiment of the invention, the droplets generated by the droplet generating unit have a diameter of 30-80 μm.

In one embodiment of the invention, the oil phase used in the droplet generating unit is a mineral oil containing 2.5% Span 80.

In one embodiment of the invention, the oil phase flow rate used in the droplet generating unit is 120 μL/h.

In one embodiment of the invention, the droplet water generating unit generates a water-in-oil droplet diameter of 50 μm.

As a preferred form of the invention, the sorting control unit performs droplet sorting by any of the following methods: dielectrophoresis, ultrasound, electric field, magnetic field, pressure extrusion or pressure suction.

As a more preferred form of the invention, the sorting control unit includes at least two dielectric electrodes for sorting droplets by dielectrophoresis.

As a preferred form of the invention, the sorting system further includes at least one droplet collecting unit for collecting and collecting the sorted droplets.

As a preferred form of the invention, the flow channel is a microfluidic chip.

As a more preferred form of the present invention, the liquid flow path has a width of 25 to 100 μm and a depth of 40 to 60 μm.

In a second aspect of the invention, a Raman-activated droplet sorting method is provided, comprising the steps of:

1) Concentration of liquid flow: Converging the cells/particles to be sorted with the buffer to form a stable single cell/particle flow;

2) Signal detection: Raman spectroscopy is performed on the single cell/particle flow one by one and the signal is collected;

3) droplet formation: after the collection is completed, the side stream is introduced into the oil phase, and water-in-oil microdroplets containing single cells/particles are formed by shearing;

4) Droplet sorting: Obtain the signal collected in the foregoing signal detecting step, perform sorting operation on the corresponding encapsulated water-in-oil droplet according to the signal, and collect the droplets of the required portion.

As a preferred form of the present invention, the sorting operation in the droplet sorting step of the above sorting method is performed by dielectrophoresis.

As a preferred form of the present invention, in the droplet sorting step of the above sorting method, there is a delay time between acquiring the acquisition signal and performing the sorting operation, and the delay time is calculated and determined by the following method: interval time = liquid flow Length/liquid flow rate, where the flow length refers to the actual distance through which the liquid detected from the signal detection point to the sorting operation point.

The invention has the following advantages and beneficial effects:

1. The present invention creatively solves the problem of signal interference caused by the Raman detection by the curved interface and the oil phase component of the droplet by the Raman detection first and the droplet encapsulation.

2. The invention realizes single cell high-throughput sorting based on Raman spectroscopy, and the flux can reach 500 cells/min, which is more than two orders of magnitude higher than other existing single cell sorting techniques based on Raman spectroscopy.

3. The present invention ensures the separation accuracy (>98%) while increasing the throughput.

4. The sorting system and method of the present invention adopts a droplet microfluidic system, which greatly reduces damage to cells and facilitates downstream processing after sorting.

In summary, the present invention provides a droplet sorting system based on Raman spectroscopy and a corresponding sorting method, which solves the problem that it is difficult to perform high-flux sorting, and has the advantages of simple and feasible, wide application range and strong expandability. Etc.

DRAWINGS

1 is a schematic diagram of the system composition of the Raman activated droplet sorting system of the present invention.

2 is a schematic structural view of an implementation of a Raman-activated droplet sorting system according to the present invention.

3 is a schematic structural view of another implementation manner of the Raman activated droplet sorting system of the present invention.

4 is a flow chart of the sorting control operation of the Raman activated droplet sorting system of the present invention.

Fig. 5 is a graph showing the results of sorting efficiency verification in the high-yield astaxanthin microalgae sorting model using the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

The overall frame design of a cell flow detection system based on Raman signals has been disclosed in the prior art, such as: CN102019277A, CN104877898A, etc., which is hereby incorporated by reference in its entirety. In the basic structure disclosed in the above documents, the basic composition of the Raman-activated droplet sorting system in the present invention is as shown in FIG. 1, which mainly includes a liquid flow trapping unit, a signal detecting unit, a droplet generating unit, and a sorting control. unit.

2 is an implementation of the Raman activated droplet sorting system of the present invention, specifically:

The flow trapping unit 1 carries at least one liquid flow channel 5 for influx of the cell/particle suspension and at least two flow channels 6 and 7 for the influent buffer for regulating the cell/particle suspension and buffering Liquid flow rate. The flow rate is generally determined by the size of the droplets desired to be produced, while also concentrating the cells/particles to be sorted in the middle of the flow channel to form a stable single cell/particle flow suitable for detection by the signal detection unit. To reduce interference with Raman signal detection, suspensions and buffers use a water-based buffer with a low Raman signal.

The signal detecting unit 2 is configured to generate a Raman laser, perform Raman laser signal detection on the single cell/particle flow formed by the liquid flow trapping unit, collect the detection signal and transmit it to the sorting control unit. These include microscopes, stages, Raman laser scattered photons, signal collection components, and high-speed CCDs. The signal alignment used for the detection may be one or a combination of unimodal contrast, multimodal contrast or full spectrum contrast. In addition, a dielectric capture unit may be additionally provided at the signal detection unit to capture the cells/particles by dielectric means to prolong the acquisition time of the Raman laser signal, and further increase the intensity of the acquired signal.

The droplet generating unit 3 includes at least one liquid flow path for flowing into the cell/particle suspension detected by the signal detecting unit, and at least one side flow inlet 8 for introducing the oil phase, which is detected by the signal detecting unit The oil phase is introduced into the single cell/particle stream to form a water-in-oil droplet. Due to the need for subsequent cell sorting, there is no more than one single cell/particle per oil-in-water droplet. In practice, it can be reduced to 0.3 single cells per particle per droplet or even lower.

The sorting control unit 4 is configured to analyze the detection signal from the signal detecting unit, and sort the droplets generated by the droplet generating unit according to the signal. The actual sorting method can be realized by any one of dielectrophoresis, ultrasonic, electric field, magnetic field, pressure extrusion or pressure suction depending on the flux requirement. Among them, dielectric electrophoretic sorting is a better way to achieve high-flux sorting requirements, that is, based on the detection signal, droplets flowing through the at least two dielectric electrodes 9 and 10 are applied with different forces. Control the flow of droplets to achieve high-speed, convenient sorting. There is a delay time between the acquisition of the acquisition signal and the sorting operation. The delay time is calculated and determined by the following method: interval time = liquid flow length / liquid flow velocity, wherein the liquid flow length refers to the time from the signal detection point to the minute Select the actual distance through which the detected liquid will flow.

The collection channel is connected to a collection container (not shown in the container), and the target droplets obtained by sorting are collected as a droplet collection unit.

3 is another form of implementing the Raman-activated droplet sorting system of the present invention. Specifically, based on the structure shown in FIG. 2 and the above description of the structure of FIG. 2, the trap electrode 11 is added to the signal detecting unit. , that is, a dielectric capture unit for capturing cells/particles by dielectric means to extend the Raman laser signal acquisition time, thereby increasing the acquired signal intensity.

Figure 4 shows a possible sorting control method.

In addition to the above units, the Raman-activated droplet sorting system of the present invention further comprises: a liquid flow driving unit for generating a driving force required for liquid flow in the sorting system; and a droplet collecting unit for The droplets after sorting are collected and recovered; and as a liquid flow carrier in the system and a flow channel into the outflow port.

As a conventional technique in the art, the liquid flow path in the system has a width of 25 to 100 μm and a depth of 40 to 60 μm.

The invention is further illustrated below in conjunction with specific examples.

Example

Microfluidic sorting chip design and manufacture

We designed a single-layer microfluidic chip with PDMS. Among them, the width for the flow passage is designed to be 50 μm. Microfluidic chips are fabricated by soft lithography and rapid prototyping techniques. Briefly, a 50 μm high SU- ^8TM mold was added to a 3 inch silicon wafer. A PDMS layer was prepared by pouring a mixture of PDMS and curing agent onto a SU-8TM mold at a mass ratio of 10:1. Curing in an oven at 70 ° C for 2 hours, the PDMS sheet with channels (~3 mm thick) was cut and peeled from the mold. The inlet and outlet ports were punched using a Harris Uni-Core biopsy punch (Electron Microscopy Sciences) 0.75 mm in diameter. After treatment with an oxygen plasma (PLASMA-PREEN II-862, Plasmatics Systems, Inc., United States), PDMS sheets were bonded with a PDMS coated glass substrate (75 mm x 25 mm x 1 mm). The sealed PDMS chip was then placed in an oven at 70 ° C for at least 12 hours to recover its hydrophobicity.

Then, the device was heated to 100 ° C on a hot plate, and a low melting point of In-Sn solder was filled into the electrode channel. A small piece of copper wire is inserted, electrically connected to the solder electrode, and further protected by the AB glue.

High-throughput sorting system

A PEEK tube (OD = 0.03 inches, ID = 0.012 inches; Cole-Parmer, USA) was used to attach the microfluidic device, a syringe mounted on the pump (LSP01-2A, Longer Pump, China) and a tube for cell collection. Cell-loaded tubing and syringes were treated with the hydrophilic agent "5% PF127" for 10 minutes and then washed in sterile deionized water for 1 minute. Mineral oil containing 2.5% (w/w) Span 80 surfactant is used to produce droplets. A high-voltage amplifier (PC 2000, Tianjin Dongwen High-Voltage Power Co., Ltd.) controlled by a digital I/O unit (DIO-1616LX-USB, CONTEC) connected to a computer is used to generate DEP trigger target droplet sorting.

The Raman microscope was performed on a custom LabRam HR micro-Raman device equipped with a Nd:YAG 532 nm laser emitter (Ventus, Laser Quantum Ltd., United Kingdom) as an excitation source to generate scattered photons for collecting Raman signals. Charge-Coupled Device (EMCCD) (Newton DU970N-BV), high-speed CCD camera for monitoring cell and droplet flow (Pike F-032, Allied Vision Technologies, China) and 60x water mirror (NA=1.0, Olympus, United Kingdom) Focus the laser beam on the sample. Measurements were performed using a 600 line/mm grating, and a 660 nm LED array was used as a source for monitoring the sorting process.

Integrate RADS devices including microfluidic devices, Raman systems and DEP systems with software designed to control electronic devices (EMCCD and high voltage amplifiers, etc.) and adjust system parameters (such as acquisition time and DEP duration) Together, in order to run automatically.

Sorting efficiency verification

Ethanol, isopropanol, Span 80, C2H3O2·Na, MgCl2·6H2O, CaCl2, FeSO4·7H2O, NaNO3, K2HPO4·3H2O, KH2PO4, NaCl, MgSO4·7H2O, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2 · Chemical reagents such as 6H2O and Na2MO4·2H2O were purchased from Sinopharm (Shanghai, China). Mineral oil, L-asparagine, yeast extract, propidium iodide (PI) PF127 and Na2EDTA·2H2O were purchased from Sigma-Aldrich (St. Louis, MO, USA). SU-8TM (GM 3025) was purchased from MicroChem (Massachusetts, USA). Polydimethylsiloxane (PDMS Sylgard) and curing agent (Sylgard 184) were purchased from Dow Corning (Midland MI USA). All reagents used in the experiments were of analytical grade. All solutions were prepared with deionized water, filtered through a 0.22 [mu]m microporous membrane prior to use or sterilized in an autoclave at 121 °C for 30 minutes.

Microalgae H. pluvialis (NIES-144) was purchased from the Microbial Culture Collection (NIES, Japan) and cultured in basal medium (1.197 g/L C2H3O2·Na, 0.357 g/L L-asparagine, 2 g/L yeast). Extract, 0.2g/L MgCl2·6H2O, 0.015g/L CaCl2, 0.01g/L FeCl3·6H2O and 20g/L agar) inoculated to liquid under continuous low light conditions (22°C, 20μmol photon m-2s-1) Base medium), shake manually once a day. To induce accumulation of astaxanthin (AXT), exponentially growing cells were resuspended in triplicate modified BBM medium (without nitrogen source and containing 10 mM NaAc) and exposed to 150 μmol photons m-2s for continuous irradiation. -1 under (generating AXT content gradient). Cells with various average AXT contents were collected, and 40 μm microporous membranes were filtered to remove debris and cell clusters, and then washed 3 times with deionized water at 3000 rpm for 3 minutes each. The cell density was measured using a cell counting plate and adjusted to about 8.02 x 106 cells/mL, and the final cell density after the clamp flow was -4.58 x 106 cells/mL). Finally, in a 2.5% Span 80 mineral oil, droplets of about 50 μm in diameter were packaged at a flow rate of 120 μL/h, with an average of 0.3 cells per droplet.

Since cell harvesting based on other sorting methods requires centrifugation or demulsification reagents such as Pico-BreakTM, all of these methods cause damage and are inefficient. We used an ultra-high porosity Parylene C membrane with a pore size of 12 μm to recover the sorted cells. In this way, the targeted cell isolation yield can be as high as 96%.

In order to verify the sorting efficiency, the cells were mixed at a specific ratio after 0 and 3 days of induction, respectively, and then subjected to Raman signal detection. As shown in Figures 5a and 5b, more than 60 cells were randomly selected and their intracellular astaxanthin content was verified, of which only one cell did not reach the preset sorting criteria. The proportion of astaxanthin-producing cells increased by an average of 98%. These results indicate that the Raman-activated droplet sorting accuracy and efficiency of the present invention are very high.

It is to be understood that various changes and modifications may be made by the skilled in the art in the form of the appended claims.

Claims (12)

1. A Raman activated droplet sorting system, characterized in that the sorting system comprises the following units in order from the first to the last in the flow direction:

   1) a liquid flow trapping unit for regulating the flow rate of the cell/particle suspension and the buffer, and concentrating the cells/particles to be sorted in the middle of the liquid flow channel to form a stable single cell/particle flow;

   2) a signal detecting unit for generating Raman scattered photons, performing Raman laser signal detection on the single cell/particle flow formed by the liquid flow trapping unit, collecting detection signals and transmitting the same to the sorting control unit;

   3) a droplet generating unit for introducing an oil phase into the single cell/particle stream detected by the signal detecting unit to form a water-in-oil type droplet;

   4) a sorting control unit, configured to analyze the detection signal from the signal detecting unit, and sort the droplets generated by the droplet generating unit according to the signal;

   The sorting system further includes a liquid flow driving unit and a liquid flow channel for generating a driving force required for liquid flow in the sorting system; the liquid flow channel is a liquid flow in the system Carrier and liquid inlet and outlet.
2. The Raman-activated droplet sorting system of claim 1 wherein said flow trapping unit comprises at least one flow channel for influx of cell/particle suspension and at least two for influx buffer The flow channel of the liquid.
3. The Raman-activated droplet sorting system according to claim 1, wherein said flow trapping unit is adapted to adjust a cell/particle suspension and a buffer flow rate to form a single cell suitable for detection by a signal detecting unit/ Particle flow.
4. A Raman-activated droplet sorting system according to claim 1, wherein said signal detecting unit comprises a microscope, a stage, a Raman laser source, a signal collecting element, and a high speed CCD.
5. The Raman-activated droplet sorting system according to claim 1, wherein the Raman laser signal comparison method used in the signal detecting unit is selected from the group consisting of one or more of the following: single peak contrast, Multi-peak comparison, full-spectral alignment.
6. The Raman-activated droplet sorting system according to claim 1, wherein said signal detecting unit comprises a dielectric trapping unit for dielectrically capturing cells/particles to extend Raman laser signal Collect time and increase the intensity of the acquired signal.
7. The Raman-activated droplet sorting system according to claim 1, wherein said droplet generating unit comprises at least one liquid flow path for flowing into a cell/particle suspension detected by the signal detecting unit, and At least one sidestream inlet for introducing an oil phase.
8. The Raman-activated droplet sorting system according to claim 1, wherein the water-in-oil droplets generated by the droplet generating unit have an average of no more than one single cell per droplet/ Particles.
9. The Raman-activated droplet sorting system according to claim 1, wherein the sorting control unit performs droplet sorting by any of the following methods: dielectrophoresis, ultrasonic, electric field, magnetic field, pressure extrusion Pressure or pressure sucking.
10. The Raman-activated droplet sorting system of claim 1 wherein said sorting system further comprises at least one droplet collecting unit for collecting and collecting the sorted droplets.
11. A Raman activated droplet sorting method comprising the following steps:

    1) Concentration of liquid flow: Converging the cells/particles to be sorted with the buffer to form a stable single cell/particle flow;

    2) Signal detection: Raman spectroscopy is performed on the single cell/particle stream one by one and the signal is collected;

    3) droplet formation: after the collection is completed, the side stream is introduced into the oil phase, and water-in-oil microdroplets containing single cells/particles are formed by shearing;

    4) Droplet sorting: obtaining the collected signal in the foregoing signal detecting step, performing a sorting operation on the corresponding encapsulated water-in-oil microdroplets according to the signal, and collecting the droplets of the desired portion.
12. The Raman-activated droplet sorting method according to claim 11, wherein in the droplet sorting step, there is a delay time between acquiring the acquisition signal and performing the sorting operation, and the delay time is calculated and determined by the following manner : Interval time = liquid flow length / liquid flow rate, wherein the liquid flow length refers to the actual distance through which the liquid detected from the signal detection point to the sorting operation point flows.

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