CN107607608A - A kind of Single cell analysis method - Google Patents

Abstract

The invention belongs to detection technique field, discloses a kind of Single cell analysis method, comprises the following steps：Syringe is driven by constant current syringe pump, cell sample and organic reagent are directed respectively into micro-fluidic chip；The cell sample and the organic reagent form microlayer model after entering the micro-fluidic chip；The microlayer model enters icp mses, and the icp mses carry out analysis detection using time-resolved mode to the microlayer model.The present invention solves carries out time resolution analysis using icp mses conventional soln nebulization sampling pattern to the element in cell in the prior art, whether each jump peak-to-peak signal obtained by can not determining is single celled signal, the problem of so as to cause testing result not accurate enough.This invention ensures that detection signal comes from unicellular, effectively analysis of the realization to unicellular middle constituent content.

Description

A single cell detection method

technical field

The invention relates to the technical field of detection, in particular to a single cell detection method.

Background technique

With the deepening of biochemical research, more and more studies have shown that cell heterogeneity exists widely and it will have different effects on the physiological process of cells. Taking cancer cells as an example, cell heterogeneity alters the phenotype and function of cancer cells through random genetic and epigenetic changes (clonal evolution) to alter cell tumorigenicity. Based on this, it is of great significance to develop simple and efficient methods for single-cell detection.

Element speciation analysis is generally achieved through the combination of efficient separation technology and highly sensitive element-specific detection technology. Among many element-specific detectors, inductively coupled plasma mass spectrometry (ICP-MS) has outstanding advantages, not only high sensitivity, low detection limit, wide linear range, but also can provide isotope-related information. Among them, the commercialized quadrupole ICP-MS has the cheapest cost and the highest popularity. Based on the time-resolved mode, the content determination of target elements in single cells can be realized. In the prior art, ICP-MS conventional solution nebulization sampling mode is usually used for time-resolved analysis of trace elements in cells. However, since the formation of aerosol is random, it is impossible to determine that each peak-hopping signal obtained is a single Cells are also signals for multiple cells. Therefore, although the probability of measuring multiple cells in time-resolved ICP-MS is low, it will still cause inaccuracy in experimental results.

Contents of the invention

The embodiment of the present application provides a single-cell detection method, which solves the problem that in the prior art, the time-resolved analysis of elements in cells using the conventional solution atomization sampling mode of an inductively coupled plasma mass spectrometer cannot be determined. Whether the peak-jumping signal is a signal of a single cell leads to the problem that the detection result is not accurate enough.

The embodiment of the present application provides a single cell detection method, comprising the following steps:

The syringe is driven by a constant flow syringe pump, and the cell samples and organic reagents are respectively introduced into the microfluidic chip;

The cell sample and the organic reagent enter the microfluidic chip to form micro-droplets;

The micro-droplets enter an inductively coupled plasma mass spectrometer, and the inductively coupled plasma-mass spectrometer uses a time-resolved mode to analyze and detect the micro-droplets.

Preferably, the constant flow syringe pump includes a first organic phase constant flow syringe pump, a cell sample constant flow syringe pump, and a second organic phase constant flow syringe pump; the syringe includes a first syringe, a second syringe, and a third syringe ;

The organic reagent is drawn into the first syringe and the third syringe, and the cell sample is drawn into the second syringe;

The first organic phase constant flow syringe pump drives the first syringe, the cell sample constant flow syringe pump drives the second syringe, and the second organic phase constant flow syringe pump drives the third syringe.

Preferably, the microfluidic chip is provided with a cross-focus channel, and the cross-focus channel includes a first microchannel, a second microchannel, a third microchannel, and a fourth microchannel; the first microchannel and The third microchannel forms the longitudinal channel of the cross-focus channel, and the second microchannel and the fourth microchannel form the transverse channel of the cross-focus channel;

One end of the first microchannel is provided with a first organic phase sampling inlet, one end of the second microchannel is provided with a cell sample sampling inlet, and one end of the third microchannel is provided with a second organic phase sampling inlet. Inlet, one end of the fourth microchannel is provided with an outlet;

The organic reagent in the first syringe is introduced into the first organic phase sampling inlet through a pump tube, the cell sample in the second syringe is introduced into the cell sample sampling inlet through a pump tube, and the third The organic reagent in the syringe is introduced into the second organic phase sampling inlet through the pump tube.

Preferably, the outlet is sealed and fixed with a quartz capillary by ethyl cyanoacrylate, the other end of the quartz capillary is fixed on the inductively coupled plasma mass spectrometer through a PEEK tube, and the formed micro-droplet passes through the The quartz capillary is introduced into the inductively coupled plasma mass spectrometer.

Preferably, the inductively coupled plasma mass spectrometer adopts a time-resolved mode to analyze and detect the micro-droplets, and the sampling interval is 1-5ms.

Preferably, the flow rate of the cell sample is 5-8 μL min ^{-1 , and the flow rate of the organic reagent is 12.5-20 μL min -1 .}

Preferably, the cell sample has a density of 5.0×10 ⁵ mL ^-1 .

Preferably, the organic reagent is hexanol.

One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

In the embodiment of this application, the injector is driven by a constant flow syringe pump, and the cell sample and organic reagent are respectively introduced into the microfluidic chip; the cell sample and the organic reagent enter the microfluidic chip to form micro-droplets; the micro-droplets enter the inductive coupling Plasma mass spectrometer, inductively coupled plasma mass spectrometer uses time-resolved mode to analyze and detect micro-droplets. In the invention, the microfluidic chip is used as a platform for generating and controlling droplets, and the droplets are used as single-cell carriers, which can effectively separate single cells. The invention combines a microfluidic chip with an inductively coupled plasma mass spectrometer to ensure that the detection signal comes from a single cell, and effectively realizes the analysis of the element content in the single cell.

Description of drawings

In order to illustrate the technical solution in this embodiment more clearly, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are an embodiment of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings without making creative efforts.

Fig. 1 is a flow chart of a single cell detection method provided by the embodiment of the present invention;

Fig. 2 is a schematic structural diagram of a device used in a single cell detection method provided in an embodiment of the present invention.

Among them, 10-first organic phase constant flow syringe pump, 11-first syringe, 20-cell sample constant flow syringe pump, 21-second syringe, 30-second organic phase constant flow syringe pump, 31-third syringe , 40-first organic phase sampling inlet, 50-cell sample sampling inlet, 60-second organic phase sampling inlet, 70-exit;

100-microfluidic chip, 200-inductively coupled plasma mass spectrometer.

detailed description

The embodiment of the present application provides a single-cell detection method, which solves the problem that in the prior art, the time-resolved analysis of elements in cells using the conventional solution atomization sampling mode of an inductively coupled plasma mass spectrometer cannot be determined. Whether the peak-jumping signal is a signal of a single cell leads to the problem that the detection result is not accurate enough.

The technical solution of the embodiment of the present application is to solve the above-mentioned technical problems, and the general idea is as follows:

A single cell detection method, comprising the following steps:

The syringe is driven by a constant flow syringe pump, and the cell samples and organic reagents are respectively introduced into the microfluidic chip;

The cell sample and the organic reagent enter the microfluidic chip to form micro-droplets;

The micro-droplets enter an inductively coupled plasma mass spectrometer, and the inductively coupled plasma-mass spectrometer uses a time-resolved mode to analyze and detect the micro-droplets.

In the invention, the microfluidic chip is used as a platform for generating and controlling droplets, and the droplets are used as single-cell carriers, which can effectively separate single cells. The invention combines a microfluidic chip with an inductively coupled plasma mass spectrometer to ensure that the detection signal comes from a single cell, and effectively realizes the analysis of the element content in the single cell.

In order to better understand the above-mentioned technical solution, the above-mentioned technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

This embodiment provides a single cell detection method, as shown in Figure 1, comprising the following steps:

Step 10: The syringe is driven by a constant flow syringe pump, and the cell sample and the organic reagent are respectively introduced into the microfluidic chip.

A schematic structural diagram of a device used in a single-cell detection method provided by an embodiment of the present invention is shown in FIG. 2 , including: a microfluidic chip 100 , an inductively coupled plasma mass spectrometer 200 , a constant-flow syringe pump, and a syringe.

The constant-current syringe pump includes a first organic phase constant-current syringe pump 10, a cell sample constant-current syringe pump 20, and a second organic-phase constant-current syringe pump 30; the syringe includes a first syringe 11, a second syringe 21, a second syringe Three syringes 31 .

The organic reagent is sucked into the first syringe 11 and the third syringe 31 , and the cell sample is sucked into the second syringe 21 .

The first organic-phase constant-current syringe pump 10 drives the first syringe 11, the cell sample constant-current syringe pump 20 drives the second syringe 21, and the second organic-phase constant-current syringe pump 30 drives the The third syringe 31 .

The microfluidic chip 100 is provided with a cross-focus channel, and the cross-focus channel includes a first microchannel, a second microchannel, a third microchannel, and a fourth microchannel; the first microchannel and the The third microchannel forms the longitudinal channel of the cross-focus channel, and the second microchannel and the fourth microchannel form the transverse channel of the cross-focus channel.

One end of the first microchannel is provided with a first organic phase sampling inlet 40, one end of the second microchannel is provided with a cell sample sampling inlet 50, and one end of the third microchannel is provided with a second organic phase. A sample inlet 60, and an outlet 70 is provided at one end of the fourth microchannel.

The organic reagent in the first syringe 11 is introduced into the first organic phase sampling inlet 40 through a pump tube, and the cell sample in the second syringe 21 is introduced into the cell sample sampling inlet 50 through a pump tube, The organic reagent in the third syringe 31 is introduced into the second organic phase sampling inlet 60 through a pump tube. The pump tubing is preferably polyethylene pump tubing.

The flow rate of the cell sample is 5-8 μL min ^-1 , and the flow rate of the organic reagent is 12.5-20 μL min ^-1 .

The cell sample has a density of 5.0×10 ⁵ mL ^-1 .

The organic reagent is hexanol. Preferred is 1% Span80 in hexanol.

Preferably, the width of the first microchannel, the second microchannel, and the third microchannel is 200 μm, and the width of the fourth microchannel is 150 μm; The heights of the second microchannel, the third microchannel and the fourth microchannel are 50 μm. The width of the intersection of the cross-focus channel is 75 μm. The total length of the longitudinal channel is

4mm, the total length of the transverse channel is 5mm.

Among them: the constant flow syringe pump is TS2-60 constant flow syringe pump (Baoding Lange Constant Flow Pump Co., Ltd., China), and the syringe is 1 mL (Shanghai Jinta Medical Equipment Co., Ltd., China). The inductively coupled plasma mass spectrometer was Xseries ICP-MS (Thermo, USA).

The processing method of the PDMS microfluidic chip: the silicon template is made by soft photolithography, using AZ-50XT photoresist. Among them, the production method of the fluid channel: mix the A component (prepolymer) and B component (curing agent) of GE RTV 615 (PDMS) at a mass ratio of 10:1, stir evenly, and place it in a vacuum desiccator using an oil pump Vacuum for 10 minutes, take it out and wait until the bubbles disappear. The PDMS sol was poured on the silicon template, cured at 75°C for 3 hours, the cured PDMS was peeled off from the silicon template, and holes were punched at the inlet and outlet of the channel. The production method of PDMS chips: place the processed PDMS and glass slides in a plasma cleaner, treat them with oxygen plasma for 1 minute, quickly bond them after taking them out, and then cure them at 75°C for 10 minutes to completely age the bonded surfaces.

Step 20: Micro-droplets are formed after the cell sample and organic reagent enter the microfluidic chip.

Using the microfluidic chip 100 as a platform for generating and controlling droplets, and using droplets as single-cell carriers, single cells can be effectively separated.

Step 30: the micro-droplets enter the ICP-MS, and the ICP-MS uses a time-resolved mode to analyze and detect the micro-droplets.

The outlet 70 is sealed and fixed with a quartz capillary by ethyl cyanoacrylate, the other end of the quartz capillary is fixed on the inductively coupled plasma mass spectrometer 200 through a PEEK tube, and the formed micro-droplet passes through the A quartz capillary is introduced into the inductively coupled plasma mass spectrometer 200 .

Preferably, the quartz capillary has a diameter of 75 μm and a length of 3 cm.

The cell sample is 5.0×10 ⁵ mL ^-1 HepG2 cell suspension.

The inductively coupled plasma mass spectrometer 200 uses a time-resolved mode to analyze and detect the micro-droplets, and the sampling interval is 1-5 ms.

The zinc element content in a single HepG2 cell was detected under optimal conditions, and its average signal intensity was 17429CPS. ZnO nanoparticles of 222nm were used in the single-cell detection device corresponding to the method provided by the invention to detect zinc in a single nanoparticle. Quantitative analysis of element content, the average signal intensity is 20653CPS. Quantitative analysis of zinc element content in a single cell by ZnO nanoparticles shows that the number of zinc atoms contained in a single cell is 2.0×10 ⁸ (equivalent to 21.7fg). In order to verify the accuracy of the obtained results, the zinc content in the cell samples was measured using a conventional acid digestion inductively coupled plasma mass spectrometer, and the results showed that the average number of zinc atoms in HepG2 cells was 2.3×10 ⁸ (equivalent to at 25.0fg), consistent with the results detected by the method of the present invention.

A single-cell detection method provided in an embodiment of the present invention includes at least the following technical effects:

In the embodiment of this application, the injector is driven by a constant flow syringe pump, and the cell sample and organic reagent are respectively introduced into the microfluidic chip; the cell sample and the organic reagent enter the microfluidic chip to form micro-droplets; the micro-droplets enter the inductive coupling Plasma mass spectrometer, inductively coupled plasma mass spectrometer uses time-resolved mode to analyze and detect micro-droplets. In the invention, the microfluidic chip is used as a platform for generating and controlling droplets, and the droplets are used as single-cell carriers, which can effectively separate single cells. The invention combines a microfluidic chip with an inductively coupled plasma mass spectrometer to ensure that the detection signal comes from a single cell, and effectively realizes the analysis of the element content in the single cell.

Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (8)

1. A kind of 1. Single cell analysis method, it is characterised in that comprise the following steps：

   Syringe is driven by constant current syringe pump, cell sample and organic reagent are directed respectively into micro-fluidic chip；

   The cell sample and the organic reagent form microlayer model after entering the micro-fluidic chip；

   The microlayer model enters icp mses, and the icp mses are using the time point Distinguish that pattern carries out analysis detection to the microlayer model.
2. 2. Single cell analysis method according to claim 1, it is characterised in that it is organic that the constant current syringe pump includes first Phase constant current syringe pump, cell sample constant current syringe pump, Second Organic Phase constant current syringe pump；The syringe includes the first injection Device, the second syringe, the 3rd syringe；

   Being drawn in first syringe and the 3rd syringe has the organic reagent, and being drawn in second syringe has The cell sample；

   The first organic phase constant current syringe pump drives first syringe, described in the cell sample constant current syringe pump driving Second syringe, the Second Organic Phase constant current syringe pump drive the 3rd syringe.
3. 3. Single cell analysis method according to claim 2, it is characterised in that the micro-fluidic chip focuses on provided with cross Type passage, the cross focus type passage include the first microchannel, the second microchannel, the 3rd microchannel, the 4th microchannel；It is described First microchannel and the 3rd microchannel form the vertical passage of the cross focus type passage, second microchannel and institute State the interconnection that the 4th microchannel forms the cross focus type passage；

   One end of first microchannel is provided with the first organic phase sample introduction entrance, and one end of second microchannel is provided with cell sample Product sample introduction entrance, one end of the 3rd microchannel are provided with Second Organic Phase sample introduction entrance, and one end of the 4th microchannel is set There is outlet；

   Organic reagent in first syringe imports the first organic phase sample introduction entrance, second injection by pump line The cell sample in device imports the cell sample sample introduction entrance by pump line, the organic reagent in the 3rd syringe The Second Organic Phase sample introduction entrance is imported by pump line.
4. 4. Single cell analysis method according to claim 3, it is characterised in that the exit passes through alpha-cyanoacrylate second Ester sealing is fixed with quartz capillary, the other end of the quartz capillary by PEEK pipes be fixed on described inductive etc. from On daughter mass spectrograph, the microlayer model of formation imports the inductivity coupled plasma mass spectrometry by the quartz capillary Instrument.
5. 5. Single cell analysis method according to claim 1, it is characterised in that the icp mses Analysis detection, sampling interval 1-5ms are carried out to the microlayer model using time-resolved mode.
6. 6. Single cell analysis method according to claim 1, it is characterised in that the flow velocity of the cell sample is 5-8 μ L min^-1, the flow velocity of the organic reagent is 12.5-20 μ L min^-1。
7. 7. Single cell analysis method according to claim 1, it is characterised in that the density of the cell sample be 5.0 × 10⁵mL^-1。
8. 8. Single cell analysis method according to claim 1, it is characterised in that the organic reagent is hexanol.