CN110687188A - Micro-fluidic chip mass spectrometry system for single cell analysis and application method thereof - Google Patents
Micro-fluidic chip mass spectrometry system for single cell analysis and application method thereof Download PDFInfo
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Abstract
A mass spectrum system of a micro-fluidic chip for single cell analysis and a using method thereof comprise a capillary tube a, a capillary tube b and a capillary tube c; the micro-flow control chip is provided with two inlets and one outlet, one end of the capillary tube a and one end of the capillary tube b are respectively connected with the outlet ends of the micro-injection pump a and the micro-injection pump b, and the other end of the capillary tube a and the other end of the capillary tube b are respectively connected with the inlet I and the inlet II of the micro-flow control chip; one end of the capillary tube c is connected with an outlet of the microfluidic chip, and the other end of the capillary tube c is connected with the inductively coupled plasma mass spectrometer through a commercial atomization system; a method for detecting single cells by a mass spectrometry system of a microfluidic chip for single cell analysis comprises the following steps: step 1, sample introduction is prepared; step 2, orderly arranging cells; step 3, forming single cell bundles; step 4, atomizing the single-cell liquid flow; step 5, quantitative analysis of single cells; and 6, repeating the measurement. Without being limited by specific fluid conditions, stable and efficient single cell arrays are formed under a wide range of flow rates.
Description
Technical Field
The invention belongs to the technical field of mass spectrometry detection equipment and analysis, and particularly relates to a microfluidic chip mass spectrometry system for single cell analysis and a using method thereof.
Background
In recent years, Inductively Coupled Plasma mass spectrometry (ICP-MS) has been used as a powerful trace element analysis technique, has the advantages of low detection limit, less interference, high sensitivity, wide analysis range and the like, and has been widely applied to the fields of biomedical science, food hygiene, chemical analysis and the like by combining with other techniques to obtain reliable and accurate qualitative and quantitative element analysis results. The microfluidic chip known as '10 inventions which greatly change the world' is developed rapidly as a new technology, has the advantages of less sample consumption, high function integration, precise scale and the like, and plays a great role in the field of cell research. The diversity and heterogeneity between homogeneous cells have important significance for researching the difference of chemical components in the cells, so that the analysis of elements in the single cells can provide important reference basis for research and development in the fields of biology, chemistry, medicine and the like. And distinguishing the diversity and heterogeneity among the cells of the same species has important significance for researching the difference of chemical components in the cells. This means that there is a large difference in the physiological response and stress expression of individual cells within the same population. To understand the heterogeneity of cells more deeply, it is of great importance for cell research. At present, single cell analysis is an important research field in recent years, and the inductively coupled plasma mass spectrometry system plays a key role in single cell detection due to its excellent trace element analysis capability.
With the development of science and technology, more and more metals play an increasingly important role in human life activities and participate in the development of life health. Nevertheless, the regulated expression of these trace metal elements in the life process is strictly controlled. Particularly, some toxic heavy metals are serious to human injuries. For example, organic mercury can break through the blood brain barrier and permanently damage the nervous system; inorganic arsenic can induce gene mutation to cause the functional structure abnormality of tissues in the organism; the toxic metal cadmium can cause pulmonary edema and kidney damage, and has the possibility of carcinogenesis, teratogenesis and mutagenesis. The research on the action mechanism of harmful metals in cells is particularly key to solving the toxic action of heavy metals, but the research on the interaction among the metals in single cells based on an inductively coupled plasma mass spectrometry system is rarely reported at present. Therefore, designing a high throughput single-cell metal analysis system is critical to elucidating the metal-on-single-cell impact.
At present, the single cell detection method based on the inductively coupled plasma mass spectrometry system mainly comprises the traditional direct sample injection of cell suspension, the droplet sample injection and the laser ablation sample injection.
Firstly, the traditional cell suspension sample injection comprises the sample injection by adopting a conventional atomizer and a total consumption atomizer, wherein the sample injection is generally to treat and dilute the incubated cells to the required concentration, and a statistical treatment method is utilized, so that a single cell is detected by an inductively coupled plasma mass spectrometry system within an integration time according to the sampling efficiency of instrument atomization, thereby realizing the direct detection of the cell. Although the method is convenient and rapid, due to the random distribution of cells in suspension and the random dispersion of atomization products, the probability of detecting two or more cells within the same integration time is obviously increased according to the calculation of a Poisson distribution formula, and a signal peak corresponding to a single cell cannot be determined, thus the original purpose of single cell detection is violated.
Secondly, the liquid drop type sample injection is the liquid drop encapsulation, the oil-water two-phase insolubilization is utilized to generate liquid drops, the cell density is adjusted, and the cells are encapsulated into the liquid drops. Then the single cell encapsulated in the liquid drop is introduced into a subsequent detector along with the liquid flow for single cell detection, and the method mainly comprises a jet printing type and a microfluidic liquid drop encapsulation type. The former relies on the action of pulse electricity to split a continuous flow phase into liquid drops (pico liter level) to realize single cell-liquid drop encapsulation with high precision, but has low detection efficiency and high cost, and is not suitable for wide application; the latter utilizes the special cross pipeline of micro-fluidic chip design, utilizes factors such as interfacial tension to realize the liquid drop encapsulation, and this method is economical, detection efficiency is high, but for the liquid drop chip reproducibility and pressure tolerance performance are not ideal and restrict its subsequent application.
And moreover, laser ablation sample introduction is based on the principle that laser micro-beams are focused on the surface of a single-cell sample to be ablated and gasified, and then the sample particles are uniformly, stably and efficiently conveyed into a subsequent detector through carrier gas to be analyzed, so that the method has the advantages of low analysis detection limit, high resolution, capability of completing space in-situ analysis and the like. But the standard substance is not easy to obtain, and special single cell sample loading equipment is needed, so that the rapid and high-flux single cell detection is limited.
Disclosure of Invention
Aiming at the problems in the existing single cell inductively coupled plasma mass spectrometry detection technology, the invention provides a micro-fluidic system for direct cell suspension sample injection and a using method thereof, which realize high-flux single cell separation sample injection based on inertia-assisted sequencing and realize element quantitative analysis without additional interfaces through ingenious design; the method is a method for detecting single cells by using a flow field effect to form a single cell beam in a sample introduction process and combining the single cell beam with inductively coupled plasma mass spectrometry; the method is suitable for inductively coupled plasma mass spectrometry of single cells or single subcellular particles; the main part of the system is a microfluidic chip prepared from Polydimethylsiloxane (PDMS) which is simple and easy to obtain, so that the single-cell sample injection efficiency is effectively ensured and the cost is saved. The single cell separation is realized from the outlet by injecting the cell suspension into the microfluidic chip from the inlet, and the single cell liquid flow is formed into single cell aerosol through an atomizing system and is introduced into an inductively coupled plasma mass spectrometer for detection. Under the optimized condition, the efficient and ordered arrangement of single cells and the subsequent high-throughput detection are ensured. Based on the inertia-assisted sequencing single-cell separation sampling system and the inductively coupled plasma mass spectrometer analysis system, stable and high-flux single-cell arrangement sampling in a large flow velocity range is realized, and the method can be used for element quantitative analysis at the single-cell level.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mass spectrum system of a micro-fluidic chip for single cell analysis comprises a micro-injection pump a, a micro-injection pump b, a micro-fluidic chip, a capillary tube a, a capillary tube b and a capillary tube c; the micro-flow control chip is provided with two inlets and an outlet, the two inlets are respectively an inlet I and an inlet II, one end of a capillary tube a is connected with the outlet end of a micro-injection pump a, the other end of the capillary tube a is connected with the inlet I of the micro-flow control chip, and the capillary tube a is used as a cell suspension liquid inlet channel; one end of the capillary tube b is connected with the outlet end of the micro-injection pump b, the other end of the capillary tube b is connected with an inlet II of the micro-fluidic chip, and the capillary tube b is used as a supplementary standard solution inlet channel; one end of the capillary tube c is connected with an outlet at the tail end of the converging channel in the microfluidic chip, the other end of the capillary tube c is connected with the inductively coupled plasma mass spectrometer through a commercial atomizing system, and the capillary tube c serves as a single-cell outlet channel.
The micro-flow control chip also comprises eight circles of spirally wound disk-shaped micron-sized single-cell separation channels, one circle of spirally wound micron-sized multifunctional channels and a micron-sized confluence channel, wherein the micron-sized single-cell separation channels and the micron-sized multifunctional channels are connected at outlets to form the micron-sized confluence channel, and micro obstacles are uniformly arranged in the micron-sized single-cell separation channels; the width of the micron-sized single cell channel is 100-500 mu m, the total length is 8.9-44.5 cm, the length of the micro-barrier in the micron-sized single cell channel is 50-250 mu m, the width is 50-250 mu m, and the total number is 52-210; the width of the micron-sized multifunctional channel is 200-1000 mu m, and the length of the micron-sized multifunctional channel is 1.0-5.0 cm; the width of the micron-sized converging channel is 200-500 mu m; the overall height is 50-100 mu m; the area of the micro-fluidic chip is 0.3cm2～4.0cm2。
A method for using a mass spectrum system of a microfluidic chip for single cell analysis to detect single cells adopts the mass spectrum system of the microfluidic chip for single cell analysis, and comprises the following steps:
step 1, sample injection preparation: providing a cell suspension liquid containing specific elements in cells by a micro-injection pump a, and connecting the cell suspension liquid to an inlet I of the microfluidic chip through a capillary a; a micro-injection pump b provides a supplementary standard solution for quantitative element analysis, and the supplementary standard solution is connected to a microfluidic chip inlet II through a capillary tube b;
step 4, single cell liquid flow atomization: the standard solution carrying the single cell beam and quantitative elemental analysis is converged at the atomizer outlet of the commercial atomization system and atomized by the carrier gas argon entering from the carrier gas inlet of the commercial atomization system;
step 5, quantitative analysis of single cells: the atomized product enters an inductively coupled plasma mass spectrometer along with carrier gas argon and is detected in a time resolution mode; capturing and recording the response of the transient signal of the element to be detected along with time, obtaining an effective signal according to an iterative algorithm, and obtaining the signal intensity-frequency distribution condition through the effective signal and Gaussian fitting; finally, quantifying the element to be detected in the single cell according to the effective signal value and the Gaussian fitting result;
step 6, repeating the determination: and (3) cleaning the mass spectrum system of the microfluidic chip for single cell analysis, repeating the steps 1-5, and carrying out sample injection detection again.
In the step 1, the preparation method of the cell suspension containing the specific element in the cell comprises the following steps:
(1) culturing cells in a first culture medium in a cell culture well plate to a logarithmic growth phase, removing the first culture medium and washing;
(2) changing a second culture medium containing a metal solution to be detected with a specific concentration into a cell culture pore plate for culture, and incubating cells;
(3) digesting the incubated cells from the cell culture well plate, performing centrifugal washing, and resuspending the centrifuged product with a phosphate buffer solution to prepare a cell suspension, wherein the phosphate particle concentration is 0.01mol/L PO4 3-，pH＝7.4；
(4) Cells in the cell suspension were counted using a hemocytometer and diluted with phosphate buffer solution to a cell number density of 103～107cells/mL。
In the step 1, elements in the supplemented standard solution are determined according to the relative atomic mass of the element to be detected, namely, the difference between the relative atomic mass of the elements to be detected and the relative atomic mass of the elements to be detected is +/-10, and the elements to be detected and the relative atomic mass of the elements to be detected are used for internal standard method quantification; the supplemented standard solution is then prepared for use according to a specified concentration.
In the step 1, the sample injection flow rate of the cell suspension is 20-1200 mu L/min, and the flow rate of the supplementary standard solution is 20-1200 mu L/min;
in the step 4, the carrier gas flow is 0.5-1.3L/min.
In the step 4, the purity of the carrier gas argon is 99.999 percent; the power of the inductively coupled plasma mass spectrometer ranges from 1250W to 1570W, and the integration time ranges from 0.1 ms to 10 ms.
Compared with the prior art, the inertial-assisted sorting single cell separation sampling system and the use method thereof in single cell detection have the advantages that:
1. compared with the traditional cell suspension sample introduction mode, the method is not limited by specific fluid conditions, stable and efficient single cell arrangement is formed under a wide range of flow rate, and more ideal single cell quantitative analysis results can be provided;
2. compared with a liquid drop generating system, the single cell detection system can avoid organic phase interference, does not need redundant cross pipeline design and greatly increases the single cell detection efficiency;
3. in the method, the trace injection pump b provides a supplementary standard solution to be introduced into the inlet II, so that the work of making a standard curve every time can be saved, a single sheath flow liquid interface of the traditional semi-quantitative detection is not needed, and the element determination is conveniently and quickly realized;
4. in the method, a micro-injection pump b provides a supplementary standard solution to be introduced into an inlet II, and the conditions of an atomization system can be matched by changing the fluid conditions and the concentration of a standard substance, so that flexible and controllable element analysis is realized;
5. in the method, the micro-injection pump b provides a supplementary standard solution to be introduced into the inlet II and converged with the single cell liquid flow, so that the single cell is effectively promoted to stably move along with the liquid flow, and the detection stability is improved.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip of the mass spectrometry system of the present invention;
FIG. 2 is a schematic diagram of a mass spectrometry system of a microfluidic chip for single cell analysis according to the present invention;
the device comprises a micro-fluidic chip 1, a micro-fluidic chip 2, capillaries a and 3, capillaries b and 4, inlets I and 5, inlets II and 6, a micron-sized single-cell separation channel 7, a micron-sized multifunctional channel 8, a micro barrier 9, a micron-sized confluence channel 10, an outlet 11, capillaries c and 12, micro injection pumps a and 13, micro injection pumps b and 14, a commercial atomization system 15 and an inductively coupled plasma mass spectrometer.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings and examples.
As shown in fig. 1 and fig. 2, a microfluidic chip mass spectrometry system for single cell analysis comprises a micro-syringe pump a12, a micro-syringe pump b13, a microfluidic chip 1, a capillary a2, a capillary b3, and a capillary c 11; the micro-flow control chip is provided with two inlets and an outlet 10, the two inlets are respectively an inlet I4 and an inlet II 5, one end of a capillary tube a2 is connected with the outlet end of a micro-injection pump a12, the other end of the capillary tube a2 is connected with the inlet I4 of the micro-flow control chip 1, and the capillary tube a2 is used as a cell suspension liquid inlet channel; one end of the capillary tube b3 is connected with the outlet end of the micro-injection pump b13, the other end of the capillary tube b3 is connected with the inlet II 5 of the micro-fluidic chip 1, and the capillary tube b3 is used as a supplementary standard solution inlet channel; one end of the capillary tube c11 is connected with the outlet 10 at the tail end of the confluence channel in the microfluidic chip 1, the other end of the capillary tube c11 is connected with the inductively coupled plasma mass spectrometer 15 through the commercial atomization system 14, the capillary tube c11 is used as a single cell outlet channel, the model of the commercial atomization system 14 is G3266-80005, the model of the inductively coupled plasma mass spectrometer 15 is 8900, the outer diameters of the capillary tube a2, the capillary tube b3 and the capillary tube c11 are all 1.6mm, and the inner diameters are both 160 mu m.
The micro-flow control chip also comprises a spiral channel, the spiral channel consists of eight circles of spirally wound disc-shaped micron-sized single-cell separation channels 6, one circle of spirally wound micron-sized multifunctional channels 7 and a micron-sized confluence channel 9, the micron-sized single-cell separation channels 6 and the micron-sized multifunctional channels 7 are connected at the outlets to form the micron-sized confluence channel 9, and micro obstacles 8 are uniformly arranged in the micron-sized single-cell separation channels 6; the width of the micron-sized single-cell separation channel 6 is 300 mu m, and the total length is 26.7 cm; the length of the micro-barriers 8 in the micron-sized single-cell channel is 150 micrometers, the width of the micro-barriers is 150 micrometers, and the number of the micro-barriers is 104; the width of the micron-sized multifunctional channel 7 is 300 mu m, the length of the micron-sized multifunctional channel is 3cm, and the width of the micron-sized confluence channel 9 is 600 mu m; the overall height is 50-100 mu m; the area of the micro-fluidic chip 1 is 1.44cm2。
Example 1
A method for using a mass spectrum system of a microfluidic chip for single cell analysis to detect single cells adopts the mass spectrum system of the microfluidic chip for single cell analysis, and comprises the following steps:
step 1, sample injection preparation: cd content was supplied from a micro syringe pump a122+The flow rate of the cell suspension of (1) was 200. mu.L/min, and the cell density was 106cells/mL, connected to microfluidic chip inlet I4 via capillary a2, will contain Cd2+The cell suspension is introduced into a micron-sized single-cell separation channel 6;
cell contains Cd2+The method for preparing the cell suspension comprises the following steps:
(1) culturing cells in a first culture medium in a cell culture well plate to a logarithmic growth phase, removing the first culture medium and washing;
(2) in the cell culture well plate, a certain Cd content is changed2+Culturing in fresh culture medium for a certain time, and incubating the cells, wherein Cd is2+The concentration of (2) is 100 mug/L;
(3) digesting the incubated cells from a cell culture well plate, performing centrifugal washing, and resuspending the centrifuged product with a phosphate buffer solution to prepare a cell suspension, wherein the concentration of phosphate particles is 0.01mol/L, and the pH value is 7.4;
(4) cells in the cell suspension were counted using a hemocytometer and diluted to the desired cell number concentration of 10 using phosphate buffer solution6cells/mL；
A micro-injection pump b13 provides a supplementary standard solution containing indium element, the flow rate is 200 mu L/min, the concentration is 100 mu g/L, the supplementary standard solution is connected to a micro-fluidic chip inlet II 5 through a capillary tube b3, and the supplementary standard solution containing indium element is led into the micron-sized multifunctional channel 7;
step 4, single cell liquid flow atomization: enters a commercial atomization system 14 and is atomized by carrier gas argon with the purity of 99.999 percent and the flow rate of 1.09L/min;
step 5, quantitative analysis of single cells: the formed atomized product is driven by carrier gas argon to enter an inductively coupled plasma mass spectrometer 15, wherein the power of the inductively coupled plasma is 1350W, and the integration time is 1 ms; detecting in a time-resolved mode; by the single-cell separation-inductively coupled plasma mass spectrometer combined analysis system, the transient signal response of cadmium and indium in a single cell along with time can be obtained, an effective signal is obtained according to an iterative algorithm, the signal intensity-frequency distribution condition is obtained through the effective signal, and the frequency distribution condition is subjected to Gaussian fitting to obtain the quantitative analysis of the cadmium in the single cell;
step 6, repeating the determination: and (3) cleaning the mass spectrum system of the microfluidic chip for single cell analysis, repeating the step 1 to the step 5, and carrying out sample injection detection again.
Example 2
A method for using a mass spectrum system of a microfluidic chip for single cell analysis to detect single cells adopts the mass spectrum system of the microfluidic chip for single cell analysis, and comprises the following steps:
step 1, sample injection preparation: a cell suspension of cells incubated in the gold-containing nanoparticle medium was supplied by a micro-syringe pump a12 at a flow rate of 200. mu.L/min and a cell density of 106cells/mL, connected to microfluidic chip inlet I4 via capillary a2, will contain Cd2+The cell suspension is introduced into a micron-sized single-cell separation channel 6;
the preparation method of the cell suspension containing gold nanoparticles in cells comprises the following steps:
(1) culturing cells in a first culture medium in a cell culture well plate to a logarithmic growth phase, removing the first culture medium and washing;
(2) in a cell culture pore plate, a fresh culture medium containing a nanoparticle solution is added for culturing for a certain time, wherein the diameter of gold nanoparticles is 12nm, the concentration of Au atoms is 1 mu mol/L, and cells are incubated;
(3) digesting the incubated cells from a cell culture well plate, performing centrifugal washing, and resuspending the centrifuged product with a phosphate buffer solution to prepare a cell suspension, wherein the concentration of phosphate particles is 0.01mol/L, and the pH value is 7.4;
(4) cells in the cell suspension were counted using a hemocytometer and diluted to the desired cell number concentration of 10 using phosphate buffer solution6cells/mL。
A platinum-containing supplementary standard solution is provided by a micro-injection pump b13, the flow rate is 200 mu L/min, the concentration is 100 mu g/L, the micro-injection pump is connected to a micro-fluidic chip inlet II 5 through a capillary tube b3, and the platinum-containing supplementary standard solution is introduced into the micro-scale multifunctional channel 7;
step 4, single cell liquid flow atomization: enters a commercial atomization system 14 and is atomized by carrier gas argon with the purity of 99.999 percent and the flow rate of 1.09L/min;
step 5, quantitative analysis of single cells: the formed atomized product is driven by carrier gas argon to enter an inductively coupled plasma mass spectrometer 15, wherein the power of the inductively coupled plasma is 1370W, and the integration time is 1 ms; detecting in a time-resolved mode; by the single-cell separation-inductively coupled plasma mass spectrometer combined analysis system, the transient signal response of gold and platinum in a single cell along with time can be obtained, effective signals are obtained according to an iterative algorithm, signal intensity-frequency distribution conditions are obtained through the effective signals, and Gaussian fitting is performed on the frequency distribution conditions to obtain quantitative analysis of the gold and platinum in the single cell. Obtaining the quality and quantity of gold nanoparticles in single cells according to the physicochemical property of gold, and obtaining the distribution profile of the gold nanoparticles in the cell subset along with time;
step 6, repeating the determination: and (3) cleaning the mass spectrum system of the microfluidic chip for single cell analysis, repeating the step 1 to the step 5, and carrying out sample injection detection again.
Example 3
A method for using a mass spectrum system of a microfluidic chip for single cell analysis to detect single cells adopts the mass spectrum system of the microfluidic chip for single cell analysis, and comprises the following steps:
step 1, sample injection preparation: cd content was supplied from a micro syringe pump a122+And Cu2+The flow rate of the cell suspension of (1) was 200. mu.L/min, and the cell density was 106cells/mL；
Cell contains Cd2+And Cu2+The cell suspension is prepared by culturing ① cells in a first culture medium to logarithmic phase in a cell culture well plate, removing the first culture medium, washing, and changing ② the cell culture well plate to a cell culture well plate containing a certain amount of Cd2+And Cu2+Culturing in fresh culture medium for a certain time, and incubating the cells, wherein Cd is2+Concentration of (2) is 1000. mu.g/L, Cu 2+③, washing the incubated cells by centrifugation, resuspending the centrifuged product in a phosphate buffer solution to give a cell suspension having a phosphate particle concentration of 0.01mol/L and a pH of 7.4, counting the cells in the cell suspension using a hemocytometer, and diluting the cell suspension with a phosphate buffer solution to a desired cell count concentration (10. mu.g/L) (④)6cells/mL)。
A micro-injection pump b13 provides a supplementary standard solution containing indium element and germanium element, the flow rate is 200 mu L/min, the concentration is 100 mu g/L, the supplementary standard solution is connected to a micro-fluidic chip inlet II 5 through a capillary b3, and the supplementary standard solution containing indium element and germanium element is led into the micron-sized multifunctional channel 7;
step 4, single cell liquid flow atomization: enters a commercial atomization system 14 and is atomized by carrier gas argon with the purity of 99.999 percent and the flow rate of 1.09L/min;
step 5, quantitative analysis of single cells: the formed atomized product is driven by carrier gas to enter an inductively coupled plasma mass spectrometer 15, wherein the power of the inductively coupled plasma is 1350W, and the integration time is 1 ms; detecting in a time-resolved mode; by the single-cell separation-inductively coupled plasma mass spectrometer combined analysis system, the transient signals of cadmium element, indium element, copper element and germanium element in a single cell can be obtained to respond with time, effective signals are obtained according to an iterative algorithm, signal intensity-frequency distribution conditions are obtained through the effective signals, Gaussian fitting is carried out on the frequency distribution conditions, and quantitative analysis of the cadmium element and the copper element in the single cell is obtained.
Step 6, repeating the determination: and (3) cleaning the mass spectrum system of the microfluidic chip for single cell analysis, repeating the step 1 to the step 5, and carrying out sample injection detection again.
Claims (8)
1. A mass spectrum system of a micro-fluidic chip for single cell analysis is characterized by comprising a micro-injection pump a, a micro-injection pump b, a micro-fluidic chip, a capillary tube a, a capillary tube b and a capillary tube c; the micro-flow control chip is provided with two inlets and an outlet, the two inlets are respectively an inlet I and an inlet II, one end of a capillary tube a is connected with the outlet end of a micro-injection pump a, the other end of the capillary tube a is connected with the inlet I of the micro-flow control chip, and the capillary tube a is used as a cell suspension liquid inlet channel; one end of the capillary tube b is connected with the outlet end of the micro-injection pump b, the other end of the capillary tube b is connected with an inlet II of the micro-fluidic chip, and the capillary tube b is used as a supplementary standard solution inlet channel; one end of the capillary tube c is connected with an outlet at the tail end of the converging channel in the microfluidic chip, the other end of the capillary tube c is connected with the inductively coupled plasma mass spectrometer through a commercial atomizing system, and the capillary tube c serves as a single-cell outlet channel.
2. The microfluidic chip mass spectrometry system for single cell analysis of claim 1, wherein: the micro-flow control chip also comprises eight circles of spirally wound disk-shaped micron-sized single-cell separation channels, one circle of spirally wound micron-sized multifunctional channels and a micron-sized confluence channel, wherein the micron-sized single-cell separation channels and the micron-sized multifunctional channels are connected at outlets to form the micron-sized confluence channel, and micro obstacles are uniformly arranged in the micron-sized single-cell separation channels; the width of the micron-sized single cell channel is 100-500 mu m, the total length is 8.9-44.5 cm, the length of the micro-barrier in the micron-sized single cell channel is 50-250 mu m, the width is 50-250 mu m, and the total number is 52-210; the width of the micron-sized multifunctional channel is 200-1000 mu m, and the length of the micron-sized multifunctional channel is 1.0-5.0 cm; the width of the micron-sized converging channel is 200-500 mu m; the overall height is 50-100 mu m; the area of the micro-fluidic chip is 0.3cm2～4.0cm2。
3. A method for using the microfluidic chip mass spectrometry system for single cell analysis to detect single cells, which uses the microfluidic chip mass spectrometry system for single cell analysis of claim 1, comprising the following steps:
step 1, sample injection preparation: providing a cell suspension liquid containing specific elements in cells by a micro-injection pump a, and connecting the cell suspension liquid to an inlet I of the microfluidic chip through a capillary a; a micro-injection pump b provides a supplementary standard solution for quantitative element analysis, and the supplementary standard solution is connected to a microfluidic chip inlet II through a capillary tube b;
step 2, ordered arrangement of cells: according to the principle of particle inertial focusing, a cell suspension is introduced into a microfluidic chip by a capillary tube a through a micro-injection pump a, under the action of adding a micro-barrier, liquid flow is continuously accelerated by secondary flow in a micron-sized single-cell separation channel, acting force generated by the secondary flow is continuously applied to cells, and disordered cells in the cell suspension are arranged into well-ordered single-cell bundles;
step 3, forming single cell bundles: in the micron-sized confluence channel, cell suspension liquid introduced into an inlet I by a micro-injection pump a through a capillary tube a and supplementary standard liquid introduced into an inlet II by a micro-injection pump b through a capillary tube b are combined to form confluence of standard solution containing single-cell liquid flow and quantitative element analysis, and the confluence is introduced into a commercial atomization system through an outlet and a capillary tube c;
step 4, single cell liquid flow atomization: the standard solution carrying the single cell beam and quantitative elemental analysis is converged at the atomizer outlet of the commercial atomization system and atomized by the carrier gas argon entering from the carrier gas inlet of the commercial atomization system;
step 5, quantitative analysis of single cells: the atomized product enters an inductively coupled plasma mass spectrometer along with carrier gas argon and is detected in a time resolution mode; capturing and recording the response of the transient signal of the element to be detected along with time, obtaining an effective signal according to an iterative algorithm, and obtaining the signal intensity-frequency distribution condition through the effective signal and Gaussian fitting; finally, quantifying the element to be detected in the single cell according to the effective signal value and the Gaussian fitting result;
step 6, repeating the determination: and (3) cleaning the mass spectrum system of the microfluidic chip for single cell analysis, repeating the steps 1-5, and carrying out sample injection detection again.
4. The use of the microfluidic chip mass spectrometry system for single cell analysis to detect single cells as claimed in claim 3, wherein: in the step 1, the preparation method of the cell suspension containing the specific element in the cell comprises the following steps:
(1) culturing cells in a first culture medium in a cell culture well plate to a logarithmic growth phase, removing the first culture medium and washing;
(2) changing a second culture medium containing a metal solution to be detected with a specific concentration into a cell culture pore plate for culture, and incubating cells;
(3) digesting the incubated cells from the cell culture well plate, performing centrifugal washing, and resuspending the centrifuged product with phosphate buffer solution to prepare cellsSuspension with phosphate particle concentration of 0.01mol/L PO4 3-，pH＝7.4；
(4) Cells in the cell suspension were counted using a hemocytometer and diluted with phosphate buffer solution to a cell number density of 103～107cells/mL。
5. The use of the microfluidic chip mass spectrometry system for single cell analysis to detect single cells as claimed in claim 3, wherein: in the step 1, the elements in the supplemented standard solution are determined according to the relative atomic mass of the element to be detected, namely, the difference between the relative atomic mass of the element to be detected and the relative atomic mass of the element to be detected is +/-10.
6. The use of the microfluidic chip mass spectrometry system for single cell analysis to detect single cells as claimed in claim 3, wherein: in the step 1, the sample injection flow rate of the cell suspension is 20-1200 mu L/min, and the flow rate of the supplementary standard solution is 20-1200 mu L/min.
7. The use of the microfluidic chip mass spectrometry system for single cell analysis to detect single cells as claimed in claim 3, wherein: in the step 4, the carrier gas flow is 0.5-1.3L/min.
8. The use of the microfluidic chip mass spectrometry system for single cell analysis to detect single cells as claimed in claim 3, wherein: in the step 4, the purity of the carrier gas argon is 99.999 percent; the power of the inductively coupled plasma mass spectrometer ranges from 1250W to 1570W, and the integration time ranges from 0.1 ms to 10 ms.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111266140A (en) * | 2020-03-09 | 2020-06-12 | 厦门大学 | Oil-free sorting-direct injection-ICPMS single cell analysis system |
| CN112067533A (en) * | 2020-09-26 | 2020-12-11 | 宁波大学 | Single cell mass spectrometry system and method |
| CN112378987A (en) * | 2021-01-18 | 2021-02-19 | 宁波华仪宁创智能科技有限公司 | Mass spectrometry system with cleaning function and method |
| CN112646701A (en) * | 2020-12-10 | 2021-04-13 | 中国科学院深圳先进技术研究院 | One-step single cell separation and distribution system |
| CN115090341A (en) * | 2022-05-13 | 2022-09-23 | 东北大学 | Micro-fluidic chip system for single-cell two-dimensional phenotype analysis and detection method |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040252584A1 (en) * | 2003-06-11 | 2004-12-16 | Agency For Science, Technology And Research | Micromixer apparatus and methods of using same |
| US20130015345A1 (en) * | 2011-07-14 | 2013-01-17 | The George Washinton University | Plume Collimation for Laser Ablation Electrospray Ionization Mass Spectrometry |
| CN105944775A (en) * | 2016-06-22 | 2016-09-21 | 苏州汶颢芯片科技有限公司 | Micro-fluidic chip for single-cell isolation |
| WO2016179291A1 (en) * | 2015-05-05 | 2016-11-10 | The Regents Of The University Of Michigan | Microfluidic photoionization detector |
| CN106128932A (en) * | 2015-05-05 | 2016-11-16 | 塞莫费雪科学（不来梅）有限公司 | For the method and apparatus that ion is expelled in electrostatic ion trap |
| CN107607608A (en) * | 2017-09-08 | 2018-01-19 | 武汉大学 | A kind of Single cell analysis method |
| CN207096166U (en) * | 2017-09-08 | 2018-03-13 | 武汉大学 | A kind of system for Single cell analysis |
| CN207877746U (en) * | 2018-01-02 | 2018-09-18 | 清华大学深圳研究生院 | A kind of micro-fluidic chip for unicellular capture and culture |
| US20180369807A1 (en) * | 2017-06-23 | 2018-12-27 | Pouya Rezai | Microfluidic centrifuge device and method for performing solution exchange and separation |
| CN109475868A (en) * | 2016-07-21 | 2019-03-15 | 新加坡科技研究局 | The equipment and its manufacturing method that outer wall for high volume fraction grain micro-filtration focuses |
| CN109596838A (en) * | 2018-12-17 | 2019-04-09 | 中国科学院电子学研究所 | The unicellular interior multiple protein simultaneous quantitative detection system of one kind and method |
-
2019
- 2019-09-29 CN CN201910932260.5A patent/CN110687188B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040252584A1 (en) * | 2003-06-11 | 2004-12-16 | Agency For Science, Technology And Research | Micromixer apparatus and methods of using same |
| US20130015345A1 (en) * | 2011-07-14 | 2013-01-17 | The George Washinton University | Plume Collimation for Laser Ablation Electrospray Ionization Mass Spectrometry |
| WO2016179291A1 (en) * | 2015-05-05 | 2016-11-10 | The Regents Of The University Of Michigan | Microfluidic photoionization detector |
| CN106128932A (en) * | 2015-05-05 | 2016-11-16 | 塞莫费雪科学（不来梅）有限公司 | For the method and apparatus that ion is expelled in electrostatic ion trap |
| CN107735682A (en) * | 2015-05-05 | 2018-02-23 | 密执安州立大学董事会 | Microfluid photoionization detector |
| CN105944775A (en) * | 2016-06-22 | 2016-09-21 | 苏州汶颢芯片科技有限公司 | Micro-fluidic chip for single-cell isolation |
| CN109475868A (en) * | 2016-07-21 | 2019-03-15 | 新加坡科技研究局 | The equipment and its manufacturing method that outer wall for high volume fraction grain micro-filtration focuses |
| US20180369807A1 (en) * | 2017-06-23 | 2018-12-27 | Pouya Rezai | Microfluidic centrifuge device and method for performing solution exchange and separation |
| CN107607608A (en) * | 2017-09-08 | 2018-01-19 | 武汉大学 | A kind of Single cell analysis method |
| CN207096166U (en) * | 2017-09-08 | 2018-03-13 | 武汉大学 | A kind of system for Single cell analysis |
| CN207877746U (en) * | 2018-01-02 | 2018-09-18 | 清华大学深圳研究生院 | A kind of micro-fluidic chip for unicellular capture and culture |
| CN109596838A (en) * | 2018-12-17 | 2019-04-09 | 中国科学院电子学研究所 | The unicellular interior multiple protein simultaneous quantitative detection system of one kind and method |
Non-Patent Citations (2)
| Title |
|---|
| LUOQUAN LI ET AL.: "Dean Flow Assisted Single Cell and Bead Encapsulation for High Performance Single Cell Expression Profiling", 《ACS SENSORS》 * |
| QIUSHI HUANG ET AL.: "Single-cell assay o n microfl uidic devices", 《ANALYST》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111266140A (en) * | 2020-03-09 | 2020-06-12 | 厦门大学 | Oil-free sorting-direct injection-ICPMS single cell analysis system |
| CN112067533A (en) * | 2020-09-26 | 2020-12-11 | 宁波大学 | Single cell mass spectrometry system and method |
| CN112067533B (en) * | 2020-09-26 | 2024-01-19 | 宁波大学 | Single cell mass spectrometry system and method |
| CN112646701A (en) * | 2020-12-10 | 2021-04-13 | 中国科学院深圳先进技术研究院 | One-step single cell separation and distribution system |
| CN112646701B (en) * | 2020-12-10 | 2023-12-26 | 中国科学院深圳先进技术研究院 | Single-step single-cell separation and distribution system |
| CN112378987A (en) * | 2021-01-18 | 2021-02-19 | 宁波华仪宁创智能科技有限公司 | Mass spectrometry system with cleaning function and method |
| CN112378987B (en) * | 2021-01-18 | 2021-05-07 | 宁波华仪宁创智能科技有限公司 | Mass spectrometry system with cleaning function and method |
| CN115090341A (en) * | 2022-05-13 | 2022-09-23 | 东北大学 | Micro-fluidic chip system for single-cell two-dimensional phenotype analysis and detection method |
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