Cell sorting chip, device and method based on dielectrophoresis induced deterministic lateral displacement

Abstract

Provided are a cell sorting chip, device, and method based on dielectrophoresis induced deterministic lateral displacement, the chip including a microfluidic channel (4), the microfluidic channel comprising a sample inlet (4.1), a straight channel, and two sample outlets; three groups of electrode array pairs are integrated at a bottom of the straight channel, the three electrode array pairs respectively being a focusing electrode group (1), a sorting electrode group (2), and a separating electrode group (3); the focusing electrode group is used for cell focusing and signal detection; the sorting electrode group is used for single cell sorting; and the separating electrode group is used for single cell separation. High-speed, precise, and lossless target cell sorting can thus be implemented.

Claims

1. A cell sorting microfluidic chip based on dielectrophoresis induced deterministic lateral displacement, comprising a micro-channel, wherein the micro-channel comprises a sample inlet, a straight channel and two sample outlets; three groups of electrode array pairs are integrated at a bottom of the straight channel, the three groups of electrode array pairs respectively being a focusing electrode group, a sorting electrode group and a separation electrode group; the focusing electrode group is used for cell focusing and signal detection; the sorting electrode group is used for single cell sorting; and the separation electrode group is used for single cell separation; wherein: the focusing electrode group comprises one or more electrode pairs, the separation electrode group comprises one or more electrode pairs, and the sorting electrode group comprises at least one electrode; each electrode in the one or more electrode pairs of the focusing electrode group has two first arms connected at a first tip point, a first centerline of the focusing electrode group passes the first tip point of each electrode in the one or more electrode pairs, an included angle between one of the two first arms and the first centerline has a value a, an included angle between the other one of the two first arms and the first centerline has a value b, an included angle between the two first arms of each electrode has a value a+b; the two first arms of different electrodes in the one or more electrode pairs of the focusing electrode group are parallel to each other, relatively, and the electrodes in the one or more electrode pairs of the focusing electrode group are distributed in a spaced manner; each electrode in the one or more electrode pairs of the separation electrode group has two second arms connected at a second tip point, a second centerline of the separation electrode group passes the second tip point of each electrode in the one or more electrode pairs, an included angle between one of the two second arms and the second centerline has a value a, an included angle between the other one of the two second arms and the second centerline has a value b, an included angle between the two second arms of each electrode has a value a+b; each electrode in the one or more electrode pairs of the separation electrode group and each electrode in the one or more electrode pairs of the focusing electrode group have a same structure, while the included angle between the two first arms of each electrode in the one or more electrode pairs of the focusing electrode group and the included angle between the two second arms of each electrode in the one or more electrode pairs of the separation electrode group are of different directions; and the at least one electrode of the sorting electrode group has a third arm, and an included angle between the third arm and the first centerline of the focusing electrode group has a value a.

2. The microfluidic chip according to claim 1, wherein the value a and b are in the range of 15-45 degrees, respectively.

3. The microfluidic chip according to claim 2, wherein the value a and b are the same.

4. The microfluidic chip according to claim 1, wherein the first centerline of the focusing electrode group and the second centerline of the separating electrode group are not in a same straight line.

5. The microfluidic chip according to claim 1, wherein the focusing electrode group, the sorting electrode group and the separation electrode group are made of ITO, carbon, graphene, or metal.

6. The microfluidic chip according to claim 1, wherein the first centerline of the focusing electrode group and the second centerline of the separating electrode group are parallel to each other with a distance c therebetween.

7. The microfluidic chip according to claim 6, wherein the distance c is in the range of 35-50 microns.

8. The microfluidic chip according to claim 1, wherein the focusing electrode group, the sorting electrode group and the separation electrode group are arranged in sequence; each electrode in the one or more electrode pairs of the focusing electrode group is of a > shape, and each electrode in the one or more electrode pairs of the separation electrode group is of a < shape.

9. The microfluidic chip according to claim 8, wherein the first centerline of the focusing electrode group and the second centerline of the separating electrode group are not in a same straight line.

10. The microfluidic chip according to claim 8, wherein the first centerline of the focusing electrode group and the second centerline of the separating electrode group are parallel to each other with a distance c therebetween.

11. A cell sorting device based on dielectrophoresis induced deterministic lateral displacement, comprising: the microfluidic chip of claim 1; a function generator, for generating sinusoidal alternating current with different frequencies and voltages; a computer, for controlling operation of a program and signal acquisition and collection of optical instruments, controlling output voltage, frequency, duty ratio of the function generator, and controlling on/off of a relay; the relay, for controlling on/off of a circuit; a fluid driving device, serving as a driving force source for driving a fluid into the micro-channel.

12. The cell sorting device based on dielectrophoresis induced deterministic lateral displacement according to claim 11, wherein the fluid driving device comprises an injection pump or a gravity driving device.

13. A cell sorting method based on dielectrophoresis induced deterministic lateral displacement, which adopts the cell sorting device of claim 11, comprising the following steps: 1) Cell sample injection: the fluid driving device is used to allow the fluid containing cells to enter the straight channel through the sample inlet of the microfluidic chip; 2) cell focusing: the focusing electrode group is connected with an output end of the function generator through a wire, and the cells in the fluid are focused to a signal monitoring point at the included angle between the two first arms by periodically applying high-frequency voltage to the focusing electrode group; 3) signal detection: an optical signal acquisition point is aligned with the signal monitoring point to perform optical signal detection, and a detected signal is processed by the program to judge whether it is a target cell or not; 4) Cell sorting: when it is a target cell, the relay is triggered to communicate with the sorting electrode group to output high-frequency voltage, so that the target cell deviates from the first centerline of the focusing electrode group along the sorting electrode group, when the program judges that it is a non-target cell, the program controls the relay not be turned on, to keep the non-target cell moving along the first centerline of the focusing electrode group without deviation; 5) Cell separation: high-frequency voltage is periodically applied to the separation electrode group to allow the non-target cells and the target cells in the fluid flow to different sample outlets.

14. A cell sorting method based on dielectrophoresis induced deterministic lateral displacement, which adopts microfluidic chip of claim 1, comprising the following steps: 1) Cell sample injection: a fluid driving device is used to allow a fluid containing cells to enter the straight channel through the sample inlet of the microfluidic chip; 2) cell focusing: the focusing electrode group is connected with an output end of a function generator through a wire, and the cells in the fluid are focused to a signal monitoring point at the included angle between the two first arms by periodically applying high-frequency voltage to the focusing electrode group; 3) signal detection: an optical signal acquisition point is aligned with the signal monitoring point to perform optical signal detection, and a detected signal is processed by a computer program to judge whether it is a target cell or not; 4) Cell sorting: when it is a target cell, a relay is triggered to communicate with the sorting electrode group to output high-frequency voltage, so that the target cell deviates from the first centerline of the focusing electrode group along the sorting electrode group; when the program judges that it is a non-target cell, the program controls the relay not be turned on, to keep the non-target cell moving along the first centerline of the focusing electrode group without deviation; 5) Cell separation: high-frequency voltage is periodically applied to the separation electrode group to allow the non-target cells and the target cells in the fluid flow to different sample outlets.

15. The cell sorting method based on dielectrophoresis induced deterministic lateral displacement according to claim 14, wherein the cells are yeast cells, Escherichia coli cells, or Hela cells.

16. The cell sorting method based on dielectrophoresis induced deterministic lateral displacement according to claim 14, wherein a flow rate of the fluid is 0.01-50 microliters per minute.

17. The cell sorting method based on dielectrophoresis induced deterministic lateral displacement according to claim 14, wherein the optical signal detection in step 3) is Raman signal detection or fluorescence signal detection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of a cell sorting chip based on dielectrophoresis induced deterministic lateral displacement. Reference numerals: 1: focusing electrode group; 2: sorting electrode group; 3: separation electrode group; 4: micro-channel; 4.1: sample inlet; 4.2: collection port; 4.3: waste liquid port; 5: signal monitoring point.

(2) FIG. 2 is a schematic diagram of various parameters of electrodes, in which a and b represent electrode included angles, d represents a distance between the electrodes, D represents a width of the electrode, c represents a distance between centerlines of electrode pairs of the two groups, and L represents a width of the pair of electrodes.

(3) FIG. 3 is a schematic structural diagram of another electrode groups.

(4) FIG. 4 is a schematic diagram of a cell sorting device based on dielectrophoresis induced deterministic lateral displacement. Reference numerals: {circle around (1)}: microfluidic chip; {circle around (2)}: function generator; {circle around (3)}: computer; {circle around (4)}: relay; {circle around (5)}: injection pump.

(5) FIG. 5 is a schematic diagram of a cell sorting process based on dielectrophoresis induced deterministic lateral displacement.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

(6) The present application will be further explained in detail with reference to the attached drawings and embodiments below. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

(7) A structure of a microfluidic chip is as shown in FIG. 1. The microfluidic chip comprises a micro-channel 4, wherein the micro-channel comprises a sample inlet 4.1, a straight channel, a collection port 4.2 and a waste liquid port 4.3; three groups of electrode array pairs are integrated at a bottom of the micro-channel 4, the three groups of electrode array pairs respectively being a focusing electrode group 1, a sorting electrode group 2 and a separation electrode group 3; the focusing electrode group is used for cell focusing and signal detection; the sorting electrode group is used for single cell sorting; and the separation electrode group is used for single cell separation.

(8) FIG. 2 is a schematic diagram of various parameters of electrodes, in which a and b represent electrode angles, d represents a distance between the electrodes, D represents a width of the electrode, c represents a distance between centerlines of electrode pairs of the two groups, and L represents a width of the electrode pairs.

(9) FIG. 3 is a schematic structural diagram of another electrode group. Wherein, the sorting electrode group has only one electrode.

Example 1: Flow-Mode Raman-Activated Cell Sorting of Yeast Cells with High Oil Yield

(10) Experiment Preparation:

(11) One milliliter of cultured yeast cells is taken into a centrifuge tube and centrifuged for 5 minutes at 5000 rpm, a supernatant is removed, one milliliter of pure water is added for resuspension, and an obtained solution is centrifuged for 5 minutes at 5000 rpm; after washing for three times is performed, one milliliter of pure water is added for resuspension. According to the concentration of fungus, it is diluted by 1,000-10,000 times, a certain concentration of surfactant (such as PF127 with the final concentration of 0.5%) is added to prevent cells from adsorbing the electrode, and after mixing uniformly, the obtained solution is injected into a syringe.

(12) A Raman instrument is turned on and a laser is turned on so that the laser is aligned with a signal monitoring point 5.

(13) The structure of the adopted microfluidic chip is as shown in FIG. 3, and the parameters of the electrodes in the microfluidic chip are as follows: the width L of the electrode pair is 1 millimeter, the width D of the electrode is 25 microns, the distance d between the electrodes is 25 microns, the electrode angles a and b are both 30 degrees, and the distance c between the centerlines of the two groups of electrode pairs is 35 microns.

(14) A microfluidic chip {circle around (1)}, a function generator {circle around (2)}, a computer {circle around (3)}, a relay {circle around (4)} and an injection pump {circle around (5)} are connected as shown in FIG. 4, and a programmed program in the computer runs, so that an output channel 2 of the function generator controls an output channel 1, the channel 2 outputs a square wave with a voltage of 20 volts and a frequency of 5 Hz, and the channel 1 outputs a sine wave with a voltage of 16 volts and a frequency of 10 MHz. At the same time, the program controls the acquisition and analysis of a Raman signal, and when a target cell is detected, the program controls the conduction of the relay {circle around (4)}.

(15) In the Experiment:

(16) The above-mentioned treated fungus liquid is pumped into the micro-channel by the injection pump {circle around (5)}, and the yeast cells in the liquid gradually move to a tip of the electrode pair under the focusing action of the focusing electrode group, wherein a flow rate of the micro-channel is about 40 microliters per minute, the electrification parameter of the electrode is 16 volts, the frequency is 10 MHz, and the focusing trap efficiency reaches over 95%.

(17) When the yeast cells move to the signal monitoring point, the Raman spectrum of them will be collected, and the program will simply analyze the collected spectrum. When it is a target cell, the Raman spectrum reflects a characteristic peak of oil, so that the program judges that it is the target cell, and the program controls the relay to be activated to allow the sorting electrode group to be turned on, and with the durations of delay and loading being set in advance, the target cell moves downwards; when it is a non-target cell, the Raman spectrum will not show the characteristic peak of oil, so that the relay will not be activated, the sorting electrode group thus has no voltage loading, and the cell continues to move along the centerline.

(18) The function of the separation electrode group is to allow the cells at both ends of the centerline of the separation electrode group to deviate from the centerline further, so that the target cells are below the channel and the non-target cells are above the channel.

(19) Finally, yeast cells with high oil yield are collected at the collection port, and yeast cells with low oil yield or no oil yield are collected at the waste liquid port.

(20) End of Experiment:

(21) The microfluidic chip channel is rinsed with absolute ethanol twice, and then put in an oven for drying and recycling.

Example 2: Flow-Mode Raman-Activated Cell Detection of Yeast Cells with High Oil Yield

(22) Experiment Preparation:

(23) One milliliter of cultured yeast cells is taken into a centrifuge tube and centrifuged for 5 minutes at 5000 rpm, a supernatant is removed, one milliliter of pure water is added for resuspension, and an obtained solution is centrifuged for 5 minutes at 5000 rpm; after washing for three times is performed, one milliliter of pure water is added for resuspension. According to the concentration of fungus, it is diluted by 1,000-10,000 times, a certain concentration of surfactant (such as PF127 with the final concentration of 0.5%) is added to prevent cells from adsorbing the electrode, and after mixing uniformly, the obtained solution is injected into a syringe.

(24) A Raman instrument is turned on and a laser is turned on so that the laser is aligned with a signal monitoring point 5.

(25) The structure of the adopted microfluidic chip is as shown in FIG. 3, and the parameters of the electrodes in the microfluidic chip are as follows: the width L of the electrode pair is 1 millimeter, the width D of the electrode is 25 microns, the distance d between the electrodes is 25 microns, the electrode angles a and b are both 30 degrees, and the distance c between the centerlines of the two groups of electrode pairs is 35 microns.

(26) A microfluidic chip {circle around (1)}, a function generator {circle around (2)}, a computer {circle around (3)}, a relay {circle around (4)} and an injection pump {circle around (5)} are connected as shown in FIG. 4, and a programmed program in the computer runs, so that an output channel 2 of the function generator controls an output channel 1, the channel 2 outputs a square wave with a voltage of 20 volts and a frequency of 5 Hz, and the channel 1 outputs a sine wave with a voltage of 16 volts and a frequency of 10 MHz. At the same time, the program controls the acquisition and analysis of a Raman signal.

(27) The above-mentioned treated fungus liquid is pumped into the micro-channel by the injection pump {circle around (5)}, and the yeast cells in the liquid gradually move to a tip of the electrode pair under the focusing action of the focusing electrode group, wherein a flow rate of the micro-channel is about 40 microliters per minute, the electrification parameter of the electrode is 16 volts, the frequency is 10 MHz, and the focusing trap efficiency reaches over 95%.

(28) When yeast cells move to the signal monitoring point, the Raman spectrum of them will be collected, and the program will simply analyze the collected spectrum.

(29) Finally, yeast cells after being detected are collected at the collection port.

(30) End of Experiment:

(31) The microfluidic chip channel is rinsed with absolute ethanol twice, and then put in an oven for drying and recycling.

(32) By analyzing the collected Raman spectra later, relevant information such as the proportion of yeast cells with high oil yield can be obtained.

Example 3: Flow-Mode Sorting of Fluorescent Cells

(33) Experiment Preparation:

(34) One milliliter of cultured Escherichia coli capable of expressing fluorescent protein is taken into a centrifuge tube and centrifuged for 5 minutes at 5000 rpm, a supernatant is removed, one milliliter of pure water is added for resuspension, and an obtained solution is centrifuged for 5 minutes at 5000 rpm; after washing for three times is performed, one milliliter of pure water is added for resuspension. According to the concentration of fungus, it is diluted by 1,000-10,000 times, a certain concentration of surfactant (such as PF127 with the final concentration of 0.5%) is added to prevent cells from adsorbing the electrode, and after mixing uniformly, the obtained solution is injected into a syringe.

(35) A fluorescence imaging instrument is turned on and a laser is turned on so that the laser is aligned with a signal monitoring point 5.

(36) A structure of the adopted microfluidic chip is as shown in FIG. 3, wherein the width L of the electrode pair is 1 millimeter, the width D of the electrode is 25 microns, the distance d between the electrodes is 25 microns, the electrode angles a and b are both 15 degrees, and the distance c between the centerlines of the two groups of electrode pairs is 50 microns.

(37) A microfluidic chip {circle around (1)}, a function generator {circle around (2)}, a computer {circle around (3)}, a relay {circle around (4)} and an injection pump {circle around (5)} are connected as shown in FIG. 4, and a programmed program in the computer runs, so that an output channel 2 of the function generator controls an output channel 1, the channel 2 outputs a square wave with a voltage of 20 volts and a frequency of 0.2 Hz, and the channel 1 outputs a sine wave with a voltage of 16 volts and a frequency of 1 MHz. At the same time, the program controls the acquisition and analysis of a fluorescence signal, and when a target cell is detected, the program controls the relay {circle around (4)} to be turned on.

(38) In the Experiment:

(39) The above-mentioned treated bacteria liquid is pumped into the micro-channel by the injection pump {circle around (5)}, and the Escherichia coli cells in the liquid gradually move to a tip of the electrode pair under the focusing action of the focusing electrode group, wherein a flow rate of the micro-channel is about 10 microliters per minute, the electrification parameter of the electrode is 16 volts, the frequency is 1 MHz, and the focusing trap efficiency reaches over 90%.

(40) When Escherichia coli cells move to the signal monitoring point, the Escherichia coli containing fluorescent protein is excited by the laser to emit a fluorescent signal that is collected by the computer, the program controls the relay to be activated to allow the sorting electrode group to be turned on, and with the durations of delay and loading being set in advance, the target cell deviates downwards; when the Escherichia coli without the fluorescent protein passes through the monitoring point, the fluorescent signal will not be excited, so that the program controls the relay not to be activated, the sorting electrode group has no voltage loading, and the cell continues to move along the centerline.

(41) The function of the separation electrode group is to allow the cells at both ends of the centerline of the separation electrode group to deviate from the centerline further, so that the target cells are below the channel and the non-target cells are above the channel.

(42) Finally, Escherichia coli with fluorescence is collected at the collection port, and non-fluorescent Escherichia coli cells are collected at the waste liquid port.

(43) End of Experiment:

(44) The microfluidic chip channel is rinsed with absolute ethanol twice, and then put in an oven for drying and recycling.

(45) It will be appreciated that various alterations and modifications of the invention will become apparent to those skilled in the art after having read the above teachings of the invention, and that such equivalents are intended to fall within the scope of the invention as defined in the appended claims.