CN110468026B

CN110468026B - Microfluidic chip for optical fiber photodynamic cell manipulation

Info

Publication number: CN110468026B
Application number: CN201910845092.6A
Authority: CN
Inventors: 苑立波; 杜佳豪
Original assignee: Guilin University of Electronic Technology
Current assignee: Guilin University of Electronic Technology
Priority date: 2019-09-07
Filing date: 2019-09-07
Publication date: 2022-10-25
Anticipated expiration: 2039-09-07
Also published as: CN110468026A

Abstract

The invention provides a microfluidic chip for optical fiber photodynamic cell manipulation. The method is characterized in that: the device comprises a base 1, a sheath liquid pool 2, a sheath liquid channel 3, a cell channel 4, a cell injection pool 5, a main channel 6, a photodynamic control area 7, a cell and waste liquid guide-out channel 8, a Tesla microvalve 9, waste liquid pools 10 and 11, cell storage pools 12 and 13, an optical fiber channel 14, a cover plate 15, a connecting hole 16 and a capillary steel pipe 17. The periphery of the photodynamic manipulation region 7 is provided with rough light reflection eliminating walls for inhibiting reflected light and eliminating the influence of the reflected light on the optical fiber manipulation cell. The invention can be used for the manipulation and sorting of cells in various modes by the optical fiber, and can be widely used in the fields of cell manipulation, sorting, analysis and the like.

Description

Microfluidic chip for optical fiber photodynamic cell manipulation

(I) technical field

The invention relates to a microfluidic chip for optical fiber photodynamic cell manipulation, which can be used for cell manipulation, sorting and analysis and belongs to the technical field of microfluidic chips.

(II) background of the invention

In 1975, a miniaturized gas chromatography apparatus was developed (Terry S C, jerman J H, angell J B.A gas chromatographic air and purifier fabricated on silicon wafer [ J ]. IEEE Transactions on Electron Devices,1979,26 (12): 1880-1886.), which laid the foundation for the miniaturization of instruments. The Concept of a miniature Total Analysis system was first proposed in the 90 s of the 20 th century by Manz et al (Manz A, graber N, widmer H M. Miniatured Total Chemical-Analysis Systems-A Novel Concept for Chemical Sensing [ J ]. Sensors and actors B Chemical,1990,1 (1-6): 244-248.).

The micro total analysis system is also called a lab-on-a-chip, also called a micro fluidic chip, and its main features are integration and miniaturization. The basic operation units of a chemical or biological laboratory are integrated on a micron-scale chip, and controllable fluid penetrates through the whole system through a microfluidic channel network, so that sample preparation, reaction, separation, detection and the like in the processes of biological, chemical and medical analysis are automatically completed.

Cells are the basic structural and functional units of an organism. It is known that all organisms except viruses are composed of cells, but vital activities of viruses must be expressed in cells. Therefore, single cell analysis plays an important role in the mechanistic interpretation of life processes within cells.

In recent years, cellular studies have been advanced to the molecular level, which can help us understand that subtle differences lead to biological phenomena, and can also help us understand diseases caused by subtle changes in cells. By studying cells on the molecular level, we can clarify the relationship and interaction between chemical components in cells, and further discover the rules of basic biological activities such as growth, differentiation, heredity, variation, etc. of organisms.

Microfluidic chips are currently an important platform for cell analysis. The micro-fluidic chip can flexibly combine different operation unit technologies and has the characteristics of integration and miniaturization.

Numerous researchers have conducted extensive studies on microfluidic cell manipulation. For example, in 2016, researchers such as Asano-Peng provided a single-cell sorting device based on a microfluidic chip, with application number of CN201620318365.3, by controlling platinum electrodes corresponding to respective cell sorting channels and whether electroosmosis flows or not through a channel sorting element, the current cells to be sorted are sorted to a required channel, and the device adopts electroosmosis driving samples without additional equipment such as an external pump, thereby realizing manipulation and sorting of the cells. In 2017, researchers such as metallurgy and the like provide a microfluidic chip detection technology for exfoliated urinary tumor cells of urothelial cancer, the application number is CN201710054628.3, a cell sorter is formed by three columnar bulges which are integrally arranged in an arc shape, gaps exist among the columnar bulges, an arc opening is used as a liquid flow inlet, the gaps on two sides of the middle columnar bulge are used as liquid flow outlets, and the two liquid flow outlets are symmetrically distributed, so that the separation and capture of various cells in urine are realized. In the same year, researchers such as Dong Hua and the like propose a microfluidic cell sorting chip and a method for coupling dielectrophoresis and spatial separation, wherein the application number is CN201711166610.9, an alternating current electric field is applied to electrodes of the sorting chip, different cells generate different dielectric power in the alternating current electric field due to the difference of dielectric properties of the cells, and thus, the cells generate offsets with different degrees in a main channel; meanwhile, the flowing cells are also subjected to lateral migration force generated by the contraction and expansion structure of the main channel, and the coupling of the two effects causes different types of cells to flow out from different outlets, so that the cells are manipulated and sorted. In 2018, chen Di et al propose a cell bidirectional dielectrophoresis single-cell control micro-fluidic chip with application number CN201811205655.7, which comprises an inlet unit, a single-cell dielectrophoresis control unit and an outlet unit which are connected in sequence. The chip realizes cell control by using the dielectrophoresis principle and aiming at the phenomenon that cells show positive and negative dielectrophoresis under different frequencies. The single cell control process is that when the cell flows through the dielectrophoresis single cell control area, the cell is captured in the micro-trap by utilizing the forward dielectrophoresis of the cell; then washing the cells which are not captured in the micro-trap by using a cell buffer solution; the electrode corresponding to the micro-trap where the target cell to be removed is located is controlled independently; individual cells in the microwell are removed from the microwell using negative dielectrophoresis of the cells. Finally, the buffer solution is used for transferring the removed cells to a cell recovery outlet for recovery, thereby realizing the manipulation of the cells.

Although the micro-fluidic chip designed based on the electroosmotic flow principle has the advantages of simple structure, easy carrying and the like, the internal structure of cells is easy to damage by a high-voltage electric field, and even cell lysis membrane inactivation is caused in severe cases, so that false negative results are caused. In addition, the generation of electroosmotic flow requires the fabrication of electrodes, increasing the complexity of the chip. The microfluidic chip for realizing cell sorting according to the physical size difference gets rid of the dependence of the conventional method on the subjective experience of pathologists, is non-invasive in the whole process, but has a complex structure, and is particularly difficult to process columnar bulges in arc-shaped arrangement. The method for sorting the cells by the dielectrophoresis method has the advantages that the cells which are not electrified per se but can be polarized in different degrees are moved laterally in the nonuniform electric field, the method is accurate in operation, cannot damage the object to be detected, is easy to integrate with other equipment, and the like, but the method is greatly influenced by the external electric field and is easy to generate errors. In addition, most of the microfluidic chips designed based on the dielectrophoresis method also need to integrate electrodes in the microfluidic chips, so that the processing difficulty is greatly increased.

The invention provides a microfluidic chip for optical fiber photodynamic cell manipulation, aiming at the defects of the background technology. A photodynamic control area is arranged between the tail part of the main channel of the microfluidic chip and the cell and waste liquid guide-out channel and is used for carrying out various controls on the cells by the optical fiber; the photodynamic manipulation area is provided with a light reflection eliminating wall for absorbing reflected light; and, tesla micro valves are integrated in the cell and waste liquid leading-out channel for inhibiting backflow. The microfluidic chip can be used for cell counting, raman spectroscopy for detecting cells, cell rotation and other manipulations. The microfluidic chip for optical fiber photodynamic cell manipulation provided by the invention can be used for manipulating and sorting cells in various modes by optical fibers, has the advantages of high sensitivity on cell manipulation, almost no damage to cell activity, simple chip structure, easiness in processing and the like, and is high in sorting efficiency when various cells are sorted.

Disclosure of the invention

The invention aims to provide a microfluidic chip for optical fiber photodynamic cell manipulation, which has the advantages of high manipulation sensitivity, almost no damage to cell activity, high sorting efficiency and easiness in chip processing.

The purpose of the invention is realized as follows:

a microfluidic chip for fiber optic photodynamic cell manipulation is characterized in that: the device comprises a base 1, a sheath liquid pool 2, a sheath liquid channel 3, a cell channel 4, a cell injection pool 5, a main channel 6, a photodynamic control area 7, a cell and waste liquid guide-out channel 8, a Tesla microvalve 9, waste

liquid pools

10 and 11,

cell storage pools

12 and 13, an optical fiber channel 14, a cover plate 15, a connecting hole 16 and a capillary steel pipe 17. Wherein, cell fluid is injected from the cell injection pool 5, sheath fluid is injected from the sheath fluid pool 2, and the cell fluid forms microflow containing cells above the main channel 6. The microflow containing cells flows into the photodynamic manipulation zone 7, and the optical fibers on the optical fiber channel 14 can manipulate the cells in the photodynamic manipulation zone 7. Finally, the manipulated cells can be arbitrarily selected and flowed into a designated cell sorting channel.

Preferably, the periphery of the photodynamic manipulation region 7 is provided with diffuse reflection eliminating walls for reducing the influence of light reflection on the photodynamic-manipulated cells.

The diffuse reflection eliminating wall is a rough surface processed. When light enters the rough surface, the surface reflects the light in all directions, so that although the incident rays are parallel to each other, the reflected rays are randomly reflected in different directions due to the fact that the normal directions of all points are not consistent, and the influence of the reflection of the light on photodynamic cell manipulation is reduced.

Tesla micro-valves 9 are provided between the cell and waste liquid discharge channel 8 and the

waste liquid reservoirs

10 and 11 and the

cell storage reservoirs

12 and 13 for suppressing backflow.

The typical tesla microvalve has a T-junction angle of 90 ° and a diverging angle of 45 °. The main performance index of the Tesla micro-valve is the one-way conduction characteristic thereof. It mainly comprises: first, in the reverse flow, there is sufficient resistance to flow; secondly, while ensuring the reverse blocking effect, the blocking effect on the forward flow is reduced as much as possible so that the forward flow performance is as good as possible.

A method for manufacturing a microfluidic chip for optical fiber photodynamic cell manipulation comprises the following steps:

according to the manufacturing of the microfluidic chip base 1 based on optical control, two base materials A and B with the same length and width and different thicknesses are taken, and the two base materials are polished smoothly. After the level gauge detects, a thick substrate A is placed in a micro-processing system, and a sheath fluid channel 3, a cell channel 4, a main channel 6, a photodynamic control area 7, a cell and waste fluid guide channel 8, a Tesla micro valve 9 and an optical fiber channel 14 are processed by the processing system.

Preferably, the width of each microfluidic channel is W μm, for example 100 μm to 250 μm; the depth is H, for example, 50 μm to 125 μm.

Then, the sheath liquid reservoir 2, the cell injection reservoir 5, the

waste liquid reservoirs

10 and 11, and the

cell storage reservoirs

12 and 13 are processed by a microfabrication system.

Preferably, the radius of each waste liquid pool and cell storage pool is R μm,200 μm to 300. Mu.m.

For convenient observation and manipulation, the light manipulation region 7 is circular and has a diameter R _d For example 2mm.

The microfluidic chip cover plate 15 based on light manipulation is manufactured by the following steps: placing the thin substrate B on a workbench of a micro-processing system, and processing a connecting hole 16;

preferably, the radius of the connection hole 16 is equal to the radius of the cell well, and is R μm, for example, 100 μm to 150 μm.

Preferably, the substrate is PMMA and quartz.

The capillary steel pipe 17 is inserted into the coupling hole 16 and fixed with glue.

The contact surface of the base 1 and the cover plate 15 is coated with a thin layer of glue, and the thin layer of glue is tightly attached to the contact surface by the enhanced pressure.

The invention has at least the following obvious advantages:

(1) A microfluidic chip for fiber optic photodynamic cell manipulation is provided. Compared with other proposed cell sorting microfluidic chips, the microfluidic chip provided by the invention has the advantages of almost no damage to cell activity, easiness in processing and the like.

(2) The optical fiber control function is integrated in the microfluidic chip, and the microfluidic chip has the advantages of high integration level, flexible operation and the like.

(3) The optical fiber cell sorter can be used for manipulating and sorting cells in various modes through the optical fiber, can simultaneously sort various cells, and is high in sorting efficiency.

(IV) description of the drawings

FIG. 1 is a top view of a base of a microfluidic chip for fiber optic photodynamic cell manipulation. The device consists of a base 1, a sheath liquid pool 2, a sheath liquid channel 3, a cell channel 4, a cell injection pool 5, a main channel 6, a photodynamic control area 7, a cell and waste liquid guide-out channel 8, a Tesla microvalve 9, waste liquid pools 10 and 11, cell storage pools 12 and 13 and an optical fiber channel 14.

FIG. 2 is a perspective view of a cover plate of a microfluidic chip for fiber optic photodynamic cell manipulation. The device consists of a cover plate 15, a connecting hole 16 and a capillary steel pipe 17.

FIG. 3 is a perspective view of a base of a microfluidic chip for fiber optic photodynamic cell manipulation. The periphery of the photodynamic manipulation region 7 is provided with diffuse reflection eliminating walls 18 for suppressing reflection of light.

FIG. 4 is a schematic plan view of a Tesla valve in a microfluidic chip for fiber optic photodynamic cell manipulation.

FIG. 5 is a cell counting device based on a microfluidic chip for fiber optic photodynamic cell manipulation. The device consists of a display 201, a CCD202, an objective 203, a coaxial double waveguide fiber 204, a coaxial double waveguide fiber connector 205, a capture light source 206, a three-terminal circulator 207, an excitation light source 208, a photomultiplier 209, an oscilloscope 210, a signal acquisition card 211, a processor 212, an objective table 213 and a microfluidic chip 214 for fiber photodynamic cell manipulation.

FIG. 6 is a schematic diagram of the optical path of the capture and excitation light and the reflected light of the coaxial double waveguide fiber. 601 is the cladding of the coaxial double-waveguide fiber, 602 is the annular core of the coaxial double-waveguide fiber, 603 is the intermediate core of the coaxial double-waveguide fiber, and 604 is the trapped cell.

Fig. 7 is a raman sorting device based on a microfluidic chip for fiber optic photodynamic cell manipulation. The device comprises a display 301, a CCD302, an objective lens 303, a coaxial double waveguide fiber 304, a coaxial double waveguide fiber connector 305, a capture light source 306, a three-terminal circulator 307, an excitation light source 308, a Raman spectrometer 309, a stage 310 and a microfluidic chip 311.

FIG. 8 is a schematic diagram of light manipulation based on a microfluidic chip for fiber optic photodynamic cell manipulation. 601 is cladding of coaxial double-waveguide fiber, 602 is annular core of coaxial double-waveguide fiber, 603 is intermediate core of coaxial double-waveguide fiber, and 604 is cell.

(V) detailed description of the preferred embodiments

The invention is further illustrated below with reference to specific examples.

Example 1:

A microfluidic chip 214 for fiber optic photodynamic cell manipulation is placed on stage 213. An objective lens 203, a CCD202 and a CCD202 are arranged above the microfluidic chip and connected with a display 201 for detecting the light manipulation condition in the microfluidic chip in real time.

A coaxial dual waveguide fiber 204 is placed in a fiber channel 14 in a microfluidic chip 214 for fiber optic photodynamic cell manipulation. The annular core of the coaxial double waveguide fiber 204 is connected to a trapping light source 206 by a coaxial double-wave optical fiber connector 205. The middle core of the coaxial dual waveguide fiber 204 is connected to the # 2 port of the three-port circulator 207 through a coaxial dual-wave optical fiber connector 205.

The end face of the coaxial double waveguide fiber 204 needs to be ground to a certain angle. The annular core is used to capture cells from the cell stream and the intermediate core transmits an excitation light source for cell counting.

In the photodynamic manipulation zone 7, the light from the capture light source 206 is passed through the annular core of the coaxial twin waveguide fiber 204 to capture cells in the single-cell stream.

An optical signal emitted by the excitation light source 208 enters the 1# port of the three-terminal circulator, is output from the 2# port and enters the middle core of the coaxial double-waveguide fiber, an optical signal reflected by a cell enters the three-terminal circulator 207 through the 2# port of the three-terminal circulator 207, a signal output from the 3# port of the circulator 207 enters the photomultiplier 209, and the photomultiplier 209 converts the output optical signal into an electrical signal and amplifies the signal. The photomultiplier 209 also splits the electrical signal into two signals, one of which is sent to the oscilloscope 210 for waveform acquisition, the other is sent to the signal acquisition card 211, and the acquired data is sent to the processor 212 for processing, thus completing cell counting.

In the microfluidic chip, because the fluid flows in a laminar flow, the captured cells can be moved to a specified laminar flow and released by moving the slight moving stage, so that the function of cell sorting is realized.

The device of example 1 performs both cell counting and cell sorting functions.

Example 2:

A microfluidic chip 311 for fiber optic photodynamic cell manipulation is placed on the stage 310. An objective lens 303, a CCD302 and a CCD302 are arranged above the microfluidic chip and connected with a display 301 for detecting the light manipulation condition in the microfluidic chip in real time.

A coaxial double waveguide fiber 304 is arranged in a fiber channel 14 in a microfluidic chip 311 for fiber optic photodynamic cell manipulation. The annular core of the coaxial double waveguide fiber 304 is connected to a trapping light source 306 through a coaxial double-wave optical fiber connector 305. The middle core of the coaxial double waveguide fiber 304 is connected to the # 2 port of the three-terminal circulator 307 by a coaxial double-wave optical fiber connector 305.

The end face of the coaxial double waveguide fiber 304 needs to be ground to a certain angle. The annular core is used to capture cells from the cell stream and the intermediate core transmits an excitation light source for exciting the raman spectrum of the cells.

In the photodynamic manipulation zone 7, the light from the capture light source 306 is passed through the annular core of the coaxial twin waveguide fiber 304 to capture cells in a single cell stream.

The optical signal emitted by the excitation light source 308 enters the 1# port of the three-terminal circulator, the optical signal is output from the 2# port and enters the middle core of the coaxial double-waveguide fiber, the optical signal reflected by the cell enters the three-terminal circulator 307 through the 2# port of the three-terminal circulator 307, and the signal output from the 3# port of the circulator 307 is received by the raman spectrometer, so that the raman spectrum of the captured cell can be measured.

As the fluid flows in a laminar flow manner on the microfluidic chip, the captured cells can be randomly selected and slightly moved by the stage according to the Raman spectrum of the captured cells, and the captured cells are moved to the designated laminar flow to be released, so that the function of cell sorting is realized.

The device of example 2 simultaneously realizes the functions of measuring the Raman spectrum of the cells and sorting the cells.

Claims

1. A microfluidic chip for fiber optic photodynamic cell manipulation is characterized in that: the cell sorting device is characterized by comprising a base (1), a sheath liquid pool (2), a sheath liquid channel (3), a cell channel (4), a cell injection pool (5), a main channel (6), a photodynamic operation and control area (7), a cell and waste liquid guide-out channel (8), a Tesla micro valve (9), a first waste liquid pool (10), a second waste liquid pool (11), a first cell storage pool (12), a second cell storage pool (13), an optical fiber channel (14), a cover plate (15), a connecting hole (16) and a capillary steel tube (17), wherein cell liquid is injected from the cell injection pool (5), sheath liquid is injected from the sheath liquid pool (2), cell liquid forms a microflow containing cells in the main channel (6), the microflow containing the cells flows into the photodynamic operation and control area (7), optical fibers on the optical fiber channel (14) can operate and control the cells in the photodynamic operation and control area (7), and finally, the operated cells can be randomly selected and made to flow into a designated cell sorting channel.

2. A microfluidic chip for fiber optic photodynamic cell manipulation according to claim 1, wherein the optical fiber is a coaxial double waveguide fiber.

3. A microfluidic chip for fiber optic photodynamic cell manipulation according to claim 2, wherein: the end face of the optical fiber is ground into a frustum shape, so that light of the annular core is converged to capture cells, and the intermediate core is used for emitting and collecting signal light.

4. A microfluidic chip for fiber optic photodynamic cell manipulation according to claim 1, wherein: rough diffuse reflection eliminating walls of light are processed on the periphery of the photodynamic manipulation area (7) and are used for eliminating the influence of light reflection on photodynamic manipulation cells.

5. A microfluidic chip for fiber optic photodynamic cell manipulation according to claim 1, wherein: tesla microvalves (9) are provided between the cell and waste liquid discharge channel (8) and the first and second waste liquid pools (10, 11) and the first and second cell holding pools (12, 13) to suppress backflow.