CN110215938B - Full-transparent spiral type acoustic wave micro-fluidic sorting chip and preparation method thereof - Google Patents
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Abstract
The invention discloses a full-transparent spiral type acoustic wave micro-fluidic sorting chip and a preparation method thereof. Under the dual action of centrifugal force and acoustic radiation force, the successful sorting of target cells without modification and damage is realized according to the density, volume and compression ratio of different cells. The device is characterized in that a micro channel meeting the standing wave generating condition is prepared from low-cost glass by utilizing a laser cutting and punching technology, and blank glass and glass with a spiral micro channel are bonded together by a thermal bonding method to form a sealed cavity. The fully transparent acoustic wave microfluidic chip prepared by the invention is simple to operate and low in cost, and is beneficial to sorting circulating tumor cells and fetal nucleated red blood cells. The device has no modification and damage to the sorted cells, does not influence the activity of the cells, is favorable for further detection of the sorted cells, and has a large application space in clinic.
Description
Technical Field
The invention belongs to the field of micro total analysis systems, and particularly relates to a full transparent spiral type acoustic wave micro-fluidic sorting chip and a preparation method thereof.
Background
The micro-fluidic chip integrates sample pretreatment, reaction, separation, detection and the like on a micron-scale chip, has the characteristics of high integration level, low reagent consumption, low manufacturing cost, high analysis efficiency and the like, and shows good application prospects in the fields of physics, chemistry, biology and the like. Conventional microfluidic chips are numerous and can be sorted based on size, magnetism, and specific binding of antigen to antibody. However, these microfluidic chips still have many problems in clinical application. For example, in the case of a filter membrane structure of a microfluidic chip, the separation efficiency is low due to the overlapping of the sizes of some cells, and in addition, other impurities in the blood can block the filter membrane during the processing of the patient blood sample. The micro-fluidic chip adopting magnetic separation has the advantages that magnetic particles are easy to agglomerate, redundant magnetic particles are difficult to remove, and the application of the magnetic particles in the aspect of medicine is limited to a certain extent. The specific combination of the antigen and the antibody can improve the capture purity and efficiency, but after the capture, the antigen and the antibody cannot be separated, and a series of modification and release have great influence on the activity of cells. However, the microfluidic chip has the disadvantage of low throughput, and sometimes cannot meet the requirement of clinical large-scale sample processing.
The proposal of acoustic tweezers technology greatly increases the flux of the sample, and utilizes acoustic waves to manipulate particles or cells. The ultrasonic wave of 0.1-10 MHz is generated through the piezoelectric effect of the piezoelectric material, the sound wave signal is adjusted through the signal generator and the power amplifier, and two lines of ultrasonic waves generated by the same sound source are superposed in the acoustic resonant cavity to form a stable standing wave sound field. Under the action of acoustic radiation force, different particles or cells produce different displacements, thereby achieving the purpose of sorting.
At present, in the preparation of bulk wave devices, dry and wet etching methods are generally adopted to prepare micro-channels on monocrystalline silicon, then glass and a silicon wafer are bonded together in an anodic bonding mode, and finally a PZT piezoelectric ceramic wafer is attached to the bottom of the silicon wafer, so that a bulk wave chip is formed. The bulk wave chip has the advantages of complex process, high material consumption, higher requirement on equipment (a thermal evaporator, inductively coupled plasma equipment and an anodic bonding instrument), and difficult observation of experimental results under a microscope due to light-proof devices. The spiral channel can sort the cells by using centrifugal force, but can not enrich.
Disclosure of Invention
Aiming at the problems of the prior micro-fluidic chip in early medical diagnosis and treatment, the invention aims to provide a full-transparent spiral type acoustic wave micro-fluidic sorting chip with low cost and simple preparation process and a preparation method thereof. The microfluidic chip prepared by the invention is assembled by adopting the fully transparent glass sheets, has low material cost and low requirement on assembly equipment, greatly reduces the production cost, can be used for sorting cells under the conditions of no modification, no damage and no influence on the activity of the cells, and is favorable for further detection of the sorted cells.
The chips prepared by the invention are all made of transparent materials. The glass is processed by laser to prepare micron-sized channels which accord with integral multiples of half-wavelength, lithium niobate single crystal with two surfaces plated with tin-doped indium oxide (ITO) conductive film layers is used as a piezoelectric material and is pasted at the bottom of an acoustic resonant cavity to be used as an acoustic wave generator, and two copper wires are led out from the two surfaces to be used as signal input leads. The fully transparent device is beneficial to observing the sorting effect of samples such as circulating tumor cells, fetal nucleated red blood cells and the like. The application of the common glass is easy to operate, and the device cost is greatly reduced. The piezoelectric materials with different frequencies correspond to channels with different depths and different fluxes, and parameters of the device can be quickly and conveniently adjusted according to clinical requirements. The chip is prepared by laser processing, so that large-scale device integration is realized conveniently. The full-transparent spiral type acoustic wave micro-fluidic chip can be used for sorting cells, the cells are not modified and damaged in the sorting process, the activity of the cells is not influenced, and the further detection of the sorted cells is facilitated. The standing wave resonant cavity generated by the piezoelectric material can enable target cells to be concentrated at the wave node, and all the target cells are concentrated on the same horizontal plane, so that the sorted cells can be conveniently collected, and the piezoelectric material has a very large application space in future clinical medicine.
The technical scheme provided by the invention is as follows:
the utility model provides a full transparent spiral acoustic wave micro-fluidic chip of selecting separately, comprises acoustic resonant cavity and the piezoelectric material that the glass piece is piled up, and its structure is as follows:
(1) the acoustic resonant cavity is formed by stacking two glass sheets;
(2) the upper glass sheet of the acoustic resonant cavity is provided with a micron-sized spiral channel for sorting circulating tumor cells and fetal nucleated red blood cells, and a hole position communicated with the channel is arranged as an inlet and outlet;
(3) the lower layer glass sheet structure of the acoustic resonant cavity is completely used for packaging the resonant cavity;
(4) the piezoelectric material is coated with conductive films on two sides and is adhered to the bottom of the acoustic resonant cavity, and two copper wires are led out from the two sides to be used as signal input leads.
Specifically, in the step (1), the channel of the upper glass plate of the acoustic resonant cavity is formed by laser processing, and the depth of the channel is in accordance with the integral multiple of half wavelength.
Specifically, the resonance frequency of the piezoelectric material used was 3.8MHz, the width of the spiral channel was 500 μm, and the corresponding depth was 200 μm.
Specifically, the glass sheets are sealed by adopting a method of low-temperature pre-bonding and muffle furnace sectional heating.
Specifically, the channel is spiral in the plane of the glass sheet. The channel is two circles of helices, and the diameter of outermost side spiral is 20 mm. Compared with a straight channel, the spiral channel can reduce the diffusion of small particles and further improve the sorting efficiency. In the spiral channel, the cells can be sorted but not enriched by only utilizing centrifugal force, and the sorting is combined with an acoustic field, so that particles or cells can be enriched while the sorting is carried out, and the sorting efficiency and purity are improved.
Specifically, the sample inlet and outlet in the step (2) is connected to the container through an adapter. Preferably, the adapter is a Peek biological adapter, and the Peek biological adapter is bonded with the punched position of the glass outlet by epoxy resin.
Specifically, in the step (4), the piezoelectric material is a lithium niobate single crystal with both surfaces plated with the tin-doped indium oxide conductive thin film layer.
Specifically, the thickness of the lithium niobate single crystal is 1mm, the cut angle is 36 degrees, and the resonance frequency of the single crystal is 3.8 MHz.
Another object of the present invention is to provide a method for preparing the above sorting chip, comprising the steps of:
(1) design template
Adjusting parameters of the micro-channel according to theoretical calculation; the resonance frequency of the used single crystal was 3.8MHz, the width of the spiral channel was 500 μm, and the corresponding depth was 200 μm; the channel is two circles of spiral lines. Preferably, the two circles of spiral lines are formed by five semicircles, the diameter of the outermost spiral line is 20mm, and from large to small, the diameters of the semicircles are 20mm,19mm,18mm,17mm and 16mm in sequence;
(2) processing glass
Cutting two pieces of glass, wherein one piece of glass is used for processing a channel, and the other piece of glass is used for packaging; preferably, the thickness of the glass sheet is 1 mm;
the glass sheet is used for processing the channel, a micron-sized channel which accords with integral multiple of half-wavelength is prepared by laser processing according to the size of the template, and then punching is carried out to be used as a sample inlet and a sample outlet;
(3) integrated device
Packaging the two glass sheets by a thermal bonding method to form a sealed cavity;
(4) structure resonant cavity
At the bottom of the channel with the depth in accordance with integral multiple of half-wavelength, lithium niobate single crystal with piezoelectric effect in certain size is pasted at the bottom, leads are led out from two sides, and the power amplifier is used for driving.
Fig. 1 to 3 show the structure of a microfluidic chip provided by the present invention.
The invention also provides application of the full-transparent spiral type acoustic wave micro-fluidic sorting chip in cell sorting under the conditions of no modification, no damage and no influence on cell activity.
The principle of the microfluidic chip for cell sorting is that centrifugal force and acoustic radiation force act together, and the hydrodynamic sorting is based on dean pull force to compete with inertia force, so that particles with different sizes are distributed at different equilibrium positions, and circulating tumor cells, white blood cells and platelets are sorted. The acoustic radiation force further enriches various sorted cells, thereby improving the sorting efficiency and purity.
FIG. 4(a) is a schematic diagram of the force applied to a cell in a channel of a chip under only centrifugal force, and FIG. 4(b) is a schematic diagram of the theory of sorting different cells under only centrifugal force;
the principle of hydrodynamic separation is to induce dean pull forces acting on particles in a curved microfluidic channel to compete with inertial forces, resulting in different size particles being distributed at different equilibrium positions. Neutrals suspended in the channels of the curve are subjected to inertial forces consisting of gradient shear forces and wall effects. The gradient shear force is generated by a velocity parabola and is related to the shear rate gradient of the velocity parabola, and the direction is directly directed to the wall from the center of the curve microchannel; wall effects are induced by asymmetric wake-up of particles near the wall and have a tendency to push the particles away from the wall. The result of the competition of the two effects is that the particles are concentrated at a specific equilibrium position. The inertial forces act to move the particles between the center of the channel and the wall, and the theoretical prediction can be expressed as:
FL＝fL(Re，xL)·ρU2a4/Dh 2 (1)
here, the coefficient fLIs Reynolds number Re (Re ═ rho UD)hMu) and the position x of the particle in the channel cross sectionL(distance from the center of the channel). U is the maximum velocity in the microchannel, ρ and μ represent the density and dynamic viscosity of the fluid, respectively, DhIs the driving diameter of the microchannel (four times the channel cross section divided by the perimeter) and a is the particle diameter. According to the formula, the particle size has a great influence on the lateral force, which is the fundamental mechanism for clearly distinguishing the equilibrium positions of particles of different sizes. Therefore, circulating tumor cells, packed cells and platelets with different sizes can be separated.
FIG. 5 is a graph showing the simulation effect of the microfluidic chip provided by the present invention on cell sorting only under the action of acoustic radiation force;
wherein, FIG. 5(a) is an overall view of the sound field; FIG. 5(b) a slice of the sound field; FIG. 5(c) sound pressure level; FIG. 5(d) sound pressure level slice;
the magnitude of the cell stress is related to the radius, density and compressibility of the cell, and the magnitude of the specific acoustic radiation force can be expressed by the following formula.
The magnitude of the acoustic radiation force may be indicated by the above notations. Where Vc is the volume of the particles and P0 is the sound pressure magnitude, defined by the above equation. The densities of the medium and the particles are respectively represented by rhocAnd ρwTwo parameters indicate that the corresponding compression factor is betawAnd betac. When phi is>At 0, the particle moves towards the nodal point; when phi is<At 0, the particle moves towards the anti-node. Thereby enriching circulating tumor cells, white blood cells and platelets at different levels.
FIG. 6 is a graph showing the simulation effect of the microfluidic chip provided by the present invention on cell sorting at different positions away from the boundary under the action of acoustic radiation force; FIG. 6(a) an overall view of the sound field away from the boundary; FIG. 6(b) is an overall view of the sound field near the boundary; FIG. 6(c) an overall graph of sound pressure level away from the boundary; FIG. 6(d) an overall graph of sound pressure level near the boundary;
in the channel with the size meeting integral multiple of half wavelength, a standing wave chamber is formed in the vertical direction, countless wave nodes form a wave node surface, and cells at different positions from the boundary can be gathered at the wave nodes, so that the cells are enriched.
Fig. 7 is a theoretical schematic diagram of the microfluidic chip provided by the invention for sorting cells under the combined action of centrifugal force and acoustic radiation force. FIG. 7(a) without any force; FIG. 7(b) centrifugal force only; FIG. 7(c) acoustic radiation force alone; FIG. 7(d) centrifugal force and acoustic radiation force act together;
(a) without any force, all cells were mixed together. (b) Separation of circulating tumor cells, leukocytes, platelets based on fluid dynamics alone, will result in the concentration of cellular particles at specific equilibrium locations. (c) The cells will be enriched at different levels by the action of acoustic radiation force alone. (d) Together, the cells will be further enriched at a specific equilibrium position.
The invention has the beneficial effects that:
1. the optical analysis and identification are carried out efficiently, quickly and in real time by adopting the full transparent material for assembly;
2. the common glass replaces the traditional monocrystalline silicon piece and the high-temperature resistant borosilicate glass, so that the production cost is greatly reduced;
3. the chip is prepared by laser cutting, so that large-scale device integration is realized conveniently;
4. the thermal bonding replaces anodic bonding, the requirement on equipment is reduced, and the processing cost is reduced;
5. the sample inlet and the sample outlet of the sample are connected by the adapter, so that the traditional leakage problem is solved;
6. the centrifugal force and the acoustic radiation force are used for sorting the cells together, so that the efficiency is higher;
7. piezoelectric materials with different frequencies correspond to channels with different depths and correspond to different fluxes, parameters of a device can be quickly and conveniently adjusted according to clinical requirements, and the problem of low flux of a traditional microfluidic chip is solved;
8. the prepared microfluidic chip can sort cells under the conditions of no modification, no damage and no influence on the activity of the cells, is favorable for further detection of the sorted cells, and provides a new idea for the application of the microfluidic chip in cell sorting;
9. the invention can realize the sorting of different cells, such as circulating tumor cells, nucleated red blood cells, myeloma cells and the like, and has important significance for the field of life science.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip provided by the present invention;
FIG. 2 is a schematic diagram of the relative positions of various parts of a microfluidic chip provided by the present invention;
FIG. 3 is a dimensional diagram of a spiral channel microfluidic chip provided by the present invention;
FIG. 4(a) is a schematic diagram of the force applied to a cell in a channel of a chip under only centrifugal force, and FIG. 4(b) is a schematic diagram of the theory of sorting different cells under only centrifugal force;
FIG. 5 is a graph showing the simulation effect of the microfluidic chip provided by the present invention on cell sorting only under the action of acoustic radiation force; wherein, FIG. 5(a) is an overall view of the sound field; FIG. 5(b) a slice of the sound field; FIG. 5(c) sound pressure level; FIG. 5(d) sound pressure level slice;
FIG. 6 is a graph showing the simulated effect of the microfluidic chip provided by the present invention on cell sorting at different positions from the boundary under the action of acoustic radiation force only;
FIG. 7 is a theoretical schematic diagram 7(a) of the microfluidic chip provided by the present invention for cell sorting under the combined action of centrifugal force and acoustic radiation force without any acting force; FIG. 7(b) centrifugal force only; FIG. 7(c) acoustic radiation force alone; FIG. 7(d) centrifugal force and acoustic radiation force act together;
reference numerals: 1-lithium niobate single crystal (36 degree Y cut, thickness 1mm) with tin-doped indium oxide conductive film layer plated on both sides; 2-ultraviolet light curing epoxy resin layer; 3-double-layer glass sheets; 4-a thermal bonding layer; 5-sample inlet; 6-sample outlet.
Detailed Description
The invention will be further explained below with reference to the drawings, to which the invention is not at all restricted.
Example 1
Preparation of full-transparent spiral type acoustic wave micro-fluidic sorting chip
The preparation steps are as follows:
1. design template
To ensure that there is a clear boundary between different sorted cells and to ensure that different sorted samples are collected at different outlets, the parameters of the microchannel are adjusted according to theoretical calculations. The resonant frequency of the single crystal of the embodiment is 3.8MHz, the width of the spiral channel is 500 μm, and the corresponding depth is 200 μm; the channel is two circles of helices, comprises five semicircles, and the diameter of outermost side helicoid is 20mm, and the diameter of big to little semicircle is 20mm,19mm,18mm,17mm,16mm in proper order. Three outlets are arranged to sort circulating tumor cells, leukocytes, and platelets based on the force exerted on the circulating tumor cells, leukocytes, and platelets in the helical channel, and the displacement from the outlet at the end of the channel. The distances from the three sample outlets to the bifurcation point are 150 μm, 300 μm and 500 μm in sequence.
2. Processing glass
And cutting two pieces of glass with the same size and the thickness of 1mm, wherein one piece of glass is used for processing a channel, and the other piece of glass is used for packaging a resonant cavity. And preparing the micron-sized channel which accords with the integral multiple of half-wavelength by utilizing laser processing according to the size of the template. The resonance frequency of the single crystal is 3.8MHz, the width of the spiral channel is 500 μm, and the corresponding depth is 200 μm; the channel is two circles of helices, and five semicircle come to constitute, and the diameter of outermost side helicoid is 20mm, and the diameter from big to little circle is 20mm,19mm,18mm,17mm,16mm in proper order. Fig. 3 shows the structure and dimensions of the spiral channel. The piezoelectric materials with different frequencies correspond to channels with different depths and different fluxes, and parameters of the device can be quickly and conveniently adjusted according to clinical requirements. After the channel is processed, holes are punched in the same piece of glass to serve as a sample inlet and a sample outlet. The sample injection ports are two, one is a blood sample injection port, and the other is a buffer solution sample injection port. Three sample outlets are provided. The structure is shown in fig. 2.
3. Integrated device
The same size of the whole glass is used as the lower glass for encapsulating the chamber. And bonding the upper and lower glass sheets by adopting a thermal bonding method to form a sealed chamber. The structure is shown in fig. 1 and 2.
The processing steps are as follows:
(1) low temperature prebonding
Cleaning the upper and lower glass sheets, sequentially cleaning with detergent for 10min, washing with tap water for 15min, washing with deionized water for more than five times, and finally, tightly attaching the two glass sheets under the action of the flow of the deionized water. The adhered upper and lower surfaces of the glass were dried by blowing, heated on a 80 ℃ baking table, and the presence or absence of diffraction fringes was observed. If diffraction stripes exist, the cleaning is incomplete, the sealing is carried out after the cleaning is needed again, if the diffraction stripes do not exist, the cleaning is carried out, and the heating is continued for 2 hours on the drying table.
(2) High-temperature bonding, and realizing high-temperature sealing between glass at 540-550 DEG C
After heating on a 80 ℃ baking table for 2h, the chip was placed in a muffle furnace. The temperature-raising program is as follows:
4. structure resonant cavity
And (2) adhering 10 mm-10 mm lithium niobate single crystals (with the thickness of 1mm and 36-degree Y-cut) with piezoelectric effect to the bottom of the lower glass sheet by using an epoxy resin ultraviolet curing mode at the bottom of the channel with the depth conforming to the integral multiple of the half-wavelength, leading out wires from two sides, driving by using a power amplifier, adjusting the frequency and the size of signals, fixing target cells at nodes and realizing the sorting effect of the same cells on the same horizontal plane.
Application example 1
Cell sorting using the microfluidic sorting chip prepared in example 1
After a patient blood sample is taken, firstly, red blood cells are removed by using a red blood cell lysate, then, different cells have different radiuses in a spiral channel under the action of centrifugal force according to the density, volume and compression ratio of the different cells, the displacement from an outlet is different, and the same cells can be gathered on the same nodal plane due to the existence of standing waves, so that the cells are sorted out, and the corresponding cells are circulating tumor cells, white blood cells and platelets from the inner side to the outer side. The target cells are not modified in the operation process, so the cells are not damaged, and the activity is not influenced.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention should be included in the scope of the present invention.
Claims (8)
1. The utility model provides a chip is selected separately to full transparent spiral sound ripples micro-fluidic comprises acoustics resonant cavity and the piezoelectric material that glass piece piles up which characterized in that:
(1) the acoustic resonant cavity is formed by stacking two glass sheets;
(2) the upper glass sheet of the acoustic resonant cavity is provided with a micron-sized spiral channel for sorting circulating tumor cells and fetal nucleated red blood cells, and a hole position communicated with the channel is arranged as an inlet and outlet; the channel is spiral on the plane of the glass sheet and is two circles of spiral lines, and the diameter of the outermost spiral line is 20 mm;
the two circles of spiral lines are formed by five semicircles, the diameter of the outermost spiral line is 20mm, and from large to small, the diameters of the semicircles are 20mm,19mm,18mm,17mm and 16mm in sequence;
the three sample outlets are arranged at positions with distances of 150 micrometers, 300 micrometers and 500 micrometers from the bifurcation point in sequence;
(3) the lower layer glass sheet structure of the acoustic resonant cavity is completely used for packaging the resonant cavity;
(4) the piezoelectric material is plated with conductive films on two sides and is adhered to the bottom of the acoustic resonant cavity, and two copper wires are led out from the two sides to be used as signal input leads; the resonant frequency of the piezoelectric material used was 3.8MHz, the spiral channel width was 500 μm, and the corresponding channel depth was 200 μm.
2. The fully transparent spiral acoustic wave microfluidic sorting chip of claim 1, wherein: the channel of the upper glass sheet of the acoustic resonant cavity is formed by laser processing, and the depth of the channel accords with integral multiple of half wavelength.
3. The fully transparent spiral acoustic wave microfluidic sorting chip of claim 1, wherein: and sealing the glass sheets by adopting a method of low-temperature pre-bonding and muffle furnace sectional heating.
4. The fully transparent spiral acoustic wave microfluidic sorting chip of claim 1, wherein: the sample inlet and outlet are connected to the container through a conversion joint.
5. The fully transparent spiral acoustic wave microfluidic sorting chip of claim 1, wherein: the piezoelectric material is a lithium niobate single crystal with two sides plated with tin-doped indium oxide conductive thin film layers.
6. The fully transparent spiral acoustic wave microfluidic sorting chip of claim 5, wherein: the thickness of the lithium niobate single crystal is 1mm, the lithium niobate single crystal is Y-cut at 36 degrees, and the resonant frequency of the single crystal is 3.8 MHz.
7. The preparation method of the full-transparent spiral type acoustic wave micro-fluidic sorting chip of claim 1 is characterized by comprising the following steps:
(1) design template
Adjusting parameters of the micro-channel according to theoretical calculation; the resonance frequency of the used single crystal was 3.8MHz, the width of the spiral channel was 500 μm, and the corresponding depth was 200 μm; the channel is two circles of spiral lines;
(2) processing glass sheets
Cutting two pieces of glass with the same size, wherein one piece of glass is used for processing a channel, and the other piece of glass is used for packaging;
the glass sheet is used for processing the channel, a micron-sized channel which accords with integral multiple of half-wavelength is prepared by laser processing according to the size of the template, and then punching is carried out to be used as a sample inlet and a sample outlet;
(3) integrated device
Packaging the two glass sheets by a thermal bonding method to form a sealed cavity;
(4) structure resonant cavity
And sticking the lithium niobate single crystal with the piezoelectric effect on the bottom of the channel with the depth conforming to the integral multiple of the half-wavelength, leading out wires from two sides, and driving by using a power amplifier.
8. The use of the fully transparent spiral acoustic wave microfluidic sorting chip according to any one of claims 1 to 6 for cell sorting without modification, damage, or influence on cell activity.
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| CN114522649B (en) * | 2022-02-15 | 2023-03-31 | 浙江大学 | Acoustic particle capturing and track control method based on magnetofluid reconstruction |
| CN115308301B (en) * | 2022-08-16 | 2023-03-10 | 中山大学 | Measuring device capable of measuring elastic modulus of cells and cell nucleuses |
| CN117019245B (en) * | 2023-08-09 | 2024-08-23 | 江南大学 | Sorting and detecting integrated sensing chip based on spiral microfluidic array and application thereof |
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