CN116786181A - Impedance flow type detection chip and preparation method and application thereof - Google Patents

Abstract

The application relates to an impedance flow type detection chip, a preparation method and application thereof. The hydrodynamic focusing region controls the cell position and the electrode detection region detects the impedance change. The sheath fluid is introduced through a sample injection hole and is equally divided into two beams, transverse focusing is realized at the junction through extrusion of two sides to the middle sample flow, and longitudinal focusing is formed through V-shaped convergence and height difference. The sheath fluid adaptively concentrates the sample at the bottom of the micro-channel transversely and longitudinally, reduces the change of the displacement height of particles, increases the signal-to-noise ratio of the impedance pulse of the particles, and improves the detection signal. Can be used for label-free high-sensitivity detection of particles, cells and the like.

Description

An impedance flow detection chip and its preparation method and application

Technical field

The invention belongs to the field of chips, and in particular relates to an impedance flow detection chip and its preparation method and application.

Background technique

Flow cytometry is an analytical technique widely used for cell counting and detection. In a typical flow cytometer configuration, samples are focused into a single arrangement under sheath flow hydrodynamics. The samples flow through the optical detector one by one to generate signals, which are detected and analyzed to contain various characteristic information. In recent years, flow cytometers have made great progress in terms of software and hardware, simplifying operations and enhancing detection diversity and accuracy. Still, these devices are expensive and bulky. Moreover, traditional flow cytometry detection is inseparable from various markers such as fluorescence, which often destroys the original state of cells.

Impedance methods based on cell electrical properties (e.g., membrane capacitance or cytoplasmic resistance) have the unique advantage of allowing cell status to be assessed in a label-free manner, without the need for sophisticated optical systems, and the instruments can be miniaturized. In recent decades, microfluidics has received increasing attention due to its unique advantages, such as less sample and reagent consumption, low cost, and compact structure. Impedance flow cytometry (IFC) based on this is an emerging single cell analysis and particle counting technology. It relies on the perturbation of the electric field by particles flowing through microfluidic channels. The perturbation signal is directly related to the electrical properties of the particle and therefore provides information about its composition and structure. The dielectric properties of cells, such as membrane capacitance and conductivity, reflect membrane morphology and function.

In traditional flow cytometers, sheath flow is introduced to focus the sample flow into a very narrow stream, forcing particles to pass through the detection area in a single sequence. This is also critical in microfluidic flow cytometry because particles also shift position during microchannel motion. Especially for impedance flow cytometry using planar electrodes, changes in the position of particles from the detection electrode lead to reduced detection consistency. Some planar devices use two-dimensional (2D) hydrodynamic focusing, in which two adjacent sheath flows squeeze the sample flow laterally (horizontally) in the middle of a microchannel. Although this method provides the ability to localize small amounts of analyte to the detection area, cells or particles in the focused flow flow at different channel heights (widths) and individual detection cannot be guaranteed.

Recently, three-dimensional (3D) focusing methods have been reported in the literature (Watkins, N., et al. "A robust electrical microcytometer with 3-dimensional hydrofocusing." Lab on A Chip 9.22 (2009): 3177-3184. Chícharo, A .,et al."Enhanced magnetic microcytometer with 3Dflow focusing for cell enumeration."Lab on a Chip 18.17(2018):2593-2603.Eluru,G.,et al."Single-layer microfluidic device to realize hydrodynamic3D flow focusing." "Lab on a Chip16.21(2016):4133-4141.). In these devices, the sample flow is surrounded by a sheath flow, focused in the lateral and vertical directions, and the sample flow is restricted to flow locally in the microchannel. By changing the flow ratio between sheath flow and sample flow, the focused flow diameter and profile can be adjusted. However, such three-dimensional focusing requires separate control of the sheath flow in the lateral and vertical directions, which increases the difficulty of operation. At the same time, due to multiple sheath flow interconnections, focusing stability is also difficult to ensure.

Contents of the invention

The technical problem to be solved by the present invention is to provide an impedance flow detection chip and its preparation method and application.

An impedance flow microfluidic chip of the present invention, the chip includes a sheath flow channel, a sample channel, and an electrode detection area; wherein the sheath flow channel is provided with a symmetrical first sheath flow channel and a second sheath flow channel. The flow channel, the second sheath flow channel and the sample channel intersect to form a hydrodynamic focusing area, and the hydrodynamic focusing area is connected to the electrode detection area through the detection channel. The height of the sheath flow channel is higher than the height of the sample channel.

The sample channel is connected with the detection channel, and the sample channel and the detection channel have the same width and the same height.

The hydrodynamic focusing area controls cell position, and the electrode detection area detects impedance changes.

The first sheath flow channel and the second sheath flow channel are located on both sides of the sample channel, and the sample channel is located in the middle. The first sheath flow channel and the second sheath flow channel form a certain included angle (0°<included angle≤180° ) are arranged and intersected. In particular, when the included angle is 90°, it is V-shaped, and when the included angle is 180°, the sheath flow channel and the sample flow channel are cross-shaped.

Furthermore, the included angle is 90°, and V-shape is the most preferred.

The hydrodynamic focusing area achieves transverse focusing at the intersection through the squeezing of the middle sample flow by the sheath flow on both sides, and longitudinal focusing through V-shaped convergence and height difference.

The chip is also provided with a sheath flow sampling port, which is connected to the first sheath flow channel and the second sheath flow channel; is provided with a sample injection port and is connected to the sample channel; and is also provided with a sample outlet port, which is connected to the electrode detection port. Regions are connected.

The electrode inspection area is provided with two pairs of planar electrodes.

For the flat electrode in the electrode detection area, the electric field is strongest in the area closest to the electrode. Such three-dimensional focusing allows the particles to be close to the electrode to increase the signal-to-noise ratio of the impedance pulse. Improves electrode detection consistency and stability by eliminating variations in particle displacement height. Based on the above-mentioned cell dielectric properties, two pairs of planar electrodes are used for downstream electrode sensing. One pair of 7 and 8 detects low frequencies, and the other pair of 12 and 13 detects high frequencies.

The ratio of the flow rates of the sheath flow to the sample flow is defined as the sheath-to-sample ratio R, and R is 1-30.

The height of the sheath flow channel is 1 μm-1mm; the width of the sample channel is 1 μm-1mm, and the height is 1 μm-1mm.

In the hydrodynamic focusing area, the sheath flow is introduced through an injection hole 10 and divided equally into two beams. At the intersection, transverse focusing is achieved by squeezing the middle sample flow from both sides, and longitudinal focusing is formed through convergence (such as V-shaped, cross-shaped convergence, etc.) and height differences. The ratio of the flow velocity of the sheath flow to the sample flow is defined as the sheath-to-sample ratio R, which determines the focusing effect of the focused flow.

Considering cell size and clogging, the width W of the detection port flow channel is 1 μm-1mm, the height H is 1 μm-1mm, and the height Hs of the sheath flow channel is 1 μm-1mm. The key point in size selection is that the sheath flow height Hs is higher than the sample channel height H. This three-dimensional focusing concept is not only applicable to microfluidic chips, but can also be used for any structure that forms a three-dimensional focusing of fluids through height differences, such as flow cytometers.

The preparation method of a resistance flow microfluidic chip of the present invention includes:

Photolithography is used to form a patterned sample channel. The height of the channel is consistent with the height of the detection channel. Then, photoresist is spin-coated on the first layer of pattern, aligned and photolithographed again to form a sheath flow channel. After mixing the PDMS prepolymer with the curing agent Pour it on the mold and release it after baking;

The detection electrode adopts a peeling process. After exposure and development with photoresist, the adhesion layer and gold layer are sputtered and then peeled and patterned; then the microchannel layer and the electrode layer are aligned and bonded through oxygen plasma, and finally the electrodes and lead,

Complete the production of microfluidic impedance detection chip.

An inspection device of the present invention, the device includes the impedance flow microfluidic chip and a lock-in amplifier.

The present invention relates to an application of the impedance flow microfluidic chip in particle and cell detection.

beneficial effects

The invention is an impedance flow cytometry detection chip and device based on three-dimensional hydrodynamic focusing. The chip includes a hydrodynamic focusing area and an electrode detection area. The hydrodynamic focusing area controls cell position, and the electrode detection area detects impedance changes. The sheath fluid is introduced through a sampling hole and divided equally into two beams. At the intersection, transverse focusing is achieved by squeezing the middle sample flow on both sides, and longitudinal focusing is formed through V-shaped convergence and height difference. The sheath liquid adaptively concentrates the sample laterally and longitudinally at the bottom of the microchannel, reducing changes in particle displacement height, increasing the signal-to-noise ratio of the particle impedance pulse, and improving the detection signal. It can be used for label-free and highly sensitive detection of particles, cells, etc.

The present invention relates to a novel, three-dimensional focusing impedance microfluidic chip, in which the sample flow passes through a set of sheath laminar flow and weir structures to achieve three-dimensional hydrodynamic adaptive focusing. Through simulation and confocal microscopy, increasing the flow ratio of sheath fluid to sample reduces the cross-sectional area of the concentrated flow at the bottom of the channel to 26.50% of that before focusing. The sheath liquid reduces the change in particle displacement height, increases the particle impedance pulse amplitude, and reduces the coefficient of variation by at least 35.85%.

The microfluidic flow cytometry system of the present invention is easy to operate and simple in structure, and provides an effective solution for monitoring cell status.

Description of the drawings

Figure 1 Schematic diagram of the three-dimensional focusing structure; including 1 glass substrate; 2 microfluidic layer; 3 sheath flow channel; 4 sample channel; 5 hydrodynamic focusing area; 6 detection channel; 7, 8, 12, and 13 two pairs of planar electrodes; 9 sample outlet; 10 sheath flow inlet; 11 sample inlet; 14 electrode detection area;

Figure 2 Microfluidic chip preparation process;

Figure 3 Three-dimensional focusing simulation results and confocal results;

Figure 4 Impedance sensing and detection device;

Figure 5 shows the detection results of 10 and 15 μm particles and cells; including (a) two-dimensional focusing 10 and 15 μm particle impedance detection, (b) three-dimensional focusing 10 and 15 μm particle impedance detection, (c) two-dimensional and three-dimensional focusing HepG2 cell impedance detection .

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Example 1

Impedance flow microfluidic chip. The chip includes a glass substrate and a microfluidic layer. The microfluidic layer is provided with a sheath flow channel, a sample channel, and an electrode detection area; wherein the sheath flow channel is provided with a symmetrical first sheath flow channel. , the second sheath flow channel, the first sheath flow channel, the second sheath flow channel and the sample channel intersect to form a hydrodynamic focusing area, and the hydrodynamic focusing area communicates with the electrode detection area through the detection channel. The height of the sheath flow channel is higher than the height of the sample channel. The chip is also provided with a sheath flow inlet and is connected to the first sheath flow channel and the second sheath flow channel. A sample inlet is provided and connected to the sample channel. Communicated; a sample outlet is also provided and communicated with the electrode detection area, and the electrode detection area is provided with two pairs of planar electrodes.

The hydrodynamic focusing area achieves transverse focusing at the intersection through the squeezing of the middle sample flow by the sheath flow on both sides, and longitudinal focusing through V-shaped convergence and height difference.

Furthermore, in the embodiment of the present invention, the width W of the detection port flow channel is 40 μm, the height H is 30 μm, the height Hs of the sheath flow channel is 50 μm, the width W of the sample channel is 40 μm, and the height H is 30 μm.

Fabrication of microfluidic chips:

The microchannel mold has a SU-8 structure and is made by two photolithography steps, including glue dispersion, pre-baking, exposure, development, and hard baking steps, as shown in Figure 2. The first photolithography creates a patterned sample channel with the channel height consistent with the detection channel height. Then a thicker photoresist is spin-coated on it, and the sheath flow channel is formed by photolithography again. Mix the PDMS prepolymer and curing agent (10:1) and pour it on the mold, bake it in an oven at 80°C for 90 minutes and then demould.

The detection electrode uses a lift-off process. After exposure and development with photoresist on BF33 borosilicate glass, a 50-nanometer chromium adhesion layer and a 200-nanometer gold layer are sputtered and then peeled off and patterned.

The microchannel layer and the electrode layer are then aligned and bonded through oxygen plasma. Finally, weld the electrodes and leads to complete the production of the microfluidic impedance detection chip.

3D focusing simulation and confocal experiments:

COMSOL multi-physics modeling software was used to numerically simulate the three-dimensional focused flow of fluid. The same geometry as the actual device was set up, and laminar flow and dilute material transport models were used to perform a steady-state solution to the concentration distribution of the sample in the microchannel.

In order to characterize the three-dimensional hydrodynamic focusing performance of the chip, confocal microscopy imaging technology was used to visualize its flow field. In the hydrodynamic focusing experiment, 1×PBS was used as the sheath flow, and 1mM fluorescein isothiocyanate solution (FITC) was used as the sample flow. The sheath flow and sample flow are controlled by precision syringe pumps to control the flow rate and flow ratio respectively. The laser confocal microscope uses a 40x objective lens and the excitation light wavelength is 488nm. The flow profile is generated by scanning the entire channel height layer by layer in the Z direction with steps of 1 μm. The scanning area is set at the intersection of the sheath and sample flow, and the software that comes with the microscope is used for image cross-section analysis.

As shown in Figure 3, a transverse slice of a microfluidic channel simulated by COMSOL with a sheath flow ratio R of 4:1 is shown. It can be seen from the simulation that the sample flow is squeezed by the two sides and top of the sheath flow at the weir structure and converges at the bottom of the channel. Figure 3 shows the simulation results and actual channel confocal images 100 μm upstream and downstream of the intersection center. Under these conditions, the sample flow cross-sectional area after focusing is significantly reduced to 26.50% of that before focusing.

Impedance flow test:

Inject the impedance flow chip onto an inverted microscope and observe with a CCD camera. Two precision syringe pumps load the sample and sheath flow respectively to adjust their respective flow rates. The signal source applies an excitation signal, and a lock-in amplifier is connected to the electrode for signal readout. The overall system test is shown in Figure 4. The first pair of electrodes downstream of the intersection applies a low-frequency signal (500kHz, 1Vpp), and the second pair of electrodes applies a slightly higher-frequency signal (5MHz, 1Vpp). The sensed impedance signal is collected to the computer, displayed and stored in the LabVIEW program, and then programmed in MATLAB to extract features for analysis. During the sampling process, the impedance signal and the particle movement video observed by the camera are simultaneously recorded, and the two can be correlated with each other in real time.

The classification test of 10 micron and 15 micron microspheres and HepG2 cells was used to verify the improvement of impedance classification ability of 3D focusing. 10 μm and 15 μm polystyrene microspheres were diluted in 1% BSA in PBS, and the concentration was controlled at around 10 ⁶ /mL. The sample flow rate was set to 1 μL/min, and the sheath flow was set according to the sheath flow ratio R=4. On chips with three-dimensional focusing structures and ordinary focusing structures, after the flow of focused microspheres has stabilized, the impedance signals of the particles are collected for comparative analysis.

Figure 5a and b show the impedance classification effects of two-dimensional focusing and three-dimensional focusing 10 micron and 15 micron microspheres respectively. The coefficients of variation of 3D focus 10 and 15 are both smaller than the respective coefficients of variation of the corresponding 2D focus, which are reduced by 35.85% and 51.46% respectively. It shows that the grouping effect of three-dimensional focusing is more obvious and each group has better aggregation. In addition, standard particles with larger diameters are more affected by 3D focusing, because the focused flow has a stronger restriction on the movement of larger diameter particles.

To further demonstrate the practical application of the developed microfluidic flow cytometer, the difference in impedance signals between 2D focusing and 3D focusing of living HepG2 cells was tested (as shown in Figure 5c). The average opacity of the latter is larger than that of the former, and the corresponding coefficient of variation CV value is smaller. It can be seen that three-dimensional focusing significantly improves the impedance signal of living cells. The coefficient of variation of the opacity of HepG2 cells during 3D focusing is higher than that of 15 micron microspheres, because the cells themselves have a larger diameter range than the microspheres.

Claims (10)

1. An impedance flow type micro-fluidic chip is characterized by comprising a sheath flow channel, a sample channel and an electrode detection area; the sheath flow channel is provided with a first sheath flow channel and a second sheath flow channel which are symmetrical, the first sheath flow channel, the second sheath flow channel and the sample channel are intersected to form a hydrodynamic focusing area, and the hydrodynamic focusing area is communicated with the electrode detection area through the detection channel.

2. The impedance flow microfluidic chip of claim 1, wherein the sheath flow channel has a height that is higher than the height of the sample channel.

3. The impedance flow microfluidic chip according to claim 1, wherein the first sheath flow channel and the second sheath flow channel are arranged at two sides of the sample channel, the sample channel is arranged in the middle, and the first sheath flow channel and the second sheath flow channel are arranged and intersected in an included angle, wherein the included angle is 0 ° < 180 °.

4. The impedance-flow microfluidic chip according to claim 1, wherein the hydrodynamic focusing region is focused laterally at the junction by extrusion of the sheath flow on both sides with respect to the intermediate sample flow, and focused longitudinally by convergence and height differences.

5. The impedance flow microfluidic chip of claim 4, wherein said convergence is a V-shaped or cross-shaped convergence.

6. The impedance flow microfluidic chip according to claim 1, wherein the chip is further provided with a sheath flow sample inlet and is in communication with the first sheath flow channel and the second sheath flow channel; a sample inlet is arranged and communicated with the sample channel; a sample outlet is also arranged and communicated with the electrode detection area; the electrode inspection area is provided with two pairs of planar electrodes.

7. The impedance flow microfluidic chip according to claim 1, wherein the ratio of the flow rate of the sheath flow to the sample flow is defined as a sheath-like ratio R, R being 1-30; the height of the sheath flow channel is 1 mu m-1mm; the width of the sample channel is 1 μm-1mm, and the height is 1 μm-1mm.

8. A preparation method of an impedance flow type micro-fluidic chip comprises the following steps:

and photoetching to form a patterned sample channel, wherein the height of the channel is consistent with that of the detection channel, then spin-coating photoresist on the first layer of pattern, aligning and photoetching again to form a sheath flow channel, mixing the PDMS prepolymer and the curing agent, pouring the mixture on a mold, baking and demolding.

9. An inspection apparatus comprising the impedance flow microfluidic chip of claim 1, a lock-in amplifier.

10. Use of the impedance flow microfluidic chip of claim 1 in particle, cell detection.