Micro-fluidic chip combining in-situ Raman detection with optical tweezers sorting and preparation method

Abstract

The invention provides a micro-fluidic chip combining in-situ Raman detection with optical tweezers sorting and a preparation method, and belongs to the technical field of micro-fluidic chips and optical detection.The micro-fluidic chip sequentially comprises a top cover layer, a middle flow channel layer and a base layer from top to bottom, and a preset micro-flow channel network is machined in the middle flow channel layer through femtosecond laser; comprising a cell injection channel, a cell sample loading channel and a sorting outlet channel, one end of the cell sample loading channel is horizontally connected and communicated with one end of the sorting outlet channel, a third conduction channel is led out from one side of a joint part of the cell sample loading channel and the sorting outlet channel and is communicated with one end of a Raman spectrum detection and optical tweezers sorting area, and the other end of the Raman spectrum detection and optical tweezers sorting area is communicated with one end of the cell injection channel; the device has the characteristics of excellent in-situ detection capability, efficient and accurate sorting function, low cost and easiness in manufacturing, and is very suitable for standardized production, popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip and optical detection technology, and in particular, it is a microfluidic chip and its preparation method that combines in-situ Raman detection with optical tweezers sorting. By integrating microfluidic channels, it can perform in-situ Raman spectroscopy detection on single cells in a liquid environment, and at the same time use optical tweezers technology to achieve precise sorting. Background Technology

[0002] Raman spectroscopy is a powerful non-destructive analytical technique that can provide molecular fingerprint information of substances. It has broad application prospects in the biomedical field, especially in cell detection, drug screening and disease diagnosis.

[0003] However, traditional Raman detection techniques face the following main challenges when detecting cells suspended in liquid environments (such as microbial cells and animal cells):

[0004] (1) Cells undergo severe Brownian motion in liquids, making it difficult to stabilize their position, which leads to difficulties in signal acquisition and a low signal-to-noise ratio;

[0005] (2) Raman signals from complex backgrounds (such as culture medium) can interfere with the signals of target cells;

[0006] (3) It is difficult to perform non-destructive and precise separation and extraction of specific target cells (such as cancer cells and stem cells) after detection, so as to carry out subsequent culture or sequencing analysis.

[0007] Currently, to solve the above problems, it is usually necessary to fix the cells on a glass slide for detection, but this changes the true physiological state of the cells and makes sorting impossible.

[0008] Some advanced technologies attempt to combine optical tweezers with Raman spectroscopy: optical tweezers can capture and stabilize single cells using a highly focused laser beam, while Raman spectroscopy simultaneously detects the captured cells; however, existing implementations typically rely on complex open liquid pools or self-made chambers, resulting in low system integration, poor stability, cumbersome operation, and high costs. These devices struggle to achieve high-throughput, automated detection and sorting, limiting their potential for clinical and industrial applications.

[0009] In addition, while existing microfluidic chips can control fluids and cells, they typically use polymers (such as PDMS) as the main material. PDMS itself has a strong Raman background signal, which will seriously interfere with the acquisition of weak Raman signals from cells, leading to detection failure. While chips made entirely of quartz have excellent optical performance, they are difficult to manufacture and extremely expensive, especially when fabricating complex three-dimensional microchannel structures.

[0010] Therefore, there is an urgent need in the field for an integrated chip that can overcome the above-mentioned shortcomings. It should have low background noise, be able to stably manipulate cells, integrate microfluidic functions to achieve high-throughput analysis, and have a simple and low-cost fabrication process. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a microfluidic chip and its fabrication method that combines in-situ Raman detection with optical tweezers sorting. The microfluidic chip, with its three-layer composite structure, cleverly combines the excellent optical transparency and low Raman background of quartz with the easy processing and encapsulation properties of PDMS material. This enables high-quality in-situ Raman detection and optical tweezers sorting of single cells in a microfluidic environment. The chip is simple to fabricate, low in cost, and suitable for widespread application.

[0012] A microfluidic chip combining in-situ Raman detection and optical tweezers sorting, and its fabrication method, wherein:

[0013] A microfluidic chip for in-situ Raman detection combined with optical tweezers sorting includes: a three-layer composite structure, from top to bottom as follows:

[0014] (The chip needs to be inverted during actual use, so the top cover layer is at the bottom.)

[0015] Top cover layer: is an ultra-thin quartz sheet. The top cover layer serves as an optical window, providing a working surface with high transmittance and high optical quality for the incident and emitting of Raman lasers and optical tweezers lasers. At the same time, its inherent low Raman background characteristics ensure a low signal-to-noise ratio of the detection signal.

[0016] As an example, the thickness of the ultrathin quartz sheet is between 100 and 200 micrometers.

[0017] Intermediate flow channel layer: a thin film structure made of polydimethylsiloxane (PDMS) material; a pre-designed microchannel network is fabricated in the thin film structure using a femtosecond laser;

[0018] The microfluidic network includes a cell injection channel, a cell loading channel, and a sorting outlet channel. The microfluidic network enables single-cell queuing and focusing of cell samples and provides operating space and path for optical tweezers sorting.

[0019] One end of the cell loading channel and one end of the sorting outlet channel are horizontally connected and conductive. A third conductive channel is led out from one side of the junction of the two, connecting to one end of the Raman spectroscopy detection and optical tweezers sorting area. Raman spectroscopy detection Measurement of optical tweezers sorting area The other end is connected to one end of the cell injection channel;

[0020] As an example, the thickness of the thin film structure is between 10 and 100 micrometers.

[0021] As an example, the third conductive channel is perpendicular to the cell loading channel and the sorting outlet channel.

[0022] Base layer: a thick quartz substrate;

[0023] The base layer provides mechanical support for the entire chip, ensuring the chip's flatness and structural stability; the thick quartz substrate is processed with a femtosecond laser to have three through holes, which serve as the chip's sample loading port, injection port, and fluid outlet, respectively; these holes are respectively connected to the other end of the cell sample loading channel, the other end of the cell injection channel, and the other end of the sorting outlet channel;

[0024] As an example, the thickness of the thick quartz substrate is 1 mm.

[0025] As an example, the three through holes are all the same size, with a diameter of 1 mm.

[0026] As an example, the Raman spectroscopy detection and optical tweezers sorting area has a circular structure.

[0027] As an example, the thick quartz substrate has two symmetrically arranged cuboid structures on both sides for processing three through holes.

[0028] The top cover layer, the intermediate flow channel layer, and the base layer are all bonded together by oxygen plasma treatment to form a closed integral structure, creating a complete microfluidic channel that can carry liquid.

[0029] A method for fabricating a microfluidic chip using in-situ Raman detection combined with optical tweezers sorting includes:

[0030] Step 1: Fabrication of the intermediate flow channel layer;

[0031] The SU-8 positive mold was fabricated on a silicon wafer using a photolithography process.

[0032] The PDMS prepolymer was mixed with a curing agent, degassed, and then poured onto an SU-8 positive mold. After heating and curing, it was peeled off to obtain a thin film structure. Then, a microfluidic network was directly fabricated on the surface of the thin film structure using a femtosecond laser direct writing method.

[0033] As an example, SU-8 is a photoresist, and the SU-8 male mold is a male film structure prepared using a specific process.

[0034] Step 2: Base layer processing;

[0035] Three identical through holes were machined on a thick quartz substrate using a femtosecond laser to serve as the sample loading hole, injection port, and fluid outlet;

[0036] Step 3: Preliminary treatment of the bonding surface;

[0037] The ultrathin quartz sheet, the intermediate layer with microfluidic network, and the thick quartz substrate are cleaned and dried; then, the bonding surfaces of the intermediate fluidic layer and the ultrathin quartz sheet are treated with oxygen plasma.

[0038] Step 4: Align and bond;

[0039] The lower surface of the processed intermediate flow channel layer is aligned and pressed with the thick quartz substrate to form a preliminary bond; then the ultra-thin quartz cover sheet is aligned and pressed with the upper surface of the intermediate flow channel layer to form the final three-layer structure chip.

[0040] As an example, the bonded three-layer chip is placed on a heating plate for post-curing to enhance bonding strength and improve overall sealing.

[0041] Step 5: Connect the interface;

[0042] The sample loading port, injection port, and fluid outlet correspond to the other ends of the cell sample loading channel, cell injection channel, and sorting outlet channel, respectively. After verification, three sets of vertically oriented PTFE capillaries are inserted into the bonding surfaces at the sample loading port, injection port, and fluid outlet, so that the sample loading port connects to the cell sample loading channel, the injection port connects to the cell injection channel, and the fluid outlet connects to the sorting outlet channel; ultimately, sample loading, rinsing, and sorting cell reception are achieved.

[0043] The beneficial effects of this invention are:

[0044] 1. Excellent in-situ detection capability: Utilizing the ultra-thin quartz window on the top layer, Raman laser can irradiate single cells captured in microchannels by optical tweezers with almost no loss and no background interference, thereby obtaining high-quality, high signal-to-noise ratio in-situ Raman spectra that truly reflect the physiological state of cells in a liquid environment.

[0045] 2. Highly efficient and precise sorting function: The chip integrates optical tweezers technology, which allows the optical tweezers laser to capture and manipulate target cells through a quartz window. Combined with the chemical information provided by Raman spectroscopy, the operator can make real-time decisions to drag specific target cells (such as cells that appear to be cancerous according to Raman spectroscopy) from the main stream to the sorting exit channel using optical tweezers, achieving non-destructive, non-contact, and highly precise single-cell sorting.

[0046] 3. Low cost and ease of fabrication: The microfluidic structure of the chip core is made of easily processed PDMS material, and the processing methods (such as molding or femtosecond laser processing) are mature, low-cost, and have short cycle times. Quartz material is used only in critical areas related to the optical path, and the use of ultra-thin quartz significantly reduces the cost of expensive materials. The three-layer bonding process is stable and reliable, making it very suitable for standardized production and widespread application. Attached Figure Description

[0047] Figure 1 This is a side view of the microfluidic chip with in-situ Raman detection combined with optical tweezers sorting according to the present invention (chip inverted state).

[0048] Figure 2 This is a schematic diagram illustrating the working principle of a microfluidic chip based on in-situ Raman detection combined with optical tweezers sorting according to the present invention.

[0049] Figure 3 This is a schematic diagram of the microchannel network structure of a microfluidic chip for in-situ Raman detection combined with optical tweezers sorting according to the present invention. Detailed Implementation

[0050] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Figures 1 to 3 As shown.

[0051] A microfluidic chip combining in-situ Raman detection and optical tweezers sorting, and its fabrication method, wherein:

[0052] A microfluidic chip for in-situ Raman detection combined with optical tweezers sorting includes: a three-layer composite structure, from top to bottom as follows:

[0053] (The chip needs to be inverted during actual use, so the top cover layer is at the bottom.)

[0054] Top cover layer 101: is an ultra-thin quartz sheet. The top cover layer 101 serves as an optical window, providing a working surface with high transmittance and high optical quality for the incident and emitting of Raman lasers and optical tweezers lasers. At the same time, its inherent low Raman background characteristics ensure a low signal-to-noise ratio of the detection signal.

[0055] As an example, the thickness of the ultrathin quartz sheet is between 100 and 200 micrometers.

[0056] Intermediate flow channel layer 102: A thin film structure made of polydimethylsiloxane (PDMS) material; a pre-designed microchannel network is fabricated in the thin film structure using a femtosecond laser;

[0057] The microfluidic network includes a cell injection channel 301, a cell loading channel 302, and a sorting outlet channel 303. The microfluidic network enables single-cell queuing and focusing of cell samples and provides operating space and path for optical tweezers sorting.

[0058] One end of the cell loading channel 302 and one end of the sorting outlet channel 303 are horizontally connected and conductive. A third conductive channel is led out from one side of the junction of the two, connecting to one end of the Raman spectroscopy detection and optical tweezers sorting area 203. pull 203 Mann spectroscopy detection and optical tweezers sorting area The other end is connected to one end of the cell injection channel 301;

[0059] As an example, the thickness of the thin film structure is between 10 and 100 micrometers.

[0060] As an example, the third conductive channel is perpendicular to the cell loading channel and the sorting outlet channel.

[0061] Base layer 103: is a thick quartz substrate;

[0062] The base layer 103 provides mechanical support for the entire chip, ensuring the flatness and structural stability of the chip; the thick quartz substrate is processed with femtosecond laser to have three through holes, which serve as the sample loading port, injection port and fluid outlet of the chip, respectively; corresponding to the other end of the cell sample loading channel, the other end of the cell injection channel and the other end of the sorting outlet channel;

[0063] As an example, the thickness of the thick quartz substrate is 1 mm.

[0064] As an example, the three through holes are all the same size, with a diameter of 1 mm.

[0065] As an example, the Raman spectroscopy detection and optical tweezers sorting area has a circular structure.

[0066] As an example, the thick quartz substrate has two symmetrically arranged cuboid structures on both sides for processing three through holes.

[0067] The top cover layer 101, the intermediate flow channel layer 102, and the base layer 103 are all bonded together as a closed whole structure through oxygen plasma treatment, forming a complete microfluidic channel that can carry liquid.

[0068] A method for fabricating a microfluidic chip using in-situ Raman detection combined with optical tweezers sorting includes:

[0069] Step 1: Fabrication of intermediate flow channel layer 102;

[0070] The SU-8 positive mold was fabricated on a silicon wafer using a photolithography process.

[0071] The PDMS prepolymer was mixed with a curing agent, degassed, and then poured onto an SU-8 positive mold. After heating and curing, it was peeled off to obtain a thin film structure. Then, a microfluidic network was directly fabricated on the surface of the thin film structure using a femtosecond laser direct writing method.

[0072] As an example, SU-8 is a photoresist, and the SU-8 male mold is a male film structure prepared using a specific process.

[0073] Step 2: Processing the base layer 103;

[0074] Three identical through holes were machined on a thick quartz substrate using a femtosecond laser to serve as the sample loading hole, injection port, and fluid outlet;

[0075] Step 3: Preliminary treatment of the bonding surface;

[0076] The ultrathin quartz sheet, the intermediate layer with microfluidic network, and the thick quartz substrate are cleaned and dried; then, the bonding surfaces of the intermediate fluidic layer and the ultrathin quartz sheet are treated with oxygen plasma.

[0077] Step 4: Align and bond;

[0078] The lower surface of the processed intermediate flow channel layer is aligned and pressed with the thick quartz substrate to form a preliminary bond; then the ultra-thin quartz cover sheet is aligned and pressed with the upper surface of the intermediate flow channel layer to form the final three-layer structure chip.

[0079] As an example, the bonded three-layer chip is placed on a heating plate for post-curing to enhance bonding strength and improve overall sealing.

[0080] Step 5: Connect the interface;

[0081] The sample loading port 202, injection port 201, and fluid outlet 204 correspond to the other ends of the cell loading channel 302, cell injection channel 301, and sorting outlet channel 303, respectively. After verification, three sets of vertically oriented PTFE capillaries are inserted into the bonding surfaces at the sample loading port 202, injection port 201, and fluid outlet 204, so that the sample loading port 202 is connected to the cell loading channel 302, the injection port 201 is connected to the cell injection channel 301, and the fluid outlet 204 is connected to the sorting outlet channel 303, thus achieving sample loading, rinsing, and cell sorting reception.

[0082] To better illustrate the design principles of this invention, specific embodiments are described below:

[0083] Example 1:

[0084] The microfluidic chip provided by this invention consists of three layers: upper, middle and lower.

[0085] The top cover layer 101 is a square ultrathin quartz sheet with a size of 24mm × 75mm and a thickness of 100 micrometers; its surface is smooth and optically polished.

[0086] The intermediate flow channel layer 102 is made of PDMS film (Sylgard 184, prepolymer to curing agent ratio 10:1). A network of interconnected microchannels is fabricated within it using femtosecond laser processing. Figure 3 ).

[0087] Cell suspension flows in through "injection port 201". After sample addition, a large number of samples are stored in "Raman spectroscopy detection and optical tweezers sorting area 203". Raman spectroscopy can be used to detect cells in "Raman spectroscopy detection and optical tweezers sorting area 203". The target cells are sorted by optical tweezers sorting function. Buffer solution flows in through "sample loading well 202". The sorted cells will flow out through "fluid outlet 204" along with the buffer solution.

[0088] The base layer 103 is a thick quartz substrate measuring 24mm × 75mm and 1mm thick, providing rigid support for the chip. Holes are drilled into the thick quartz substrate to pre-drill sample loading holes, continuous flow inlet holes, and continuous flow outlet holes.

[0089] During preparation, the bonding surface between the PDMS intermediate flow channel layer 102 and the ultrathin quartz top cover layer 101 was first treated with oxygen plasma (power 50W, time 50s).

[0090] Then, first align and press the PDMS layer 102 with the base layer 103, then cover it with the top cover layer 101 and press it gently. Finally, place it on an 80°C hot plate and heat it for 1 hour to cure for permanent bonding.

[0091] Finally, PDMS blocks with pre-drilled holes are bonded to the sample loading port, injection port, and fluid outlet port, and then PTFE capillary tubes are connected, thus completing the chip fabrication.

[0092] Example 2:

[0093] The working process of a chip;

[0094] See Figure 1 When using the chip, the cell suspension is injected into the sample well using an external syringe. The cells will stabilize in the "Raman spectroscopy detection and optical tweezers sorting area 203". At this time, the external injection pump injects buffer into the chip through the sample well 202 at a flow rate of 20 μL / min. Single-cell sorting can be achieved through Raman spectroscopy detection and optical tweezers sorting. The sorted cells will flow into the collection tube from the fluid outlet 204.

[0095] Example 3:

[0096] Design of sorting equipment;

[0097] A confocal Raman spectrometer is integrated with an optical tweezers system. The objective lens is located above the chip, directly facing the ultrathin quartz cap layer 1. A 1064nm laser beam is used to form the optical tweezers, and a 532nm laser beam is used to excite the Raman signal.

[0098] Select the cell to be tested in the “Raman spectroscopy detection / optical tweezers sorting area 203”, turn on the Raman laser to irradiate it, and the spectrometer collects its Raman scattered light and generates a spectrum.

[0099] The computer performs real-time analysis of the acquired spectra. If the cell's spectrum matches the target characteristics (e.g., the peak intensity of a specific biomarker exceeds a threshold), the system controls optical tweezers to drag the cell out of the "Raman spectroscopy detection and optical tweezers sorting area 203" by moving the laser focus or the sample stage, and into the fluid outlet 204, where it is finally collected into a test tube. This enables in-situ detection of mixed cell populations and precise sorting based on chemical information.

[0100] It should be noted that, for the sake of simplicity, the method embodiments are all described as a series of actions. However, those skilled in the art should understand that this application is not limited to the described order of actions, because according to this application, some steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also understand that the embodiments described in the specification are all optional embodiments, and the actions involved are not necessarily required by this application.

[0101] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

[0102] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed in this application can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0103] Those skilled in the art will understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0104] The above description is only a preferred embodiment of the present invention. It should be understood that the above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention, and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the idea and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microfluidic chip for in-situ Raman detection combined with optical tweezer sorting, characterized in that, From top to bottom in turn includes: Top cover layer: a layer of ultra-thin quartz sheet; The middle flow channel layer: thin film structure made of polydimethylsiloxane material; by femtosecond laser in the sheet structure processing out the preset micro-channel network; The micro-channel network includes: cell injection channel, cell loading channel and sorting outlet channel; one end of the cell loading channel and one end of the sorting outlet channel are horizontally connected and conductive, and a third conductive channel is led out on one side of the joint part, which is connected with one end of the Raman spectrum detection and optical tweezers sorting area, and the other end of the Raman spectrum detection and optical tweezers sorting area is connected with one end of the cell injection channel; The base layer: a layer of thick quartz substrate; The thick quartz substrate is processed by femtosecond laser to have three through holes, which are used as sample hole, injection port and fluid outlet of the chip respectively; corresponding to the other end of the cell loading channel, the other end of the cell injection channel and the other end of the sorting outlet channel respectively; The top cover layer, the middle flow channel layer and the base layer are bonded into a closed whole structure by oxygen plasma treatment, forming a complete and liquid-bearing microfluidic channel.

2. The microfluidic chip for in-situ Raman detection combined with optical tweezer sorting according to claim 1, wherein, The thickness of the ultra-thin quartz sheet is between 100-200 microns.

3. The microfluidic chip of claim 1, wherein, The thickness of the thin film structure is between 10-100 microns.

4. The microfluidic chip of claim 1, wherein, The third conductive channel is perpendicular to the cell loading channel and the sorting outlet channel.

5. The microfluidic chip of claim 1, wherein, The thickness of the thick quartz substrate is 1mm.

6. The microfluidic chip of claim 1, wherein, The three through holes are equal in size, with a diameter of 1mm.

7. The microfluidic chip of claim 1, wherein, The Raman spectrum detection and optical tweezers sorting area is a circular structure.

8. The microfluidic chip of claim 1, wherein, Two square structures are symmetrically arranged on both sides of the thick quartz substrate for processing the three through holes.

9. A method for fabricating a microfluidic chip for in-situ Raman detection combined with optical tweezer sorting, characterized in that, It includes: Step one, processing and manufacturing of the middle flow channel layer; SU-8 positive mold is prepared on the silicon wafer by photolithography process; after mixing and degassing, the PDMS prepolymer is poured on the SU-8 positive mold, heated and cured, and then peeled off to obtain a thin film structure; then the micro-channel network is directly processed on the surface of the thin film structure by femtosecond laser direct writing method; Step two, base layer processing; Three identical through holes are processed on the thick quartz substrate by femtosecond laser, which are used as sample hole, injection port and fluid outlet; Step three, preliminary treatment of the bonding surface; The ultra-thin quartz sheet, the middle layer with micro-channel network and the thick quartz substrate are cleaned and dried; then the bonding surface of the middle flow channel layer and the bonding surface of the ultra-thin quartz sheet are treated by oxygen plasma; Step four, alignment bonding; The lower surface of the treated middle flow channel layer is aligned with the thick quartz substrate and pressed to form a preliminary bonding; Then the ultra-thin quartz cover sheet is aligned with the upper surface of the middle flow channel layer and pressed to form the final three-layer structure chip; Step five, interface communication; The sample hole, injection port and fluid outlet correspond to the other end of the cell loading channel, the other end of the cell injection channel and the other end of the sorting outlet channel respectively; after detection, three groups of vertical ptfe capillary tubes are inserted into the bonding surface of the sample hole, injection port and fluid outlet to realize the connection of the sample hole with the cell loading channel, the injection port with the cell injection channel and the fluid outlet with the sorting outlet channel; finally, the sample loading, washing and receiving of sorted cells are realized.

10. The method according to claim 9, wherein the microfluidic chip is prepared by in-situ Raman detection combined with optical tweezer sorting. The SU-8 male mold is a male film structure under a preparation process, and the three-layer structure chip after bonding is placed on a heating plate to perform a post-curing operation, so as to enhance the bonding strength and improve the overall sealing performance.

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