Bedside rapid detection chip of biochemical parameters based on microfluidic electrochemical sensing and preparation process

Abstract

The application discloses a biochemical parameter bedside rapid detection chip based on micro-fluidic electrochemical sensing and a preparation process, and aims to solve the problems of single index and insufficient anti-complex biological matrix interference capability of the existing bedside rapid detection chip. Technical scheme points are as follows: the chip substrate, the micro-fluid channel and the electrochemical sensor module are integrated in the chip substrate, the micro-fluid channel comprises a sample inlet, a filter area, a mixing section, a detection area and a waste liquid outlet which are sequentially communicated, the inner cavity of the filter area is flat and is provided with a plurality of micro columns which are uniformly distributed, the spacing between the micro columns is between 15 and 30 mu m, the mixing section is coated with soluble dry reagents, and the detection end of the electrochemical sensor module is exposed to the detection area.

Description

Technical Field

[0001] This invention relates to an electrochemical rapid detection chip, and more specifically, to a bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing and its fabrication process. Background Technology

[0002] Intracranial infections pose a significant challenge to neurosurgery and neurocritical care, with their pathological processes often accompanied by substantial changes in the biochemical composition of cerebrospinal fluid (CSF). Against this backdrop, CSF testing provides crucial evidence for the clinical diagnosis of intracranial infectious diseases, offers objective indicators for assessing disease severity and treatment response, and provides real-time information for dynamic monitoring and treatment plan adjustments. With the advancements in neurocritical care and precision medicine, clinical demands for CSF testing have increased: not only are faster testing speeds and more comprehensive information required, but also higher monitoring frequencies. Consequently, the testing paradigm is gradually shifting from single-indicator analysis to multi-parameter comprehensive assessment. Currently, cerebrospinal fluid (CSF) testing technology mainly follows two technical paths: one is a standardized, high-precision testing system centered on a central laboratory, encompassing various analytical methods such as biochemistry, cytology, microbiology, and molecular diagnostics. This system can achieve highly sensitive detection of multiple indicators, but it relies on large-scale instruments and specialized experimental environments, resulting in complex testing procedures. Samples need to undergo ex vivo transport and centralized analysis, leading to problems such as long testing cycles and high costs. The other type is point-of-care testing technology aimed at shortening testing time. It features relatively simple operation and fast testing speed, but it generally faces limitations such as single detection indicators, limited quantitative capabilities, and insufficient resistance to interference from complex biological matrices (cell debris, large molecular protein aggregates, and other impurities). Moreover, it often relies on external testing equipment, and its testing stability and result repeatability still need to be improved.

[0003] In summary, existing cerebrospinal fluid (CSF) testing technologies still struggle to balance accuracy and timeliness, failing to simultaneously meet clinical needs for rapid response and reliable multi-parameter detection. Therefore, there is an urgent need to develop a new testing technology to achieve rapid and reliable detection of key CSF indicators under limited sample conditions. Summary of the Invention

[0004] This invention provides a bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing and its fabrication process; it solves the problems of existing bedside rapid detection chips having limited indicators and insufficient resistance to interference from complex biological matrices (cell debris, macromolecular protein aggregates, and other impurities).

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing, comprising a chip substrate, a microfluidic channel, and an electrochemical sensor module, wherein the microfluidic channel and the electrochemical sensor module are both integrated within the chip substrate; the microfluidic channel includes a sample inlet, a filtration zone, a mixing section, a detection zone, and a waste outlet connected in sequence; the inner cavity of the filtration zone is flat and has formed a plurality of uniformly distributed micropillars, the spacing between the micropillars being between 15 and 30 μm; the mixing section is coated with a soluble dry reagent; the detection end of the electrochemical sensor module is exposed within the detection zone.

[0006] Furthermore, the chip substrate is also provided with a waste liquid pool that is connected to the waste liquid outlet.

[0007] Furthermore, the chip substrate includes a PDMS microfluidic device and a flexible substrate. The PDMS microfluidic device is composed of a top PDMS thin layer, a middle PDMS thin layer, and a bottom PDMS thin layer. The sample inlet and the filtration zone are located on the top PDMS thin layer, and a main channel connects the sample inlet and the filtration zone. The mixing section is located on the middle PDMS thin layer, and at least two sections are provided in parallel. A branch channel connects the filtration zone and the mixing section. Several detection zones are provided and located on the bottom PDMS thin layer. The waste liquid outlet starts on the bottom PDMS thin layer and passes through the middle PDMS thin layer and the top PDMS thin layer successively.

[0008] Furthermore, a waste liquid pool is located on the top PDMS thin layer and is connected to the waste liquid outlet. The waste liquid pool is an airbag structure with elastic recovery.

[0009] This invention also provides a fabrication process for a bedside rapid detection chip: To fabricate a flexible electrochemical sensing array, a flexible polyimide substrate is first prepared on a silicon wafer. Then, a metal electrode layer is deposited and patterned on the flexible polyimide substrate to form three working electrodes. Next, the nanostructure of the working electrodes is modified and specific recognition molecules are immobilized. Finally, the working electrodes are encapsulated with Nafion. To fabricate PDMS microfluidic devices, a top-layer positive mold, a middle-layer positive mold, and a bottom-layer positive mold are first fabricated using the SU-8 positive mold fabrication process. The three positive molds are then subjected to surface silanization treatment. The top-layer, middle-layer, and bottom-layer PDMS thin layers are then fabricated using the positive molds. Finally, the three PDMS thin layers are aligned and pre-bonded. The mixing section within the PDMS microfluidic device is pre-coated with a dry reagent, a dry reagent precursor solution is precisely injected into the mixing section, and then vacuum dried. Chip alignment and bonding integration involves placing the underlying PDMS thin layer on the PDMS microfluidic device face-to-face with the flexible polyimide substrate in a thermoforming bonding machine, heating and pressurizing for an appropriate time to form a permanent seal bond, and then performing edge sealing and reinforcement.

[0010] In summary, the present invention has the following beneficial effects: 1. Through the design of multi-layer PDMS structure, parallel hybrid section and multiple detection areas, multiple biochemical parameters can be detected simultaneously and rapidly on the same bedside detection chip, solving the problem of single detection index of traditional chips; 2. The filtration area adopts a flat inner cavity and a uniformly distributed micropillar structure (spacing 15-30 μm), which can effectively trap impurities such as cell debris and large protein aggregates, significantly improving the chip's anti-interference ability against complex biological samples. 3. Sample injection, filtration, mixing, reaction, detection, and waste collection are all integrated into the same chip, eliminating the need for additional instruments. Sample pretreatment and detection are completed in one step, truly achieving rapid point-of-care testing. 4. The chip adopts a combination of multi-layer PDMS and flexible substrate, which is small in size and highly integrated; the waste liquid pool adopts an elastic airbag structure, which can realize active collection of waste liquid without the need for external negative pressure / pump, making it more convenient and safer to use and less prone to leakage. Attached Figure Description

[0011] Figure 1 This is a schematic diagram showing the overall structure of the present invention disassembled. Figure 2 for Figure 1 A diagram from another perspective. Detailed Implementation

[0012] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0013] In the description of this invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top / bottom", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0014] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "set up / connected," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0015] The present invention will now be described in detail with reference to the accompanying drawings.

[0016] Example 1: See Figure 1 and Figure 2 A bedside rapid detection chip for detecting biochemical parameters of cerebrospinal fluid based on microfluidic electrochemical sensing includes a chip substrate 100, a microfluidic channel, and an electrochemical sensor module. The microfluidic channel and the electrochemical sensor module are both integrated in the chip substrate. The microfluidic channel includes a sample inlet 10, a filtration zone 20, a mixing section 30, a detection zone 40, and a waste outlet 50 connected in sequence. The sample inlet, located at one end of the chip substrate, is used for direct injection of cerebrospinal fluid samples (≤500μL) and adopts a conical funnel structure for easy manual sample addition or docking with a micro-injection pump. The inner cavity of the filtration zone is flat and has several uniformly distributed micropillars 21. The spacing between the micropillars is between 15 and 30 μm, and the diameter of the micropillars is between 40 and 60 μm. They are also staggered. The mixing zone is pre-coated with dry pH buffers (such as phosphate, Tris-HCl, etc.). As the sample flows through the mixing zone, the dry buffers are rapidly reconstituted, adjusting the sample pH to the optimal range for the electrochemical reaction (e.g., pH 7.4). The dry buffers also contain supporting electrolytes (such as KCl, potassium ferricyanide), providing the necessary ionic conductivity environment for the subsequent electrochemical reaction. The mixing section features a serpentine or fishbone-shaped microchannel structure to enhance chaotic convection between the sample and the reconstituted reagents, ensuring uniform pH adjustment and electrolyte distribution. The cross-sectional area of ​​the channels within the mixing section ranges from 15,000 to 30,000 μm. 2 Between 20 and 50 mm in length, ensure that the sample stays in the mixing section for more than 5 seconds to allow for full reconstitution of the dry reagent and complete pH adjustment; The detection end of the electrochemical sensor module is exposed in the detection area, which contains multiple independent detection areas. Each chamber corresponds to three working electrodes. The chamber volume is 10-100μL and is in direct contact with the working electrodes to ensure the stability of the electrochemical reaction area. Specific recognition molecules are pre-encapsulated on the working electrodes of the detection area. Each detection chamber is independent of each other and supports the simultaneous detection of multiple parameters such as glucose, lactate, and total protein. The chip substrate is also equipped with a waste liquid pool 70 connected to the waste liquid outlet, which is used to discharge the waste liquid after the reaction, maintain the pressure balance in the microchannel, and realize fully enclosed detection to avoid the risk of biological contamination.

[0017] Specifically, the chip substrate includes a PDMS microfluidic device 110 and a flexible substrate 120. The PDMS microfluidic device consists of a top PDMS thin layer 111, a middle PDMS thin layer 112, and a bottom PDMS thin layer 113. The sample inlet and filtration zone are located on the top PDMS thin layer, and a main channel 80 connects the sample inlet and the filtration zone. The mixing section is located on the middle PDMS thin layer, and at least two sections are connected in parallel. A branch channel 90 connects the filtration zone and the mixing section. There are three detection zones, located on the bottom PDMS thin layer. The waste liquid outlet starts on the bottom PDMS thin layer and passes through the middle PDMS thin layer and the top PDMS thin layer successively. Based on the PDMS thin layer stacking molding, the structural design facilitates mold processing, bonding, and large-scale production, making it suitable for the industrial application of point-of-care rapid testing products.

[0018] Specifically, the waste liquid tank is located on the top PDMS thin layer and is connected to the waste liquid outlet. The waste liquid tank is an airbag structure with elastic recovery.

[0019] This embodiment also discloses a fabrication process for a bedside rapid detection chip: The first step is to fabricate a flexible electrochemical sensing array. (1) Preparation of flexible substrate: spin-coat a sacrificial layer material (such as polymethyl methacrylate, PMMA) with a thickness of 2 μm on a clean silicon wafer surface and bake at 180°C for 2 min to cure; spin-coat a polyimide precursor solution with a thickness of about 2-10 μm at a speed of 2000 rpm; place the silicon wafer in a nitrogen-protected environment for high-temperature curing to form a flexible polyimide substrate; cool naturally to room temperature for later use.

[0020] (2) Metal electrode layer deposition and patterning: a titanium adhesion layer and a copper conductive layer are sequentially deposited on the surface of a flexible substrate using magnetron sputtering. Positive photoresist is spin-coated onto the surface of the metal layer, and after pre-baking, ultraviolet lithography is performed through a mask to form an electrode pattern. Copper etchant and titanium etchant are sequentially used for wet etching to form a three-electrode array containing multiple independent working electrodes, counter electrodes and reference electrodes. The remaining photoresist is removed, and the substrate is cleaned with deionized water and dried with nitrogen.

[0021] (3) Modification of the working electrode nanostructure: The working electrode was immersed in HAuCl4 solution in a three-electrode system by electrochemical deposition. The deposition potential and deposition time were controlled to deposit gold nanoparticles on the surface of the working electrode, and the particle size was controlled within the range of 10-100 nm. The electrode was then cleaned with deionized water and dried with nitrogen.

[0022] (4) Immobilization of specific recognition molecules: Using micro-spotting technology, different specific recognition molecules are immobilized on the surface of different working electrodes: glucose detection electrode: glucose oxidase or glucose dehydrogenase is immobilized; lactate detection electrode: lactate oxidase is immobilized; total protein detection electrode: anti-human albumin antibody is immobilized; after spotting, the electrodes are incubated in a humidified chamber at 4°C for 12 hours to allow the recognition molecules to bind stably to the electrode surface; the electrodes are gently washed with phosphate buffer (PBS, pH 7.4) to remove unbound recognition molecules; and the electrodes are dried naturally or by blowing with nitrogen gas.

[0023] (5) Preparation of Nafion encapsulation layer: 0.5% Nafion solution is locally spin- / drop-coated in the working electrode area and dried at room temperature to form an encapsulation layer with a thickness of about 100 nm, which is used to enhance anti-interference ability and stabilize the recognition molecular layer.

[0024] The second step is to fabricate PDMS microfluidic devices. (1) Preparation of SU-8 male molds: Three male molds were prepared, namely: The top positive mold has a molded structure with an inlet, main channel, filtration zone, waste liquid outlet, and alignment mark; the middle positive mold has a molded structure with a branch channel, serpentine mixing channel, waste liquid outlet, and alignment mark; the bottom positive mold has a molded structure with a detection zone, waste liquid outlet, and alignment mark; after hard baking, it is naturally cooled and then surface silanized.

[0025] (2) PDMS casting and curing: PDMS raw material preparation, aluminum foil dam is made on silanized SU-8 positive mold; PDMS mixture is slowly poured, and cured after standing and leveling; demolding, cooling and cutting along the dam, carefully peeling off PDMS to obtain three independent PDMS thin layers (top layer, middle layer and bottom layer).

[0026] (3) Alignment and pre-bonding of multilayer PDMS: Place the three layers of PDMS in the order of bottom layer, middle layer and top layer on the microscope alignment stage; precisely adjust the relative positions of the three layers through the alignment marks (cross marks) so that the top layer sample inlet is aligned with the middle layer filter area inlet; the middle layer mixing area outlet is aligned with the bottom layer detection chamber inlet; there is no misalignment between the detection chamber and the contact area of ​​the subsequent electrodes; inject a small amount of deionized water through the alignment mark hole to temporarily fix it with the aid of surface tension; stack the aligned three layers of PDMS and place them in the oven for hot pressing to achieve reversible bonding; finally clean and dry, and store for later use.

[0027] The third step is to pre-coat the mixing section within the PDMS microfluidic device with dry reagents. (1) Before the PDMS microfluidic layer is bonded to the electrode substrate, a dry reagent precursor solution is precisely injected into the mixing zone channel of the PDMS microfluidic layer; the reagent precursor solution formulation (taking general detection as an example): pH buffer pair: 0.1 M phosphate buffer (PBS, pH 7.4); supporting electrolyte: 0.1 M KCl, 1 mM potassium ferricyanide; Injection method: A micro-injection pump is used to slowly inject the solution through the injection port or a specially designed infusion port, ensuring that the injection volume completely fills the mixing zone channel; (2) Immediately after injection, the PDMS microfluidic layer is placed in a vacuum drying oven and dried at room temperature to remove moisture. After drying, the pH buffer pair and supporting electrolyte are attached to the inner wall of the mixing zone channel in the form of a uniform dry film. The dried PDMS microfluidic layer is stored in a desiccator for later use.

[0028] The fourth step is chip alignment and bonding integration. (1) Oxygen plasma surface treatment: The PDMS microfluidic layer (bonding surface facing up) and the flexible substrate (electrode surface facing up) are placed in a plasma cleaner for surface activation treatment. During the treatment, the mixing area and three-electrode area of ​​PDMS can be shielded to avoid plasma contact with dry reagents and specific recognition molecules.

[0029] (2) Microscopic alignment and bonding: Place the treated PDMS microfluidic layer on the microscopic alignment stage with the bonding surface facing up; place the flexible substrate on the moving platform with the electrode surface facing down; under the microscope, accurately align the detection chamber and the working electrode and make them contact each other; place it in the hot press bonding machine, heat and pressurize for an appropriate time to form a permanent seal bond; let it cool naturally to room temperature.

[0030] (3) Edge sealing and reinforcement: Apply a small amount of uncured PDMS to the chip edge and heat it again to cure it to enhance the edge sealing; check the appearance of the chip to ensure that there are no bubbles, no misalignment and no leakage.

[0031] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing, comprising a chip substrate, a microfluidic channel, and an electrochemical sensor module, wherein the microfluidic channel and the electrochemical sensor module are both integrated within the chip substrate, characterized in that: The microfluidic channel includes an inlet, a filtration zone, a mixing section, a detection zone, and a waste outlet connected in sequence; the inner cavity of the filtration zone is flat and has several micropillars evenly distributed among each other, with the spacing between the micropillars between 15 and 30 μm; the mixing section is coated with soluble dry reagents; the detection end of the electrochemical sensor module is exposed in the detection zone.

2. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 1, characterized in that: The micropillars have a diameter between 40 and 60 μm and are interspersed with each other.

3. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 1, characterized in that: The mixing section is a serpentine microchannel structure or a fishbone-shaped micromixing structure.

4. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 3, characterized in that: The cross-sectional area of ​​the channel within the mixing section is between 15,000 and 30,000 μm. 2 Between 20 and 50 mm in length, ensure that the sample stays in the mixing section for more than 5 seconds.

5. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 1, characterized in that: The chip substrate is also provided with a waste liquid pool that is connected to the waste liquid outlet.

6. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 5, characterized in that: The chip substrate includes a PDMS microfluidic device and a flexible substrate. The PDMS microfluidic device is composed of a top PDMS thin layer, a middle PDMS thin layer, and a bottom PDMS thin layer. The sample inlet and the filtration zone are located on the top PDMS thin layer, and a main channel connects the sample inlet and the filtration zone. The mixing section is located on the middle PDMS thin layer, and at least two sections are provided in parallel. A branch channel connects the filtration zone and the mixing section. Several detection zones are provided and located on the bottom PDMS thin layer. The waste liquid outlet starts on the bottom PDMS thin layer and passes through the middle PDMS thin layer and the top PDMS thin layer successively.

7. The bedside rapid detection chip for biochemical parameters based on microfluidic electrochemical sensing according to claim 6, characterized in that: The waste liquid tank is located on the top PDMS thin layer and is connected to the waste liquid outlet. The waste liquid tank is an airbag structure with elastic recovery.

8. A fabrication process for a bedside rapid detection chip, characterized in that: To fabricate a flexible electrochemical sensing array, a flexible polyimide substrate is first prepared on a silicon wafer. Then, a metal electrode layer is deposited and patterned on the flexible polyimide substrate to form three working electrodes. Next, the nanostructure of the working electrodes is modified and specific recognition molecules are immobilized. Finally, the working electrodes are encapsulated with Nafion. To fabricate PDMS microfluidic devices, a top-layer positive mold, a middle-layer positive mold, and a bottom-layer positive mold are first fabricated using the SU-8 positive mold fabrication process. The three positive molds are then subjected to surface silanization treatment. The top-layer, middle-layer, and bottom-layer PDMS thin layers are then fabricated using the positive molds. Finally, the three PDMS thin layers are aligned and pre-bonded. The mixing section within the PDMS microfluidic device is pre-coated with a dry reagent, a dry reagent precursor solution is precisely injected into the mixing section, and then vacuum dried. Chip alignment and bonding integration involves placing the underlying PDMS thin layer on the PDMS microfluidic device face-to-face with the flexible polyimide substrate in a thermoforming bonding machine, heating and pressurizing for an appropriate time to form a permanent seal bond, and then performing edge sealing and reinforcement.

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