25-hydroxyvitamin D dissociation-neutralization-quantification integrated micro-fluidic chip

Abstract

The invention is applicable to the technical field of in-vitro diagnosis and immunodetection, and provides a 25-hydroxyvitamin D dissociation-neutralization-quantification integrated micro-fluidic chip which comprises a closed shell, a PET (polyethylene terephthalate) film layer and a PMMA (polymethyl methacrylate) substrate layer which are sequentially assembled from top to bottom, the PET film layer is provided with a continuous micro-channel network, the micro-channel network is formed by alternately connecting straight channels and serpentine channels in series, and an alkalization dissociation area, an acidification neutralization area, a delay diffusion area and a detection area are sequentially integrated in the liquid flow direction; according to the invention, the chip is self-driven by capillary force and is combined with the mixing enhancement and driving force supplement effects of the micro-column array, so that efficient dissociation, rapid neutralization and sequential competitive immunodetection of 25-hydroxyvitamin D in a sample are realized, an external pump valve or artificial liquid transfer is not needed, and full-automatic and rapid quantitative detection of'sample input-result output 'is achieved; the problems of complicated operation, difficulty in miniaturization and large detection error in the prior art are solved, and the method is suitable for real-time detection scenes.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and immunoassay technology, and particularly relates to an integrated microfluidic chip for the dissociation, neutralization and quantification of 25-hydroxyvitamin D. Background Technology

[0002] Existing 25-hydroxyvitamin D detection technologies are mainly divided into liquid chromatography-tandem mass spectrometry (the gold standard) and immunoassay (widely used in clinical practice). Both methods require treating the sample with strong acid or strong alkali to release 25-hydroxyvitamin D from vitamin D-binding protein (VDBP) (dissociation step), followed by pH adjustment through acid-base neutralization, and finally transfer to the detection platform for quantitative detection (detection step). Although microfluidic technology has integration advantages, existing microfluidic integration solutions are difficult to adapt to the synergistic requirements of "slow dissociation, rapid neutralization, and non-equilibrium competitive detection," failing to achieve truly integrated detection.

[0003] The existing technology has the following problems: The operation is cumbersome and cannot be fully automated: the existing detection process requires multiple independent steps of "dissociation-neutralization-detection", which relies on manual or semi-automatic sample addition, incubation, transfer and other operations. Even on large automated analyzers, the detection sequence will be disrupted, the detection time of a single sample will be extended, the equipment throughput will be reduced, and full automation cannot be achieved. The system is complex and difficult to miniaturize and integrate: Existing solutions rely on multiple independent functional modules to complete each step, resulting in large and expensive equipment that cannot meet the needs of miniaturization and integration in real-time testing scenarios such as primary healthcare, bedside or home, thus limiting the expansion of application scenarios. Multiple sources of error and poor reproducibility: Repeated manual and mechanical interventions significantly increase the risk of cross-contamination, pipetting errors, and fluctuations in reaction conditions, directly affecting the accuracy of test results and resulting in poor reproducibility, making it difficult to guarantee the reliability of test data; Therefore, an integrated microfluidic chip for the dissociation, neutralization, and quantification of 25-hydroxyvitamin D is needed to solve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide an integrated microfluidic chip for the dissociation, neutralization, and quantification of 25-hydroxyvitamin D to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A microfluidic chip integrating 25-hydroxyvitamin D dissociation-neutralization-quantification comprises a sealed shell, a PET film layer, and a PMMA substrate layer assembled from top to bottom. The PET film layer is characterized by a continuous microfluidic network, which is formed by alternating series of straight and serpentine channels, and sequentially integrates an alkalization dissociation zone, an acidification neutralization zone, a delayed diffusion zone, and a detection zone along the liquid flow direction. The PMMA substrate layer has a micropillar array at the specific channel junctions corresponding to the microfluidic network, forming a microvalve; The enclosed shell is provided with a sample injection hole and a transparent detection window. The starting end of the microfluidic network is connected to the sample injection hole, and the end is set to the transparent detection window, so as to realize the integration of sample dissociation, neutralization and quantitative detection. The multi-layer assembly structure (enclosed shell, PET film layer, PMMA substrate layer) ensures the chip's sealing and structural stability. The alternating "straight channel-serpentine channel" microfluidic network enables the orderly connection of each functional area. The micro-pillar array micro-valve works in conjunction with the flow channels to solve the problems of low mixing efficiency and capillary drive force attenuation in long flow paths in microfluidics. The whole system achieves integrated dissociation, neutralization, and detection without the need for external driving equipment, laying the structural foundation for fully automated detection.

[0006] In a further technical solution, the alkaline decomposition zone includes a first straight channel and a first serpentine channel, wherein a dry alkaline reagent is pre-placed in the middle of the first straight channel; The acidification and neutralization zone includes a second straight channel and a second serpentine channel, with a dry acidic neutralization buffer pre-placed in the middle of the second straight channel; The first straight channel of the alkaline dissociation zone is pre-filled with dry alkaline reagent to ensure rapid dissolution and formation of a strongly alkaline environment as the sample flows through. The first serpentine channel extends the reaction time and promotes thorough mixing by increasing the flow path length and inducing Dean flow, ensuring complete denaturation and dissociation of VDBP. The second straight channel of the acidification and neutralization zone is pre-filled with dry acidic neutralization buffer. The second serpentine channel enables rapid and uniform mixing of acid and base, avoiding the strong alkaline environment from disrupting subsequent immune reactions and ensuring that the pH of the system is quickly adjusted to the neutral range.

[0007] In a further technical solution, the delayed diffusion region includes a third straight channel and a third serpentine channel, and the PMMA substrate layer corresponding to the region of the third straight channel is pre-positioned with a dried 25-hydroxyvitamin D competitive antigen labeled with signal molecules; The third straight channel in the delayed diffusion region contains a pre-placed dried labeled competitive antigen. The third serpentine channel extends the flow path length, providing sufficient time for the dissolution and diffusion of the competitive antigen. Molecular diffusion achieves the difference in movement rates between the competitive antigen and the analyte antigen, creating conditions for sequential competitive detection.

[0008] In a further technical solution, the lower surface of the PET film layer corresponding to the fourth straight channel is pre-coated with a limited amount of anti-25-hydroxyvitamin D capture antibody; The fourth straight channel of the detection area is pre-packaged with a limited amount of captured antibodies to ensure the specific binding of antibodies to antigens. The limited design is adapted to the principle of competitive immunoassay, providing a basis for quantitative analysis. The transparent detection window facilitates signal reading and ensures the availability of test results.

[0009] In a further technical solution, the micropillars of the micropillar array have a diameter of 50-100μm and a spacing of 20-30μm. The microvalve is not located between the third straight channel and the third serpentine channel, but only at the junction of the remaining straight channels and serpentine channels. The staggered micropillar array enhances the mixing effect by disturbing the liquid flow and improves the capillary driving force through the microporous structure, compensating for the attenuation of capillary force in long processes; the microvalve position design avoids premature mixing of labeled reagents, ensures the realization of the sequential competition mechanism, and further optimizes the detection accuracy.

[0010] In a further technical solution, when the PET film layer and the PMMA substrate layer are in natural contact, a gap of 1-10 μm is formed. After the liquid enters, the PET film layer is lifted up to form a microchannel with a depth of 20-50 μm. The width of all microchannels is 1 mm. The dynamic formation of microchannels adapts to the needs of liquid flow, and the depth range of 20-50μm balances the stability of liquid flow and reaction efficiency. The 1mm channel width ensures sufficient contact between reagents and samples, providing a suitable spatial environment for each reaction step.

[0011] In a further technical solution, the PET film layer is provided with symmetrically distributed semi-circular positioning holes on both sides at a distance of 3mm from the starting end, and the PMMA substrate layer is provided with cylindrical positioning posts that are adapted to the positioning holes, the diameter of the positioning posts being slightly smaller than the diameter of the positioning holes; The matching design of positioning holes and positioning posts enables precise alignment of the PET film layer and the PMMA substrate layer, ensuring that the microfluidic network and each functional area are matched in position, avoiding fluid flow obstruction or reaction failure due to assembly deviation, and improving the assembly accuracy and consistency of the chip.

[0012] In a further technical solution, the drying alkaline reagent is NaOH or KOH, and the drying acidic neutralization buffer is HCl or citrate buffer. After drying, both the alkaline reagent and the acidic neutralization buffer form circular reagent dots with an area of ​​1 mm². The selection of specific alkaline reagents and acidic neutralization buffers ensures effective dissociation and neutralization. The dried circular reagent dots ensure uniform reagent distribution. The 1 mm² area design is adapted to the microchannel size, ensuring sufficient contact and rapid dissolution of the reagent and sample.

[0013] In a further technical solution, the signal molecule is a fluorescent microsphere, a fluorescent dye, or a quantum dot; The adaptability design of multiple signal molecules broadens the ways to read detection signals, meets the adaptation requirements of different detection instruments, and at the same time ensures the stability and sensitivity of the signal, providing a reliable signal basis for quantitative detection.

[0014] In a further technical solution, the PMMA substrate layer is also provided with a waste liquid area, which is made of a water-absorbing material and is connected to the end of the microchannel network; The waste liquid area, made of absorbent material, allows for the collection of liquid after testing, preventing liquid from overflowing and contaminating the testing area or the external environment, thus ensuring the safety of chip use and the cleanliness of the testing environment.

[0015] Through the aforementioned structural synergy, the three independent steps of 25-hydroxyvitamin D detection—dissociation, neutralization, and sequential competitive detection—are integrated into a single chip's continuous microchannels, achieving fully automated "sample in, result out" detection. The multi-layered structure and microchannel design ensure the orderly connection of each functional area. The micropillar array microvalve solves the core challenges of mixing efficiency and driving force in microfluidic technology. The sequential competitive mechanism improves detection sensitivity and linear range. The overall structure is compact, low-cost, and easy to operate, effectively overcoming the shortcomings of existing technologies and meeting the needs of point-of-care testing scenarios. Compared with the prior art, the beneficial effects of the present invention are: This invention is highly integrated, achieving full automation and improving detection efficiency: By using a “straight-snake” alternating microfluidic network, the dissociation, neutralization, and detection functional areas are integrated into a single chip. Combined with capillary self-drive and micropillar array assisted drive, no manual or mechanical liquid transfer is required, avoiding interference from multi-step operations on the detection sequence, significantly shortening the detection time of a single sample, increasing equipment throughput, and solving the problems of cumbersome operation and inability to achieve full automation in existing technologies. This invention features a compact structure that is easy to miniaturize and integrate, expanding its application scenarios: the chip adopts a multi-layer assembly structure, with all functional units integrated on a chip of just a few square centimeters, eliminating the need for independent functional modules, reducing the size and cost of the device, and meeting the needs of miniaturization and integration in real-time testing scenarios such as primary healthcare, bedside, and home use, thus solving the problems of complexity and difficulty in miniaturization of existing technology systems; This invention reduces sources of error and improves detection accuracy and reproducibility: the integrated design reduces manual and mechanical intervention, avoiding cross-contamination, pipetting errors, and fluctuations in reaction conditions; the synergistic effect of the microcolumn array and the serpentine channel ensures uniform mixing of the reaction system, and the sequential competition mechanism optimizes reaction kinetics, improving detection sensitivity and linear range, thus solving the problems of numerous errors and poor reproducibility in existing technologies.

[0016] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0017] Figure 1 This is a three-dimensional exploded view of the structure of the present invention, wherein: a) is a top view of the chip's enclosed shell, showing the overall structural layout of the enclosed shell, clearly marking the specific locations of the sample injection port and the transparent detection window; b) is a top view of the microchannel network of the PET film layer, presenting a continuous microchannel network formed by alternating straight channels and serpentine channels, while also indicating the distribution and corresponding range of the alkalization zone, dissociation zone, acidification zone, neutralization zone, delay zone, and detection zone on the microchannel network; c) is a top view of the dimensions and positions of each functional area and structural component of the chip, clearly marking the length dimensions of the alkalization zone, dissociation zone, acidification zone, neutralization zone, delay zone, and detection zone, while also showing the placement positions of the positioning pillars, marking area, waste liquid area, and microvalve; d) is a top view of the micropillar array, presenting the staggered arrangement of the micropillars, and the corresponding positional relationship between the micropillar array and the microvalve. Figure 2 This is a schematic diagram of the layered assembly and fixing method of the present invention, focusing on the fitting method of the positioning hole and the positioning post; Figure 3 This is a schematic diagram of the transparent detection window structure of the closed shell of the present invention, with its dimensions of 60mm in length and 6mm in width marked, clearly showing the corresponding position of the detection window and the detection area of ​​the microfluidic network below, demonstrating the structural basis for reading the detection signal. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0020] Example 1 like Figure 1-3 As shown, this embodiment of the invention provides an integrated microfluidic chip for the dissociation, neutralization, and quantification of 25-hydroxyvitamin D, comprising a sealed outer shell, a PET film layer, and a PMMA substrate layer assembled from top to bottom. The PET film layer has a continuous microfluidic channel network with alternating "straight-snake" patterns. The PMMA substrate layer has a micropillar array, positioning columns, and a waste liquid area. The sealed outer shell has a sample injection port and a transparent detection window (60 mm long and 6 mm wide).

[0021] In this embodiment, the chip fabrication and principle verification steps are as follows: Material preparation: Select a PET film with a thickness of 100μm, a PMMA substrate with pre-set cylindrical positioning columns (1.5mm high, 0.18mm in diameter), microcolumn array (microcolumn diameter 50-100μm, column spacing 20-30μm, staggered arrangement) and water-absorbing waste liquid area, and prepare a closed shell with sample injection hole and transparent detection window.

[0022] Processing of the PET film layer: A continuous microchannel network is processed using a high-precision laser cutting machine, consisting of the following channels: a first straight channel (15mm long), a first serpentine channel (containing 4 bends, 7mm long in the straight direction), a second straight channel (10mm long), a second serpentine channel (containing 2 bends, 4mm long in the straight direction), a third straight channel (10mm long), a third serpentine channel (containing 3 bends, 5.5mm long in the straight direction), and a fourth straight channel (15mm long). All channels are 1mm wide. Symmetrical semi-circular positioning holes (0.2mm in diameter) are machined on both sides of the PET film 3mm from the starting end. The lower surface of the PET film is plasma-treated to make it hydrophilic.

[0023] Chip assembly: The positioning holes of the PET film are inserted into the positioning posts of the PMMA substrate to achieve precise alignment. Then, the sealing cover is covered and encapsulated by snap-fit ​​to ensure airtightness.

[0024] Validation protocol and data recording: Prepare a mixture of blue dye and PBS, inject 50 μL into the sample injection well, and record the time it takes for the liquid to flow through each channel. The results are shown in the table below: Table 1. Time required for liquid to flow through each microchannel Microchannel Time required (s) First Straight Passage 30 First serpentine passage 180 Second Straight Lane 20 Second serpentine passage 60 Third Straight Lane 20 Third serpentine passage 120 Fourth Straight Lane 25

[0025] The verification results show that the liquid flow can stably flow through all channels under the assistance of capillary force and micropillar array, and the residence time of each channel meets the design requirements of "slow dissociation" and "delayed diffusion", indicating that the chip structure is reasonable.

[0026] Example 2 The difference between this embodiment and Embodiment 1 is that the PET film layer and PMMA substrate layer were pre-coated with specific reagents, and a control group chip was added to verify the detection performance of 25-hydroxyvitamin D and its consistency with existing clinical methods.

[0027] In this embodiment, the detection and performance verification steps are as follows: Materials preparation: Prepare streptavidin, biotin-labeled anti-25-hydroxyvitamin D antibody (concentration 30 μg / mL), 1 M KOH solution, 1 M citric acid solution, fluorescent microsphere-labeled 25-hydroxyvitamin D (concentration 10 μg / mL, as a competitive antigen), serum sample to be tested, Roche chemiluminescence immunoassay system, and fluorescence analyzer.

[0028] Reagent pre-coating: PET film layer: 1.5 μL of streptavidin was spotted in the fourth straight channel detection area, incubated for 1 h, washed and dried, and then 1.6 μL of biotin-labeled anti-25-hydroxyvitamin D antibody was spotted, incubated for 1 h, washed and dried; 1 μL of 1M KOH solution was spotted in the first straight channel alkalization area, and 1 μL of 1M citric acid solution was spotted in the second straight channel acidification area, and dried at 37℃ and 20% relative humidity for 15 min.

[0029] PMMA substrate layer: 1.2 μL of fluorescent microsphere-labeled 25-hydroxyvitamin D was spotted in the labeling area corresponding to the third straight channel and dried at 37°C and 20% relative humidity for 15 min.

[0030] Chip assembly: The assembly method is the same as in Example 1, ensuring accurate positioning and sealed packaging.

[0031] Control group chip fabrication: The structure is the same as the experimental group, except that the labeling area and the detection area are both set in the PET film layer (on the same layer), and a synchronous competition mode is adopted. The remaining steps are the same as the experimental group.

[0032] Detection procedure: 50 μL of serum to be tested was injected into the experimental group chip. After the liquid completed alkaline dissociation, acidification neutralization, delayed diffusion and sequential competitive immune reaction, the fluorescence signal intensity of the detection area was read by a fluorescence analyzer, and the quantitative result was calculated by the built-in calibration curve. The control group chip was used to detect the same sample and the synchronous competitive signal was recorded. The same sample was detected by the Roche chemiluminescence system as a reference.

[0033] The comparison results of sequential and synchronous competition are shown in the table below: Table 2. Synchronous Competition and Sequential Competition Sample number Synchronous competition result (RLU) Randomized Competition Result (RLU) S1 5600 6200 S2 320 1550 S3 2000 3000 The results show that the signal strength in the sequential competition mode is significantly higher than that in the synchronous competition mode, proving that the delayed diffusion design and sequential competition mechanism of the present invention effectively improve the detection sensitivity.

[0034] The comparison results with Roche's chemiluminescence system are shown in the table below: Table 3 Comparison of measurement results between Roche chemiluminescence immunoassay system and the present invention. sample Roche results (ng / ml) Results of this invention (ng / ml) deviation(%) 1 8.00 8.15 -1.88 2 9.65 9.80 -1.55 3 7.95 8.60 -8.18 4 15.10 15.79 -4.57 5 17.10 17.45 -2.05 Comparison with the Roche chemiluminescence system shows that the detection results of this invention have small deviations and good consistency with the Roche system, which can meet the needs of clinical testing.

[0035] The above embodiments demonstrate that the microfluidic chip structure of the present invention is reasonably designed, enabling integrated detection of 25-hydroxyvitamin D, and that the detection sensitivity and accuracy meet clinical requirements, thus possessing practical application value.

[0036] Working principle and usage process of this invention: Sample loading and actuation: Add 50 μL of the sample to be tested into the sample injection hole of the closed shell. When the sample comes into contact with the micron-sized slit entrance formed by the PET film layer and the PMMA substrate layer, it quickly enters the microchannel under the drive of capillary force. The PET film layer is pushed up by the liquid to form a stable microchannel with a depth of 20-50 μm. Alkali dissociation: The liquid flow first passes through the alkalization zone of the first straight channel, dissolving the pre-placed dry alkaline reagent and forming a local strong alkaline environment; when it flows through the microcolumn array microvalve at the channel junction, the liquid flow is initially disturbed, and then enters the first serpentine channel. The serpentine bend induces the generation of Dean's vortex secondary flow, which works with the microvalve to promote the thorough and uniform mixing of alkaline solution and sample. VDBP rapidly denatures and dissociates under strong alkaline conditions, releasing free 25-hydroxyvitamin D. Some denatured and aggregated proteins are deposited in the low-flow-rate region of the serpentine channel. Acidification and neutralization: The alkaline mixture that has completed dissociation continues to move forward and enters the acidification zone of the second straight channel, where the pre-placed dry acidic neutralization buffer is dissolved. Then it enters the second serpentine channel, where the acid and alkaline mixtures are rapidly and uniformly mixed under the synergistic effect of the Dean vortex and the microcolumn array, and the reaction system is neutralized to a neutral range suitable for the immune response. Delayed diffusion: The neutralized liquid flow (containing free 25-hydroxyvitamin D) enters the delayed diffusion zone composed of the third straight channel and the third serpentine channel. As it flows through, it dissolves the pre-placed dry-labeled competitive antigen in the PMMA substrate layer. Since the microchannel is in a laminar flow state, the competitive antigen mainly relies on molecular diffusion to migrate from the bottom of the channel to the PET film layer (top of the channel). The diffusion rate is much lower than the liquid transport rate, thus realizing the difference in movement rate between the competitive antigen and the antigen to be tested. Sequential competitive immunoassay: Free analyte antigens first arrive at the detection zone of the fourth straight channel with the main liquid flow and preferentially bind to the limited capture antibody pre-coated on the lower surface of the PET film layer; subsequently, the delayed-diffusion labeled competitive antigens arrive at the detection zone one after another and bind to the remaining antibody sites, completing the sequential competitive immunoassay. Quantitative detection: Through a transparent detection window, the analyzer scans the signal in the detection area (signal intensity is negatively correlated with the concentration of 25-hydroxyvitamin D in the sample), and combined with the built-in calibration curve, to achieve quantitative detection of 25-hydroxyvitamin D; the liquid after detection flows into the waste liquid area of ​​the PMMA substrate layer and is absorbed.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

A 1,25-hydroxyvitamin D dissociation-neutralization-quantification integrated microfluidic chip, comprising a sealed outer shell, a PET film layer, and a PMMA substrate layer assembled sequentially from top to bottom, characterized in that: The PET film layer is provided with a continuous microchannel network, which is formed by alternating series of straight channels and serpentine channels, and sequentially integrates an alkali decomposition zone, an acidification neutralization zone, a delayed diffusion zone and a detection zone along the liquid flow direction. The PMMA substrate layer has a micropillar array at the specific channel junctions corresponding to the microfluidic network, forming a microvalve; The enclosed shell is provided with a sample injection hole and a transparent detection window. The starting end of the microfluidic network is connected to the sample injection hole, and the end is set to the transparent detection window, so as to realize the integrated dissociation, neutralization and quantitative detection of the sample.

2. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The alkaline decomposition zone includes a first straight channel and a first serpentine channel, with a dry alkaline reagent pre-placed in the middle of the first straight channel; The acidification and neutralization zone includes a second straight channel and a second serpentine channel, with a dry acidic neutralization buffer pre-placed in the middle of the second straight channel.

3. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The delayed diffusion region includes a third straight channel and a third serpentine channel, and the PMMA substrate layer corresponding to the region of the third straight channel is pre-positioned with a dried, labeled 25-hydroxyvitamin D competitive antigen containing signal molecules.

4. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The lower surface of the PET film layer corresponding to the fourth straight channel is pre-coated with a limited amount of anti-25-hydroxyvitamin D capture antibody.

5. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The micropillars of the micropillar array have a diameter of 50-100μm and a spacing of 20-30μm. The microvalve is not located between the third straight channel and the third serpentine channel, but only at the junction of the remaining straight channels and serpentine channels.

6. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: When the PET film layer comes into natural contact with the PMMA substrate layer, a gap of 1-10 μm is formed. After the liquid enters, the PET film layer is lifted up, forming a microchannel with a depth of 20-50 μm. The width of all microchannels is 1 mm.

7. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The PET film layer has symmetrically distributed semi-circular positioning holes on both sides at a distance of 3mm from the starting end, and the PMMA substrate layer has cylindrical positioning posts that are adapted to the positioning holes. The diameter of the positioning posts is slightly smaller than the diameter of the positioning holes.

8. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 2, characterized in that: The dried alkaline reagent is NaOH or KOH, and the dried acidic neutralization buffer is HCl or citrate buffer. After drying, both the alkaline reagent and the acidic neutralization buffer form circular reagent dots with an area of ​​1 mm².

9. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 3, characterized in that: The signaling molecules are fluorescent microspheres, fluorescent dyes, or quantum dots.

10. The integrated microfluidic chip for 25-hydroxyvitamin D dissociation-neutralization-quantification according to claim 1, characterized in that: The PMMA substrate layer also has a waste liquid area, which is made of a water-absorbing material and is connected to the end of the microchannel network.

Images