Quantum dot microfluidic immunodetection chip and kit for detecting female genital tract chlamydia trachomatis

Abstract

The invention focuses on a quantum dot microfluidic immunodetection chip and a kit for detecting female genital tract chlamydia trachomatis. The chip comprises a substrate and a cover plate, a sample loading area, a pretreatment area, a reaction area, a detection area and a waste liquid area which are sequentially communicated are formed on the substrate, a plurality of layers of polyelectrolyte-nano magnetic bead self-assembled films are formed in the pretreatment area, an electromagnetic coil is embedded in the cover plate and is aligned with the center of the pretreatment area, and the electromagnetic coil is embedded in the cover plate. A quantum dot anti-MOMP antibody is fixed on the inner wall of a channel of the reaction area, and a photonic crystal microcavity is formed in the detection area. The kit comprises the chip. According to the chip, rapid, high-sensitivity and high-specificity detection of CT is realized through three core innovative designs of a fluorescence enhancement region, an active in-situ pretreatment and enrichment structure and a space-time resolution antibody arrangement structure; meanwhile, a kit containing the chip is provided, the detection convenience and the automation level are further improved, and the application requirements of basic medical institutions are met.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic detection technology, specifically relating to a quantum dot microfluidic immunoassay chip integrating dynamic fluorescence enhancement, active sample pretreatment, and a high-efficiency immunoreaction interface, as well as a kit containing this chip for rapid detection of Chlamydia trachomatis in the female genital tract. This invention is suitable for clinical medical institutions, especially primary healthcare institutions, for rapid, highly sensitive, and highly specific detection of Chlamydia trachomatis infection in the female genital tract. It enables integrated operation of sample loading, automatic pretreatment, immunoreaction, and signal detection, providing technical support for the early diagnosis and intervention of Chlamydia trachomatis infection. Background Technology

[0002] Chlamydia trachomatis (CT) is one of the most common sexually transmitted pathogens worldwide. If left untreated, infection in women can lead to cervicitis, endometritis, pelvic inflammatory disease, and in severe cases, fallopian tube obstruction, ectopic pregnancy, and even infertility, posing a significant threat to women's reproductive health. Clinical practice shows that early and accurate detection is crucial for reducing the incidence of infectious complications. However, existing detection technologies have significant limitations: cell culture methods are time-consuming and cumbersome; nucleic acid amplification methods require expensive equipment and complex sample pretreatment; and traditional immunoassays have low sensitivity and are susceptible to sample interference. In recent years, the combination of microfluidics and quantum dot technology has brought new ideas to immunoassay, but its application in female reproductive tract samples still faces three major challenges: sample blockage, insufficient sensitivity, and low immunoreaction efficiency. Existing patents attempt to optimize these methods, but have failed to simultaneously solve all the above problems, necessitating a dedicated detection chip and reagent kit. Summary of the Invention

[0003] This invention provides a quantum dot microfluidic immunoassay chip, which achieves rapid, high-sensitivity, and high-specificity detection of Chlamydia trachomatis through three core innovative designs: active in-situ pretreatment and enrichment structure, spatiotemporally resolved antibody arrangement structure, and fluorescence enhancement region. At the same time, it provides a reagent kit containing this chip to further improve the convenience and automation of the detection.

[0004] Technical solution: (I) This invention provides a quantum dot microfluidic immunoassay chip The chip adopts a layered integration design, which includes a substrate and a cover plate.

[0005] The substrate is made of transparent quartz glass, which has good optical transmittance and mechanical stability and can withstand laser processing and vacuum evaporation processes.

[0006] The microfluidic channel is formed on the substrate surface by photolithography and etching processes. It includes a sample loading area, a pretreatment area, a reaction area, a detection area and a waste liquid area that are connected in sequence. Each area is connected by a smoothly transitioned microchannel to avoid fluid stagnation.

[0007] The cover is made of polydimethylsiloxane and has a sample injection port and a waste liquid outlet, which are sealed to the microfluidic channel.

[0008] The inner wall of the pretreatment zone channel has multiple layers of self-assembled films formed by polyelectrolytes and magnetic nanobeads. Specifically: the first layer is polydiallyldimethylammonium chloride (PDDA, concentration 1-2 mg / mL, pH 7.4), modified for 10-15 minutes, and bonded to the inner wall of the channel through electrostatic interaction (the quartz surface is negatively charged after activation with hydrofluoric acid); the second layer is carboxylated magnetic nanobeads (diameter 180-220 nm, preferably 200 nm, concentration 0.5-1 mg / mL) with surface-coupled hydrophobic groups (dodecyltrimethoxysilane), modified for 15-20 minutes, and bonded through electrostatic interaction between the positively charged polyelectrolyte and the negatively charged magnetic nanobeads; the above steps are repeated 4-6 times (preferably 5 times) to form a 5-layer self-assembled film, with a magnetic bead loading ≥ 1 × 10⁻⁶. 6 It can efficiently adsorb mucus and cell debris in samples.

[0009] An electromagnetic coil is embedded in the cover plate and aligned with the center of the pretreatment area. The electromagnetic coil is connected to a portable controller and can generate an alternating magnetic field with a frequency of 50-100Hz and a magnetic field strength of 50-100mT. This drives the nano-magnetic beads in the channel to perform vortex motion (rotation speed 500-1000rpm, the rotation speed of the nano-magnetic beads is controlled by the frequency of the alternating magnetic field, 50Hz corresponds to about 500rpm, 100Hz corresponds to about 1000rpm, ensuring that the impurity adsorption rate is ≥95%). This achieves the integrated function of dynamic stirring, impurity adsorption and in-situ enrichment, with a pretreatment efficiency of ≥95% and no risk of channel blockage. It abandons the traditional filter membrane design and greatly enhances the regenerative performance of the chip.

[0010] The reaction zone contains a channel wall covalently bound with a quantum dot-labeled Chlamydia trachomatis-specific antibody. This antibody targets the major outer membrane protein of Chlamydia trachomatis (MOMP, which is highly conserved and a common antigen for all serotypes). The quantum dots have a CdSe / ZnS core-shell structure (particle size 3-5 nm, emission wavelength 580 nm) and are coupled via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) with a coupling efficiency ≥90% and an antibody concentration of 8-12 μg / mL (preferably 10 μg / mL).

[0011] Quantum dot-labeled antibodies are arranged in a spatially fluid-flow density gradient, with the density gradually decreasing from the inlet to the outlet of the reaction zone, forming alternating stripes on the circumferential inner wall of the channel; this achieves a synergistic effect of rapid antigen capture at the inlet and high-affinity binding at the outlet. For example, the density at the inlet is 800-1000 antibodies / μm. 2 The density at the outlet is 300-500 particles / μm. 2 Meanwhile, the quantum dot-labeled antibodies, arranged in alternating stripes on the cross-section of the reaction zone, induce micro-vortices in the fluid, breaking the laminar diffusion limitation, increasing the effective collision frequency between antigen and antibody, and improving the efficiency of the immune response.

[0012] The inner wall of the detection zone channel (fluorescent detection window area, 1×2 mm) is modified with a secondary antibody against Chlamydia trachomatis. This secondary antibody and the quantum dot-labeled antibody recognize different antigenic epitopes of the main outer membrane protein, avoiding competitive binding. The concentration of the secondary antibody is 5-8 μg / mL (preferably 6 μg / mL), and it is fixed by amino-carboxyl covalent binding. The CT antigen in the sample first binds to the quantum dot-labeled antibody in the reaction zone to form a "quantum dot-antibody-antigen" complex, which then flows to the detection zone and binds to the secondary antibody to form a "secondary antibody-antigen-quantum dot-labeled antibody" complex. The quantum dots emit fluorescence under excitation light (488 nm laser) and are detected.

[0013] Furthermore, the chip also includes a photonic crystal microcavity formed inside the substrate below the detection area.

[0014] The photonic crystal microcavity has a face-centered cubic structure with a period of 280-320 nm (preferably 300 nm). Its bandgap center wavelength matches the quantum dot emission wavelength (580 nm), and its Q-value (quality factor) is ≥1000, enabling resonant enhancement of the fluorescence signal. The photonic crystal microcavity can be fabricated in situ inside the substrate (50-100 μm directly below the detection area) using femtosecond laser two-photon direct writing technology. Femtosecond laser two-photon direct writing technology: A femtosecond laser with a wavelength of 800 nm and a pulse width of 100 fs is focused 80 μm directly below the detection area inside the quartz substrate. The laser power is 50 mW, and the scanning speed is 10 μm / s. Through a 'dot array-layer stacking' path, a face-centered cubic photonic crystal microcavity with a period of 300 nm (Q-value ≥1000) is formed.

[0015] (II) This invention provides a kit for detecting Chlamydia trachomatis in the female reproductive tract. The kit includes a quantum dot microfluidic immunoassay chip, a portable controller and reader, and matching reagents.

[0016] Quantum dot microfluidic immunoassay chip: 1 chip / box, sealed in an aluminum-plastic packaging bag containing desiccant, shelf life 6 months at 4℃.

[0017] The portable control and reading instrument includes an alternating magnetic field generation module and a fluorescence detection module. The alternating magnetic field generation module provides a magnetic field of 50-100Hz and 50-100mT for the pretreatment area, while the fluorescence detection module provides an excitation wavelength of 488nm and an emission wavelength of 580nm for the detection area. The detection sensitivity is ≤0.1pg / mL. It can automatically collect fluorescence signals and calculate intensity values, and display "positive / negative" results on the screen.

[0018] The supporting reagents include: Sample diluent: containing 0.01 mol / L PBS (pH 7.4), 0.5% Triton X-100 (to lyse epithelial cells and release CT antigen), and 0.1% sodium azide (preservative), 10 mL / bottle; Washing buffer: containing 0.01 mol / L PBS (pH 7.4), 0.1% Tween-20 (to remove unbound impurities), and 0.05% sodium azide, 10 mL / bottle; Positive control: CT standard strain (serotype D, the most common clinical serotype) containing 10³ copies / mL, 1mL / vial, stored at -20℃; Negative control: containing sterile physiological saline (containing 0.1% sodium azide), 1 mL / vial, stored at 4℃; Auxiliary accessories: sterile sampling swabs (5 swabs / box), centrifuge tubes (10 swabs / box, 1.5mL), instruction manual (1 copy / box).

[0019] (III) Detection Principle This invention is based on the principle of synergistic detection of active preprocessing and spatiotemporally resolved immune response, and the specific steps are as follows: Sample loading: Immerse the female reproductive tract swab sample (cervical swab) in the sample diluent, vortex for 10 minutes (to lyse cells), and inject 10 μL of supernatant into the chip sample loading area; Pretreatment: The portable controller activates the micro electromagnetic coil to generate an alternating magnetic field, which drives the nanomagnetic beads in the pretreatment area to vortex and adsorb impurities such as mucus and cell debris in the sample (pretreatment time is 2 minutes). The purified sample (containing CT antigen) flows into the reaction area under capillary action. Reaction Zone: Purified samples enter the reaction zone, where antigens first rapidly bind to high-concentration quantum dot-labeled antibodies at the inlet (binding efficiency ≥80%), and then flow with the fluid to the outlet, where they further bind to low-concentration antibodies (reducing non-specific binding). Simultaneously, alternating stripe antibodies induce local micro-vortices in the fluid, breaking the laminar diffusion limitation and increasing the effective collision frequency between antigens and antibodies by 3-5 times, shortening the reaction time, reducing the length of the reaction zone, and decreasing the chip size. Detection: The quantum dot-antibody-antigen complex flows to the detection area and binds to the secondary antibody to form a double antibody sandwich complex. The quantum dots emit fluorescence under excitation light (580nm) and are detected. Result interpretation: The controller collects the fluorescence intensity of the detection area. If the fluorescence intensity of the sample to be tested is ≥80% of the fluorescence intensity of the positive control, it is judged as positive (CT infection exists); if it is ≤120% of the fluorescence intensity of the negative control, it is judged as negative (no infection). The entire detection process takes ≤15 minutes.

[0020] Chip regeneration: Low-concentration urea / guanidine hydrochloride (denaturant) disrupts the hydrophobic and hydrogen bonding interactions between the antigen and the quantum dot anti-MOMP antibody without affecting the spatial structure of the secondary antibody, allowing the complex to detach rapidly and achieving chip regeneration. The regeneration reagent is PBS at pH 7.4 containing 2-4M urea and 0.05% Tween-20. The regeneration process is as follows: Introduce the regeneration reagent through the chip sample injection port at a flow rate of 2 μL / min, filling the detection area and allowing it to stand for 1-2 minutes. Urea permeates the complex, disrupting non-covalent bonds. Introduce washing buffer at a flow rate of 5 μL / min, rinsing for 3 minutes to remove the detached complex and residual urea. Introduce PBS at pH 7.4, allowing it to stand for 1 minute to restore the pH of the detection area and ensure the activity of the secondary antibody. Introduce a positive control (10³ copies / mL CT standard strain) and detect fluorescence intensity to verify the activity of the secondary antibody (fluorescence intensity should recover to more than 90% of the initial detection).

[0021] (iv) Beneficial effects of the invention This invention addresses the three major drawbacks of existing technologies—sample clogging, low sensitivity, and low reaction efficiency—through the dynamic magnetic bead structure in the pretreatment zone, the spatial arrangement of antibodies in the reaction zone, and the fluorescence enhancement structure in the detection zone. It achieves a detection limit as low as 10¹ copies / mL (the concentration of the major outer membrane protein (MOMP) of the standard Chlamydia trachomatis (CT) strain (serotype D) at 10¹ copies / mL is approximately 0.05 pg / mL, meeting the detection sensitivity requirement of ≤0.1 pg / mL for portable controllers and readers), 100% specificity, a detection time ≤15 minutes, and an anti-clogging success rate ≥95%. Its overall performance far surpasses existing quantum dot microfluidic chips and traditional detection methods.

[0022] This invention further integrates the chip with other reagents to form a reagent kit, achieving integrated sample addition, detection, and reading. It is easy to operate (no professional technicians are required), and the portable controller weighs ≤500g. It can be used in primary healthcare institutions, community health service centers, and other scenarios to meet the clinical needs for early, rapid, and accurate detection of female reproductive tract CT infections. Attached Figure Description

[0023] Figure 1This is a schematic diagram of the overall structure of a quantum dot microfluidic immunoassay chip. Figure 2 This is a schematic diagram showing the direction of antibody flow in the reaction zone.

[0024] Figure 3 This is a schematic diagram of the circumferential arrangement of antibodies in the reaction zone.

[0025] Figure 4 This is a schematic diagram of the fabrication process of a quantum dot microfluidic immunoassay chip.

[0026] Substrate 100, sample loading area 11, sample injection port 110, pretreatment area 12, pretreatment area injection port 120, self-assembled film 121, magnetic positioning part 122, reaction area 13, reaction area injection port 130, gradient stripe 131, alternating stripe 132, detection area 14, detection area injection port 140, photonic crystal microcavity 141, waste liquid area 15, waste liquid outlet 150, cover plate 200, electromagnetic coil 21, hydrophobic isolation strip 16. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this invention are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0028] I. Quantum Dot Microfluidic Immunoassay Chip Structure like Figure 1 As shown, the quantum dot microfluidic immunoassay chip includes a glass substrate and a cover plate.

[0029] PDMS (polydimethylsiloxane) cover sheet 200: includes sample injection port 110, pretreatment zone injection port 120, reaction zone injection port 130, detection zone injection port 140, and waste liquid outlet 150 (1mm in diameter), used to seal the channel and provide a fluid interface. Thickness 500μm, dimensions 20mm×30mm.

[0030] Glass substrate 100: Microfluidic channels are formed by etching on the front side. The microfluidic channels include a sample loading area 11, a pretreatment area 12, a hydrophobic isolation zone 16, a reaction area 13, a detection area 14, and a waste liquid area 15.

[0031] The inner wall of the pretreatment zone 12 channel is electrostatically bonded with carboxylated magnetic nanobeads through polydiallyldimethylammonium chloride modified on the surface of the channel inner wall. Magnetic positioning portions 122 are coated on the inner walls between the pretreatment zone 12 and the sample loading zone 11, and between the pretreatment zone 12 and the reaction zone 13. The magnetic positioning portions 122 and the magnetic nanobeads are magnetically repelled. The material of the magnetic positioning portions 122 (e.g., ferrite, nickel alloy) and its magnetic strength (e.g., 10-20 mT) are specified. The repulsive force (15 mT) of the magnetic positioning portions is only considered.

[0032] An electromagnetic coil 21 is embedded in the cover plate 200 and aligned with the center of the pretreatment area 12. The electromagnetic coil 21 is used to generate an alternating magnetic field around the nano magnetic beads to drive the magnetic beads to vortex motion.

[0033] like Figure 2 and Figure 3 As shown, quantum dot anti-MOMP antibodies are covalently fixed on the inner wall of the reaction zone 13 channel, and the density of the quantum dot anti-MOMP antibodies decreases in a gradient direction along the liquid flow direction to form gradient stripes 131, and alternating stripes 132 are formed on the circumferential direction of the inner wall of the channel.

[0034] The inner wall of the detection zone 14 is modified with a secondary antibody against Chlamydia trachomatis. A photonic crystal microcavity 141 is formed on the back side of the glass substrate of the detection zone, corresponding to the position of the detection zone. The photonic crystal microcavity 141 adopts a periodic air hole structure to enhance the fluorescence localization effect.

[0035] In the sample to be tested, the Chlamydia trachomatis antigen forms a quantum dot-antibody-antigen complex with the quantum dot anti-MOMP antibody in the reaction area. The complex then flows to the detection area and binds with the secondary antibody to form a secondary antibody-antigen-quantum dot labeled antibody complex. The quantum dots emit fluorescence under excitation light and are detected.

[0036] The substrate 100 is also etched with a hydrophobic isolation region 16 that connects the pretreatment region and the reaction region, and the inner wall of the hydrophobic isolation region 16 is formed with a functional area hydrophobic surface.

[0037] II. Chip Fabrication like Figure 4 As shown, the fabrication process of the quantum dot microfluidic immunoassay chip provided by this invention includes: Pre-treatment of the substrate; A face-centered cubic photonic crystal microcavity is formed on the back side of the substrate at the position corresponding to the detection area; A sample loading area, a pretreatment area, a reaction area, a detection area, and a waste liquid area are sequentially connected and etched on the front side of the substrate. A hydrophobic isolation zone is formed between the pretreatment zone and the reaction zone; Magnetic positioning parts are formed by magnetron sputtering at the junctions of the pretreatment zone and the sample loading zone, and the pretreatment zone and the reaction zone. PDMS cover plates with embedded electromagnetic coils are formed by 3D printing; Carboxylated magnetic nanobeads with hydrophobic groups coupled to the inner wall of the channel in the pretreatment zone were modified with polydiallyl dimethyl ammonium chloride. Gradient stripes of decreasing antibody concentration are formed in the reaction zone along the liquid flow direction, as well as alternating stripes formed in the circumferential direction by the alternating laminar flow of antibody solution and buffer solution; Secondary antibodies are modified in the detection area; The substrate and PDMS cover sheet, after undergoing the above steps, are cleaned with oxygen plasma, bonded together, and then baked and sealed.

[0038] 1. A photonic crystal microcavity is formed on the back side of the glass substrate. 1) Glass substrate pretreatment Select a 20mm×30mm×1mm quartz glass sheet (transmittance ≥90%, resistant to high temperature corrosion); immerse it in the following solutions in sequence: ① 5% sodium hydroxide solution (60℃, ultrasonic cleaning for 10 minutes to remove grease); ② deionized water (ultrasonic cleaning for 5 minutes × 3 times); ③ 10% hydrofluoric acid (soaking at room temperature for 3 minutes to activate the surface); dry with nitrogen gas, bake in a 120℃ oven for 30 minutes, and set aside.

[0039] 2) Backside electron beam exposure and development Cover the non-microcavity area on the back of the substrate with high-temperature resistant tape, exposing only the 1mm×2mm microcavity area (the reaction area corresponds to the projected position). Electron beam photoresist (ZEP520A, 200nm thickness) was spin-coated onto the back of a glass substrate at the projection position corresponding to the reaction area, and pre-baked at 180°C for 3 minutes; electron beam exposure (accelerating voltage 50kV, dose 300μC / cm²) was performed to form a circular air hole array (diameter 200nm, depth 150nm) with a pattern of 300nm period at the projection position corresponding to the reaction area, corresponding to the detection area position (1mm×2mm). Develop with developer (ZED-N50) for 90 seconds, rinse with deionized water to form a photoresist mask; Inductively coupled plasma (ICP) etching: use CF4 / O2 (ratio 4:1) as the etching gas, power 200W, etching depth 150nm to form a photonic crystal structure; remove residual photoresist with acetone, and dry with nitrogen.

[0040] 2. Etching of microfluidic channels on the front side of the glass substrate 1) Photoresist coating Positive photoresist AZ1500 (suitable for glass etching) was used and applied by spin coater: ① Low speed: 500rpm / 10s (uniform spreading); ② High speed: 4000rpm / 30s (thickness control, final photoresist layer thickness 5μm, ensuring etching accuracy). Pre-baking: 65℃ for 10 minutes, then 95℃ for 20 minutes, gradually increasing the temperature to avoid air bubbles in the adhesive layer, then allow to cool naturally to room temperature.

[0041] 2) Photolithography and pattern transfer A first mask is provided, which is a high-precision chrome-plated quartz template (size matched with the glass substrate) containing the complete pattern of microfluidic channels. It is used to transfer the pattern onto the photoresist on the glass surface during photolithography with an accuracy of ±1μm.

[0042] Align the mask pattern with the glass substrate using a photolithography alignment system (accuracy ±2μm) to ensure that the hydrophobic isolation zone is located between the pretreatment area and the reaction area; UV exposure: wavelength 365nm, energy 100mJ / cm², exposure time 8 seconds (to expose the photoresist).

[0043] Development: Immerse the glass slide in AZ300MIF developer (25°C, 60 seconds) to remove the photoresist in the exposed areas and expose the glass surface to be etched; rinse with deionized water for 10 seconds to stop development, and blow dry with nitrogen. Hardening: Bake in a 120℃ oven for 20 minutes to enhance the adhesion between the unexposed photoresist and the glass.

[0044] 3) Wet etching forms microfluidic channels The etching solution uses a hydrofluoric acid buffer (BOE): 40% hydrofluoric acid: 40% ammonium fluoride = 1:6 (volume ratio), and the etching rate is about 10 μm / min (to facilitate depth control).

[0045] The glass slide was immersed in BOE etching solution (25°C, stand for 5 minutes) and the etching depth reached 50μm (consistent with the channel design depth). Remove the glass slide and immediately rinse it three times with deionized water to stop the etching process. Remove residual photoresist: Immerse in acetone (ultrasonic cleaning for 5 minutes), then rinse with isopropanol and dry with nitrogen. At this point, a recessed microfluidic channel is formed on the glass surface.

[0046] The channel depth was measured by a step gauge: 50±2μm, and the width was 250±5μm; optical microscopy showed that the channel edges were smooth, without serrated defects, and the functional area boundaries were clear.

[0047] 3. Fabrication of PDMS cover plate with embedded electromagnetic coil 1) Electromagnetic coil prefabrication (copper foil etching process) Select a 50μm thick polyimide-based copper foil (high temperature resistant and good insulation), cut it to 20mm×30mm size; ultrasonically clean with isopropanol for 5 minutes to remove the surface oxide layer, and then blow dry with nitrogen.

[0048] 2) Photolithographic coil pattern AZ1500 photoresist (5μm thickness) is spin-coated onto the copper foil surface, and then a "coil-specific mask" (directly above the pretreatment area, with a spiral coil diameter of 3mm, a line width of 50μm, and 120 turns) is applied after pre-baking; ultraviolet exposure (parameters as before) is performed, and the copper foil area to be etched is exposed after development, followed by hard film treatment.

[0049] 3) Copper foil etching and insulation Immerse in ferric chloride etching solution (40℃, 10 minutes) to remove copper foil not protected by photoresist and form a spiral coil; ultrasonically clean with acetone for 5 minutes to remove photoresist and expose the coil pattern; spin-coat the coil surface with polydimethylsiloxane (PDMS) prepolymer (100μm thickness) and cure at 80℃ for 1 hour, leaving only the pads (1mm in diameter) at both ends of the coil for subsequent wiring.

[0050] 4) 3D printing to create cover plate molds Includes a sample inlet protrusion (1 mm in diameter and 500 μm in height) and a coil positioning groove (50 μm in depth, matching the coil size); the mold surface is sprayed with a release agent to facilitate subsequent PDMS peeling.

[0051] 5) Coil positioning and PDMS casting Place the prefabricated electromagnetic coil into the positioning groove of the mold (ensure that the coil is directly above the pretreatment area), and align the solder pads with the wiring holes reserved in the mold. Mix PDMS and curing agent at a ratio of 10:1 (by mass), stir for 3 minutes, and then degas under vacuum for 20 minutes (vacuum degree ≤ 10 Pa). Slowly pour the degassed PDMS into the mold (covering the coil, with a total thickness of 500μm), and cure it in an oven at 80℃ for 2 hours.

[0052] 6) Drilling and processing of cover plates Peel off the PDMS cover and use a 1mm diameter stainless steel punch to drill holes at the raised positions on the mold. ① Sample injection port (corresponding to the pretreatment area inlet); ②Auxiliary injection port in the pretreatment area (1 port); ③ Reaction zone antibody injection well (1 well), buffer injection well (2 wells); ④ Waste liquid outlet (corresponding to waste liquid area); Clean the cover plate with isopropyl alcohol for 5 minutes, dry it with nitrogen, and check if the coil is intact (measure the resistance with a multimeter; the normal resistance value is 10±1Ω).

[0053] 4. Surface chemical partitioning modification (1) Preparation of hydrophobic isolation zone Recoat the AZ1500 photoresist onto the glass slide with the etched channels (steps as above). A special mask for the isolation zone (exposing only a 0.5 mm long channel area between the pretreatment area and the reaction area) is used for UV exposure and development to expose the isolation zone area to be modified. The special mask for the isolation zone only contains a narrow pattern of 0.5 mm long between the pretreatment area and the reaction area, which matches the shape of the hydrophobic isolation zone and precisely covers the location where the hydrophobic isolation zone needs to be prepared; the other areas are light-shielding chrome-plated layers to ensure that only this narrow area is exposed during photolithography.

[0054] The glass slide was immersed in an anhydrous ethanol solution of 1% perfluorooctyltriethoxysilane (PFOTS) (containing 0.1% acetic acid as a catalyst) and left to stand at room temperature for 30 minutes to allow the siloxy groups of PFOTS to react with the hydroxyl groups on the glass surface to form a hydrophobic layer (with the -CF3 groups facing outwards). The slide was then ultrasonically cleaned with acetone for 5 minutes, rinsed with isopropanol to remove the photoresist, and dried with nitrogen. At this point, the contact angle of the isolation zone area was ≥120°, while other areas remained hydrophilic (contact angle ≤30°).

[0055] Contact angle measurement: 125±3° for the isolation zone, 25±2° for the pretreatment / reaction zone, with clear boundaries (transition zone ≤50μm); Fluorescence verification: Injecting fluorescent aqueous solution into the channel, it is visible that the liquid forms a distinct meniscus at the isolation zone and cannot diffuse across the boundary naturally.

[0056] (2) Preparation of magnetic positioning part A magnetic coating is deposited at the boundary of the pretreatment zone (where it connects with the sample loading zone and the reaction zone) to form a magnetic positioning part of the magnetic confinement boundary.

[0057] A "magnetic positioning part special metal mask" is used: the mask is made of 20mm×30mm stainless steel, and hollow windows are only opened at the "connection between the pretreatment area and the sample loading area" and "connection between the pretreatment area and the reaction area" (window shape: long strip matching the channel, length 500μm, width 250μm, consistent with the channel size of the pretreatment area), and other areas are completely covered to avoid the magnetic coating being deposited incorrectly; Mask alignment: The mask cutout window is aligned with the two boundary channels of the pretreatment area on the substrate using the photolithography alignment system (accuracy ±2μm). This ensures that the magnetic positioning part exactly covers the inner wall of the channel at the entrance (near the sample loading area) and the exit (near the reaction area) of the pretreatment area, perfectly matching the functional position of "limiting magnetic bead diffusion".

[0058] Sputtering: A magnetron sputtering system (model: JGP-450C) was used, with ferrite (Ni-Zn ferrite, with stable magnetic strength and weak magnetic properties suitable for the "weak repulsion" requirement) as the target material, and a vacuum level of ≤5×10⁻ 4Pa (to ensure coating purity and prevent impurity interference); sputtering power of 150W (to avoid excessive power causing substrate overheating and affecting the prepared hydrophobic isolation band); argon flow rate of 15sccm (to provide a stable sputtering atmosphere and ensure coating uniformity); deposition time of 3 minutes (to control the coating thickness to 50nm, ensuring a magnetic strength of 15mT while avoiding excessive coating thickness that would lead to narrow channels); Deposition: The substrate aligned with the mask is placed into the sputtering machine, and the sputtering process is started. Ferrite coating is deposited only in the cut-out window area to form the magnetic positioning part.

[0059] Mask Removal: After sputtering is complete, wait for the substrate to cool to room temperature (about 10 minutes), then carefully peel off the metal mask, avoiding scratching the magnetic coating; Coating reinforcement: The substrate is placed in a 120℃ oven and baked for 20 minutes to enhance the adhesion between the ferrite coating and the glass substrate (adhesion ≥3N / cm², tested by cross-cut method to ensure that it will not fall off during subsequent magnetic bead modification and fluid rinsing). Cleaning verification: Gently wipe the non-positioning area with an isopropyl alcohol swab to confirm that there is no magnetic coating residue (observed under an optical microscope, there is no obvious reflection in the non-positioning area, and the reflection in the positioning area is uniform), to avoid affecting the hydrophilicity / hydrophobicity of other areas.

[0060] (3) Carboxyl group activation in the pretreatment zone Inject 0.1 mol / LEDC / NHS mixture (10 μL, just enough to fill the pretreatment zone channel) through the dedicated injection port (to be drilled later) in the pretreatment zone using a pipette, and incubate at room temperature for 20 minutes; EDC activates the hydroxyl groups on the glass surface, which react with NHS to form active esters, facilitating the subsequent binding of amino groups (PDMS is positively charged); rinse three times with deionized water to remove unreacted reagents.

[0061] (4) Secondary activation of the reaction zone After the pretreatment zone is modified and blocked, inject fresh EDC / NHS mixture (15 μL, filling the reaction zone) and incubate at room temperature for 20 minutes; rinse 3 times with deionized water and set aside (to ensure that only the reaction zone has antibody-coupled activity).

[0062] (5) Activation of amino groups in the detection zone (adaptation to secondary antibody) The detection area on the front of the substrate is covered with a metal mask (with a 1mm × 2mm window cut out) and immersed in a 3% 3-aminopropyltriethoxysilane (APTES) ethanol solution (room temperature, 30 minutes); rinsed with deionized water to remove the mask and unreacted APTES, forming amino groups (-NH2) on the surface of the detection area, which enhances the binding force of the secondary antibody.

[0063] 5. Preparation of polyelectrolyte-magnetic nanobead composite film in the pretreatment zone (1) Modification with polydiallyldimethylammonium chloride Inject a 1.5 mg / mL polydiallyldimethylammonium chloride solution (pH 7.4, positively charged, 10 μL) into the pretreatment zone and incubate at room temperature for 12 minutes. The solution will bind to the negatively charged glass surface through electrostatic adsorption. Inject 50 μL of deionized water (slowly push to avoid the liquid from passing through the isolation zone) into the pretreatment zone inlet and dry with nitrogen.

[0064] (2) Modification of carboxylated magnetic nanobeads Inject a suspension of 0.8 mg / mL carboxylated magnetic nanobeads (negatively charged, 200 nm in diameter, 10 μL in volume) into the pretreatment zone and incubate at room temperature for 18 minutes to bind PDMS via electrostatic adsorption. Inject 30 μL of deionized water from the isolation zone side to remove residual magnetic beads at the boundary and dry with nitrogen.

[0065] (3) Preparation of multilayer composite membranes The modification was repeated 5 times to form a 5-layer alternating composite film (total thickness of about 1 μm); 1% BSA solution (10 μL, incubated at room temperature for 30 minutes) was injected to block the active sites of unbound magnetic beads and avoid non-specific adsorption; the film was dried in a vacuum drying oven at 30℃ (10 Pa) for 15 minutes to completely remove moisture.

[0066] 6. Antibody flow direction gradient and alternating stripe modification in the reaction zone (1) Hydrophobic protection of the pretreatment area Inject 0.5% PFOTS ethanol solution (10 μL, incubate at room temperature for 15 minutes) into the pretreatment zone to form a hydrophobic layer (contact angle ≥115°) on the surface of the magnetic beads to prevent subsequent adsorption of antibody solution; rinse with ethanol (30 μL) and vacuum dry.

[0067] (2) Density gradient modification in the direction of fluid flow This step mainly involves controlling the flow rate ratio of antibody solution to buffer solution to create a linear difference in the adsorption concentration of antibody at different locations along the flow direction of the channel. This results in a high proportion of antibody solution and a high adsorption density at the inlet of the reaction zone, and a high proportion of buffer solution and a low adsorption density at the outlet.

[0068] Dual-channel infusion pump configuration: A Harvard Instruments PHD2000 four-channel infusion pump was used. Channel 1 was loaded with 10 μg / mL quantum dot-labeled antibody solution (CdSe / ZnS core-shell structure, emission wavelength 580 nm, concentration verified by BCA protein quantification), and channel 2 was loaded with 0.01 mol / L PBS (pH 7.4, containing 0.05% Tween-20 to reduce non-specific adsorption). Fluid flow path connection: Y-type polytetrafluoroethylene (PTFE) connector (inner diameter 200μm) is used to connect the tubing of channel 1 and channel 2 to the inlet of the reaction zone, ensuring that there is no dead volume in the connector (dead volume will cause antibody solution residue and affect gradient uniformity). Table 1 Flow rate program (lasting 30 minutes) Initial stage (0-10 minutes): The antibody solution flows at a high rate (1.2 μL / min), becoming the dominant solution at the Y-connector (accounting for 30%), and rapidly flows through the inlet section of the reaction zone (first 5 mm). Initial stage (0-10 minutes): The antibody solution has a high flow rate (1.2 μL / min) and dominates at the Y-type connector (accounting for 30%). It flows rapidly through the inlet section of the reaction zone (first 5 mm). At this time, the activated carboxyl groups (EDC / NHS activated) on the inner wall of the channel react rapidly with the antibody amino groups to form a high-density antibody layer (800-1000 cells / μm²). Intermediate phase (10-20 minutes): The antibody flow rate decreases to 0.8 μL / min, the buffer content increases to 80%, the concentration of the antibody solution in the middle of the channel (5-10 mm) decreases, and the adsorption density decreases to 500-650 cells / μm². Terminal stage (20-30 minutes): The antibody flow rate is only 0.4 μL / min. In the channel outlet section (10-15 mm), the antibody solution is diluted by a large amount of buffer, and only a small amount of antibody binds to carboxyl groups, with the density dropping to 300-500 cells / μm². Key control: Keep the chip horizontal during incubation (tilt angle ≤ 0.5°) to avoid gravity causing the antibody solution to shift towards the outlet, which would affect the gradient linearity.

[0070] Fluorescence imaging: Using a laser confocal microscope (excitation wavelength 488nm, emission wavelength 580nm), take 5 points (inlet, 2mm, 5mm, 10mm, and outlet) along the liquid flow direction in the reaction zone, measure the fluorescence intensity, and plot the fluorescence intensity-distance curve. If it shows a linear decrease (R²≥0.98), the gradient is qualified. Quantitative calculation: The fluorescence intensity of each point was analyzed using ImageJ software. Based on the known antibody concentration-fluorescence intensity standard curve, the antibody density of each point was calculated to ensure that the density ratio of the inlet to the outlet is approximately 3:1 (1000:300).

[0071] (3) Alternating stripe modification (visualization of four-strand laminar flow focusing) Core principle: Based on the gradient of liquid flow direction, by adding two additional buffers containing fluorescein, an alternating laminar flow of "antibody solution-buffer solution-antibody solution-buffer solution" is formed in the cross-section of the channel. The antibody is adsorbed only in the area in contact with the antibody solution, forming a striped pattern.

[0072] Table 2 Equipment Expansion and Piping Connections

[0073] Four-channel infusion pump configuration: In addition to the original dual channels, channels 3 and 4 are added, both loaded with PBS containing 0.1% sodium fluorescein (fluorescein is used to observe the laminar flow interface and does not affect antibody binding). Fabrication of the laminar flow focusing adapter: A quartz cross-shaped adapter (250 μm inner diameter, matching the channel width) is used. Channel 1 (antibody solution, 0.4 μL / min), channel 2 (buffer, 3.6 μL / min), channel 3 (fluorescein buffer, 1 μL / min), and channel 4 (fluorescein buffer, 1 μL / min) are connected to the four inlets of the adapter, and the outlet is connected to the middle of the reaction zone (5 mm away) to ensure that the four liquid streams converge in the adapter to form parallel laminar flow. Flow rate settings: The total flow rate remains at 4 μL / min, and the flow rate ratio of the four liquid streams is: antibody solution: buffer solution: fluorescein buffer 1: fluorescein buffer 2 = 0.4:3.6:1:1, to ensure that the width of each laminar flow is uniform (approximately 250 μm / 4 = 62.5 μm; in reality, due to the fluid viscosity effect, the antibody layer width is approximately 50 μm, and the buffer layer width is approximately 75 μm, which does not affect the formation of stripes).

[0074] Laminar flow start-up: First open channels 2, 3, and 4 (buffer solution and fluorescein solution), and wait for the flow to stabilize (about 2 minutes) before opening channel 1 (antibody solution) to avoid initial flow disturbance from disrupting the laminar flow interface; Interface observation: Using an inverted fluorescence microscope (excitation wavelength 490nm, emission wavelength 520nm), the cross-section of the reaction area was observed, and four clear parallel laminar flows were visible: green fluorescent bands (fluorescein buffer) and colorless bands (antibody solution / buffer) were arranged alternately, with no diffusion (the diffusion distance at the laminar flow interface is ≤2μm, much smaller than the stripe width). Stripe adsorption process: During 30 minutes of incubation at room temperature, the quantum dot antibodies in the antibody solution layer covalently bind to the carboxyl groups on the inner wall of the channel, while the fluorescein buffer layer contains no antibodies. Therefore, an alternating pattern of "colorless antibody stripes - green fluorescent blank stripes" is formed in the cross-section. Key precautions: Avoid vibrating the chip during incubation (vibration can cause the laminar flow interface to fuse), and control the laboratory temperature at 25±1℃ (temperature changes can affect fluid viscosity and cause fluctuations in stripe width).

[0075] Observation of stripe morphology: Cross-sectional images of the reaction area were taken using a confocal microscope. The stripe width was 10±1μm and the spacing was 10±1μm. There was no fusion or breakage. Multi-directional rinsing: First, inject 50 μL PBS (flow rate 2 μL / min) into the inlet of the reaction zone to rinse away residual antibody solution; then inject 50 μL PBS (flow rate 2 μL / min) into the outlet to remove residual fluorescein solution at the linker; finally, blow dry with nitrogen (flow rate 3 L / min) and vacuum dry for 10 minutes (30℃, 10 Pa). Final verification: Observation was performed again using a confocal microscope. If the fluorescence intensity in the striped area was uniform (CV≤5%) and the blank area had no fluorescence (intensity≤5a.u.), then the stripe modification was qualified.

[0076] Through the above refined operations, antibody arrangement with "linearly controllable gradient of fluid flow direction and clear boundary of alternating stripes" can be clearly achieved, laying the foundation for subsequent rapid and efficient immune response.

[0077] 7. Secondary antibody modification in the detection area The inner wall of the detection window is covalently immobilized with a secondary anti-MOMP antibody (6 μg / mL), which recognizes the MOMP epitopes at positions 85-100 and does not compete with the antibody in the reaction region (positions 25-40); a "secondary antibody-antigen-quantum dot antibody" sandwich structure is formed, with a detection limit of 10¹ copies / mL.

[0078] 8. Chip Packaging The substrate and PDMS cover sheet, after the above steps, are placed in an oxygen plasma cleaner with the following parameters: power 100W, oxygen flow rate 20sccm, and processing time 60 seconds. After removal, they are immediately aligned and bonded, and baked at 60℃ for 30 minutes to achieve permanent sealing, thus obtaining the quantum dot microfluidic immunoassay chip.

[0079] III. Reagent Kit Assembly and Performance Validation 1. Reagent kit assembly Assemble the prepared chips (sealed in an aluminum-plastic packaging bag containing desiccant), sample diluent (10 mL / bottle), washing buffer (10 mL / bottle), positive control (1 mL / vial, 10³ copies / mL CT standard strain), negative control (1 mL / vial, sterile physiological saline), sterile sampling swabs (5 swabs), centrifuge tubes (10 swabs, 1.5 mL each) and instruction manual (1 copy) into a kit. The portable control and reading instrument is packaged separately and sold as a set.

[0080] 2. Sensitivity Experiment The CT standard strain (serotype D) was diluted to 10... 0 10¹, 10², 10³, 10 4 The kit was used to detect fluorescence intensity at gradient concentrations of copies / mL, with three replicates for each concentration.

[0081] When the concentration is ≥10¹copies / mL, the fluorescence intensity is significantly higher than that of the negative control (P<0.01), and the limit of detection is 10¹copies / mL, which is 10 times that of existing quantum dot microfluidic chips (limit of detection 10²copies / mL), meeting the needs of early infection detection.

[0082] 3. Specificity test Common pathogens of the female reproductive tract (Neisseria gonorrhoeae, Ureaplasma urealyticum, Candida albicans, Trichomonas vaginalis) and vaginal secretions from healthy individuals were selected, with three replicates for each sample, and the kit was used for detection.

[0083] All non-CT samples had fluorescence intensities ≤ 110% of the negative control fluorescence intensity, and were therefore considered negative, with no cross-reaction and a specificity of 100%.

[0084] 4. Anti-clogging performance test Thirty cervical swab samples with high viscosity (mucus content ≥50%) were selected and tested using this kit and a traditional filter membrane pretreatment chip (CN116643040A filter membrane pore size 0.22μm, static filtration pretreatment). The channel clogging rate was statistically analyzed. The pretreatment success rate of this kit was 96.7% (29 / 30), with no channel clogging; the pretreatment success rate of the traditional filter membrane chip was 56.7% (17 / 30), with 13 samples showing clogging. The anti-clogging performance of this invention is significantly better than that of existing technologies.

[0085] 5. Testing time and stability Detection time: From sample loading to result interpretation, the average time is 12.5 minutes (2 minutes of preprocessing + 8 minutes of reaction + 2.5 minutes of detection), which is much shorter than existing technologies (25-60 minutes).

[0086] Stability: The chip was sealed and stored at 4℃ for 0, 1, 2, 3 and 6 months, and the fluorescence intensity of 10³ copies / mL CT standard samples was detected. The results showed that the fluorescence intensity change rate was ≤8% within 6 months, indicating good stability.

[0087] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A quantum dot microfluidic immunoassay chip, characterized in that, The substrate includes a substrate and a cover plate, wherein the substrate is etched with a sample loading area, a pretreatment area, a reaction area, a detection area and a waste liquid area that are sequentially connected. The inner wall of the pretreatment zone channel is electrostatically and dynamically bonded with carboxylated magnetic nanobeads by polydiallyldimethylammonium chloride modified on the surface of the inner wall of the channel. Magnetic positioning parts are coated on the inner walls between the pretreatment zone and the sample loading zone and between the pretreatment zone and the reaction zone. The magnetic positioning parts and the magnetic nanobeads are magnetically repelled. An electromagnetic coil is embedded in the cover plate and aligned with the center of the pretreatment area. The electromagnetic coil is used to generate an alternating magnetic field around the nanomagnetic beads to drive the magnetic beads to vortex motion. The quantum dot anti-MOMP antibody is covalently fixed on the inner wall of the reaction zone channel, and the density of the quantum dot anti-MOMP antibody decreases in a gradient along the liquid flow direction to form gradient stripes, and alternating stripes are formed on the circumferential inner wall of the channel. The inner wall of the detection area channel is modified with secondary antibodies against Chlamydia trachomatis. In the sample to be tested, the Chlamydia trachomatis antigen forms a quantum dot-antibody-antigen complex with the quantum dot anti-MOMP antibody in the reaction area. The complex then flows to the detection area and binds with the secondary antibody to form a secondary antibody-antigen-quantum dot labeled antibody complex. The quantum dots emit fluorescence under excitation light and are detected.

2. The quantum dot microfluidic immunoassay chip according to claim 1, characterized in that, The nanobeads have a diameter of 180-220 nm and a magnetic bead loading of ≥1×10⁻⁶. 6 The adsorption rate of mucus impurities is ≥95% per mm².

3. The quantum dot microfluidic immunoassay chip according to claim 1, characterized in that, The quantum dots have a CdSe / ZnS core-shell structure, a diameter of 3-5 nm, and an emission wavelength of 580 nm.

4. The quantum dot microfluidic immunoassay chip according to claim 1, characterized in that, The electromagnetic coil has 100-150 turns, a wire diameter of 50μm, and a resistance of 9-11Ω.

5. The quantum dot microfluidic immunoassay chip according to claim 1, characterized in that, The substrate is also etched with a hydrophobic isolation region connecting the pretreatment region and the reaction region, and the inner wall of the hydrophobic isolation region is formed with a functional area hydrophobic surface.

6. The fabrication process of the quantum dot microfluidic immunoassay chip as described in any one of claims 1 to 5, characterized in that, include: Pre-treatment of the substrate; A face-centered cubic photonic crystal microcavity is formed on the back side of the substrate at the position corresponding to the detection area; A sample loading area, a pretreatment area, a reaction area, a detection area, and a waste liquid area are sequentially connected and etched on the front side of the substrate. A hydrophobic isolation zone is formed between the pretreatment zone and the reaction zone; Magnetic positioning parts are formed by magnetron sputtering at the junctions of the pretreatment zone and the sample loading zone, and the pretreatment zone and the reaction zone. PDMS cover plates with embedded electromagnetic coils are formed by 3D printing; Carboxylated magnetic nanobeads with hydrophobic groups coupled to the inner wall of the channel in the pretreatment zone were modified with polydiallyl dimethyl ammonium chloride. Gradient stripes of decreasing antibody concentration are formed in the reaction zone along the liquid flow direction, as well as alternating stripes formed in the circumferential direction by the alternating laminar flow of antibody solution and buffer solution; Secondary antibodies are modified in the detection area; The substrate and PDMS cover sheet, after undergoing the above steps, are cleaned with oxygen plasma, bonded together, and then baked and sealed.

7. A kit for detecting Chlamydia trachomatis in the female reproductive tract, characterized in that, Includes the quantum dot microfluidic immunoassay chip, portable controller and reader, and matching reagents as described in any one of claims 1 to 5; The portable control and reading instrument includes an alternating magnetic field generating module and a fluorescence detection module. The alternating magnetic field generating module provides a magnetic field of 50-100Hz and 50-100mT to the pretreatment area, and the fluorescence detection module provides an excitation wavelength of 488nm and an emission wavelength of 580nm to the detection area.

8. The reagent kit according to claim 7, characterized in that, The supporting reagents include: The sample diluent is PBS at pH 7.4 containing 0.5% Triton X-100 and 0.1% sodium azide; Washing buffer, wherein the washing buffer is PBS with pH 7.4 containing 0.1% Tween-20 and 0.05% sodium azide; Positive control, wherein the positive control is a CT standard strain containing 10³ copies / mL; The negative control is sterile physiological saline containing 0.1% sodium azide.

9. Use of the quantum dot microfluidic immunoassay chip according to any one of claims 1 to 5 in the preparation of a kit for the detection of Chlamydia trachomatis in the female reproductive tract.

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