Portable pressing type magnetoelastic microbial sensor based on 3D printing, preparation method and detection method

Abstract

The invention discloses a portable pressing type magnetoelastic microbial sensor based on 3D printing, a preparation method and a detection method, and belongs to the technical field of biosensors. The sensor comprises a shell, a sensing chip, a detection coil and a signal acquisition module; an air pump cavity, a micro-channel and a reaction cavity are arranged in the shell; the sensing chip is embedded in the reaction cavity, and the detection coil surrounds the outer side of the reaction cavity and is electrically connected with the signal acquisition module; one end of the micro-channel is communicated with the air pump cavity, and the other end is communicated with the reaction cavity; the micro-channel is provided with an external sampling interface; the shell is prepared by adopting a 3D printing technology, a sample is driven by pressure generated by pressing the air pump cavity to enter the reaction cavity along the micro-channel, and then the sewage sample is detected; according to the invention, the integration and portability problems are solved, and on-site real-time detection is adapted; the whole-process closed operation does not need sample transfer, and the reliability of detection data is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically a portable press-type magnetic microbial sensor, its preparation method, and its detection method. Background Technology

[0002] Against the backdrop of rapid industrialization and urbanization, the disorderly discharge of industrial wastewater and domestic sewage is becoming increasingly prominent. Excessive levels of pathogenic microorganisms, heavy metal ions, and organic pollutants in water bodies are frequent, severely disrupting the aquatic ecological balance and threatening human health through drinking water and the food chain. Relevant testing institutions have a rigid demand for rapid water quality testing—it needs to meet the high efficiency requirements of routine water quality screening while also being adaptable to on-site emergency monitoring of sudden water pollution incidents. However, current traditional water quality testing technologies (such as laboratory chromatography and biochemical culture methods) have significant shortcomings, failing to simultaneously meet the requirements of speed, accuracy, portability, and low cost. Current water quality testing technologies face the following core problems: (i) Insufficient testing efficiency and practicality, making it difficult to meet on-site needs. Traditional wastewater testing methods (such as biochemical culture for microbial detection, which requires 24-72 hours, and chromatographic methods for pollutant detection, which require pre-treatment of samples) have long testing cycles and cannot meet the "instant response" requirements of emergency monitoring. At the same time, the operation procedures are cumbersome, requiring professional personnel to perform sample pretreatment, reagent addition, instrument debugging, and other steps. Moreover, the testing process is highly dependent on the laboratory environment and has weak resistance to interference from impurities in the water body and temperature fluctuations. In addition, the purchase cost of large-scale testing instruments (such as liquid chromatographs and mass spectrometers) can reach hundreds of thousands of yuan, and the operation and maintenance costs are high, which is difficult for ordinary grassroots units and emergency scenarios to afford. (ii) Low level of integration, poor portability and field adaptability Existing magnetoelastic sensors generally adopt a separate design of "detection coil + detection chip + external sampling device + data processing terminal". The modules are connected by pipes and cables, resulting in a large overall device (usually larger than 30cm×20cm×15cm) and heavy (≥5kg), which is inconvenient to carry and operate in mobile scenarios such as the field, sewage wells, and sewage outlets. At the same time, the detection process requires an external power supply (220V AC) and large data acquisition equipment, which is severely limited by site conditions and cannot be promoted and applied in sewage detection scenarios in areas without power supply or in remote areas. (iii) Sample consumption and contamination are prominent issues, making it difficult to guarantee the accuracy of testing. 1. High sample volume requirement: Traditional sensors require 5-10 mL of wastewater sample per detection. For scarce samples (such as trace amounts of polluted water or samples from specific pollution sources) or scenarios where large-scale collection is difficult (such as narrow sewage pipes), collection is extremely challenging, and detection may even be impossible. 2. High risk of secondary and cross-contamination: During the detection process, samples must be transferred from the collection container to a pretreatment device and then to the detection chip, exposed to air throughout. This exposes the samples to secondary pollution as harmful microorganisms and pollutants in the wastewater can diffuse into the environment. Furthermore, aerosol diffusion can easily lead to cross-contamination between different samples, resulting in false positives and severely impacting data reliability. 3. Significant human error: Sampling, sample addition, and sample transfer all require manual intervention. Operator proficiency directly affects detection accuracy, and the cumbersome process can easily lead to omissions and errors, further reducing detection accuracy. (iv) The preparation process is complex, costly, and difficult to scale up. 1. High mold and processing costs: Key components of traditional magnetoelastic sensors, such as microchannels, reaction chambers, and detection modules, require mold fabrication using precision machining (e.g., CNC milling) or photolithography. The processing cycle is 7-15 days, resulting in low design flexibility and an inability to quickly adjust structural dimensions according to different detection targets (e.g., different microorganisms, contaminants). Furthermore, the purchase cost of precision machining and photolithography equipment is high, with a single mold costing over 10,000 yuan. 2. Stringent fabrication conditions: The fabrication process requires a Class 100 cleanroom environment and relies on large-scale precision machining equipment (e.g., photolithography exposure machines, precision milling machines), leading to high operating and maintenance costs that are difficult for ordinary manufacturers to meet. 3. Difficulty in large-scale production: The complex process (involving more than ten steps including mold processing, component assembly, calibration, and debugging) and poor compatibility between stages easily lead to component assembly deviations and performance inconsistencies. This makes standardized, mass production difficult, resulting in high unit production costs (over 2,000 yuan per unit), hindering its market promotion and widespread adoption. Summary of the Invention

[0003] This invention overcomes the shortcomings of existing technologies and proposes a portable, press-type magnetic elastic microbial sensor based on 3D printing, its preparation method, and its detection method. Through the core design of "integrated structure + 3D printing mold preparation + magnetic elastic immunosensing", this invention solves the pain points of existing technologies and achieves rapid, accurate, and portable detection of sewage samples.

[0004] This invention is achieved through the following technical solution: A portable, press-type magnetic elastic microbial sensor based on 3D printing includes a housing, a sensing chip, a detection coil, and a signal acquisition module. The housing contains an air pump chamber, a microchannel, and a reaction chamber. The sensing chip is embedded in the reaction chamber, and the detection coil is wrapped around the outside of the reaction chamber and electrically connected to the signal acquisition module. One end of the microchannel is connected to the air pump chamber, and the other end is connected to the reaction chamber. The microchannel is equipped with an external sampling interface. The pressure generated by pressing the air pump chamber drives the sample to enter the reaction chamber along the microchannel.

[0005] Furthermore, the housing is made of PDMS material, and the air pump cavity, microchannel, and reaction chamber are integrally formed by molding.

[0006] Furthermore, the mold is prepared by 3D printing.

[0007] Furthermore, the substrate of the sensor chip is an iron-nickel-based amorphous metal alloy thin film, with a gold layer sputtered on the surface of the thin film. A mercaptoethylamine self-assembled film is connected to the surface of the gold layer through Au-S bonds, and a specific antibody is fixed on the surface of the self-assembled film through amide bonds.

[0008] Furthermore, the sensor chip is fixed in the center of the reaction chamber, with the surface of the sensor chip facing the microchannel outlet.

[0009] The method for fabricating the portable press-type magnetic elastic microbial sensor based on 3D printing includes the following steps: S1. A mold is prepared by 3D printing, and the mold integrates microchannels, reaction chambers, and air pump chambers. S2. Slowly inject the pre-mixed PDMS liquid into the mold; place the mold after injection into a vacuum heating box for heating and heat preservation, so that the PDMS can be completely cured; then demold to obtain the shell; S3. The detection coil is wrapped tightly around the outside of the reaction chamber of the housing; S4. Prepare the sensor chip and embed and fix the sensor chip in the reaction chamber; S5. Connect the leads of the detection coil to the signal input terminal of the signal acquisition module.

[0010] Furthermore, the mold is pretreated before being injected with PDMS pre-prepared liquid; the pretreatment involves immersing the mold in anhydrous ethanol, ultrasonically cleaning it, and then drying it, followed by uniformly spraying a release agent onto the inner surface of the mold.

[0011] Furthermore, the fabrication methods of the sensor chip include: First, the iron-nickel-based amorphous metal alloy film with a gold layer sputtered on its surface is completely immersed in a mercaptoethylamine solution, so that mercaptoethylamine molecules form a dense self-assembled film on the gold layer surface through Au-S bonds. Then, the specific antibody against the target analyte is activated, and the modified iron-nickel-based amorphous metal alloy film is completely immersed in the activated antibody working solution, so that the activated antibody can be firmly bound to the amino groups on the surface of the self-assembled membrane through amide bonds. Finally, the film is removed, and the unbound antibodies on the film surface are removed; after air drying, the sensor chip is obtained.

[0012] A wastewater sample detection method, employing the aforementioned portable, press-type magnetic elastic microbial sensor based on 3D printing, includes the following steps: The first step is to align the microchannel sampling interface with the wastewater sample, ensuring that the interface is completely immersed in the sample; The second step is negative pressure sampling: Press the air pump chamber with your finger to expel the air inside the air pump chamber; then slowly release your finger, and a negative pressure difference will be formed inside the air pump chamber, which will drive the sewage sample to be automatically drawn into the microchannel and transported to the reaction chamber through the microchannel; after sampling is completed, remove the sensor from the sample. The third step is to place the sensor statically so that the target microorganisms / pollutants in the sewage sample can fully combine with the specific antibodies on the surface of the sensor chip to form an antigen-antibody complex. This complex causes changes in the mass and stiffness of the sensor chip, which in turn causes a shift in the resonant frequency. The fourth step is signal detection and result analysis: the signal acquisition module is activated to acquire the resonant frequency signal of the sensor chip and calculate the concentration of the target microorganisms / pollutants in the sewage sample.

[0013] The beneficial effects of this invention compared to the prior art are as follows: I. Solving the integration and portability issues to adapt to on-site real-time testing Through an integrated design of "press-pump-microchannel-detection chip", the press-pump is directly molded inside the microchannel PDMS mold for the first time. The detection coil tightly surrounds the reaction chamber, reducing the overall size of the sensor to a portable range (50mm×30mm×20mm) and weighing ≤100g. It has no external cables and supports handheld operation and use in scenarios without power supply (built-in battery life ≥8 hours). It completely eliminates the dependence of traditional sensors on large instruments, external power supplies and laboratory environments, and can be flexibly adapted to mobile detection scenarios such as field, sewage wells, and sewage outlets, filling the technical gap of rapid on-site detection. II. Solving sample contamination and consumption problems and improving detection accuracy 1. Fully enclosed operation: The entire process of "sampling-addition-detection" requires no sample transfer. The sample only flows in the closed channel and reaction chamber, which completely avoids secondary pollution caused by sample exposure and multiple transfers. At the same time, it eliminates cross-contamination caused by aerosol diffusion, effectively reduces the risk of false positive test results, and improves the reliability of test data by more than 90%. 2. Low sample consumption: Only 100-200μL of sewage sample is needed for a single test, which is much lower than that of traditional sensors (5-10mL), making it suitable for scenarios with scarce samples and micro-volume collection. 3. Reduce human error: The automatic sampling method with press-type sampling eliminates the need for manual sample transfer and addition. The operation steps are simplified to "immerse sample - press-to-absorb sample - let stand for detection", reducing human error by more than 80% and significantly improving detection accuracy. III. Solve the problem of complex preparation processes and reduce costs and barriers to large-scale production. 1. 3D printed molds replace traditional precision machining: The processing cycle of 3D printed molds is shortened by 90% (from 7-15 days to 1.5 hours), the mold cost is reduced by 95% (from tens of thousands of yuan to 50 yuan), and it can be reused more than 50 times, which greatly reduces R&D and production costs; 2. Simplified process flow: The core preparation steps are only 3, requiring no large-scale precision equipment or Class 100 cleanroom laboratories, allowing ordinary production enterprises to quickly start production; 3. Feasible for large-scale production: Standardized 3D printing and PDMS molding processes ensure consistent sensor performance across batches (error ≤5%), enabling mass production and reducing the production cost per sensor to below 500 yuan (compared to ≥2000 yuan per traditional sensor), facilitating market promotion and widespread adoption. (iv) Improve testing efficiency and scenario adaptability to meet diverse needs. 1. Significantly improved testing efficiency: Single sample testing time ≤ 20 minutes, enabling on-site immediate results, far faster than traditional testing methods (24-72 hours), meeting the needs of "rapid response" for emergency monitoring and "efficient advancement" for routine screening; 2. Easy to operate: No professional skills training is required. A single person can complete the entire process, making it suitable for different user groups such as primary healthcare, environmental supervision, and sewage treatment plants. 3. Strong stain resistance: PDMS material is corrosion resistant and flexible, and can adapt to the complex environment of wastewater testing sites (acids, alkalis, impurities, temperature fluctuations). The sensor can be reused ≥50 times, reducing the cost of use. 4. Wide detection range: By changing different specific antibodies, it can be adapted to microorganisms such as Escherichia coli and Salmonella, as well as various target substances such as heavy metal ions and organic pollutants, to meet diverse water quality testing needs. Attached Figure Description

[0014] Figure 1 This is a physical image of the magnetic elastic microbial sensor described in this invention; Figure 2 This is a schematic diagram of the structure of the magnetic elastic microbial sensor described in this invention; Figure 3It is a design drawing of a T-shaped press air pump mold. Detailed Implementation

[0015] To make the technical problems to be solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The technical solutions of this invention are described in detail below with reference to the embodiments and accompanying drawings, but the scope of protection is not limited thereto. Example 1

[0016] This embodiment proposes a portable, press-type magnetic elastic microbial sensor based on 3D printing. (See [link to documentation]). Figures 1 to 3 The aforementioned magnetic elastic microbial sensor is a miniaturized handheld detection device with an overall size controlled within 50mm×30mm×20mm and a weight ≤100g. It includes a housing, a sensing chip, a detection coil, and a signal acquisition module. The housing is an integrated PDMS structure, which serves as the core supporting substrate. The integrated PDMS structure includes an air pump chamber, a microchannel, and a reaction chamber. The sensing chip is embedded in the reaction chamber, and the detection coil surrounds the outside of the reaction chamber and is electrically connected to the signal acquisition module. All components work together to achieve sample delivery, specific identification, and signal acquisition functions. I. Integrated PDMS Structure Specifically, the integrated PDMS structure is made of PDMS material (SYLGARD 184) and is molded in one piece. It is the core skeleton of the sensor, integrating three key functional areas. Each area is seamlessly connected and cannot be separated. The specific structural parameters are as follows: Air pump chamber: 5mm×5mm×2mm (width×height×depth), providing a power chamber for sample delivery, with a reserved interface for docking with microchannels (5mm×2mm, tolerance ±0.05mm). Microchannel: linear structure, dimensions 1mm×1mm×20mm (width×height×length), one end precisely docks with the air pump chamber interface, the other end extends into the reaction chamber, with a reserved external sampling interface (1mm in diameter) to ensure precise delivery of the sample from the sampling port to the reaction chamber; Reaction chamber: 1.5mm×5.5mm×3mm (width×height×depth), providing a closed space for the reaction between the sample and the sensor chip. A detection coil is wound around the outside of the reaction chamber, and the sensor chip is fixed in the center inside. The surface of the sensor chip is aligned with the microchannel outlet to ensure full contact between the sample and the sensor chip.

[0017] The air pump chamber, microchannel, and reaction chamber are integrally molded together. The 5mm×2mm interface of the air pump chamber is precisely aligned with one end of the microchannel (with a tolerance of ±0.05mm) to ensure leak-free sample delivery. The other end of the microchannel extends directly into the reaction chamber, forming a sample delivery path of "sampling interface → microchannel → reaction chamber". The pressure generated by pressing or squeezing the air pump chamber can drive the sample into the reaction chamber along the microchannel.

[0018] II. Sensor Chip The sensor chip substrate is a thin film of iron-nickel-based amorphous metal alloy (Metglas2826) (10mm × 5mm × 20μm), with a 50nm gold layer sputtered on the surface. A self-assembled mercaptoethylamine (SAM) film is connected to the gold layer surface via Au-S bonds. Specific antibodies (targeting the analyte) are immobilized on the surface of the SAM via amide bonds, forming a complete specific recognition layer. The sensor chip is a thin sheet and is embedded inside the reaction chamber using a fixation method.

[0019] The sensor chip is fixed to the center of the PDMS reaction chamber using medical double-sided adhesive (0.1mm thick). After fixing, the chip surface is precisely aligned with the microchannel outlet, ensuring that the sample delivered by the microchannel can directly contact the specific antibody layer on the chip surface, achieving specific recognition of the target analytes in the sample. The fixing method is adhesive, with no mechanically detachable structure, ensuring the chip's positional stability during the detection process.

[0020] 3. The detection coil is made of tightly wound 0.1mm diameter copper wire, which is wrapped around the outside of the PDMS reaction chamber with 60 turns. The coil spacing is ≤0.2mm, and 5mm long leads are reserved at both ends. The leads are fixed with hot melt glue to prevent them from falling off. IV. Signal Acquisition Module A miniature impedance analyzer (30mm×20mm×10mm, weight ≤50g) is used as the core for signal reception and processing, with a reserved signal input terminal. The 5mm long leads reserved at both ends of the detection coil are directly connected to the signal input terminal of the miniature impedance analyzer. The interface is sealed with insulating tape to prevent short circuits of the leads and reduce external interference, ensuring that the resonant signal collected by the coil is stably transmitted to the signal acquisition module. The sensor assembly is based on an integrated PDMS structure. After the sensor chip and detection coil are connected to the PDMS structure, a sealing test is performed on the key connections: pressing the air pump chamber and injecting deionized water inside, checking for leaks at the connection between the air pump chamber and the microchannel, and at the sealing surface of the reaction chamber. Once no leaks are confirmed, the overall integration is completed. This ultimately forms a complete functional chain: "signal acquisition module → detection coil → reaction chamber (embedded sensor chip) → microchannel → air pump chamber / sampling interface." All components are stably connected without loosening, meeting the requirements for handheld operation. Example 2

[0021] This embodiment proposes a method for fabricating a portable press-type magnetic elastic microbial sensor based on 3D printing, used for the fabrication of the sensor described in Example 1; specifically: Module 1: Design and manufacturing of T-shaped press-type air pump mold (including integrated molding of microchannels and reaction chamber) This module uses 3D printing to create a high-precision mold, which is then used to form an integrated structure of "air pump cavity - microchannel - reaction chamber" made of PDMS material, laying the foundation for subsequent integration. The specific steps are as follows: 1. 3D Modeling and Export: Using 3DMax 2024 3D modeling software, the T-shaped press-type air pump mold was designed with the following core parameters: The overall mold dimensions are 20mm × 15mm × 10mm, integrating a 1mm × 1mm × 20mm (width × height × length) linear microchannel, a 1.5mm × 5.5mm × 3mm (width × height × depth) reaction chamber, and a 5mm × 5mm × 2mm (width × height × depth) air pump chamber. One end of the microchannel connects to the reaction chamber, and the other end precisely aligns with the 5mm × 2mm interface of the air pump chamber (with a tolerance of ±0.05mm) to ensure smooth sample delivery. During the modeling process, the mold was divided into two core modules: an upper mold and a lower mold, with reserved positioning pin holes (2mm in diameter) and demolding grooves (1mm in width) for easy demolding and removal of PDMS molded parts. After modeling, an STL format model file adapted for DLP photopolymerization 3D printing was exported, with model accuracy controlled within ±0.02mm. 2. 3D Printing and Mold Post-processing: High-temperature resistant photosensitive resin was selected as the printing material. The STL model was imported into slicing software (such as Chitubox), and the printing parameters were set as follows: layer thickness 0.05mm, exposure time 8s / layer, bottom exposure time 30s, and printing speed 50mm / h. After setting the parameters, the model was imported into the 3D printer, and the printing program was started to complete the mold blank formation (printing time approximately 1.5 hours). After printing, the mold blank was removed and placed in an ultrasonic cleaning tank containing isopropyl alcohol (99.7% concentration) for 10 minutes at a frequency of 40kHz to remove uncured resin from the surface. Then, the mold blank was placed in... Post-curing in a UV curing chamber (wavelength 365nm, power 30W) for 15 minutes improves mold hardness and dimensional stability. After removal, the support structure (support density 20%) is manually peeled off, and then the inner surface of the mold (microchannel, reaction chamber, and air pump chamber) is polished using a plasma polisher (power 50W, argon atmosphere) for 5 minutes to achieve an inner surface roughness Ra≤0.1μm. Finally, the mold dimensions are checked (measured with calipers and a microscope) to ensure that key parameters such as microchannel diameter and reaction chamber size meet design requirements, resulting in a smooth and dimensionally accurate T-shaped press air pump mold (upper mold + lower mold). 3. PDMS molding (preparation of pump cavity, microchannel, and reaction chamber): ① Prepare PDMS prepreg: Add 10g of PDMS A glue (SYLGARD 184 base glue) to a clean test tube, followed by 1g of PDMS B glue (SYLGARD 184 curing agent). Use a glass stir bar to stir thoroughly at 500rpm for 10min to ensure that A glue and B glue are evenly mixed. Let the mixed gel stand for 4-5 hours (room temperature 25℃, humidity 50%), or place it in a vacuum drying oven (-0.09MPa) for 30min to degas and completely remove internal air bubbles, forming a bubble-free PDMS prepreg. Seal and set aside for later use. ② Mold pretreatment: Soak the mold in anhydrous ethanol (concentration 99.7%) for 5 min, ultrasonically clean (40 kHz, 5 min) and blow dry. Then, evenly spray the mold release agent (model: CRC03035) on the inner surface of the mold (microchannel, reaction chamber, air pump chamber area), with the spray thickness controlled at 1-2 μm. Let it stand for 10 min to allow the mold release agent to fully adhere. ③ PDMS casting and curing: Slowly inject the pre-treated PDMS liquid into the pre-treated mold (the casting height is slightly higher than the upper surface of the mold cavity by 0.5mm) to avoid air bubbles; place the mold after injection into a vacuum heating box, set the temperature to 70℃ and the vacuum degree to -0.08MPa, heat for 4 hours, then turn off the heating switch and continue to keep it warm for 3 hours using the residual heat (the temperature slowly drops below 40℃) to ensure that the PDMS is completely cured; ④ Demolding and Molded Part Inspection: Remove the mold and allow it to cool to room temperature (25℃). Gently pry the upper and lower molds through the demolding groove. Use tweezers to slowly remove the cured PDMS molded part (including the air pump cavity, microchannels, and reaction chamber) from the mold, avoiding damage to the microchannel structure. Check whether the surface of the molded part is smooth and undamaged, and whether the microchannels are unobstructed (this can be verified by injecting deionized water). Discard unqualified products and keep qualified products for future use. 4. Detection Coil Winding: Select 0.1mm diameter copper wire and tightly wind it around the outside of the reaction chamber of the PDMS molded part. The number of turns is 60, and the coil spacing is ≤0.2mm. Avoid short circuits of the copper wire during the winding process. Leave 5mm long leads at both ends of the coil (for connecting the signal acquisition module). Fix the leads with hot melt glue to prevent them from being pulled off. After winding, check the coil conductivity (measured with a multimeter, the resistance value is controlled within 50-80Ω) to ensure that the resonant signal of the magnetoelastic sensing element can be stably acquired. Module 2: Fabrication of Sensor Chips The core objective of this module is to modify specific antibodies onto the Metglas2826 substrate to achieve specific identification of target microorganisms / pollutants in wastewater, while ensuring the chip's detection sensitivity and stability. The specific steps are as follows: 1. Substrate cleaning and preparation of self-assembled membranes (SAM): ① Take a Metglas 2826 thin film (10mm×5mm×20μm) with a 50nm gold layer sputtered on its surface, place it in an ultrasonic cleaning tank filled with deionized water, and ultrasonically clean it at a frequency of 40kHz for 5 minutes to remove surface oil, dust and other impurities; after cleaning, place it on clean filter paper and air dry it naturally at room temperature (25℃) in a windless environment (about 30 minutes) to avoid wiping and causing damage to the gold layer; ② The air-dried Metglas2826 film was completely immersed in a 40 mM / mL mercaptoethylamine solution and placed at room temperature (25°C) in the dark for 12 hours to allow mercaptoethylamine molecules to form a dense self-assembled film (SAM) on the gold layer surface through Au-S bonds (gold-mercapto reaction). ③ Remove the film that has formed SAM, use a pipette to remove the residual mercaptoethylamine solution on the surface, and then place it in a container containing PBS buffer (0.01M, pH7.4) and soak for 30 min (room temperature 25℃, gentle shaking, 50 rpm) to remove mercaptoethylamine molecules that are not completely fixed on the surface; after soaking, remove the film and let it air dry for later use. 2. Antibody activation treatment: ① Select a specific antibody against the target analyte (such as a monoclonal antibody for detecting Escherichia coli), and dilute it to 10 μg / mL with PBS buffer (0.01 M, pH 7.4) to obtain the antibody working solution; ② Add 1 mL of antibody working solution to a centrifuge tube, followed by 200 μL of a mixed solution containing 4 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 4 mg / mL NHS (N-hydroxysuccinimide). Place the tube in a constant temperature shaker and shake at room temperature (25℃) and 200 rpm for 30 min to activate the carboxyl group (-COOH) in the antibody molecule into NHS ester, thereby improving its binding efficiency with the amino group (-NH2) on the surface of the self-assembled membrane. 3. Antibody fixation and blocking treatment: ① The SAM-modified Metglas2826 membrane was completely immersed in the activated antibody working solution and placed in a constant temperature incubator. It was then allowed to stand at 37°C for 1 hour to allow the activated antibody to firmly bind to the amino groups on the surface of the self-assembled membrane via amide bonds. ② Remove the membrane and use a pipette to remove any residual antibody solution on the surface. Then place the membrane in a container containing PBS buffer (0.01M, pH 7.4) at a dilution ratio of 1:10 (membrane to buffer volume ratio). Incubate at 37°C for 0.5 hours to remove unbound antibodies that are physically adsorbed onto the membrane surface. ③ Remove the film, rinse the surface twice with deionized water, and air dry naturally to obtain the magnetostrictive immunosensor chip product; place the product in a sealed bag and store it at 4°C for a shelf life of no less than 3 months. Module 3: Sensor Assembly The prepared PDMS integrated structure (pump chamber-microchannel-reaction chamber) was integrated and assembled with the sensing chip and signal acquisition module (miniature impedance analyzer, size 30mm×20mm×10mm, weight ≤50g): ① Fix the sensor chip in the center of the reaction chamber with medical double-sided tape (0.1mm thick, with good biocompatibility) to ensure that the chip surface is aligned with the microchannel outlet, so as to facilitate full contact between the sample and the chip; ② Connect the lead wire of the detection coil to the signal input terminal of the miniature impedance analyzer, and wrap the interface with insulating tape to prevent short circuits and signal interference; ③ Perform a sealing test on the connection between the air pump chamber and the microchannel, and the sealing surface of the reaction chamber (connect the air pump chamber to the press air pump, inject deionized water through the press air pump, and observe whether there is any leakage). After ensuring that there is no leakage, complete the overall assembly of the sensor; after assembly, the overall size of the sensor is controlled within 50mm×30mm×20mm, and the weight is ≤100g, supporting handheld operation. Example 3

[0022] This embodiment proposes a wastewater sample detection method, employing the portable press-type magnetic elastic microbial sensor based on 3D printing as described in Embodiment 1, specifically comprising the following steps: 1. Sample collection preparation: Align the microchannel sampling interface (1mm in diameter) of the sensor with the sewage sample (such as sewage well, sewage outlet, water sample container), and ensure that the interface is completely immersed in the sample (immersion depth ≥2mm) to prevent air from entering the channel. 2. Negative pressure sampling: Gently press the air pump chamber with your finger (pressing stroke 1-2mm, moderate force to avoid damaging the PDMS structure) to expel the air inside the air pump, and hold the press for 3 seconds; then slowly release your finger, and a negative pressure difference will be formed inside the air pump (negative pressure value approximately -0.02MPa), driving the sewage sample to be automatically drawn into the microchannel and transported to the reaction chamber through the microchannel. The sample aspiration volume is controlled at 100-200μL (adjusted by the air pump pressing pressure); after sampling is completed, remove the sensor from the sample and wipe the surface of the sampling interface with lint-free paper to remove any remaining sample. 3. Reaction incubation: Place the sensor statically (horizontally to avoid sample spillage) and incubate at room temperature (20-30℃) for 20 minutes to allow the target microorganisms / pollutants in the wastewater sample to fully bind with the specific antibodies on the surface of the sensor chip, forming an antigen-antibody complex. This complex will cause changes in the mass and stiffness of the sensor chip, thereby causing a shift in the resonant frequency. 4. Signal Detection and Result Analysis: Start the miniature impedance analyzer, set the detection parameters (frequency range 1-10MHz, resolution 1Hz), and acquire the resonant frequency signal of the sensor chip; record the resonant frequency values ​​before and after detection, and calculate the frequency shift change (Δf = frequency after detection - frequency before detection); according to the preset calibration curve (pre-calibrated using standard concentration samples, Δf is linearly related to the target concentration), substitute the frequency shift to calculate the concentration of the target microorganism / pollutant in the wastewater sample; after detection, the sample in the reaction chamber can be discharged by pressing the air pump, and deionized water can be injected to clean the microchannel and reaction chamber (clean 2-3 times). The sensor can be reused (reusable ≥50 times). It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0023] The detailed descriptions listed above are merely specific descriptions of feasible implementation methods of this application and are not intended to limit the scope of protection of this application. All equivalent implementation methods or modifications made without departing from the spirit of the art of this application should be included within the scope of protection of this invention.

Claims

1. A portable, press-type magnetic elastic microbial sensor based on 3D printing, characterized in that, It includes a housing, a sensor chip, a detection coil, and a signal acquisition module; the housing contains an air pump chamber, a microchannel, and a reaction chamber; the sensor chip is embedded in the reaction chamber, and the detection coil is wrapped around the outside of the reaction chamber and electrically connected to the signal acquisition module; one end of the microchannel is connected to the air pump chamber, and the other end is connected to the reaction chamber; the microchannel is provided with an external sampling interface; the sample is driven into the reaction chamber along the microchannel by the pressure generated by pressing or squeezing the air pump chamber.

2. The portable press-type magnetic elastic microbial sensor based on 3D printing according to claim 1, characterized in that, The housing is made of PDMS material, and the air pump cavity, microchannel, and reaction chamber are integrally formed by molding.

3. The portable press-type magnetic elastic microbial sensor based on 3D printing according to claim 2, characterized in that, The mold is prepared by 3D printing.

4. The portable press-type magnetic ballistic microbial sensor based on 3D printing according to claim 1, characterized in that, The substrate of the sensor chip is an iron-nickel-based amorphous metal alloy thin film. A gold layer is sputtered on the surface of the thin film. A mercaptoethylamine self-assembled membrane is connected to the surface of the gold layer through Au-S bonds. Specific antibodies are fixed on the surface of the self-assembled membrane through amide bonds.

5. The portable press-type magnetic ballistic microbial sensor based on 3D printing according to claim 1, characterized in that, The sensor chip is fixed in the center of the reaction chamber, with the surface of the sensor chip facing the microchannel outlet.

6. The method for fabricating a portable press-type magnetic elastic microbial sensor based on 3D printing according to any one of claims 1-5, characterized in that, Includes the following steps: S1. A mold is prepared by 3D printing, and the mold integrates microchannels, reaction chambers, and air pump chambers. S2. Slowly inject the pre-mixed PDMS liquid into the mold; place the mold after injection into a vacuum heating box for heating and heat preservation, so that the PDMS can be completely cured; then demold to obtain the shell; S3. The detection coil is wrapped tightly around the outside of the reaction chamber of the housing; S4. Prepare the sensor chip and embed and fix the sensor chip in the reaction chamber; S5. Connect the leads of the detection coil to the signal input terminal of the signal acquisition module.

7. The method for fabricating a portable press-type magnetic elastic microbial sensor based on 3D printing according to claim 6, characterized in that, The mold is pretreated before being injected with PDMS pre-prepared liquid; the pretreatment involves immersing the mold in anhydrous ethanol, ultrasonically cleaning it, and then blowing it dry, followed by uniformly spraying a release agent onto the inner surface of the mold.

8. The method for fabricating a portable press-type magnetic elastic microbial sensor based on 3D printing according to claim 6, characterized in that, Methods for fabricating sensor chips include: First, the iron-nickel-based amorphous metal alloy film with a gold layer sputtered on its surface is completely immersed in a mercaptoethylamine solution, so that mercaptoethylamine molecules form a dense self-assembled film on the gold layer surface through Au-S bonds. Then, the specific antibody against the target analyte is activated, and the modified iron-nickel-based amorphous metal alloy film is completely immersed in the activated antibody working solution, so that the activated antibody can be firmly bound to the amino groups on the surface of the self-assembled membrane through amide bonds. Finally, the film is removed, and the unbound antibodies on the film surface are removed; after air drying, the sensor chip is obtained.

9. A method for detecting wastewater samples, characterized in that, The detection is performed using the portable, press-type magnetic elastic microbial sensor based on 3D printing as described in any one of claims 1-5, and includes the following steps: The first step is to align the microchannel sampling interface with the wastewater sample, ensuring that the interface is completely immersed in the sample; The second step is negative pressure sampling: Press the air pump chamber with your finger to expel the air inside the air pump chamber; then slowly release your finger, and a negative pressure difference will be formed inside the air pump chamber, which will drive the sewage sample to be automatically drawn into the microchannel and transported to the reaction chamber through the microchannel; after sampling is completed, remove the sensor from the sample. The third step is to place the sensor statically so that the target microorganisms / pollutants in the sewage sample can fully combine with the specific antibodies on the surface of the sensor chip to form an antigen-antibody complex. This complex causes changes in the mass and stiffness of the sensor chip, which in turn causes a shift in the resonant frequency. The fourth step is signal detection and result analysis: the signal acquisition module is activated to acquire the resonant frequency signal of the sensor chip and calculate the concentration of the target microorganisms / pollutants in the sewage sample.

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