Giant magnetoresistive sensor combined with microfluidic technology and manufacturing method and application of giant magnetoresistive sensor

Abstract

The invention discloses a giant magnetoresistive sensor combined with a micro-fluidic technology and a manufacturing method and application thereof, and belongs to the cross technical field of sensor technologies, micro-fluidic technologies and marine organism detection. Introduction, reaction and washing separation of a seawater sample are realized through a microfluidic channel, functional magnetic nanoparticles are introduced to carry out magnetic labeling and magnetic enrichment on target marine microorganisms, and a giant magnetoresistance chip array is used for carrying out high-sensitivity electrical detection on magnetic signals. The rapid, sensitive and specific recognition on marine bacteria, microalgae, fungi and viruses is realized. The method has the advantages of being high in seawater matrix interference resistance, high in sensitivity, small in sample dosage, capable of achieving multi-target parallel detection, suitable for on-site deployment and the like, and is suitable for the fields of marine ecological monitoring, harmful algal bloom early warning, mariculture disease monitoring, marine environment safety early warning and the like.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of sensor technology, microfluidics technology and marine biological detection technology, specifically relating to a giant magnetoresistive sensor combining microfluidics technology, its manufacturing method and application. Background Technology

[0002] Marine microorganisms, including bacteria, archaea, fungi, microalgae, and viruses, are an important component of marine ecosystems, playing a crucial role in marine carbon and nitrogen cycles and energy flow. However, some marine microorganisms, such as red tide algae, pathogenic bacteria, and viruses, can cause algal blooms, aquaculture diseases, and public health risks, seriously impacting marine ecological security, fisheries production, and human health. Therefore, rapid, sensitive, and field-deployable molecular identification and detection of marine microorganisms is of great significance.

[0003] Currently, marine microbial detection mainly relies on technologies such as microscopic observation, culture and identification, immunoassay, nucleic acid amplification (PCR, qPCR, LAMP), and high-throughput sequencing. Microscopic and culture methods are cumbersome to operate, have long detection cycles, and are difficult to achieve real-time monitoring; nucleic acid amplification and sequencing methods have high sensitivity but expensive equipment, complex procedures, and high requirements for experimental conditions, making them unsuitable for rapid detection in scenarios such as offshore platforms, aquaculture sites, and buoy systems; traditional immunological detection methods (such as ELISA and colloidal gold) are easily affected by high salinity, high turbidity, and autofluorescence interference in seawater, resulting in poor detection stability and repeatability.

[0004] Microfluidics integrates sample transport, mixing, washing, separation, and detection within microscale channels, offering advantages such as small sample volume, high reaction efficiency, and ease of automation and parallelization. However, seawater samples are prone to channel contamination and non-specific adsorption due to high salt ion concentrations, abundant organic matter, and colloidal particles. Furthermore, the concentration of target microorganisms is often low, making it difficult to meet early warning detection requirements when effective enrichment methods are lacking.

[0005] Magnetic nanoparticles, as biomarkers, offer advantages such as good biocompatibility, strong magnetic response, and low background interference. Giant magnetoresistance (GMR) sensors exhibit extremely high sensitivity to changes in weak magnetic fields, enabling chip-level integration and array-based detection, and their electrical readout methods are easily miniaturized and power-efficient. However, existing GMR biodetection platforms are mostly used for relatively clean samples such as serum. When directly applied to complex seawater samples, they still suffer from problems such as insufficient target capture efficiency, severe non-specific adsorption, and limited multi-target detection capabilities.

[0006] Therefore, there is an urgent need for a technical solution that organically combines the advantages of microfluidic sample processing, the enrichment capacity of magnetic nanoparticles, and the high sensitivity of GMR detection, for the rapid molecular identification and quantitative detection of marine microorganisms in complex seawater matrices. Summary of the Invention

[0007] Technical problem to be solved: In view of the above-mentioned technical problems, the present invention provides a giant magnetoresistive sensor that combines microfluidics technology, its manufacturing method and application, making it suitable for molecular recognition and detection of marine microorganisms, and solving the problems of complex seawater samples, low detection sensitivity, difficulty in field deployment and insufficient multi-target detection capability in the prior art.

[0008] Technical solution: In the first aspect, the present invention provides an application of a giant magnetoresistive sensor combined with microfluidic technology in the identification and detection of marine microbial molecules. The application includes the following steps: sample introduction, magnetic labeling reaction, washing and separation, magnetic enrichment and GMR readout.

[0009] Secondly, the present invention provides a giant magnetoresistive (GMR) sensor combining microfluidic technology. The GMR sensor, from top to bottom, includes a top cover, a photoresist layer, and a GMR chip substrate. The top cover has a fluid inlet and a fluid outlet. The photoresist layer includes a first photoresist layer and a second photoresist layer, with the second photoresist layer disposed inside the first photoresist layer. An annular perforation is formed between the first and second photoresist layers, and adhesive is provided within the annular perforation. Two through-holes are provided on the second photoresist layer, and the two through-holes are connected to form a detection area. The fluid inlet and fluid outlet are respectively aligned with the centers of the two through-holes and have equal radii. A GMR chip array is provided on the upper surface of the detection area of ​​the GMR chip substrate. The GMR chip array includes a plurality of GMR chips, each of which is a multilayer film structure, and its upper surface is fixed with trapped molecules.

[0010] Preferably, the capture molecule is an antibody, aptamer, nucleic acid probe, polypeptide, or combination thereof targeting marine microorganisms or their molecular markers; the detection molecule used in conjunction with the capture molecule is functionalized with biotin, and the biotin is linked to magnetic nanoparticles through streptavidin to form a sandwich or bridging structure of "capture molecule-target microorganism-magnetic nanoparticle".

[0011] Preferably, an external magnetic field source is provided at the corresponding position of the detection area to magnetically enrich the target marine microorganisms labeled with magnetic nanoparticles, thereby improving the detection sensitivity of low-abundance targets and reducing non-specific background; the external magnetic field source is one or more of a permanent magnet, an electromagnetic coil, or an integrated micro-electromagnetic structure.

[0012] Preferably, the surface of the giant magnetoresistive chip and / or the inner wall of the microfluidic channel are provided with an anti-non-specific adsorption sealing layer to adapt to the complex matrix environment of seawater.

[0013] Furthermore, the sealing layer is bovine serum albumin, casein, polyethylene glycol, zwitterionic polymer, or a combination thereof.

[0014] Thirdly, the present invention provides a method for manufacturing a giant magnetoresistive sensor incorporating microfluidic technology, comprising the following steps: S1. A giant magnetoresistive multilayer film structure is deposited on a silicon wafer using magnetron sputtering technology to obtain a wafer; S2. A giant magnetoresistive stripe sensor structure is formed on a wafer by photolithography and dry etching, and electrodes and passivation layers are prepared to obtain a giant magnetoresistive chip array. S3. A photoresist layer structure is prepared by photolithography and bonded to the substrate of the giant magnetoresistive chip, and then the top cover is attached to form a microfluidic channel. S4. Immobilize the molecules required for marine microbial detection on the surface of the giant magnetoresistive chip and perform a sealing process.

[0015] Beneficial effects: 1) Magnetic signal detection does not rely on optical readout and has strong resistance to seawater turbidity and autofluorescence interference; 2) The combination of microfluidics, magnetic enrichment, and GMR array enables highly sensitive and rapid detection; 3) Small sample volume and miniaturized device, suitable for deployment on ships, buoys, and in aquaculture sites; 4) Parallel detection of multiple marine microorganisms can be achieved through array design; 5) The manufacturing process is mature, which facilitates large-scale production and application. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the overall structure of the giant magnetoresistive sensor combining microfluidic technology according to the present invention; Figure 2 This is a schematic diagram of the microfluidic channel and detection area structure; Figure 3 A schematic diagram illustrating the principle of magnetic nanoparticle labeling of marine microorganisms; Figure 4 A schematic diagram illustrating the principle of magnetic signal detection using a giant magnetoresistive chip array. The numbers in the diagram are: 1. Top cover, 2. Photoresist layer, 2-1. First photoresist layer, 2-2. Second photoresist layer, 2-3. Circular cutout, 2-4. Detection area, 3. Giant magnetoresistive chip substrate, 3-1. Giant magnetoresistive chip, 4. Magnetic nanoparticles. Detailed Implementation

[0017] The present invention will be described in detail below with reference to specific embodiments: Example 1

[0018] like Figure 1As shown, the giant magnetoresistive sensor of the present invention, which incorporates microfluidic technology, includes, from top to bottom, an upper cover 1, a photoresist layer 2, and a giant magnetoresistive chip substrate 3. The upper cover 1 is provided with a fluid inlet 1-1 and a fluid outlet 1-2; the photoresist layer 2 includes a first photoresist layer 2-1 and a second photoresist layer 2-2, with an annular perforation 2-3 formed between the first photoresist layer 2-1 and the second photoresist layer 2-2 and sealed with adhesive; the second photoresist layer 2-2 is provided with two through holes, which are connected to form a detection area 2-4; the giant magnetoresistive chip substrate 3 is located below the detection area 2-4, and its upper surface is provided with a giant magnetoresistive chip array 3-1. Example 2

[0019] A method for manufacturing a giant magnetoresistive sensor incorporating microfluidic technology includes the following steps: A multilayer film structure consisting of Ta, NiFeCr, PtMn, CoFe, Ru, CoFe, NOL, CoFe, Cu, CoFe, NiFe, Cu, and Ta was sequentially deposited on a silicon wafer using magnetron sputtering. The stripe sensor structure was formed and electrodes were fabricated by photolithography and dry etching. A Si3N4 passivation layer was deposited by PECVD. Subsequently, a photoresist layer was prepared by photolithography and bonded to the chip substrate. A top cover was then attached to form a microfluidic channel.

[0020] Example 3: Marine Microbial Detection Using red tide algae as the detection target, seawater samples are introduced into a microfluidic channel through a fluid inlet after simple filtration; magnetic nanoparticles with anti-red tide algae antibodies immobilized on their surface are added and incubated in the reaction zone to form a complex; unbound material is removed in the washing zone; an external magnetic field is applied in the detection zone for magnetic enrichment; the magnetic signal is detected by a GMR chip array and the results are output, realizing rapid identification of red tide algae.

[0021] Example 4: Parallel Detection of Multiple Targets Capture molecules targeting Vibrio, Aeromonas hydrophila, and marine viruses are fixed at different pixels in a giant magnetoresistive chip array, enabling parallel detection of the three pathogenic microorganisms, which is suitable for monitoring diseases in marine aquaculture.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of a giant magnetoresistive sensor combining microfluidics technology in the identification and detection of marine microbial molecules.

2. The application according to claim 1, characterized in that: The giant magnetoresistive sensor combining microfluidics technology comprises, from top to bottom, a top cover (1), a photoresist layer (2), and a giant magnetoresistive chip substrate (3). The top cover (1) has a fluid inlet (1-1) and a fluid outlet (1-2). The photoresist layer (2) includes a first photoresist layer (2-1) and a second photoresist layer (2-2). The second photoresist layer (2-2) is disposed inside the first photoresist layer (2-1), and an annular cutout (2-3) is formed between the first photoresist layer (2-1) and the second photoresist layer (2-2). The hollowed-out area (2-3) contains adhesive, and the second photoresist layer (2-2) has two through holes. The two through holes are connected to form a detection area (2-4). The fluid inlet (1-1) and fluid outlet (1-2) are respectively aligned with the center of the two through holes and have the same radius. The giant magnetoresistive chip substrate (3) is provided with a giant magnetoresistive chip array on the upper surface of the detection area (2-4). The giant magnetoresistive chip array includes several giant magnetoresistive chips (3-1). The giant magnetoresistive chip (3-1) is a multilayer film structure, and the upper surface is fixed with trapped molecules.

3. The application according to claim 2, characterized in that: The capture molecule is an antibody, aptamer, nucleic acid probe, polypeptide, or combination thereof targeting marine microorganisms or their molecular markers; the detection molecule used in conjunction with the capture molecule is functionalized with biotin, which is linked to magnetic nanoparticles via streptavidin.

4. The application according to claim 2, characterized in that: An external magnetic field source is provided at the corresponding position of the detection area (2-4). The external magnetic field source is one or more of a permanent magnet, an electromagnetic coil, or an integrated micro-electromagnetic structure.

5. The application according to claim 2, characterized in that: The surface of the giant magnetoresistive chip (3-1) and / or the inner wall of the microfluidic channel are provided with an anti-non-specific adsorption sealing layer.

6. The application according to claim 5, characterized in that: The sealing layer is bovine serum albumin, casein, polyethylene glycol, zwitterionic polymer, or a combination thereof.

7. The application according to claim 2, characterized in that, The manufacturing method of the giant magnetoresistive sensor combining microfluidics technology includes the following steps: S1. A giant magnetoresistive multilayer film structure is deposited on a silicon wafer using magnetron sputtering technology to obtain a wafer; S2. A giant magnetoresistive stripe sensor structure is formed on a wafer by photolithography and dry etching, and electrodes and passivation layers are prepared to obtain a giant magnetoresistive chip array. S3. A photoresist layer structure is prepared by photolithography and bonded to the substrate of the giant magnetoresistive chip, and then the top cover is attached to form a microfluidic channel. S4. Immobilize the molecules required for marine microbial detection on the surface of the giant magnetoresistive chip and perform a sealing process.

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