Square capillary outer wall coupled optical fiber biosensor device and false positive detection method

Abstract

The application discloses a square capillary outer wall coupling optical fiber biosensor device and an anti-false positive detection method, relates to the technical field of sensing, and the device comprises a square capillary, a first outer wall coupling optical fiber, a second outer wall coupling optical fiber, a third outer wall coupling optical fiber, a functional microbead bed area, a porous limiting plug and an optical fiber fixing layer; the square capillary is internally provided with a microfluidic channel, and a reference microbead bed area, a detection microbead bed area and a confirmation microbead bed area are sequentially arranged; porous limiting plugs are arranged between the bed areas and on both sides to prevent bead migration and allow solution to pass. The three outer wall coupling optical fibers are fixed to the same side outer wall of the square capillary through the optical fiber fixing layer and correspond to the bed areas; the optical fibers are coupled into the bed areas through the outer wall. The application significantly reduces the false positive risk and improves the detection repeatability and structural stability.

Description

Technical Field

[0001] This application relates to the field of sensor technology, and in particular to a square capillary outer wall coupled fiber optic biosensor and a method for detecting false positives. Background Technology

[0002] With the development of fields such as biomedical detection, environmental monitoring, food safety, drug development, and rapid on-site diagnosis, the demand for rapid, sensitive, and in-situ detection of low-concentration biomarkers, antigens and antibodies, nucleic acid molecules, viral particles, and small molecule metabolites is constantly increasing. Fiber optic biosensors, due to their advantages such as small size, resistance to electromagnetic interference, corrosion resistance, long-distance transmission capability, ease of miniaturization, and integration with microfluidic systems, have broad application prospects in the field of biological detection.

[0003] Fabry-Perot interferometric fiber optic sensors are an important type of fiber optic sensor. These sensors typically utilize multiple reflective interfaces to form a low-precision interference cavity. Changes in the refractive index, temperature, cavity length, or interface conditions of the analyte will cause changes in the peak position, phase, intensity, or contrast of the reflected interference spectrum. By demodulating the spectral signal, the detection of the analyte physical quantity or biochemical parameter can be achieved.

[0004] Existing capillary fiber optic sensors typically utilize the hollow structure of the capillary itself as a microfluidic channel, enabling light to interact with the analyte liquid through side illumination or end-face coupling. However, the following problems still exist in some situations: Conventional capillary fiber optic sensors are mostly based on a single detection area, and can only obtain single-point or single-region responses. When there are bubbles, particulate turbidity, temperature fluctuations, non-specific adsorption, or complex matrix interference in the sample, a single detection area is difficult to distinguish between the actual target binding signal and the background interference signal, which can easily lead to false positive results.

[0005] Traditional capillary biosensors typically rely on modifications to the inner wall of the capillary to recognize molecules. Due to the limited specific surface area of ​​the inner wall, the target enrichment capacity is insufficient, and the binding efficiency of low-concentration target substances with recognition molecules is limited, thus affecting detection sensitivity and response speed.

[0006] If functional microbeads are added directly into the capillary, the microbeads are prone to migration, aggregation, or being washed out of the detection area with the liquid flow, making it difficult to form a stable detection window; different functional microbeads are also prone to mixing, making it impossible to clearly distinguish between the reference, detection, and confirmation areas.

[0007] In existing fiber optic biosensor structures, the coupling position between the fiber and the microfluidic channel and the corresponding detection area are not stable enough, making it difficult to achieve parallel or sequential detection of multiple functional areas in the same capillary, which limits its application in high-reliability biosensing. Summary of the Invention

[0008] The purpose of this application is to provide a square capillary outer wall coupled fiber optic biosensor and a method for detecting false positives, so as to significantly reduce the risk of false positives and improve detection repeatability and structural stability.

[0009] To achieve the above objectives, this application provides the following solution: In a first aspect, this application provides a square capillary outer wall coupled optical fiber biosensor, comprising: a square capillary, a first outer wall coupled optical fiber, a second outer wall coupled optical fiber, a third outer wall coupled optical fiber, a functional microbead bed region, a porous limiting plug, and an optical fiber fixing layer. The square capillary tube has a hollow internal region that forms a microfluidic channel for the flow of the solution to be tested. The functional microbead bed region includes at least a reference microbead bed region, a detection microbead bed region, and a confirmation microbead bed region; the reference microbead bed region, the detection microbead bed region, and the confirmation microbead bed region are arranged sequentially along the length direction of the microfluidic channel; The porous limiting plug is disposed between adjacent functional microbead bed regions and / or on both sides of the functional microbead bed region to restrict the migration of microbeads in the functional microbead bed region along the direction of the microfluidic channel, while allowing the test solution to pass through. The first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber are disposed on the same outer wall of the square capillary and correspond to the reference microbead bed area, the detection microbead bed area, and the confirmation microbead bed area, respectively. The fiber fixing layer is used to fix the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to the outer wall of the square capillary, and to enable the light emitted by the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to be coupled into the corresponding functional microbead bed region through the outer wall of the square capillary.

[0010] In one embodiment, the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber are all single-mode fibers, multimode fibers, or fiber probes.

[0011] In one embodiment, the optical fiber fixing layer is a UV-curable adhesive layer, a refractive index matching adhesive layer, or an optically transparent adhesive layer.

[0012] In one embodiment, the porous limiting plug has a porous liquid-permeable structure; the pore size of the porous limiting plug is smaller than the particle size of the microbeads in the functional microbead bed region, so that the test solution can pass through the porous limiting plug, while the microbeads are confined within the corresponding functional microbead bed region.

[0013] In one embodiment, the microbeads in the reference microbead bed region are inert microbeads, blocked microbeads, nonspecific immunoglobulin modified microbeads, bovine serum albumin modified microbeads, or polyethylene glycol modified microbeads; the microbeads in the reference microbead bed region are used to characterize the background response generated by the sample matrix, temperature drift, bubbles, turbidity, or nonspecific adsorption.

[0014] In one embodiment, the surface of the microbeads in the detection microbead bed is modified with a target recognition molecule; the target recognition molecule includes one or more of antibodies, antigens, aptamers, nucleic acid probes, enzymes, molecularly imprinted layers, or receptor proteins.

[0015] In one embodiment, the surface of the microbeads in the confirmation microbead bed region is modified with a confirmation recognition molecule that is different from that in the detection microbead bed region; the confirmation recognition molecule is used to provide positive confirmation of the response generated by the detection microbead bed region.

[0016] In one embodiment, the light emitted from the first outer wall coupled fiber, the second outer wall coupled fiber, or the third outer wall coupled fiber enters the square capillary through the corresponding fiber fixing layer, generating multiple reflected beams at the interface of the outer wall, inner wall, and microfluidic channel of the square capillary. The multiple reflected beams return to the first outer wall coupled fiber, the second outer wall coupled fiber, or the third outer wall coupled fiber to form a reflection interference spectrum.

[0017] In one embodiment, when the target object binds to the target recognition molecule in the detection microbead bed region, the equivalent refractive index, scattering loss, absorption characteristics, or reflection interface conditions of the detection microbead bed region change, causing changes in the peak position, intensity, phase, or interference contrast of the reflection interference spectrum.

[0018] Secondly, this application provides a method for detecting false positives, which is based on the aforementioned square capillary outer wall coupled fiber optic biosensor; the method for detecting false positives includes: The sample to be tested is injected into the microfluidic channel of a square capillary. The first reflection interference spectrum of the reference microbead bed region is acquired by the first outer wall coupled optical fiber; the second reflection interference spectrum of the detection microbead bed region is acquired by the second outer wall coupled optical fiber; and the third reflection interference spectrum of the microbead bed region is acquired and confirmed by the third outer wall coupled optical fiber. Based on the first reflection interference spectrum, the second reflection interference spectrum, and the third reflection interference spectrum, the first spectral response parameters of the reference microbead bed region, the second spectral response parameters of the detection microbead bed region, and the third spectral response parameters of the confirmation microbead bed region are extracted respectively; the spectral response parameters include peak position shift, reflection intensity change, interference contrast change, and phase change; Based on the second spectral response parameter, determine whether the response of the detection microbead bed region exceeds the detection threshold; If the response of the detected microbead bed region does not exceed the detection threshold, it is determined to be a negative result; if the response of the detected microbead bed region exceeds the detection threshold, it is determined whether the response of the reference microbead bed region is within the background allowable range based on the first spectral response parameter. If the response of the reference microbead bed region exceeds the background allowable range, it is determined to be an interference signal or a false positive risk; if the response of the reference microbead bed region is within the background allowable range, it is determined, based on the third spectral response parameter, whether the response of the confirmed microbead bed region and the response of the detected microbead bed region meet a preset confirmation relationship; the preset confirmation relationship includes one or more of the following: the response directions of the detected microbead bed region and the confirmed microbead bed region are consistent, the response amplitude ratio is within a preset range, the response time sequence meets a preset condition, or the correlation between the two responses is higher than a preset threshold. If the response of the microbead bed region and the response of the detection microbead bed region meet the preset confirmation relationship, it is determined to be a true positive; if the preset confirmation relationship is not met, it is determined to be a false positive or an interference signal.

[0019] According to the specific embodiments provided in this application, this application has the following technical effects: This application provides a square capillary outer wall coupled fiber optic biosensor and a method for preventing false positives. The device includes a square capillary, a first outer wall coupled fiber optic cable, a second outer wall coupled fiber optic cable, a third outer wall coupled fiber optic cable, a functional microbead bed region, a porous limiting plug, and a fiber fixing layer. The square capillary has a hollow internal region that forms a microfluidic channel for the flow of the solution to be tested. The functional microbead bed region includes at least a reference microbead bed region, a detection microbead bed region, and a confirmation microbead bed region. The reference microbead bed region, the detection microbead bed region, and the confirmation microbead bed region are arranged sequentially along the length of the microfluidic channel. The porous limiting plug is disposed between adjacent functional microbead bed regions and / or functional microbead bed regions. On both sides of the bead bed region, there are structures to restrict the migration of microbeads within the functional microbead bed region along the microfluidic channel direction, while allowing the test solution to pass through. The first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber are disposed on the same outer wall of the square capillary, and correspond to the reference microbead bed region, the detection microbead bed region, and the confirmation microbead bed region, respectively. The fiber fixing layer is used to fix the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to the outer wall of the square capillary, and to enable the light emitted by the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to be coupled into the corresponding functional microbead bed region through the outer wall of the square capillary. This application constructs a reference microbead bed region, a detection microbead bed region, and a confirmation microbead bed region within the same square capillary, transforming a single detection signal into a multi-regional correlation judgment signal. This significantly reduces the risk of false positives caused by bubbles, turbidity, temperature drift, and non-specific adsorption in complex samples. Furthermore, it utilizes porous limiting plugs to spatially limit the functional microbead bed, enabling liquid to pass through while microbeads cannot, thus stably maintaining the functional microbead bed within the corresponding optical fiber detection window and improving detection repeatability and structural stability. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the overall appearance and structure of the biosensor device of this application; Figure 2 This is a cross-sectional view of the internal functional area of ​​the square capillary tube in this application; Figure 3 This is a schematic diagram of the optical path of a single outer wall-coupled fiber detection site in this application; Figure 4 This is a flowchart of the method for detecting false positives in this application; Figure 5 This is a schematic diagram of the spectral response relationship of the three functional regions in this application.

[0022] Reference numerals: 1. Square capillary; 2. Microfluidic channel; 3. First outer wall coupled optical fiber; 4. Second outer wall coupled optical fiber; 5. Third outer wall coupled optical fiber; 6. Reference microbead bed area; 7. Detection microbead bed area; 8. Confirmation microbead bed area; 11. UV-curable adhesive fixing layer; 12. Porous limiting plug; 13. Incident light; 14. First reflected light; 15. Second reflected light; 16. Third reflected light. Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] This application belongs to the interdisciplinary field of fiber optic sensing technology, microfluidic detection technology, and biosensing technology. It utilizes the flat outer wall of a square capillary as the coupling interface for the optical fiber. Multiple functional microbead beds are arranged inside the square capillary, and porous limiting plugs are used to spatially limit and functionally partition these different microbead beds, allowing the sample solution to flow sequentially through the reference microbead bed, the detection microbead bed, and the confirmation microbead bed. Multiple outer-wall coupled optical fibers respectively perform reflection interference spectral acquisition corresponding to different functional microbead beds. By comparing the spectral response relationships of different functional areas, the detection of target biomolecules is achieved, and false positive signals caused by non-specific adsorption, bubbles, turbidity, temperature drift, and other interference factors are identified and eliminated. This solves the problems of existing capillary fiber optic biosensors, such as single functional areas, insufficient target enrichment capacity, difficulty in stable microbead confinement, and susceptibility to false positives in the detection of complex samples.

[0026] In one exemplary embodiment, such as Figure 1 and Figure 2 As shown, a square capillary outer wall coupled optical fiber biosensor is provided, comprising: a square capillary 1, a first outer wall coupled optical fiber 3, a second outer wall coupled optical fiber 4, a third outer wall coupled optical fiber 5, a functional microbead bed region, a porous limiting plug 12, and an optical fiber fixing layer.

[0027] The square capillary 1 has a hollow region extending along its length, which forms a microfluidic channel 2 for the flow of the test solution, cleaning solution, buffer solution or reaction reagent.

[0028] In this embodiment, the square capillary 1 has at least one flat outer wall, which allows the outer wall-coupled optical fiber to be stably attached to or close to the outer wall of the capillary, and allows the incident light beam to enter the interior of the capillary more vertically, reducing the lens effect and alignment error caused by the curved surface of the circular capillary.

[0029] The square capillary 1 is preferably a fused silica square capillary 1, which has good flatness on both its outer and inner walls. Compared with a circular capillary, the flat outer wall of the square capillary 1 is conducive to the alignment, bonding and fixation of the optical fiber coupled to the outer wall, so that the incident light can enter the interior of the capillary more stably.

[0030] The functional microbead bed region includes at least a reference microbead bed region 6, a detection microbead bed region 7, and a confirmation microbead bed region 8; the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8 are arranged sequentially along the length of the microfluidic channel 2. The reference microbead bed region 6 is used to record background responses and non-specific interferences; the detection microbead bed region 7 is used to capture target objects and generate the main detection response; the confirmation microbead bed region 8 is used to further confirm the positive response generated by the detection microbead bed region 7.

[0031] In this embodiment, the microbead matrix in the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8 may be the same or different. The microbead matrix includes one or more of silica microbeads, polystyrene microbeads, magnetic microbeads, hydrogel microbeads, agarose microbeads, or functionalized polymer microbeads.

[0032] In one embodiment, the microbeads in the reference microbead bed region 6 are inert microbeads, blocked microbeads, nonspecific immunoglobulin modified microbeads, bovine serum albumin modified microbeads, or polyethylene glycol modified microbeads; the microbeads in the reference microbead bed region 6 are used to characterize the background response generated by the sample matrix, temperature drift, bubbles, turbidity, or nonspecific adsorption.

[0033] In one embodiment, the surface of the microbeads in the detection microbead bed region 7 is modified with a target recognition molecule (first target recognition molecule), such as a capture antibody or aptamer corresponding to a target antigen; the target recognition molecule includes one or more of antibodies, antigens, aptamers, nucleic acid probes, enzymes, molecularly imprinted layers, or receptor proteins.

[0034] In one embodiment, the surface of the microbeads in the confirmatory bead bed region 8 is modified with a confirmatory recognition molecule (second target recognition molecule) that is different from that in the detection bead bed region 7, such as an antibody or another confirmatory aptamer targeting a different epitope of the same target; the confirmatory recognition molecule is used to positively confirm the response generated by the detection bead bed region 7. Thus, when the target is actually present, both the detection bead bed region 7 and the confirmatory bead bed region 8 can produce spectral responses that conform to a preset relationship, while the reference bead bed region 6 should not produce a significantly abnormal response.

[0035] In one specific preparation method, the functional microbead bed region can be formed according to the following steps: First, a square capillary tube 1 is horizontally fixed on a micromanipulation platform, and a first porous limiting plug is formed at a predetermined position within the microfluidic channel 2; then, a reference functional microbead suspension is injected from one end of the square capillary tube 1, causing the reference functional microbeads to be blocked by the first porous limiting plug and aggregated in a predetermined reference region; then, a second porous limiting plug is formed on the other side of the reference functional microbead bed, thereby confining the reference microbead bed region 6 between the first and second porous limiting plugs. Afterwards, a detection functional microbead suspension is injected, and corresponding porous limiting plugs 12 are formed on both sides to form a detection microbead bed region 7; then, a confirmation functional microbead suspension is injected, and corresponding porous limiting plugs 12 are formed on both sides to form a confirmation microbead bed region 8. Thus, the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8 are distributed sequentially along the length of the microfluidic channel 2, and the microbeads in each functional region are limited by the porous limiting plug 12, allowing the liquid to pass through each functional region sequentially along the microfluidic channel 2.

[0036] The porous limiting plug 12 is disposed between adjacent functional microbead bed areas and / or on both sides of the functional microbead bed area to restrict the migration of microbeads in the functional microbead bed area along the direction of the microfluidic channel 2, while allowing the solution to be tested to pass through.

[0037] In one embodiment, the porous limiting plug 12 has a porous liquid-permeable structure. The pore size of the porous limiting plug 12 is smaller than the particle size of the microspheres in the functional microsphere bed region, thus allowing liquid, target molecules, and small-sized reaction components to pass through. Specifically, the equivalent pore size of the porous limiting plug 12 is 5% to 80% of the particle size of the functional microspheres, allowing the test solution to pass through the porous limiting plug 12 while the microspheres are confined within the corresponding functional microsphere bed region. This ensures that different functional microsphere bed regions are stably maintained in predetermined spatial positions, preventing the microspheres from being washed away or mixing. The porous limiting plug 12 is formed of photocurable hydrogel, porous polymer, porous glass, porous silica gel, microporous membrane, sintered microsphere structure, or porous ceramic material.

[0038] The length of the porous limiting plug 12 can be 20μm to 1000μm, preferably 50μm to 500μm, in order to reduce fluid resistance while ensuring the limiting ability of the microbeads.

[0039] In a preferred embodiment, the porous limiting plug 12 can be formed in situ by photocuring hydrogel or photocuring porous polymer. Specifically, a hydrogel precursor solution or porous polymer precursor solution containing a photoinitiator is injected into a predetermined position in the microfluidic channel 2, and the predetermined area is cured by masking, focused ultraviolet light, or localized illumination to form a porous limiting plug 12 fixed to the inner wall of the square capillary 1. The uncured precursor solution is then washed away with a buffer solution. The formed porous limiting plug 12 has a connected pore structure with a pore size smaller than the particle size of the functional microbeads, allowing sample solution and target molecules to pass through. In another embodiment, the porous limiting plug 12 can also be pre-prepared from a microporous membrane, sintered microspheres, porous glass plug, or porous polymer plug and then placed into the microfluidic channel 2 of the square capillary 1.

[0040] The first outer wall coupling fiber 3, the second outer wall coupling fiber 4, and the third outer wall coupling fiber 5 are disposed on the same outer wall of the square capillary 1, and correspond to the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8, respectively, so that the incident light beam can pass through the outer wall of the square capillary 1 perpendicularly or approximately perpendicularly and enter the corresponding functional microbead bed region. Each outer wall coupling fiber can serve as a transmission channel for incident light and return light.

[0041] In one embodiment, the first outer wall coupling fiber 3, the second outer wall coupling fiber 4, and the third outer wall coupling fiber 5 are all single-mode fibers, multi-mode fibers, or fiber probes, preferably single-mode fibers.

[0042] The length of a single functional microbead bed region is 0.2mm to 10mm, and the center-to-center distance between the detection sites corresponding to the adjacent outer wall coupled optical fibers is 1mm to 20mm.

[0043] In this embodiment, the first outer wall coupled fiber 3, the second outer wall coupled fiber 4, and the third outer wall coupled fiber 5 are respectively fixed to the same flat outer wall of the square capillary 1 by a UV-curable adhesive fixing layer 11. The UV-curable adhesive fixing layer 11 is located on the outer wall surface of the square capillary 1 and does not enter the interior of the microfluidic channel 2. The UV-curable adhesive fixing layer 11 is not only used for mechanical fixation, but also can use a refractive index matching adhesive to reduce the interface reflection loss between the fiber end face and the outer wall of the capillary. The angle between the exit end face of the outer wall coupled fiber and the outer wall of the square capillary 1 is preferably 90°, so that the incident light can enter the square capillary 1 perpendicularly or approximately perpendicularly.

[0044] After the formation of each functional microbead bed region, the first outer wall coupling fiber 3, the second outer wall coupling fiber 4 and the third outer wall coupling fiber 5 are moved to the position above or on the corresponding side wall of the reference microbead bed region 6, the detection microbead bed region 7 and the confirmation microbead bed region 8, respectively. The optimal coupling position is determined by real-time monitoring of the intensity of the reflection spectrum or the interference contrast. Then, the UV curing adhesive fixing layer 11 is used for curing and fixing.

[0045] In this embodiment, the detection microbead bed region 7 and the confirmation microbead bed region 8 are respectively modified with a first recognition molecule and a second recognition molecule targeting different epitopes of the same target, so that when the target is present, the detection microbead bed region 7 and the confirmation microbead bed region 8 produce correlated spectral responses.

[0046] In one embodiment, the light emitted from the first outer wall coupled fiber 3, the second outer wall coupled fiber 4, or the third outer wall coupled fiber 5 enters the square capillary 1 through the corresponding fiber fixing layer, generating multiple reflected beams at the interface of the outer wall, inner wall, and microfluidic channel 2 of the square capillary 1. The multiple reflected beams return to the first outer wall coupled fiber 3, the second outer wall coupled fiber 4, or the third outer wall coupled fiber 5 to form a reflection interference spectrum.

[0047] In one embodiment, when the target object specifically binds to the target recognition molecule in the detection microbead bed region 7, the equivalent refractive index, scattering loss, absorption characteristics, or reflection interface conditions of the detection microbead bed region 7 change, thereby causing changes in the peak position, intensity, phase, or interference contrast of the reflection interference spectrum in the detection region. By further comparing the responses of the reference microbead bed region 6 and the confirmatory microbead bed region 8, false positive signals caused by factors such as bubbles, turbidity, temperature changes, and non-specific adsorption can be eliminated.

[0048] like Figure 3 As shown, taking the detection microbead bed region 7 corresponding to the second outer wall coupled optical fiber 4 as an example, the incident light 13 emitted by the outer wall coupled optical fiber enters the square capillary 1 through the UV-curable adhesive fixing layer 11. The incident light 13 generates a first reflected light 14, a second reflected light 15, and a third reflected light 16 at the relevant interfaces of the outer wall, inner wall, and microfluidic channel 2 of the square capillary 1. The first reflected light mainly comes from the interface near the outer wall of the optical fiber fixing layer and the outer wall of the square capillary 1, the second reflected light mainly comes from the medium interface between the inner wall of the square capillary 1 and the microfluidic channel 2, and the third reflected light mainly comes from the inner wall on the other side of the microfluidic channel 2 or the relevant equivalent reflection interface of the microbead bed. After the first reflected light 14, the second reflected light 15, and the third reflected light 16 return to the outer wall coupled optical fiber, they superimpose with each other to form a reflection interference spectrum.

[0049] The outer wall-coupled optical fiber of this application can be connected to a broadband light source, an optical fiber circulator, an optical switch, a spectrometer, and a computer. The microfluidic channel 2 of the square capillary 1 can be connected to a peristaltic pump or a syringe pump and a waste liquid collection container to complete sample injection, spectral acquisition, and data processing.

[0050] The detection microbead bed region 7 is located within the optical detection area through which the incident light 13 passes. After the target object binds to the recognition molecules on the surface of the microbeads in the detection microbead bed region 7, it will change the equivalent refractive index, scattering loss, absorption characteristics, or reflection interface conditions of the detection microbead bed region 7, thereby causing changes in the peak position, intensity, phase, or interference contrast of the reflection interference spectrum.

[0051] The optical fiber fixing layer is used to fix the first outer wall coupling fiber 3, the second outer wall coupling fiber 4 and the third outer wall coupling fiber 5 to the outer wall of the square capillary 1, and to enable the light emitted by the first outer wall coupling fiber 3, the second outer wall coupling fiber 4 and the third outer wall coupling fiber 5 to be coupled into the corresponding functional microbead bed region through the outer wall of the square capillary 1.

[0052] In one embodiment, the optical fiber fixing layer is a UV-curable adhesive layer (UV-curable adhesive fixing layer 11), a refractive index matching adhesive layer, or an optically transparent adhesive layer. The optical fiber fixing layer is located on the outer wall surface of the square capillary 1 and does not enter the interior of the microfluidic channel 2.

[0053] The square capillary 1 is a fused silica square capillary with an outer side length of 100 μm to 1000 μm and an inner side length of 50 μm to 800 μm; more preferably, the outer side length of the square capillary 1 is 300 μm to 700 μm and the inner side length is 100 μm to 500 μm. The particle size of the microbeads in the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8 is 1 μm to 100 μm, preferably 5 μm to 50 μm. The equivalent pore size of the porous limiting plug 12 is smaller than the particle size of the microbeads, preferably 5% to 80% of the particle size, more preferably 10% to 50% of the particle size, to ensure that the sample solution, target molecules, and buffer solution can pass through the porous limiting plug 12, while the functional microbeads cannot pass through the porous limiting plug 12. The length of a single functional microbead bed region is 0.2mm to 10mm, preferably 0.5mm to 5mm; the center-to-center distance between the detection sites corresponding to adjacent outer wall coupling optical fibers is 1mm to 20mm, preferably 3mm to 10mm.

[0054] In one exemplary embodiment, this embodiment provides a method for preventing false positives, which is based on the aforementioned square capillary outer wall coupled fiber optic biosensor; the method for preventing false positives includes: S1: Inject the sample to be tested into the microfluidic channel 2 of the square capillary 1.

[0055] S2: The first reflection interference spectrum of the reference microbead bed region 6 is acquired through the first outer wall coupling optical fiber 3; the second reflection interference spectrum of the detection microbead bed region 7 is acquired through the second outer wall coupling optical fiber 4; and the third reflection interference spectrum of the confirmation microbead bed region 8 is acquired through the third outer wall coupling optical fiber 5.

[0056] S3: Based on the first reflection interference spectrum, the second reflection interference spectrum and the third reflection interference spectrum, extract the first spectral response parameter of the reference microbead bed region 6, detect the second spectral response parameter of the microbead bed region 7 and confirm the third spectral response parameter of the microbead bed region 8 respectively; the spectral response parameters include peak position shift, reflection intensity change, interference contrast change and phase change.

[0057] S4: Based on the second spectral response parameter, determine whether the response of the detection microbead bed region 7 exceeds the detection threshold.

[0058] S5: If the response of the microbead bed region 7 does not exceed the detection threshold, it is determined to be a negative result; if the response of the microbead bed region 7 exceeds the detection threshold, it is determined whether the response of the reference microbead bed region 6 is within the background allowable range based on the first spectral response parameter.

[0059] S6: If the response of the reference microbead bed region 6 exceeds the background allowable range, it is determined to be an interference signal or a false positive risk; if the response of the reference microbead bed region 6 is within the background allowable range, it is determined, according to the third spectral response parameter, whether the response of the confirmation microbead bed region 8 and the response of the detection microbead bed region 7 meet the preset confirmation relationship; the preset confirmation relationship includes one or more of the following: the response direction of the detection microbead bed region 7 and the confirmation microbead bed region 8 is consistent, the response amplitude ratio is within the preset range, the response time sequence meets the preset conditions, or the correlation between the two responses is higher than the preset threshold.

[0060] S7: If the response of the microbead bed region 8 and the response of the detection microbead bed region 7 meet the preset confirmation relationship, it is determined to be a true positive; if the preset confirmation relationship is not met, it is determined to be a false positive or an interference signal.

[0061] like Figure 4 As shown, the false-positive detection method of this embodiment first injects the sample into the microfluidic channel 2 of the square capillary 1, allowing the sample to flow through the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8. Subsequently, the reflection interference spectra of the three functional regions are acquired respectively, and the corresponding response parameters are extracted. The response parameters can be the spectral peak shift, the change in reflection intensity, the change in interference contrast, or the phase change.

[0062] Let the response in the reference region be ΔS1, the response in the detection region be ΔS2, and the response in the confirmation region be ΔS3. If ΔS2 is less than the detection threshold T2, it is determined to be negative; if ΔS2 is greater than or equal to the detection threshold T2, and ΔS1 is greater than the background threshold T1, it is determined to be an interference signal or a false positive risk; if ΔS2 is greater than or equal to the detection threshold T2, ΔS1 is less than or equal to the background threshold T1, and ΔS3 and ΔS2 satisfy a consistency relationship, it is determined to be a true positive. The consistency relationship includes the ratio of ΔS3 to ΔS2 being within a preset range [a, b], or the correlation between the responses of ΔS3 and ΔS2 being greater than or equal to a preset correlation coefficient C.

[0063] If the response of the detection bead bed region 7 does not exceed the detection threshold, the result is considered negative. If the response of the detection bead bed region 7 exceeds the detection threshold, the response of the reference bead bed region 6 is further assessed to determine if it is within the acceptable background range. If the response of the reference bead bed region 6 is abnormal, it indicates the possible presence of interfering factors such as bubbles, turbidity, temperature drift, or non-specific adsorption, and is considered an interference signal or a false positive risk. If the response of the reference bead bed region 6 is within the acceptable background range, the response of the confirmation bead bed region 8 is further assessed to determine if it satisfies a preset confirmation relationship with the response of the detection bead bed region 7. If it does, the result is considered a true positive; otherwise, it is considered a false positive or an interference signal.

[0064] In one embodiment, the preset confirmation relationship can be that the peak position drift direction of the microbead bed region 8 and the detection microbead bed region 7 is consistent, and the ratio of their response amplitudes is within a preset range. In another embodiment, the preset confirmation relationship can also be that the response time sequence, response amplitude correlation, or response curve shape of the microbead bed region 8 and the detection microbead bed region 7 meet preset conditions.

[0065] like Figure 5 As shown, during the detection process, the responses of the reference microbead bed region 6, the detection microbead bed region 7, and the confirmation microbead bed region 8 can be compared and analyzed. When the target object is actually present, the detection microbead bed region 7 produces a significant response, the confirmation microbead bed region 8 produces a confirmation response that is consistent with or related to it, while the reference microbead bed region 6 only produces a low background response. When interference factors are present, the reference microbead bed region 6 may show a significant response, or the detection microbead bed region 7 and the confirmation microbead bed region 8 may not satisfy the confirmation relationship. In this case, it can be determined as a false positive or interference signal.

[0066] The false-positive detection method of this application has the following advantages: This application constructs a reference microbead bed region, a detection microbead bed region, and a confirmation microbead bed region within the same square capillary, transforming a single detection signal into a multi-regional correlation judgment signal. This can significantly reduce the risk of false positives caused by bubbles, turbidity, temperature drift, and non-specific adsorption in complex samples.

[0067] This application utilizes a porous limiting plug to spatially limit the functional microbead bed, achieving the function of "liquid can pass through, but microbeads cannot pass through", so that the functional microbead bed is stably kept within the corresponding optical fiber detection window, improving detection repeatability and structural stability.

[0068] This application utilizes functional microbeads as carriers for recognizing molecules. Compared with simple capillary wall modification, it can significantly increase the specific surface area and improve the target enrichment capacity and biobinding efficiency.

[0069] This application utilizes the flat outer wall of a square capillary for outer wall coupling, which facilitates fiber positioning and fixation, and reduces the lens effect and coupling instability problems caused by the curved surface of a circular capillary.

[0070] This application allows for flexible modification of the surface of different functional microbead bed regions according to detection needs, thereby adapting to various application scenarios such as antigen and antibody detection, nucleic acid detection, aptamer detection, enzyme-catalyzed reaction detection, and environmental pollutant detection.

[0071] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. Furthermore, those skilled in the art will recognize that, based on the ideas of this application, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A square capillary outer wall coupled fiber optic biosensor, characterized in that, include: A square capillary tube, a first outer wall coupled optical fiber, a second outer wall coupled optical fiber, a third outer wall coupled optical fiber, a functional microbead bed region, a porous limiting plug, and an optical fiber fixing layer; The square capillary tube has a hollow internal region that forms a microfluidic channel for the flow of the solution to be tested. The functional microbead bed region includes at least a reference microbead bed region, a detection microbead bed region, and a confirmation microbead bed region; the reference microbead bed region, the detection microbead bed region, and the confirmation microbead bed region are arranged sequentially along the length direction of the microfluidic channel; The porous limiting plug is disposed between adjacent functional microbead bed regions and / or on both sides of the functional microbead bed region to restrict the migration of microbeads in the functional microbead bed region along the direction of the microfluidic channel, while allowing the test solution to pass through. The first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber are disposed on the same outer wall of the square capillary and correspond to the reference microbead bed area, the detection microbead bed area, and the confirmation microbead bed area, respectively. The fiber fixing layer is used to fix the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to the outer wall of the square capillary, and to enable the light emitted by the first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber to be coupled into the corresponding functional microbead bed region through the outer wall of the square capillary.

2. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The first outer wall coupling fiber, the second outer wall coupling fiber, and the third outer wall coupling fiber are all single-mode fibers, multi-mode fibers, or fiber probes.

3. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The optical fiber fixing layer is a UV-curable adhesive layer, a refractive index matching adhesive layer, or an optically transparent adhesive layer.

4. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The porous limiting plug has a porous liquid-permeable structure; the pore size of the porous limiting plug is smaller than the particle size of the microbeads in the functional microbead bed region, so that the test solution can pass through the porous limiting plug, while the microbeads are confined within the corresponding functional microbead bed region.

5. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The microbeads in the reference microbead bed are inert microbeads, blocked microbeads, non-specific immunoglobulin modified microbeads, bovine serum albumin modified microbeads, or polyethylene glycol modified microbeads; the microbeads in the reference microbead bed are used to characterize the background response generated by the sample matrix, temperature drift, bubbles, turbidity, or non-specific adsorption.

6. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The surface of the microbeads in the detection microbead bed is modified with target recognition molecules; the target recognition molecules include one or more of antibodies, antigens, aptamers, nucleic acid probes, enzymes, molecularly imprinted layers, or receptor proteins.

7. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The surface of the microbeads in the confirmation microbead bed region is modified with confirmation recognition molecules that are different from those in the detection microbead bed region; the confirmation recognition molecules are used to provide positive confirmation of the response generated by the detection microbead bed region.

8. The square capillary outer wall coupled fiber optic biosensor device according to claim 1, characterized in that, The light emitted from the first outer wall coupled fiber, the second outer wall coupled fiber, or the third outer wall coupled fiber enters the square capillary through the corresponding fiber fixing layer, generating multiple reflected beams at the interface of the outer wall, inner wall, and microfluidic channel of the square capillary. The multiple reflected beams return to the first outer wall coupled fiber, the second outer wall coupled fiber, or the third outer wall coupled fiber to form a reflection interference spectrum.

9. The square capillary outer wall coupled fiber optic biosensor device according to claim 8, characterized in that, When the target object binds to the target recognition molecule in the detection microbead bed region, the equivalent refractive index, scattering loss, absorption characteristics, or reflection interface conditions of the detection microbead bed region change, causing changes in the peak position, intensity, phase, or interference contrast of the reflection interference spectrum.

10. A method for detecting false positives, characterized in that, The method for preventing false positives is based on the square capillary outer wall coupled fiber optic biosensor as described in any one of claims 1-9; the method for preventing false positives includes: The sample to be tested is injected into the microfluidic channel of a square capillary. The first reflection interference spectrum of the reference microbead bed region is acquired by the first outer wall coupled optical fiber; the second reflection interference spectrum of the detection microbead bed region is acquired by the second outer wall coupled optical fiber; and the third reflection interference spectrum of the microbead bed region is acquired and confirmed by the third outer wall coupled optical fiber. Based on the first reflection interference spectrum, the second reflection interference spectrum, and the third reflection interference spectrum, the first spectral response parameters of the reference microbead bed region, the second spectral response parameters of the detection microbead bed region, and the third spectral response parameters of the confirmation microbead bed region are extracted respectively; the spectral response parameters include peak position shift, reflection intensity change, interference contrast change, and phase change; Based on the second spectral response parameter, determine whether the response of the detection microbead bed region exceeds the detection threshold; If the response of the detected microbead bed region does not exceed the detection threshold, it is determined to be a negative result; if the response of the detected microbead bed region exceeds the detection threshold, it is determined whether the response of the reference microbead bed region is within the background allowable range based on the first spectral response parameter. If the response of the reference microbead bed region exceeds the background allowable range, it is determined to be an interference signal or a false positive risk; if the response of the reference microbead bed region is within the background allowable range, it is determined, based on the third spectral response parameter, whether the response of the confirmed microbead bed region and the response of the detected microbead bed region meet a preset confirmation relationship; the preset confirmation relationship includes one or more of the following: the response directions of the detected microbead bed region and the confirmed microbead bed region are consistent, the response amplitude ratio is within a preset range, the response time sequence meets a preset condition, or the correlation between the two responses is higher than a preset threshold. If the response of the microbead bed region and the response of the detection microbead bed region meet the preset confirmation relationship, it is determined to be a true positive; if the preset confirmation relationship is not met, it is determined to be a false positive or an interference signal.

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