TSP-1 detection device

Abstract

The invention provides TSP-1 detection equipment. The TSP-1 detection equipment is characterized by comprising a sensing core module, according to the sensing core module, a Metglas alloy is used as a substrate, and after chromium and gold modification is carried out on the surface, a TSP-1 antibody is attached to the substrate. The equipment is a novel detection scheme with high sensitivity, high specificity, low cost and portability, rapid and accurate detection of TSP-1 is achieved, the detection limit (12.857 ngmL <-1 >) and the linear range (12.857-100 ngmL <-1 >) of the equipment completely meet the clinical diagnosis requirement of OA, meanwhile, interference of external factors such as electromagnetic interference and light source influence is avoided, the equipment can be directly applied to actual joint fluid sample detection of OA patients, and the detection efficiency is improved. An efficient technical means is provided for OA early diagnosis and disease prognosis monitoring, the detection cost is reduced, and the convenience and practicability of clinical detection are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a TSP-1 detection device, and more specifically, to TSP-1 magnetoelastic test paper. Background Technology

[0002] The current approach to early diagnosis of osteoarthritis (OA) faces several challenges. Traditional imaging techniques can only detect advanced OA and cannot achieve early screening. Early diagnosis of OA relies on the highly sensitive detection of the biomarker platelet-reactive protein (TSP-1). However, existing TSP-1 detection methods (such as enzyme-linked immunosorbent assay) suffer from drawbacks including complex operation, high preparation costs, time-consuming testing, and a linear range that does not match clinical needs. These limitations make it difficult to meet the clinical demands for convenient and accurate testing. Specifically:

[0003] The existing method is enzyme-linked immunosorbent assay (ELISA). This method has a cumbersome operation procedure, a long detection cycle, and high equipment and reagent costs, making it unsuitable for rapid detection and on-site applications.

[0004] The existing method two, immunohistochemistry and biochemical assays, cannot capture low concentrations of biomarkers in early-stage patients and is completely incapable of early screening.

[0005] The existing methods described above suffer from several drawbacks. Firstly, they lack portability, relying on large laboratory equipment and specialized operating environments, making them unsuitable for on-site clinical testing, primary healthcare screening, or postoperative bedside monitoring. Secondly, they exhibit a mismatch in practicality, such as a disconnect between the detection range (linear range, detection limit) and the clinical diagnostic needs of osteoarthritis (OA). They either fail to detect low-concentration early biomarkers or cannot cover medium- to high-concentration pathological states, thus failing to meet the needs of monitoring the entire disease course. Furthermore, existing solutions suffer from weak anti-interference capabilities; some fiber optic and electrochemical sensors are susceptible to external factors such as electromagnetic interference, light source fluctuations, and environmental corrosion, compromising detection accuracy and reliability. Finally, the imbalance between cost and efficiency is a key factor hindering the widespread adoption of these methods. In other words, existing technologies are either prohibitively expensive or time-consuming, failing to balance the economic and rapid requirements of clinical applications. Summary of the Invention

[0006] This invention aims to overcome the above-mentioned deficiencies and provide a novel detection solution that combines high sensitivity, high specificity, low cost, and portability, enabling rapid and accurate detection of TSP-1. Its detection limit (12.857 ng・mL⁻¹) and linear range (12.857-100 ng・mL⁻¹) perfectly match the clinical diagnostic needs of OA, while avoiding interference from external factors such as electromagnetic interference and light source effects. It can be directly applied to the detection of actual joint fluid samples from OA patients, providing an efficient technical means for early diagnosis and prognostic monitoring of OA, reducing detection costs, and improving the convenience and practicality of clinical testing.

[0007] This invention provides a TSP-1 detection device, characterized in that it includes a sensing core module;

[0008] The sensing core module uses Metglas alloy as a base, and after chromium and gold modification on the surface, the TSP-1 antibody is attached to it.

[0009] Furthermore, the TSP-1 detection device provided by the present invention is characterized in that: chromium and gold are sequentially deposited on the substrate surface to form a chromium layer and a gold layer.

[0010] Furthermore, the TSP-1 detection device provided by the present invention is characterized in that the thickness of the chromium layer and the gold layer is 50-150nm.

[0011] Furthermore, the TSP-1 testing device provided by the present invention is characterized in that: the surface-modified Metglas alloy is subjected to an amination treatment.

[0012] Furthermore, the TSP-1 detection device provided by the present invention is characterized in that: the TSP-1 antibody is activated and then combined with the modified alloy.

[0013] Furthermore, the TSP-1 detection device provided by the present invention is further characterized in that: the specific preparation method of the sensing core module is as follows:

[0014] S1.ME chip pretreatment and modification:

[0015] S1.1. Cut the Metglas alloy material to the target size;

[0016] S1.2. A chromium layer and a gold layer are sequentially deposited on the surface of the bare chip using plasma sputtering technology;

[0017] S1.3. Clean the chip to remove surface impurities;

[0018] S2.TSP-1 antibody fixation:

[0019] S2.1. Amination treatment: The chip modified by S1 is horizontally immersed in the amination reagent and incubated at room temperature to form amino active sites on the chip surface;

[0020] S2.2. Antibody activation: TSP-1 antibody is activated by an antibody activation reagent to obtain an activated antibody solution;

[0021] S2.3. Antibody fixation: The chip after S2.1 amination is immersed in the activated antibody solution formed in S2.2, and the antibody is fixed by incubation to allow the amino group to covalently bind to the activated carboxyl group;

[0022] S2.4. Cleaning and Blocking: After rinsing the chip with buffer to remove non-specifically bound antibodies, immerse the chip in blocking reagent to block non-specific binding sites.

[0023] Furthermore, the TSP-1 detection device provided by the present invention is further characterized in that it also includes a signal detection module;

[0024] The signal detection module includes: a glass tube encasing the coil, an analyzer, and a DC bias magnet;

[0025] The core sensing module is housed inside the glass tube;

[0026] A coil wound around a glass tube is electrically connected to the analyzer to generate an alternating magnetic field;

[0027] A DC bias magnet is placed on the outside of the glass tube to maximize the vibration amplitude of the sensor.

[0028] Furthermore, this invention also provides the application of the aforementioned TSP-1 detection device in the quantitative detection of TSP-1 in ex vivo samples. The ex vivo samples are ex vivo serum or synovial fluid samples.

[0029] Furthermore, this invention also suggests the application of the aforementioned TSP-1 detection device as an early diagnostic device for OA.

[0030] Functions and effects of this invention:

[0031] The device of this invention offers precise detection performance tailored to clinical needs, achieving a balance between high sensitivity and a wide linear range: Its detection limit is as low as 12.857 ng·mL⁻¹, accurately capturing low concentrations of TSP-1 markers in early-stage OA patients, overcoming the limitations of traditional immunohistochemical and biochemical assays for early screening; simultaneously, its linear range covers 12.857-100 ng·mL⁻¹, perfectly matching the concentration range required for clinical OA diagnosis, thus meeting the needs of monitoring the entire OA disease course.

[0032] The device of this invention is highly specific and effectively avoids cross-interference: through the specific binding design of TSP-1 antibody and antigen, it exhibits high specificity in the identification of target biomarkers. Experimental verification shows that, at the same concentration, the resonance frequency shift induced by TSP-1 is 7 times that of other biomolecules such as lysozyme (LZM) and C-reactive protein (CRP), which can effectively eliminate interference from extraneous proteins in complex biological samples and overcome the shortcomings of some existing detection technologies, such as insufficient specificity and susceptibility to false positive results.

[0033] The device of this invention is simple to prepare, convenient to operate, and fast to detect: the sensor is based on Metglas alloy 2826MB and can be prepared by simple processes such as plasma sputtering of a chromium-gold layer, amination, and antibody immobilization, without the need for complex and precise processing procedures, thus solving the problem of complex manufacturing processes for fiber optic sensors; the detection process uses wireless signal transmission, and there is no physical connection between the sensor and the instrument. The detection can be completed by reacting the sample with the sensor for only 40 minutes. The operation process is simple and the time consumption is much shorter than that of traditional enzyme-linked immunosorbent assay (ELISA), overcoming the shortcomings of existing technologies such as cumbersome operation and long detection cycle.

[0034] The device of this invention is low-cost, highly portable, and suitable for multiple application scenarios: The Metglas alloy 2826 MB material has the advantages of low cost and lightweight, and the overall structure of the sensor is small. It does not rely on large laboratory equipment and can realize clinical on-site detection, primary medical screening or postoperative bedside monitoring. It solves the limitations of most existing detection methods, such as high cost, poor portability, and only being usable in professional laboratories, and significantly reduces the application threshold of OA detection.

[0035] The device of this invention has strong anti-interference ability and high detection stability: relying on the excellent magnetostrictive effect of Metglas alloy, the sensor detection is not affected by factors such as ambient light fluctuations and fiber optic connection loss, and there is no need to worry about electromagnetic interference or acid and alkali corrosion. Compared with non-grating fiber optic sensors and electrochemical sensors, its anti-interference ability is more outstanding. In the experiment, through three repeated calibration verifications, it showed good repeatability, and the detection results were accurate and reliable, overcoming the shortcomings of some existing technologies that are easily affected by the external environment and have insufficient detection stability.

[0036] The device of this invention is highly practical and can be directly used for clinical sample testing. See the application examples, which demonstrate that this device was successfully applied to the actual testing of serum samples (n=10) from OA patients, and the results were compared one-to-one with ELISA test results. The results showed no statistically significant difference between the mean values ​​of the magnetoelastic test strip and the ELISA test results, with a correlation coefficient of 0.857, indicating good consistency between the two detection methods. This verifies its applicability in complex clinical samples, overcoming the shortcomings of some existing technologies that can only perform tests under ideal laboratory conditions and cannot adapt to actual clinical samples, providing direct and effective technical support for the early diagnosis and prognosis of OA. Attached Figure Description

[0037] Figure 1 Electron microscopy characterization results;

[0038] Figure 2 A. A graph showing the relationship between TSP-1 concentration and the resonance shift of magnetoelastic test paper;

[0039] Figure 2 B. Standard curve of the relationship between TSP-1 concentration and resonance shift of magnetoelastic test paper;

[0040] Figure 3 A. There was no statistically significant difference between the mean values ​​of the magnetoelastic test strip results and the ELISA results;

[0041] Figure 3 B. Correlation diagram between the results of magnetoelastic test paper and ELISA test results. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1. TSP-1 Portable Detection Device

[0044] The TSP-1 portable testing device in this embodiment is in the shape of a test strip and includes a sensing core module and a signal detection module.

[0045] Among them, the core sensing module: the core is Metglas Alloy 2826 MB (composition Fe). 40 Ni 38 Mo4B 18The ME chip, fabricated from [material name], measures 5mm × 1mm × 28μm.

[0046] A 100nm chromium layer and a 100nm gold layer are sequentially deposited on the chip surface.

[0047] The chromium layer acts as an adhesive layer to enhance the adhesion of the gold film, while also forming an electrochemical protective layer to prevent the substrate from dissolving in a salt environment.

[0048] The gold layer is used to achieve covalent immobilization of biomolecules. The TSP-1 antibody is immobilized on the surface of the gold layer as a capture probe, and non-specific binding sites are blocked by bovine serum albumin (BSA).

[0049] The specific fabrication method of this core sensing module is as follows:

[0050] S1.ME chip pretreatment and modification:

[0051] S1.1. Cut the Metglas alloy 2826 MB material to obtain a bare chip with dimensions of 5mm × 1mm × 28μm;

[0052] S1.2. A chromium layer and a gold layer are sequentially deposited on the surface of the bare chip using plasma sputtering technology;

[0053] The desired effect can be achieved with the thickness of the chromium and gold layers ranging from 50-150 nm. In an optimal experimental example, a 100 nm chromium layer and a 100 nm gold layer are deposited sequentially on the surface. In the experimental examples of this invention, it was found that the 100 nm chromium layer is optimal, as it can fully cover the substrate surface, forming a uniform and dense adhesive layer, ensuring a strong bond between the gold film and the substrate (no peeling after repeated rinsing); it effectively prevents substrate dissolution and maintains structural stability even after immersion in clinical samples (salt environment) for more than 40 minutes; the 100 nm gold layer optimally forms a continuous and flat surface, providing sufficient reaction sites for amination treatment (mercaptoethylamine), ensuring uniform amino density on the chip surface, and subsequent antibody binding rate ≥85% (as required in the experimental example); the thickness is moderate, so as not to change the chip's inherent resonant frequency due to excessive weight (still maintained at around 440 kHz), ensuring that the detection instrument can accurately capture frequency shift signals. When the thickness is less than 50 nm, it will lead to insufficient stability and antibody loading. However, when the surface deposit is larger than 150nm, the excessive thickness of the surface deposit can lead to poor product stability and the inability of the detector to identify the target.

[0054] In other experimental cases, we used ME chips to directly manufacture the products and found that antibody loading was difficult and the stability was poor. This made the products completely unable to meet the clinical testing needs. In other words, modification is a prerequisite for subsequent antibody immobilization and stable sensor operation.

[0055] S1.3. Clean the chip three times alternately with methanol and deionized water to remove surface impurities.

[0056] S2.TSP-1 antibody fixation

[0057] S2.1. Amination treatment: The chip modified by S1 is horizontally immersed in a 40mM mercaptoethylamine solution and incubated at room temperature for 12h to form amino active sites on the chip surface;

[0058] In other experimental examples, we also used 3-aminopropyltriethoxysilane (APTES), ethylenediamine, cysteine, etc. to perform amino activation, which achieved the same effect.

[0059] In some experimental cases, we omitted the amination step and found that it significantly reduced the antibody loading.

[0060] S2.2. Antibody activation: Add 10-50 μg / mL -1 A solution of TSP-1 antibody (source: ABclonal, type: human, catalog number: A2125) at pH 7.4 and 4 mg / mL -1 EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 4 mg / mL -1 The NHS (N-hydroxysuccinimide) mixture was reacted at 37°C for 30 min to obtain an activated antibody solution;

[0061] The optimal concentration of the antibody is 25 μg / mL. -1 This is the optimal concentration for "balancing sensitivity and specificity." Experiments have shown that deviations from this concentration directly affect detection performance, especially when the antibody concentration is too low, such as < 10 μg / mL. -1 When the total amount of activated antibody is insufficient, the antibody density immobilized on the chip surface is low, and the probability of binding to TSP-1 antigen in the sample decreases; when the antibody concentration is too high, such as >50 μg / mL -1 However, this can lead to wasted resources because excess antibodies cannot be fully fixed on the chip surface, and the excess is washed away, increasing preparation costs. In addition, the increase in non-specific binding and excessive antibody aggregation on the chip surface can easily form non-specific binding sites, leading to false positive results. Furthermore, the excessive antibody increases the chip surface load, which may change the inherent resonant frequency and affect the accuracy of the detection calibration curve.

[0062] In other experimental cases, carbodiimide hydrochloride derivatives were used for activation, which ensured that the binding rate of the activated antibody to the amination chip was ≥85%.

[0063] S2.3. Antibody fixation: The chip after S2.1 amination is immersed in the activated antibody solution formed in S2.2 and incubated at 25-40℃ for 0.5-2h. Antibody fixation is achieved through covalent binding of amino groups and activated carboxyl groups.

[0064] The optimal incubation temperature is 37℃, which is close to the physiological temperature of the human body. This is the suitable temperature for the covalent binding of amino groups (on the chip surface) and activated carboxyl groups (on the antibody surface), ensuring both reaction efficiency and preventing antibody denaturation. In some experimental cases, it was found that when the temperature is too low (<25℃, such as room temperature of 20℃), the covalent binding reaction rate slows down, and sufficient binding cannot be completed within 1 hour, resulting in insufficient antibody fixation and decreased sensitivity. When the temperature is too high (>40℃, such as 45℃), the TSP-1 antibody (protein) will denature and lose its specificity for binding to the TSP-1 antigen, leading to complete detection failure.

[0065] The optimal reaction time is 1 hour, which ensures that the amino group and the activated carboxyl group react fully, and the antibody is stably fixed on the chip surface with a binding rate of ≥85%. In some experimental cases, it was found that when the time is too short (<30 min): the binding is insufficient, the amount of antibody fixed is small, the resonance shift signal is weak, and the sensitivity is insufficient; when the time is too long (>2 h): there is no additional gain, and it may lead to excessive antibody aggregation, increased non-specific binding, and prolonged preparation cycle, reducing efficiency.

[0066] S2.4. Cleaning and Blocking: Rinse the chip 5 times with PBS buffer (pH=7.4) to remove non-specifically bound antibodies. Then, immerse the chip in 0.1% BSA solution (pH=7.4) at 25-40℃ for 20-60 min to block non-specific binding sites. Finally, rinse 5 times with PBS buffer and vacuum dry to obtain the ME biosensor for TSP-1 detection.

[0067] The optimal reaction temperature is 37°C, which ensures that BSA (or other blocking agents) can efficiently bind to sites on the chip surface that are not occupied by antibodies. In some experimental cases, it was found that when the temperature is too low (<25°C) the blocking is insufficient, and when the temperature is too high (>40°C) the BSA denatures, non-specific binding will increase.

[0068] The optimal reaction time is 30 min, which can fully block non-specific sites. In some experimental cases, it was found that when the time is too short (<20 min), the blocking is incomplete and the non-specific binding rate is >5%; when the time is too long (>60 min), BSA is over-adsorbed, which may block the fixed antibody binding sites and affect the antigen-antibody specific binding.

[0069] In other experimental cases, other biological blocking agents such as casein and gelatin were used to complete the blocking. The results showed that conventional blocking agents could also achieve a non-specific binding rate of ≤5% after blocking.

[0070] The signal detection module includes: a glass tube encasing the coil, a vector network analyzer (such as an Agilent AV3620A), and a DC bias magnet;

[0071] The ME chip is placed inside a glass tube.

[0072] The coil is connected to a vector network analyzer via a cable to generate an alternating magnetic field.

[0073] A DC bias magnet is used to maximize the vibration amplitude of the sensor, enabling wireless detection of the resonant frequency.

[0074] The specific setup and debugging method for this signal detection module:

[0075] The prepared ME sensor is placed inside a glass tube, and a coil is wound around the outside of the glass tube. The coil is connected to a vector network analyzer via a cable. A DC bias magnet is placed outside the coil, and the position of the magnet is adjusted to maximize the vibration amplitude of the sensor. The vector network analyzer is calibrated, and the detection frequency range is set to cover the sensor's natural resonant frequency (approximately 440kHz) to ensure accurate capture of frequency offset signals.

[0076] In other test cases, the testing instrument can be replaced by other wireless signal analyzers with resonant frequency monitoring capabilities, such as the Keysight E5063A vector network analyzer or the Rohde & Schwarz ZNB20 vector network analyzer, as long as the instrument's detection accuracy is ≥0.1kHz and the frequency range covers the sensor's inherent resonant frequency (430-450kHz).

[0077] Based on the above design, Metglas alloy 2826 MB was selected as the sensing substrate. Its high magnetostriction coefficient, lightweight and wireless sensing characteristics ensure the high sensitivity and portability of the sensor.

[0078] Chromium-gold dual-layer surface modification process: It not only solves the adhesion problem between the gold film and the iron-based alloy substrate, but also forms an electrochemical protective layer, improving the stability of the sensor in biological samples (salt environment);

[0079] EDC / NHS activation-mercaptoethylamine amination covalent fixation strategy: to achieve efficient and stable fixation of TSP-1 antibody, reduce antibody detachment, and ensure the specificity of antigen-antibody binding;

[0080] The wireless resonant frequency detection system avoids interference from contact detection by eliminating the physical connection between the sensor and the detection instrument. At the same time, relying on the magnetostrictive effect, it is unaffected by external factors such as ambient light sources and electromagnetic interference.

[0081] Detection performance precisely matched to clinical needs: detection limit as low as 12.857 ng·mL -1 Linear range 12.857-100 ng·mL -1 It fully covers the TSP-1 concentration range required for OA diagnosis.

[0082] Example 2. Use of the TSP-1 portable testing device

[0083] 2.1. Detection method:

[0084] S1. Take the sample to be tested (e.g., serum, synovial fluid) and drop it onto the surface of the ME sensor inside the glass tube, ensuring that the sample is in full contact with the sensor;

[0085] S2. Start the vector network analyzer and record the resonance frequency every 5 minutes, continuously monitoring for 40 minutes until the reaction reaches equilibrium;

[0086] S3. Using the frequency offset corresponding to the lowest value of the resonance frequency as the detection signal, the concentration of TSP-1 in the sample is calculated in combination with the calibration curve;

[0087] S4. After the test is completed, rinse the glass tube and sensor 5 times with PBS buffer at pH 7.4. The tubes can be reused (if necessary).

[0088] The detection principle of this TSP-1 portable testing device is as follows:

[0089] Based on the excellent magnetostrictive effect of the Metglas alloy 2826 MB: Under the combined action of an alternating magnetic field and a DC bias magnetic field, the ME chip will undergo longitudinal tensile vibration at its natural resonant frequency (approximately 440kHz in air), which satisfies the formula: f0 is the natural resonant frequency, L is the chip length, E is the elastic modulus, ρ is the density, and ν is the Poisson's ratio.

[0090] When the TSP-1 antibody on the chip surface specifically binds to TSP-1 in the sample, the chip surface loading mass (Δm) increases, resulting in a decrease in the resonance frequency. The frequency shift is positively correlated with the TSP-1 concentration. By monitoring this shift, the TSP-1 content can be quantitatively analyzed.

[0091] Example 3. Characterization and performance testing of the TSP-1 portable testing device

[0092] 3.1. Electron Microscopy Characterization (SEM)

[0093] like Figure 1 As shown, scanning electron microscopy revealed that the surface of the bare ME chip was smooth and free of impurities; after amination treatment, uniform precipitates appeared on the chip surface, confirming successful amination; after antibody fixation, uniformly dispersed antibody aggregates were visible on the chip surface; and after binding with TSP-1, a large number of spherical aggregates formed on the chip surface, indicating that the antigen-antibody specific binding was effective.

[0094] 3.2. Calibration Curve

[0095] like Figure 2 As shown in Figure A, the sensor is effective for TSP-1 concentrations in the range of 12.857-100 ng / mL. -1 It exhibits a good linear response within the range. As the TSP-1 concentration increases, the resonance shift of the magnetoelastic paper also increases.

[0096] like Figure 2 As shown in B, the linear equation is y = 0.0304x - 14.104, r 2 The value was 0.9119, and the detection limit was 12.857 ng·mL. -1 .

[0097] 3.3. Relevance to ELISA Method

[0098] The magnetoelastic test paper of this invention was used in conjunction with the ELISA method to detect the TSP-1 concentration in serum samples from 8 OA patients, and the results were compared one-to-one with the ELISA results. The results showed that, as Figure 3 As shown in Figure A, there was no statistically significant difference between the mean values ​​of the magnetoelastic test strip results and the ELISA results. Figure 3 As shown in B, the correlation coefficient between the two is 0.857, confirming the reliability of the detection results of this invention.

Claims

1. A TSP-1 testing device, characterized in that: Includes a core sensing module; The sensing core module uses Metglas alloy as a substrate, and after chromium and gold modification on the surface, the TSP-1 antibody is attached to it.

2. The TSP-1 testing device as described in claim 1, characterized in that: The chromium and gold are deposited sequentially on the substrate surface to form a chromium layer and a gold layer.

3. The TSP-1 testing device as described in claim 1, characterized in that: The thickness of the chromium and gold layers is 50-150 nm.

4. The TSP-1 testing device as described in claim 1, characterized in that: The surface-modified Metglas alloy was subjected to amination treatment.

5. The TSP-1 testing device as described in claim 1, characterized in that: The TSP-1 antibody was activated and then combined with the modified alloy.

6. The TSP-1 testing device as described in claim 1, characterized in that: The specific fabrication method of the sensing core module is as follows: S1.ME chip pretreatment and modification: S1.

1. Cut the Metglas alloy material to the target size; S1.

2. A chromium layer and a gold layer are sequentially deposited on the surface of the bare chip using plasma sputtering technology; S1.

3. Clean the chip to remove surface impurities; S2.TSP-1 antibody fixation: S2.

1. Amination treatment: The chip modified by S1 is horizontally immersed in the amination reagent and incubated at room temperature to form amino active sites on the chip surface; S2.

2. Antibody activation: TSP-1 antibody is activated by an antibody activation reagent to obtain an activated antibody solution; S2.

3. Antibody fixation: The chip after S2.1 amination is immersed in the activated antibody solution formed in S2.2, and the antibody is fixed by incubation to allow the amino group to covalently bind to the activated carboxyl group; S2.

4. Cleaning and Blocking: After rinsing the chip with buffer to remove non-specifically bound antibodies, immerse the chip in blocking reagent to block non-specific binding sites.

7. The TSP-1 testing device as described in claim 1, characterized in that: It also includes a signal detection module; The signal detection module includes: a glass tube encasing the coil, an analyzer, and a DC bias magnet; The core sensing module is housed inside the glass tube; The coil wound around the glass tube is electrically connected to the analyzer to generate an alternating magnetic field; The DC bias magnet is positioned on the outside of the glass tube to maximize the vibration amplitude of the sensor.

8. The application of a TSP-1 detection device as described in any one of claims 1-7 in the quantitative detection of TSP-1 in ex vivo samples.

9. The application as described in claim 8, characterized in that: The ex vivo sample is an ex vivo serum or synovial fluid sample.

10. The application of a TSP-1 detection device as described in any one of claims 1-7 as an early diagnostic device for OA.

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