Parathyroid hormone detection probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical detection and immunoassay, and particularly discloses a parathyroid hormone detection probe as well as a preparation method and application thereof. The parathyroid hormone detection probe provided by the invention comprises a capture probe and a signal probe, the capture probe is a magnetic particle of which the surface is fixed with a PTH specific antibody 1, and the signal probe is a PTH specific antibody 2 of which the surface is marked with a photosensitizer. The immunodetection probe provided by the invention can solve the problems of unstable enzyme activity, high detection cost, complex operation and the like in the existing enzyme labeling immunodetection technology. Different from the traditional chemiluminescence or enzyme catalysis signal amplification mechanism, the photosensitization oxidation reaction and the double-antibody sandwich immune recognition are creatively combined, and the color change of the chromogenic substrate is catalyzed by utilizing the characteristic that the photosensitizer efficiently generates active oxygen under the irradiation of visible light, so that the high-sensitivity and high-specificity detection on the to-be-detected object PTH is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and immunoassay technology, and in particular to a parathyroid hormone detection probe, its preparation method, and its application. Background Technology

[0002] Parathyroid hormone (PTH) is a single-chain polypeptide hormone synthesized and secreted by the chief cells of the parathyroid glands. It plays a central role in regulating calcium and phosphorus metabolism homeostasis in the human body. Abnormal PTH levels are closely related to a variety of diseases, such as primary hyperparathyroidism, secondary hyperparathyroidism (common in patients with chronic kidney disease), hypoparathyroidism, and osteoporosis. Therefore, rapid and accurate detection of PTH concentration in the blood is of crucial clinical significance for the diagnosis, differential diagnosis, treatment efficacy evaluation, and prognosis of these diseases.

[0003] Currently, the mainstream methods for detecting PTH in clinical laboratories are mainly based on immunoassay principles, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and electrochemiluminescent immunoassay. These methods typically rely on enzyme-labeled (e.g., horseradish peroxidase, HRP) or chemiluminescently labeled secondary antibodies to catalyze the substrate to generate color, light, or electrical signals for detection. Although these technologies are relatively mature, they still have some inherent limitations: First, the activity of enzyme-labeled substances is easily affected by environmental temperature, pH, and storage conditions, resulting in relatively poor stability and potentially batch-to-batch variability; second, while chemiluminescence methods offer high sensitivity, they usually require expensive equipment and specialized reagents, leading to high detection costs; furthermore, some methods involve cumbersome procedures and long detection times, making them unsuitable for rapid detection in clinical emergencies or during surgery.

[0004] In recent years, researchers have begun exploring non-enzyme-dependent signal amplification strategies in search of more stable and convenient detection methods. Among these, enzyme-mimicking catalysis, particularly nanomaterials with peroxidase activity, has shown promising application prospects. However, the catalytic efficiency, stability, and biocompatibility of these enzyme-mimicking materials still have room for improvement. Photosensitive oxidation, as an emerging signal generation mechanism, utilizes photosensitizers to generate reactive oxygen species under specific wavelengths of light, which then oxidize colorless chromogenic substrates (such as TMB) to produce color changes. This process is enzyme-independent and has potential advantages such as mild reaction conditions, ease of control, and signal stability. If photosensitive oxidation can be combined with highly specific immunorecognition reactions to construct a novel PTH detection probe, it is expected to overcome some of the shortcomings of traditional enzyme labeling techniques and provide a superior new method for PTH detection in clinical practice.

[0005] Therefore, developing a new PTH immunoassay method based on photosensitive oxidation reaction that has high sensitivity, high specificity, good stability and is easy to operate has become a worthy research direction in this field. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention provides a parathyroid hormone (PTH) immunoassay probe based on a photosensitive oxidation reaction, its preparation method, and its detection application. The immunoassay probe provided by this invention can solve the problems of unstable enzyme activity, high detection cost, and complex operation in existing enzyme-labeled immunoassay techniques. Unlike traditional chemiluminescence or enzyme-catalyzed signal amplification mechanisms, this invention innovatively combines a photosensitive oxidation reaction with double-antibody sandwich immunorecognition. It utilizes the property of photosensitizers to efficiently generate reactive oxygen species under visible light irradiation, catalyzing a color change in the chromogenic substrate, thereby achieving highly sensitive and specific detection of the analyte PTH.

[0007] The present invention also provides a method for preparing a PTH detection probe.

[0008] This invention also provides applications of PTH detection probes.

[0009] In a first aspect, the present invention provides a parathyroid hormone detection probe, the probe comprising a capture probe and a signal probe; the capture probe is a magnetic microparticle with a PTH-specific antibody 1 immobilized on its surface, and the signal probe is a PTH-specific antibody 2 with a photosensitizer labeled on its surface.

[0010] According to specific embodiments of the present invention, the probe provided by the present invention innovatively combines the photosensitive oxidation signal amplification mechanism with the classic double-antibody sandwich immunorecognition principle to construct a novel non-enzyme-dependent detection system. Unlike conventional immunoassays that rely on biological enzymes such as horseradish peroxidase, the present invention utilizes the characteristic of photosensitizers to efficiently generate ROS under visible light irradiation, directly catalyzing the oxidation and colorimetric reaction of colorless chromogenic substrates (such as TMB), thereby achieving quantitative detection of the target analyte PTH. This design fundamentally avoids the inherent defects of unstable enzyme labeling activity and easy inactivation, significantly improving the long-term stability and batch-to-batch reproducibility of the detection system.

[0011] According to some embodiments of the present invention, the PTH-specific antibody 1 is a C-terminal recognition antibody of PTH, and the PTH-specific antibody 2 is an N-terminal recognition antibody of PTH.

[0012] This invention is the first to directly label a photosensitizer onto the N-terminal recognition antibody of PTH and construct a "magnetic capture-photosensitive signal" bifunctional probe system with a C-terminal recognition antibody immobilized on magnetic microparticles. This design not only retains the high specificity of immune recognition but also achieves non-enzyme-dependent signal amplification through photosensitive oxidation, significantly improving the stability and reproducibility of detection. More importantly, this invention can also achieve a synergistic improvement in detection performance by systematically optimizing multiple key variables such as the coupling ratio of photosensitizer and antibody, coupling chemical methods, reaction system pH, temperature, and light parameters. These optimized combinations are not easily foreseen by those skilled in the art or obtained through limited experiments, demonstrating the inventiveness of this invention.

[0013] According to some embodiments of the present invention, the surface of the magnetic microparticles is modified with functional groups, including at least one of carboxyl, amino, or streptavidin; such functional groups facilitate the immobilization of antibodies on the magnetic microspheres.

[0014] According to some embodiments of the present invention, the magnetic particles are selected from at least one of magnetic iron oxide microspheres, nickel magnetic microspheres, or cobalt magnetic microspheres; preferably, the magnetic particles are superparamagnetic iron oxide microspheres with a particle size range of 50~500 nm.

[0015] According to some embodiments of the present invention, the photosensitizer is selected from at least one of phthalocyanine compounds, porphyrin compounds, phenothiazine dyes, xanthan dyes, or natural photosensitizers.

[0016] According to some embodiments of the present invention, the phthalocyanine compounds include zinc phthalocyanine or aluminum phthalocyanine; the porphyrin compounds include hematoporphyrin, protoporphyrin IX, or tetraphenylporphyrin; the phenothiazine dyes include methylene blue or toluidine blue; the xanthan dyes include rose red, Bengal rose red, or erythrosine; and the natural photosensitizers include riboflavin or curcumin.

[0017] According to some preferred embodiments of the present invention, the photosensitizer is selected from Bengal rose red or methylene blue; these dye molecules have moderate molecular weight and good water solubility, and have minimal impact on the spatial conformation and immunobinding activity of the antibody after conjugation. At the same time, they have strong absorption and high intersystem crossing efficiency in the visible light region, and can maximize the conversion of light energy into chemical energy (generating reactive oxygen species).

[0018] A second aspect of the present invention provides a method for preparing a parathyroid hormone detection probe as described in the first aspect of the present invention, comprising the following steps:

[0019] S1. Mix magnetic microparticles with PTH-specific antibody 1 in buffer solution in the presence of activator, collect the product by magnetic separation, and obtain the capture probe after blocking.

[0020] S2. PTH-specific antibody 2 and photosensitizer are mixed and reacted in buffer in the presence of activator, and the resulting signal probe is purified.

[0021] In step S2, the molar ratio of the PTH-specific antibody 2 to the photosensitizer is 1:(4~7).

[0022] Through experiments with strict control of variables, this invention has found that the signal probe prepared with a molar ratio of PTH-specific antibody 2 to photosensitizer in the range of 1:4 to 1:7 exhibits the best performance; among which, 1:6 is the optimal ratio. Within this ratio range, the amount of photosensitizer labeled on each antibody molecule reaches an ideal balance point: providing sufficient photosensitizing active sites to generate a strong signal while avoiding impaired antibody immune activity or non-specific aggregation due to excessive labeling.

[0023] According to some embodiments of the present invention, in step S1, the activator includes at least one of a carbodiimide crosslinking agent, glutaraldehyde, and genipin; the carbodiimide crosslinking agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N,N'-diisopropylcarbodiimide; the buffer solution includes at least one of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer, and borate buffer, and the pH value of the buffer solution is 5.0~9.0; the temperature of the mixing reaction is 4~37°C, and the time is 0.5~4h.

[0024] According to some embodiments of the present invention, in step S2, the activator includes at least one of the following: carbodiimide / succinimide system, glutaraldehyde, and sulfonyl-SMCC; the buffer solution includes at least one of the following: carbonate buffer solution, phosphate buffer solution, and borate buffer solution; the pH value of the buffer solution is 7.0 to 9.5; the mixing reaction is carried out under light-protected conditions; the reaction temperature is 4 to 25°C; and the reaction time is 1 to 6 hours.

[0025] According to some preferred embodiments of the present invention, the activator is sulfonyl-SMCC. The present invention explored and compared different coupling chemical strategies, finding that coupling using sulfonyl-SMCC crosslinking agents is more suitable for the signal probes of the present invention compared to other methods such as traditional EDC / NHS or glutaraldehyde methods. It allows for better control of coupling orientation and degree, and the prepared signal probes exhibit superior performance in terms of storage stability and batch consistency.

[0026] According to some embodiments of the present invention, in step S2, the purification method includes at least one of dialysis, gel filtration chromatography, and ultrafiltration centrifugation.

[0027] A third aspect of the present invention provides the application of the parathyroid hormone detection probe as described in the first aspect of the present invention in detecting parathyroid hormone in a sample, wherein the method for detecting parathyroid hormone in a sample comprises the following steps:

[0028] S10. Immune reaction: Mix the capture probe with the sample solution and incubate to allow PTH to bind to specific antibody 1; add the signal probe to form a sandwich immune complex.

[0029] S20. Separation and washing: Perform magnetic separation and washing to remove unbound substances; greatly reducing background interference.

[0030] S30, Photosensitive color development: Add a color development substrate to the washed complex and carry out a photosensitive oxidation reaction under visible light irradiation;

[0031] S40. Detection and Analysis: Measure the absorbance value of the reaction system and calculate the concentration of PTH in the sample according to the standard curve.

[0032] The detection method of this invention is simple to operate and can be completed with only conventional magnetic separation device and ultraviolet-visible spectrophotometer, making it easy to promote in clinical laboratories.

[0033] According to some embodiments of the present invention, the chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3-amino-9-ethylcarbazole (AEC) or 3,3'-diaminobenzidine (DAB).

[0034] According to some preferred embodiments of the present invention, the chromogenic substrate is TMB (3,3′,5,5′-tetramethylbenzidine). The present invention has found that among various commonly used chromogenic substrates, TMB exhibits good compatibility with this photosensitive oxidation system. Since the preferred photosensitizers in this system (such as Bengal rose red and methylene blue) primarily generate singlet oxygen, and TMB possesses extremely high reactivity and selectivity for singlet oxygen, with its oxidation product exhibiting strong and stable characteristic absorption at 652 nm, the PTH detection probe system constructed in this invention, using TMB as the substrate, achieves a low detection limit of 0.18 ng / mL, significantly outperforming other substrates such as ABTS and OPD.

[0035] According to some embodiments of the present invention, in step S10, the incubation temperature is 28~32℃ and the incubation time is 10~60min; the buffer solution is phosphate buffer or Tris-HCl buffer with a pH value of 7.2-7.6.

[0036] According to some embodiments of the present invention, in step S30, the wavelength of the visible light is 400~700 nm, and the illuminance is 50~90 mW / cm². 2 The irradiation time is 20-25 minutes; the temperature of the photosensitive oxidation reaction is 15-40°C; preferably, the light intensity is 60-80 mW / cm². 2 ,

[0037] According to some embodiments of the present invention, in step S40, the signal change is a change in absorbance value or a color change; when measuring the absorbance value, the measurement wavelength is 370~700 nm, and the specific wavelength is determined according to the characteristic absorption peak of the oxidation product of the selected chromogenic substrate; the standard curve is plotted by measuring the signal values ​​of a series of PTH standard solutions.

[0038] In a fourth aspect, the present invention provides a parathyroid hormone detection kit comprising the parathyroid hormone detection probe described in the first aspect of the present invention.

[0039] This invention focuses on exploring the influence of reaction conditions on the detection performance of PTH detection probes, and determines the optimal pH and temperature for the immunoreaction, as well as the optimal light intensity and time for photosensitive color development. Through the synergistic optimization of these optimal parameters, the high sensitivity, high specificity and good operational stability of PTH detection are ensured.

[0040] The beneficial effects of this invention are:

[0041] 1) This invention creatively combines the photosensitive oxidation signal amplification mechanism with the classic double antibody sandwich immunorecognition principle to construct a novel non-enzyme-dependent detection system. It utilizes the characteristic of photosensitizers to efficiently generate reactive oxygen species under visible light irradiation to catalyze the color change of the chromogenic substrate, thereby realizing the detection of PTH in the test sample.

[0042] 2) In order to achieve higher sensitivity and specificity in detection performance, this invention explores and obtains the photosensitizer suitable for the PTH detection probe and the optimal molar ratio range of the photosensitizer and the labeled PTH-specific antibody through experiments and analysis, so as to achieve the best balance of detection performance, which can provide sufficient photosensitizing active sites to generate a strong signal, while avoiding the damage to antibody immune activity or non-specific aggregation due to excessive labeling.

[0043] 3) This invention also achieves comprehensive optimization of the working conditions of the PTH detection probe, from the conditions of the capture probe reacting with the sample PTH to the light intensity and light time during the photosensitizer photo-oxidation reaction, and the selection of the best chromogenic substrate. Through the synergistic effect of the optimized parameters from multiple perspectives, the high sensitivity, high specificity and good operational stability of PTH detection are further guaranteed.

[0044] 4) The PTH detection probe prepared by this invention can achieve highly sensitive detection of PTH concentration over a wide range by relying on its excellent specificity and signal amplification capability, and has excellent sensitivity and selectivity; moreover, the preparation process of this probe is simple and reliable, the raw materials are readily available, the preparation process does not require complex equipment, and it can be mass-produced.

[0045] 5) The probe of this invention is simple to operate for PTH detection, requiring no complicated sample pretreatment. PTH quantitative detection can be completed with the help of a conventional ultraviolet spectrophotometer, which greatly reduces equipment investment and maintenance costs.

[0046] 6) The solution of the present invention has no significant cross-response to common interfering substances in serum, can work stably in a variety of biological samples such as serum and plasma, and is suitable for multiple scenarios such as clinical testing, disease diagnosis and health screening, showing broad application prospects.

[0047] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0049] Figure 1 This is a schematic diagram illustrating the detection principle of the PTH detection probe of the present invention;

[0050] Figure 2 The detection results of the detection probe prepared in Example 3 of the present invention on PTH under different antibody and photosensitizer labeling molar ratios are shown in Figure a. Figure a shows the absorption spectrum of the detection system when the detection probe with different labeling molar ratios is used to measure PTH standard. Figure b shows the bar chart of the absorbance value of the detection system at 652 nm as a function of different labeling molar ratios. Figure c shows the actual photographs of the detection probe with different labeling ratios detecting PTH.

[0051] Figure 3 The results of PTH detection using capture probes prepared with different photosensitizers under the same detection conditions are shown in Example 4 of the present invention. Figure a shows the absorption spectrum of the detection system when measuring PTH standard using capture probes prepared with different photosensitizers; Figure b shows the bar chart of absorbance at 652 nm of the detection system when using capture probes prepared with different photosensitizers; and Figure c shows an actual photograph of PTH detection using capture probes prepared with different photosensitizers.

[0052] Figure 4The results of PTH detection using signal probes prepared with different activators under the same detection conditions are shown in Example 5 of the present invention. Figure a shows the absorption spectrum of the detection system when PTH standard is measured using signal probes prepared with different activators; Figure b shows the bar chart of absorbance at 652 nm of the detection system when signal probes prepared with different activators are used; Figure c shows an actual photograph of PTH detection using signal probes prepared with different activators.

[0053] Figure 5 The detection results of the detection probe prepared in Example 6 of the present invention on PTH under different light intensities are shown in Figure a. Figure a shows the absorption spectrum of the detection system when measuring PTH standard under different light intensities; Figure b shows the bar chart of the absorbance value of the detection system at 652 nm as a function of different light intensities; and Figure c shows the actual photographs of the detection probe detecting PTH under different light intensities.

[0054] Figure 6 The detection results of the detection probe prepared in Example 7 of the present invention on PTH under different illumination times are shown in Figure a. Figure a shows the absorption spectrum of the detection system when measuring PTH standard under different illumination times; Figure b shows the dotted line graph of the absorbance value of the detection system at 652nm as a function of different illumination times; and Figure c shows the actual photographs of the detection probe detecting PTH under different illumination times.

[0055] Figure 7 The detection results of the detection probe prepared in Example 8 of the present invention on PTH under different temperature conditions are shown in Figure a. Figure a shows the absorption spectrum of the detection system when measuring PTH standard at different temperatures. Figure b shows the dotted line graph of the absorbance value of the detection system at 425 nm as a function of different temperatures. Figure c shows the actual photographs of the detection probe detecting PTH at different temperatures.

[0056] Figure 8 The detection results of the detection probe prepared in Example 9 of the present invention on PTH under different pH conditions are shown in Figure a. Figure a shows the absorption spectrum of the detection system when PTH standard is measured at different pH values. Figure b shows the dotted line graph of the absorbance value of the detection system at 425 nm as a function of different pH values. Figure c shows the actual photographs of the detection probe detecting PTH at different pH values.

[0057] Figure 9The detection results of the detection probe prepared in Example 10 of this invention under different chromogenic substrates for PTH detection are shown in Figure a. Figure a is the standard curve of the detection system with chromogenic substrate TMB when measuring PTH standard; Figure b is the standard curve of the detection system with chromogenic substrate ABTS when measuring PTH standard; Figure c is the standard curve of the detection system with chromogenic substrate OPD when measuring PTH standard; Figure d is the standard curve of the detection system with chromogenic substrate DAB when measuring PTH standard; Figure e is the standard curve of the detection system with chromogenic substrate AEC when measuring PTH standard.

[0058] Figure 10 The detection results of the detection probe prepared in Example 11 of this invention for PTH are shown in Figure a. Figure a shows the ultraviolet absorption spectrum of the detection system in different concentrations of PTH, and Figure b shows the linear relationship between the absorption intensity of the detection system at 652 nm and the PTH concentration (the inset is an actual photograph of the detection system solution at different PTH concentrations).

[0059] Figure 11 The bar chart shows the absorption intensity of the detection system at 652 nm in the presence of other analytes, using the detection probe prepared in Example 11 of this invention. Detailed Implementation

[0060] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0061] The C-terminal and N-terminal recognition antibodies for PTH used in the examples were both goat anti-human PTH polyclonal antibodies, purchased from Fitzgerald Industries International, catalog numbers 70-XG67 and 70-XG68, respectively. Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer were followed. All reagents and instruments used, unless otherwise specified, were commercially available products.

[0062] Example 1

[0063] This embodiment provides a method for preparing a parathyroid hormone (PTH) detection probe based on a photosensitive oxidation reaction. The probe comprises two parts: a capture probe and a signal probe. A schematic diagram illustrating its PTH detection principle is shown below. Figure 1 As shown, the specific preparation steps are as follows:

[0064] 1) Preparation of the capture probe:

[0065] Take 1.0 mg of carboxyl-modified superparamagnetic iron oxide nanoparticles (200 nm in diameter) and place them in a 1.5 mL centrifuge tube; add 1 mL of 0.1 mol / L MES buffer (pH 6.0), vortex to mix, place on a magnetic separator, discard the supernatant, and wash twice; add 1 mL of freshly prepared activator solution (MES buffer containing 0.4 mg EDC and 0.6 mg NHS), and react at 800 rpm for 30 min at room temperature; remove the supernatant by magnetic separation, and wash once with MES buffer; add 1 mL of MES buffer containing 50 μg PTH primary antibody, and react at 1000 rpm for 2 h at 25 °C; add 100 μL of 1% BSA solution for blocking, and react at room temperature for 1 h; collect the product by magnetic separation, wash three times with PBS buffer (pH 7.4, containing 0.05% Tween-20), and finally resuspend in 1 mL of PBS buffer (pH 6.0). In step 7.4), the capture probe suspension was obtained and stored at 4°C for later use.

[0066] 2) Preparation of signal probes:

[0067] Dissolve 1.0 mg of Bengal rose red (RB) in 0.5 mL of water to prepare a 2 mg / mL stock solution. Take 250 μL of the above stock solution, add 0.3 mg of EDC and 0.4 mg of NHS, and activate at 500 rpm for 30 min under light-protected conditions at room temperature. Slowly add the activated rose red solution dropwise to 1 mL of carbonate buffer (pH 9.0) containing 100 μg of PTH secondary antibody, controlling the dropwise addition rate at 10 μL / min. Under light-protected conditions at 4 °C, slowly shake at 800 rpm for 4 h. Transfer the reaction solution to a dialysis bag with a molecular cutoff of 10 kDa, and dialyze with PBS buffer (pH 7.4) for 24 h under light-protected conditions at 4 °C, changing the dialysate 6 times during this period. Collect the solution in the dialysis bag, centrifuge at 12000 rpm for 10 min at 4 °C, and take the supernatant as the signal probe solution. Aliquot and store at 4 °C under light-protected conditions.

[0068] Example 2

[0069] This embodiment provides another method for preparing a PTH detection probe based on a photosensitive oxidation reaction. This probe consists of two parts: a capture probe and a signal probe. The specific preparation steps are as follows:

[0070] 1) Preparation of the capture probe:

[0071] 1.2 mg of amino-modified superparamagnetic iron oxide nanoparticles (180 nm in diameter) were washed twice with 0.1 M phosphate buffer (pH 7.4). 1 mL of 2.5% glutaraldehyde solution was added, and the mixture was shaken at 800 rpm for 2 h at 25 °C to ensure complete reaction between the amino groups on the surface of the magnetic particles and the glutaraldehyde. After the reaction, the aldehyde-modified magnetic particles were collected by magnetic separation and washed three times with phosphate buffer to remove unreacted glutaraldehyde. The aldehyde-modified magnetic particles were then mixed with 60 μg of PTH primary antibody in HEPES buffer (pH 7.2) and reacted at 37 °C for 1.5 h. This reaction condition utilizes the dual-aldehyde property of glutaraldehyde, where one end binds to the amino group of the magnetic particle, and the other end forms a Schiff base structure with the amino group of the antibody. After the reaction, 100 μL of 1% glycine solution was added to block the unreacted aldehyde groups, and the reaction was stopped at room temperature for 30 min. Finally, the product was collected by magnetic separation, washed three times with PBS buffer (pH 7.4), resuspended in 1 mL PBS, and stored at 4°C.

[0072] 2) Preparation of signal probes:

[0073] A sulfonyl-SMCC crosslinking method was used. 100 μg of PTH secondary antibody was dialyzed with HEPES buffer (pH 7.5). 0.5 mg of sulfonyl-SMCC crosslinking agent was added, and the reaction was carried out at 4°C in the dark for 1 h, allowing the amino groups on the antibody molecule surface to react with the NHS ester groups of the crosslinking agent, introducing maleimide groups. After the reaction, the antibody was dialyzed in PBS buffer for 4 h using a dialysis bag with a molecular cutoff of 10 kDa to remove unreacted sulfonyl-SMCC. Separately, 1.0 mg of protoporphyrin IX photosensitizer was dissolved in DMSO, and 0.3 mg of Traut's reagent was added. The reaction was carried out at 25°C in the dark for 30 min, introducing thiol groups onto the photosensitizer molecule. The modified photosensitizer and the activated antibody were mixed in PBS buffer (pH 7.2) and reacted at 4°C in the dark for 4 h, achieving conjugation of the photosensitizer and antibody through the specific reaction of maleimide and thiol groups. Purification was performed using gel filtration chromatography with a Sephadex G-25 column and PBS buffer as the mobile phase. The target probe was collected and stored at 4°C in the dark.

[0074] Example 3

[0075] This embodiment provides PTH detection probes prepared with different antibody and photosensitizer labeling ratios, and systematically compares their detection performance.

[0076] 1) Preparation of signal probes: Five parallel experiments were set up, with the amount of the secondary antibody fixed at 100 μg. The amount of methylene blue added was varied to make the molar ratio of antibody to MB 1:2, 1:4, 1:6, 1:8, and 1:10, respectively. The specific preparation steps are as follows: Weigh 0.43 μg, 0.85 μg, 1.28 μg, 1.70 μg, and 2.13 μg of MB, respectively, and dissolve them in 0.5 mL of water. Add the corresponding amounts of EDC (0.3 mg) and NHS (0.4 mg) to each group, and activate at room temperature in the dark for 30 min. Add the activated MB solution dropwise to carbonate buffer (pH 9.0) containing 200 μg of PTH secondary antibody. React at 4℃ in the dark for 4 h, and dialyze in PBS for 24 h using a dialysis bag with a molecular cutoff of 10 kDa to remove unreacted MB, obtaining signal probes with different labeling ratios.

[0077] 2) Preparation of capture probes: Take 5 aliquots of 1.0 mg carboxyl-modified superparamagnetic iron oxide nanoparticles, wash twice with 1 mL of 0.1 M MES buffer (pH 6.0), and add 1 mL of freshly prepared activator solution (MES buffer containing 0.4 mg EDC and 0.6 mg NHS) to each aliquot. Activate at room temperature for 25 min, remove the supernatant by magnetic separation, add 50 μg of PTH primary antibody to each aliquot, react at 30 °C for 2 h, and block with 100 μL of 1% BSA solution for 1 h. Collect the product by magnetic separation, wash three times with PBS buffer (pH 7.4), and resuspend in 1 mL of PBS.

[0078] 3) Performance Testing: Take 100 μL of each captured probe prepared in each group and mix it with 20 μL of standard solution containing 1.0 μg / mL PTH. After incubation at 37℃ for 30 min, add 50 μL of the corresponding signal probe and continue incubation for another 30 min. Remove the supernatant by magnetic separation and wash three times with PBS. Add 200 μL of TMB substrate solution (0.5 mg / mL) and 100 μL of acetate buffer (pH 4.0), and incubate under visible light (wavelength 525 nm, light intensity 50 mW / cm²). 2 Immediately after irradiation for 10 minutes, the absorbance value at 652 nm was measured, and each group was measured three times.

[0079] Test results are as follows Figure 2As shown, the absorbance of the detection system at 652 nm reaches its maximum when the molar labeling ratio of antibody to MB is 1:6. Analysis of these results suggests that when the labeling molar ratio is below 1:6, the insufficient number of photosensitizer molecules coupled to each antibody results in limited reactive oxygen species, leading to a weak detection signal. Conversely, when the labeling molar ratio is above 1:6, although the number of photosensitizer molecules increases, excessively high labeling density may cause changes in antibody spatial conformation, obscure antigen binding sites, or lead to probe aggregation, thus decreasing the detection signal and increasing non-specific adsorption.

[0080] Therefore, considering factors such as detection performance, stability, and economy, the present invention preferably uses a labeling molar ratio of antibody to photosensitizer of 1:(4~8), with 1:6 being the optimal labeling ratio.

[0081] Example 4

[0082] This embodiment explores the performance differences of different types of photosensitizers when applied to the PTH detection probe of this invention.

[0083] The capture probes were prepared according to the method in Example 1. Five photosensitizers—Bengalis rose red, methylene blue, zinc phthalocyanine, hematoporphyrin, and riboflavin—were selected to prepare signal probes. During preparation, the amount of PTH secondary antibody was fixed at 100 μg, and the molar ratio of antibody to photosensitizer was controlled at 1:6. Coupling was performed using the sulfonyl-SMCC crosslinking method described in Example 2. After the reaction, each reaction solution was purified by dialyzing in PBS buffer (pH 7.4) at 4°C in the dark for 24 hours to obtain five different signal probe solutions for later use.

[0084] Five detection systems were prepared, each containing 100 μL of capture probe and 20 μL of 1.0 μg / mL PTH standard solution. The capture probe and PTH standard solution were mixed and incubated at 37°C for 30 min. Subsequently, 50 μL of each of the five different signal probe solutions was added to each of the five systems, and incubation was continued for 30 min to form complete sandwich immune complexes. After the immunoreaction, magnetic separation was performed, and the complexes were washed three times with PBS buffer containing 0.05% Tween-20. 200 μL of TMB substrate solution (0.5 mg / mL) and 100 μL of acetate buffer (pH 4.0) were added to each washed complex. All reaction systems were placed at a wavelength of 525 nm and an intensity of 70 mW / cm². 2 Irradiate the system under a visible light source for 20 minutes, and then immediately measure the absorbance at 652 nm using an ultraviolet absorption spectrometer. Each group is measured three times.

[0085] Experimental results are as follows Figure 3As shown, the detection system using Bengal rose red and methylene blue as photosensitizers exhibits significantly higher absorbance at 652 nm compared to the other three photosensitizer systems. Based on these experimental results, this embodiment confirms that in the PTH photosensitizing detection system constructed in this invention, Bengal rose red and methylene blue, as photosensitizers, significantly outperform other types of photosensitizers such as phthalocyanines, porphyrins, and natural photosensitizers in terms of detection performance, and are therefore preferred photosensitizers in this invention.

[0086] Example 5

[0087] This embodiment systematically explores the performance differences of different activators used for coupling photosensitizers and antibodies (i.e., preparing signal probes), and screens and verifies the optimal activation strategy through comparative experiments.

[0088] The capture probes were prepared according to the method in Example 1. Bengal rose red was selected as the model photosensitizer, the amount of PTH secondary antibody was fixed at 100 μg, and the molar ratio of antibody to photosensitizer was fixed at 1:6. Five different activation strategies were selected to prepare signal probes, including: EDC / NHS system, sulfonyl-SMCC heterobifunctional crosslinking agent, glutaraldehyde, genipin, and succinimide octanoate (DSS). Specific reaction conditions were consistent with those in Example 1.

[0089] Five detection systems were prepared, each containing 100 μL of capture probe and 20 μL of 1.0 μg / mL PTH standard solution. The capture probe and PTH standard solution were mixed and incubated at 37°C for 30 min. Subsequently, 50 μL of signal probe solutions prepared with the five different activators were added to each of the five systems, and incubation was continued for 30 min to form complete immune complexes. After the reaction, magnetic separation and washing were performed. 200 μL of TMB substrate solution and 100 μL of acetate buffer (pH 4.0) were added to each complex. All reaction systems were incubated at 525 nm wavelength and 70 mW / cm². 2 Irradiate the sample under a visible light source with high intensity for 20 min, and then immediately measure the absorbance at 652 nm.

[0090] Experimental results are as follows Figure 4 As shown, the performance of signal probes prepared using different activators varies significantly. The probe prepared using sulfonyl-SMCC exhibits the best performance, with the highest detection signal intensity. In contrast, the probe prepared using the EDC / NHS system has a slightly lower signal intensity. The probe prepared using the DSS method performs reasonably well, but its signal intensity and stability are slightly lower than the former two. Probes prepared using glutaraldehyde or genipin methods show significantly weaker detection signal intensity.

[0091] Based on the above experimental results, this embodiment confirms that sulfonyl-SMCC is the preferred activator for achieving efficient and controllable coupling between antibodies and photosensitizers when preparing the photosensitive signal probe of the present invention. This method can maximize the preservation of biological and photocatalytic activities, significantly improve the detection performance of the probe, and thus ensure the high sensitivity, high specificity, and good reproducibility of the entire detection system.

[0092] Example 6

[0093] This embodiment investigates the effect of light intensity on the detection performance of the PTH detection probe during the photosensitive oxidation reaction.

[0094] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 1, and the preparation conditions shall be kept consistent.

[0095] 2) Prepare 6 detection systems, each containing 100 μL of capture probe and 20 μL of PTH standard solution with a concentration of 1.0 μg / mL.

[0096] 3) Mix the capture probe with the PTH standard solution and incubate at 37°C for 30 min to allow the PTH antigen to fully bind to the primary antibody. Then add 60 μL of the signal probe and continue incubation at the same temperature for 30 min to form a complete sandwich immune complex. After the immune reaction, remove unbound material using magnetic separation technology and wash three times with PBS buffer containing 0.05% Tween-20.

[0097] 4) The six experimental groups were subjected to photosensitive oxidation reactions under different light intensities. The light intensity gradients were set to 10, 30, 50, 70, 90, and 110 mW / cm². 2 All groups were irradiated with visible light at a wavelength of 525 nm for a uniform duration of 10 minutes. The photoreaction was carried out at room temperature to ensure consistent environmental conditions.

[0098] After the photoreaction was completed, 200 μL of TMB substrate solution (0.5 mg / mL) and 100 μL of acetate buffer (pH 4.0) were immediately added to each system. The absorbance at 652 nm was then measured using a UV absorption spectrometer. Each group was measured three times.

[0099] Experimental results are as follows Figure 5 As shown, with the light intensity increasing from 10 mW / cm 2 Increased to 80 mW / cm 2 The absorbance value showed a significant upward trend. When the light intensity continued to increase to 150 mW / cm², the absorbance value increased significantly. 2At that time, the absorbance value actually decreased. This test result indicates that moderate light intensity can ensure that the photosensitizer can fully generate reactive oxygen species while avoiding background signal enhancement caused by excessive light.

[0100] Based on the above experimental results, this embodiment determines that the suitable light intensity range for the photosensitive oxidation reaction is 50~90mW / cm². 2 70 mW / cm 2 This is the optimal choice. Under these conditions, both detection sensitivity and a good signal-to-noise ratio can be maintained.

[0101] Example 7

[0102] This embodiment investigates the effect of illumination time on the detection performance of the PTH detection probe during the photosensitive oxidation reaction.

[0103] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 2, and the preparation conditions shall be kept consistent.

[0104] 2) Prepare 7 detection systems, each containing 100 μL of capture probe and 20 μL of PTH standard solution with a concentration of 1.0 μg / mL.

[0105] 3) Incubate the capture probe with PTH standard solution at 37°C for 30 min, then add 50 μL of signal probe and continue incubation for another 30 min. After the immune reaction is complete, remove unbound components using magnetic separation technology and wash three times with washing buffer.

[0106] 4) The fixed light intensity is 50 mW / cm². 2 The illumination time gradients were set to 5, 10, 15, 20, 25, and 30 min. All experimental groups were illuminated at a wavelength of 550 nm, while maintaining other reaction conditions consistently. The photoreaction was carried out at room temperature to ensure the stability of environmental parameters.

[0107] Immediately after the photoreaction was completed, 200 μL of TMB substrate solution and 100 μL of Tris-HCl buffer were added to each system, and the absorbance at 652 nm was measured using a UV absorption spectrometer.

[0108] Experimental results are as follows Figure 6 As shown, during the illumination period of 5-20 minutes, the absorbance value increases rapidly with the extension of illumination time. After the illumination time exceeds 20 minutes, the increase in absorbance value tends to slow down and enters a plateau period.

[0109] In this embodiment, the optimal illumination time for the photosensitive oxidation reaction is determined to be 20-25 minutes, with 20 minutes being the optimal choice. Under this condition, background interference can be effectively controlled while ensuring detection sensitivity, achieving rapid and accurate detection.

[0110] Example 8

[0111] This embodiment investigates the effect of temperature on the detection performance of the PTH detection probe of the present invention.

[0112] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 1, and the preparation conditions shall be kept consistent.

[0113] 2) Prepare 5 detection systems, each containing 200 μL of capture probe and 20 μL of PTH standard solution with a concentration of 1.0 μg / mL.

[0114] 3) Mix the capture probe with the PTH standard solution, and place the samples in different temperatures for immunoreaction. The temperature gradients were set at 20℃, 25℃, 30℃, 35℃, and 40℃, with a uniform reaction time of 30 min. During this process, a precision thermostatic shaker was used to ensure accurate temperature control, with fluctuations controlled within ±0.5℃. After the immunoreaction was complete, 70 μL of the signal probe was added, and the temperature was set to 37℃ for a further 30 min.

[0115] 4) After the immunoassay was completed, all samples underwent a standardized magnetic separation and washing process using pre-cooled washing buffer, maintaining consistent operating conditions. Subsequently, a photosensitive oxidation reaction was performed, with the temperature conditions consistent with the immunoassay temperature and the light intensity fixed at 50 mW / cm². 2 Irradiation time: 10 minutes.

[0116] Immediately after the photoreaction was completed, 200 μL of OPD substrate solution and 100 μL of Tris-HCl buffer were added to each system, and the absorbance value at 450 nm was measured using a UV absorption spectrometer.

[0117] Experimental results are as follows Figure 7 As shown, the detection signal was strongest at an immune response temperature of 30℃, significantly higher than other temperature groups. When the temperature was below 30℃, the immune response was incomplete, and the signal intensity decreased significantly; when the temperature was above 30℃, antibody activity began to be affected, non-specific binding increased, and the signal-to-noise ratio decreased.

[0118] Example 9

[0119] This embodiment investigates the effect of system pH on detection performance during PTH detection using the present invention.

[0120] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 1, and the preparation conditions shall be kept consistent.

[0121] 2) Prepare 5 detection systems, each containing 200 μL of capture probe and 20 μL of PTH standard solution with a concentration of 1.0 μg / mL.

[0122] 3) Prepare phosphate buffer systems with pH values ​​of 6.4, 6.8, 7.2, 7.6, 8.0, and 8.4, with a fixed concentration of 10 mM, ensuring consistent ionic strength at each pH. The immunoreaction temperature is uniformly controlled at 37℃, and the reaction time is 30 min. During this process, a precision pH meter is used to confirm the actual pH value of each buffer solution, with deviations controlled within ±0.03.

[0123] 4) During the immune reaction, all samples underwent the first stage of antigen-antibody binding under the corresponding pH conditions. After the reaction, the samples were uniformly adjusted to pH 7.2 PBS buffer for signal probe binding reaction, and the temperature was maintained at 37℃ for 30 min.

[0124] 5) After the immunoassay was completed, all samples underwent a standardized magnetic separation and washing process using a pH 7.2 buffer solution. Subsequently, a photosensitive oxidation reaction was performed, with conditions uniformly controlled in an acetate buffer system at pH 7.0 and a light intensity of 50 mW / cm². 2 Irradiation time: 10 minutes, temperature: 30℃.

[0125] Immediately after the photoreaction was completed, 100 μL of OPD substrate solution and 50 μL of Tris-HCl buffer were added to each system, and the absorbance at 450 nm was measured using a UV absorption spectrometer.

[0126] Experimental results are as follows Figure 8 As shown, the detection signal is most stable and has the highest intensity within the pH range of 7.2–7.6, significantly better than other pH conditions. When the pH is below 6.8, the antigen-binding activity of the antibody is significantly inhibited, the efficiency of immune complex formation decreases, resulting in a weakened signal intensity. Conversely, when the pH is above 7.8, the absorption intensity decreases.

[0127] Example 10

[0128] This embodiment presents a systematic comparative study on the performance of different chromogenic substrates in the PTH probe photosensitive oxidation detection system of this invention.

[0129] Five chromogenic substrates were selected for the study, including TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), DAB (3,3'-diaminobenzidine), and AEC (3-amino-9-ethylcarbazole).

[0130] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 2, and the preparation conditions shall be kept consistent.

[0131] 2) Prepare 5 detection systems, each containing 100 μL of capture probe and 20 μL of PTH standard solution with a concentration of 1.0 μg / mL.

[0132] 3) The capture probe and PTH standard solution were incubated at 37°C for 30 min, followed by the addition of 50 μL of signal probe and incubation for another 30 min. After the immunoreaction was complete, unbound material was removed by magnetic separation, and the sample was washed three times with PBS buffer containing 0.05% Tween-20. This process ensured that all experimental groups were subjected to completely identical conditions before the colorimetric reaction.

[0133] 4) During the colorimetric reaction stage, working solutions of the corresponding substrates were added to each experimental group. The TMB group received 200 μL of 0.5 mg / mL TMB solution and 100 μL of acetate buffer (pH 4.0); the ABTS group received 300 μL of 1 mg / mL ABTS solution; the OPD group received 300 μL of 0.4 mg / mL OPD solution; the DAB group received 300 μL of 0.5 mg / mL DAB solution; and the AEC group received 300 μL of 0.3 mg / mL AEC solution. All experimental groups were treated under a light intensity of 50 mW / cm². 2 The photosensitive oxidation reaction was carried out under standard conditions of 20 min of light exposure.

[0134] 5) Immediately after the reaction, the absorbance values ​​of each group were measured. The absorbance at 652 nm was measured for the TMB group, at 414 nm for the ABTS group, at 425 nm for the OPD group, at 450 nm for the DAB group, and at 490 nm for the AEC group. Simultaneously, a corresponding blank control was set up for each group to calculate the signal-to-noise ratio and detection background.

[0135] Experimental results are as follows Figure 9As shown, different chromogenic substrates exhibited significant differences in the detection system. Calculations revealed that the detection limits (LODs) for TMB substrate were 0.3 ng / mL, ABTS substrate 0.8 ng / mL, OPD substrate 1.2 ng / mL, and DAB and AEC substrates 2.5 ng / mL and 3.0 ng / mL, respectively. Linearity testing showed that TMB substrate exhibited good linearity in the range of 1–500 ng / mL, significantly superior to other substrates.

[0136] Based on the above experimental results, this invention determines TMB as the optimal chromogenic substrate for the photosensitive oxidation detection system. Its superior performance includes high sensitivity, a wide linear range, and a good signal-to-noise ratio, meeting the requirements for PTH detection in clinical samples. Furthermore, TMB substrates also possess advantages such as good stability and safety, making them suitable for widespread application under routine laboratory conditions.

[0137] Example 11

[0138] This embodiment uses a parathyroid hormone probe based on a photosensitive oxidation reaction to detect PTH. The specific steps are as follows:

[0139] 1) The preparation of the capture probe and signal probe shall be carried out in accordance with the method of Example 1, and the preparation conditions shall be kept consistent.

[0140] 2) Test the response of the detection probe to different concentrations of PTH:

[0141] S1. Place 100 μL of the capture probe into a 2 mL centrifuge tube, and add 20 μL of solutions containing 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 μg / mL PTH, respectively. Vortex to mix for 10 seconds. Place the mixture in a 37°C constant temperature shaker and incubate at 800 rpm for 30 min to allow PTH to fully bind to the capture probe.

[0142] S2. Add 50 μL of signal probe solution to the above reaction system and gently vortex to mix. Continue to incubate at 37°C with shaking at 600 rpm for 25 min to form a complete "magnetic bead-first antibody-PTH-second antibody-photosensitizer" sandwich complex.

[0143] S3. Place the reaction tube on the magnetic separator and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully aspirate the supernatant. Add 1 mL of pre-cooled washing buffer, gently resuspend the magnetic beads by pipetting, and perform magnetic separation again, then discard the supernatant. Repeat this washing step three times to ensure thorough removal of unbound impurities.

[0144] S4. Add 200 μL of TMB substrate working solution and 100 μL of acetate buffer (pH 7.0) to the washed magnetic bead-immune complex, and vortex to mix. Place the reaction tube under a halogen tungsten lamp at a wavelength of 525 nm and an induction wavelength of 50 mW / cm². 2 Irradiation under light intensity induces a photosensitive oxidation reaction.

[0145] S5. Dilute the reacted solution to 2 mL with acetate buffer and then test it in a UV absorption spectrometer.

[0146] Experimental results are as follows Figure 10 As shown, the solution color gradually deepens with increasing PTH concentration, the absorption peak of the test solution at 652 nm increases significantly, and there is a good linear relationship between the absorbance at 652 nm and the PTH concentration.

[0147] 2) Test the selectivity of the detection probe for PTH:

[0148] S10. Place 100 μL of the capture probe working solution into a 2 mL centrifuge tube. Add 20 μL of 1.0 μg / mL PTH, calcitonin, insulin, growth hormone, glucagon, thyroid-stimulating hormone, prolactin, hemoglobin, angiotensin, glutathione, albumin, and ascorbic acid solution. Vortex to mix for 10 seconds. Incubate the mixture in a 37°C shaker at 600 rpm for 30 minutes to allow PTH to fully bind to the capture probe.

[0149] S20. Add 50 μL of signal probe solution to each of the above reaction systems and mix gently. Continue incubation at 37°C with shaking at 600 rpm for 30 min.

[0150] S30. Place the reaction tube on the magnetic separator and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully aspirate the supernatant. Add 1 mL of pre-cooled washing buffer, gently pipette to resuspend the magnetic beads, perform magnetic separation again, and discard the supernatant. Repeat three times.

[0151] S40. Add 200 μL of TMB substrate working solution and 100 μL of acetate buffer (pH 7.0) to the washed magnetic bead-immune complex, and vortex to mix. Place the reaction tube under a halogen tungsten lamp at a wavelength of 525 nm and an induction wavelength of 50 mW / cm². 2 Irradiation under light intensity induces a photosensitive oxidation reaction.

[0152] S50. After diluting the reacted solution to 2 mL with acetate buffer, place it in a UV absorption spectrometer for testing.

[0153] Experimental results are as follows Figure 11As shown, calcitonin, insulin, growth hormone, glucagon, thyroid-stimulating hormone, prolactin, hemoglobin, angiotensin, glutathione, albumin, and ascorbic acid do not significantly alter the absorption of TMB at 652 nm, and the solution color does not change significantly. This result indicates that the detection probe provided by this invention has high specificity and excellent selectivity for detecting PTH.

[0154] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A parathyroid hormone detection probe, characterized in that, The probe comprises a capture probe and a signal probe; the capture probe is a magnetic microparticle with PTH-specific antibody 1 immobilized on its surface, and the signal probe is a PTH-specific antibody 2 with a photosensitizer labeled on its surface.

2. The parathyroid hormone detection probe according to claim 1, characterized in that, The PTH-specific antibody 1 is a C-terminal recognition antibody of PTH, and the PTH-specific antibody 2 is an N-terminal recognition antibody of PTH.

3. The parathyroid hormone detection probe according to claim 1, characterized in that, The surface of the magnetic microparticles is modified with functional groups, including at least one of carboxyl, amino, or streptavidin; the magnetic microparticles are selected from at least one of magnetic iron oxide microspheres, nickel magnetic microspheres, or cobalt magnetic microspheres. The photosensitizer is selected from at least one of phthalocyanine compounds, porphyrin compounds, phenothiazine dyes, xanthan dyes, or natural photosensitizers.

4. The method for preparing the parathyroid hormone detection probe according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Mix magnetic microparticles with PTH-specific antibody 1 in buffer solution in the presence of activator, collect the product by magnetic separation, and obtain the capture probe after blocking. S2. PTH-specific antibody 2 and photosensitizer were mixed and reacted in buffer in the presence of activator, and the signal probe was obtained after purification. In step S2, the molar ratio of the PTH-specific antibody 2 to the photosensitizer is 1:(4~7).

5. The preparation method according to claim 4, characterized in that, In step S1, the activator includes at least one of carbodiimide crosslinking agents, glutaraldehyde, and genipin; the buffer includes at least one of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer, and borate buffer, and the pH of the buffer is 5.0 to 9.0; the temperature of the mixing reaction is 4 to 37°C, and the time is 0.5 to 4 hours.

6. The preparation method according to claim 4, characterized in that, In step S2, the activator includes at least one of the following: carbodiimide / succinimide system, glutaraldehyde, and sulfonyl-SMCC; the buffer solution includes at least one of the following: carbonate buffer, phosphate buffer, and borate buffer; the pH value of the buffer solution is 7.0~9.5; the mixing reaction is carried out under light-protected conditions; the reaction temperature is 4~25℃; and the reaction time is 1~6h.

7. The application of the parathyroid hormone detection probe according to any one of claims 1 to 3 in detecting parathyroid hormone in a sample, characterized in that, The method for detecting parathyroid hormone in a sample includes the following steps: S10. Immune reaction: Mix the capture probe with the sample solution and incubate to allow PTH to bind to specific antibody 1; add the signal probe to form a sandwich immune complex. S20. Separation and washing: Perform magnetic separation and washing to remove unbound substances; S30, Photosensitive color development: Add a color development substrate to the washed complex and carry out a photosensitive oxidation reaction under visible light irradiation; S40. Detection and Analysis: Measure the absorbance value of the reaction system and calculate the concentration of PTH in the sample according to the standard curve.

8. The application according to claim 7, characterized in that, The chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3-amino-9-ethylcarbazole (AEC) or 3,3'-diaminobenzidine (DAB).

9. The application according to claim 7, characterized in that, In step S10, the incubation temperature is 28~32℃, and the incubation time is 10~60min; the buffer solution is phosphate buffer or Tris-HCl buffer, with a pH value of 7.2~7.

6. In step S30, the wavelength of the visible light emitted is 400~700 nm, and the light intensity is 50~90 mW / cm². 2 The irradiation time is 20-25 minutes; the temperature of the photosensitive oxidation reaction is 15-40℃.

10. A parathyroid hormone detection kit, characterized in that, It includes the parathyroid hormone detection probe according to any one of claims 1 to 3.

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