A highly sensitive detection method for free testosterone

Abstract

The present invention provides a highly sensitive detection method for free testosterone, which is used for quantitatively detecting the concentration of free testosterone in the human body. It belongs to the technical field of molecular detection and solves the technical problem of difficult quantitative detection of low-concentration free testosterone in the body. The technical solution is as follows: It includes the following steps: 1) Preparation of the detection substrate: Using a microplate as a carrier, through a photonic crystal template, an inverse opal structure photonic crystal is prepared as the detection substrate; 2) Detection of the target: The sample and the SERS nanolabel are sequentially added into the microholes, and by testing the Raman characteristic peak intensity of the label on the detection substrate, the concentration of free testosterone is calculated. The beneficial effect of the present invention is that the detection method of free testosterone in the present invention combines the advantages of the high specific surface area of the inverse opal structure photonic crystal and the highly sensitive detection of the SERS nanolabel, meeting the requirements for trace free testosterone detection.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly relates to a highly sensitive detection method for free testosterone. Background Art

[0002] Testosterone (T) is a steroid hormone and is the main component of male androgen. Testosterone can be secreted by the testicles of men or the ovaries of women. For men, 95% of testosterone is secreted by Leydig cells in the testicles and 5% is secreted by the adrenal cortex. Its release mode is pulsatile, with a circadian rhythm and is regulated seasonally. For women, 5-25% of testosterone is secreted by the ovaries, 5-25% comes from the adrenal glands, and 50-70% is converted from peripheral androstenedione. Since there are androgen receptors in the reproductive, urinary, skin, bone, muscle, hematopoiesis, cardiovascular, and nervous systems, etc., testosterone can act on these target organs. Testosterone has many effects on men, such as stimulating the development of male sexual organs, stimulating and maintaining secondary sexual characteristics, promoting and maintaining sexual function, promoting sperm production, and promoting protein synthesis, etc. In addition, testosterone also plays an important role in women, such as promoting protein synthesis in women, making women's muscles not flabby, enhancing memory, reducing fat, and increasing bone hardness, etc. If the testosterone content in the human body increases, its clinical significance is idiopathic male precocious puberty, familial male precocious puberty, adrenocortical hyperplasia, adrenocortical tumors (significantly increased in adenocarcinoma, often increased in adenoma), testicular tumors, testicular feminization, polycystic ovary syndrome, ovarian virilizing tumors, pineal tumors, idiopathic hirsutism, or hypothyroidism. If the testosterone content in the body decreases, the occurrence of diseases such as Down syndrome, uremia, myotonic dystrophy, liver insufficiency, cryptorchidism, primary or secondary hypogonadism, male testicular hypoplasia, eunuchoidism, hypothalamic or pituitary hypogonadism, etc. should be considered. Therefore, abnormal testosterone content will seriously affect the quality of life of men and women.

[0003] In medical tests, radioimmunoassay is commonly used to measure testosterone. It is a quantitative assay method for trace substances in liquid phase, which combines the high sensitivity and precision of radioactive isotopes with the specificity of antigen-antibody reaction. This method has the advantages of high sensitivity, strong specificity, good precision and small sample volume. However, it requires the introduction of radioactive isotope labeling, so it has relatively high requirements for the operation of testers and the instrument equipment. Approximately 54% of testosterone in the circulatory system binds to albumin to form a complex (Alb-T), but this binding is relatively loose; about 44% of testosterone binds to sex hormone-binding globulin to form a complex, and this binding is relatively firm. Only 2% of testosterone exists in the circulatory system in a free form, which is called free testosterone (FT). Among them, the testosterone with relatively firm binding has no biological activity; Alb-T is easily dissociated in the tissue capillary bed and releases testosterone again. Therefore, Alb-T and FT are collectively called bioavailable testosterone. Clinically, detecting free testosterone has more biological significance. Existing commercial free testosterone detection methods include liquid chromatography-tandem mass spectrometry (LC-MS / MS) and kit detection methods. LC-MS / MS requires professional testers, professional sites and complex sample pretreatment processes, and the instruments required for testing are relatively expensive; the prices of the kits required for the kit detection method are generally high, and corresponding reagents need to be added in multiple times; in addition, the detection limits of kit detection are all at the pg / mL level. Therefore, these testing methods all have certain limitations in actual clinical operations. Therefore, developing a free testosterone detection method with simple operation, low cost, rapidity and high sensitivity is an urgent need in clinical practice. Summary of the Invention

[0004] The purpose of the present invention is to provide a highly sensitive detection method for free testosterone. This method uses the inverse opal structure photonic crystal in a microplate as the detection substrate and SERS nanolabels as the detection labels to achieve the quantitative detection of free testosterone in the human body. The inverse opal photonic crystal structure and SERS nanolabels respectively have the advantages of high specific surface area and high Raman signal intensity, which provide the possibility for the quantitative detection of trace substances in the body, can simultaneously meet the clinical requirements of low cost, simple operation and rapidity, and have important significance for guiding the diagnosis of diseases caused by abnormal testosterone content and the prevention of related diseases.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions: A highly sensitive detection method for free testosterone, comprising the following steps:

[0006] 1) Preparation of the detection substrate: Using a microplate as a carrier, through a photonic crystal template, an inverse opal structure photonic crystal is prepared as the detection substrate;

[0007] 2) Target detection: The sample and SERS nanolabels are added to the micro-wells in sequence. By measuring the intensity of the Raman characteristic peaks of the labels on the detection substrate, the concentration of free testosterone is calculated.

[0008] As a further optimized scheme of a highly sensitive detection method for free testosterone provided by the present invention, the specific preparation steps of the detection substrate in step 1) are as follows:

[0009] ① By means of vertical deposition or electrostatic self-assembly, a monodisperse colloidal particle solution is placed in the micro-wells of a microplate to prepare an opal-structured photonic crystal template.

[0010] ② Prepare a hydrogel prepolymer solution, pour it into the photonic crystal template in the micro-well, and heat or cure it by ultraviolet light.

[0011] ③ By adding an etching solution to the micro-well to remove the template particles by etching; or removing the template by heating to obtain an inverse opal-structured photonic crystal.

[0012] ④ Using magnetron sputtering, evaporation coating, electroless plating or atomic layer deposition methods, uniformly deposit a metal or metal oxide on the surface of the inverse opal-structured photonic crystal to form a nanoscale metal or metal oxide coating, and obtain an inverse opal-structured photonic crystal with a metal or metal oxide coating.

[0013] ⑤ By means of electrostatic adsorption or covalent bonding, the substance for capturing the target is modified on the surface of the inverse opal-structured photonic crystal with a coating to finally obtain the detection substrate.

[0014] As a further optimized scheme of a highly sensitive detection method for free testosterone provided by the present invention, the monodisperse colloidal particles in step ① are polymethyl methacrylate, porous silicon, polystyrene, polyethylene, silica, calcium carbonate or polyethyl acrylate;

[0015] The diameter of the monodisperse colloidal particles in step ① is 10 nm to 10 μm; the concentration of the monodisperse colloidal particle solution is 5 to 90 wt%.

[0016] The material of the microplate in step ① is polystyrene, polypropylene, stainless steel, polyethylene, plexiglass, polyvinyl chloride, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polymethacrylate, polytetrafluoroethylene or polyamide;

[0017] The number of holes in the microplate in step ① is 4, 6, 8, 12, 24, 48, 96, 384 or 1536 holes.

[0018] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the hydrogel prepolymer solution in step ② is nitrocellulose, cellulose acetate, alginic acid, hyaluronic acid, chitosan, collagen, poly-L-lysine, poly-L-glutamic acid, acrylic acid, polyvinylidene fluoride, polyacrylic acid, polymethacrylic acid, polyacrylamide, poly-N-substituted acrylamide or agarose;

[0019] The concentration of the hydrogel prepolymer solution in step ② is 1-60%; the heating and curing temperature is 30-70 °C, and the time is 5-30 min; the ultraviolet light wavelength is 200-450 nm, the power is 5-50 W, and the time is 10 s-20 min.

[0020] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the etching solution in step ③ is dimethylformamide, hydrofluoric acid or tetrahydrofuran;

[0021] The heating temperature in step ③ is 200-600 °C.

[0022] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the metal in step ④ is gold, silver, copper or platinum;

[0023] The metal oxide in step ④ is titanium dioxide, zinc oxide or molybdenum dioxide;

[0024] The thickness of the metal or metal oxide coating in step ④ is 1-100 nm.

[0025] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the substance for capturing the target in step ⑤ is a capture antibody, aptamer or ligand corresponding to free testosterone.

[0026] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the preparation steps of the SERS nanolabels in step 2) are as follows:

[0027] ① Fix the Raman dye molecules on the surface of the nanoparticles by electrostatic adsorption or covalent bonding;

[0028] ② Modify the detection antibody, aptamer or ligand corresponding to free testosterone on the surface of the Raman dye-functionalized nanoparticles by electrostatic adsorption or covalent bonding; or use the Raman dye-functionalized nanoparticles as the core, synthesize a shell layer on its surface to form core-shell structured nanoparticles, and then modify the detection antibody, aptamer or ligand corresponding to free testosterone on the surface of the nanoparticles by electrostatic adsorption or covalent bonding to finally form SERS nanolabels.

[0029] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the Raman dye molecule in step ① is 5,5'-dithiobis(2-nitrobenzoic acid), p-aminothiophenol, water-soluble 3H-indocyanine-type bioluminescent labeling dye, crystal violet, methylene blue, rhodamine 6G, 3-aminothiophenol, 4-aminothiophenol, 4-nitrobenzenethiol, 2-naphthalenethiol, p-fluorothiophenol, Nile blue A, 4-mercaptobenzonitrile, toluidine blue, p-mercaptothiophenol, 4-mercaptobenzoic acid, rhodamine B isothiocyanate, malachite green isothiocyanate, 4,4'-bipyridine, 4-hydroxythiophenol, methyl blue, p-mercaptoaniline, 4-chlorothiophenol, 1,4-benzenedithiol, p-aminothiophenol or 4-mercaptopyridine;

[0030] The nanoparticles in step ① are nanospheres, nanorods, nanostars, nanosheets, nanocubes or nanoflowers;

[0031] The material of the nanoparticles in step ① is gold, silver, copper, platinum, carbon, titanium dioxide, zinc oxide, molybdenum dioxide or silicon dioxide; the size of the nanoparticles is 1-400 nm.

[0032] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the core-shell materials of the core-shell structured nanoparticles in step ② are all gold, silver, copper, platinum, carbon, titanium dioxide, zinc oxide, molybdenum dioxide or silicon dioxide; the core and shell can be composed of the same material or different materials;

[0033] The size of the core of the core-shell structured nanoparticles in step ② is 1-300 nm, and the thickness of the shell is 1-50 nm.

[0034] As a further optimized solution for the highly sensitive detection method of free testosterone provided by the present invention, the method for testing the signal of the SERS nanotag on the detection substrate in step 2) is to collect it by a confocal micro-Raman spectrometer, a portable Raman spectrometer or a handheld Raman spectrometer.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. The carrier of the free testosterone detection system of the present invention is a microplate, which can achieve high-throughput and rapid target detection.

[0037] 2. The free testosterone detection method of the present invention combines the advantages of the high specific surface area of the inverse opal structure photonic crystal and the highly sensitive detection of the SERS nanotag, meeting the requirement for trace free testosterone detection.

[0038] 3. The inverse opal-structured photonic crystal in the present invention has a light modulation effect, which can further enhance the signal of the SERS nanolabel, facilitating the detection of low-abundance substances in vivo.

[0039] 4. The system constructed by the method of the present invention includes a detection substrate with an inverse opal-structured photonic crystal and an SERS nanolabel, both of which have relatively low production costs, thus effectively reducing the detection cost and facilitating operation.

[0040] 5. Compared with the traditional liquid chromatography-tandem mass spectrometry and kit detection methods, the free testosterone detection method of the present invention has more performance advantages, price advantages, and operation advantages, and is of great significance for guiding the diagnosis of diseases caused by abnormal testosterone levels and the prevention of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram for preparing the detection substrate in the present invention.

[0042] Figure 2 It is a schematic diagram of the free testosterone detection process in the present invention.

[0043] Among them, the reference numerals are: 1, microplate; 2, self-assembly of monodisperse colloidal particles into a photonic crystal template in the micropores; 3, perfusion of the hydrogel prepolymer solution into the photonic crystal template; 4, cured hydrogel-photonic crystal structure; 5, removal of the template by heating or etching; 6, inverse opal-structured photonic crystal; 7, formation of a metal or metal oxide coating by magnetron sputtering, evaporation plating, electroless plating, or atomic layer deposition; 8, inverse opal-structured photonic crystal with a metal or metal oxide coating;

[0044] 9, inverse opal-structured photonic crystal modified with a capture substance corresponding to the target; 10, addition of the sample; 11, sample solution; 12, inverse opal-structured photonic crystal bound to the target; 13, addition of the SERS nanolabel; 14, SERS nanolabel; 15, inverse opal-structured photonic crystal bound to the target and the SERS nanolabel; 15, laser; 16, Raman spectrum. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. Of course, the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] Example 1

[0047] A highly sensitive detection method for free testosterone provided by the present invention is applied to the detection of free testosterone, including the following steps:

[0048] 1) Preparation of the detection substrate:

[0049] ① By means of vertical deposition, a 5 wt% monodisperse colloidal silica nanoparticle solution was placed in the micropores of a polystyrene 4-well plate to prepare an opal-structured photonic crystal template, where the diameter of the silica nanoparticles was 10 nm;

[0050] ② A 1% polyacrylamide hydrogel prepolymer solution was prepared and poured into the photonic crystal template in the micropores, and irradiated under ultraviolet light with a wavelength of 200 nm and a power of 5 W for 20 min to cure it;

[0051] ③ Hydrofluoric acid was added to the micropores to etch away the photonic crystal template, obtaining an inverse opal-structured photonic crystal;

[0052] ④ By means of magnetron sputtering, a 1-nm-thick gold layer was uniformly deposited on the surface of the inverse opal-structured photonic crystal to obtain an inverse opal-structured photonic crystal with a gold coating;

[0053] ⑤ By means of electrostatic adsorption, the capture antibody corresponding to free testosterone was modified on the surface of the inverse opal-structured photonic crystal with a gold coating, finally obtaining the detection substrate.

[0054] 2) Target detection: The sample and the SERS nanolabel were sequentially added to the micropores, and the Raman spectrum of the label on the detection substrate was collected by a confocal micro-Raman spectrometer. By calculating the intensity of the Raman characteristic peak, the concentration of free testosterone was obtained.

[0055] Among them, the preparation steps of the SERS nanolabel are as follows:

[0056] ① The Raman dye molecule 5,5'-dithiobis(2-nitrobenzoic acid) was fixed to the surface of gold nanospheres with a diameter of 1 nm by means of covalent linkage;

[0057] ② By means of electrostatic adsorption, the detection antibody corresponding to free testosterone was modified on the surface of the 5,5'-dithiobis(2-nitrobenzoic acid)-functionalized gold nanospheres, finally forming the SERS nanolabel.

[0058] Example 2

[0059] A highly sensitive detection method for free testosterone provided by the present invention is applied to the detection of free testosterone, including the following steps:

[0060] 1) Detection substrate preparation:

[0061] ① By means of electrostatic self-assembly, a 90 wt% monodisperse colloidal polystyrene particle solution was placed in the micropores of a polypropylene 24-well plate to prepare an opal-structured photonic crystal template, where the diameter of the polystyrene particles was 10 μm;

[0062] ② Prepare a cellulose nitrate hydrogel prepolymer solution with a concentration of 60%, pour it into the photonic crystal template in the micropores, and heat it at 30 °C for 30 min to cure it;

[0063] ③ Add tetrahydrofuran to the micropores to corrode away the photonic crystal template and obtain an inverse opal structure photonic crystal;

[0064] ④ Use the atomic layer deposition method to uniformly deposit a titanium dioxide layer with a thickness of 100 nm on the surface of the inverse opal structure photonic crystal to obtain an inverse opal structure photonic crystal with a titanium dioxide coating;

[0065] ⑤ Modify the aptamer corresponding to free testosterone onto the surface of the inverse opal structure photonic crystal with a titanium dioxide coating by covalent bonding to finally obtain the detection substrate.

[0066] 2) Target detection: Add the sample and the SERS nanolabel to the micropores in sequence, collect the Raman spectrum of the label on the detection substrate by a handheld Raman spectrometer, and calculate the concentration of free testosterone by calculating the intensity of the Raman characteristic peak.

[0067] Among them, the preparation steps of the SERS nanolabel are as follows:

[0068] ① Fix the Raman dye molecule Nile Blue A on the surface of silver nanospheres with a diameter of 30 nm by electrostatic adsorption;

[0069] ② Synthesize a 1-nm-thick gold shell on the surface of the Nile Blue A-functionalized silver nanospheres to form core-shell nanoparticles; then modify the aptamer corresponding to free testosterone on the surface of the core-shell nanoparticles by covalent bonding to finally form the SERS nanolabel.

[0070] Example 3

[0071] A highly sensitive detection method for free testosterone provided by the present invention is applied to the detection of free testosterone, including the following steps:

[0072] 1) Detection substrate preparation:

[0073] ① By vertical deposition, place a 50 wt% monodisperse colloidal polymethyl methacrylate nanoparticle solution in the micropores of a stainless steel 1536-well plate to prepare an opal structure photonic crystal template, where the diameter of the polymethyl methacrylate nanoparticles is 20 nm;

[0074] ②Prepare a polyvinylidene fluoride hydrogel prepolymer solution with a concentration of 30%, pour it into the photonic crystal template in the micropores, and heat it at 70 °C for 5 min to cure it;

[0075] ③Add dimethylformamide to the micropores to corrode away the photonic crystal template and obtain an inverse opal structure photonic crystal;

[0076] ④Using the method of evaporation deposition, uniformly deposit a silver layer with a thickness of 50 nm on the surface of the inverse opal structure photonic crystal to obtain an inverse opal structure photonic crystal with a silver coating;

[0077] ⑤By means of electrostatic adsorption, modify the ligand corresponding to free testosterone on the surface of the inverse opal structure photonic crystal with a silver coating, and finally obtain the detection substrate.

[0078] 2) Target detection: Sequentially add the sample and the SERS nanolabel to the micropores, collect the Raman spectrum of the label on the detection substrate by a portable Raman spectrometer, and calculate the concentration of free testosterone by calculating the intensity of the Raman characteristic peak.

[0079] Among them, the preparation steps of the SERS nanolabel are as follows:

[0080] ①Fix the Raman dye molecule 4-mercaptobenzoic acid on the surface of the silica nanosphere by covalent bonding, and the diameter of the silica nanosphere is 300 nm;

[0081] ②Synthesize a silver shell with a thickness of 50 nm on the surface of the 4-mercaptobenzoic acid-functionalized silica nanosphere to form core-shell nanoparticles; then modify the ligand corresponding to free testosterone on the surface of the core-shell nanoparticles by electrostatic adsorption to finally form the SERS nanolabel.

[0082] Example 4

[0083] A highly sensitive detection method for free testosterone provided by the present invention is applied to the detection of free testosterone, including the following steps:

[0084] 1) Detection substrate preparation:

[0085] ①By means of electrostatic self-assembly, place a 30 wt% monodisperse colloidal calcium carbonate nanoparticle solution in the micropores of a polytetrafluoroethylene 96-well plate to prepare an opal structure photonic crystal template, wherein the diameter of the calcium carbonate nanoparticles is 100 nm;

[0086] ②Prepare a poly-N-polyacrylamide hydrogel prepolymer solution with a concentration of 50%, pour it into the photonic crystal template in the micropores, and irradiate it under an ultraviolet lamp with a wavelength of 450 nm and a power of 50 W for 10 s to cure it;

[0087] ③ Add hydrofluoric acid into the micropores to etch away the photonic crystal template, obtaining a photonic crystal with an inverse opal structure;

[0088] ④ Adopt the electroless plating method to uniformly deposit a gold layer with a thickness of 60 nm on the surface of the photonic crystal with an inverse opal structure, obtaining a photonic crystal with an inverse opal structure having a gold coating;

[0089] ⑤ Modify the capture antibody corresponding to free testosterone onto the surface of the photonic crystal with an inverse opal structure having a gold coating by means of covalent linkage, finally obtaining the detection substrate.

[0090] 2) Target detection: Sequentially add the sample and the SERS nanolabel into the micropores, collect the Raman spectrum of the label on the detection substrate through a confocal micro-Raman spectrometer, and calculate the concentration of free testosterone by calculating the intensity of the Raman characteristic peaks.

[0091] Among them, the preparation steps of the SERS nanolabel are as follows:

[0092] ① Fix the Raman dye molecule methylene blue onto the surface of the gold nanorods by electrostatic adsorption, and the size of the gold nanorods is 400 nm;

[0093] ② Modify the detection antibody corresponding to free testosterone on the surface of the methylene blue-functionalized gold nanorods by electrostatic adsorption method, finally forming the SERS nanolabel.

[0094] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any partial changes to the formulations and processes based on this should be within the protection scope of the present invention.

Claims

1. A highly sensitive detection method for free testosterone, characterized in that, It includes the following steps: 1) Preparation of the detection substrate: Using a microplate as a carrier, through a photonic crystal template, an inverse opal structure photonic crystal is prepared as the detection substrate; 2) Target detection: The sample and SERS nanolabels are sequentially added into the micro-wells, and by testing the Raman characteristic peak intensity of the labels on the detection substrate, the concentration of free testosterone is calculated; The preparation steps of the SERS nanolabels in step 2) are as follows: ① Fix Raman dye molecules on the surface of nanoparticles by electrostatic adsorption or covalent bonding; ② Modify the detection antibody, aptamer or ligand corresponding to free testosterone on the surface of the Raman dye-functionalized nanoparticles by electrostatic adsorption or covalent bonding; or use the Raman dye-functionalized nanoparticles as the core, synthesize a shell layer on its surface to form core-shell structured nanoparticles, and then modify the detection antibody, aptamer or ligand corresponding to free testosterone on the surface of the nanoparticles by electrostatic adsorption or covalent bonding to finally form SERS nanolabels; The Raman dye molecules in step ① are 5,5'-dithiobis(2-nitrobenzoic acid), p-aminothiophenol, water-soluble 3H-indocyanine-type bioluminescent labeling dyes, crystal violet, methylene blue, rhodamine 6G, 3-aminothiophenol, 4-aminothiophenol, 4-nitrobenzenethiol, 2-naphthalenethiol, p-fluorobenzenethiol, Nile blue A, 4-mercaptobenzonitrile, toluidine blue, p-mercaptothiophenol, 4-mercaptobenzoic acid, rhodamine B isothiocyanate, malachite green isothiocyanate, 4,4'-bipyridine, 4-hydroxythiophenol, methyl blue, p-mercaptoaniline, 4-chlorobenzenethiol, 1,4-benzenedithiol, p-aminothiophenol or 4-mercaptopyridine; The nanoparticles in step ① are nanospheres, nanorods, nanostars, nanosheets, nanocubes or nanoflowers; The material of the nanoparticles in step ① is gold, silver, copper, platinum, carbon, titanium dioxide, zinc oxide, molybdenum dioxide or silicon dioxide; the size of the nanoparticles is 1 - 400 nm.

2. The highly sensitive detection method of free testosterone according to claim 1, characterized in that, The specific preparation steps of the detection substrate in step 1) are as follows: ① By vertical deposition or electrostatic self-assembly, place a monodisperse colloidal particle solution in the micro-wells of a microplate to prepare an opal structure photonic crystal template; ② Prepare a hydrogel prepolymer solution, pour it into the photonic crystal template in the micro-wells, and heat or cure it by ultraviolet light; ③ Remove the template particles by corrosion by adding a corrosive solution to the micro-wells; or remove the template by heating to obtain an inverse opal structure photonic crystal; ④ Use magnetron sputtering, evaporation coating, electroless plating or atomic layer deposition methods to uniformly deposit metal or metal oxide on the surface of the inverse opal structure photonic crystal to form a nanoscale metal or metal oxide coating, and obtain an inverse opal structure photonic crystal with a metal or metal oxide coating; ⑤ Modify the substance that captures the target on the surface of the inverse opal structure photonic crystal with a coating by electrostatic adsorption or covalent bonding to finally obtain the detection substrate.

3. The highly sensitive detection method of free testosterone according to claim 2, characterized in that, The monodisperse colloidal particles in step ① are polymethyl methacrylate, porous silicon, polystyrene, polyethylene, silicon dioxide, calcium carbonate or polyethyl acrylate; The diameter of the monodisperse colloidal particles in step ① is 10 nm to 10 μm; the concentration of the monodisperse colloidal particle solution is 5 to 90 wt%; The material of the microplate in step ① is polystyrene, polypropylene, stainless steel, polyethylene, plexiglass, polyvinyl chloride, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polymethacrylate, polytetrafluoroethylene or polyamide; The number of holes in the microplate in step ① is 4, 6, 8, 12, 24, 48, 96, 384 or 1536 holes.

4. The highly sensitive detection method for free testosterone according to claim 2, wherein The hydrogel prepolymer solution in step ② is nitrocellulose, cellulose acetate, alginic acid, hyaluronic acid, chitosan, collagen, poly-L-lysine, poly-L-glutamic acid, acrylic acid, polyvinylidene fluoride, polyacrylic acid, polymethacrylic acid, polyacrylamide, poly-N-substituted acrylamide or agarose; The concentration of the hydrogel prepolymer solution in step ② is 1 to 60%; the temperature for heat curing is 30 to 70 °C, and the time is 5 - 30 min; the ultraviolet light wavelength is 200 to 450 nm, the power is 5 to 50 W, and the time is 10 s to 20 min.

5. The highly sensitive detection method for free testosterone according to claim 2, characterized in that, The etching solution in step ③ is dimethylformamide, hydrofluoric acid or tetrahydrofuran; The heating temperature in step ③ is 200 to 600 °C.

6. The highly sensitive detection method for free testosterone according to claim 2, wherein The metal in step ④ is gold, silver, copper or platinum; The metal oxide in step ④ is titanium dioxide, zinc oxide or molybdenum dioxide; The thickness of the metal or metal oxide coating in step ④ is 1 to 100 nm.

7. A highly sensitive detection method for free testosterone according to claim 2, characterized in that, The substance for capturing the target in step ⑤ is a capture antibody, aptamer or ligand corresponding to free testosterone.

8. The highly sensitive detection method for free testosterone according to claim 1, characterized in that, The core and shell materials of the core-shell structured nanoparticles in step ② are one or several of gold, silver, copper, platinum, carbon, titanium dioxide, zinc oxide, molybdenum dioxide or silicon dioxide; The size of the core of the core-shell structured nanoparticles in step ② is 1 to 300 nm, and the thickness of the shell is 1 to 50 nm.

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