Free testosterone detection kit based on SERS (Surface Enhanced Raman Scattering) and preparation method and detection method thereof

Abstract

The invention discloses a free testosterone detection kit based on SERS (Surface Enhanced Raman Scattering) as well as a preparation method and a detection method of the kit. The kit consists of an SERS nano-label based on a gold core and a gold shell and functionalized magnetic beads, the structure of the SERS nano-label based on the gold core and the gold shell comprises the gold core, a Raman dye modified on the surface of the gold core, the gold shell coated on the surface, a sulfydryl polyethylene glycol carboxyl (HS-PEG-COOH) and sulfydryl polyethylene glycol (PEG-SH) protective layer outside the gold shell, and a free testosterone antigen. The method is simple and convenient to operate, can be used for analyzing trace free testosterone in a sample, has the advantages in the aspects of simplicity and convenience in operation, detection period, analysis cost and the like, and has important significance on rapid diagnosis of diseases caused by abnormal content of free testosterone.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical analysis and detection, and particularly relates to a free testosterone detection kit based on SERS, as well as a preparation method and a detection method of such a kit. Background Art

[0002] Testosterone (T), as a steroid hormone, widely exists in the bodies of both men and women and plays important roles, such as stimulating the development of male sexual organs, stimulating and maintaining secondary sexual characteristics, promoting and maintaining sexual function, promoting sperm production, promoting protein synthesis, enhancing muscle strength, improving memory, reducing fat, and increasing bone hardness. In the human circulatory system, approximately 54% of testosterone binds to albumin to form a relatively loosely structured complex (Alb-T); approximately 44% of testosterone binds to sex hormone-binding globulin to form a relatively firmly structured complex; only 2% of testosterone exists in the circulatory system in a free form, which is called free testosterone (FT). Among them, the testosterone with a 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 referred to as bioavailable testosterone. Clinically, detecting free testosterone can directly reflect the progression of related diseases and has more physiological significance.

[0003] The existing methods for detecting free testosterone mainly include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Among them, the analysis sensitivity of ELISA is generally not high and cannot meet the requirements for detecting trace free testosterone in clinical practice; RIA needs to use radioactive isotopes as detection labels, there are certain radiation safety hazards, and its operation is relatively complex; CLIA is easily affected by other matrices in the sample and its sensitivity is not high either; although HPLC-MS / MS has high detection sensitivity, its equipment is expensive, the operation is complex, it requires professional technical personnel, and the detection cycle is long, and it is usually used in clinical testing laboratories or high-precision diagnoses, which limits the further expansion of its application scope.

[0004] In recent decades, due to its advantages such as high sensitivity, no need for complex sample pretreatment, non-destructive sample detection, and simple operation, surface-enhanced Raman scattering (SERS) spectroscopy has been widely used in many fields such as analytical chemistry, material analysis, food safety, biomedicine, and customs inspection. Magnetic beads have unique advantages in sample separation due to their easy separation characteristics demonstrated by their own magnetic properties. Therefore, the present invention discloses a SERS-based free testosterone detection kit and detection method, which can simultaneously meet clinical requirements such as low cost, simple operation, and rapid analysis, and is of great significance for the rapid diagnosis of diseases caused by abnormal free testosterone content and the prevention of related diseases.

[0005] Bing Liu et al. published a paper on SERS-based free testosterone detection in *Colloids and Surfaces B: Biointerfaces* in June 2022. It involves using commercially purchased carboxyl-functionalized magnetic beads to connect anti-free testosterone antibodies and using them as a detection carrier; preparing silver-core gold-shell nanoparticles as an enhancement substrate for SERS nanolabels, modifying Nile blue A at the core-shell gap as a signal label, and finally connecting free testosterone antigen to the outermost layer of the particles to form the final SERS nanolabel. When detecting samples, the SERS nanolabel and the sample are added to the magnetic bead carrier solution at the same time, and the content of free testosterone in the sample is quantitatively calculated by collecting the Raman spectrum on the magnetic beads after the reaction. However, this detection system has certain limitations, mainly manifested in the following aspects: First, the magnetic beads used are commercially purchased, especially carboxyl-functionalized magnetic beads, which are not cheap in price; Second, the enhancement substrate of the SERS nanolabel, silver-core gold-shell nanoparticles, is difficult to synthesize because it is not easy to synthesize silver nanoparticles with uniform particle size; Third, the connection method between free testosterone antigen and silver-core gold-shell nanoparticles is electrostatic adsorption, which is not as stable as covalent connection; In addition, due to the easy oxidation of silver materials in the external environment, the stability of silver-core gold-shell nanoparticles is lower than that of gold-core gold-shell nanoparticles; and only free testosterone antigen and BSA are modified on the outer layer of the silver-core gold-shell, and due to the existence of steric hindrance, the non-dense coating layer also reduces the stability of silver-core gold-shell nanoparticles. Therefore, developing a free testosterone detection system with low raw material cost and high stability is beneficial to its further application in clinical diagnosis. Summary of the Invention

[0006] The purpose of the present invention is to provide a SERS-based free testosterone detection kit.

[0007] The second purpose of the present invention is to provide a preparation method of the above-mentioned kit.

[0008] The third purpose of the present invention is to provide a detection method of the above-mentioned kit.

[0009] To this end, the present invention adopts the following technical solution: a free testosterone detection kit based on SERS, characterized in that it consists of a SERS nanolabel based on a gold core-gold shell and functionalized magnetic beads. The structure of the SERS nanolabel based on the gold core-gold shell includes a gold core, a Raman dye modified on the surface of the gold core, a gold shell coated on the surface, a mercapto poly(ethylene glycol) carboxyl (HS-PEG-COOH) and mercapto poly(ethylene glycol) (PEG-SH) protective layer outside the gold shell, and a free testosterone antigen.

[0010] Further, the gold core is a spherical particle with a particle size of 35 - 40 nm.

[0011] Further, the Raman dye is a dye with Raman activity, selected from one of Nile blue A (NBA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (4-MBA), 4-mercaptoaniline (4-MA), 6-mercaptopurine (6-MP), mercaptoacetic acid (MMA), and mercaptopropionic acid (MPA).

[0012] Further, the thickness of the gold shell is 3 nm.

[0013] Further, the structure of the functionalized magnetic beads includes magnetic nanoparticles and a monoclonal antibody against free testosterone modified on the surface.

[0014] Further, the magnetic nanoparticles are spherical particles made of ferrite (magnetite), with a particle size of 300 nm.

[0015] The present invention also adopts the following technical solution: A preparation method of a free testosterone detection kit based on SERS, including the steps of preparing SERS nanolabels, preparing functionalized magnetic beads, and the step of combining and packaging them; wherein: The preparation of SERS nanolabels specifically includes the following steps: S1, Take a certain amount of chloroauric acid (HAuCl4) solution in a three-necked round-bottom flask, and dilute it with water to 50 mL. The three-necked round-bottom flask is equipped with a condensation reflux device, and then the diluted solution is heated to boiling.

[0016] S2, Under vigorous stirring, quickly add a reducing agent to the solution in step S1, keep the solution boiling for 5 minutes, and then let it cool to room temperature.

[0017] S3. Take a certain amount of the solution from step S2, dilute it (10 mL), place it in a three-necked round-bottom flask, stir vigorously, and simultaneously inject HAuCl4 solution and a mixed solution of ascorbic acid and trisodium citrate respectively, heat the mixed solution to boiling, and maintain it at this temperature for about 30 minutes, then cool to room temperature.

[0018] S4. Add cetyltrimethylammonium chloride (CTAC) to the mixed solution from step S3, mix well and then centrifuge. Then, under ultrasonic conditions, dropwise add the Raman dye solution into the particle solution. After incubating for 20 minutes, let it stand for 3 hours. Finally, centrifuge the reaction solution, and resuspend the precipitate in 10 mL of ultrapure water.

[0019] S5. Inject HAuCl4 and hydroxylamine hydrochloride into the solution from step S4 simultaneously, and under vigorous stirring, let the mixture react for 45 minutes. Then centrifuge, and resuspend the precipitate in ultrapure water.

[0020] S6. First add HS-PEG-COOH solution to the solution from step S5, stir and react for 20 minutes, then add PEG-SH solution, stir evenly, incubate for 4 hours, and finally centrifuge, and resuspend the precipitate in ultrapure water.

[0021] S7. Add EDC and NHS solutions to the solution from step S6 successively, stir and react for 20 minutes, then centrifuge, and resuspend the precipitate in 2 mL of PBS.

[0022] S8. Add free testosterone antigen to the solution from step S7, react at 25 °C for 4 hours, then add BSA and react for 2 hours. Centrifuge, disperse the precipitate in 0.5 mL of PBS solution, and store it refrigerated.

[0023] The preparation of functionalized magnetic beads specifically includes the following steps: L1. Dissolve ferric chloride in the reducing agent ethylene glycol, add sodium acetate and polyethylene glycol to the solution under vigorous stirring, and then stir at room temperature for 30 minutes.

[0024] L2. Transfer the mixed solution into a stainless-steel autoclave reactor with a Teflon coating, seal it, control the temperature at 200 °C, and react for 10 hours.

[0025] L3. After the solution cools, a precipitate is formed. Separate the magnetic precipitate and the solution with a magnet. Wash the magnetic precipitate 3 times with ethanol, then vacuum dry it at 65 °C for 6 hours, and store it refrigerated.

[0026] L4. Disperse the magnetic particles in ultrapure water, sonicate, add acetic acid, adjust the pH value of the reaction solution to 8 with ammonia water, react at 50 °C for 5 hours, and after separation, resuspend it in 2 mL of ultrapure water.

[0027] L5. Add EDC and NHS solutions to the L4 magnetic bead solution successively. After stirring for 20 minutes, perform magnetic separation, and resuspend the magnetic beads in 2 mL of PBS.

[0028] L6. Add the anti-free testosterone monoclonal antibody to the solution in step L5, react at 25 °C for 4 hours, then add BSA and react for 2 hours. Centrifuge, disperse the precipitate in 0.5 mL of PBS solution, and store it refrigerated.

[0029] Further, in the step S1, the concentration of the HAuCl4 solution is 0.2% (w / v), and the volume is 2.5 mL.

[0030] Further, in the step S2, the reducing agent is a 1% (w / v) sodium citrate solution containing 0.05% (w / v) citric acid, and the volume of the reducing agent is 2 mL.

[0031] Further, in the step S3, take 6 mL of the solution in step S2, the concentration of the HAuCl4 solution is 0.2% (w / v), and the volume is 2 mL, dilute it with water to 10 mL; the concentration of ascorbic acid is 0.1% (w / v), and the volume is 0.5 mL, the concentration of trisodium citrate is 0.1% (w / v), and the volume is 0.25 mL. After mixing the ascorbic acid and trisodium citrate solutions, dilute it with water to 10 mL. The injection time is 45 minutes.

[0032] Further, in the step S4, the concentration of CTAC is 0.1 M, and the volume is 10 mL; the concentration of the Raman dye is 2 mM, and the volume is 0.45 mL.

[0033] Further, in the step S5, the concentration of HAuCl4 is 0.465 mM, and the concentration of hydroxylamine hydrochloride is 6.25 mM; the injection speed is 2 mL / min, and the injection time is 80 seconds. The volume of the resuspended ultrapure water is 1 mL.

[0034] Further, in the step S6, the concentration of HS-PEG-COOH is 50 μM, and the volume is 0.04 mL, the concentration of PEG-SH is 50 μM, and the volume is 0.4 mL. The volume of the resuspended ultrapure water is 2 mL.

[0035] Further, in the step S7, the concentration of EDC is 20 mg / mL, and the volume is 200 μL, the concentration of NHS is 15 mg / mL, and the volume is 200 μL.

[0036] Further, in the step S8, the concentration of the free testosterone antigen is 5 mg / mL, and the volume is 1 μL, the concentration of BSA is 1%, and the volume is 3 mL.

[0037] Further, in the step L1, the mass of ferric chloride is 1.3 g, the volume of ethylene glycol is 40 mL, the mass of sodium acetate is 3.55 g, and the mass of polyethylene glycol is 1 g.

[0038] Further, in the step L4, the concentration of acetic acid is 0.5 M and the volume is 30 mL.

[0039] Further, in the step L5, the concentration of EDC is 20 mg / mL, the volume is 200 μL, the concentration of NHS is 15 mg / mL, and the volume is 200 μL.

[0040] Further, in the step L6, the concentration of the anti-free testosterone monoclonal antibody is 5 mg / mL, the volume is 1 μL, the concentration of BSA is 1%, and the volume is 3 mL.

[0041] The present invention also adopts the following technical solution: A detection method for a free testosterone detection kit based on SERS includes the following steps: T1, adding the sample solution and the SERS nanolabel into the functionalized magnetic bead solution in the microplate well simultaneously; T2, oscillating and reacting at 25 °C for 30 minutes, and then washing the functionalized magnetic beads 5 times with PBS buffer; T3, collecting the Raman spectrum on the functionalized magnetic beads by a Raman spectrometer to quantitatively detect the free testosterone in the sample.

[0042] The technical effects of the present invention are as follows: 1. The detection carrier of the free testosterone detection kit of the present invention is a nanomagnetic bead, which can quickly separate the magnetic beads after the immune reaction without the support of a centrifugation device, greatly simplifying the operation steps.

[0043] 2. The detection label of the free testosterone detection kit of the present invention is an SERS nanolabel, which has a high signal-to-noise ratio and can be used for the analysis of trace free testosterone in a sample.

[0044] 3. The enhancement substrate of the SERS nanolabel in the free testosterone detection kit of the present invention is a gold core-gold shell nanoparticle, which has high stability and provides a guarantee for the reliable detection of free testosterone.

[0045] 4. The outer protective layer of the gold core-gold shell nanoparticle, which is the enhancement substrate of the SERS nanolabel in the free testosterone detection kit of the present invention, is mercapto polyethylene glycol carboxyl and mercapto polyethylene glycol. Since they are high molecular polymers and have a chain structure, they can form a dense protective layer on the surface of the nanoparticles, protecting the nanomaterials from the interference of the external environment, thereby further improving the stability of the SERS nanolabel.

[0046] 5. The free testosterone detection kit and detection method of the present invention can collect the Raman spectra on magnetic beads through a handheld or portable Raman spectroscopy device, which provides the possibility for on-site rapid detection.

[0047] 6. Compared with the traditional liquid chromatography-tandem mass spectrometry, the free testosterone detection kit and detection method of the present invention have advantages in terms of operation simplicity, detection cycle, analysis cost, etc., and are of great significance for the rapid diagnosis of diseases caused by abnormal free testosterone content. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the operation schematic diagram of the free testosterone detection kit in the present invention; Figure 2 is the preparation schematic diagram of the SERS nanolabel in the present invention; Figure 3 is the preparation schematic diagram of the functionalized magnetic beads in the present invention; Figure 4 is the schematic diagram of the SERS-based free testosterone detection method in the present invention (with free testosterone in the sample); Figure 5 is the schematic diagram of the SERS-based free testosterone detection method in the present invention (without free testosterone in the sample); Figure 6 is the TEM image of the SERS nanolabel in the present invention; Figure 7 is the SEM image of the functionalized magnetic beads in the present invention; Figure 8 is the Raman spectrum of the SERS nanolabel in the present invention within 90 days; Figure 9 is the statistical chart of the intensity of the characteristic Raman signal (1330 cm-1) of the SERS nanolabel in the present invention within 90 days; Figure 10 is the ultraviolet-visible light absorption spectrum of the SERS nanolabel in the present invention within 90 days; Figure 11 is the linear relationship diagram obtained by analyzing free testosterone samples with different concentrations using the free testosterone detection kit in the present invention.

[0049] Among them, the attached drawing reference numerals are: 1. SERS nanolabel based on gold core and gold shell; 2. Functionalized magnetic beads; 3. Gold core; 4. Raman dye; 5. Gold shell; 6. HS-PEG-COOH; 7. PEG-SH; 8. Free testosterone antigen; 9. Magnetite magnetic nanoparticles; 10. Carboxyl group; 11. Monoclonal antibody against free testosterone; 12. Sample solution; 13. Free testosterone; 14. Microplate; 15. Laser with a wavelength of 785 nm; 16. Raman spectrum. Detailed implementation mode

[0050] 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 attached drawings and 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. Embodiment 1

[0051] As Figure 2 shown, the structure of the SERS nanolabel 1 based on gold core and gold shell includes a gold core 3, a Raman dye 4 modified on the surface of the gold core 3, a gold shell 5 coated on the surface, HS-PEG-COOH 6 and PEG-SH 7 protective layers, and a free testosterone antigen 8. The specific preparation process is as follows: S1. Take 2.5 mL of 0.2% (w / v) HAuCl4 solution in a three-necked round-bottom flask, and dilute it with water to 50 mL. The three-necked round-bottom flask is equipped with a condensation reflux device, and then the diluted solution is heated to boiling.

[0052] S2. Under vigorous stirring, quickly add 2 mL of 1% (w / v) sodium citrate (containing 0.05% (w / v) citric acid) to the solution in step S1, keep the solution boiling for 5 minutes, and then cool it to room temperature.

[0053] S3. Take 6 mL of the solution in step S2 after dilution (10 mL) and place it in a three-necked round-bottom flask, and stir vigorously; take 2 mL of 0.2% (w / v) HAuCl4 and add water to 10 mL, take 0.5 mL of 0.1% (w / v) ascorbic acid and 0.25 mL of 0.1% (w / v) trisodium citrate, and add water to 10 mL; at the same time, inject the HAuCl4 solution and the mixed solution of ascorbic acid and trisodium citrate respectively, and set the injection time to 45 minutes. After the injection is completed, heat the mixed solution to boiling and keep it at this temperature for about 30 minutes, and then cool it to room temperature.

[0054] S4. Add 10 mL of 0.1 M CTAC to the mixed solution in step S3, mix well and then centrifuge. Next, under ultrasonic conditions, gradually add 450 μL of 2 mM DTNB solution dropwise to the particle solution. After incubating for 20 minutes, let it stand for 3 hours. Finally, centrifuge the reaction solution, and resuspend the precipitate in 10 mL of ultrapure water.

[0055] S5. Inject HAuCl4 and hydroxylamine hydrochloride into the solution in step S4 simultaneously. The concentrations of HAuCl4 and hydroxylamine hydrochloride are 0.465 mM and 6.25 mM respectively, and the injection rates and times are 2 mL / min and 80 seconds respectively. Under vigorous stirring, let the mixture react for 45 minutes, then centrifuge, and resuspend the precipitate in 1 mL of ultrapure water.

[0056] S6. First add 40 μL of 50 μM HS-PEG-COOH solution to the solution in step S5, stir and react for 20 minutes, then add 400 μL of 50 μM PEG-SH solution, stir evenly, incubate for 4 hours, and finally centrifuge. Resuspend the precipitate in 2 mL of ultrapure water.

[0057] S7. Add 200 μL of 20 mg / mL EDC and 200 μL of 15 mg / mL NHS solution to the solution in step S6 successively. After stirring and reacting for 20 minutes, centrifuge, and resuspend the precipitate in 2 mL of PBS.

[0058] S8. Add 5 mg / mL and 1 μL of free testosterone antigen to the solution in step S7, react at 25 °C for 4 hours, then add 3 mL of 1% BSA and react for 2 hours. Centrifuge, disperse the precipitate in 0.5 mL of PBS solution, and store it refrigerated. After the above steps, the SERS nanolabel 1 as shown in Figure 6 is prepared. Its particle size is about 43 nm, and as can be seen from Figure 8 、 Figure 9 and Figure 10 , the coefficient of variation of the signal of SERS nanolabel 1 within 90 days is 10.79%. Therefore, it can be concluded that the prepared SERS nanolabel 1 has high stability. Example 2

[0059] As Figure 3 shown, the structure of the functionalized magnetic bead 2 includes magnetic nanoparticles 9, carboxyl groups 10 modified on the surface, and anti-free testosterone monoclonal antibody 11. Its specific preparation process is as follows: L1. Dissolve 1 g of ferric chloride in 40 mL of reducing agent ethylene glycol, and add 4 g of sodium acetate and 1 g of polyethylene glycol to the solution under vigorous stirring.

[0060] L2. Put the mixed solution into a microwave reactor with a magnetic stir bar, control the temperature at 200 °C, and stir for 60 minutes.

[0061] L3. After the solution cools, a precipitate is formed. Separate the magnetic precipitate and the solution using a magnet. Wash the magnetic precipitate 3 times with ethanol, then dry it at 65 °C and store it refrigerated.

[0062] L4. Disperse the magnetic particles in ultrapure water, sonicate, add 30 mL of 0.1 M acetic acid, adjust the pH of the reaction solution to 7 with ammonia water, react at 40 °C for 4 hours, and after separation, resuspend in 2 mL of ultrapure water.

[0063] L5. Add 200 μL of 20 mg / mL EDC and 200 μL of 15 mg / mL NHS solution to the magnetic bead solution in L4 successively. After stirring and reacting for 20 minutes, perform magnetic separation, and resuspend the magnetic beads in 2 mL of PBS.

[0064] L6. Add 5 mg / mL of anti-free testosterone monoclonal antibody with a volume of 1 μL to the solution in step L5, react at 25 °C for 4 hours, then add 3 mL of 1% BSA and react for 2 hours. Centrifuge, disperse the precipitate in 0.5 mL of PBS solution, and store it refrigerated. After the above steps, the functionalized magnetic beads 2 as shown in Figure 7 are prepared. Its material is magnetite, and the particle size is approximately 300 nm. Example 3

[0065] As Figure 1 shown, a detection method for a free testosterone detection kit based on SERS includes the following operation steps: T1. Add sample solution 12 and SERS nanotag 1 simultaneously to the solution of functionalized magnetic beads 2 in microplate 14; T2. Shake and react at 25 °C for 30 minutes, then wash the functionalized magnetic beads 2 five times with PBS buffer; T3. Collect the Raman spectrum of the functionalized magnetic beads 2 using a portable Raman spectrometer to detect free testosterone in the sample.

[0066] Qualitative result interpretation: 1) As Figure 4 shown, when the sample solution 12 contains free testosterone 13, it competes with SERS nanotag 1 for the reaction sites on the functionalized magnetic beads 2 simultaneously. Then the amount of SERS nanotag 1 bound to the functionalized magnetic beads 2 will decrease. Collect the Raman spectrum 16 of the functionalized magnetic beads 2, and the characteristic Raman signal intensity is weak; 2) As Figure 5As shown, when the sample solution 12 does not contain free testosterone 13, the SERS nanolabels 1 will directly bind to the reaction sites on the functionalized magnetic beads 2, so the amount of SERS nanolabels 1 bound to the functionalized magnetic beads 2 will increase. By collecting the Raman spectrum 15 of the functionalized magnetic beads 2, the characteristic Raman signal intensity is strong. Example 4

[0067] Similarly, the Raman active dye DTNB can be replaced with NBA. According to the steps of Example 1, SERS nanolabels 1 embedded with NBA are prepared; and the detection of free testosterone is carried out according to the following steps: T1, Prepare sample solutions 12 with free testosterone 13 concentrations of 10 pg / mL, 40 pg / mL, 70 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, and 500 pg / mL respectively. After they are fully mixed with the SERS nanolabels 1, they are simultaneously added to the solution of functionalized magnetic beads 2 in the microplate 14; T2, Shake and react at 25 °C for 30 minutes, and then wash the functionalized magnetic beads 2 five times with PBS buffer; T3, Collect the Raman spectrum of the functionalized magnetic beads 2 with a handheld Raman spectrometer, and obtain a Figure 11 linear relationship diagram as shown, which fully conforms to the characteristics of competitive immunoreaction.

[0068] Quantitative result interpretation: Based on the relationship between the magnitude of the collected characteristic Raman signal value and the concentration of free testosterone, a standard curve is established to calculate the detection limit. As Figure 11 shown, as the content of free testosterone 13 in the sample solution 12 increases, after the immunoreaction, the intensity of the collected characteristic Raman signal gradually weakens, which also conforms to the principle of competitive immunoreaction. The linear relationship diagram shows that the free testosterone 13 concentration has a good linear relationship in the range of 10 - 500 pg / mL, and the linear equation is: y = -17.78x + 20217.42, R2 = 0.988. According to the calculation formula of the detection limit (LOD): LOD = VMean + V3*SD (VMean is the characteristic Raman signal value corresponding to the negative sample, and SD is the standard deviation of 5 repetitions of the negative sample), the detection limit of this detection kit is calculated to be 35.96 pg / mL.

[0069] 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 SERS-based free testosterone detection kit, characterized in that: The invention is composed of a SERS nano-tag based on a gold core and a gold shell and functionalized magnetic beads. The structure of the SERS nano-tag based on a gold core and a gold shell includes a gold core, a Raman dye modified on the surface of the gold core, a gold shell coated on the surface, a mercapto polyethylene glycol carboxyl group and a mercapto polyethylene glycol protective layer outside the gold shell, and free testosterone antigen.

2. The SERS-based free testosterone detection kit according to claim 1, characterized in that: The gold core is a spherical particle with a particle size of 35-40 nm; the Raman dye is a dye with Raman activity, selected from one of Nile Blue A, 5,5'-dithiobis(2-nitrobenzoic acid), 4-mercaptobenzoic acid, 4-mercaptoaniline, 6-mercaptopurine, thioglycolic acid, and mercaptopropionic acid; the thickness of the gold shell is 3 nm.

3. The SERS-based free testosterone detection kit according to claim 1, characterized in that: The structure of the functionalized magnetic beads comprises magnetic nanoparticles and anti-free testosterone monoclonal antibodies modified on the surface; the magnetic nanoparticles are spherical particles made of ferroferric oxide and have a particle size of 300 nm.

4. A method for preparing a SERS-based free testosterone detection kit, comprising the steps of preparing a SERS nanotag and preparing functionalized magnetic beads, and combining and packaging them.

5. The method for preparing a SERS-based free testosterone detection kit according to claim 4, characterized in that: The preparation of SERS nanotags specifically includes the following steps: S1, take chloroauric acid solution, dilute it with water, and then heat the diluted solution to boiling; S2, adding a reducing agent to the solution in step S1, keeping the solution boiling for 5 minutes, and then cooling it to room temperature; S3, take 10 mL of the solution in step S2, stir vigorously, and inject HAuCl4 solution and a mixed solution of ascorbic acid and trisodium citrate respectively, heat to boiling, and keep for 30 minutes, and then cool to room temperature; S4, adding hexadecyltrimethylammonium chloride (CTAC) to the mixed solution of step S3, mixing thoroughly and centrifuging, then adding the Raman dye solution dropwise to the particle solution under ultrasound, incubating for 20 minutes, standing for 3 hours, and finally centrifuging the reaction solution, and resuspending the precipitate in ultrapure water; S5, injecting HAuCl4 and hydroxylamine hydrochloride into the solution of step S4 simultaneously, allowing the mixture to react for 45 minutes, followed by centrifugation, and resuspending the precipitate in ultrapure water; S6, first add HS-PEG-COOH solution to the solution in step S5, stir and react for 20 minutes, then add PEG-SH solution, stir evenly, incubate for 4 hours, centrifuge and resuspend the precipitate in ultrapure water; S7, adding EDC and NHS solution to the solution in step S6, stirring and reacting for 20 minutes, centrifuging, and resuspending the precipitate in PBS; S8, adding free testosterone antigen to the solution of step S7, reacting at 25°C for 4 hours, then adding BSA to react for 2 hours, centrifuging, dispersing the precipitate in PBS solution, and storing in a refrigerator.

6. The method for preparing a SERS-based free testosterone detection kit according to claim 4, characterized in that: The preparation of functionalized magnetic beads specifically includes the following steps: L1, dissolving ferric chloride in ethylene glycol, a reducing agent, adding sodium acetate and polyethylene glycol to the solution, followed by stirring at room temperature for 30 minutes; L2, transfer the mixed solution into an autoclave reactor, seal it, and control the temperature at 200 °C for 10 hours; L3, after the solution is cooled, a precipitate is formed, and the magnetic precipitate and the solution are separated by a magnet, and the magnetic precipitate is ethanol, then vacuum dried for 6 hours and stored in a refrigerator; L4, disperse the magnetic particles in ultrapure water, sonicate, add acetic acid, adjust the pH value of the reaction solution to 8 with ammonia water, react at 50 °C for 5 hours, separate and resuspend in ultrapure water; L5, add EDC and NHS solution to the magnetic bead solution of L4, stir and react for 20 minutes, separate by magnetic separation, and resuspend the magnetic beads in PBS; L6, add anti-free testosterone monoclonal antibody to the solution in step L5, react at 25°C for 4 hours, then add BSA to react for 2 hours, centrifuge, disperse the precipitate in PBS solution, and store in a refrigerator.

7. The method for preparing a SERS-based free testosterone detection kit according to claim 5, characterized in that: In step S1, the concentration of the HAuCl4 solution is 0.2% w / v and the volume is 2.5 mL; In step S2, the reducing agent is a 1% w / v sodium citrate solution containing 0.05% w / v citric acid, and the volume of the reducing agent is 2 mL; In the step S3, the volume of the solution in step S2 is 6 mL, the concentration of HAuCl4 solution is 0.2% w / v, the volume is 2 mL, and water is added to dilute to 10 mL; the concentration of ascorbic acid is 0.1% w / v, the volume is 0.5 mL, the concentration of trisodium citrate is 0.1% w / v, the volume is 0.25 mL, the ascorbic acid and trisodium citrate solutions are mixed, and then water is added to dilute to 10 mL; the injection time is 45 minutes; In step S4, the concentration of CTAC is 0.1 M and the volume is 10 mL; the concentration of Raman dye is 2 mM and the volume is 0.45 mL; In step S5, the concentration of HAuCl4 is 0.465 mM, the concentration of hydroxylamine hydrochloride is 6.25 mM; the injection speed is 2 mL / min, the injection time is 80 seconds; the volume of resuspending ultrapure water is 1 mL; In step S6, the concentration of HS-PEG-COOH is 50 μM, the volume is 0.04 mL, the concentration of PEG-SH is 50 μM, the volume is 0.4 mL. The volume of resuspended ultrapure water is 2 mL; In step S7, the concentration of EDC is 20 mg / mL, the volume is 200 μL, the concentration of NHS is 15 mg / mL, the volume is 200 μL; In step S8, the concentration of free testosterone antigen is 5 mg / mL, the volume is 1 μL, the concentration of BSA is 1%, and the volume is 3 mL.

8. The method for preparing a SERS-based free testosterone detection kit according to claim 6, characterized in that: In step L1, the mass of ferric chloride is 1.3 g, the volume of ethylene glycol is 40 mL, the mass of sodium acetate is 3.55 g, and the mass of polyethylene glycol is 1 g; In step L4, the concentration of acetic acid is 0.5 M and the volume is 30 mL; In step L5, the concentration of EDC is 20 mg / mL, the volume is 200 μL, and the concentration of NHS is 15 mg / mL, the volume is 200 μL; In step L6, the concentration of the anti-free testosterone monoclonal antibody is 5 mg / mL, the volume is 1 μL, and the concentration of BSA is 1%, the volume is 3 mL.

9. A detection method of a free testosterone detection kit based on SERS, characterized in that: The steps include: T1, the sample solution and SERS nanotags are added simultaneously to the functionalized magnetic bead solution in the microplate wells; T2, shake reaction at 25 °C for 30 min, followed by washing the functionalized magnetic beads 5 times with PBS buffer; T3, the Raman spectra on the functionalized magnetic beads were collected by Raman spectrometer to quantitatively detect the free testosterone in the sample.

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