REAGENT KIT FOR DETECTING SEX HORMONE AND METHOD FOR DETECTING SEX HORMONE USING SAME

Abstract

The present disclosure provides a reagent kit for detecting a sex hormone, which contains a first reagent containing a metal nanoprobe in which a sex hormone and a Raman reporter are immobilized and a second reagent containing a magnetic particle in which an antibody for detecting the sex hormone is immobilized, and a method for detecting a sex hormone using the same.

Claims

1. A SERS-based method for detecting a sex hormone comprising the steps of: preparing a sample solution comprising a sex hormone; preparing a metal nanoprobe in which the sex hormone is bound to a Raman reporter; preparing a magnetic particle in which a primary antibody and a secondary antibody for detecting the sex hormone are immobilized; adding the metal nanoprobe and the magnetic particle in which the primary antibody and the secondary antibody are immobilized to the sample solution at the same time; forming an immunocomplex with the magnetic particle through a competitive immunoreaction of each of the sex hormone in the sample solution and the sex hormone of the metal nanoprobe with the primary antibody immobilized in the magnetic particle; separating the magnetic particle in which the immunocomplex is formed using magnetism; irradiating a laser light to the separated magnetic particle; and detecting the sex hormone by measuring a surface-enhanced Raman scattering (SERS) signal after the irradiation of the laser light.

2. The SERS-based method for detecting a sex hormone according to claim 1, wherein, in the step of forming the immunocomplex, the intensity of the SERS signal is decreased as the concentration of the sex hormone in the sample solution is higher because the amount of the metal nanoprobe forming the immunocomplex with the magnetic particle is decreased; or the intensity of the SERS signal is increased as the concentration of the sex hormone in the sample solution is lower because the amount of the metal nanoprobe forming the immunocomplex with the magnetic particle is increased.

3. The SERS-based method for detecting a sex hormone according to claim 1, wherein the sex hormone is estrogen or testosterone.

4. The SERS-based method for detecting a sex hormone according to claim 3, wherein the estrogen is one or more selected from a group consisting of estradiol, estrone and estriol.

5. The SERS-based method for detecting a sex hormone according to claim 1, wherein the sample solution is selected from a group consisting of a tissue extract, a cell lysate, a whole blood, a blood plasma, a blood serum, a saliva, an ocular fluid, a cerebrospinal fluid, a sweat, a urine, a milk, an ascitic fluid, a synovial fluid, a peritoneal fluid and a dried blood spot.

6. The SERS-based method for detecting a sex hormone according to claim 1, wherein the secondary antibody is an anti-mouse antibody immobilized on the magnetic particle and the primary antibody is an anti-sex hormone antibody which binds to the secondary antibody and specifically immunoreacts with the sex hormone.

7. The SERS-based method for detecting a sex hormone according to claim 1, wherein the detectable concentration of the sex hormone is 0.1-1,000 pg/m L.

8. The SERS-based method for detecting a sex hormone according to claim 1, wherein the limit of detection of the sex hormone is 0.1 pg/mL.

9. The SERS-based method for detecting a sex hormone according to claim 1, wherein the detection time of the sex hormone is 2 hours or shorter.

10. A method for diagnosis of precocious puberty of a subject comprising the steps of: extracting a sample solution from a subject; detecting a sex hormone for the extracted sample solution by the method for detecting a sex hormone according to claim 1; and determining the concentration of the detected sex hormone.

11. The method according to claim 10, wherein the concentration of the detected sex hormone is 0.1-1000 pg/m L.

12. The method according to claim 10, which further comprises, after the step of determining the concentration of the detected sex hormone, a step of diagnosing as precocious puberty when the concentration of the detected sex hormone is higher than 10 pg/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1 schematically illustrates a first reagent and a second reagent of the present disclosure.

[0078] FIG. 2 schematically illustrates a method for detecting a sex hormone according to the present disclosure.

[0079] FIG. 3 shows transmission electron microscopic (TEM) images (a) and dynamic light scattering (DLS) data (b) of synthesized gold nanoprobes.

[0080] FIG. 4 shows TEM images of immunocomplexes formed through a competitive immunoreaction according to the present disclosure (the numerical values on the upper left-hand corners are the concentrations of estradiol in sample solutions).

[0081] FIGS. 5a-5c show a result of measuring Raman signals using a method for detecting estradiol according to an exemplary embodiment of the present disclosure.

[0082] FIGS. 6a-6c show a result of measuring Raman signals using a method for detecting testosterone according to an exemplary embodiment of the present disclosure.

[0083] FIG. 7 shows a result of detecting estradiol by a SERS-based detection method according to an exemplary embodiment of the present disclosure and a result of detecting estradiol by ELISA analysis as a comparative example.

[0084] FIG. 8 shows a result of detecting testosterone by ELISA analysis as a comparative example.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0085] Hereinafter, specific examples are provided to help understanding of the present disclosure. However, the following examples only exemplify the present disclosure and it will be obvious to those of ordinary skill in the art that various changes and modifications can be made within the scope and technical idea of the present disclosure and such changes and modifications are included within the scope of the appended claims.

Example 1: Preparation of First Reagent (Metal Nanoprobe)

[0086] Chloroauric acid (HAuCl.sub.4), trisodium citrate, poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (HS-PEG-COOH, MW ˜3500), poly(ethylene glycol) methyl ether thiol (HS-PEG, MW ˜2000), EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were purchased from Sigma-Aldrich. Malachite green isothiocyanate (MGITC) was purchased from Invitrogen and estradiol-ovalbumin conjugate (E2-OVA) was purchased from Cusabio.

[0087] For synthesis of metal nanoprobes, spherical gold nanoparticles were synthesized (Frens, 1973, Nature Physical Science 241, 20-22.). 50 mL of a 0.01% HAuCI4 solution was heated to boiling and 0.5 mL of a 1% trisodium citrate solution was added dropwise. At first, the color of the HAuCI4 aqueous solution turned blue as nanoparticles (seeds) were formed. Then, the solution turned red gradually with time as the nanoparticles grew. After the color of the nanoparticles to be synthesized was confirmed, the mixture was boiled further for 15 minutes and the reaction was terminated. Then, the gold nanoparticles were aged for 4 hours or longer while cooling to room temperature. It was confirmed through transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements that gold nanoprobes were synthesized stably with uniform sizes of about 40-50 nm, as seen from FIG. 3. Subsequently, for use as a SERS substrate, the Raman reporter malachite green isothiocyanate (MGITC) was coated on the gold nanoparticles. After adding the Raman reporter to 1 mL of 0.12 nM 40-nm gold nanoparticles dropwise so that the final concentration was 50 nM and then adding 60 μL of 10 μM poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (HS-PEG-COOH, MW 3500) and 120 μL of 10 μM poly(ethylene glycol) methyl ether thiol (HS-PEG, MW 2000) dropwise, the mixture was incubated for 3 hours in order to introduce carboxyl functional groups onto the surface of the gold nanoparticles. Then, estradiol-ovalbumin (E2-OVA) conjugate or testosterone-BSA (bovine serum albumin) conjugate was immobilized using the carboxyl functional groups on the surface of the gold nanoparticles. For this, after adding 5 μL of 25 mM EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) dropwise and mixing for 15 minutes, 2 μL of 1 mg/mL estradiol-ovalbumin was added dropwise and the mixture was reacted at room temperature for 2 hours. Then, the reaction mixture was incubated at 4° C. for 12 hours. Unbound residues were removed through three centrifugations (7200 rpm, 10 minutes).

Example 2: Preparation of Second Reagent (Magnetic Particle)

[0088] Secondary antibodies (anti-mouse IgG (Fc-specific) antibody produced in goat), EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were purchased from Sigma-Aldrich. Magnetic microparticles (Dynabeads MyOne™) and PBS buffer (0.1 mM, pH 7.4) were purchased from Invitrogen. Primary antibodies (anti-17β estradiol antibody or mouse anti-testosterone monoclonal antibody) were purchased from Abcam.

[0089] The secondary antibodies (anti-mouse antibody) were immobilized using the carboxyl functional groups on the surface of the magnetic microparticles. For this, the carboxyl functional groups on the surface of the magnetic microparticles were activated by adding 5 μL of 0.1 M EDC and NHS dropwise for 30 minutes. Then, after adding 2 mg/mL secondary antibodies (anti-mouse antibody) dropwise and incubating at room temperature for 2 hours, residues not bound to the surface of the magnetic microparticles were removed using magnetism. The separated magnetic particles were dissolved in PBS (10 mM, pH 7.4) and stored at 4° C. Subsequently, the preparation of magnetic particles for detecting sex hormones was completed by adding 25 μL of 0.55 g/mL anti-estradiol antibodies (or anti-testosterone antibodies) as the primary antibodies to 25 μL of the magnetic particles in which the secondary antibodies were immobilized and incubating at room temperature for 90 minutes. After the reaction, residues not bound to the surface of the magnetic microparticles were removed using magnetism and the separated magnetic particles were dissolved in PBS (10 mM, pH 7.4).

Example 3: Detection of Sex Hormones Based on Surface-Enhanced Raman Scattering

[0090] 3-1: Detection of Estradiol

[0091] First, blood containing estradiol was prepared as a sample solution. Then, 25 μL of the magnetic particles in which the secondary antibodies and the primary antibodies (anti-estradiol antibody) are immobilized, which was synthesized in Example 2, and 50 μL of the gold nanoprobes prepared in Example 1 were added at the same time to 25 μL of the sample solution. A total of 90 minutes was spent for the detection.

[0092] FIG. 4 shows TEM (transmission electron microscopy) images of immunocomplexes formed through a competitive immunoreaction according to the present disclosure (the numerical values on the upper left-hand corners are the concentrations of estradiol in sample solutions). As can be seen from FIG. 4, the amount of the metal nanoprobes bound to the magnetic particles decreased as the concentration of the sex hormone in the sample solution was higher.

[0093] Then, the magnetic particles in which the first and second immunocomplexes were formed were separated using magnetism and Raman analysis was conducted for the separated magnetic particles. The Raman analysis was performed as follows. The Renishaw Invia Raman spectrometer (Renishaw, UK) was used and the Spectra Physics He—Ne laser operating at 632.8 nm was used as a light source. The Rayleigh line was removed using a holographic notch filter located in the collection path. All the peak positions were calibrated by measuring the peak position of silicon as reference at 520 cm.sup.−1 before measurement. Raman spectra were collected in the range of 630-1730 cm.sup.−1 with an exposure time of 1 second using a laser with an output wavelength of 633 nm and an output power of 20 mW. A 20× objective lens was used to focus the laser spot. Baseline correction of all the Raman spectra was performed using the WiRE 4.0 software (Renishaw, UK). Quantitative analysis of the sex hormone was performed for the peak at 1613 cm.sup.−1 which showed the strongest intensity.

[0094] The Raman signals were obtained as shown in FIGS. 5a-5c. The limit of detection was 0.1 pg/mL.

[0095] 3-2: Detection of Testosterone

[0096] First, blood containing testosterone was prepared as a sample solution. Then, 25 μL of the magnetic particles in which the secondary antibodies and the primary antibodies (anti-testosterone antibody) are immobilized, which was synthesized in Example 2, and 50 μL of the gold nanoprobes prepared in Example 1 were added at the same time to 25 μL of the sample solution. A total of 90 minutes was spent for the detection.

[0097] Raman analysis was performed in the same manner as described in Example 3-1. The Raman signals were obtained as shown in FIGS. 6a-6c. The limit of detection was 0.1 pg/mL.

Comparative Example: Comparison with ELISA Analysis

[0098] 1: ELISA Analysis for Detection of Estradiol

[0099] Enzyme immunoassay (ELISA) is a commonly employed diagnostic method based on the color change of blood to which an antigen is added due to enzymatic action. To assess the detection sensitivity of the sex hormone analyzing technique based on surface-enhanced Raman spectroscopy according to the present disclosure, it was compared with an analysis result using the Abnova estradiol detection kit (based on ELISA). This diagnosis method performs quantitative analysis through competitive reaction of estradiol in an analyte sample with estradiol labeled with a luminescence-inducing material. After attaching antibodies that can immobilize estradiol onto a plate and adding the analyte sample, the antibodies conjugated with the luminescence-inducing material were allowed to be bound to the immobilized antigens. Diagnosis was made by measuring color change depending on the content of the luminescence-inducing material. The substances used in the analysis are described in Table 2.

TABLE-US-00002 TABLE 2 Component Amount Goat Anti-Rabbit IgG-coated microtiter wells 96 wells Estradiol Reference standards: 0, 10, 30, 100, 0.5 ml each 300, and 1000 pg/ml. Liquid, ready to use. Rabbit Anti-Estradiol Reagent (pink color) 7 ml Estradiol-HRP Conjugate Reagent 12 ml (blue color) Estradiol control 1, Liquid, Ready to use 0.5 ml Estradiol Control 2, Liquid, Ready to use 0.5 ml TMB Reagent (One-Step) 11 ml Stop Solution (1N HCl) 11 ml

[0100] FIG. 7 compares the results of detecting the sex hormone based on surface-enhanced Raman scattering (a) and ELISA analysis (b). To compare the two detection methods, for the SERS-based detection method according to the present disclosure, the detectable range was 0.1-1,000 pg/mL and the detection limit was 0.1 pg/mL. In contrast, for the ELISA analysis method, the detectable range was 5-1,000 pg/mL and the detection limit was 5 pg/mL.

[0101] 2: ELISA Analysis for Detection of Testosterone

[0102] The substances used in the ELISA analysis are described in Table 3.

TABLE-US-00003 TABLE 3 Component Amount Testosterone-Coated Wells: microtiter wells coated 1 plate, with testosterone-BSA conjugates 96 wells Reference Standard Set: Contains 0, 1, 5, 10, 25, 50, 0.5 ml/vial 75 and 100 ng/ml testosterone, liquid, ready to use. Mouse Anti-Testosterone Reagent: Contains mouse 7 ml anti-testosterone in bovine serum albumin (BSA) buffer Goat Anti-Mouse IgG HRP Conjugate Reagent: 12 ml Contains goat anti-mouse IgG conjugated to HRP Washing Buffer (PBS-Tween 20, 0.1%, v/v) 12 ml TMB Reagent: Contains 3, 3′, 5, 5′~TMB 11 ml stabilized in buffer solution Stop Solution: Diluted hydrochloric acid (1N HCl) 11 ml

[0103] FIG. 8 compares the result of detecting testosterone by ELISA analysis. For the SERS-based detection method according to the present disclosure, the detectable range was 0.1-1,000 pg/mL and the detection limit was 0.1 pg/mL. In contrast, for the ELISA analysis method, the detectable range was 1-100 pg/mL and the detection limit was 0.18 ng/mL (=180 pg/mL).

[0104] From these results, it can be seen that the SERS-based detection method according to the present disclosure allows analysis of the sample at concentration ranges of 0.1-5 pg/mL, which is impossible with the ELISA analysis method. In particular, the present disclosure meets the requirement of detection of the sex hormone at low concentrations of below 10 pg/mL, which is necessary for the diagnosis of precocious puberty. In contrast, detection of the sex hormone at low concentrations of below 10 pg/mL is impossible with the ELISA analysis method. Accordingly, it can be seen that the SERS-based detection method according to the present disclosure is an analysis method capable of detecting the sex hormone with high sensitivity, which is necessary for the diagnosis of precocious puberty.

Example 4: Evaluation of Clinical Applicability

[0105] 30 blood samples were analyzed by the Architect's estradiol assay (automated assay). The Architect's estradiol detection method is an immunoassay using a chemiluminescent material and is capable of quantitative analysis based on chemiluminescence signals depending on the amount of the sex hormone present in blood. The detectable range of this method is 10-1000 pg/mL and the detection limit is 10 pg/mL. Accordingly, analysis is impossible for samples at concentrations below 10 pg/mL. Table 4 shows the result of testing the concentration of estradiol in the 30 blood samples using the Architect's assay system. And, the result of analyzing the same blood samples using the SERS-based detection method according to the present disclosure is compared in Table 5. From the analysis result given in Table 5, it can be seen that very significantly results are attained for the 30 blood samples with the SERS-based detection method as compared to the Architect's estradiol detection method. In particular, it can be seen that even the samples with estradiol concentrations lower than 10 pg/mL could be analyzed accurately, which was impossible with the Architect's assay system. Through this, the clinical applicability of the SERS-based detection method according to the present disclosure was verified. In particular, the method is very superior in analyzing blood samples with sex hormone concentrations lower than 10 pg/mL as compared to the existing analysis equipment. Accordingly, it can be seen that the SERS-based detection method according to the present disclosure is a very suitable method for diagnosis of precocious puberty through detection of sex hormones in blood with high sensitivity.

TABLE-US-00004 TABLE 4 Number Gender Age E2conc.(pg/mL) 101 F 13 39 102 F 10 27 103 F 8 15 104 F 8 <10 105 F 12 56 106 F 8 18 107 F 8 <10 108 F 8 <10 109 F 8 <10 110 F 8 14 111 F 8 <10 112 F 9 <10 113 F 13 29 114 F 10 <10 115 F 8 17 116 M 15 22 117 F 7 <10 118 F 8 <10 119 F 10 <10 120 F 10 89 121 F 9 <10 122 F 9 12 123 F 9 17 124 M 10 11 125 F 8 16 126 F 10 19 127 F 8 15 128 F 8 12 129 F 9 13 130 F 9 26

TABLE-US-00005 TABLE 5 CMI assay SERS Assay Grade Sample No. (pg/mL) (pg/mL) Negative 104 <10  6.2 107 <10  5.3 108 <10  2.4 109 <10  7.9 111 <10  4.1 112 <10  8.8 114 <10  8.6 117 <10  9.9 118 <10  6.7 119 <10  9.6 121 <10  3.9 Low positive 103  15 18.8 106  18 21.2 110  14 19.1 115  17 25.3 122  12 16.7 123  17 20.1 124  11 15.9 125  16 24.9 126  19 22.7 127  15 16.1 128  12 13.9 129  13 17.1 Positive 101  39 43.8 102  27 31 105  56 52.9 113  29 33.2 116  22 24.4 120  89 98.3 130  26 27.1