KR20200037697A - Bio prove for detection of hormone and method of detecting target analyte thereof - Google Patents

Abstract

The present invention relates to a bio-probe for detecting hormones, comprising: magnetic microparticles (MMPs) where a first ligand specifically binding to a target analyte binds; and metal nanoparticles where a second ligand specifically binding to a target analyte binds. According to the present invention, antibody-coupled magnetic particles can separate a target hormone, so that a sex hormone in the urine can be non-invasively detected based on molecular barcode analysis without a pre-treatment step of a sample. Therefore, it is possible to save time required for the diagnosis of precocious puberty.

Description

Bio probe for hormone detection and method of detecting target analyte using the same {Bio prove for detection of hormone and method of detecting target analyte thereof}

A magnetic microparticle (MMP) bound with a first ligand that specifically binds to a target analyte; And a second ligand-specific metal nanoparticle that specifically binds to the target analyte.

If the appearance of secondary sexual characteristics or menarche begins before age 8, it is diagnosed as premature ejaculation. In recent years, the number of adolescents with precocious puberty has increased significantly, especially in girls, more than 10 times more frequently. Mechanisms of premature ejaculation have not been known, but genetic diseases, early menarche, unbalanced nervous system, and changes in nutritional status are considered as major causes. This developmental imbalance leads to high health risks such as abnormal physical, biological and nutritional pathways. In addition, premature ejaculation also affects the physiological and psychological health of adulthood associated with endocrine-related cancer, metabolic and mental disorders.

Early diagnosis is important because the prognosis for premature ejaculation disease varies greatly depending on the timing of treatment. In this regard, various approaches have been developed to improve the detection sensitivity of premature ejaculation. After confirming sex hormones, magnetic resonance imaging (MRI) is performed to perform ultrasound and brain imaging to diagnose premature ejaculation. In the case of male children, the range of puberty is determined by the level of plasma testosterone in the morning, and in females, premature ejaculation is diagnosed by the level of estradiol. That is, the detection of estradiol has a low sensitivity in diagnosing premature ejaculation because the serum estradiol level is very irregular. Testing for extreme sensitivity to gonadotropins is the most common method for diagnosing premature ejaculation. The above method is to measure gonadotropin after stimulation by GnRH or GnRH releasing hormone agonist, and blood should be collected by repeating 5 to 8 times at intervals of 90 to 120 minutes. Therefore, not only is the entire examination process painful and inconvenient for children, but also the cost is relatively high and the time is long.

Meanwhile, urine contains hormones and proteins, which are used for non-invasive diagnosis. Sensitive mass spectrometry and sandwich ELISA kits are the most common methods of analysis because steroid concentrations in the urine are more diluted than plasma concentrations. However, sample preparation and time consuming pretreatment steps are not only required for the process, but in some cases radioactive molecules are used as hormone-labeled compounds for analysis, which has the disadvantage of being expensive and unstable. Recently, nanotechnology has been combined with hormone diagnostics to improve the detection system. Sensitivity, detection of multiple targets in the analysis remains a problem. Therefore, there is a need to develop a method capable of simultaneously detecting multiple target substances present in a low concentration of urine without pre-treatment of the sample.

One aspect is a magnetic microparticle (MMP) bound to a first ligand that specifically binds to a target analyte; And it is to provide a bio-probe for hormone detection comprising a metal nanoparticle bound to a second ligand that specifically binds to the target analyte.

Another aspect is to provide a composition for diagnosing premature ejaculation, adult disease, or cancer, or a drug test comprising the bio probe.

Other aspects include magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to a target analyte with a sample; And it provides a method for detecting a bio-barcode-based target analyte comprising the step of performing a bio-barcode analysis of the complex.

Other aspects include magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to a target analyte with a sample; And it provides a method for providing information for the diagnosis of premature ejaculation, adult disease, or cancer, or drug testing, comprising performing a bio-barcode analysis of the complex.

One aspect is a magnetic microparticle (MMP) bound to a first ligand that specifically binds to a target analyte; And a metal nanoparticle to which a second ligand specifically binding to the target analyte is provided.

The term "target analyte" in the present specification refers to a substance that is a target to confirm the presence or absence of a biological sample. The target analyte can be a hormone and can be, for example, estradiol, testosterone, progesterone, corticosteroids, androgens, androsterones, epitestosterones, ethiocolanolones, or combinations thereof.

The target analyte that can be applied to the bio probe according to one embodiment may be a material having specific binding ability to each other such as protein, DNA or RNA. Specifically, it may be a material capable of forming a magnetic microparticle-target analyte-metal nanoparticle complex by specifically binding the first ligand on the magnetic microparticle and the second ligand on the metal nanoparticle.

Further, the first ligand and / or the second ligand may be protein, DNA or RNA, and may be selected from, for example, aptamer, antibody, and barcode DNA. In this case, the aptamer may be composed of nucleic acids that can simultaneously act as barcode DNA. The bio probe according to an embodiment may include two or more of the first ligand and / or the second ligand.

The first ligand can bind to the magnetic microparticles directly or through a linker, and the second ligand can bind to metal nanoparticles directly or through a linker. When the first ligand and / or the second ligand are proteins, the terminal may be introduced to the surface of magnetic microparticles or metal nanoparticles using a coupling reaction between carboxylic acid and amine. In addition, the metal nanoparticles may further include a water-soluble low-molecular substance that forms a covalent bond with the metal nanoparticles. In this case, the low molecular substance may include a thiol group, and the thiol group is, for example, 2-thiazoline-2-thiol, 1- [2- (dimethylamino) ethyl] -1H-tetrazol-5-thiol ( 1- [2- (Dimethylamino) ethyl] -1H-tetrazole-5-thiol, MTDT), 2-thiazoline 2-thiol, PEG-thiol, or the like It can be a combination. That is, since the thiol group forms a relatively stable bond through covalent bonding with the metal nanoparticles, it may be stably attached without falling off during the reaction of the aptamer. On the other hand, when the first ligand and / or the second ligand is DNA, the terminal may be modified with a reactive functional group to bind to magnetic microparticles or metal nanoparticles, respectively. For example, DNA whose terminal is modified with a thiol group has a binding ability to metal nanoparticles, and the binding can be broken by adding a substituent capable of forming a stronger bond on the metal surface.

When the target analyte is a nucleic acid capable of binding to a DNA having a complementary sequence such as DNA or RNA, the barcode DNA may have a form in which a sequence corresponding to a second ligand is connected to the terminal. At this time, the metal nanoparticle including the barcode DNA does not require a separate second ligand for complex formation with the target analyte.

The specific binding between the target analyte and the ligand may be antigen-antibody binding, hybridization between complementary sequences, or ligand-receptor binding. For example, when the target analyte is a protein such as an antigen or a receptor for a specific ligand, an antibody or ligand that specifically binds to each is selected as a first ligand or a second ligand, and by specific binding between them. It can be made to form a complex. In addition, when the target analyte is a nucleic acid such as DNA and RNA, DNA having a sequence complementary to a part of the target analyte may be selected as a second ligand to form a complex by hybridization therebetween.

The bio probe according to an embodiment may be for diagnosing premature ejaculation, adult disease, or cancer, or for drug testing. Specifically, the cancer may be urinary system cancer or gynecological cancer. The urinary tract cancer may be, for example, bladder cancer, prostate cancer, penile cancer, ureter cancer or kidney cancer, and the gynecological cancer may be, for example, breast cancer, uterine cancer or ovarian cancer.

As described above, the bio probe according to one aspect may detect a target analyte in a sample by forming a magnetic microparticle-target analyte-metal nanoparticle complex. At this time, when the target analyte is a sex hormone, it can be usefully used as a non-invasive diagnostic method for the diagnosis of premature ejaculation. In addition, it can be applied to drug tests, diagnosis of adult diseases and other diseases according to the type of the target analyte.

Another aspect provides a composition for diagnosing premature ejaculation, adult disease or cancer, or a drug test comprising the bio probe. The details of the bio probe are as described above.

Other aspects include magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to a target analyte with a sample; And it provides a method for detecting a bio-barcode-based target analyte comprising the step of performing a bio-barcode analysis of the complex. The method has an advantage in that a target analyte present in a small amount in a sample can be detected without an amplification process.

Another aspect of the method according to an embodiment includes magnetic microparticles having a first ligand bound specifically to a target analyte; And mixing a metal nanoparticle with a second ligand specifically binding to a target analyte with a sample to form a magnetic microparticle-target analyte-metal nanoparticle complex. The target analyte may be a hormone, etc., specifically, a pituitary hormone, a steroid-based hormone, or a thyroid hormone. The complex may be formed by antigen-antibody binding, hybridization between complementary sequences, or ligand-receptor binding between the first ligand bound to the magnetic microparticles and the target analyte and between the second ligand bound to the metal nanoparticles and the target analyte. Can be formed. For example, when the target analyte is estradiol, the antigen-antibody bond between the antibody bound to the magnetic microparticles and estradiol and the ligand-receptor bond between the anti-estradiol aptamer and estradiol bound to the metal nanoparticles The complex can be formed. In addition, when the target analyte is testosterone, a complex can be formed by antigen-antibody binding between an antibody bound to magnetic microparticles and testosterone and antigen-antibody binding of an anti-testosterone antibody bound to metal nanoparticles and testosterone. have.

The method according to one embodiment includes performing a bio-barcode analysis of the complex. The bio-barcode analysis can be performed by mass spectrometry. In general, bio-barcode analysis has been performed using ELISA, electrophoresis, or fluorophores. However, when using the ELISA method, since the sensitivity is not only low, but the ELISA method through a competitive reaction is generally used, there is a problem that the cost of using the antibody increases and the limit of the detection limit exists. The competition reaction is to determine the color development by competing two antibodies (HRP attached antibody and HRP unattached antibody) that detect the same hormone, and the higher the hormone concentration, the lower the signal. In addition, in the case of electrophoresis, DNA having a complementary sequence to barcode DNA must be prepared separately, and there is a problem that accurate quantification is impossible due to the characteristics of electrophoresis. In addition, in the case of analysis using a fluorescence chromophore, there is a problem in that the cost is increased because an additional fluorescence material needs to be modified, and the detection limit is not good. However, bio-barcode analysis by mass spectrometry can not only realize the effect of signal amplification compared to conventional mass spectrometry through nanoparticles, but also improves sensitivity by 1 million times or more and compares quantitatively compared to conventional ELISA. There is an advantage.

In addition, the step of introducing a low molecular material containing a thiol group in the metal nanoparticles may be further included. At this time, the thiol group included in the low-molecular substance is 2-thiazoline-2-thiol, 1- [2- (dimethylamino) ethyl] -1H-tetrazol-5-thiol, 2-thiazoline 2-thiol or PEG- Thiol and the like. For example, when the low-molecular substance containing the thiol group is 2-thiazoline-2-thiol or MTDT, the substance is a molecule exhibiting high ionization efficiency and is water-soluble and capable of covalently binding to gold, a mass spectrometer Improve the signal. That is, the combination of barcode reagents (DNA and compound) and gold nanoparticles can amplify the final detection signal that contributes to the high sensitivity of the analysis system, so that the level of detection (100 ag / ml) is much lower than the detection value reported in the previous studies. Target analytes can be detected.

Other aspects include magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to the target analyte with a sample. And it provides a method for providing information for the detection of a target analyte comprising the step of performing a bio-barcode analysis of the complex. The details of the target analyte are as described above.

Another aspect of the method according to an embodiment includes magnetic microparticles having a first ligand bound specifically to a target analyte; And mixing a metal nanoparticle with a second ligand specifically binding to a target analyte with a sample to form a magnetic microparticle-target analyte-metal nanoparticle complex. The details of the complex are as described above. The sample may be blood, tissue, urine, mucus, saliva, tears, plasma, serum, or a combination thereof.

Urine contains hormones, proteins, etc., and is used for non-invasive diagnosis. Steroid hormones present in urine are present at a lower concentration than those present in plasma, and should be detected using a special detection device with high sensitivity. do. In particular, in children, there is a problem of suffering in order to collect plasma. Therefore, when the sample is urine, it is possible to detect non-invasive sex hormones in the urine based on bio-barcode analysis without pre-treatment of the sample, so the target analyte, even though the hormone concentration in the urine of the child is lower than the serum For example, it can be used for the diagnosis of bar premature ejaculation, which is easy to detect sex hormones.

The method according to one embodiment includes performing a bio-barcode analysis of the complex. The details of the step of performing the bio-barcode analysis are as described above. As described above, in the bio probe according to one aspect, about 70 to 9 million molecules are loaded on the surface of the metal nanoparticles, and the mass spectrometer signal is greatly improved, and the LOD concentration of 100 ag / 1000 times lower than that of ELISA ㎖ can be detected, it is possible to detect multiple target hormones present in a low concentration in the urine at the same time, as well as early diagnosis and prognosis prediction of sex premature ejaculation, it can solve the problem of pain caused during diagnosis .

Urinary tract analysis generally requires additional sample preparation steps to eliminate many urinary components that affect false positive signals. However, in the case of the probe according to one aspect, since the magnetic particle to which the antibody is bound can separate the target hormone, it is possible to non-invasively detect the sex hormone in urine based on molecular barcode analysis without the pretreatment step of the sample. It can save time for diagnosing premature ejaculation.

In children, the concentration of hormone in the urine is lower than that in serum, so there is a limit in measuring the hormone in the urine. However, in the case of the probe according to one aspect, the final detection signal can be amplified with high sensitivity by the combination of the barcode reagent and gold nanoparticles, so that multiple target hormones can be simultaneously detected even at low concentrations of urine. And the diagnosis of premature ejaculation, such as collecting samples with pain.

1 is a diagram showing an outline of a barcode analysis for measuring sex hormones according to one aspect.
2 is a graph showing the results of mass spectrometry analysis of 2-thiazoline-2-thiol for the detection of estradiol.
3 is a graph showing the results of detecting estradiol (A) and testosterone (B) using a commercial ELSIA kit (n = 4).
Figure 4 shows the results of bio-barcode analysis based on gel electrophoresis (A: estradiol, B; testosterone).
Figure 5 shows the results of the detection of multiple targets using bio-barcode analysis based on gel dry motion.
6 is a fluorescence signal graph of barcode DNA. (A) is the result of detecting a single target in urine, and (B) is the result of simultaneously detecting multiple hormones (2 types).

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Preparation example]

Preparation of urine

Six children (3 males, 3 females) of 6 to 10 year olds suspected of premature ejaculation were prepared by receiving 10 ml of auto urine in a sample cup.

Preparation of control urine

Covalent urine (hormone-free urine) was prepared by adsorbing hormones using styrene-divinylbenzene copolymer resin (SERDOLIT PAD-I, 0.1-0.2 mm, Serva, Germany).

[Example]

Example 1 Functionalization of Antibodies to Magnetic Particles

Anti-estradiol and testosterone polyclonal antibodies (Abs) are magnetic particles (micro magnetic) through a carboxylic acid and amine coupling reaction using 1-ethyl-3 (-3-dimethylaminopropyl) carboimide (EDC) reagent. particle, MMP). After incubating the magnetic particles and the antibody, the reaction was stopped by adding Tris-HCl buffer. After washing the antibody conjugated-MMP (Abs-MMP) three times with Tris buffer solution, Abs-MMP was re-calibrated with PBS solution. The antibody was bound to MMP according to the standard procedure provided by Invitogen. Subsequently, the loading capacity of MMP was calculated using a bicinchoninic acid (BCA) protein assay according to Equation 1 below.

[Calculation formula 1]

Amount of binding protein = initial protein concentration-unbound protein concentration in the supernatant

Example 2. Preparation of gold nanoparticle probe (AuNP)

2-1. Preparation of gold nanoparticle probes for estradiol

Since the size of the aptamer is smaller than the size of the antibody, a large amount of molecules can be loaded onto the nanoparticles, which has advantages such as cost effectiveness and stability. In addition, the binding affinity, cross-linking and specificity of anti-estradiol aptamers have already been verified. Thus, a probe for detecting estradiol was prepared using aptamer (45 bp) instead of an anti-estradiol monoclonal antibody.

The 5 'end of the anti-estradiol DNA aptamer was modified with a disulfide group. At this time, the disulfide group forms a relatively stable bond by covalently bonding with AuNP, and may be stably attached without falling off during the reaction of the aptamer. The base sequences of the anti-17-β-estradiol aptamer and the anti-17-β-estradiol aptamer modified with a fluorophore are shown in Table 1 below.

Anti-17-β-estradiol aptamer 5'AAAAAAAAAAAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTG 3 ' Anti-17-β-estradiol aptamer modified with fluorophore 5'AAAAAAAAAAAAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTG-Alexa 647

High performance liquid chromatography (HPLC) -purified aptamer was dissolved in Milipore water (18.2 mm 2) to obtain a 100 μm solution. To 100 μL of aptamer solution, 100 μL of 100 nM 1,2-dithiothreitol (DTT) (Sigma-Aldrich) was added to decompose disulfide bonds. After 1 hour, DTT was removed and the separated thiolated aptamer was removed using a NAP-5 column. The concentration of thiolated aptamer was calculated by measuring absorbance at a wavelength of 260 nm. DNA solution was added to 50 nm AuNP (final concentration ratio of DNA: AuNS = 10,000: 1). A phosphate buffer solution containing pH 7 and 10% SDS was added for pH adjustment, and then 6 aliquots of 2M sodium chloride solution (Sigma-Aldrich) were added to a final concentration of 0.3M. Thereafter, the resulting solution was incubated overnight at room temperature. The aptamer-functionalized AuNP was centrifuged at 8,500 rpm for 15 minutes and concentrated in 1/10 volume of deionized water of the initially used AuNP solution.

2-2. Preparation of gold nanoparticle probes for testosterone

For testosterone detection, probes were prepared using monoclonal antibodies. The monoclonal antibody was implanted on the surface of a gold nanoparticle probe as testosterone barcode DNA. 10 μl of anti-testosterone monoclonal antibody was added to 1 ml of 50-nm AuNP to adjust the pH to 9.2. Since the gold-sulphide bond is used to bind the barcode DNA with AuNP, a thiol group was added to the 5 'end of the barcode DNA, and cysteine-protein G was incubated with AuNP for 30 minutes for directional antibody conjugation with AuNP. At this time, the cysteine containing a thiol functional group directly binds to AuNP, and a part of protein G binds to the Fc region of the antibody. The disulfide group reduced the barcode DNA added to the Ab-AuNP solution. Barcode DNA sequences are shown in Table 2 below;

Testosterone barcode dna 5'TTTTTTTTTTTTAGGGGTACAGGGTGTTCT 3 ' Fluorochrome-modified barcode DNA 5'TTTTTTTTTTTTAGGGGTACAGGGTGTTCT-Alexa488

Thereafter, the mixture was left at 10 ° C for a few minutes, and then 2M sodium chloride solution was added to the final concentration of 0.15M. Antibody and barcode DNA-functionalized AuNPs were centrifuged at 4 ° C., 8,500 rpm for 15 minutes and concentrated in 1/10 volume of deionized water of the AunP solution used initially.

2-3. Preparation of gold nanoparticle probes for mass spectrometry

2-thiazoline-2-thiol and 1- [2- (dimethylamino) ethyl] -1H-tetrazol-5-thiol (1- [2- (Dimethylamino)) with high ionization efficiency to improve mass spectrometer signal ethyl] -1H-tetrazole-5-thiol (MTDT) was grafted onto the 50 nm AuNP surface with aptamer or antibody. The 2-thiazoline-2-thiol and MTDT, which are water-soluble and capable of covalent bonding with gold, represent estradiol and testosterone, respectively. For the detection of estradiol, the anti-estradiol aptamer was first incubated with AuNP and then 2-thiazoline-2-thiol (molar ratio of 2-thiazoline-2-thiol to AuNP = 10 ⁸ : 1) was added. . In addition, in order to detect testosterone, an anti-testosterone monoclonal antibody was mixed with AuNP and then incubated with an MTDT solution (molar ratio of MTDT to AuNP = 10 ⁸ : 1).

Example 3. Confirmation of the properties of the AuNP probe

The properties of the probes prepared in Examples 2-1 and 2-2 were confirmed. First, in order to quantify the amount of barcode DNA / aptamer per AuNP, fluorescence chromophore-labeled barcode DNA / aptamer-functionalized AuNP was centrifuged at 8.500 rpm for 15 minutes and resuspended in 1 ml of deionized water to unbound DNA. / The process of removing the aptamer was repeated twice. After further centrifugation, the pellet of the fluorochrome-labeled AuNP probe was treated with 50 μl of 25 mM potassium cyanide (KCN) (Sigma-Aldrich) and the mixture was incubated at room temperature until AuNP was completely dissolved. The fluorescence intensity of the chromophore-labeled barcode DNA / aptamer was measured using Infinite® F200 Pro (Tecan, Zurich, Switzerland) to obtain a standard curve. Samples were serially diluted in 10-fold increments ranging from 1 μM to 1 nM. Finally, the fluorescence intensity of the fluorescence-labeled DNA / aptamer released from AuNP was measured and compared to a standard curve to determine the number of barcode DNA / aptamer cross-linked to AuNP.

As a result of measuring the barcode DNA fluorescence signal of testosterone, it was confirmed that about 80 barcode DNAs can be fixed at 50 nm. In addition, as a result of measuring the fluorescence signal of aptamer of estradiol, it was confirmed that about 2,500 aptamers may be functionalized target hormones.

Next, the amount of antibody against AuNP was quantified using BCA analysis. The Au core of Ab-DNA-AuNP was dissolved with KCN solution. Then, the BCA analysis solution was added to the solution. After culturing the sample, the absorbance was measured using a reader Infinite® F200 Pro (Tecan).

As a result of measuring the barcode DNA fluorescence signal, it was confirmed that about 80 barcode DNAs can be fixed at 50 nm. In addition, it was confirmed by BCA analysis that about 7 antibodies were present in 60 nm AuNP. As a result of confirming the polyclonal antibody loading capacity of MMP, it was confirmed that the initial antibody amount was 150 μg, and only 10 μg remained on the antibody after conjugation and separation. That is, it can be seen that the antibody has been successfully introduced to the particle surface.

Example 4. Bio-barcode analysis using mass spectrometry

The barcode analysis procedure was performed in the same manner as above except for the final detection portion. After the analysis, 50 mM of 100 mM KCN was added to the sample to dissolve AuNP in an ionized form, and then 10 μl of the sample was diluted with 90 μl of water. Each 10 µl of the diluted sample was injected into the column. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using a UFLC XR series liquid chromatography system (Shimadzu, Japan) with a Kinetex C18 column (100 mm Х 2.1 mm ID, 2.6 μm particle size, Phenomenex, Torrance, CA) and This was done using a Q exactive plus tandem mass spectrometer from Thermo Scientific (San Jose, USA). The mobile phase included 0.1% formic acid aqueous solution for mobile phase A and 0.1% formic acid methanol for mobile phase B. The mobile phase elution gradient was applied at a flow rate of 0.5 mL / min, 2% mobile phase B was maintained for 0.5 min, ramped to 95% B over 8.5 min, and maintained until 9.0 min. Subsequently, re-equilibration was performed at 2% B for 1 minute so that the total execution time was 10 minutes. All analytes were analyzed in cation mode, and the capillary temperature was set at 300 ° C. The spray voltage was 4000 v, and the mass spectrum was collected through an ion scan mode generated from molecular ions, which was called parallel-response monitoring. Sheath gas, ion sweep gas, and auxiliary gas-flow rate were 53 arb (arbitrary unit), 3 arb, and 14 arb, respectively.

2 is a graph showing the results of mass spectrometry analysis of 2-thiazoline-2-thiol for estradiol detection.

2, the graph confirmed that the molecular barcoding method successfully detected 100 ag / ml of estradiol. Although a large amount of barcode DNA in AuNP enhanced the mass spectrometer signal, this effect was not dramatic. Because the molecular weight of barcode DNA is relatively high (anti-estradiol aptamer (barcode): 14000.1 g / mol, testosterone barcode: 9261 g / mol), therefore, the ionization efficiency is lower than estradiol or testosterone. In addition, multiple charge distributions generated by electron spray ionization (ESI) and various isotopic patterns induce multiple ion signals in the mass spectrum of barcode DNA and cause relatively low sensitivity in mass spectrometry by ESI. In fact, as a result of detecting barcode DNA using a mass spectrometer, it was confirmed that although 2,000 barcode DNAs were loaded into AuNP, the signal was improved 10 times compared to direct hormone detection. Based on the standard curve, the loading capacity of AuNPs for small molecules was calculated and it was confirmed that about 70-90 million 2-thiazoline-2-thiols and MTDTs bind to a single AuNP. Hormones were determined based on the peaks of 2-thiazoline-2-thiol and MTDT in the final detection step. This indicates that the signal from the mass spectrometer can be amplified and allows detection of high sensitivity even at very low hormone levels. From the above results, it can be confirmed that this approach can detect 1 ag / ml of hormone, 10 ⁵ times lower than the LOD of the previous study (10 fg / ml). That is, the conventional 17-β-estradiol mass spectrometer was able to detect 300 fg / mL (detection limit = 300 fg / mL), but the method using a probe according to one aspect was 17-β-estradiol 100 ag / Ml was successfully detected in urine.

[Comparative example]

Comparative Example 1. Immunoassay (ELISA)

For standard ELISA analysis for estradiol and testosterone, commercial ELISA kits (anti-testosterone = ab108666 and anti-17-β-estradiol = ab108667, Abcam) were used. Diluted samples, standards and controls were added to the wells and then 100 μl of HRP-conjugate was added to each well. At this time, a blank well was left for the substrate blank. After incubating for 1 hour at 37 ° C for testosterone and 2 hours at 37 ° C for estradiol, each well was washed 3 times with 300 μl of 1 × wash buffer. After the last wash, the remaining fluid was removed cleanly and 100 μl of TMB substrate solution was added to all wells. After further incubation at 37 ° C. for 15 minutes, 100 μl of stop solution was added to all wells. Finally, the absorbance of the sample was measured at 450 nm for a reference wavelength of 620-630 nm within 30 minutes after adding the stop solution or within 5 minutes for the blank.

3 is a graph showing the results of detecting estradiol (A) and testosterone (B) using a commercial ELSIA kit (n = 4).

As shown in FIG. 3, it was confirmed that the detection limit (LOD) of the hormone in the PBS buffer condition was about 10-20 pg / ml, and it was confirmed that the urine increased to 20-70 pg / ml. That is, since urine contains various urinary tract substances such as ions and proteins, it can block the binding of hormones and antibodies, so the sensitivity of analysis of the ELISA kit in urine may be reduced compared to buffer conditions.

Comparative Example 2. Bio-barcode analysis using electrophoresis

Bio-barcode gel analysis was applied to detect estradiol and testosterone. Barcode DNA with different lengths of testosterone and estradiol (30 and 45 bp respectively) can separate native polyacrylamide gels during electrophoresis, so a bio-barcode gel analysis was performed to detect the two hormones. First, bio barcode analysis was performed at various urine hormone concentrations ranging from 100 μg / ml to 1 pg / ml.

After analysis, 10 mM of 100 mM KCN was added to the sample to dissolve AuNP in ionized form. In addition, double-stranded barcode DNA (bar code dsDNA) was formed by adding 1 μL of 1 μM oligonucleotide (hybrid DNA) complementary to barcode DNA and 2 μl of 1 M sodium chloride solution (S7653, Sigma-Aldrich). After incubating the solution at 95 ° C. for 5 minutes, barcode dsDNA was checked for the length of barcode DNA using polyacrylamide gel electrophoresis. The 15% negative polyacrylamide gel solution was 30% acrylamide / bis solution (29: 1) (BIO-RAD), 10 x Tris / Boric Acid / EDTA (BIO-RAD), ammonium sulfate (Sigma-Aldrich) and tetra Prepared with methylethylenediamine (BIO-RAD). The final DNA sample mixed with 10 x loading buffer (TaKaRa) was placed in a gel, and electrophoresis was performed at 120v for 1 hour. After electrophoresis, the gel was stained with SYBR® Green I Nucleic Acid Stain (Lonza) to visualize double-stranded DNA (dsDNA). The gel was washed twice with deionized water and imaged using a gel documentation system Scinomics, Daejeon, South Korea). The fluorescence intensity of each DNA band was analyzed using ImageJ, and the signal was normalized to a negative control (NC) band.

4 shows the electrophoresis results of bio-barcode analysis to detect two individual target hormones in urine.

As shown in Fig. 4, in accordance with the expected molecular weight (testosterone: 30 bp, estradiol: 45 bp), barcode DNA of different lengths corresponding to each target gene could be identified on the natural polyacrylamide gel. The analysis result for detecting a single hormone showed a detection limit of 1 pg / ml.

Figure 5 shows the results of multiple target detection using bio-barcode analysis based on gel electrophoresis.

As shown in FIG. 5, each hormone could be determined by gel analysis, but it was confirmed that the sensitivity was decreased. The graph showed that the intensity had a weak linear correlation, so this approach was unable to quantify the absolute amount of hormone.

Comparative Example 3. Bio-barcode analysis using fluorescence chromophore

Target hormones (17-β-estradiol and testosterone) were serially diluted 10-fold at a wide range of concentrations (100 μg / ml to 10 ag / ml). The solution used for the sample was prepared by dilution of a PBS solution containing DMSO (20%) or covariate urine (control urine). 10 μl magnetic probe and 10 μl AuNP probe were added to 100 μl of serially diluted samples. The mixture was incubated for 1 hour at room temperature. The MMP-target-AuNP complex was briefly magnetically separated and washed twice with PBS-DMSO solution. To separate barcode DNA with a fluorescence chromophore, the sample was resuspended in 100 μl of 50 mM KCN solution. Thereafter, the sample was heated at 95 ° C. for 5 minutes and a magnetic field was applied to the signal to obtain a liquid sample excluding particles. Through this, the magnetic particles used in the reaction were removed, and only barcode DNA detached from the AuNP probe was selectively separated. The solution containing barcode DNA and ionized AuNP was transferred to 96 wells with a black transparent bottom. Finally, the fluorescence intensity of each plate was measured at 488 and 647 nm using a plate reader Infinite® F200 Pro (Tecan).

6 is a fluorescence signal graph of barcode DNA.

As shown in Figure 6a, it was confirmed that the detection limit of estradiol is 10 pg / ml, and the detection limit of testosterone is 100 ng / ml. In addition, fluorescence-based bio-barcode analysis was able to determine two hormones in a single analysis and was successfully detected within 1 hour (FIG. 6B).

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that it can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Bio prove for detection of hormone and method of detecting target          analyte thereof <130> PN121905 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> anti-17-beta-estradiol aptamer <400> 1 aaaaaaaaaa aagggatgcc gtttgggccc aagttcggca tagtg 45 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> testosterone barcode DNA <400> 2 tttttttttt ttaggggtac agggtgttct 30

Claims (18)

A magnetic microparticle (MMP) bound with a first ligand that specifically binds to a target analyte; And a second ligand-specific metal nanoparticle that specifically binds to the target analyte. The bio probe according to claim 1, wherein the target analyte is selected from the group consisting of estradiol, testosterone, progesterone, corticosteroid, androgen, androsterone, epitestosterone, and thiocholanolone. The bio probe according to claim 1, wherein the first ligand is an antibody. The bio probe according to claim 1, wherein the second ligand is selected from aptamer, antibody, and barcode DNA. The bio probe of claim 3, wherein the first ligand is bound to magnetic microparticles directly or through a linker. The method according to claim 4, The second ligand is a bio probe that is bound to the metal nanoparticles directly or through a linker. The method according to claim 1, wherein the metal nanoparticle is a bio probe that further comprises a low-molecular material. The bio probe according to claim 7, wherein the low molecular material is a water-soluble material that forms a covalent bond with metal nanoparticles. The bio probe according to claim 7, wherein the low molecular substance comprises a thiol group. The method according to claim 9, wherein the thiol group is 2-thiazoline-2-thiol, 1- [2- (dimethylamino) ethyl] -1H-tetrazol-5-thiol (1- [2- (Dimethylamino) ethyl] -1H -Bio probe selected from the group consisting of tetrazole-5-thiol, MTDT), 2-thiazoline 2-thiol and PEG-thiol. The bio probe according to claim 1, wherein the specific binding between the target analyte and the ligand is an antigen-antibody binding, hybridization between complementary sequences, or a ligand-receptor binding. The method according to claim 1, The probe is a bio-probe for the diagnosis or drug test of premature ejaculation, adult disease, or cancer. A composition for diagnosing premature ejaculation, adult disease, or cancer, or a drug test comprising the bio probe of claim 1. Magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to a target analyte with a sample; And
A method of detecting a bio-barcode-based target analyte, comprising performing bio-barcode analysis of the complex. The method of claim 10, wherein the target analyte is selected from the group consisting of estradiol, testosterone, progesterone, corticosteroid, androgen, androsterone, epitestosterone, and thiocholanolone. The method of claim 10, wherein the bio-barcode analysis is performed by mass spectrometry. The method of claim 14, further comprising introducing a low molecular material comprising a thiol group to the metal nanoparticles. Magnetic microparticles bound with a first ligand that specifically binds to a target analyte; And forming a magnetic microparticle-target analyte-metal nanoparticle complex by mixing a metal nanoparticle bound with a second ligand specifically binding to a target analyte with a sample; And
Method for providing information for the detection of a target analyte comprising the step of performing bio-barcode analysis of the complex.

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