KR101631581B1 - FRET sensor fused nano-particles and detecting method for phytoestrogen using the same - Google Patents

Abstract

The present invention relates to a FRET sensor in which nanoparticles are fused and more particularly to a FRET sensor in which nanoparticles are fused to an estrogen receptor ligand binding domain to which donor fluorescent protein and acceptor fluorescent protein are bound, energy transfer sensors and methods for detecting phytoestrogens present in food and biological samples.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a FRET sensor fused with nanoparticles and a method for detecting phytoestrogens using the same,

The present invention relates to a FRET sensor in which nanoparticles are fused and more particularly to a FRET sensor in which nanoparticles are fused to an estrogen receptor ligand binding domain to which donor fluorescent protein and acceptor fluorescent protein are bound, energy transfer sensors, and methods for detecting phytoestrogens present in food and biological samples.

Phytoestrogen is a plant compound that is structurally or functionally similar to the estrogen hormone for human and related active metabolism. Phytoestrogens occur naturally in high concentrations in many plants, such as beans, fruits, and cabbage. Especially, the beans are famous for their abundance of phytoestrogens, which are isoflavones including genistein and daidzein. These phytoestrogens are nutrients used for the treatment of osteoporosis and hyperlipidemia. It is in the limelight. In addition, many of these therapeutic compounds have been selected as candidates for cancer prevention compounds and undergo clinical testing. Resveratrol is found in the red grape skin and red wine ingredient, and due to the biological activity of this material, a variety of researches are currently under way on various animal and human effects.

It has been reported that asymptomatic cases of breast cancer and prostate cancer, which are related to hormones, are less common among Oriental populations, in which phytoestrogen-containing foods are relatively consumed. Thus, phytoestrogens are presumed to play a role in the prevention of various hormone-related diseases. For example, phytoestrogen intake is associated with alterations in plasma lipid risk factors, emphasized in the nutritional and pharmaceutical importance of natural chemical compounds, antioxidant protection against LDL, antiproliferative and vascular reactivity effects I think.

It is very important to monitor and quantify the amount of phytoestrogens in food, and it is also known to be a daily intake for infants and growing children. In the case of adolescent girls, phytoestrogens in diet foods are said to be associated with advancing the menstrual cycle because they play a similar role to natural estrogen hormones. Thus, various physiochemical techniques such as HPLC (high performance liquid chromatography), gas chromatography (GC), capillary electrophoresis, and mass spectroscopy have been applied to detect and quantify phytoestrogens in food and biological samples come. However, most of these detection methods are time consuming and require a high degree of equipment.

FRET (fluorescence resonance energy transfer) is a technique for measuring the interaction between two fluorescent molecules. It is a technique for accepting excited donor fluorophore molecules from an excited donor fluorophore molecule through the interaction between electric dipoles, And energy transfer was calculated by using an acceptor fluorophore molecule. The efficiency of FRET depends on the spatial distance between the donor and acceptor molecules, and the efficiency drops to R ⁶ , the distance between the two molecules. The broad application of FRET in biology has greatly facilitated the detection and visualization of protein interactions, structural changes, and protein-ligand interactions. Therefore, the fluorescent protein was used as a biological sensor to detect the structural changes caused by the binding of protein ligands, especially the various receptor proteins.

The estrogen receptor (ER) has been studied as a potential drug target for more than 10 years. The tertiary structure of the ligand binding domain (LBD) to ER has been found to be a typical mechanism of structural changes in response to the binding of small ligand molecules. The carboxyl terminal helix 12 (H12) of LBD undergoes structural changes, serves as a molecular switch for the binding of various ligands, It is known that ER and secondary suppressor protein interact. The reaction of this nucleic acid receptor to the ligand and the structural changes that occur after the reaction led to the development of detection methods for monitoring ligand-protein interactions based on FRET.

In a recent FRET-related study, various compounds and hormone analogs were reported for affinity and binding ability, and the FRET system was introduced to study the estrogen receptor (ER). Although FRET is commonly used in the medical and industrial fields, some studies report only the practical aspects of industrial fluorescent probes.

Accordingly, the present inventors have made intensive efforts to develop a FRET sensor capable of detecting phytoestrogen with a simple, rapid and high sensitivity. As a result, they have found that phytoestrogens can be detected with high sensitivity and stability by combining a nanoparticle material and a FRET ER probe And confirmed the present invention.

One object of the present invention is to provide a FRET sensor in which nanoparticles are fused to an estrogen receptor ligand binding domain to which donor fluorescent protein and acceptor fluorescent protein are bound.

It is another object of the present invention to provide a method for detecting phytoestrogens using a FRET sensor using a probe in which nanoparticles are fused.

In order to achieve the above object, the present invention relates to a fluorescence resonance energy transfer (FRET) sensor in which nanoparticles are fused to an estrogen receptor ligand binding domain to which donor fluorescent protein and acceptor fluorescent protein are bound.

The term "FRET (fluorescence resonance energy transfer)" in the present invention is called fluorescence resonance energy transfer. When an energy donor, which is a short wavelength protein, absorbs energy from the outside, Refers to a phenomenon in which only long wavelength fluorescence of a receptor is released by being transmitted without radiation to an energy acceptor, which is a longer wavelength excitation protein, instead of being emitted. The energy donor and acceptor should be located within a very short distance (<10 nm) in order to generate receptor fluorescence by FRET or photobleaching of donor emitting light. When a donor, which is a fluorescent protein that emits light of a specific wavelength, and a receptor that is a fluorescent protein capable of absorbing energy due to resonance with the emission energy are within a predetermined distance, light energy irradiated from the donor The receptor is transmitted without radiation to reduce the emission of the intrinsic wavelength of the donor and FRET phenomenon occurs in which the receptor that receives energy from the donor emits light in its own wavelength band.

As used herein, the term "donor fluorescent protein" refers to a substance that transmits energy absorbed from the outside, and is a fusion protein fused with a short wavelength fluorescent protein or short wavelength fluorescent protein in principle of FRET. Refers to a fusion protein bound to a long wavelength fluorescent protein or a long wavelength fluorescent protein.

Examples of such fluorescent proteins include green phosphor protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (eGFP), red phosphor protein (RFP), enhanced red phosphor (EFP), a blue fluorescent protein (BFP), an enhanced blue fluorescent protein (eBFP), a yellow fluorescent protein (YFP), an enhanced yellow fluorescent protein (eYFP) (eCFP), but the present invention is not limited thereto. Among them, a fluorescent protein of a short wavelength is a donor fluorescent protein, and a fluorescent protein of a long wavelength is used as a receiving fluorescent protein.

In the present invention, the terms "short wavelength" and "long wavelength" are relatively determined. That is, the wavelength of the light emitted by the fluorescent protein used in the donor fluorescent protein means that the fluorescent protein used in the receiving fluorescent protein has a relatively shorter wavelength than the wavelength of the light emitted by the fluorescent protein. Preferably, the enhanced blue fluorescent protein (eCFP) is used as the donor fluorescent protein and the enhanced yellow fluorescent protein (eYFP), which is longer in wavelength than the indigo fluorescent protein, is used as the receiving fluorescent protein.

The term "estrogen receptor ligand binding domain" in the context of the present invention encompasses, without limitation, a moiety having the ability to bind to a ligand in an estrogen receptor, such as the estrogen receptor ligand binding domain in two forms: estrogen receptor alpha receptor alpha, ER alpha) ligand binding domain or an estrogen receptor beta (ER beta) ligand binding domain, preferably the estrogen receptor alpha ligand binding domain may be used, more preferably the estrogen receptor beta Receptor alpha ligand binding domain. Preferably, the protein from which the estrogen receptor ligand binding domain can exhibit activity may have 70%, 80%, 90% or 95% homology with SEQ ID NO: 1.

Preferably, in the present invention, the donor fluorescent protein and the acceptor fluorescent protein are bound to an estrogen receptor ligand binding domain, wherein the donor fluorescent protein is linked to the N-terminal of the receptor ligand binding domain of estrogen, A FRET sensor linked to the C-terminus of the estrogen receptor ligand binding domain can be constructed.

According to one embodiment of the present invention, the FRET sensor is typically composed of 301 to 533 amino acid probes (SEQ ID NO: 1), and was constructed based on the ligand binding domain (LBD). Carboxyl terminal helix 12 (H12) in the ligand binding domain has been known to undergo structural changes and acts as a molecular switch for binding of various ligands (FIG. 3).

In addition, the probe can be used in a tagged form for purification. Any known tag can be used without limitation. Examples thereof include a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA) (NEB, USA), FLAG (IBI, USA) or a histidine tag (His-tag), preferably a histidine tag (His-tag). The histidine tag sequence is a sequence in which several histidine residues are continuous and has a strong binding ability with a metal ion. Thus, the product in which a target protein is fused with a histidine tag sequence is obtained by a single step . &Lt; / RTI &gt;

In addition, a linker can be used to bind the donor and acceptor fluorescent protein to an estrogen receptor ligand. The linker is generally composed of several to several tens of amino acids, and the type and number of amino acids are well known Preferably, when linking the donor fluorescent protein to the N-terminus of the estrogen receptor ligand binding domain, a linker consisting of two amino acid residues (AS) may be used, and the acceptor fluorescent protein may be linked to an estrogen receptor ligand binding domain , A linker composed of 12 amino acids (LGIEGRISEPGS) can be used.

In the FRET sensor of the present invention, detection efficiency can be improved by manufacturing nanoparticles through fusion. The nanoparticles can be used for increasing detection efficiency, such as quantum dots containing a carboxyl group, a polymer, a magnetic substance, Particles may be used without limitation.

The quantum dots are mainly composed of cadmium selenide (CdSe), cadmium telluride (CdTe), and cadmium (CdS) in the center of about 2 to 10 nm in size. The shells are about 10 to 15 nm But are not limited to, quantum dots encoded with zinc sulfide (ZnS) in size.

Examples of the polymer include polypyrrole, polyaniline, polyvinyl alcohol (PVA), polystyrene, GOx, prussian Blue, protonic acid, graphite oxide, etc.). However, the present invention is not limited thereto. Preferably, polystyrene may be used, and more preferably, carboxylated polystyrene may be used.

According to one embodiment of the present invention, the donor fluorescent protein is fused to carboxylated polystyrene nanoparticles by binding an NHS-ester and a carboxyl group of an estrogen receptor ligand binding domain to which the acceptor fluorescent protein is bound.

On the other hand, the magnetic nanoparticles are ferromagnetic or superparamagnetic particles generally having a size of about 10 nm. Ferrites such as CoFe ₂ O ₄ and MnFe ₂ O ₄ , which are iron oxides such as Fe ₂ O ₃ and Fe ₃ O _4, and Fe ₃ O _4, where one Fe is converted to another magnetic-related atom, , FePt, CoPt, etc., alloyed with a noble metal to enhance conductivity and stability, but are not limited thereto.

In yet another aspect, the present invention relates to a method for detecting phytoestrogens using the FRET sensor.

The method for detecting phytoestrogen using the FRET sensor is to measure and detect a signal generated due to binding of the FRET estrogen receptor probe to which the nanoparticles are bound and the phytoestrogen.

The term "phytoestrogen" in the present invention means a substance known to have a physiological activity similar to estrogen, a female hormone, derived from a plant, and examples thereof include flavones, But are not limited to, flavonois, league lignans, coumestan, and isoflavones, but may be isoflavones.

The term "flavones" in the present invention refers to all yellow pigments including, but not limited to, chrysin, primechin, apigenin and luteolin having a skeleton like flavone.

The term "flavonoids" in the present invention includes, but is not limited to, vitamin P and citrin.

In the present invention, the term "turbulent turbulence" is used to refer to any of the following: pinoresinol, podophyllotoxin, steganacin, lariciresinol, secoisolariciresinol, but are not limited to, matairesinol, hydroxymatairesinol, syringaresinol or sesamin.

The term "cumesters" in the present invention includes, but is not limited to, coumestrol.

Examples of the isoflavones include genistein, daidzein, glycitein, resveratrol, irilone, orobol, pseudobaptigenin, But it is not limited thereto, and preferably it can be genistein, daidzein, resveratrol, and the like.

As a method for measuring the signal, a fluorescence signal can be measured for binding of the estrogen receptor alpha-ligand binding domain of the phytoestrogen to the FRET probe to which nanoparticles are bound. In addition, detection of the phytoestrogen can be detected using the ratio of the difference between the ratio of eYFP (550 nm) to eCFP (475 nm) in the presence of the ligand and the ratio of Δ in the same ratio in the absence of the ligand.

According to one embodiment of the present invention, Δ ratio values for genistein, resveratrol and daidzein were 0.30, 0.50 and 0.26, respectively, and in the DMSO control sample without phytoestrogen It was confirmed that the signal was not detected and the Δ ratio value was not obtained, so that the sensitivity was high. Also, the fusion probe in which the nanoparticles were fused yielded a fluorescence signal having a Δ ratio of 1.65, 2.60 and 1.37 for genistein, resveratrol and daidzein, respectively, And about six times higher than that of each FRET probe (Fig. 5).

As described above, the present invention relates to a method for detecting phytoestrogens, which is superior to a conventional method for detecting phytoestrogens using a FRET sensor having nanoparticles incorporated therein. More particularly, the present invention relates to a method for detecting phytoestrogens, Can be used for accurate detection with high sensitivity, and is expected to be suitably modified for use in food and medicine fields.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an illustration of an estrogen receptor alpha (a) probe.
Figure 2 shows that an estrogen receptor alpha (?) Probe was purified using Ni-NTA chromatography.
Figure 3 shows that two modified structures of helix 12 (red label) in the ligand binding domain represent an agonistic ligand (top) and an antagonistic ligand (bottom).
Figure 4 shows the chemical structure of genistein, daidzein, and resveratrol belonging to isoflavin among phytoestrogens.
Figure 5 shows a comparison of FRET signals for FRET probes for genistein, daidzein, and resveratrol versus probes (black bars) coupled with nanoparticles.
FIG. 6 shows the relative Δ ratio of the FRET signal of genistein to the probe to which the probe and the nanoparticle are bound.
Figure 7 shows the relative Δ ratio of the FRET signal of resveratrol to the probes to which the probe and the nanoparticles are bound.
Figure 8 shows the relative Δ ratio of the FRET signal of the daidzein to the probes to which the probe and the nanoparticles are bound.
Figure 9 shows the value of the Δ ratio over time for the control, genistein, daidzein, and resveratrol.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these examples.

Example  1: Experimental material

Genistein, resveratrol, and daidzein were purchased from Sigma-Aldrich (St. Louis, Mo.). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). eCFP and eYFP genes were donated by the Microbiology Research Center of the Korea Biotechnology Research Institute. ^{Poly-Prep ® Chromatography Column (1} × 4 ㎝, Ni-NTA superflow) the column and the Ni-NTA resin packed in was purchased from (Hilden, Germany) Qiagen, Res Q (1 ㎖) PD-10 desalting column and the G -500 resin was purchased from GE Healthcare (Piscataway, NJ). Polystyrene nanoparticles with carboxylated surfaces were purchased from Micromode (100 nm Micromer, 25 mg / ml) and NHS (N-hydroxysuccinimide) and EDC (1-Ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride were purchased from Sigma-Aldrich. The buffer solutions often used for the connection of sensor proteins to activated nanoparticles are buffer A (25 mM MES, pH 6.0), buffer B (1 × phosphate buffered saline; pH 7.4), buffer C (250 mM sodium bicarbonate buffer, pH 8.0).

Example  2 : CFP - ERa - YFP Construct

The construct of the estrogen receptor alpha (gene ID BC 128573) was prepared based on the ligand binding domain (LBD), with the LBD having an amino acid residue of 301 to 553 (SEQ ID NO: 1) Had the same binding affinity as LBD. The FRET-based phytoestrogen sensor was genetically engineered to have Linker Molecule Linker 1 (L1) and Linker 2 (L2) with eCFP and eYFP at the N-terminus and C-terminus, respectively. The shorter linker moieties produce a higher signal than the longer connection, which supports the fact that the connection plays an important role in producing larger signals. Genetic modification was accomplished by inserting the eYFP gene from the original pEYFP-N1 vector (Clontech) into the pET21a vector ( BamHI / HindIII ) and by inserting the eCFP gene from the pECFP-N1 vector (Clontech) ( NdeI / HindIII ). The ER gene was inserted between the cleavage sites of BamHI and NheI after PCR amplification from the original copy using primers 5'CGCGCTAGCTCTAAGAAGAACAGCCTGGCCTTG3 '(SEQ ID NO: 2) and 3'CGCGGATCCACTGGGCGCATGTAGGCGGTGGGC5' (SEQ ID NO: 3). The N-terminus of the linker 1 (L1) of the eCFP and the estrogen receptor ligand binding domain was a dipeptide (AS) and the linker 2 (L2) was a decapeptide (LGIEGRISEPGS) between the eYFP and the C-terminus.

Example  3: Protein expression and purification

Recombinant cells were transformed with Luria Bertani broth [1% (w / v) bacto-tryptone, 0.5% (w / v) yeast extract, 0.5% (w / v) NaCl] containing ampicillin (100 g / The cells were cultured at 25 ° C, and the cells were collected by centrifugation. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole) and resuspended in ice bath (VC-600, Sonics & Materials Inc., USA). Cell lysates were applied to Poly-Prep ^® chromatography columns (1 × 4 cm, Ni-NTA superflow, Qiagen, Hilden, Germany) using Ni-TNA resin. The resin was washed with wash buffer (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM imidazole). Elution of the protein was further purified in elution buffer (50 mM Tris-HCl (pH 7.5), 50 mM NaCl and 250 mM imidazole) and the protein was applied to a Res Q (1 ml) cation exchange column . Protein was dialyzed against 50 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10 mM beta-mercaptoethanol and concentrated to 20 mg / ml. The final protein was estimated to have a purity and homogeneity of over 96% by SDS-PAGE.

Example  4: Fusion of sensor protein with activated polystyrene nanoparticles

For protein fusion, the carboxylated polystyrene nanoparticles were activated by modification of the carboxyl group and the NHS-ester surface. Briefly, 500 [mu] l of nanoparticles were mixed with 500 [mu] l of 0.4 M EDC and 0.8 M NHS dissolved in buffer A. [ The mixture was incubated at room temperature for 1 hour, mixed with Buffer B, and passed through a PD-10 column to remove excess EDC and NHS. The sensor protein was added to the activated nanoparticles adjusted to a final concentration of 400 [mu] g / ml, pH 8.0 buffer solution C. [ After incubation at room temperature with a solution of the nanoparticles and the sensor protein, a blocking agent, ethanolamine (pH 8.5) was added at a concentration of 100 mM. The excess unbound protein was removed by S-500 column size exclusion chromatography and the NP-protein fusion was collected in buffer A at a volume of 1 ml each. Respectively.

Example  5: FRET  analysis

To distinguish between agonist and antagonist binding, the sensor protein was incubated in a buffer containing 10 mM Tris-Cl (pH 7.5) 50 mM NaCl and 5 mM beta-mercaptoethanol in a buffer containing 3 x ^10-8 M &lt; / RTI &gt; To detect other phytoestrogens using the FRET, the ligand was dissolved in DMSO (dimethysulfoxide) and added to a final concentration of 10 &lt; ^-5 &gt; M. To confirm the ligand binding affinity, the final concentration of the ligand was added to 10 ^-15 to 10 ^-4 M. All reactions were carried out at 16 ° C for 30 minutes. All experiments were performed on a 1 ml test system for nanoparticle fusion probes. In order to identify other phytoestrogens, phytoestrogens were used at a concentration of 10 ^-5 M, and in experiments to determine the ligand binding affinity, the concentration of the ligand was performed in a concentration range of 10 ^-22 to 10 ^-4 M. The FRET analysis was carried out with an LS 45 luminescence spectrometer (Perkin Elmer instruments) at 433 nm by eCFP stimulation. The emission of eYFP was detected at 525 nm and the eCFP was detected at 475 nm. FRET values were expressed as the ratio of eYFP to eCFP signal. FL WinLab (Perkin-Elmer Corporation) was used for data acquisition.

Experiment result

Experimental Example  One. FRET  Estrogen receptor Of the probe  Confirm production

FRET sensor probes for the estrogen receptor α were constructed based on the ligand binding domain (LBD) and consisted of amino acids 301-553. The carboxyl terminal helix 12 (H12) of the LBD undergoes a structural change and acts as a molecular switch due to the binding of various ligands. As such, the helix was already tested with a FRET probe (Fig. 1). To make probes effective for phytoestrogen detection, eCFP (enhanced CFP) protein was attached to the N-terminus of LBD using an additional linker of two amino acid residues (AS), and eYFP (enhanced YFP) And ligated to the C-terminus of the LBD using a linker residue (LGIEGRISEPGS). A His-tag was added to the C-terminus of eYFP for efficient purification of the probe. Upon ligand binding, the change in the position of eYFP linked to helix 12 led to the transfer of energy from eCFP to eYFP, resulting in a change in the decrease accompanied by the release of eYFP at 525 nm and the release of eCFP at 475 nm.

The His-tag fusion protein is known as E. internally expressed in E. coli (RIL), sequenced by Ni-NTA chromatography, and 98% pure protein was generated using cation exchange chromatography (measured by SDS-PAGE, Figure 2). To prepare nanoparticle-fused probes, the purified protein probes were immobilized on the surface of 100 nm diameter carboxylated polystyrene nanoparticles. The carboxyl groups around the nanoparticles are activated by EDC (N-ethyl-N '- (3-dimethyllaminopropyl) carbodiimide / NHS (N-hydroxysuccinimide) coupling reagent and the surface NHS- Amino acid groups were used. Various EDC / NHS concentrations were used as coupling reagents and experiments were conducted to produce fusion probes in which stable and effective nanoparticles were fused.

Experimental Example  2. FRET ER In the probe  by Phytoestrogenic  detection

Estrogen receptor a has been shown to adapt to various structures depending on the chemistry and polarity of ligand binding to LBP. These structural changes from other origins of helix 12 lead to binding (Figure 3). Compounds such as genistein and daidzein belonging to the isoflavone family of compounds have a diphenolic core and a hydroxyl moiety that are similar to the natural agonists of the estrogen receptor (eg, 17-β estradiol) ).

Resveratrol has a stilbenoid structure (Figure 4) and is similar to 4-OHT (4-hydroxytamoxifen), a known antagonist used in chemotherapy. To investigate the phytochemical binding principle for the probes of the present invention, FRET reactions were measured after addition of each ligand, and the ratio of Δ for each sample was calculated. The ratio refers to the difference between the ratio of YFP (525 nm) to CFP (475 nm) in the presence of the ligand and the same ratio in the absence of the ligand.

FRET data clearly detects phytoestrogenic binding and has a Δ ratio to 0.30, 0.50, and 0.26 for genistein, resveratrol and daidzein, respectively, and does not detect signals in DMSO control samples (Fig. 5). In addition, regarding the principle of action site binding of resveratrol, the Δ ratio of resveratrol was about 1.5 times greater than that of genistein and daidzein. Notably, the fusion probe fused with nanoparticles yielded a fluorescence signal with a Δ ratio of 1.65, 2.60 and 1.37 for genistein, resveratrol, and daidzein, respectively, Fold higher than for each unfused FRET probe (Figure 5).

In the present invention, all the FRET? Ratios are average values for three experiments using a ligand concentration of 10 ^-5 M and a probe concentration of 3 × 10 ^-8 M. A significant increase in nanoparticle fusion FRET detoxification can be interpreted as the sensor protein is efficiently fused and concentrated on the surface of nanoparticles that amplify the FRET signal associated with binding to the ligand.

The FRET profile shown at the classical isobestic point in the present invention suggests that the detected signal is a reliable value for the interaction between the probe and the ligand.

Experimental Example  3. On phytoestrogen  Dose response curves for

The sensitivity and binding principle for FRET probes were determined with statistical precision. FRET ER probes were reacted with increasing phytoestrogen concentration, and the ratios were determined for each ligand.

The calculated EC ₅₀ values of genistein, resveratrol and daidzein for FRET probes were 8 uM, 10 uM and 21 uM, respectively (Figs. 6, 7 and 8). Such EC ₅₀ values were obtained by an average of 5 experiments based on three parameter sigmoidal curves generated by extrapolation of the Y axis (Δ ratio) to the X axis (log ^{ligand concentration} ) .

In vitro , the ligand binding domain of ER determined by luciferase assay had a genistein-binding EC ₅₀ of 15 uM and an analysis of full length protein with an EC ₅₀ of 0.46 uM. The EC ₅₀ values for the binding of the estradiol to the estrogen receptor a for the antagonist hydroxytamoxiphene were 15 × 10 ^-6 M and 20 × 10 ^-6 M, respectively.

Collectively, the data presented for the binding principle of individual FRET ER probes for phytoestrogens was consistent with previously reported values. The binding curve showed a linear pattern in the range of 10 ^-10 to 10 ^-7 M ligand concentration for each FRET probe. The limit of detection (LOD) can be defined as the lowest detectable ligand concentration and is calculated by adding the ratio to the control with 5 times the standard deviation.

A comparison of the respective FRET probe, the nanoparticles are fused probe genistein (genistein), resveratrol (resveratrol) and the id for each agent ^{(daidzein) 9.6 × 10 -10,} 9.0 × 10 -10, 9.2 × 10 - It showed the EC ₅₀ value of ^10. The nanoparticle-fused probe is more sensitive than the FRET probe determined by 10 ^-22 M LOD. The dynamic working range had a concentration range of 10 to 90% of the FRET signal obtained and increased to 10 ^-18 to 10 ^-1 M for the three phytoestrogen ligands. The shape of the sigmoid curve showed a low slope and was enlarged according to the ligand concentration (Figs. 6, 7 and 8).

Experimental Example  4. FRET ER Of the probe  Decision on stability

Development of a probe for ligand detection requires a probe that produces a stable response to the ligand in the sample over an extended period of time. To investigate the FRET response to ligands with time-out, the fusion protein (3 x 10 ^-8 M) was reacted with phytoestrogen ligand (10 ^-5 M) and FRET ratios were measured at various time intervals.

Interestingly, the FRET reaction was linear and rapidly increased within 30 minutes and reached its maximum density after 6 hours of addition of genistein or daidzein (Fig. 9). Once the peak level was reached, the FRET signal remained stable for more than 30 hours at 16 ° C without reduction in density.

In contrast, a significant decrease in the FRET signal for unbound probes appeared at the same time-out, and the ligand-binding form was more stable than the unbound. The rapid increase in signal within 30 min of incubation was consistent with previous FRET studies on 4-OHT ligand. However, the FRET assay system showed a gradual increase in maximum values [genistein (after 6 hours), resveratrol (after 12 hours), daidzein (after 6 hours)].

At room temperature, each of the estrogen receptor probes showed a stable signal at and after 8 hours, and it was observed to agglomerate slowly in solution. These results indicate that the FRET sensor based on the estrogen receptor could bind the phytoestrogen ligand without significant loss in the natural twist of the protein over time.

In addition, nanoparticle fusion increased the stability of the estrogen receptor protein, and the ligand binding protein was more stable than the unbound protein.

The above results suggest that the FRET sensor fused with the nanoparticles of the present invention not only exhibits a stable reaction but also can detect phytoestrogens at a strength of 6 times or more higher than that of a conventional FRET sensor.

<110> Korea Research Institute of Biosciences and Biotechnology <120> FRET sensor fused nano-particles and detecting method for          phytoestrogen using the same <130> PA100341 / KR <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> estrogen receptor alpha ligand binding domain <400> 1 Ser Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val   1 5 10 15 Ser Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp              20 25 30 Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn          35 40 45 Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val      50 55 60 Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu  65 70 75 80 Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met                  85 90 95 Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg             100 105 110 Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu         115 120 125 Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu     130 135 140 Phe Val Cys Leu Lys Ser Ile Leu Leu Asn Ser Gly Val Tyr Thr 145 150 155 160 Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His                 165 170 175 Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys             180 185 190 Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu         195 200 205 Leu Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu His     210 215 220 Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu 225 230 235 240 Leu Glu Met Leu Asp Ala His Arg Leu His Ala Pro Thr                 245 250 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgcgctagct ctaagaagaa cagcctggcc ttg 33 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgcggatcca ctgggcgcat gtaggcggtg ggc 33

Claims (11)

FRET (fluorescence resonance energy transfer) sensor in which nanoparticles are fused to an estrogen receptor ligand binding domain to which donor fluorescent protein and acceptor fluorescent protein are bound.
The method of claim 1, wherein the phosphor protein is selected from the group consisting of a green phosphor protein (GFP), a modified green fluorescent protein (mGFP), an enhanced green fluorescent protein (eGFP), a red phosphor protein ), Enhanced red fluorescent protein (eRFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) And an enhanced blue fluorescent protein (eCFP).
The FRET sensor according to claim 1, wherein the donor fluorescent protein is an enhanced blue fluorescent protein and the acceptor fluorescent protein is an enhanced yellow fluorescent protein.
2. The FRET sensor of claim 1, wherein the donor fluorescent protein is linked to the N-terminus of an estrogen receptor ligand binding domain and the acceptor fluorescent protein is linked to the C-terminus of an estrogen receptor ligand binding domain.
7. The FRET sensor of claim 1, wherein the estrogen receptor ligand binding domain is an estrogen receptor alpha ligand binding domain or an estrogen receptor beta ligand binding domain.
6. The FRET sensor of claim 5, wherein the estrogen receptor ligand binding domain is the estrogen receptor alpha ligand binding domain of SEQ ID NO: 1.
The FRET sensor according to claim 1, wherein the nanoparticles are selected from the group consisting of quantum dots containing a carboxyl group, a polymer, a magnetic substance, and gold.
8. The FRET sensor of claim 7, wherein the nanoparticles are carboxylated polystyrene nanoparticles.
A method for detecting phytoestrogen using the FRET sensor of any one of claims 1 to 8.
10. The composition of claim 9, wherein the phytoestrogen is selected from the group consisting of flavones, flavonois, lignan, coumestan, and isoflavones. Way.
11. The method of claim 10, wherein said isoflavones are genistein, resveratrol or daidzein.

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