KR101338136B1 - Multiplex Assay for Target Substance With Enhanced Signal Amplification Using Gold Nanoparticles and System for the Same - Google Patents

Abstract

Disclosed are a method and a system for analyzing multiplexing treatment of a target material using gold nanoparticles to improve signal amplification. The analysis system of this target material includes a support, a capturer, gold nanoparticle complex, a fluorescent RNA detector, and ribonuclease H, which is fixed to the support and selectively binds to the target material. In this complex, the detector binds to the surface of the gold nanoparticles as a different material from the trapper, selectively binding to the target material. In the other region of the surface of the gold nanoparticle, a plurality of single strands of the same deoxyribonucleotide are connected. The fluorescent RNA detector is unique to the detector-gold nanoparticle complex and comprises a single stranded ribonucleotide linked to a fluorescent source at the end and a quencher for the fluorescent source, the ribonucleotide complementary to the deoxyribonucleotide Inclusive sequence. When the target material of the trapper and the detector is present in the sample, hybridization of the deoxyribonucleotide and the ribonucleotide occurs, and the ribonuclease H is hydrolyzed to release fluorescence inhibition to generate and amplify a fluorescence signal.

Description

Multiplex Assay for Target Substance With Enhanced Signal Amplification Using Gold Nanoparticles and System for the Same

The present invention relates to a method for detecting a target substance capable of multiplexing treatment and an analysis system thereof. More specifically, the present invention relates to a novel signal amplification method and system for detecting and quantifying target substances such as biomolecules using gold nanoparticles.

Since most biomolecules, that is, biomarkers, which are important clues for the diagnosis of diseases, are present at very low concentrations in the body or in a sample, an ultra-high sensitivity detection method is required. Preferred ultra-sensitivity biomarker quantification methods are not limited to any particular molecule and can be generalized to multiple diseases and samples, eliminating expensive devices or complex laboratory procedures, and eliminating false positive signals resulting from non-selective adsorption. And quantitative analysis of different biomarkers can be performed simultaneously, and the cost of such an assay should not be excessive.

Among the existing methods of measuring biomolecules or labels, immunoassays are close to the above criteria, which are very sensitive to detection, extremely selective, and capable of producing antibodies without the need for cumbersome pretreatment of the sample. In theory, it has advantages in terms of generality, which can theoretically analyze anything. The most representative of these immunoassays is an enzyme-linked immunosorbent assay (ELISA). In general, enzyme immunoassay involves the detection of an analyte or target substance using an enzyme-labeled antibody and a substrate that develops through this enzymatic reaction.

One of the features that distinguish ELISA from general immunoassay is the presence or absence of signal amplification through enzymes. Most immunoassay methods do not undergo an amplification process by enzymes. Therefore, immunoassay methods without signal amplification usually have relatively high sensitivity using fluorescence detection. The use of fluorescence signals in immunoassays allows for the labeling of antibodies that target different targets with fluorescent materials that fluoresce at different wavelengths, which facilitates multiplex signal analysis where multiple substances must be detected simultaneously. For this reason, the existing multiplexing method is mainly based on immunoassay using fluorescence. In order to perform multiplexed analysis by ELISA, different enzymes should be used for different target materials. In other words, each target should be equipped with an antibody, in addition to enzymes that selectively react to different substrates to produce distinct absorbance or fluorescence changes. For this reason, ELISA, which requires the discovery and use of various enzyme-substrate pairs as the types of analytes in a sample increase, has many technical difficulties in implementing a multiplexed analysis.

The most widely known method without enzymes using multiplexed immunoassays is the xMAP technology developed by Luminex. The technique analyzes the target material by fluorescence of the beads by immobilizing unique antibodies to the target material on microspheres containing different fluorescent materials. However, this method requires an expensive flow cytometer, which is inefficient in terms of cost compared to ELISA, which is relatively easy to miniaturize and commercialize, such as a pregnant self- kit. Apart from this, several research groups have proposed a method by which multiple immunoassays can be performed on one microplate. Swarzman et al. Immobilized an anti-immunoglobulin antibody (anti-IgG) on a polystyrene bead of about 6 μm in diameter, and then used a binding antibody of an analyte to perform immunoassay ( Anal Biochem . 1999, 271, pp. 143-151). As a means for multiplexing analysis, this method used various fluorescent substances attached to the detection antibody of the analyte. Nichkova et al. Have multiplexed fluorescence immunoassays that perform simultaneous immunofluorescence analysis of multiple substances in a sample by fluorescence intensity analysis by connecting Qdots emitting light at different wavelengths to each antibody against multiple substances to be analyzed. (multiplex-FLISA (fluorescence-linked immunosorbent assay)) was (Anal watching Lett . 2007, 40, 1423--1433). However, these methods have the disadvantage that the detection limit is less sensitive than the conventional ELISA method because there is no enzyme signal amplification process. GenTel has provided multiple immunoassay methods for analyzing fluorescence intensity by attaching different fluorescent materials directly to each antibody.

An ELISA-based multiplex assay, an enzyme-based immunoassay, is provided by Quansys Biosciences, which creates a 20 nanoliter spot array at the bottom of one well of a 96-well microplate. One point was shown to indicate the result of ELISA analysis for one target material. In this method, multiple ELISA (multiplex-ELISA) format is provided by quantifying the degree of luminescence generated by the reaction of peroxidase and substrate. This method has the disadvantage of using a plate that is pre-pointed in the microplate well and expensive equipment for image acquisition and analysis. Another multiple ELISA method is an enzyme signal amplification method that uses two antibodies, each linked to alkaline phosphatase and peroxidase, to analyze two types of substances in a sample. In this method two substances were detected simultaneously (US Published Publication 20040241776A1). In principle, this method is meaningful as a multiple ELISA method, but if more than two kinds of simultaneous analytes in a real sample increase, it is necessary to continuously find enzymes that catalyze independent reactions in addition to the peroxidase and phosphatase. Not only are there difficulties, but even the findings leave uneconomical problems with the use of different enzymes.

In short, there is still a need for multiplexed analyte quantitation methods that can simultaneously quantify different analytes while maintaining the generality, superior sensitivity, and costly analytical advantages found in ELISA. The situation is not effective.

One of the technical problems of the present invention is to provide a multiplexed analysis method capable of multiplexing multiple target materials, providing a level of sensitivity comparable to an enzyme immunoassay, a wide range of applications, and relatively inexpensive analysis. Another object of the present invention is to provide an analysis system for this analysis method.

In order to solve the above technical problem, an aspect of the present invention provides a kit for detecting a target substance. The kit includes a detector-gold nanoparticle complex and a fluorescent RNA probe unique to the detector-gold nanoparticle complex. In this kit, the detector-gold nanoparticle complex of the kit has a detector that selectively binds to the target material, which is bound to the surface of the gold nanoparticle, and the other region of the surface of the gold nanoparticle in the complex is a single strand of the same deoxyribo. A plurality of nucleotides are linked. The aforementioned fluorescent RNA detector includes a single stranded ribonucleotide sequence complementary to the deoxyribonucleotide, which is linked to a fluorescent source and a quencher for the fluorescent source. . In one embodiment, the fluorescent source is located at either end of this ribonucleotide and the quencher is located at the other end of the ribonucleotide.

When a sample containing the desired target material is added to this detection kit and selective binding of the target material and the detector is made, a hybrid double strand is formed between the deoxyribonucleotide sequence and the ribonucleic acid sequence of the fluorescent RNA detector by adding a fluorescent RNA detector. can do. Hydrolysis of the ribonucleic acid sequence in this hybrid double strand with ribonuclease H (RNase H) separates the fluorescent source interacting with the quencher in the fluorescent RNA detector, thereby emitting a fluorescent signal. On the other hand, in the state where the hydrolysis of the ribonucleic acid sequence by the ribonuclease H does not occur, the fluorescence signal is suppressed by the action of the quencher.

In one embodiment of the target detection kit, since the kit contains different types of detector-gold nanoparticle complexes, it is possible to simultaneously analyze as many kinds of target substances as the types of the complexes. Such a plurality of target material detection kits contain different types of detector-nanoparticle complexes, and each type of complex contains a fluorescent RNA detector unique to it. At this time, there is no coincidence between the detectors and the deoxyribonucleotides between the detector-gold nanoparticle complexes and the different detector-gold nanoparticle complexes of different types in the target detection kit. In addition, two fluorescent RNA detectors unique to different types of detector-gold nanoparticles, respectively, are not the same between ribonucleotides and each other.

In another aspect of the invention, a system for analyzing a target material is provided. The system comprises a support, a capturer immobilized on the support and selectively binding to a target material, the aforementioned detector-gold nanoparticle complex, the aforementioned fluorescent RNA detector and ribonuclease H do. In this assay system the trap is a substance which is not the same as the detector. In one specific embodiment of the invention said capture and detector are different antibodies that selectively bind to the same target substance.

Another aspect of the invention provides a method for analyzing a target substance in a sample. This method comprises (1) a capture step of selectively binding to the target substance to be detected and contacting the sample with a capture antibody immobilized on a surface thereof, and (2) the detector from the detector-gold nanoparticle complex described above. A binding step of binding the phosphorus detection antibody-gold nanoparticle complex to the surface subjected to the capture step, (3) a washing step of washing the surface subjected to the binding step, and (4) the aforementioned fluorescent RNA detection unique to the target material A hybridization step of adding a ruler to the surface subjected to the washing step under conditions suitable for forming double strands between the ribonucleotide and the deoxyribonucleotide, (5) a hydrolysis step of adding RNase H to the surface subjected to the hybridization step, and (6) the And a fluorescence measurement step of irradiating the surface subjected to the cutting step with light of an excitation wavelength band of a fluorescence source and measuring fluorescence intensity. .

In another aspect of the present invention, there is provided a method of calibration of target substance concentration and fluorescence generation relationship. This calibration method comprises the steps of (a) preparing a plurality of sample solutions of the target substance having known concentrations, and performing the above-described method of analyzing the target substance for each of the sample solutions, and (b) Calculating a calibration curve from the fluorescence measurement value and the concentration of the known target substance.

The target material is not particularly limited in the present invention, but in one specific embodiment, the target material is a biomolecule.

Using the target material analysis system and analysis method of the present invention, multiplexing of multiple target materials can be performed with sensitivity, accuracy, and reproducibility comparable to ELISA. In addition, the target material analysis system and analysis method of the present invention enable general multiplexed analysis of target materials without being limited to any specific design or some target materials.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram which shows the operation principle of the multiple target substance analysis system which concerns on one Embodiment of this invention. In FIG. 1, the capturer and the detector are different antibodies to the target material and generate distinct fluorescent signals using RNA fluorescence detectors unique to the target material, AFP and h-FABP, respectively.
Figure 2 is a target material analysis method (right) and prior art enzyme immunoassay (left) according to an embodiment of the present invention, RNase H signal using a biotinylated detection antibody and streptavidin instead of the detector-gold nanoparticle complex It is a schematic diagram which shows the structure of the amplification method (middle).
3 is a deoxyribonucleotide concentration and the deoxyribonucleotide concentration showing the intensity of fluorescence emitted from the fluorescent source isolated from the fluorescent RNA detector by incubating the fluorescence RNA detector according to the present invention after culturing the fluorescent RNA detector according to the present invention Calibration curve of fluorescence intensity.
Fig. 4 is a quantitative curve showing the concentration and the intensity of fluorescence detected using AFP 4a or h-FABP 4b as a target substance. In Figure 4, the triangle is an analysis method according to an embodiment of the present invention, the hollow circle is a RNase H signal amplification method using a biotinylated detection antibody and streptavidin instead of the detector-gold nanoparticle complex, the hollow circle is Enzyme immunoassay is shown.
FIG. 5 shows a quantitative curve between the concentration of the target substance and the measured fluorescence intensity at the same time quantifying AFP and h-FABP by the assay method according to one embodiment of the present invention. Circles in FIG. 5 represent AFP and triangles represent h-FABP, respectively.

Hereinafter, the present invention will be described in more detail. In interpreting the terms or words used in the present specification and claims, it is to be understood that the interpretation of terms or words used herein is necessarily conventional, based on the principle that the inventor can properly define the concept of the term to describe its invention in the best way It is to be understood that the invention is not to be interpreted as being limited only by the precautionary sense, but should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention as described in the present specification.

The present invention relates to an analysis method and a target analysis system capable of simultaneously analyzing various target substances in one sample by using signal amplification by gold nanoparticles and ribonuclease H. It also discloses a target detection kit that forms an integral part of the assay system.

In one aspect of the invention, a kit for detecting a target is provided. The target detection kit includes a detector-gold nanoparticle complex and a fluorescent RNA probe inherent in the detector-gold nanoparticle complex. It includes. In addition, a plurality of single strands of the same deoxyribonucleotide are connected to the surface of the gold nanoparticle constituting the complex in a region different from where the detector is bound.

In the present invention, the detector is a constituent of the detector-gold nanoparticle complex, which is optionally, preferably specific, with any target material in both when present as part of the complex within the complex or alone alone with the complex. It is a substance that can be combined. For example, the detector may be an antibody to the target substance, an enzyme or derivative thereof based on the target substance, a receptor having the ligand as the target substance, a small organic molecule such as a ligand that selectively binds to the target substance, or the like. to be.

The target material to be analyzed in the present invention is not particularly limited to small organic materials, inorganic materials, polymer materials, metal ions and the like. In one embodiment the target substance is a biomolecule. In the case of biomolecules, there are various examples showing specific binding properties, which is one example that is a good target material of the present invention. Among the biomolecules, there are no limitations and all biomolecules are possible, including but not limited to autoantibodies, ligands, natural extracts, peptides, antigens, antibodies, non-antibodies, carbohydrates, carbohydrate-protein conjugates. ), A lipid, a synthetic drug, a natural drug, a metabolite, a genome, a virus and a virus-producing material, and a bacterium and a virus-producing material, but may not be limited thereto. Biomolecules as such target substances may be included alone, in mixtures or with several other target substances in any form of sample, such as cell lysate, patient blood samples, tissue samples, and the like. have.

In one specific embodiment of the invention said target substance is an antigen of an antibody.

In one preferred embodiment of the invention the target material is immobilized for effective analysis of the target material. The fixing of the target material encompasses the case where the target material itself is directly fixed to the surface of the solid phase, or the target material is fixed to the surface of the solid phase through any medium. It can also be used by pretreatment which fixes a sample to the surface of a solid phase. In one embodiment this medium selectively binds to the target material and is a material different from the detector, which is immobilized on the surface of the solid phase. For this purpose, the surface of the solid phase may be modified. The means for immobilizing the target material is preferably specific, but is not necessarily specific as long as the detector-gold nanoparticle complex of the invention can selectively, preferably specifically bind to the target material. The method of fixing the target material on the surface of the solid phase forms a covalent bond between the target material and the surface or the modified surface, or by using a non-covalent bond (for example, a bond using an intermolecular force such as hydrogen bond). Either way.

When the target material is immobilized, the detector-gold nanoparticle complex of the present invention can be immobilized by binding to the target material as well. Thus, non-selective binding or adsorbate can be removed by washing after fixing the complex. This has the advantage of greatly reducing false positives. The use of immobilized target material can be particularly effective in diagnosing liquid samples, such as body fluids, as compared to when the target material is present in the form of a mixture in a sample in solution. This is because a biological sample solution such as a bodily fluid may have ribonuclease (RNase), and thus, a fluorescent RNA detector may be cleaved to give a false positive signal regardless of whether or not it specifically binds to a deoxyribonucleotide in the complex.

The detector may be used as long as it selectively binds to the target material under conditions complexed with gold nanoparticles. The selective bond may be a non-covalent bond such as van der Waals attraction, an electrostatic attraction, hydrogen bonding, salt bridge formation or a covalent bond such as disulfide linkage. Any target material and detector capable of binding by specific interactions, for example by hydrogen bonding, or by highly selective chemical reactions, is the subject of the present invention. In one embodiment of the invention, the selective binding of the detector to the target substance is non-covalent. Some examples of combinations of detectors for the aforementioned target substances include antigens and antibodies, substrates (and derivatives thereof) and enzymes (and enzyme derivatives), enzymes and inhibitors, ribozymes and substrates, and non-peptide small organic substances and combinations thereof. Protein, a pair of complementary nucleic acids, an aptamer and a target molecule to which the aptamer binds, a receptor and its ligand, a binding protein that recognizes the nucleic acid sequence and the nucleic acid sequence, a binding protein that recognizes the peptide sequence and the peptide sequence There is this.

In one embodiment of the kit of the invention the detector is an antibody against a target substance. In one embodiment of the kit of the invention the detector is a protein, not an antibody, or a small organic molecule. For example, a small organic molecule such as biotin and avidin (avidin), and a small organic molecule as a target of the protein receptor, and the detector may be a non-antibody protein that specifically binds to it. Of course, it is also possible to configure the target material as a non-antibody protein and the detector as a small organic molecule. For example, a combination of FKBP12 protein and a small organic molecule, the immunosuppressant tacrolimus, cyclophilin protein and drugs cyclosporin, glutathione and glutathione-S-transferase (glutathione S) -transferase). Furthermore, triple pairs such as the FKBP12 protein, the small organic molecule rapamycin, and the rapamycin target protein (target-of-rapamycin, TOR) that bind to the rapamycin-FKBP12 complex, can be extended to the capturer described below. Also can be mentioned.

Alternatively, in another embodiment of the present invention, the target material is an inorganic ion, such as a crown ether or a cryptand, and a cation specifically binding thereto, and the detector may be composed of organic molecules. In addition, in the case of enzymes and their irreversible inhibitors (e.g., organophosphorus compounds such as sarin and acetylcholinesterase), specific chemical reactions occur between the detector and the target material, resulting in covalent bonds. It can also be configured to be connected to each other through.

In another embodiment of the detection kit of the present invention, the detector is a sugar molecule or a nucleic acid.

In each pair of various combinations described above, whether the detector and the target substance correspond to any of the two substances in the pair does not depend on the order in which the pairs are described. In other words, for example, a combination of a receptor and a ligand may be a detector or a ligand may be a detector. The average person skilled in the art can specifically select the appropriate combination in the above-described order according to the conditions such as cost and constraints of realistic technical means such as chemical reactions required for the preparation of the complex, desired detection sensitivity and accuracy.

Gold nanoparticles in the present invention is a particle of gold having a diameter of a nanometer scale, the surface may be modified as described later herein. These gold nanoparticles can be combined with a detector described later to produce a detector-gold nanoparticle complex. Using gold nanoparticles makes it easier to use a variety of means to amplify a signal. In addition, gold nanoparticles can be easily manufactured with a narrow particle size distribution, as well as a large number of molecules for signal transmission on the surface thereof, thereby increasing the sensitivity of the analytical method.

In the present invention, the shape and size of the gold nanoparticles are not particularly limited and may be any shape and size commonly used in the art. In addition, the gold nanoparticles used in the detector-gold nanoparticles of the present invention may be prepared by a method commonly used in the art, and it is appropriate to have such a conventional particle size distribution and distribution of shapes. For example, the gold nanoparticles may be spherical. For example, the size of the gold nanoparticles may range from about 2 nm to about 100 nm on average. In a more specific embodiment the size of the gold nanoparticles can range from about 5 nm to about 50 nm. In even more specific embodiments the size of the gold nanoparticles may range from about 10 nm to about 30 nm. The size of the gold nanoparticles may be appropriately defined according to the shape of the nanoparticles. For example, if the gold nanoparticles are spherical, their diameters are large, and if the gold nanoparticles are non-spherical, they have the longest dimension. Can be defined

In one specific embodiment of the detection kit of the invention the gold nanoparticles in the detector-gold nanoparticle complex are spherical. In more specific embodiments, the spherical gold nanoparticles have an average diameter of about 5-30 nm.

In the detector-gold nanoparticle complex, the detector is associated with gold nanoparticles. The interaction between the detector and the gold nanoparticles may be covalent or non-covalent. In one specific embodiment of the present invention, the detector binds to the gold nanoparticles of the complex in an attractive interaction rather than covalently. The attractive interaction of such non-covalent bonds can be adsorption, for example. In one specific embodiment of the invention, the detector is an antibody and binds to the gold nanoparticles in a complex, rather than covalently, in a manpower interaction. The binding of antibodies to attractive gold interactions on gold nanoparticle surfaces is well known in the art and is not described here.

The gold nanoparticles constituting the detector-gold nanoparticle complex of the present invention have a detector bound to the surface thereof, and deoxyribonucleotides are linked to other regions of the surface not bound to the detector. In the detection kit of the present invention, such deoxyribonucleotides have a unique base sequence for each detector, so that the detector-gold nanoparticle complex of the present invention, together with a detector that selectively binds to the target material, is identified to be unique for each target material. It acts like a barcode. Thanks to the presence of deoxyribonucleotides that are unique for each detector (and therefore unique for each target material), the present invention enables simultaneous multiplexing of multiple target materials. In addition, since the deoxyribonucleotide forms a DNA-RNA hybrid strand with a fluorescent RNA detector to be described later, it forms a condition for cleavage by RNase H and generation of a fluorescent signal.

In one embodiment, the deoxyribonucleotides of the detector-gold nanoparticle complex of the present invention may further have a functional group that is not present in the natural nucleotide to link to the gold nanoparticle surface. And the deoxyribonucleotide may further comprise a linking portion for connecting between the functional group and the deoxyribonucleotide base sequence. In one embodiment, the surface of the gold nanoparticles of the composite may be modified with the above-described linking functional group. For example, at one end of the deoxyribonucleotide, a functional group known to form a bond by modifying the surface of the gold nanoparticle such as a thiol group may be provided. For example, the thiol group can be connected to the deoxyribonucleotide directly or through the linkage to the base sequence of the deoxyribonucleotide in the form of an alkylenethiol group. In this case, the deoxyribonucleotide is linked to the surface of the gold nanoparticles by a gold (Au) -sulfur (S) bond.

In one specific embodiment, the linking portion of the deoxyribonucleotide may include a functional group selected from an alkylene group, an ether group, an arylene group, an aralkylene group, and an alkaliylene group. In a more specific embodiment, the linking portion of the deoxyribonucleotide comprises an ethylene glycol repeat unit. In a more specific embodiment, the connecting portion is polyethylene glycol having 6 to 18 repeating units.

In the detector-gold nanoparticle complex of the present invention, the length of the deoxyribonucleotide nucleotide sequence is greater than or equal to a length that can serve as a "barcode", and thus can be distinguished, and the DNA-RNA under the active conditions of the fluorescent RNA detector and RNase H. The length which can form a hybrid double strand is sufficient and it does not specifically limit. For example, the length of the nucleotide sequence is preferably 6 to 20 nucleotides. More specifically, the length of the nucleotide sequence is preferably 10 to 15 nucleotides. If the base sequence is less than 6 nucleotides in number, there is a possibility that the discrimination may be inferior or hybrid duplex formation may not be smooth, and even more than 20, the effect of further improving the discrimination and hybrid duplex formation is insignificant compared to 20 cases. Do.

In order to prevent a reaction inhibitory effect that is hindered by RNase H action due to the proximity between the gold nanoparticle and the deoxyribonucleotide, the deoxyribonucleotide is 5 to 5 'at the 5' end, in addition to the site of hybridization with the fluorescent RNA detector. 50, preferably 10 to 30 bases (A, T, C, or G) may further comprise repeated base repeat sequences. In this case, since the G repeat sequence may form an unwanted secondary structure such as a G-quadruplex in G-4, thereby inhibiting the enzymatic reaction, the nucleotide repeat sequence may consist of only A, T or C. May be, but is not limited thereto.

The deoxyribonucleotide density on the surface of the gold nanoparticles constituting the detector-gold nanoparticle complex of the present invention is at least one per gold nanoparticle, which is linearly or nearly linearly proportional to the effective amplification of the fluorescence signal and the concentration of the target substance. As the fluorescent signal can be expressed and the density is higher, the amplification of the fluorescent signal is increased. Two or more deoxyribonucleotides may be contained on the surface to further increase the amplification of the signal.

The fluorescent RNA detector in the target substance detection kit of the present invention is paired with the complex and comprises a single stranded ribonucleotide sequence complementary to the base sequence of the deoxyribonucleotide in the detector-gold nanoparticle complex. Fluorescent RNA detectors can thus form DNA-RNA hybrid double strands with the deoxyribonucleotides under appropriate conditions. In addition, the fluorescent RNA detector has a quencher and a fluorescence source connected to the ribonucleotide, so that the fluorescent source does not fluoresce even when the fluorescent source receives an excitation wavelength, but the ribonucleotide sequence is destroyed and emits a fluorescent signal when the quencher and the fluorescent source are separated. It also plays the role of signal generator and sensor. Destruction of such ribonucleotide sequences can be accomplished using ribonuclease H (RNase H), which hydrolyzes ribonucleotide portions in a DNA-RNA hybrid double strand. The ribonucleotide sequence of the fluorescent RNA detector is unique to either detector-gold nanoparticle complex and can thus be designed to have a unique sequence for each target material and thus emit a different fluorescence wavelength than the target material.

In one embodiment of the invention, the fluorescence source and quencher connected to the fluorescent RNA detector are located at both ends with ribonucleotides in between. For example, if the fluorescent source is located at the 5 'end of the ribonucleotide sequence of the fluorescent RNA detector, the quencher may be located at the 3' end.

Fluorescent source in the fluorescent RNA detector of the present invention may be used as commonly used in the art, there is no particular limitation. Some examples of suitable fluorescence sources are the coumarin series (eg 7-hydroxycoumarin, 4-methyl-7-hydroxycoumarin, etc.), the xanthene series (eg ROX, TET, Texas Red, Fluoresce). Phosphorus (tetramethylodamine, etc.), cyanine (e.g., Cy3, Cy5, etc.), Bodipy series (e.g., Bodipy FL, Bodipy 530, Bodipy R6G, Bodipy TMR, etc.), Alexa Fluoride Alexa Fluor series (e.g., Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 546, Alexa Fluor 350, Alexa Fluor 555, Alexa Fluor 532, etc.), and DyLight Fluor Series of fluorescent sources (eg, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, etc.).

As a quencher in the fluorescent RNA detector of the present invention is commonly used in this field, as long as it can effectively suppress the emission of the adopted fluorescent source, it is not particularly limited. Two fluorescent RNA detectors using different fluorescence sources may use the same quencher as long as their wavelengths overlap. Some examples of the quencher include dabsyl compounds, QSY-based materials (eg QSY-35, QSY-7, QSY-9, QSY-21, etc.), BHQ-based materials (eg BHQ-1, BHQ- 2, BHQ-3, etc.) and fluorescence resonance energy transfer (FRET) materials (e.g. cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), green fluorescent protein ( green fluorescent protein (GFP), etc.).

In one embodiment of the present invention, different types of detector-gold nanoparticle complexes can be used to quantitate a plurality of target substances simultaneously, thus employing a unique fluorescent RNA detector for each complex. In this case, the different types of the detector-gold nanoparticles are different from each other when the two different types of detector-nanoparticle complexes are not identical to each other, and deoxyribonucleotides are not identical to each other. Means that. And if there are two different types of detector-gold nanoparticle complexes, the ribonucleotide sequences and the fluorescent sources do not coincide between the two fluorescent RNA detectors unique to each type. The above-described detectors, deoxyribonucleotide sequences, ribonucleotide sequences and fluorescers may not be identical between two heterogeneous complexes or between two detectors and may result in cross-hybridization of nucleic acid strands that may generate false positive signals. It is preferable that there is sufficient discrimination power, that is, sequence difference or wavelength difference so that wavelength overlap between fluorescence sources does not occur.

Another aspect of the present invention provides an analysis system of a target material including a detector-gold nanoparticle complex of the detection kit and a fluorescent RNA detector.

This assay system comprises a support, a capturer that is fixed to the support and selectively binds to the target material, the aforementioned detector-gold nanoparticle complex, the aforementioned fluorescent RNA detector and ribonuclease H (RNase H). In the target substance analysis system of the present invention, the capturer is a substance which is not the same as the detector.

In the present invention, the capturer is not particularly limited as a substance which selectively binds to the target substance, preferably specifically, and can bind to the same target substance as the detector, but is fixed to the support as a substance which is not the same as the detector. It can be anything that can be done. In one embodiment of the invention the capturer selectively binds to a region different from the detector in the target material.

The target material that can be analyzed in the target material analysis system of the present invention is the same as described above for the target material detection kit.

As for the capturer in the target substance analysis system of the present invention, the above description of the detector of the target substance detection kit is applied as it is.

For example, the combination of the target substance and the capture agent or the combination of the capture agent and the target substance may include antigens and antibodies, substrates and enzymes, enzymes and inhibitors, ribozymes and substrates, non-peptide small organic substances and their binding proteins, complementary Pairs of nucleic acids, aptamers and their target molecules, receptors and their ligands, nucleic acid sequences and binding proteins that recognize the nucleic acid sequences, peptide sequences and binding proteins that recognize the peptide sequences. In one embodiment of the invention the capturer is an antibody against a target substance.

In the target substance analysis system of the present invention, the support provides a fixed surface capable of fixing the trap. Thanks to this support and the trapper, the target material in the liquid sample can be fixed to prevent false positive signal generation as described above. In the present invention, the support is a solid phase, characterized in that the capturer can be fixed to the surface through covalent or non-covalent bond.

In the analysis system of the present invention, a material of the support may be a conventional material, that is, glass, silicon, metal, semiconductor, or plastic. In one embodiment the support may be any solid support commonly used in solid based immunoassays. For example, a well of a microtiter plate widely used in the conventional ELISA method may be used as a support, for example, a flat bottom plate or a U-shaped bottom plate depending on the shape. bottom plate). There are avidin coated plates, streptavidin-coated plates, glutathione coated plates, anti-glutathione-S-transferase (anti-GST) coated plates, etc., depending on whether the ligand or protein is coated. Polystyrene plate, polypropylene plate and the like can be used, but is not limited thereto.

In the present invention, the method of fixing the capturer to the support is not particularly limited. For example, all methods commonly used in solid state immunoassay methods are possible. For example, in one specific embodiment of the present invention it can be fixed by coating the trap on the support.

One embodiment of the assay system of the present invention does not require any (pre) treatment of the sample to determine whether the sample contains such a target substance or to measure the concentration of the target substance in the sample. In other words, the sample can be used as is without the need for (pre) treatment of the sample for analysis, or can be analyzed immediately by pretreatment only by diluting the sample in a suitable buffer solution. For example, when the target material is an antibody and the detector is an antigen against it, such as when the target material is a ligand and the detector is its receptor.

In the assay system of the present invention, RNase H may be extracted or purified from cells or expressed and purified in the form of recombinant proteins. Some examples of RNase H include, but are not limited to, RNase H (gene accession number: V00337, coding region: 243-707, protein accession number: CAA23620) derived from E. coli.

In order to simultaneously multiplex the plurality of target substances, the assay system may contain different capture agents or more, therefore, detector-gold nanoparticle complexes, and fluorescent RNA detectors according to the complexes. That is, each of the capturer, the detector, and the deoxyribonucleotide depends on the type of the target substance, and the capture antibodies that differ from the target substance, the detectors, between the detector and the trapper, the deoxyribonucleotides, and ribonucleotides. The differences are different. By doing so, the intensity of the fluorescence signal in any one wavelength band can be designed so as to be proportional to the amount of one target material.

Another aspect of the present invention provides a method for analyzing a target substance in a sample. This method of analysis

(A) a capture step of selectively binding to the target material to be detected and contacting the sample with a capture antibody immobilized on a surface;

(B) a detection antibody-gold nanoparticle complex in which a detection antibody that selectively binds to the target material at a different site than the capture antibody is bound to the surface of the gold nanoparticles, wherein the detection antibody-gold nanoparticle complex is present at different regions of the surface of the gold nanoparticles; A binding step of binding the detection antibody-gold nanoparticle complex having a plurality of identical single stranded deoxyribonucleotides linked to the surface subjected to the capture step;

(C) a washing step of washing the surface passed through the bonding step;

(D) a fluorescent RNA detector comprising a single stranded ribonucleotide sequence complementary to said deoxyribonucleotide, said fluorescent RNA detector comprising a fluorescent source at one end thereof and a quencher for said fluorescent source at the other end thereof; A hybridization step applied to the surface subjected to the washing step under conditions suitable for double strand formation between the ribonucleotide and the deoxyribonucleotide;

(E) nucleotide hydrolysis step of adding RNase H to the surface subjected to the hybridization step; And

(F) a fluorescence measurement step of irradiating the surface subjected to the cutting step with light of the excitation wavelength band of the fluorescence source and measuring the fluorescence intensity.

Steps (a) and (b) can be regarded as a special case where both the trap and the detector are antibodies in the method for analyzing a target substance to which the assay system of the present invention is applied.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram explaining one specific embodiment of the analysis method of this invention. This embodiment is equipped with a unique detector-gold nanoparticle complex, deoxyribonucleotide and fluorescent RNA detector for each target material so that both target materials can be analyzed simultaneously. On the support indicated by the black horizontal bar in Fig. 1, a capture antibody (capture antibody), which is an antibody to the target material, is immobilized, and the surface of the support on which the capture antibody is not immobilized is used to inhibit an inhibitory protein to prevent nonspecific binding. For example, bovine serum albumin (BSA). Ellipses labeled BSA in FIG. 1 represent bovine serum albumin. In the assay system of FIG. 1, the target substances are alpha fetoprotein (AFP) and heart fatty acid binding protein (h-FABP), each of which is depicted as being captured in the corresponding capture antibody. The assay system of FIG. 1 detects fluorescent RNA detectors (first fluorescent RNA detector and second fluorescent RNA detector) that emit fluorescence signals (first fluorescence and second fluorescence in the wavelength band) distinguished for AFP and h-FABP. And deoxyribonucleotides (first and second deoxyribonucleotides) complementary to these fluorescent RNA detectors. Selective binding of the target material to the capture antibody immobilizes the conjugate of the capture antibody and the target material on the support. This conjugate can be detected with a detector-gold nanoparticle complex according to the invention. In FIG. 1, the detector is a detection antibody that is an antibody like the capture agent. Single strands of deoxyribonucleotides extend on the surface of the gold nanoparticles (red spheres) to which the detection antibody binds. In the embodiment shown in FIG. 1, this deoxyribonucleotide is linked with gold-sulfur bonds to the gold nanoparticles of the detection antibody-gold nanoparticle complex, as seen in the enlarged left-left view of FIG. 1. In one more specific embodiment of the invention such deoxyribonucleotides may take the structure of a base sequence-linking-alkylenethiol functional group, for example polyethylene glycol having six ethylene glycol repeating units may be used as the linking portion.

When the detector-gold nanoparticle complex binds to the target material fixed with the trap, the non-specifically bound materials can be washed off by washing the support.

In one embodiment of the analytical method of the present invention, the step of breaking the gold-sulfur bond between the nanoparticle and the deoxyribonucleotide by adding a reducing agent to the deoxyribonucleotide linked to the detector-gold nanoparticle complex through a thiol group at either end as described above. You can put more. When this gold-sulfur bond is broken, the deoxyribonucleotide is released from the detection antibody-gold nanoparticle complex. The reduction treatment of this deoxyribonucleotide can be carried out before or simultaneously with the nucleotide hydrolysis step of (e) above. In other words, if the deoxyribonucleotide is removed from the surface of the gold nanoparticles before the (e) step by treatment with a reducing agent, hybridization with the fluorescent RNA detector or RNase H hydrolysis may be prevented by the gold nanoparticles or nearby deoxyribonucleotides connected to the surface of the gold nanoparticles. Can be prevented. On the other hand, deoxyribonucleotide release from gold nanoparticles may be performed after the addition of RNase H in step (e). Alternatively, RNase H treatment, DNA-RNA hybrid strand formation, and deoxyribonucleotide release can occur simultaneously.

DTT treated with an arrow on the upper portion of the gold nanoparticles of FIG. 1 stands for dithiothreitol, a reducing agent, and indicates the treatment of such a reducing agent. To burn. As an example of DTT, dithiothreitol, β-mercaptoethanol, and mixtures thereof may be used as a reducing agent for deoxyribonucleotide cleavage.

Deoxyribonucleotides released from the surface of gold nanoparticles by reducing agent treatment can form hybrid double strands with fluorescent RNA detectors. At this time, the concentration of the fluorescent RNA detector is not particularly limited as long as it can generate a measurable fluorescent signal, and may take any value. If typical, for example, a concentration of RNA detector of 100 nM or more is appropriate. In the hybrid double strand of the deoxyribonucleotide and the fluorescent RNA detector described above, the fluorescent source labeled with the fluorescent RNA detector is labeled in the same detector, and fluorescence is quenched due to the quencher located close to the fluorescent source. Addition of an RNA degrading enzyme (a pie slice cut out in FIG. 1), such as RNase H, to trigger a hydrolysis reaction of an RNA site in the hybrid double strand, and the fluorescent material and the quencher are separated from each other. . The RNA degrading enzyme may be activated using an appropriate buffer solution, and the buffer solution may be used as is commonly known in the art.

In other words, the amplified fluorescent signal can be obtained by treating the hybrid double strand with a sufficient amount of RNase H for a sufficient reaction time. In Fig. 1, the circuit diagrams of the right and upper left parts show the fluorescence signal amplification cycle by RNase H. Multiple hybrid double strands will form from a plurality of deoxyribonucleotides present in each detector-gold nanoparticle complex, and fluorescence will occur whenever RNase H hydrolyzes the hybrid strand. If sufficient fluorescent RNA detector is added, the cycle of such hybridization-RNA hydrolysis-luminescence is repeated as shown in FIG.

Since the amount of the target substance bound to the detector is proportional to the amount of the target substance in the sample, the removal of nonspecific binding by washing or separation may make the aforementioned hybrid double strand formation proportional to the amount of the target substance in the sample. In other words, the fluorescence signal intensity obtained by the RNase H treatment can be proportional to the amount of the target substance in the sample, so that the detection kit of the present invention can also quantify the target substance in the sample.

In one embodiment of the present invention, said binding cleavage step of deoxyribonucleotide by reducing agent treatment is performed before the hybridization step of (d). The hybridization step of (d) and the addition of RNase H and (d) treatment of (e) may occur simultaneously.

In a more specific embodiment of the present invention, the addition of the fluorescent RNA detector of step (d) and the addition of RNase H of step (e) may occur simultaneously. Fluorescent RNA detectors can be included in the enzyme action buffer of RNase H to allow both ribonucleotide cleavage and DNA-RNA hybridization to occur simultaneously. In even more specific embodiments, the addition of the fluorescent RNA detector, RNase H and the reducing agent may occur simultaneously. In other words, both the fluorescent RNA detector and the reducing agent can be added to the enzyme action buffer of RNase H.

The analytical method of the present invention may have an additional washing step in addition to the washing step (c) described above for more accurate detection. E.g

(A) between the capture step and the binding step,

(B) between the hybridization step and the hydrolysis step,

(C) a washing step may be further included in at least one of the hydrolysis step and the measuring step.

Another aspect of the present invention provides a calibration method for the relationship between target substance concentration and fluorescence generation. This calibration method

(1) preparing a plurality of sample solutions of the target substance having a known concentration, and performing the above-described method of analyzing the target substance for each sample solution to obtain fluorescence measurements;

(2) calculating a calibration curve from the fluorescence measurement value and the concentration of the known target substance.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Experimental Examples. The following examples are intended to illustrate the invention in detail and are not intended to limit the scope of the invention in any way.

Alpha fetoprotein (AFP) and heart fatty acid binding protein (h-FABP) were used as targets to verify the analytical method of the present invention, and the detector was used as a monoclonal antibody against these proteins. It was analyzed by an analysis method according to one embodiment of the present invention.

AFP and anti-AFP monoclonal antibodies were purchased from Fitzgerald, USA, Acton, Mass., And h-FABP and anti-h-FABP monoclonal antibodies, purchased from Meridian Life Science, Memphis, TN, USA. . Among the monoclonal antibodies, 10C-CR1007M4 (for AFP) and H01314M (for h-FABP) were used as captors, and 10C-CR1007M5 (for AFP) and H01315M (for h-FABP) were used as detectors.

Preparation Example 1 Preparation of Detector-Gold Nanoparticle Complex

Complexes were prepared using the detector as an anti-AFP monoclonal antibody or an anti-h-FABP monoclonal antibody. 1 mL of 10 nm gold colloid (British Biocell International, Cardiff, UK) was incubated with 14 μg of the corresponding monoclonal antibody for 30 minutes at room temperature with gentle shaking. Tween 20 detergent and pH 8.0 phosphate buffer were added to a final concentration of 0.05% and 10 mM, respectively, and 100 μL of 5'-terminal thiolated deoxyribonucleotide was added to the gold nanoparticles at 4 ° C. The surface was modified by gentle shaking. This 5'-terminal thiolated deoxyribonucleotide had a linkage in which six ethylene glycol units were inserted between the 5'-terminal thiol group and the deoxyribonucleotide sequence, and the sequence was 5'-AGCGTTGTAG-3 'for AFP detection and h-FABP detection. The dragon was 5'-AGCGTTGTAG-3 '.

20 μL of 5 M NaCl was added to the gold nanoparticle solution incubated for 1 hour, and then gently shaken at 4 ° C. overnight. 100 μL of 10% bovine serum albumin (BSA) was added to the surface modified detector-gold nanoparticle complex to stabilize for one hour. The solution was then centrifuged at 4 ° C., 18,000 × g for 50 minutes, after which the supernatant was lifted off. This centrifugation process was repeated and the final pellet was suspended in 1 mL of Tween 20 containing Phosphate buffer containing 0.1% BSA. The concentration of gold nanoparticles was measured by absorbance (wavelength 522 nm, absorbance coefficient 1.024 × 10 ⁸ M ⁻¹ cm ⁻¹ ).

Preparation Example 2 Preparation of Fluorescent RNA Detector

Fluorescent RNA detector was synthesized by Bioneer (Daejeon). Fluorescent RNA detectors for AFP were 5'-TAMRA- with Tetramethyltamine (TAMRA) as the fluorescent source at the 5 'end and Black Hole Quencher 2 (BHQ2) (BHQ2) from the US Biosearch Technologies as a quencher at the 3' end. CCCAUAGAGU-BHQ2-3 'was used, and 5'-Cy5-CUACAACGCU-BHQ2-3' with Cy5 as the fluorescent source at the 5 'end and BHQ2 as the quencher at the 3' end was used as the fluorescent RNA detector for h-FABP. Used.

Preparation Example 3 Deoxyribonucleotide Number Measurement of Detector-Gold Nanoparticle Complex

The number of deoxyribonucleotides present on the surface of one gold nanoparticle in the deoxyribonucleotide-linked detector-gold nanoparticle complex was measured using the RNase H reaction.

100 μL of RNase H reaction solution (100 nM of fluorescent RNA detector of Preparation Example 2, Protector RNase inhibitor (PRI) (Roche, Mannheim, Germany) 0.4 unit, RNase H 6 unit, Tris-HCl 40 mM, MgCl ₂ 4 mM , DTT 10 mM, BSA 0.003%, pH 7.7), serially diluted deoxyribonucleotide or gold nanoparticle complexes were incubated at 37 ° C for 15 minutes, followed by fluorescence intensity with Appliskan (Thermo Scientific, Woltan, Mass.) The excitation / luminescence filters were set at 544/589 and 590/675 nm, respectively, for TAMRA and Cy5. The released deoxyribonucleotide concentration was obtained from this standard linear calibration curve. The average number of nucleotides per gold nanoparticle was obtained by dividing the concentration of the deoxyribonucleotide by the gold nanoparticle concentration.

3 is a calibration curve calculated from the experimental results of Preparation Example 3. FIG. This calibration curve was obtained by performing an RNase H cleavage reaction on a deoxyribonucleotide and a fluorescent RNA detector of known concentration, and measuring the fluorescence intensity. The calibration curve can be used to determine the number of deoxyribonucleotides that are actually bound to the detector-gold nanoparticle complex. The hybrid double strands of fluorescent RNA detector and deoxyribonucleotide are separated from the complex by a reducing agent treatment method. After measuring the intensity of fluorescence generated by reacting with RNase H, the deoxyribonucleotide concentration corresponding to the fluorescence intensity can be determined by estimating the calibration curve. The number of deoxyribonucleotides covalently linked to 5 'sulfur atom and gold on the surface of gold nanoparticles having an average size of 10 nm obtained in Preparation Example 1 was about 75 per gold nanoparticle.

Target material analysis method according to an embodiment of the present invention using the detector-gold nanoparticle complex prepared as described above, the ELISA of the prior art, and the detector and RNase H without the detector-gold nanoparticle complex as a reference example. Signal amplification analysis method using a comparison was compared. 2 is a schematic diagram showing the configuration of three methods used in this comparative experiment. The signal amplification analysis method using the detector and RNase H without the detector-gold nanoparticle complex (in the middle of FIG. 2, “Reference Example Analysis Method”) is a biotinylated detection antibody to streptavidin instead of the detector-gold nanoparticle. And a conjugated biotinylated deoxyribonucleotide was used. In all three methods, the detection signal was measured by the intensity of the fluorescence signal.

Detector -Analysis using gold nanoparticle complex

Phosphate buffered saline (PBS) of the trapper (10C-CR1007M4 (for AFP) and H01314M (for h-FABP)) for each well of a Maxisorp Black® 96-well microplate (Thermo Scientific, USA) 100 μL of solution (2 μg / mL) was added and incubated overnight at 4 ° C. Each well was then washed three times with phosphate buffered saline (300 μL), 200 μL of blocking buffer was added and then incubated for 1 hour at room temperature. Wash three times with 100 μL of Tween 20-containing phosphate buffered saline (PBST) (0.05% Tween 20 in PBS), and then analyze the target substances with different concentrations (0, 12.5, 25, 50, 100, 200 pg / mL). 100 μL of the solution dissolved in buffer (1% BSA in PBST) was added per well and incubated for 1 hour. The microplates were washed with PBST (3 × 300 μL) and then diluted detector-gold nanoparticle complexes in assay buffer (final absorbance 0.025, final concentration about 320 pM) and incubated at room temperature for 1 hour. After washing with PBST (3 × 300 μL), 100 μL of diluted fluorescent RNA detector was added to the assay buffer and incubated for 30 minutes at room temperature. After washing with PBST (3 × 300 μL), 100 μL of RNase H reaction solution was added and incubated at 37 ° C. for 30 minutes. Fluorescence intensity was measured by Appliskan (Thermo Scientific, Woltan, Mass.), With excitation / emission filter settings of 544/589 and 590/675 nm. All assays were repeated three times.

Comparative Example 1 ELISA Experiment

100 μL of 3 μg / mL capture antibody (10C-CR1007M4 (for AFP) and H01314M (for h-FABP)) was added to each well of a clear Maxisorp 96-well microplate, incubated overnight at 4 ° C, and then in phosphate buffer. Washed (3 × 300 μL). 200 μL of blocking buffer (3% BSA in PBS) was added to the wells coated with capture antibody, incubated for 1 hour at room temperature, and then washed three times with 300 μL of PBST. 100 μL of assay buffer (0.05% Tween 20 in PBST) diluted with target material at various concentrations (0, 0.625, 1.25, 2.5, 5, 10 ng / mL) was added to the wells and incubated for 1 hour at room temperature. Plates were then washed with PBST (3 × 300 μL), then 100 μL of biotinylated detection antibody (3 μg / mL of assay buffer dilution) was added and incubated for 1 hour at room temperature. Biotinylated detection antibody was incubated for 1 hour at room temperature with 70 μL of 1 mg / mL detection antibody diluted with PBS with 0.7 μL of 20 mM EZ-Link NHS-PEG12-Biotin (Rock Scientific, Rockford, Ill.) This was obtained by purification with Zeba Spin decontamination column (molecular weight exclusion point 40 kDa) of the same company. The concentration of biotinylation detection antibody was quantified by Bradford assay (Bradford assay solution from Bio-Rad, Hercules, Calif.). Subsequently, 100 μL of streptavidin-horseradish peroxidase (streptavidin-HRP, Cambridge Abcam, UK) diluted to 2 μg / mL in assay buffer after PBST washing of microplates (3 × 300 μL) was added at room temperature. Incubated for hours. This microplate was further washed with PBST (3 × 300 μL) and 100 μL of TMB substrate solution (GenDepot, Barker, Texas, USA) was added per well and incubated for 10 minutes at room temperature. 100 μL of TMB reaction termination solution (GenDepot, Barker, Texas, USA) was added per well and immediately measured for absorbance at 450 nm with a 96-well microplate reader (SpectraMax Plus ™ from Molecular Devices, Sunnyvale, CA). It was.

Reference Example 1 RNase H Signal Amplification Experiment Without Detector-Gold Nanoparticle Complex

The RNase H signal amplification experiment without the detector-gold nanoparticle complex was performed using a biotinylated detection antibody, streptavidin and biotinylated deoxyribonucleotide instead of the detector-gold nanoparticle complex (final absorbance 0.025) diluted with the assay buffer. Except for the same procedure as in Example 1.

Specifically, 100 μL of the biotinylated detection antibody diluted (3 μg / mL) in the assay buffer instead of the detector-gold nanoparticle complex after the addition of the assay buffer solution and the PBST washing of the target material of Example 1 was added to the well for 1 hour. Incubated at room temperature. As the biotinylated detection antibody, the same biotinylated detection antibody as in Comparative Example 1 was used. Subsequently, 100 μL of streptavidin, washed with PBST (3 × 300 μL), diluted with assay buffer (2 μg / mL), was added and incubated for 30 minutes at room temperature. Streptavidin was used as S4762 Sigma-Aldrich. After washing with PBST (3 × 300 μL), 100 μL of biotinylated deoxyribonucleotides (200 nM) diluted with assay buffer were added and incubated for 30 minutes at room temperature. This biotinylated deoxyribonucleotide has the same base sequence as the 5'-terminal thiolated deoxyribonucleotide of Preparation Example 1 described above, except that the thiol group is changed to biotin in its structure. Subsequently, RNase H cleavage was performed in the same manner as in Example 1.

4 and 5 show the results of comparing the three methods with respect to analysis of only one target material of AFP or h-FABP. 4 shows the correlation between the concentration of the target substance put into the experiment and the intensity of fluorescence detected. 4A is a result of measuring fluorescence intensity using AFP and FIG. 4B as a target material, respectively. In FIG. 4, the triangle shows the result of analysis of Example 1 according to an embodiment of the present invention, and the hollow circle amplifies the RNase H signal of Reference Example 1 using a biotinylated detection antibody and streptavidin instead of the detector-gold nanoparticle complex. In the method, a hollow circle represents the enzyme immunoassay of Comparative Example 1. As shown in FIG. 4A, the target material analysis method according to the present invention of Example 1 was linearly proportional to the concentration of the fluorescence signal in the same manner as the ELISA of the prior art or the reference example 1 method. In Figure 4a this linear proportional interval was from about 12.5 pg / mL to about 200 pg / mL. And such a linear proportional relationship can be seen in Figure 4b.

Comparing the limits of detection for each method based on the data shown in FIGS. 4A and 4B, it is shown in Table 1 below.

From the results shown in Table 1, the analytical method according to one embodiment of the present invention is about 70 to 100 times higher than the ELISA of the prior art, 50 depending on the same RNase H signal amplification as the present invention, but 50 than the reference example without using gold nanoparticles. You can see that it is about 150 times more sensitive.

Detector Multiplex Analysis of Target Material Using -Gold Nanoparticle Complex

The assay method of the present invention tested whether both AFP and h-FABP can be analyzed simultaneously. Capture antibodies of AFP and h-FABP were immobilized together on a microplate and were tested in the same manner as the single target substance analysis method of Example 1 except that the detector-gold nanoparticle complexes for each of them were used together.

5 is a graph showing the results of fluorescence measurements in Example 2. FIG. Circles in FIG. 5 represent AFP and triangles represent h-FABP, respectively. It can be seen from the data of FIG. 5 that the fluorescent signal is well linearly proportional to the concentration even when both target materials are simultaneously analyzed by the analysis method of the present invention.

The detection limit was measured from the experimental results of FIG. 5 to examine whether the detection limit is changed when performing multiplexing analysis on a plurality of target materials and when analyzing a single target material. The results are summarized in Table 2.

The results shown in Table 2 show that the target material analysis method of the present invention can exhibit a sensitivity similar to that of a single analysis of only the target material even when multiple target materials are simultaneously multiplexed.

Analysis of Human Serum Samples

In order to evaluate the precision and accuracy of the analytical method of the present invention, AFP and h-FABP in human serum samples were multiplexed. A percentage coefficient of variation (% CV) was used as an indicator of precision, and a percentage recovery was used as an indicator of accuracy. Eighteen spiked samples were prepared by adding 1, 3, and 9 ng / mL of AFP and h-FABP to healthy human serum, respectively, and analyzed simultaneously by the method of Example 2. The precision and accuracy of AFP and h-FABP measured on human serum samples are summarized in Table 3 and Table 4, respectively.

As can be seen from Table 3, the analytical method of the present invention was less than 10% for both AFP and h-FABP at all concentrations measured by the percent change coefficient. This indicates that the precision of the analytical method of the present invention is suitable for use in clinical diagnostics.

The accuracy of the analytical method of the present invention, expressed as a percentage recovery, ranged from 101.2% to 106.1%, indicating a suitable accuracy for use in actual analytical applications (Table 4).

Using the target material analysis method of the present invention from the above examples and comparative examples, it was confirmed that multiplexed analysis of multiple target materials can be performed without sacrificing sensitivity, accuracy, and reproducibility of the analysis.

While the present invention has been particularly shown and described with reference to certain exemplary embodiments thereof, it should be understood that the same is by way of illustration and example only and is not to be taken by way of limitation. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. .

Accordingly, all equivalents of the claims and their equivalents, as well as the embodiments described hereinabove and the appended claims, are also within the scope of the inventive concept.

Claims (25)

Antibody-gold nanoparticle complexes
As a kit for detecting a target substance comprising a fluorescent RNA probe inherent in the antibody-gold nanoparticle complex,
The antibody-gold nanoparticle complex of the kit is composed of a single strand in which an antibody selectively binding to the target material binds to the surface of the gold nanoparticle, and a plurality of deoxyribonucleotides are connected to another region of the surface of the gold nanoparticle. The polynucleotides bind,
The fluorescence RNA detector includes a single strand of polynucleotide having a fluorescence source and a quencher for the fluorescence source and a plurality of ribonucleotides connected to a terminal thereof, and a plurality of ribonucleotides connected to a plurality of ribonucleotides. And a polynucleotide comprises a sequence complementary to a single strand of polynucleotide having a plurality of deoxyribonucleotides linked thereto. The method of claim 1, wherein the target detection kit contains heterogeneous antibody-gold nanoparticle complexes, and each kind of the antibody-gold nanoparticle complex contains a fluorescent RNA detector unique to it.
At this time, any one antibody-nanoparticle complex in the target detection kit and another antibody-nanoparticle complex having different kinds thereof match each other, and a single strand of polynucleotides in which a plurality of deoxyribonucleotides are connected to each other. Not
The two fluorescent RNA detectors unique to the different kinds of antibody-gold nanoparticles are target detection kits, characterized in that the single strands of polynucleotides in which a plurality of ribonucleotides are connected to each other and the fluorescent sources are not identical to each other. . The target detection kit of claim 1, wherein the target substance is an antigen that specifically binds to the antibody. The target detection kit of claim 1, wherein the combination of the target substance and the antibody is an antigen and an antibody. The single-stranded polynucleotide having a plurality of deoxyribonucleotides linked thereto has a thiol group at either end and is connected to the surface of the gold nanoparticle by a gold-sulfur bond through a thiol group. Target Detection Kit. The linking portion of claim 1, wherein the deoxyribonucleotide comprises a functional group selected from an alkylene group, an ether group, an arylene group, an aralkylene group, and an alkaliylene group between a nucleotide region and a thiol group of a plurality of single-stranded polynucleotides. Target detection kit, characterized in that the presence. The target detection kit of claim 6, wherein the linking unit is an ethylene glycol-derived repeating unit. The method of claim 1, wherein the fluorine source is coumarin (coumarin), xanthene series, cyanine series, Bodipy (Bodipy) series, Alexa Fluor series, and DyLight Fluor (DyLight Fluor) Target detection kit, characterized in that selected from the group consisting of fluorescence source. The method of claim 1, wherein the quencher is selected from the group consisting of a dabsyl compound, a QSY-based material, a BHQ-based material, and a fluorescence resonance energy transfer (FRET) material. Kit. The target material detection kit according to claim 1, wherein the antibody binds to the gold nanoparticles by attraction interaction rather than covalent bonds. The method of claim 1, wherein the gold nanoparticles are spherical, the target material detection kit, characterized in that the average diameter of 5 to 30 nm. The kit of claim 11, wherein the surface of the gold nanoparticles has two or more single strands of polynucleotides linked by a plurality of deoxyribonucleotides to each other. delete delete delete delete As a method of analyzing a target substance in a sample,
A capture step of selectively binding to the target substance to be detected and contacting the sample with a capture antibody immobilized on a surface;
A detection antibody-gold nanoparticle complex in which a detection antibody that selectively binds to the target substance at a different site than the capture antibody is bound to the surface of the gold nanoparticle, wherein a plurality of deoxyribonucleotides are formed in different regions of the surface of the gold nanoparticle. A binding step of binding the detection antibody-gold nanoparticle complex to which the dog-linked single-stranded polynucleotide is bound to the surface subjected to the capture step;
A washing step of washing the surface subjected to the bonding step;
A fluorescent RNA detector comprising a single strand of polynucleotides having a plurality of ribonucleotides complementary to a single strand of polynucleotides linked to a plurality of deoxyribonucleotides, the fluorescent source at one end and the fluorescent source at the other end The fluorescence RNA detector comprising a quencher for the washing step under conditions in which a double strand can be formed between a single strand of polynucleotide linked to a plurality of ribonucleotides and a single strand of polynucleotide linked to a plurality of deoxyribonucleotides Hybridization step of applying to the rough surface;
A ribonucleotide hydrolysis step of adding RNase H to the surface subjected to the hybridization step; And
And a fluorescence measurement step of irradiating a surface subjected to the hydrolysis step to light of the excitation wavelength band of the fluorescence source and measuring fluorescence intensity. 18. The method of claim 17, wherein a single strand of polynucleotide to which a plurality of deoxyribonucleotides are linked has a thiol group at one end thereof, and is connected to the surface of the gold nanoparticle by a gold-sulfur bond through the thiol group.
The analysis method further comprises a cleavage step of breaking the gold-sulfur bond with a reducing agent treatment before the hydrolysis step. 19. The method of claim 18, wherein said cleaving step is performed before said hybridization step. 18. The method of claim 17, wherein the addition of the fluorescent RNA detector and RNase H occur simultaneously. 19. The method of claim 18, wherein the addition of the fluorescent RNA detector, RNase H, and a reducing agent occur simultaneously. The method of claim 18, wherein the reducing agent is selected from dithiothreitol, β-mercaptoethanol, and mixtures thereof. 18. The method of claim 17, further comprising washing the surface in at least one of (a) to (c) below:
(A) between the capture step and the binding step,
(B) between the hybridization step and the hydrolysis step,
(C) between the hydrolysis step and the measurement step. 18. The method of claim 17, wherein the fluorescent RNA detector concentration is at least 100 nM. As a calibration method of the relationship between target substance concentration and fluorescence generation,
Preparing a plurality of sample solutions of the target substance having a known concentration, and performing a target substance analysis method of claim 17 on each sample solution to obtain fluorescence measurements; And
Computing a calibration curve from the fluorescence measurement value and the concentration of the known target material.

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