KR20160067540A - Simultaneous Detection Methods of Multiple Targets in a Sample and Uses Thereof - Google Patents

Abstract

The present invention relates to a method for simultaneously detecting multiple targets within a sample, and a kit using the method. One purpose of the present invention is to provide a kit for simultaneously detecting double targets within a sample, and other purpose of the present invention is to provide a method for simultaneously detecting double targets within a sample. The method and the kit of the present invention detect two targets at the same time using (a) (i) first metal nanoparticles for target detection to surface areas of which a first targeting moiety and a first detecting single ply oligonucleotide barcode are each independently coupled and (ii) second metal nanoparticles for target detection to surface areas of which a second targeting moiety and a second detecting single ply oligonucleotide barcode are each independently coupled; and (b) two types of fluorescent oligonucleotide probes which are capable of hybridized with each of the first and second detecting single ply oligonucleotide barcode. The detection is multiply performed at the same time through a fluorescence analysis obtained through decomposition of the formed double ply oligonucleotide. Therefore, the method and the kit of the present invention are useful in molecular diagnosis of infectious diseases (particularly, virus-induced infectious diseases), and, above all, are capable of simultaneously analyzing multiple targets in a sample of interest (for instance, a small amount of blood sample collected from a patient) such that the method and kit are capable of being applied to medical uses such as in vitro diagnosis simply and effectively.

Description

[0002] Simultaneous Detection Methods of Multiple Targets in a Sample [

The present invention relates to a method for simultaneously detecting multiple targets in a sample and a kit using the same.

Although many techniques for multiple immunoassays have been developed so far, no technology has been reported to simultaneously quantitatively measure each antigen and antibody against various viral infections on a microwell plate. Although ELISA is widely used as a quantitative analysis method of microwell plate method, simultaneous multiplex analysis is impossible and there is a limit to the dynamic range for measurement. In addition, since there are various antibodies that bind to a secondary antibody such as anti-human IgG in the human body, it is impossible to generate a signal specific to the target, so that the present invention can perform a simultaneous multiple measurement by modifying the indirect assay method I want to present it.

Methods commonly used to detect disease targets, such as viral infection in blood, include nucleic acid amplification testing methods such as a branched-chain DNA signal amplification assay, a transcription-mediated amplification assay, a PCR-based nucleic acid amplification assay, and the like NAT). However, while these methods are very sensitive detection platforms, there is a disadvantage in that pre-amplification DNA preparation is essential and expensive equipment and well-trained experimenters are required. In addition, special attention should be paid to the design of the primer to prevent amplification of undesired targets. For this reason, the sample is subjected to a pre-screening process using an ELISA, and it is difficult to detect one or more targets with a general ELISA system. To overcome these problems, a multiplexed micro-bead immunoassay (MMIA) method using micro-beads is used to perform a more effective and improved screening process, but this method also requires an expensive flow cytometry analyzer. Therefore, there is an urgent need in the art for a method for simultaneously analyzing multiple biomarkers in a very simple and easy-to-execute manner in order to overcome the above-mentioned problems.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a method capable of simultaneously detecting an antigen and an antibody. As a result, the present inventors have succeeded in capturing target molecules by using metal nanoparticles (for example, gold nanoparticles) in which a targeting moiety and a single strand oligonucleotide barcode for detection are independently bound to each other on a surface thereof and then complementary to the oligonucleotide barcode The target molecule can be easily and efficiently detected by fluorescence using a fluorescent oligonucleotide probe (specifically, a fluorescent RNA probe), and two or more kinds of metals produced using different targeting moieties and oligonucleotide bar codes When two or more types of fluorescent probes complementary to the nanoparticles and the barcode are used, two or more target molecules (for example, hepatitis viruses A, B, C, D, etc.) in the objective sample are simultaneously detected or identified with high sensitivity The present invention has been completed .

Accordingly, an object of the present invention is to provide a kit for simultaneous detection of double targets in a sample.

It is another object of the present invention to provide a method for simultaneous detection of double targets in a sample.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for detecting a first target (a) a first targeting moiety and a first detectable single strand oligonucleotide barcode, Metal nanoparticles; (b) a second metal nanoparticle for target detection, wherein the second targeting moiety and the second detectable single-stranded oligonucleotide barcode are independently bonded to the surface region; And (c) two fluorescent oligonucleotide probes capable of hybridizing to the first or second detectable single strand oligonucleotide barcode, respectively, for simultaneous detection of a double target in a sample.

According to another aspect of the present invention, the present invention provides a method for simultaneous detection of a double target in a sample comprising the steps of:

(a) contacting a sample of interest with two metal nanoparticles, wherein the metal nanoparticles have (i) a first targeting moiety and a first detectable single stranded oligonucleotide barcode independently of each other (Ii) a second targeting moiety and a second detectable single-stranded oligonucleotide barcode bonded to the surface region, and (ii) a second metal nanoparticle for target detection coupled to the surface region independently of each other ;

(b) reacting the mixture of step (a) with a substrate coated with two antibodies capable of binding the first target and the second target, respectively;

(c) hybridizing the complex captured on the substrate of step (b) with two kinds of fluorescent oligonucleotide probes, wherein the two kinds of fluorescent oligonucleotide probes are hybridized with the first or second detectable single strand oligonucleotide barcode Respectively; And

(d) hydrolyzing two double-stranded oligonucleotide molecules formed by hybridization on the metal nanoparticles in the complex of step (c) to detect the probe-derived fluorescence signal.

The present inventors have sought to develop a method capable of simultaneously detecting an antigen and an antibody. As a result, the present inventors have succeeded in capturing target molecules by using metal nanoparticles (for example, gold nanoparticles) in which a targeting moiety and a single strand oligonucleotide barcode for detection are independently bound to each other on a surface thereof and then complementary to the oligonucleotide barcode The target molecule can be easily and efficiently detected by fluorescence using a fluorescent oligonucleotide probe (specifically, a fluorescent RNA probe), and two or more kinds of metals produced using different targeting moieties and oligonucleotide bar codes When two or more types of fluorescent probes complementary to the nanoparticles and the barcode are used, two or more target molecules (for example, hepatitis viruses A, B, C, D, etc.) in the objective sample are simultaneously detected or identified with high sensitivity .

Conventional immunoassay methods are either direct or indirect methods using antigen-antibody binding, and it is difficult to simultaneously detect the antigen and the antibody in a sample in which both the antigen and the antibody are present. Briefly, when the substance to be detected is an antibody or an antigen, the substance immobilized on the substrate is an antigen or an antibody, respectively. When the substance immobilized on the substrate and the substance to be detected bind to each other to form a complex, a primary detection antibody is finally used to detect the complex (i.e., antibody-antigen or antigen-antibody complex). Therefore, since the same secondary antibody (for example, anti-human IgG) is used to identify the primary detection antibody, there is a problem in that detection signals between the antibody (antigen-antibody complex and antigen-antibody complex) It is difficult to apply it to diagnosis of diseases. To overcome this and overcome more efficient detection or diagnostic methods, the present inventors devised an indirect gold nanoparticle-oligonucleotide-linked immunosorbent assay (OLISA) method and used it in the same sample Antigen and antibody could be detected / identified simultaneously (see Figures 1 and 2).

First, metal nanoparticle composites were prepared (see Fig. 1). The metal nanoparticle complex is a metal nanoparticle complex, each of which is individually associated with a unique targeting moiety and a detectable single strand oligonucleotide barcode, wherein each metal nanoparticle complex specifically binds to a different target. For example, when two kinds of targets to be detected are used, two types of metal nanoparticle composites are used, and the first targeting moiety and the second targeting moiety in the metal nanoparticle composite have a binding ability and specificity to different targets I have.

As a first step of the simultaneous detection method of the present invention, a target sample is contacted with metal nanoparticle composites. As used herein, the term " sample of interest " refers to any biological sample used to detect a target using the metal nanoparticle complex, including, for example, blood, plasma, serum, But are not limited to, saliva, sputum, urine, lymph, bone marrow, saliva, milk, urine, feces, ophthalmic solution, semen, brain extract, spinal fluid, joint fluid, thymic fluid, no. More specifically, it includes blood, plasma and serum.

When two kinds of targets are simultaneously detected in a target sample, two kinds of metal nano-particle composites having different structures are used. More specifically, the two types of metal nanoparticle complexes are composed of (i) a first metal nanoparticle for target detection combined with a first targeting moiety and a first detectable single-stranded oligonucleotide barcode independently of each other in a surface region, and ii) a second targeting moiety and a second detectable single-stranded oligonucleotide barcode are independently comprised of metal nanoparticles for target detection coupled to the surface region, and more specifically, (i) an antigen-targeting moiety And (ii) an antibody-targeting moiety and a second detectable single-stranded oligonucleotide barcode, wherein the first detectable single-stranded oligonucleotide barcode is bound to the surface region independently of each other, and And a second metal nanoparticle for target detection coupled to the surface region . For example, when simultaneous detection of hepatitis B and C viruses in a sample of interest, the two metal nanoparticle complexes are (i) an antigen-targeting moiety of HCV (hepatitis C virus) and a first detectable single- (Ii) a detection antibody for hepatitis B virus (HBV) - a targeting moiety and a second detectable single stranded oligonucleotide probe, wherein the oligonucleotide barcode is attached to the surface region independently of each other; The nucleotide barcode may be composed of a second metal nanoparticle for target detection coupled to the surface region independently of each other, and conversely, the HCV antibody-targeting moiety and the HBV antigen-targeting moiety may be used, respectively.

In some embodiments of the present invention, when detecting three targets simultaneously in the kit and method of the present invention, it is possible to use a third metal nanoparticle complex having a different configuration, more specifically, a third targeting moiety And a third detectable single-stranded oligonucleotide barcode are independently bound to the surface region, and a fluorescent oligonucleotide probe complementary to the third detectable single-stranded oligonucleotide barcode.

Although the kit and method of the present invention can be applied to detect three or more targets depending on the kind of the metal nanoparticle complex, the following description will be made on the case where two kinds of metal nanoparticle complexes are used, unless otherwise specified.

The term " nanoparticles " as used herein refers to particles of various materials having diameters in the nanometer range, specifically 8-100 nm, more specifically 10-50 nm, and most particularly 20-30 nm in diameter . The nanoparticles are not particularly limited as long as they are nano-sized particles. For example, nanocarbons, metal nanoparticles, metal oxide particles, quantum dots, polymer latexes, polymer nanospheres, and the like can be given. The nanoparticles used in the present invention refer to metal nanoparticles. As used herein, the term " metal nanoparticles " refers to gold, silver nanoparticles having a diameter in nanometers, specifically 8-100 nm, more specifically 10-50 nm, and most specifically 20-30 nm in diameter , And metal particles such as platinum, copper, palladium, and iron, all of which have the property of a metal. This small particle size facilitates efficient binding of the metal nanoparticles of the present invention to a target target (e.g., an antigen or antibody to a virus).

In some embodiments of the invention, the metal nanoparticles used in the invention are gold, platinum, silver, copper or palladium nanoparticles, and more specifically gold nanoparticles. The gold nanoparticles are easy to manufacture in the form of stable particles, they can be easily controlled in size, and have high biocompatibility, unlike the heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel and beryllium . For instance, gold nano-particles used in the present invention may be prepared as follows: the HAuCl ₄ with gold source, which was a sodium citrate and a reducing agent the reduction of HAuCl ₄ can be prepared gold nanoparticles. In this case, the size of the gold nanoparticles can be controlled by varying the citrate addition. That is, as the amount of citrate added increases, nucleation becomes more frequent, so that the size of gold nanoparticles may decrease. When gold nanoparticles are larger than 100 nm in diameter, their properties as nanoparticles are extinguished. In addition, since gold nanoparticles have a weak bond with a functional group such as a thiol group, It is difficult to prepare particles bound with universal binding partner molecules. Therefore, the diameter of the gold nanoparticles of the present invention is specifically 8-100 nm, more specifically 10-50 nm, and most particularly 20-30 nm.

The universal binding partner molecule used in the present invention includes a targeting moiety and a detectable single-stranded oligonucleotide barcode that are independently bound to the surface of metal nanoparticles (specifically, gold nanoparticles).

As used herein, the term " targeting moiety " refers to a known or novel material that is bonded to the surface of metal nanoparticles and can bind to the target (i.e., the target molecule). In some embodiments of the invention, the targeting moiety is an antibody, antigen, peptide, protein or fragment thereof, more specifically an antibody, antigen, peptide or fragment thereof, and more particularly an antibody, antigen or fragment thereof to be. For example, a targeting moiety that may be used in the present invention may include the F _ab and F _c sites of an antibody or antibody, and non-covalently binds to the antigen of the target via the F _ab site, The branch may be a partial region of the general electric field F _ab region within the range. As used herein, the term " F _ab site " refers to one site of an antibody that interacts with the antigen of the target. Generally, the F _ab sites of all antibodies in one class exhibit nearly identical properties.

When the targeting moiety is an antibody, a full-length antibody or a portion thereof may be used. The full-length antibodies include polyclonal antibodies and monoclonal antibodies and can be prepared by methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference.

The term " single strand oligonucleotide barcode " used in the present invention is a moiety used for detection of metal nanoparticles bound to a target, and includes a fluorescent oligonucleotide probe of the present invention (for example, RNA Oligonucleotide having a 'barcode' function capable of forming a double stranded oligonucleotide (for example, a DNA-RNA complex) hybridized with a metal nanoparticle (probe) at a terminal (concretely, 5'- Functional groups. In some embodiments of the present invention, the single-stranded oligonucleotide has a thiol group or an amine group bound to its 5'-terminal, more specifically, a thiol group is bonded thereto. In certain embodiments of the invention, the length of the detectable single stranded oligonucleotides of the invention is from 6 to 20 bases, more specifically from 10 to 15 bases. Generally, when the number of nucleotide sequences is less than 6, the discrimination power is low, or when the hybridized double strand is not easily formed, and when the number of nucleotide sequences is more than 20, there is little improvement in discrimination power and double strand formation, It is also possible. In the present invention, the detectable single strand oligonucleotide of the present invention is exemplified by SEQ ID NO: 1 and SEQ ID NO: 2, but is not limited thereto. Furthermore, the detectable single-stranded oligonucleotides of the present invention may be modified at one or more bases, sugars, or backbone positions for stability, as long as they do not inhibit (De Mesmaeker et al., Curr Opin Struct Biol . , 5 (3): 343-55, 1995).

The direct binding of the detectable single stranded oligonucleotide barcode to the metal nanoparticles results in the binding (duplex formation) with a fluorescent oligonucleotide probe and the subsequent activity of ribonucleolytic enzymes (e. G., RNase H) on the hybridized double strand A linker (or spacer) may be interposed between the metal nanoparticle and the detectable single stranded oligonucleotide barcode, taking into account that the metal nanoparticle may have a disruptive effect. The linker not only allows the detectable single-stranded oligonucleotide barcode to bind to nanoparticles at a high density, but also can function to facilitate bonding with the probe of the fluorescent oligonucleotide. The linker may be an oligonucleotide of a suitable length, specifically an oligo- A nucleotide, an oligo- T nucleotide, an oligo- C nucleotide, or an oligonucleotide in which A, T or C is combined, and in the case of oligo- G nucleotides, G-quadruplex can induce a secondary structure such as an enzyme to inhibit the activity of the enzyme. However, the G-base may be included in the linker by forming an oligonucleotide in combination with A, T or C.

Next, in order to separate a binding complex formed between the target in the target sample and the metal nanoparticle complex, a substrate coated with an antibody capable of binding to a first target and a second target, The resulting mixture is allowed to react.

In some embodiments of the present invention, the substrate may be a suitable rigid or semi-rigid support, such as a plate, membrane, filter, chip, slide, wafer, fiber, magnetic bead or non-magnetic bead, gel, tubing, Microparticles and capillaries, more specifically plates, and most particularly microwell plates.

As a final step, two fluorescent oligonucleotide probes capable of hybridizing with a detectable single stranded oligonucleotide barcode in a metal nanoparticle complex associated with a target immobilized on a substrate are added to form a double stranded oligonucleotide. The term " hybridization " as used herein means that two single-stranded nucleic acids form a duplex structure by pairing complementary base sequences. Hybridization can occur either in perfect match between single stranded nucleic acid sequences or in the presence of some mismatching nucleotides. The degree of complementarity required for hybridization can vary depending on hybridization reaction conditions, and can be controlled by temperature. The terms " annealing " and " hybridization " are not different and are used interchangeably herein.

In some embodiments of the present invention, the fluorescent oligonucleotide probe comprises fluorophore and quencher molecules at both ends, respectively. As used herein, the term " fluorescent oligonucleotide probe " refers to a nucleic acid sequence that hybridizes to the detectable oligonucleotide barcode with a complementary sequence, more specifically an RNA sequence, with the detectable oligonucleotide barcode sequence to form a double stranded oligonucleotide Refers to a molecule that forms a nucleotide sequence, and specifically, it is shown in SEQ ID NO: 3 and SEQ ID NO: 4, but it is not limited thereto.

Then, ribonucleolytic enzyme H (RNase H) is added to decompose the double-stranded oligonucleotide so as to detect fluorescence derived from the end of the degraded fluorescent nucleotide probe, thereby quantitatively confirming the presence of the two kinds of targets in the target sample do.

In some embodiments of the present invention, both ends of the probe of the fluorescent oligonucleotide of the present invention are selected from the group consisting of rhodamine, coumarin, cyanine, EvoBlue, oxazine, carbopyronin, naphthalene, biphenyl, anthracene, Fluorescent moieties such as fluorescent dyes or derivatives thereof having a basic backbone such as threne, pyrene, carbazole, etc. can be selectively conjugated. For example, the fluorescent moieties of the present invention can be synthesized using ATTO 390 ™ (479), ATTO 425 ™ (484), ATTO 465 ™ (508), ATTO 488 ™ (523), ATTO 495 ™ (527), ATTO 520 ™ 538), ATTO 532 ™ (553), ATTO Rho6G ™ (570), ATTO 550 ™ (576), ATTO 565 ™ (592), ATTO Rho3B ™ 565, ATTO Rho11 ™ 608, ATTO Rho12 ™ 532 ), ATTO Thio12 (579), ATTO 610 (634), ATTO 611 X (681), ATTO 620 (643), ATTO Rho14 (625), ATTO 633 (657), ATTO 647 ATTO 647 N (669), ATTO 655 (684), ATTO Oxa 12 (663), ATTO 700 (719), ATTO 725 (752), ATTO 740 (764), BODIPY TMR , BODIPY 558/568 (568), BODIPY 564/570 (570), EvoBlue 10 ™, EvoBlue 30 ™, MR121, Cy2 ™ 506, YO-PRO ™ -1 509, YOYO ™ -1 509, Calcein 517 ), FITC 518, FluorX 519, Alexa 520, Rhodamine 110 520, 5-FAM 522, Oregon Green 500 (522), Oregon Green 488 (524 ), RiboGreen (525), Rhodamine Green (TM) 527, Rhodamine 123 (529), Magnesium Green (531), Calcium Green ™ (533), TO-PRO ™ -1 533, TOTO1 533, JOE 548, BODIPY 530/550 550, Dil 565, Cy3 ™ 570, Rhodamine B (575), Calcium Orange (576), Pyranine Y (580), Rhodamine B (576), Alexa ™ 546 (570), TRITC 572, Magnesium Orange 575, Picoeritrin R & B 575, Rhodamine Phalloidin 580), TAMRA 582, Rhodamine Red 590, Cy3.5TM 596, ROX 608, Calcium CrimsonTM 615, Alexa 594 615, Texas Red 615, Nile Red (628), YO-PRO ™ -3 (631), YOYO ™ -3 (631), R-ficocyanin (642), C-ficocyanin (648), TO- TOTO3 (660), DiD DilC (5) (665), Cy5 (670), Tiadicarbocyanine (671), Cy5.5 (694), Biosearch Blue (447), CAL Fluor Gold 540 CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM 520, Fluorescein 520, Fluorescein- 650 (566), Quasar 570 (667), Quasar 670 (705), Quasar 705 (610) and derivatives or conjugates thereof, Information that is not. The number in parentheses is the maximum emission wavelength in nanometers. In addition, the fluorescent moiety derivative of the present invention may further comprise a free carboxyl group, an ester (e.g., N-hydroxysuccinimide (NHS) ester) or a maleimide derivative, and the conjugate of the fluorescent moiety Tolidine, biotin, paloidine, amine, azide or iodoacetamide conjugates. The term " link " or " conjugation " as used herein means connecting two components through at least one chemical bond, wherein the connection or conjugation is a covalent bond, , Ionic bonds, coordination bonds, and the like, and is specifically made through covalent bonds.

In some embodiments of the invention, the 5'-end of the fluorescent oligonucleotide probe of the invention is labeled with a ROX or Cy5.5 fluorescent material, and the 3'-end is labeled with BHQ-2.

The method of the present invention may further comprise the step of dissociating two double-stranded oligonucleotide molecules formed between the first or second detectable single-stranded oligonucleotide barcode and the fluorescent oligonucleotide probe from the metal nanoparticles , The process may be performed before or simultaneously with the hybridization step. In some embodiments of the invention, the dissociation process may be carried out by treatment of a reducing agent, more specifically by treatment with DTT (dithiothreitol), beta-mercaptoethanol, or a mixture thereof.

A fluorescent signal obtained from a target sample using the metal nanoparticle complex of the present invention can be effectively used for detection of two or more kinds of targets, and in particular, a signal effectively separating two kinds of targets (for example, HCV antigen and HBV detection antibody) (Note: Fig. 1). In addition, the fluorescence signal analysis can be performed using various methods known in the art. That is, the fluorescence signal is read and processed by a suitable apparatus available in the art. For example, protocols and procedures known in the art may be used, including fluorescence analyzers, microplate readers (e.g., affixes), automated processing using robotic devices, laser scanning systems.

As used herein, the term " coefficient of variation (CV) " is an average value of standard deviations in a statistic. It indicates how each average value is distributed, and the smaller the CV value, the higher the reliability. That is, when the nano-metal particle composite of the present invention is used, the deviation (% CV value) of the measured fluorescence values is in a very low range, and the targets (for example, two kinds of targets) can be separated with a remarkably high sensitivity . As used herein, the term " recovery " refers to the actual efficiency of the analysis process, and may be determined by performing an analysis run on a sample of known quantity / concentration (i.e., Concentration / concentration ratio) to evaluate the reliability of the analysis process. In some embodiments of the present invention, the metal nanoparticle composites exhibit a very low, more specifically 20% CV, and more specifically a 2-15% CV value, and more specifically at least 90% More specifically greater than or equal to 95%, and most specifically greater than or equal to 96% (see Table 2).

In some embodiments of the invention, the target to which the kits and methods of the invention may be applied includes viruses, fungi, bacteria or other microorganisms, and more specifically, viruses. Therefore, the kit and method of the present invention can be applied to the identification and diagnosis of an infectious disease induced by a target to be detected. The term " infectious disease " as used herein refers to a disease or condition associated with the presence of a subject or an organism (infectious agent; e.g., a virus) in or in contact with the subject. The term " diagnosis " as used herein includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis (e.g., identifying an infectious disease or condition, determining responsiveness to treatment of the disease, and determining the effect thereof) of an object that is afflicted with the disease (e. G., Information about therapeutic efficacy Lt; / RTI > to monitor the state of the object in order to provide < RTI ID = 0.0 > For example, when the detection subject is hepatitis B and C virus, it is useful for diagnosis of diseases induced by the viruses. The method comprising reacting a sample (e.g., blood) derived from a subject / patient with two metal nanoparticles of the present invention (more specifically, gold nanoparticles) The presence or absence of two different viruses (for example, hepatitis B and hepatitis C virus) can be confirmed. Thus, the kit and method of the present invention are simple and effective for the identification or diagnosis of diseases caused by microorganisms, more specifically diseases caused by viruses, and more particularly diseases caused by hepatitis viruses . Depending on the metal nanoparticle complex used, the microbial-induced infectious diseases to which the present invention may be applied may be varied, and more specifically, virus-induced liver diseases and bacterial infectious diseases (e.g., bacterial infectious disease, and more particularly liver disease and respiratory virus-infectious disease caused by hepatitis virus. In some embodiments of the present invention, the pharmaceutical composition of the present invention includes a hepatitis induced by hepatitis viruses (for example, hepatitis A, B, C, D, E viruses), hepatitis, liver cirrhosis and liver cancer, (COPD), emphysema, pneumonia, chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), asthma, allergic and seasonal rhinitis, pharyngitis, croup, , Respiratory diseases caused by lower respiratory tract infections of pneumonia, and inflammatory airways diseases.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for simultaneously detecting multiple targets in a sample and a kit using the same.

(b) a method and kit of the present invention comprises the steps of: (a) providing a first target moiety and a first detectable single strand oligonucleotide barcode, wherein (i) the first targeting moiety and the first detectable single strand oligonucleotide barcode are independently ii) a second metal nanoparticle for target detection, wherein the second targeting moiety and the second detectable single stranded oligonucleotide barcode are bonded to the surface region independently of each other; And (b) two fluorescent oligonucleotide probes capable of hybridizing to the first or second detectable single strand oligonucleotide barcode, respectively.

(c) the detection is carried out simultaneously with multiplexing through fluorescence analysis obtained through degradation of the double-stranded oligonucleotides formed.

(d) Thus, the methods and kits of the present invention are useful for the molecular diagnosis of infectious diseases (particularly virus-induced infectious diseases) and, above all, multi-target blood samples from a subject sample Since multiple targets can be analyzed at the same time, they can be easily and effectively applied to medical uses such as in vitro diagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of a multiplex GNP OLISA method for simultaneously measuring multiple antigens and antibodies: A panel, targeting moiety, and detectable single strand oligonucleotide. Combination of gold nanoparticles coupled with a barcode to a target Complex; And panel B, fluorescence signal detection through reaction of the complex with a fluorescent RNA probe after immobilization on the substrate.
FIG. 2 is a graph showing the simultaneous multiplex measurement of anti-HCV antibody and HBV antigen using the GNP OLISA method. Concentration of abscissa, antibody or antigen; And vertical axis, Delta fluorescence intensity.
FIG. 3A and FIG. 3B are graphs showing that when the anti-HCV antibody and the HBV antigen are simultaneously measured, they react with each other without interference. Concentration of abscissa, antibody or antigen; And vertical axis, Delta fluorescence intensity.
4A and 4B are graphs showing the anti-HCV antibody and the HBV antigen measured by the ELISA method, respectively. Concentration of abscissa, antibody or antigen; And vertical axis,? Absorbance.
5A and 5B are graphs showing the anti-HCV antibody and HBV antigen measured in the serum using the GNP OLISA method, respectively. Concentration of abscissa, antibody or antigen; And vertical axis, Delta fluorescence intensity.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One: Ag - GNP - DNA  or dAb - GNP - DNA  Composite manufacturing

HCV antigen (Meridian Life Science, Memphis, TN, USA) and HBV detection antibody (Meridian Life Science, Memphis, TN, USA) were added to 500 ml of colloid of 20 nm gold nanoparticles (British Biocell International, Cardiff, UK) After incubation for about 20 minutes, the cells were blocked with BSA (Sigma-Aldrich, St. Louis, Mo., USA). Tween-20 (Sigma-Aldrich, St. Louis, Mo., USA) and phosphate buffer (pH 8.0) were added to final concentrations of 0.05% and 10 mM, DNA oligonucleotide (synthesized on Oligocam) was added and allowed to react overnight at 4 < 0 > C with vigorous stirring. Then, 15 ml of 5 M NaCl (Sigma-Aldrich, St. Louis, MO, USA) was added and salting was performed at room temperature for 1 hour. To the functionalized GNP, 250 ml of 3% BSA was added, -25 < 0 > C) for 30 minutes. After all the reactions were completed, centrifugation was performed twice for 20 minutes at 18,000 g for purification, and the precipitated conjugates were stored at 4 ° C in a 10 mM phosphate solution containing 0.1% BSA.

The oligonucleotides used for simultaneous multiplex measurement of HBV antigen and anti-HCV IgG groups and sequences of complementary RNA probes (synthesized in biona) are listed in Table 1.

Oligonucleotide order Biomarker DNA2
F-RNA2-Q 5'-ACTCTATGG-3 '
ROX-5'-CCCAUAGAG-3'-BHQ2 HCV Ab DNA3
F-RNA2-Q 5'-AGCGTTGTA-3 '
Cy5-5'-CUACAACGC-3'-BHQ2 HCV Ag

Nanoparticle bound DNA sequence and RNA probe sequence used for its detection.

Example  2: Multiplex GNP OLISA

(HBc cAb) or anti-immunoglobulin antibody (anti-IgG) (Abcam, Cambridge, UK) were diluted in PBS and coated onto black 96-well microplates at a concentration of 1 μg / ml or 4 μg / I did it. After reacting at room temperature for 1 hour, it was blocked with 3% BSA solution for 1 hour. For the quantification, the GNP conjugates prepared above and the anti-HCV or HBV antigen were mixed for each concentration, reacted for 1 hour, and centrifuged at 18,000 g for 20 minutes to obtain a precipitate. The precipitate was suspended in assay buffer (1% BSA in PBST), blocked, and placed in well-completed well plates. After incubation at room temperature for 1 hour, fluorescent signals amplified by RNase H reaction were amplified by excitation / emission of 544/589 nm and 633/675 nm, respectively, using an Applispan (Thermoscientific, Walthan, Mass., USA) And the results are shown in Fig. Between all the steps, the plates were washed three times with 300 [mu] l of PBST.

As can be seen in Fig. 2, it can be seen that each target is simultaneously and specifically measured multiple times independently (i.e., without interference) for different targets.

Example 3: Mutual interference test when detecting biomarkers using multiplex GNP OLISA (identification of specificity)

For quantification, the remaining steps are similar to the multiplex GNP OLISA except for mixing the prepared GNP conjugates and the anti-HCV or HBV antigen, respectively, by concentration. However, in order to confirm that there is no mutual interference, anti-HCVs are mixed and reacted with each other under the absence of HBV antigen (FIG. 3A), and in the opposite manner, HBV antigens are mixed in concentration in the absence of anti- (Fig. 3b). It was confirmed whether the two kinds of RNA probes used in the multiplex reaction specifically reacted with anti-HCV or HBV antigen. All the other reactions and measurement methods are the same as in Example 2.

As can be seen in Figures 3A and 3B, it can be seen that each target is simultaneously and specifically measured multiple times independently (i.e., without interference) for different targets.

Example  4: Conventional ELISA Lt; RTI ID = 0.0 > anti- HCV  And HBV  antigen

4-1. term- HCV  Indirectly for measurement ELISA

The HCV antigen was diluted in PBS and 100 [mu] l at a concentration of 4 [mu] g / ml was coated on a black 96-well microplate. After reacting at room temperature for 1 hour, the plate was washed with 300 μl of PBS three times, 200 μl of 3% BSA solution was added, and the mixture was blocked for 1 hour. After washing three times with 300 [mu] l of PBST, anti-HCV was mixed with concentration in assay buffer for quantitation and reacted for 1 hour. HRP-conjugated anti-goat IgG (Abcam, Cambridge, UK) was then additionally reacted at a concentration of 0.4 [mu] g / ml for 1 hour. After washing three times with 300 μl of PBST, 100 μl of TMB substrate solution (Sigma-Aldrich, St. Louis, Mo., USA) was added and reacted for 10 minutes. 100 μl of TMB stop solution (Sigma-Aldrich, St. Louis, Mo., USA) was added additionally. Then, the absorbance was measured at 450 nm in a 96-well microplate reader (SpectraMax Plus ^TM, Molecular Devices, Sunnyvale, CA, USA).

4-2. HBV  Sandwich for antigen measurement ELISA

The HBV-captured antibody was diluted in PBS and 100 [mu] l at a concentration of 4 [mu] g / ml was coated on a black 96-well microplate. After reacting at room temperature for 1 hour, the plate was washed with 300 μl of PBS three times, 200 μl of 3% BSA solution was added, and the mixture was blocked for 1 hour. After washing three times with 300 [mu] l of PBST, the HBV antigen was mixed in concentration to the assay buffer for quantitation and reacted for 1 hour. Biotin-labeled HBV detection antibody was further reacted at a concentration of 1 μg / ml for 1 hour. After washing three times with 300 [mu] l of PBST, HRP-conjugated streptavidin was further reacted at a concentration of 1 [mu] g / ml for 1 hour. After washing three times with 300 μl of PBST, 100 μl of TMB substrate solution (Sigma-Aldrich, St. Louis, MO, USA) was added and reacted for 10 minutes. Later, TMB stop solution (Sigma-Aldrich, St. Louis, MO, USA) was added to 100 additional ㎕ and a 96-well microplate reader, the absorbance at ^{450 nm (SpectraMax Plus TM, Molecular} Devices, Sunnyvale, CA, USA) .

As can be seen from Figs. 4A and 4B, it was confirmed that the absorbance of each of the targets increased in a concentration-dependent manner.

Example  5: GNP OLISA Lt; RTI ID = 0.0 > anti- HCV  And HBV  antigen

5-1. term- HCV  Indirectly for measurement GNP OLISA

Anti-chlorine IgG was diluted in PBS and 100 ㎕ at a concentration of 4 / / ml was coated on a black 96-well microplate. After reacting at room temperature for 1 hour, the plate was washed with 300 μl of PBS three times, 200 μl of 3% BSA solution was added, and the mixture was blocked for 1 hour. After washing three times with 300 ㎕ of PBST, the prepared GNP conjugates and anti-HCV were mixed by concentration for 1 hour, and centrifuged at 18,000 g for 20 minutes to obtain a precipitate. The precipitate was suspended in assay buffer (1% BSA in PBST), blocked, and placed in well-completed well plates. After incubation at room temperature for 1 hour, the fluorescence signal amplified by RNase H reaction was read out with an excitation / emission filter setting of 544/589 nm using an Appliscan (Thermoscientific, Walthan, Mass., USA) The results are illustrated in FIG. 5A.

5-2. HBV  Sandwich for antigen measurement GNP OLISA

The HBV-captured antibody was diluted in PBS and 100 [mu] l at a concentration of 4 [mu] g / ml was coated on a black 96-well microplate. After reacting at room temperature for 1 hour, the plate was washed with 300 μl of PBS three times, 200 μl of 3% BSA solution was added, and the mixture was blocked for 1 hour. After washing three times with 300 ㎕ of PBST, the prepared GNP conjugates and HBV antigen were mixed by concentration for 1 hour for quantification, and centrifuged at 18,000 g for 20 minutes to obtain a precipitate. The precipitate was suspended in assay buffer (1% BSA in PBST), blocked, and placed in well-completed well plates. After incubation at room temperature for 1 hour, the fluorescence signal amplified by RNase H reaction was read out with an excitation / emission filter setting of 633/675 nm using an Appliscan (Thermoscientific, Walthan, MA, USA) The results are illustrated in FIG. 5B.

As can be seen from Figs. 5A and 5B, it was confirmed that each of the targets was increased in fluorescence intensity in a concentration-dependent manner.

Example  6: Multiplex GNP OLISA And serum Spy king ( spiking Each of the biomarkers (anti- HCV and HBV antigens)  analysis

A commercially available human serum was mixed with two markers at various concentrations (50, 100, or 200 ng / ml) and the serum solution was analyzed to actually perform multi-OLISA on human serum. Multiplex GNP In the same way as OLISA, a standard curve is created based on the amount of each marker analyzed and quantified, and the detection standard deviation (% CV) and the recovery (recovery) (Table 2).

Biomarker Item density( ng / ml ) 50 100 200 Anti-HCV
% CV (deviation) 5.025 15.493 8.360 accuracy(%) 99.5 105.9 98.1 HBV antigen
% CV 2.237 3.642 3.524 accuracy(%) 108.2 106.3 96.2

Multiplex GNP OLISA Spiking with Indirect and Sandwich ELISA in Serum.

As shown in Table 2, each of the targets has a deviation of about 2-15% and an accuracy of 96-108%, and it is confirmed that the target is a condition that can be utilized in actual human serum analysis.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, 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. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Institute of Science and Technology <120> Simultaneous Detection Methods of Multiple Targets in a Sample          and Uses Thereof <130> K07446 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> DNA2 <400> 1 actctatgg 9 <210> 2 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> DNA3 <400> 2 agcgttgta 9 <210> 3 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> RNA2 <400> 3 cccauagag 9 <210> 4 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> RNA3 <400> 4 cuacaacgc 9

Claims (17)

(a) a first metal nanoparticle for target detection, wherein a first targeting moiety and a first detectable single stranded oligonucleotide barcode are independently bonded to a surface region; (b) a second metal nanoparticle for target detection, wherein the second targeting moiety and the second detectable single-stranded oligonucleotide barcode are independently bonded to the surface region; And (c) two fluorescent oligonucleotide probes each capable of hybridizing to the first or second detectable single strand oligonucleotide barcode. 2. The kit of claim 1, wherein the targeting moiety is an antibody or antigen to a virus. The kit according to claim 1, wherein the first metal nanoparticles for target detection and the second metal nanoparticles for target detection have binding capacities for different targets. The kit of claim 1, wherein the metal nanoparticles are gold, platinum, silver, copper, or palladium nanoparticles. The kit according to claim 1, wherein the fluorescent oligonucleotide probe is labeled at both ends with a fluorophore and a quencher molecule. The kit according to claim 1, wherein the two kinds of fluorescent oligonucleotide probes exhibit different fluorescence. The method according to claim 1, wherein the sample is selected from the group consisting of blood, plasma, serum, tissue, cells, saliva, sputum, urine, lymph, bone marrow, saliva, milk, urine, feces, Thymus, ascites, amniotic fluid or cell tissue fluid. The kit according to claim 1, wherein the kit further comprises ribonucleolytic enzyme H (RNase H). 2. The kit of claim 1, wherein the kit further comprises a metal nanoparticle having a third targeting moiety and a third detectable single-stranded oligonucleotide barcode attached to the surface region independently of each other, and a second nanoparticle complementary to the third detectable single-stranded oligonucleotide barcode Wherein the three targets are simultaneously identifiable when the probe further comprises a fluorescent oligonucleotide probe. A method for simultaneous detection of a double target in a sample comprising the steps of:
(a) contacting a sample of interest with two metal nanoparticles, wherein the metal nanoparticles comprise (i) a first targeting moiety and a first detectable single stranded oligonucleotide barcode (ii) a second targeting moiety and a second detectable single-stranded oligonucleotide barcode are bound to the surface region independently of each other, and (ii) a second target moiety and a second detectable single- A metal nanoparticle for detecting a target;
(b) reacting the mixture of step (a) with a substrate coated with two antibodies capable of binding to the first target and the second target, respectively;
(c) hybridizing the complex captured on the substrate of step (b) with two kinds of fluorescent oligonucleotide probes, wherein the two kinds of fluorescent oligonucleotide probes are hybridized with the first or second detectable single strand oligonucleotide barcode Respectively; And
(d) hydrolyzing two double-stranded oligonucleotide molecules formed by hybridization on the metal nanoparticles in the complex of step (c) to detect the probe-derived fluorescence signal. The simultaneous detection method according to claim 10, wherein the first metal nanoparticles for target detection and the second metal nanoparticles for target detection have binding capacities for different targets. 11. The method of claim 10, wherein the substrate is a microwell plate. 11. The method of claim 10, wherein the simultaneous detection method comprises: (c-1) contacting two double-stranded oligonucleotide molecules formed between the first or second detectable single-stranded oligonucleotide barcode and a fluorescent oligonucleotide probe to the metal nanoparticle Further comprising the steps of: 14. The method of claim 13, wherein said dissociation is accomplished through treatment of a reducing agent. 11. The method of claim 10, wherein the simultaneous detection method comprises the steps of: (a) combining a third targeting moiety and a third detectable single-stranded oligonucleotide barcode with metal nanoparticles bound to the surface region independently of each other; and Wherein the three targets can be identified simultaneously if they additionally comprise a complementary fluorescent oligonucleotide probe. 11. The method of claim 10, wherein the target comprises a virus. 17. The method of claim 16, wherein the virus is hepatitis virus.

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