KR20200042843A - Kit for detecting multiple biomarkers and detection method - Google Patents

Abstract

The present invention relates to a kit for simultaneously detecting multiple bio-markers, which comprises: a substrate; an N-type docking strand attached to N-type detection antibody; and a donor strand which complementarily bonds with the N-type docking strand, wherein the docking strand or the donor strand comprises a fluorescent molecule, and simultaneously identifies an N-type bio-marker using FRET efficiency by the fluorescent molecule, and to a detection method using the same.

Description

Kit for detecting multiple biomarkers and detection method {Kit for detecting multiple biomarkers and detection method}

A substrate, N docking strands attached to N detection antibodies, and donor strands complementarily binding to the N docking strands, wherein the docking strand or donor strand comprises a fluorescent molecule, and the fluorescence Disclosed is a kit for simultaneous detection of multiple biomarkers and a detection method using the same, which simultaneously discriminates N biomarkers using a fret efficiency by molecules.

Biomarkers are biological indicators that can detect changes in the body using proteins, DNA, RNA, and metabolites. As a method of detecting a biomarker contained in a specific sample derived from a living organism, it is possible to check the normal or pathological condition of the living organism, and objectively measure the degree of response to drugs. Recently, it has been spotlighted by the medical community in that it is an effective method for diagnosing various intractable diseases such as cancer, heart disease, stroke, and dementia.

Commonly used biomarker detection technology is mainly an ELISA-based technology, and is known to have a maximum sensitivity of several tens to tens of pg / ml. Therefore, in order to detect a biomarker, a biological sample of a certain amount or more proportional to the sensitivity is required.

In the case of determining whether tumors are generated by using a biomarker, in most cases, one biomarker is determined in one test and the test is repeated several times. However, when a tumor is generated, not only the value of one biomarker is changed, but also the value of two or more biomarkers is changed. Therefore, in order to confirm whether or not a tumor has occurred, detecting multiple types of biomarkers at the same time can increase the accuracy of discrimination.

In order to detect a large number of biomarkers with current technology, an amount of blood and a kit proportional to the number of biomarkers to be detected are required. This not only induces the psychological and physical burden of the subject due to a large amount of blood collection and the economic burden of using a plurality of detection kits, but also may cause difficulties in early diagnosis of the disease, and thus it is necessary to simultaneously diagnose N biomarkers. .

In order to simultaneously detect N types of biomarkers using a small amount of biological samples, a high sensitivity and a technique capable of multiple diagnosis is required.

Korean Registered Patent No.1431062, “Multi breast biomarker set for breast cancer diagnosis, detection method thereof, and breast cancer diagnostic kit including antibodies therefor” (published on September 23, 2013) Korean Patent Publication No. 20170096619, "Method and kit for simultaneous detection of multiple diseases based on 3D protein nanoparticle probe" (published on August 24, 2017)

An object of the present invention includes a substrate, N kinds of docking strands attached to N kinds of detection antibodies, and donor strands complementarily binding to the N kinds of docking strands, wherein the docking strand or donor strand is a fluorescent molecule. It is to provide a kit for simultaneous detection of multiple biomarkers that includes, and simultaneously identifies N types of biomarkers using fret efficiency by the fluorescent molecule.

Another object of the present invention relates to a method for simultaneously detecting multiple biomarkers, wherein N types of biomarkers are attached to a substrate, and N types of detection antibodies to which N types of docking strands are attached, respectively. The step of binding to each of the N kinds of biomarkers, the step of each donor strand (Donor strand) is coupled to the N kinds of docking strands, measuring the fret efficiency generated by the fluorescent molecule contained in the docking strand or the donor strand It is to provide a method for simultaneous detection of multiple biomarkers, including determining the type of the biomarkers of the N types using the fret efficiency and measuring the biomarker concentration from the number of biomarkers per unit area.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, the substrate; N types of docking strands attached to N types of detection antibodies; And a donor strand that complementarily bonds with the N-type docking strand. Included, wherein the docking strand or donor strand includes a fluorescent molecule, using the fret efficiency of the fluorescent molecule to discriminate N kinds of biomarkers simultaneously, there is provided a kit for simultaneous detection of multiple biomarkers.

According to one side, the kit may further include a fret strand labeled with a fluorescent molecule.

According to one side, the fret strand may be complementary to the N-type docking strand.

According to one side, the fluorescence molecule is a donor strand or a donor strand or a specific base of a docking strand, a donor strand or a fret strand, 3'- or 5'-end, or on the backbone. It can act as an acceptor.

According to one side, the kit may further include a capture antibody.

According to another embodiment of the present invention, a method for simultaneously detecting multiple biomarkers, comprising: attaching N biomarkers to a substrate; Binding each of the N types of detection antibodies to which the N types of docking strands are attached to the N types of biomarkers; Donor strand (Donor strand) is coupled to each of the N kinds of docking strands; Measuring fret efficiency generated by the fluorescent molecules included in the docking strand or the donor strand; Including, by using the fret efficiency to determine the type of the N-type biomarker, and measuring the biomarker concentration from the number of biomarkers per unit area, a method for simultaneous detection of multiple biomarkers is provided.

According to one side, the detection method may further include the step of binding the fluorescent molecule labeled FRET strand to the N-type docking strand.

According to one side, the fret strand may be complementary to the N-type docking strand.

According to one side, the fluorescent molecule is a donor or billion on a specific base, 3'- or 5'-end, or backbone of a docking strand, donor strand or fret strand. It can act as an acceptor.

According to one side, the detection method may further include attaching a capture antibody to the substrate.

The present invention can detect two or more types of biomarkers at the same time using fret efficiency, and can distinguish types of biomarkers of low concentration included in a small amount of samples.

Specifically, the present invention is characterized in that the fluorescent molecule-labeled strand is temporarily bonded, so even when the fluorescent molecule acting as a donor or acceptor becomes photobleached, the fluorescent molecule that is not photobleached in the process of repeating the binding and separation The labeled donor strand or fret strand can be combined with the docking strand to measure fret efficiency, so that N kinds of biomarkers can be detected simultaneously regardless of photobleaching.

In the case of using a fret strand, a fluorescent molecule on the fret strand shines only when the donor strand and the fret strand are simultaneously bonded to the docking strand, and when the fret strand is not used, only when the donor strand is coupled to the docking strand. Since the fluorescent molecules on the docking strand emit light, it is possible to measure the fluorescent molecules that emit light temporarily without interference from the surrounding fluorescent molecules, so that the position of the fluorescent molecules can be measured with high precision of less than 10 nm, which results in one screen. It has a high dynamic range that can measure a large number of biomarkers simultaneously.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 schematically shows a kit for simultaneous detection of multiple biomarkers according to an embodiment of the present invention.
2 schematically shows a kit for simultaneous detection of N biomarkers according to an embodiment of the present invention.
Figure 3 schematically shows a kit for simultaneous detection of N types of biomarkers according to another embodiment of the present invention.
4 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.
5 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.
6 schematically shows a kit for simultaneous biomarker detection according to an embodiment of the present invention.
7 schematically illustrates a kit for detecting micro RNA according to an embodiment of the present invention.
8 shows absorbance spectrum and emission spectrum data according to an embodiment of the present invention.
9 schematically shows the principle of simultaneous detection of multiple biomarkers according to an embodiment of the present invention.
10 is a fluorescence image measured by varying the concentration of the donor strand according to an embodiment of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and the scope of the patent application right is not limited or limited by these embodiments. It should be understood that all modifications, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described herein, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

The terms used in the present specification will be briefly described.

In this specification, "biomarker" includes all biomaterials capable of confirming the normal or pathological condition of a living organism.

As used herein, “antibody” is used in a broad sense to include “detection antibody”, and specifically, monoclonal antibodies (including monoclonal antibodies and full-length monoclonal antibodies) and polyclonal antibodies (polyclonal antibodies). , Multispecific antibodies (e.g. bispecific antibodies), and antibody fragments (e.g., variable regions and other portions of the antibody that exhibit the desired biological activity). In addition, the antibody of the present invention includes both monoclonal antibodies and polyclonal antibodies, and includes chimeric antibodies, humanized antibodies, and human antibodies. Preferably, may include Fab, F (ab) 2, Fab ', F (ab') 2, Fv, diabody, nanobody or scFv, more preferably scFv, Fab, Nanobodies and immunoglobulin molecules.

As used herein, “strand” means a nucleic acid in a state capable of attaching a fluorescent molecule on a specific base or a single-stranded or double-stranded nucleic acid in a state in which a fluorescent molecule is already attached.

As used herein, “fluorescent molecule” refers to a fluorescent dye compound that can be detected by being labeled on the strand.

In this specification, the term “fret efficiency” refers to a different energy transfer efficiency according to the distance between fluorescent molecules by using a phenomenon in which energy of a fluorescent molecule excited by light is transferred to other adjacent fluorescent molecules. And, it may be to include all of the meaning of ordinary fret efficiency recognized by those skilled in the art.

In the present specification, “donor” refers to a fluorescent molecule that transmits energy when excited by light, and when two or more fluorescent molecules are adjacent to each other, means a fluorescent molecule that absorbs or emits light of a relatively short wavelength.

As used herein, “acceptor” refers to a fluorescent molecule that receives energy from a donor in an excited state, and when two or more fluorescent molecules are adjacent to each other, means a fluorescent molecule that absorbs or emits light having a relatively long wavelength.

In the present specification, “capture antibody” may be any one included in the above-mentioned term “antibody”, and specifically refers to an antibody that serves to fix a biomarker on a substrate.

According to an embodiment of the present invention, the substrate; N types of docking strands attached to N types of detection antibodies; And a donor strand that complementarily bonds with the N-type docking strand. Included, wherein the docking strand or donor strand includes a fluorescent molecule, using the fret efficiency of the fluorescent molecule to discriminate N kinds of biomarkers simultaneously, there is provided a kit for simultaneous detection of multiple biomarkers.

According to one side, the kit may further include a FRET strand labeled with a fluorescent molecule.

According to one side, the fret strand may be complementary to the N-type docking strand.

According to one side, the fluorescence molecule is a donor strand or a donor strand or a specific base of a docking strand, a donor strand or a fret strand, 3'- or 5'-end, or on the backbone. It can act as an acceptor.

According to one side, the kit may further include a capture antibody.

According to another embodiment of the present invention, a method for simultaneously detecting multiple biomarkers, comprising: attaching N biomarkers to a substrate; Binding each of the N types of detection antibodies to which the N types of docking strands are attached to the N types of biomarkers; Donor strand (Donor strand) is coupled to each of the N kinds of docking strands; Measuring fret efficiency generated by the fluorescent molecules included in the docking strand or the donor strand; Including, by using the fret efficiency to determine the type of the N-type biomarker, and measuring the biomarker concentration from the number of biomarkers per unit area, a method for simultaneous detection of multiple biomarkers is provided.

According to one side, the detection method may further include the step of binding the fluorescent molecule labeled FRET strand to the N-type docking strand.

According to one side, the fret strand may be complementary to the N-type docking strand.

According to one side, the fluorescent molecule is a donor or billion on a specific base, 3'- or 5'-end, or backbone of a docking strand, donor strand or fret strand. It can act as an acceptor.

According to one side, the detection method may further include attaching a capture antibody to the substrate.

The biomarker of the present invention may be a polypeptide, peptide, nucleic acid, protein or metabolite that can be detected in biological fluids such as blood, saliva, urine, but is not limited thereto, AFP (alpha fetoprotein), CA15-3, CA27-29, CA19-9, CA-125, calcitonin, calretinin, carcinoembryonic antigen (CEA), CD34, CD99, MIC-2, CD117, chromogranin, cytokeratin ( cytokeratin, various types: TPA, TPS, Cyfra21-1), desmin, epihelial membrane antigen (EMA), Factor VIII, CD31, FL1, glial fibrillary acidic protein (GFAP), GCDFP-15 (gross cystic disease fluid) protein, HMB-45, human chorionic gonadotropin (hCG), immunoglobulin, inhibin, keatin, various types of keratin, lymphocyte marker, MART-1 (Melan-A) ), Myo D1, MSA (muscle-specific actin), neurofilament (neurofilament), NSE (neuron-specific enolase), PLAP (placental alkaline phosphatase), PSA ( prostate-specific antigen), PTPRC (CD45), S100 protein, smooth muscle actin (SMA), synaptophysin, TK (thymidine kinase), Tg (thyroglobulin), TTF-1 (thyroid transcription factor-1), M2-PK, vimentin, interleukin, CD24, CD40, integrin, cystatin, interferon, TNF (tumor necrosis factor), MCP, VEGF, GLP, ICA, HLA-DR, ICAM, EGFR, FGF, BRAF, GFEB, FRS, LZTS, CCN, mucine, lectin, apolipoprotein, tyrosine, neuronal adhesion molecule-like protein, fibronectin, glucose, uric acid , Carbonic anhydrase or cholesterol.

The biomarker to be detected in the present invention can be used as long as it is used in the general scientific or medical field, such as bioprocessing, pathogenicity, and measurement or evaluation of a pharmacological process for treatment, and is not particularly limited. Specifically, the present invention detects N biomarkers simultaneously by using two or more fluorescent molecules labeled on a docking strand or a fret strand and a measurement value of fret efficiency measured by a donor labeled on a donor strand. It relates to a kit and a detection method.

The detection antibody is for detecting a biomarker using an antigen-antibody reaction, and it is preferable to use a heterogeneous type of biomarker to be detected. In addition, in order to measure fret efficiency at the time of detection, each detection antibody uses a docking strand labeled with two or more fluorescent molecules, or a docking strand capable of complementarily binding to a fret strand labeled with two or more fluorescent molecules. It is preferred to use. The docking strand, donor strand or fret strand may preferably use nucleic acids such as DNA, RNA, PNA, LNA, MNA, GNA, TNA or analogs thereof.

The “fret efficiency” is a phenomenon in which energy of a fluorescent molecule excited by light is transferred to other fluorescent molecules adjacent to each other, and indicates a different energy transfer efficiency according to a distance between fluorescent molecules, specifically calculated as follows. Mean value.

Fret efficiency = (intensity of light emitted by acceptor) / (sum of intensity of light emitted by donor and acceptor)

At this time, the light intensity of the donor or acceptor may be affected by the distance between the donor and the acceptor, but the donor and acceptor are marked on a specific base, 3'- or 5'-end, or basic skeleton By controlling the distance between the donor and the acceptor, fret efficiency can be controlled.

At this time, the fret efficiency is that the acceptor is excited by the light to excite the donor, and the donor that is not completely separated by the optical component (dichroic mirror) used to separate the emitted light, the donor and the light emitted by the acceptor. It may be a value of correcting a difference in detection efficiency of the image sensor according to a difference in wavelength of a portion of light emitted by the acceptor or a light emitted by the donor and the acceptor.

For example, if the interval is 1 when the number of bases existing between the donor and the acceptor is 1, the interval between the donor and the acceptor is 1, 2, 3,… Two or more fret pairs (n) are prepared. The fret efficiency for the interval is measured by measuring the fret efficiency for the produced n kinds of fret pairs. The same type of detection antibody is attached with the same type of docking strand, and the other type of detection antibody is attached with a different type of docking strand. N pairs of frets may be directly labeled on the docking strand. Thereafter, when the reaction with the sample containing the biomarker proceeds, the specific biomarker is bound to a specific detection antibody. At this time, the fret efficiency is calculated by measuring the intensity of light emitted by the donor and acceptor of each molecule. For example, when a donor strand labeled with a fluorescent molecule acting as donor 1 is bound to the docking strand, the fret efficiency can be measured by a pair of frets consisting of donor 2 and an acceptor absorbing light from the excited donor 1. .

The fluorescent molecules are rhodamine, Alexa, fluorescein, fluorescein isothiocyanate (FITC), 5-carboxy fluorescein (FAM), Atto, BODIPY, CF, CY, DyLight Fluor and Texas Red (Texas Red) is preferably selected from the group consisting of, but is not necessarily limited thereto.

Additionally, when further comprising fret strands, the fret pair is labeled on the fret strand and not on the docking strand. In this case, it is possible to measure fret efficiency at the moment when both the donor strand and the fret strand are coupled to the docking strand.

By comparing the database value with the measured value, the type of detection antibody used for detection can be confirmed, and finally the type of biomarker can be confirmed.

In one embodiment, assuming that the interval capable of distinguishing the fret efficiency by the above-described content is an integer of 4 or more and 20 or less, there are 17 types of fret pairs that can be produced by one donor and one acceptor. . If two pairs of donors and acceptors exist under the same condition, there are 289 types, and if three pairs exist, there are 4,913 types. In addition, there are 289 kinds of fret pairs that can be produced with two donors and one acceptor, and there are 4,913 kinds of fret pairs that can be produced with three donors and one acceptor. A variety of biomarkers can be detected using the fluorescent molecule type of.

1 schematically shows a kit for simultaneous detection of multiple biomarkers according to an embodiment of the present invention.

First, referring to FIG. 1A, a configuration of a kit including a detection antibody, a docking strand attached to a detection antibody, and a donor strand that complementarily binds N kinds of docking strands is exemplarily illustrated. The donor strand is labeled with a donor 1 fluorescent molecule, and the docking strand is labeled with a fret pair of donor 2 and acceptor fluorescent molecules. In this case, the fret efficiency can be measured at the moment when the donor strand is coupled to the docking strand.

According to one side, the kit may further include a FRET strand.

In the case of using a fret strand, even if some fluorescent molecules are photo-bleached, there is an advantage in that the fret efficiency can be measured by repeating the binding and separation of the donor strand and the fret strand labeled with the non-photo-bleached fluorescent molecule to the docking strand. .

Referring to FIG. 1B, a configuration of a kit including a detection antibody, a docking strand attached to a detection antibody, a donor strand complementarily binding to N kinds of docking strands, and a fret strand complementarily binding to a specific docking strand are exemplary. It is shown as. The donor strand is labeled with a donor 1 fluorescent molecule, and the fret strand is labeled with a fret pair consisting of donor 2 and acceptor fluorescent molecules. In this case, it is possible to measure fret efficiency at the moment when both the donor strand and the fret strand are coupled to the docking strand.

Referring to Figure 1c, the configuration of a kit comprising a detection antibody, a docking strand attached to the detection antibody, a donor strand that complementarily binds N kinds of docking strands, and a fret strand that complementarily binds to a specific docking strand are exemplary. It is shown as. The donor strand is labeled with a donor 1 fluorescent molecule, and the fret strand is labeled with a fret pair consisting of donor 2, donor 3 and acceptor fluorescent molecules. By labeling two or more fluorescent molecules on the fret strand, it is possible to detect multiple biomarkers using the minimum fluorescent molecule type.

2 schematically shows a kit for simultaneous detection of N biomarkers according to an embodiment of the present invention.

Referring to FIG. 2, a configuration of a kit including a detection antibody, a docking strand attached to the detection antibody, and a donor strand that complementarily binds N kinds of docking strands is illustrated. The donor strand is labeled with a donor 1 fluorescent molecule, and the docking strand is labeled with a fret pair of donor 2 and acceptor fluorescent molecules. The docking strand attached to detection antibody 1 is labeled with donor 2 and acceptor remotely to have low fret efficiency, and the docking strand attached to detection antibody N is labeled close to donor 2 and acceptor to have high fret efficiency.

The reason why the kit of the present invention is constructed as described above is that if the same fret efficiency is measured from the docking strand of N detection antibodies combined with N biomarkers, the type of the detected biomarkers cannot be specified.

Therefore, in order to obtain a measurement value of fret efficiency that can be distinguished, it is preferable to use N kinds of docking strands and N kinds of fret pairs that are different by the number of biomarkers to be detected. In this case, it is preferable that all donor strands that bind to the N kinds of docking strands use the same species.

Specifically, when two or more fluorescent molecules are directly labeled on each of the docking strands, the N kinds of docking strands may be different in the number of bases present between two or more fluorescent molecules to be labeled. More specifically, the N-type docking strand may be a pair of frets having different fret efficiency. When two or more fluorescent molecules are labeled on the fret strand, the N kinds of fret strands may be different in the number of bases present between the two or more fluorescent molecules to be labeled. More specifically, the N-type fret strand may be a pair of frets having different fret efficiency. At this time, two or more fluorescent molecules labeled on the docking strand or fret strand may act as donors or acceptors.

Figure 3 schematically shows a kit for simultaneous detection of N types of biomarkers according to another embodiment of the present invention.

Referring to FIG. 3, the configuration of a kit including a detection antibody, a docking strand attached to a detection antibody, a donor strand complementarily binding to N kinds of docking strands, and a fret strand complementarily binding to a specific docking strand are exemplary It is shown as. In the donor strand, the donor 1 fluorescent molecule is labeled with the donor 2 and the acceptor remotely so as to have low fret efficiency in the fret strand 1 that complementarily binds the docking strand attached to the detection antibody 1, and the docking attached to the detection antibody N The fret strand N which complementarily couples with the strand is marked with the donor 2 and the acceptor closely to have high fret efficiency. The configuration and principle are the same as in FIG. 2 except that the fret pair is marked on the fret strand rather than the docking strand.

4 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.

Referring to Figure 4, the configuration of the kit comprising a detection antibody, a docking strand attached to the detection antibody, a donor strand that complementarily binds N kinds of docking strands, and two fret strands that complementarily bind to a specific docking strand. Illustratively shown. The donor strand is labeled with a donor 1 fluorescent molecule, and a pair of donor 2 and acceptor frets are labeled on the fret strands 1-1 and 1-2, respectively, which complementarily bind to the docking strand attached to the detection antibody 1. In this case, it is preferable to label each fret pair so that the fret efficiency is different.

In addition, fret strands having the same fret efficiency may have the same base sequence. Thus, one fret strand can be complementary to several docking strands. As an example, three fret strands with fret efficiency of 0.25, 0.5, and 0.75 are designed, biomarker 1 has fret efficiency of 0.25 and 0.5, biomarker 2 has fret efficiency of 0.25 and 0.75, and biomarker 3 is fret. When designed to have an efficiency of 0.5 and 0.75, a fret strand having a fret efficiency of 0.25 can be complementarily coupled to a docking strand of biomarker 1 and biomarker 2, and a fret strand having a fret efficiency of 0.5 is biomarker 1 and biomarker 3 Can be complementarily coupled to the docking strand, and a fret strand having a fret efficiency of 0.75 can be complementarily coupled to the docking strand of biomarker 2 and biomarker 3. In this case, the fret strand that complementarily binds to the N-type docking strand may be N or less.

5 schematically shows a kit for simultaneous detection of multiple biomarkers according to another embodiment of the present invention.

Referring to FIG. 5, the configuration of a kit including a detection antibody, a docking strand attached to a detection antibody, a donor strand complementarily binding to N kinds of docking strands, and a fret strand complementarily binding to a specific docking strand are exemplary It is shown as. The donor strand is labeled with a donor 1 fluorescent molecule, and a pair of donor 2 and acceptor frets is labeled on a fret strand 1 that complementarily binds a docking strand attached to the detection antibody 1. At this time, when the docking strand is configured such that two or more joining sites capable of complementarily joining the same donor strand and the fret strand are present, when the donor strand and the fret strand are simultaneously joined to at least one joining part, a fret Since the efficiency can be measured, it is possible to analyze more than twice as fast as the configuration shown in FIG. 3 described above.

In addition, the docking strand or fret strand may be a plurality of donors or acceptors are labeled. In this case, two or more donors may be different types of fluorescent molecules from the acceptor, and two or more donors may be different types of fluorescent molecules. In another case, the two or more acceptors may be different types of fluorescent molecules from the donor, and the two or more acceptors may be different types of fluorescent molecules.

For example, a plurality of docking strands may be attached to one detection antibody. At this time, the docking strands to be attached are of the same type, and the donor strands and fret strands that are coupled to each docking strand are the same. This configuration is schematically illustrated in FIG. 6.

Referring to FIG. 6, the configuration of a kit including a detection antibody, a docking strand attached to a detection antibody, a donor strand complementarily binding to N kinds of docking strands, and a fret strand complementarily binding to a specific docking strand are shown. have. Specifically, two or more docking strands may be attached to one detection antibody. The donor strand is marked with a donor 2 and an acceptor fret pair in a fret strand 1 in which a donor 1 fluorescent molecule is complementarily coupled to a docking strand attached to the detection antibody 1, and a donor strand and a fret in any one of the two or more docking strands. When the strands are combined at the same time, the fret efficiency can be observed, and analysis more than twice as fast as the above-described configuration of FIG. 3 is possible. That is, when using a kit configured as shown in FIG. 6, an effect similar to that of FIG. 5 can be obtained.

As another example, a kit for simultaneous detection of multiple biomarkers made by combining two or more of the configurations shown in FIGS. 1 to 6 may be used for simultaneous detection of multiple biomarkers.

As another example, the kit for simultaneous detection of multiple biomarkers of the present invention may be used for detecting micro RNA, as shown in FIG. 7.

Referring to FIG. 7, a configuration of a kit for detecting nucleic acids such as DNA and RNA including micro RNA is exemplarily illustrated. An N-type nucleic acid to be detected is immobilized on a substrate by attaching a binding material and a binding-compatible material of the substrate to one end of the N-type nucleic acid to be detected, or by coating a nucleic acid complementary to a portion of each nucleic acid in advance on the substrate. Since the base sequence of N kinds of nucleic acids to be detected is determined, each nucleic acid can be discriminated using N kinds of donor strands and fret strands complementary thereto.

FIG. 8 shows data obtained by measuring the absorption and emission spectra of the donor 1 (Alexa Fluor 488), the donor 2 (Cy5), and the acceptor (Cy7) according to an embodiment of the present invention.

Figure 8 (a) shows the absorption spectrum of the fluorescent molecules. As an example, the black vertical dotted line in (a) shows a 488 nm wavelength used to excite donor 1, wherein donor 1 is excited, but donor 2 and acceptor are not excited. In this case, since the donor 2 and the acceptor can emit light only when energy is received from the donor 1 by the fret, the donor strand labeled with the donor 1 is coupled to the docking strand to measure the fret efficiency of the fret pair. When the fret strand is used, the donor strand and the fret strand must be simultaneously coupled to the docking strand to measure the fret efficiency of the fret pair.

Figure 8 (b) shows the emission spectrum of the fluorescent molecules. As an example, based on the wavelength indicated by the black vertical dotted line in (b), the brightness of the donor 2 is measured by combining the light intensity of the wavelength band between the left and right dotted lines using a dichroic mirror, which is longer than the right dotted line. The fret efficiency of a fret pair can be calculated by measuring the brightness of the acceptor by summing the light intensity of the wavelength band. In this case, it is preferable to measure the intensity of light between the donor 2 and the acceptor after the fret efficiency between donor 1 and donor 2 is close to 1 and the signal of donor 1 is removed.

In addition, by adjusting the distance between donor 1 labeled on the donor strand and donor 2 labeled on the fret strand, the fret efficiency between donor 1 and donor 2 is adjusted, and the light intensity of donor 1 and donor 2 is measured. By calculating fret efficiency, multiple biomarkers can be detected.

9 schematically shows the principle of simultaneous detection of multiple biomarkers according to an embodiment of the present invention.

N kinds of biomarkers are attached to a chamber having a substrate as a bottom, and N kinds of detection antibodies to which N kinds of docking strands are attached are combined with the N kinds of biomarkers, and then donor strands are included. Filling the chamber with a buffer causes the donor strand to bind complementarily to the docking strand. At this time, by measuring the light intensity of the fret pair generated, fret efficiency can be measured and multiple biomarkers can be discriminated. At this time, the fret efficiency can be calculated by measuring the intensity of light by wavelength using an optical filter (dichroic mirror) or by measuring the relative spectral size of fluorescent molecules using a prism or grating.

9 shows a method of measuring the intensity of light emitted by the donor 2 and the acceptor using an optical filter (dichroic mirror).

Referring to FIG. 9, (a) shows a state in which two types of biomarkers 1 and 2 are fixed to a substrate. Biomarker 1 has detection antibody 1 with docking strand 1 attached thereto, and biomarker 2 has detection antibody 2 with docking strand 2 attached thereto. (b) shows the state where the donor strand is coupled to the docking strand 2 and the fret pair emits light. At this time, the fret efficiency of the biomarker 2 is calculated by measuring the brightness of the donor 2 and the acceptor. In addition, (c) shows a state in which the donor strand is separated from the docking strand 2 so that the fret pair does not emit light, and (d) shows the state in which the donor strand is coupled to the docking strand 1 to emit light. At this time, the fret efficiency of the biomarker 1 is calculated by measuring the brightness of the donor 2 and the acceptor. Thereafter, (e) represents a state in which the donor strand is separated from the docking strand 1 so that the fret pair does not emit light, and (f) represents the state in which the donor strand recombines with the docking strand 1 to emit light. In this case, too, the fret efficiency of the biomarker 1 is calculated by measuring the brightness of the donor 2 and the acceptor. After the fret efficiency of all biomarkers in the observation area is measured at least once, the type of biomarker is determined using the fret efficiency, and then the biomarker concentration is measured from the number of biomarkers per unit area.

By controlling the type of donor strand (DNA, RNA, PNA, etc.), the length of the donor strand that binds the docking strand, and the ion concentration of the buffer, it is possible to control the time it takes for the donor strand to separate after binding to the docking strand. By extending this time, the measurement time can be reduced by allowing the donor strand to be attached to most docking strands. Alternatively, by shortening this time, it is possible to prevent donor strands from being bonded to most docking strands. If the donor strand is temporarily coupled to either docking strand to make the fret pair light, most of the docking strands in the vicinity do not emit light, so fret efficiency can be measured without interference from other fret pairs. Therefore, the position of the fret pair can be measured with high precision of less than 10 nm. In addition, this allows a large number of biomarkers to be simultaneously measured on one screen, and thus has a high dynamic range. The same can be applied to fret strands. At this time, it is preferable that the time at which the donor strand is separated is short within a few seconds, and the time at which the fret strand is separated is longer than a few seconds.

In addition, since the speed at which the donor strand or fret strand bonds to the docking strand is proportional to the concentration of the donor strand or fret strand, the higher the concentration, the higher the analysis speed.

10 is a fluorescence image measured by varying the concentration of the donor strand according to an embodiment of the present invention.

As shown in FIG. 8 (b), a portion of the donor 1 spectrum has a section overlapping a portion of the donor 2 spectrum, and this overlapping section affects the brightness measurement of donor 2. FIG. 10 shows a screen obtained by measuring the brightness of donor 2 through an imaging camera, wherein the concentrations of donor strands present in the buffer are (a) 100 nM, (b) 500 nM, and (c) 1000 nM. It is an image when. As shown in Fig. 10, as the concentration of the donor strand increases, the background noise of the donor 2 image increases, which degrades the accuracy of the donor 2 brightness measurement. That is, as the concentration of the donor strand increases, the analysis speed increases, but the accuracy of fret efficiency measurement decreases, so the concentration of the donor strand included in the buffer is preferably 1 nM to 1 uM to satisfy both speed and accuracy.

As another example, when a plurality of fret strands are coupled to one docking strand, the donor strand may be combined as many as the number of the fret strands. When the acceptors labeled on the plurality of fret strands are the same, the labeled donors may use the same or different types from each other, but it is preferable that they are different types from the acceptors.

Specifically, when a plurality of fret strands are coupled to one docking strand, the donor and acceptor labeled on the fret strand may be combined in the following four cases.

i) The donors labeled on the plurality of fret strands may be the same fluorescent molecules, and the acceptors may be the same fluorescent molecules.

ii) The donors labeled on the plurality of fret strands may be fluorescent molecules different from each other, and the acceptors may be the same fluorescent molecules.

iii) The donors labeled on the plurality of fret strands may be the same fluorescent molecules, and the acceptors may be different fluorescent molecules.

iv) The donors labeled on the plurality of fret strands may be different fluorescent molecules, and the acceptors may be different fluorescent molecules.

According to one side, the kit for simultaneous detection of multiple biomarkers may further include a capture antibody. However, this is according to an embodiment of the present invention, and depending on the situation, the biomarker may be directly attached to the substrate. This is for speed, economy, and convenience of the measurement process, and if necessary, biomarkers can be detected through antigen-antibody reaction with a capture antibody attached to a substrate to increase the accuracy of the measurement result.

Therefore, the substrate included in the kit of the present invention may be a surface treated to detect a biomarker so that the biomarker is directly attached to the surface of the substrate or a capture antibody capable of capturing the biomarker is attached. . When attached to the detection process, it is preferable that the substrate is coated with a capture antibody and a binding specific substance. Specifically, it is preferable to apply a coating that is specific to the capture antibody and is non-specific to the detection antibody or strand.

Alternatively, the capture antibody may be pre-attached to the surface of the substrate. At this time, the substrate may be any one selected from the group consisting of slide glass, cover slip, quartz, plastic, etc., but is not limited thereto.

In addition, the kit of the present invention may be a research use only (RUO), an investigational use only (IUO) kit, or an in vitro diagnostic (IVD).

According to one side, the fluorescent molecule may be a specific base of the strand, 3'- or 5'-end, or act as a donor or acceptor on the backbone. Preferably, the fluorescent molecule acting as a donor or acceptor may be labeled on a specific base of a docking strand, a donor strand or a fret strand to measure fret efficiency.

The method for simultaneous detection of multiple biomarkers of the present invention may be a detection method using the above-described multiple biomarker detection kit, or may include a method described below.

In using the method of the present invention, a step of preparing a fret efficiency database described below is preceded. The distinguishable fret efficiency reference value can be measured in advance and databased, and the type of biomarker can be confirmed by comparing the measured fret efficiency measurement value after the reaction.

The fret efficiency database is produced through the following process.

By constructing a FRET pair with donors and acceptors for different fluorescent molecules A and B, respectively, A and B are marked on a specific base on the DNA basic skeleton, and DNA with the different types of fret pairs labeled as required To produce.

For example, if 50 kinds are required on the database, 50 kinds of labeled DNAs with different distances between labeled A and B are prepared. At this time, at one end of each DNA, a material that is applied to the substrate and a binding-friendly material are attached, so that it can be fixed to the substrate. If necessary, when the donor and acceptor to be used are commercially available fluorescent molecular materials, the production process may be omitted and commercially available DNA may be used with the donor and acceptor attached.

However, in determining the fluorescent molecule used as the donor and acceptor, it is preferable that the wavelength of the maximum value of the emission spectrum of the fluorescent molecule used as the donor is shorter than the wavelength of the maximum value of the absorption spectrum of the fluorescent molecule used as the acceptor. Do.

When two or more types of DNA are prepared, DNA having a distance of 1 fret pair is fixed to a substrate and the fret efficiency of the distance is measured. Subsequently, the same procedure as described above is repeated in sequence from DNA having a distance between two fret pairs to obtain a fret efficiency reference value, and this is databased by distance.

Subsequently, by comparing the fret efficiency measurement value measured in the biomarker detection process with the fret efficiency reference value, the distance between the donor and the acceptor of the fret pair used for the detection can be known, which indicates the type of the detected biomarker. Able to know.

However, the fret efficiency measurement may have an error due to noise, so the fret efficiency of all distances may not be distinguishable. For example, if the fret efficiency when the distance is 1 is 0.95 (average) ± 0.05 (error), and the fret efficiency when the distance is 2 is 0.94 ± 0.05, the distances 1 and 2 cannot be distinguished by the fret efficiency. . Therefore, when detecting an actual biomarker, it is preferable to use only a distance in which fret efficiency is clearly distinguished among values in the database.

When the fret efficiency reference value is secured, a step of injecting a sample containing the biomarker and attaching the biomarker to the substrate is performed to detect the biomarker.

According to one side, the method for simultaneously detecting multiple biomarkers may further include attaching a capture antibody to a substrate.

The method of adsorbing the capture antibody to the substrate is, for example, diluting the capture antibody with a carbonate buffer solution or a bicarbonate buffer solution of 0.06M with a pH of 9.5, and adsorbing the diluted antibody by contacting the substrate at a constant temperature for a predetermined time. It may be. Alternatively, the surface of the substrate is coated with biotin-bound PEG (polyethylene glycol) or BSA (bovine serum albumin), combined with streptavidin, and adsorbed by binding to the biotin-bound capture antibody. May be Subsequently, when the capture antibody adsorbed on the substrate is processed with a sample or a processed sample on the substrate, a biomarker included in the sample is formed. Additionally, after formation of the conjugate, it may be desirable to wash with a wash buffer containing TWEEN 20, Triton X-100, etc. for the purpose of removing non-specifically bound antibodies or removing contaminants.

Strands used in the present invention may use nucleic acids such as DNA, RNA, PNA, LNA, MNA, GNA, TNA, or analogs, respectively. In addition, the fluorescent molecule used in the present invention, rhodamine (rhodamine), Alexa (Alexa), fluorescein (fluorescein), FITC (fluorescein isothiocyanate), FAM (5-carboxy fluorescein), Atto, BODIPY, CF, CY , DyLight Fluor and Texas Red (Texas Red) is preferably selected from the group consisting of, but is not limited thereto.

In addition, the sample used in the present invention may be selected from the group consisting of blood, plasma, serum, saliva, tissue fluid, and urine, but only as long as the biomarker that needs to be detected is included depending on the type or characteristics of the diagnosis. It is not limited.

Subsequently, the detection of N biomarkers using the detection method of the present invention will be described later in more detail through examples.

Hereinafter, the present invention will be described in detail through examples.

Example 1. Preparation of fret efficiency database

Different fluorescent molecules A, B, and C are donor 1, donor 2, and acceptor, respectively, and donor 1 is marked on the donor strand and donor 2 and acceptor are marked on the fret strand. The distance between two fluorescent molecules A and B is determined so that the fret efficiency between donor 1 and donor 2 is maximized. B and C are each composed of a fret pair with a donor 2 and an acceptor, to be marked on the fret strand, and a fret strand having a different type of fret pair is produced as necessary.

For example, if 50 types are required on the database, 50 different fret strands with different distances between labeled B and C are prepared. If necessary, when the donor 1, donor 2, and acceptor used are commercially available fluorescent molecular materials, the production process may be omitted and a commercially available fret strand with donor 1, donor 2, and acceptor attached may be used. .

However, in determining the fluorescent molecules used as the donor 1, donor 2 and acceptor, it is preferable that the wavelength of the maximum value of the emission spectrum of each fluorescent molecule is the shortest donor 1 and the longest acceptor.

When two or more types of fret strands are prepared, a fret strand having a distance between donors 2-acceptors and a donor strand is coupled to a docking strand, and then the fret efficiency of the distance is measured. Thereafter, from the fret strand having a distance between two donor 2-acceptor fret pairs, the same procedure as described above is repeated in order to obtain a fret efficiency reference value, and this is databased by distance.

Subsequently, by comparing the fret efficiency measurement value measured in the biomarker detection process with the fret efficiency reference value, the distance between the donor 2-acceptor of the fret pair used for detection can be known, and the type of the biomarker detected therefrom. You can check

However, the fret efficiency measurement may have an error due to noise, so the fret efficiency of all distances may not be distinguishable. For example, if the fret efficiency when the distance is 1 is 0.95 (average) ± 0.05 (error), and the fret efficiency when the distance is 2 is 0.94 ± 0.05, the distances 1 and 2 cannot be distinguished by the fret efficiency. . Therefore, when detecting an actual biomarker, it is preferable to use only a distance in which fret efficiency is clearly distinguished among values in the database. Thereafter, in the embodiments described below, it is assumed that 10 of the fret efficiency reference values of distances 1 to 50 are clearly distinguishable, and the detection example will be described.

Example 2. Fabrication of N kinds of docking strands and N kinds of fret strands

Docking strands can be manufactured in the number of types of biomarkers to be actually detected.

Since the fret efficiency that can be clearly distinguished by one pair of donor 2-acceptor fret pairs in Example 1 is assumed to be 10, if there are 10 types of biomarkers to be detected, 1 pair of donor 2-acceptor fret pairs It is possible to detect sufficiently by itself. Therefore, it is manufactured so that one fret strand can be coupled to the docking strand.

If, when the type of biomarkers to be detected exceeds 10, two or more fret pairs composed of donor 2-acceptors may be used. Therefore, it is manufactured such that two or more fret strands can be coupled to the docking strand. If two fret pairs are used, the clearly distinguishable fret efficiency will be up to 55, and if three fret pairs are used, the clearly distinguishable fret efficiency will be up to 205. In theory, if the fret efficiency that can be divided into one fret pair is 10 at maximum, it may be determined that the fret efficiency that can be divided into two fret pairs can be calculated as 100, but the fret pair 1 and the fret pair If the fret efficiency of 2 is i) a, b and ii) b, a, respectively, the cases of i) and ii) cannot be distinguished from each other, so only one of i) or ii) is docked strand If used as, there are practically 55 cases where fret efficiency can be distinguished. However, if the types of fluorescent molecules of the donor 2 or acceptor of the fret pair 1 and the fret pair 2 are different, the fret efficiency that can be divided into two pairs may be 100, and the three pairs may be 1000.

Example 3. Measurement of fret efficiency and discrimination of biomarkers

Kits were constructed as follows to detect two types of biomarkers, biomarkers 1 or 2.

Docking strand 1 was attached to detection antibody 1 binding to biomarker 1, and docking strand 2 was attached to detection antibody 2 binding to biomarker 2. The docking strands 1 and 2 were manufactured so that two types of fret strands can be combined. The docking strand 1 was manufactured such that a fret strand having a fret efficiency of 0.2 and 0.4, respectively, and a fret strand having a fret efficiency of 0.6 and 0.8, respectively, could be combined with the docking strand 2.

Biomarkers 1 and 2 were fixed to the substrate, and detection antibodies 1 and 2 were bound to each. After injecting into the chamber a buffer containing four types of fret strands having donor strands and fret efficiency of 0.2, 0.4, 0.6, and 0.8, respectively, the fret efficiency of the fret strand bound to the docking strand was measured. As described above with reference to FIG. 9, the positions of the molecules 1 and 2 are observed as intermittent fluorescent signals on the imaging camera observing the brightness of the donor 2 and the acceptor, and the brightness of the donor 2 and the acceptor is measured to measure the fret. Efficiency was analyzed. As a result of the measurement, since the fret efficiency of 0.2 and 0.4 was measured at the position of molecule 1, it was confirmed that molecule 1 is biomarker 1. With the same principle, fret efficiency of 0.6 and 0.8 was measured at the position of molecule 2, and it was confirmed that molecule 2 is biomarker 2.

The biomarkers included in the sample have a very wide concentration range from fg / ml to mg / ml depending on the type. When the biomarker having an excessively high concentration is fixed to a substrate, the number of dots appearing on one screen is too large, and overlapping occurs between the dots. As a result, the fret efficiency of individual molecules cannot be measured. Samples can be diluted in various proportions to detect molecules.

If biomarkers having a similar concentration range are detected, the analysis can be performed after diluting the biomarkers at the same ratio. For example, when diluting biomarkers having a concentration of about 1 ng / ml to 1: 100, if an appropriate number of biomarker molecules are detected on one screen, biomarkers having a concentration of about 100 ng / ml are typically 1: Analysis can be performed after dilution to 10000.

As described above, although the embodiments have been described by the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and / or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (11)

Board;
N kinds of docking strands attached to N kinds of detection antibodies; And
A donor strand complementarily binding to the N-type docking strand;
Including,
The docking strand or donor strand contains a fluorescent molecule,
A kit for simultaneous detection of multiple biomarkers that simultaneously identifies N kinds of biomarkers using fret efficiency by the fluorescent molecule. According to claim 1,
The kit is a kit for simultaneous detection of multiple biomarkers further comprising a FRET strand labeled with a fluorescent molecule. According to claim 2,
The fret strand is a kit for simultaneous detection of multiple biomarkers that complementarily binds to a docking strand. The method according to claim 1 or 2,
The fluorescent molecule acts as a donor or acceptor on a specific base, 3'- or 5'-end, or backbone of a docking strand, donor strand or fret strand. Kit for simultaneous detection of multiple biomarkers. According to claim 1,
The kit is a kit for simultaneous detection of multiple biomarkers, which further comprises a capture antibody. A method for simultaneously detecting multiple biomarkers in a sample,
Attaching N biomarkers to the substrate;
Binding each of the N types of detection antibodies to which the N types of docking strands are attached to the N types of biomarkers;
Donor strand (Donor strand) is coupled to each of the N kinds of docking strands; And
Measuring fret efficiency generated by the fluorescent molecules included in the docking strand or the donor strand;
Including,
Simultaneous detection method of multiple biomarkers for determining the type of the N biomarkers using the fret efficiency. The method of claim 6,
A step in which a fluorescent molecule-labeled FRET strand binds to the N-type docking strand;
Simultaneous detection method of multiple biomarkers further comprising a. The method of claim 7,
The fret strand is a method for simultaneous detection of multiple biomarkers that are complementary to the N-type docking strand. The method of claim 6 or 7,
The fluorescent molecule acts as a donor or acceptor on a specific base, 3'- or 5'-end, or backbone of a docking strand, donor strand or fret strand. Simultaneous detection method of multiple biomarkers. The method of claim 6,
The method of detecting multiple biomarkers simultaneously, further comprising attaching a capture antibody to the substrate. The method of claim 6,
The method of detecting multiple biomarkers simultaneously, further comprising measuring the concentration of the biomarkers in the sample from the number of biomarkers per unit area.

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