Patent Publication Number: US-2021189471-A1

Title: Molecular beacon-based optical gene biosensor employing retro-reflection and quantitative analysis method of nucleic acid molecule

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims a benefit under 35 U.S.C. § 119(a) of Korean Patent Application No. 10-2017-0053397 filed on Apr. 26, 2017, on the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference for all purposes. 
     BACKGROUND 
     1. Field 
     The present disclosure relates to an optical gene biosensor capable of quantitative analysis of a target nucleic acid molecule using an optical scheme and an analytical method using the same. 
     2. Description of Related Art 
     The molecular beacon refers to an oligonucleotide with a stem-loop structure with self-complementary sections. The beacon includes a stem section positioned at each of both ends thereof and having a self-complementary sequence, and a loop section between the two stem sections and having a complementary sequence to a nucleic acid molecule (DNA, RNA, etc.) to be detected. 
     In a conventional biosensor using the molecular beacon, typically, one end of the oligonucleotide is modified with a fluorophore, while the other end thereof is modified with a quencher which can absorb and quench fluorescence derived from the fluorophore. 
     When no target nucleic acid molecule is present, the stem sections with the self-complementary sequences hybridize with each other, and thus the molecular beacon exhibits a hairpin structure. In this structure, the fluorophore and the quencher defining both distal ends of the molecular beacon are adjacent to each other. As a result, the fluorescence from the fluorophore is transferred to the quencher via fluorescence resonance energy transfer and thus is quenched. In contrast, when a complementary target nucleic acid molecule is present, the target nucleic acid molecule is hybridized with the loop section of the molecular beacon to form a double helix structure. As a result, a torsional force acts to separate the stem sections of the molecular beacon from each other. As a result, a distance between the fluorophore and the quencher becomes larger, such that the energy transfer phenomenon between the fluorophore and the quencher is eliminated, and thus a fluorescence signal unique to the fluorophore is emitted. Since an intensity of the fluorescence signal emitted under this condition is proportional to a concentration of the target nucleic acid molecule, presence or absence of the target nucleic acid molecule or the concentration thereof can be quantitatively/quantitatively analyzed by tracking the fluorescence signal change. 
     As described above, the conventional molecular beacon-based biosensor may use the fluorophore. Thus, spectroscopy-based fluorescence analysis equipment is required. 
     Specifically, since the fluorophore provides a valid signal only related to excitation light having a particular wavelength, the conventional biosensor uses essentially a combination of a halogen lamp that provides all wavelengths as a light source and a monochromator that can select and irradiate light having a specific wavelength from the all wavelengths. Alternatively, the conventional biosensor uses essentially a high power laser device and LEDs for supplying laser having short wavelengths. In this case, since these light sources are very expensive and require high power consumption, it may be difficult to miniaturize, carry and commercialize the biosensor. 
     Further, according to the conventional biosensor, an emission filter is required to specifically detect and separate the excitation light irradiated with the fluorescent signal derived from the fluorophore. In this connection, expensive light receiving equipment such as a photomultiplier tube (PMT) is essential for sensitive detection of the fluorescence signal. In addition, there is a problem that a very sophisticated arrangement and assembly between these optical components is required. 
     SUMMARY 
     This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter. 
     One purpose of the present disclosure is to provide an optical gene biosensor that is capable of detecting presence, a concentration, and the like of a target nucleic acid molecule in a non-spectral manner using a retro-reflective optical marker. 
     Another purpose of the present disclosure is to provide a method for performing quantitative analysis of a target nucleic acid molecule using the optical gene biosensor. 
     Purposes of the present disclosure are not limited to the above-mentioned purpose. Other purposes and advantages of the present disclosure as not mentioned above may be understood from following descriptions and more clearly understood from embodiments of the present disclosure. Further, it will be readily appreciated that the purposes and advantages of the present disclosure may be realized by features and combinations thereof as disclosed in the claims. 
     A first aspect of the present disclosure proposes an optical gene biosensor comprising: a substrate; a molecular beacon anchored to the substrate, wherein the molecular beacon includes an oligonucleotide specifically binding to a target nucleic acid molecule and a first compound bound to a first terminal of the oligonucleotide; an optical marker specifically binding to the first compound, wherein the optical marker is configured to retro-reflect irradiated light; a light source for irradiating the optical marker with light; and a light-receiver for receiving light retro-reflected from the optical marker. 
     In one implementation of the first aspect, the oligonucleotide includes: a loop section having base sequences complementary to base sequences of the target nucleic acid molecule; a first stem section extending from a first terminal of the loop section and having a terminal coupled to the first compound; and a second stem section extending from a second terminal of the loop section opposite to the first terminal of the loop section and having base sequences complementary to base sequences of the first stem section, wherein the second stem is bound to the substrate. 
     In one implementation of the first aspect, the loop section includes 20 to 40 base sequences, wherein each of the first and second stem sections includes 4 to 8 base sequences. 
     In one implementation of the first aspect, the first compound includes at least one selected from a group consisting of biotin, dinitrophenyl (DNP), and digoxigenin (DIG). 
     In one implementation of the first aspect, the molecular beacon further includes a first spacer compound connecting the first compound to a terminal of the first stem section, wherein the first spacer compound includes an alkyl chain having 4 to 10 carbons or mini-poly(ethylene glycol) having about 4 to 10 repeating units. 
     In one implementation of the first aspect, the molecular beacon further includes a functional group covalently bonding to the substrate. 
     In one implementation of the first aspect, the molecular beacon further includes a second spacer compound connecting the functional group to a terminal of the second stem section, wherein the second spacer compound includes an alkyl chain having 4 to 10 carbons or mini-poly(ethylene glycol) having about 4 to 10 repeating units. 
     In one implementation of the first aspect, the optical marker includes: a transparent core particle; a total-reflection inducing layer to cover a portion of a surface of the core particle, wherein the total-reflection inducing layer is made of a material having a refractive index lower than a refractive index of the core particle in a visible light wavelength range of 360 nm to 820 nm; a to-be-modified layer formed on the total-reflection inducing layer; and a second compound coupled to the to-be-modified layer, wherein the second compound specifically binds to the first compound. 
     In one implementation of the first aspect, the core particle is made of one selected from a group consisting of silica, glass, polystyrene, and polymethyl methacrylate having a refractive index of 1.4 or greater in the visible wavelength range. 
     In one implementation of the first aspect, the total-reflection inducing layer is made of at least one selected from a group consisting of aluminum (Al), copper (Cu), gold (Au), silver (Ag), and zinc (Zn). 
     In one implementation of the first aspect, the core particle has an average diameter of 600 nm or greater and 2 μm or smaller, wherein the total-reflection inducing layer covers 30% to 70% of the surface of the core particle. 
     In one implementation of the first aspect, the total-reflection inducing layer has a thickness of 10 to 100 nm. 
     In one implementation of the first aspect, the to-be-modified layer is made of at least one selected from a group consisting of platinum (Pt), gold (Au), and silver (Ag). 
     In one implementation of the first aspect, the first compound includes at least one selected from a group consisting of biotin, dinitrophenyl (DNP), and digoxigenin (DIG), wherein when the first compound includes the biotin, the second compound includes at least one selected from a group consisting of avidin, streptavidin and neutraavidin specifically binding to the biotin, wherein when the first compound includes the dinitrophenyl, the second compound includes an anti-DNP antibody compound specifically binding to the dinitrophenyl (DNP), wherein when the first compound includes the digoxigenin (DIG), the second compound includes an anti-DIG antibody compound specifically binding to the digoxigenin (DIG). 
     In one implementation of the first aspect, the optical marker further includes a magnetic layer made of magnetic material, wherein the magnetic layer is placed between the total-reflection inducing layer and the to-be-modified layer. 
     In one implementation of the first aspect, the light-source is placed above the optical marker, and irradiates the optical marker with mixed light of a plurality of wavelengths or light of a single wavelength, wherein the light-receiver is placed above the optical marker and is spaced apart from the light-source, wherein the light-receiver receives light generated from the light-source and then irradiated to the optical marker and then retro-reflected from the optical marker. 
     In one implementation of the first aspect, the light-source emits light in an inclined manner in a range of 0° exclusive to 45° inclusive with respect to a propagating direction of the retro-reflected light. 
     In one implementation of the first aspect, the light-receiver includes: an image generator for receiving the retro-reflected light and for imaging the light into an image signal; and an image analyzer for analyzing the image signal generated from the image generator. 
     In one implementation of the first aspect, the image analyzer counts a number of the optical markers based on the image signal and analyzes a concentration of the target nucleic acid molecule based on the number. 
     In one implementation of the first aspect, the image analyzer measures an amount of light retro-reflected from the optical marker based on the image signal, and analyzes a concentration of the target nucleic acid molecule based on the amount. 
     A second aspect of the present disclosure proposes a method for quantitatively analyzing a nucleic acid molecule, the method comprising: a first step of reacting a target nucleic acid molecule with a molecular beacon immobilized on a substrate; a second step of specifically binding an optical marker to the molecular beacon to which the target nucleic acid molecule is bound; a third step of irradiating light to the optical marker coupled to the molecular beacon; and a fourth step of generating and analyzing an image corresponding to light retro-reflected from the optical marker. 
     In one implementation of the second aspect, the first step includes: applying a solution sample containing the target nucleic acid molecule onto the substrate to which the molecular beacon is immobilized; and reacting the target nucleic acid molecule with a loop section of the molecular beacon, wherein the loop section has base sequences complementary to base sequences of the target nucleic acid molecule. 
     In one implementation of the second aspect, the second step includes: applying the optical markers onto the substrate on which the solution sample is present; and specifically binding the optical markers to the molecular beacon to which the target nucleic acid molecule is bound. 
     In one implementation of the second aspect, the method further comprises, after the second step and before the third step, removing non-reacted optical markers not bound to the molecular beacon. 
     In one implementation of the second aspect, in the third step, white light is irradiated to the optical marker, wherein in the fourth step, quantitative analysis of the target nucleic acid molecule is performed based on an image corresponding to retro-reflected white light. 
     Effects of the present disclosure are as follows but are not limited thereto. 
     According to the present disclosure, after binding the target nucleic acid molecule to the molecular beacon, the optical marker is bound to the molecular beacon. Thus, the quantitative analysis of the target nucleic acid molecule is performed using light retro-reflected from the optical marker. Thus, the target nucleic acid molecule can be quantitatively analyzed at a high sensitivity without use of expensive and bulky optical equipment, compared to the conventional biosensor using the fluorophore. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated in and form a part of this specification and in which like numerals depict like elements, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. 
         FIG. 1  is a schematic diagram for describing an optical gene biosensor according to an embodiment of the present disclosure. 
         FIG. 2  is a schematic view for describing a molecular beacon shown in  FIG. 1 . 
         FIG. 3 a    and  FIG. 3 b    are cross-sectional views for describing an optical marker shown in  FIG. 1 . 
         FIG. 4  is a schematic diagram for describing a positional relationship between a light-source and a light-receiver shown in  FIG. 1 . 
         FIG. 5  is a flow chart to describe a method of quantitative analysis of a nucleic acid molecule according to an embodiment of the present disclosure. 
         FIG. 6  is a graph showing a result of gene analysis according to Present Example 2. 
         FIG. 7  is a graph showing the results of gene analysis according to Present Example 2. 
     
    
    
     DETAILED DESCRIPTIONS 
     For simplicity and clarity of illustration, elements in the figures. are not necessarily drawn to scale. The same reference numbers in different figures. denote the same or similar elements, and as such perform similar functionality. Also, descriptions and details of well-known steps and elements are omitted for simplicity of the description. Furthermore, in the following detailed description of the present disclosure, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be understood that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present disclosure. 
     Examples of various embodiments are illustrated and described further below. It will be understood that the description herein is not intended to limit the claims to the specific embodiments described. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the present disclosure as defined by the appended claims. 
     The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. As used herein, the singular forms “a” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprising”, “includes”, and “including” when used in this specification, specify the presence of the stated features, integers, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, operations, elements, components, and/or portions thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expression such as “at least one of” when preceding a list of elements may modify the entire list of elements and may not modify the individual elements of the list. 
     It will be understood that, although the terms “first”, “second”, “third”, and so on may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section described below could be termed a second element, component, region, layer or section, without departing from the spirit and scope of the present disclosure. 
     In addition, it will also be understood that when a first element or layer is referred to as being present “on” a second element or layer, the first element may be disposed directly on the second element or may be disposed indirectly on the second element with a third element or layer being disposed between the first and second elements or layers. It will be understood that when an element or layer is referred to as being “connected to”, or “coupled to” another element or layer, it can be directly on, connected to, or coupled to the other element or layer, or one or more intervening elements or layers may be present. In addition, it will also be understood that when an element or layer is referred to as being “between” two elements or layers, it can be the only element or layer between the two elements or layers, or one or more intervening elements or layers may be present. 
     Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. 
       FIG. 1  is a schematic diagram for describing an optical gene biosensor according to an embodiment of the present disclosure.  FIG. 2  is a schematic view for describing a molecular beacon shown in  FIG. 1 .  FIG. 3 a    and  FIG. 3 b    are cross-sectional views for describing an optical marker shown in  FIG. 1 .  FIG. 4  is a schematic diagram for describing a positional relationship between a light-source and a light-receiver shown in  FIG. 1 . 
     Referring to  FIG. 1  to  FIG. 4 , an optical gene biosensor  100  according to an embodiment of the present disclosure may quantitatively analyze a target nucleic acid molecule  10  to be detected in an optical manner. In one embodiment, the optical gene biosensor  100  may include a molecular beacon  110 , a substrate  120 , an optical marker  130 , a light-source  140 , and a light-receiver  150 . 
     The molecular beacon  110  may include an oligonucleotide  111  that specifically binds to a target nucleic acid molecule  10  and a first compound  112  that specifically binds to the optical marker  120 . 
     The oligonucleotide  111  may include a loop section  111   a , a first stem section  111   b , and a second stem section  111   c.    
     The loop section  111   a  may specifically bind to the target nucleic acid molecule  10 . For example, the loop section  111   a  may have a base sequence complementary to the target nucleic acid molecule  10  and thus hybridize with the target gene  10 . In one embodiment, the loop section  111   a  may include about 20 to about 40 base sequences in consideration of base sequences of the target nucleic acid molecule  10 . 
     The first stem section  111   b  may include a plurality of first base sequences extending from a first terminal of the loop section  111   a . The second stem section  111   c  may include a plurality of second bases extending from a second terminal of the loop section  111   a  opposite the first terminal. The first base sequences of the first stem section  111   b  may be complementary to the second base sequences of the second stem section  111   c . Thus, when the target nucleic acid molecule  10  is not bound to the loop section  111   a , the oligonucleotide  111  may have a hairpin structure in which the first and second stem sections  111   b  and  111   c  are hybridized with each other, as shown on a left side of  FIG. 3 . In one embodiment, each of the first and second stem sections  111   b  and  111   c  may include about 4 to 8 complementary base sequences. 
     A terminal of the first stem section  111   b  may be coupled to the first compound  112 . A terminal of the second stem section  111   c  may be fixed to the substrate  130 . This will be described later. 
     The first compound  112  may be coupled to the terminal of the first stem section  111   b  and may be specifically combined with the optical marker  120 . Specifically, when the first and second stem sections  111   b  and  111   c  are hybridized with each other, the optical marker  120  cannot bind to the first compound  112  due to steric hindrance. However, when the target nucleic acid molecule  10  hybridizes with the loop section  111   a , the hybridization between the first and second stem sections  111   b  and  111   c  is disabled, such that the optical marker  120  may be coupled to the first compound  112 . 
     The first compound  112  is not limited to a specific type as long as the compound  112  can specifically bind to a specific compound of the optical marker  120 . In one embodiment, the first compound  112  may include a ligand molecule. For example, the first compound  112  may include one or more selected from a group consisting of biotin, dinitrophenyl (DNP), digoxigenin (DIG), and the like. 
     In one embodiment, the first compound  112  may be coupled to the terminal of the first stem section  111   b  via a first spacer compound  114 . That is, the first compound  112  may be coupled to one terminal of the first spacer compound  114 , while the other terminal of the first spacer compound  114  may be coupled to the terminal of the first stem section  111   b . Otherwise, the first compound  112  may be directly bonded to the terminal of the first stem section  111   b . In this case, structural and steric hindrance caused by the molecular beacon  110  may reduce a bond yield between the first compound  112  and a second compound  135  of the optical marker  130 . However, when coupling the first compound  112  to the terminal of the first stem section  111   b  via the first spacer compound  114 , the first compound  112  is spaced further away from the substrate  120  due to the presence of the first spacer compound  114 . Thus, not only the first compound  112  may be exposed to a sample solution containing the target nucleic acid molecule  10  but also fluidity thereof in the sample solution is improved, thereby to improve the bond yield between the first compound  112  and the second compound  135  of the optical marker  130 . 
     In one embodiment, the first spacer compound  114  may include an alkyl chain having about 4 to 10 carbons or mini-poly(ethylene glycol) having about 4 to 10 repeating units. 
     In one example, the terminal of the second stem section  111   c  may be modified with a functional group  113  to be fixed to the substrate  130 . The functional group  113  is not limited to a specific type as long as the functional group  113  forms a chemical bond with the substrate  130  to fix the molecular beacon  110  to the substrate  130 . For example, the functional group  113  may include an amine group —NH 2 . In one embodiment, the functional group  113  may be coupled to the terminal of the second stem section  111   c  via a second spacer compound  115 . When the molecular beacon  110  includes the second spacer compound  115 , the oligonucleotide  111  may be spaced farther away from the substrate  120  and thus may be exposed to the sample solution containing the target nucleic acid molecule  10  as well as may have improved flow ability in the sample solution, thereby improving the hybridization yield between the loop section  111   a  of the oligonucleotide  111  and the target nucleic acid molecule  10 . Further, the second spacer compound  115  may act in a similar manner to the first spacer compound  114  to improve the bond yield between the first compound  112  and the second compound  135  of the optical marker  130 . 
     In one embodiment, the second spacer compound  115  may include an alkyl chain having about 4 to 10 carbons or about 4 to 10 repeating units-containing mini-poly(ethylene glycol). 
     The substrate  120  is not limited to a specific type as long as the molecular beacon  110  may be fixed thereto. For example, the substrate  120  may be made of glass, silicon, polymer, metal, etc., and may have a flat top surface to which the molecular beacon  110  is bonded. The polymer may include polystyrene (PS), polymethyl methacrylate (PMMA), olefin copolymer (COC), polydimethylsiloxane (PDMS), etc. The metal may include noble metals such as gold (Au), platinum (Pt), and silver (Ag). 
     In one embodiment, when the substrate  120  is made of one of a glass, a silicone, polymer and the functional group  113  of the molecular beacon  110  is an amine group, the surface of the substrate  120  may be activated by a plasma treatment or ultraviolet light exposure thereto, and then 3-aminopropyltriethoxysilane (APTES) is applied to the active surface thereof to expose the amine group. Subsequently, an amine reactive crosslinker such as glutaraldehyde is applied thereto. This may cause covalent bonding between the amine group which is a functional group of the molecular beacon  110  and an amine group of the 3-aminopropyltriethoxysilane. In this way, the molecular beacon  110  may be fixed to the substrate  120 . 
     In another embodiment, when the substrate  120  is made of a noble metal and the functional group  113  of the molecular beacon  110  is an amine group, an amine-reactive self-assembled monolayer (SAM) is formed using a material such as DTSSP (3,3′-dithiobis(sulfosuccinimidyl propionate) or DTSP (dithiobis(succinimidyl propionate) to expose a succinimide group on the surface of the substrate  120 . Then, the amine functional group  113  of the molecular beacon  110  and the succinimide group may be covalently bonded to each other via an amide bond. In this way, the molecular beacon  110  may be fixed to the substrate  120 . 
     In one example, in the two cases, after combining the molecular beacon  110  to the substrate  120 , a molecule including an amine group such as ethanolamine may be used to block reaction residues remaining on the surface of the substrate, such as aldehyde or succinimide group. 
     The optical marker  130  may specifically bind to the first compound of the molecular beacon  110  and may retro-reflect the light emitted from the light-source  140  toward the light-source  140 . 
     In one embodiment, as shown in  FIG. 3 a   , the optical marker  130  may include a transparent core particle  131 , a total-reflection inducing layer  132  covering a portion of the transparent core particle  131 , a to-be-modified layer  133  disposed on the total-reflection inducing layer  132 , and a second compound  135  coupled directly or indirectly to the to-be-modified layer  133 . 
     The core particle  131  may have a spherical shape. As used herein, the term ‘spherical’ means not only a perfect sphere with the same radius from a center to every point on a surface thereof but also an actual sphere whose difference between a maximum radius and a minimum radius is smaller than about 10%. 
     In one embodiment, the core particle  131  may have an average diameter of about 600 nm or greater and 2 μm or smaller, in consideration of a relationship between the diameter and a wavelength of light emitted from the light-source  140 , bio sensing ability, and the like. 
     In one embodiment, the core particle  131  may be made of a transparent material that may transmit incident light. For example, the core particle  131  may be made of a transparent oxide, a transparent polymer material, or the like. The transparent oxide may include, for example, silica, glass, etc. The transparent polymer material may include, for example, polystyrene, poly methyl methacrylate, and the like. 
     The total-reflection inducing layer  132  may be formed to cover a portion of the surface of the core particle  131  and may totally reflect at least a portion of light traveling inside the core particle  131  to increase an amount of light as retro-reflected toward the light-source  140 . 
     In one embodiment, the total-reflection inducing layer  132  may be formed on the surface of the core particle  131  to cover an area of at least about 30% and at most 70% of the surface of the core particle  131 . When the total-reflection inducing layer  132  covers an area smaller than 30% of the surface of the core particle  131 , there is a problem that a sensitivity of the gene biosensor  100  is deteriorated since a large amount of the light incident inside the core particle  131  is not retro-reflected but is leaked. To the contrary, when the total-reflection inducing layer  132  covers an area greater than 70% of the surface of the core particle  131 , there may be a problem that the sensitivity of the gene biosensor  100  is reduced since the amount of light incident to the core particle  131  decreases. Thus, in one embodiment, the total-reflection inducing layer  132  may be formed on the surface of the core particle  131  to cover at least about 40% or at most 60% of the surface of the core particle  131 . 
     In one embodiment, to totally reflect at least a portion of the light propagating inside the core particle  131  to increase the amount of light retro-reflected toward the light-source  140 , the total-reflection inducing layer  132  may be made of a material with a lower refractive index than that of the core particle  131 . In one embodiment, the core particle  131  may be made of a material having a refractive index of at least about 1.4 in a visible wavelength range of at least 360 nm to 820 nm, while the total-reflection inducing layer  132  may be made of a material having a refractive index lower than that of the core particle  131 . Specifically, when the core particle  131  is made of a transparent oxide or transparent polymer material having a refractive index of about 1.4 or greater in the visible region, the total-reflection inducing layer  132  may be made of a metallic material having a refractive index lower than 1.4. For example, the total-reflection inducing layer  132  may be made of at least one selected from gold (Au) having a refractive index of about 0.22 with respect to light at a wavelength of 532 nm, silver (Ag) having a refractive index of about 0.15 with respect to light at a wavelength of 532 nm, aluminum (Al) having a refractive index of about 1.0 with respect to light at a wavelength of 532 nm, copper (Cu) with a refractive index of about 0.4 with respect to light at a wavelength of 532 nm, zinc (Zn) with a refractive index of about 1.2 with respect to light at a wavelength of 532 nm. 
     In one embodiment, to improve adhesion between the total-reflection inducing layer  132  and the core particle  131 , chromium (Cr) may be applied to the surface of the core particle  131  and then the total-reflection inducing layer  132  may be formed thereon. In this case, in order to prevent retro-reflection ability from deteriorating, the chromium is preferably applied at a thickness of about 2 nm to 5 nm. Alternatively, in another embodiment, the total-reflection inducing layer  132  itself may be made of a material that is highly adhesive with the core particle  131 . For example, when the core particle  131  is made of a transparent oxide, the total-reflection inducing layer  132  may be made of aluminum (Al) or copper (Cu). 
     In one embodiment, the total-reflection inducing layer  132  may have a thickness of about 10 to 100 nm to prevent light leakage due to light transmission and to improve dispersibility of the optical marker  130 . When the thickness of the total-reflection inducing layer  132  is smaller than 10 nm, a portion of the light incident inside the core particle  131  may pass through the total-reflection inducing layer  132  and may leak. When the thickness of the total-reflection inducing layer  132  exceeds 100 nm, a weight of the optical marker  130  may increase, resulting in a decrease in the dispersibility of the optical marker  130  in the liquid. 
     The to-be-modified layer  133  may be formed on the surface of the total-reflection inducing layer  132 . The to-be-modified layer  133  may be made of a metallic material that can be easily combined with biological substances. For example, the to-be-modified layer  133  may be made of noble metals such as platinum (Pt), gold (Au), silver (Ag), etc., which are easily modified with biological substances and have excellent anti-oxidation ability. 
     In one embodiment, the to-be-modified layer  133  may be formed as a separate layer independent of the total-reflection inducing layer  132 . For example, when the total-reflection inducing layer  132  may be made of a metal material other than noble metals, while the to-be-modified layer  133  may be made of a noble metal material layer covering the total-reflection inducing layer  132 . 
     Alternatively, in another embodiment, the to-be-modified layer  133  and the total-reflection inducing layer  132  may be integrally formed with each other. For example, when the total-reflection inducing layer  132  is made of noble metals with a lower refractive index than that of the core particle, such as gold (Au) or silver (Ag), the total-reflection inducing layer  132  may function as the to-be-modified layer  133 . 
     In one embodiment, the to-be-modified layer  133  may have a thickness of about 100 nm or smaller to prevent the dispersibility decrease, and thus aggregation within the liquid of the optical marker  130 . 
     The second compound  135  may be bonded directly or indirectly to the to-be-modified layer  133  and may bind specifically to the first compound  112  of the molecular beacon  110 . A type of the second compound  135  may vary depending on the first compound  112 . The second compound  135  may include one or more selected from proteins, nucleic acids, ligands, and the like. For example, when the first compound  112  of the molecular beacon  110  includes a biotin, the second compound  135  may include one or more protein compounds selected from avidin, streptavidin, neutravidin, etc., which may specifically bind to the biotin. In another example, when the first compound  112  of the molecular beacon  110  includes dinitrophenyl (DNP), the second compound  135  may include an anti-DNP antibody compound that may specifically bind to the dinitrophenyl (DNP). In another example, when the first compound  112  of the molecular beacon  110  includes digoxigenin (DIG), the second compound  135  may include an anti-DIG antibody compound that may bind to the digoxigenin (DIG). 
     In one example, the second compound  135  may be bound only to a surface of the to-be-modified layer  133  and may not bond to an exposed surface of the core particle  131 . Thus, when specifying a location of the second compound  135  with respect to the core particle  131  coated by the total-reflection inducing layer  132  and the to-be-modified layer  133 , an exposed portion of the core particle  131  is directed to the light-source  140  when the maker  130  is bound to the molecular beacon  110 , thereby inducing a more powerful retro-reflective signal. As a result, the sensitivity of the gene biosensor  100  can be significantly improved. 
     In one embodiment of the present disclosure, as shown in  FIG. 3 b   , the optical marker  130  may further include a magnetic layer  134  made of a magnetic material and disposed between the total-reflection inducing layer  132  and the to-be-modified layer  133 . The magnetic layer  134  may be made of a magnetic material such as, for example, iron (Fe), nickel (Ni), manganese (Mn), fired bodies or oxides thereof. When the optical marker  130  further includes the magnetic layer  134 , a magnetic field may be externally applied thereto, such that an orientation of the optical marker  130  may be adjusted to not only induce a more powerful retro-reflective signal, but also the external magnetic field may be used to remove optical markers  130  which are not combined with the molecular beacon  110 . 
     The light-source  140  may be placed above the optical marker  130 . The optical marker  110  coupled to the molecular beacon  110  may be irradiated with light from the light source. A light source type of the light-source  140  is not limited to a specific type. For example, the light source type of the light-source  140  may include a light source type for generating light in which light-beams of various wavelengths are mixed, or a light source type for generating monochromatic light of a specific wavelength. Specifically, the light source type of the light-source  140  may include a halogen lamp, a mercury lamp, a fluorescent lamp, a light emitting diode (LED), or a laser. However, the present disclosure is not limited thereto. 
     The light-receiver  150  may be placed above the optical marker  130  and be spaced apart from the light-source  140 . The light-receiver  150  may receive the light emitted from the light-source  140  and irradiated to the optical marker  130  and then retro-reflected from the optical marker  130  and may quantitatively analyze information about the presence or absence and concentration of the target nucleic acid molecule  10  based on the received light. As long as the light receiver  150  could receive the retro-reflected light and analyze the information about the target nucleic acid molecule  10  based on the retro-reflected light, a configuration of the light-receiver  150  is not particularly limited. In one embodiment, the light-receiver  150  may include an image generator for imaging the retro-reflected optical signal and an image analyzer for analyzing image information generated by the image generator. In this case, the image generator may include any known image generating device such as a digital camera without limitation. As the image analyzer, a known image analysis system may be used without limitation. 
     In one embodiment, the light-receiver  150  may quantify the concentration of the target nucleic acid molecule  10  by the image analyzer counting the number of the optical markers  130  based on the image generated by the image generator. 
     In another embodiment, the light-receiver  150  may quantify the concentration of the target nucleic acid molecule  10  by the image analyzer measuring the amount of light retro-reflected from the optical marker  130  based on the image produced by the image generator. 
     In one embodiment, as shown in  FIG. 4 , to minimize the degradation of the sensitive due to interference between the light irradiated from the light-source  140  and the retro-reflected light from the optical marker  130 , and to prevent degradation of the sensitivity due to light mirror-reflected from the substrate  120 , the light-receiver is oriented to receive retro-reflected light within an angle range of about −10° to +10° relative to a normal line to a surface of the substrate  121  while the light-source  140  is oriented to irradiate light in an inclined manner at an angle of about 0° exclusive to 45° inclusive with respect to an advancing direction of the retro-reflected light. 
       FIG. 5  is a flow chart for describing the quantitative analysis method of nucleic acid molecule according to an embodiment of the present disclosure. The quantitative analysis method of the nucleic acid molecule according to an embodiment of the present disclosure may be performed using the optical gene biosensor  100  as described with reference to  FIGS. 1 to 4 . 
     Referring to  FIG. 5  along with  FIG. 1  to  FIG. 4 , the quantitative analysis method of the nucleic acid molecule according to an embodiment of the present disclosure includes a first step S 110  of reacting the target nucleic acid molecule  10  with the molecular beacon  110  immobilized on the substrate  120 ; a second step S 120  of specifically coupling the optical marker  130  to the molecular beacon  110  to which the target nucleic acid molecule  10  is bound; a third step S 130  of irradiating light to the optical marker  130  coupled to the molecular beacon  110 ; and a fourth step S 140  of generating and analyzing an image of the retro-reflected light from the optical marker  130 . 
     In the first step S 110 , a solution sample containing the nucleic acid molecule  10  is applied onto the substrate  120  to which the molecular beacon  110  is immobilized. Thus, the step S 110  may combine the loop section  111   a  of the molecular beacon  110  with the nucleic acid molecule  10 . As previously described, the loop section  111   a  has a base sequence complementary to a base sequence of the nucleic acid molecule  10  and thus may specifically bind to the nucleic acid molecule. To facilitate the binding between the molecular beacon  110  and the nucleic acid molecule  10 , the solution sample containing the nucleic acid molecule  10  may be heated to a temperature of about 35° C. to 45° C. However, the heating temperature may vary depending on a length of the base sequence of each of the molecular beacon  110  and the target nucleic acid molecule  10  and percentages of guanine (G) and cytosine (C) present in the base sequence. 
     In one example, after the solution sample containing the nucleic acid molecule  10  is applied onto the substrate  120  to induce the binding of the molecular beacon  110  to the nucleic acid molecule  10 , the solution sample may be cooled to about 1 to 5° C. for a predetermined time duration and then maintained at room temperature in order to convert molecular beacons  110  non-reacted with the nucleic acid molecule  10  to a hairpin structure. 
     In the second step S 120 , the optical markers  130  may be applied onto the substrate  120  on which the solution sample is present to specifically couple the optical markers  130  to the molecular beacon  110  to which the target nucleic acid molecule  10  is bound. In this case, the first compound  112  of the molecular beacon  110  may bind to the second compound  135  of the optical marker  130  in a specific manner. 
     As described above, the oligonucleotide  111  of each of the molecular beacons  110  has the first and second stem sections  111   b  and  111   c  formed at both terminals of the loop section  111   a  respectively and having complementary base sequences with each other. Thus, the oligonucleotide  111  bound to the nucleic acid molecule  10  maintains a linear structure in which the terminal thereof coupled to the first compound  112  becomes a top terminal away from the substrate  120 . To the contrary, in the oligonucleotide  111  to which the nucleic acid molecule  10  is not bound, the first and second stem sections  111   b  and  111   c  bind to each other such that the oligonucleotide has a hairpin structure. As a result, the terminal thereof coupled to the first compound  112  may face the substrate  120 . Thus, when applying the optical markers  130  on the substrate  120  where the solution sample is present, the optical markers  130  may be readily accessible to the first compound  112  of the linear oligonucleotide  111  to form a bond  135  between the first compound  112  of the oligonucleotide  111  and the second compound of the optical marker  130 . However, the first compound  112  of the oligonucleotide  111  with the hairpin structure may have difficulty in accessing to the optical marker  130  due to the steric hindrance. As a result, the bond between the first compound  112  of the oligonucleotide  111  and the second compound  135  of the optical marker  130  may not occur. 
     In one example, after coupling the optical marker  130  to the molecular beacon  110  to which the target nucleic acid molecule  10  is bound, the optical markers  130  as non-reacted with the beacon may be removed. The removal of the non-reacted optical markers  130  may be performed using a washing solution to remove a solvent of the solution sample and other non-reacted materials. 
     In the third step  130 , the light-source  140  may be used to irradiate the optical marker  130  coupled to the molecular beacon  110  with light. In this case, the second compound  135  of the optical marker  130  is bound only to the to-be-modified layer  133  surface and not to the exposed surface of the core particle  131 . Thus, when the optical marker  130  is bound to the molecular beacon  110 , the exposed surface of the core particle  131  of the optical marker  130  is oriented toward the light-receiver  150 . As a result, the receiver  150  may generate a stronger retro-reflective signal in response to the light irradiation from the light-source  140 . 
     In the fourth step  140 , the light-receiver  150  generates and analyzes an image of the substrate  120  to which the optical marker  110  is coupled, while the light-source  140  illuminates light. For example, the light-receiver  150  may generate an image of the substrate  120  using the image generator. The image may be analyzed by the image analyzer counting the number of the optical markers  130  based on the image. 
     According to the present disclosure, after binding the target nucleic acid molecule to the molecular beacon, the optical marker is bound to the molecular beacon. Thus, the quantitative analysis of the target nucleic acid molecule is performed using light retro-reflected from the optical marker. Thus, the target nucleic acid molecule can be quantitatively analyzed at a high sensitivity without use of expensive and bulky optical equipment, compared to the conventional biosensor using the fluorophore. 
     Hereinafter, Present Examples of the present disclosure will be described in detail. The following Present Examples are only some embodiments of the present disclosure, and the present disclosure should not be construed as limited to the following Examples. 
     Present Example 1 
     To demonstrate applicability of the molecular beacon-based gene bio-sensing scheme using the retro-reflection principle developed in accordance with the present disclosure to actual nuclear acid marker measurement and to verify analytical capability thereof, gene sensing of a FemA gene of antibiotic-resistant  Staphylococcus aureus  as an infectious microorganism was performed. Specifically, a cDNA fragment of a FemA gene having a base sequence of 5′-CTATGAGTTAAAGCTTGCTGAAGGTTATGA-3′ was synthesized and employed as a target nucleic acid molecule. 
     The molecular beacon structure was designed to have 5′-NH 2 —(CH 2 ) 6 -GTGAGCTCATAACCTTCAGCAAGCTTTAACTCATAGGCTCAC-(CH 2 ) 6 -Biotin-3′. The molecular beacon has a stem section including 6×2 base pairs (bps) and a loop section including 30 base pairs (bps). 
     As a substrate on which the molecular beacon is fixed, a chip for particle counting in which a gold thin film is patterned on a glass substrate is used. Specifically, the gold thin film pattern was formed such that 16 squares, each having an area of 340 μm×340 μm, are patterned. 
     The surface of the gold thin film pattern on which the amine reactive SAM was formed using DTSSP was modified with the molecular beacons. Then, CDNA fragments of the FemA gene at various concentrations of 0 nM, 0.01 nM, 0.1 nM, 1.0 nM, 10.0 nM and 100 nM were reacted with the molecular beacons. Subsequently, optical marker particles modified with streptavidin that specifically bind to the biotin of the molecular beacon are bound to the molecular beacons. Then, the optical marker particles were irradiated with white light using a white LED light source. At this state, an image of the substrate was generated using a digital camera connected to an objective lens having a numerical aperture of 0.065. The image was then analyzed by counting the number of the optical marker particles using the Image J program. 
     Experimental Example 1 
       FIG. 6  is a graph showing a result of gene analysis according to Present Example 1. This graph indicates an average of three repeated experiment results. 
     Referring to  FIG. 6 , it may be seen that as the concentration of cDNA fragments increases, the number of optical marker particles in the image increases. That is, it may be seen that when quantitative analysis of the target nucleic acid molecule according to Present Example 1 is performed, the concentration of the target nucleic acid molecule can be measured accurately. 
     In one example, when detecting the target nucleic acid molecule based on a retro-reflective signal according to Present Example 1, a limit of detection (LOD) was calculated to be 34 pM. This may indicate a significant improvement over a limit of detection (LOD) of 10 nM of a conventional molecular beacon-based bio-sensing scheme employing the fluorophore disclosed in a following cited article: 
     Cited article: Sensitivity and Specificity of Metal Surface-Immobilized “Molecular Beacon” Biosensors, J. Am. Chem. Soc., 2005, vol. 127, pp. 7932-7940 
     From the above results, it may be seen that the method in accordance with the present disclosure may allow the target nucleic acid molecule to be measured more accurately than the bio-sensing method using the conventional expensive optical equipment while the method in accordance with the present disclosure requires the use of minimal optical devices such as the non-spectral white light source and the digital camera. 
     Present Example 2 
     To determine whether the retro-reflection principle-based gene biosensing scheme as developed in accordance with the present disclosure is applicable to analysis of single nucleotide polymorphism (SNP) as a major application field of the molecular beacon, following two genes: ‘target gene’ and ‘verification gene’ were quantitatively analyzed in the same manner as in Present Example 1 using the same molecular beacon and optical marker as in Present Example 1. 
     
       
         
           
               
               
            
               
                 (i) 
                 target nucleic acid molecule (target gene): 
               
               
                   
                 5′-CTATGAGTTAAAGCTTGCTGAAGGTTATGA-3′ 
               
               
                   
               
               
                 (ii) 
                 verification nucleic acid molecule 
               
               
                   
                 (verification gene): 
               
               
                   
                 5′-CTATGCGTTAAAGCGTGCTGAAGATTATGA-3′ 
               
            
           
         
       
     
     The verification nucleic acid molecule refers to DNA having three base pairs differing from those of the target nucleic acid molecule. 
     Experimental Example 2 
       FIG. 7  is a graph showing a result of gene analysis according to Present Example 2. Table 1 is a result showing a ratio of a signal intensity for the verification nucleic acid molecule to a signal intensity for the target nucleic acid molecule based on the concentration calculated from  FIG. 7 . 
     
       
         
           
               
               
             
               
                   
                 TABLE 1 
               
             
            
               
                   
                   
               
               
                   
                 Concentration 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 0.01 nM 
                 0.1 nM 
                 1 nM 
                 10 nM 
                 100 nM 
                 Average 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Intensity 
                 24.0% 
                 20.0% 
                 43.6% 
                 48.4% 
                 53.4% 
                 37.9% 
               
               
                 ratio 
               
               
                   
               
            
           
         
       
     
     Referring to  FIG. 6  and Table 1, it may be seen that although only three base pairs differ between the ‘target gene’ and ‘verification gene’, the intensity of the signal for the verification nucleic acid molecule is significantly reduced compared to the intensity for the signal of the target nucleic acid molecule. Specifically, the retro-reflective signal for the verification nucleic acid molecule was only about 38% of the retro-reflective signal for the target nucleic acid molecule. 
     From these results, it may be seen that the optical gene biosensor according to the present disclosure may be used in the field of gene mutation screening. 
     Although the present disclosure has been described with reference to the preferred embodiments of the present disclosure, those skilled in the art will appreciate that the present disclosure may be modified in various ways without departing from the spirit and scope of the present disclosure set forth in the claims below.