Patent Application
20190086401
The present invention relates to a biosensor and a sample analysis method using the biosensor.
Common methods for analyzing biological samples such as nucleic acids and proteins can be divided into two major areas as follows. The first one is a method of analyzing the concentration of a sample by measuring optical absorbance using an ultraviolet-visible spectroscopic method. In the method, an absorbance is measured by passing light of a certain intensity through a sample and then comparing intensity of light before and after the passage. Such an optical absorbance measurement method measures only the concentration of a specific functional group contained in the sample. Therefore, there exists inconvenience of applying an additional analytical method or more in order to quantitatively analyze the reactivity and activity of a specific binding substance in a biological reaction. Furthermore, the method offers a low analytical sensitivity of 10-6M and thus it is not suitable for analyzing a biological sample that typically requires a high analytical sensitivity of 10-12M.
The second one is to utilize enzyme immunoassay, as disclosed in the patent documents of the following prior art documents. Enzyme immunoassay is a method commonly used for quantitatively analyzing the reactivity and activity of a specific sample at a high analytical sensitivity of 10-12M. The enzyme immunoassay uses a quantitative analysis method in which a sample is analyzed using an enzyme-labeled antibody formed by chemical binding of an enzyme such as peroxidase or galactosidase with an antibody in a target-specific antigen-antibody reaction. Alternatively, fluorescence immunoassay can be used in which a sample is analyzed using an antigen or antibody labeled with a fluorescent dye such as fluorescein and rhodamine and a fluorescence analyzer.
These analytical methods are widely used because they permit to analyze, with an excellent detection sensitivity, the reactivity and activity of the reaction between a reactant and a target analyte in a sample. However, they still have problems of a long assay time and high assay cost because of complicated sample processing, labeling of a sample or target analyte with a fluorescent dye, or use of an expensive analyzer. In particular, enzyme immunoassay or fluorescence immunoassay has difficulties in rapid screening of a large number of libraries during drug development or biomarker development due to a long assay time and necessity of using a separate target-specific antibody depending on the target analyte.
Therefore, there is a desperate need for a solution to the problem of the conventional sample analysis method.
The present invention is intended to solve the aforementioned problems of the conventional arts. One aspect of the present invention is to form a thin film layer in which conductive nanoparticles or nanostructures are dispersedly disposed on at least one of one surface and the other surface of the substrate and to provide a biosensor that detects a sample by inducing an LSPR phenomenon.
A different aspect of the present invention is to provide a biosensor that has a narrow portion having a relatively narrow width at a predetermined position of a substrate so as to prevent a sample from rising along a gap between two immediate substrates or between a substrate and an inner wall of a cuvette.
Another aspect of the present invention is to provide a relatively simple and inexpensive sample analysis method using a biosensor utilizing an LSPR phenomenon without a separate sample pretreatment process.
A biosensor according to an embodiment of the present invention comprises a substrate having a predetermined length and a thin film layer, formed by dispersing and arranging conductive nanoparticles or nanostructures on at least one of the both sides of the substrate to cause localized surface plasmon resonance (LSPR) phenomenon, which is immersed in a target sample to bind a target analyte in the target sample; and a gripping unit connected to one end of the substrate and gripped by a user.
In addition, a biosensor according to an embodiment of the present invention further comprises a cap connected to the substrate and the gripping unit and releasably inserted into a cuvette accommodating the target sample.
In addition, a biosensor according to an embodiment of the present invention further comprises a fixing unit which is arranged on an outer surface of the cap and has deformable resilience capable of being close contact with an inner circumferential surface of the cuvette when the cap is inserted into the cuvette.
In addition, in a biosensor according to an embodiment of the present invention, the fixing unit is formed to be extended and bent from an outer surface of the cap.
In addition, in a biosensor according to an embodiment of the present invention, an outer surface portion of the cap facing the fixing unit is recessed.
In addition, in a biosensor according to an embodiment of the present invention the substrate comprises a narrow portion having a relatively narrow width at a predetermined height with respect to the other end of the substrate.
In addition, in a biosensor according to an embodiment of the present invention, the narrow portion is formed as an ascend prevention groove dented concavely from the side of the substrate.
In addition, in a biosensor according to an embodiment of the present invention, the ascend prevention grooves are formed on both sides of the substrate.
In addition, in a biosensor according to an embodiment of the present invention, a plurality of the ascend prevention grooves are formed along the sides and spaced apart in the lengthwise direction of the substrate.
In addition, in a biosensor according to an embodiment of the present invention, a plurality of the sensing units are spaced apart from one another and placed side by side.
In addition, a biosensor according to an embodiment of the present invention further comprises a pair of guards, disposed opposite to each other with the sensing unit therebetween, to protect the sensing part.
In addition, a biosensor according to an embodiment of the present invention comprises an adaptor, disposed under bottom surface of a cuvette accommodating the target sample thereinto, to adjust a height of the cuvette.
In addition, in a biosensor according to an embodiment of the present invention, the adaptor is formed in a block shape, and comprises an engaging groove on the outer surface of the adaptor for binding to a bottom surface of the cuvette.
In addition, in a biosensor according to an embodiment of the present invention, the sensing unit is immersed in the target sample, and then the target sample is analyzed by irradiating the cuvette with light.
In addition, in a biosensor according to an embodiment of the present invention the analysis of the target sample is protein assay, immunoassay, kinetic analysis, or small molecule detection.
In another aspect, a sample analysis method using a biosensor according to an embodiment of the present invention comprises (a) preparing the biosensor according to any one of the claims 1 to 15; (b) immersing a sensing unit of the biosensor in a detection sample containing a detection substance and immobilizing the detection substance; and (c) immersing the sensing unit in a target analyte by inserting the sensing unit on which the detection substance is immobilized in a cuvette accommodating the target analyte which specifically binds to the detection substance.
In addition, a sample analysis method using a biosensor according to an embodiment of the present invention may further comprise a step of immersing the sensing unit of the biosensor in a rinsing solution between the steps (b) and (c).
In addition, a sample analysis method using a biosensor according to an embodiment of the present invention may further comprise a step of measuring absorbance, after the (c), by arranging the biosensor of which the sensing unit is inserted in the cuvette in a spectroscopic analyzer.
In addition, in a sample analysis method using a biosensor according to an embodiment of the present invention the sensing unit is inserted and immersed in a cuvette containing the detection substance in the (c), and may further comprise a step of measuring absorbance by placing the biosensor of which the sensing unit is immersed in the cuvette containing the detection substance into a spectroscopic analyzer between the steps (b) and (c).
In addition, in a sample analysis method using a biosensor according to an embodiment of the present invention may further comprise a step of measuring absorbance by placing the biosensor of which the sensing unit is inserted and immersed in a cuvette containing a rinsing solution into a spectroscopic analyzer.
The features and advantages of the present invention will become more apparent from the following description with reference to the appended drawings.
Prior to this, it should be understood that the terms and words used in the present specification and the claims are not to be construed as having common and dictionary meanings but are construed as having meanings and concepts corresponding to the technical spirit of the present invention in view of the principle that the inventor can define properly the concept of the terms and words in order to describe his/her invention with the best method.
A biosensor of the present invention quantitatively detects a sample by inducing an LSPR phenomenon on the thin film layer of metal nanoparticles or nanostructures dispersedly disposed on at least one of one surface and the other surface of the substrate. The biosensor can easily induce the reaction between biological samples or between biological and non-biological samples, without a separate sample pretreatment process.
In addition, in a biosensor of the present invention, the sensing unit, immersed in a cuvette containing a sample, for analyzing the sample has a narrow portion having a relatively narrow width formed at a predetermined position of the substrate constituting the sensing unit. The narrow portion offers an advantage of preventing the sample from rising by a capillary force along a gap between two immediately adjacent substrates in parallel or between the substrate and the inner surface of the cuvette.
In addition, a sample analysis method using a biosensor of the present invention is based on a LSPR phenomenon and thus does not necessitate chromophore labeling, unlike enzyme immunoassay that requires a complicated step of labeling a sample molecule with a fluorescent dye. Therefore, the biosensor permits to quantitatively analyze a sample through a simple detection process only with a visible light spectroscopic analyzer.
The objectives, specific advantages, and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments with reference to the appended drawings. It should be noted that the same reference numerals are denoted to the elements of the drawings in the present specification with the same numerals as possible, even if they are displayed in other drawings. Also, the terms “the first”, “the second” and the like are used to distinguish one element from another and thus the element is not limited thereto. Hereinafter, in the description of the present invention, a detailed explanation of related known arts which may unnecessarily obscure the gist of the present invention will be omitted.
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the appended drawings.
As illustrated in
A biosensor according to an embodiment of the present invention relates to a sensor for detecting a sample utilizing an LSPR phenomenon, and comprises a sensing unit (10) and a gripping unit (20).
Surface plasmon resonance (SPR) refers to a phenomenon of the propagation of surface plasmon polaritons which are generated on or near the surface of conductive materials by coupling of electrons and photons having a specific wavelength. In general, SPR is a phenomenon of the collective oscillation of conduction band electrons propagating along the interface between a metal with a negative dielectric constant and a medium with a positive dielectric constant. SPR results in enhanced intensity in comparison with an incident electromagnetic wave and shows characteristics of an evanescentwave which exponentially decays as getting far-off perpendicularly from the interface.
SPR can be classified as a propagating surface plasmon resonance (PSPR) observed at the interface between a dielectric material and a 10-200 nm-thick flat metal surface; and localized surface plasmon resonance (LSPR) observed from nanoparticles or nanostructures. A biosensor based on LSPR detects a change in the LSPR wavelength showing a maximum absorption or scattering which depends on a change of the chemical and physical environment on the surface (for example, a change in refractive index of a medium near the surface) of the nanoparticles or nanostructure. The detection of the LSPR wavelength change permits to distinguish specific molecules or to analyze concentration of specific molecules in a medium; LSPR is highly sensitive to the change of refractive index and that allows label-free detection. A biosensor according to the present invention is fabricated such that LSPR is applied.
At this time, LSPR occurs in the sensing part (10) which comprises the substrate (11) and the thin film layer (13).
Here, the substrate (11) is a plate-shaped part having a predetermined length. The substrate (11) is not necessarily limited to a flat plate, but may be formed in various shapes such as a curved shape or a convexo-concave (Ha) shape. However, hereinafter, the description assumes a flat plate.
On the other hand, the substrate (11) may be an optically transparent or opaque substrate (11), but the optically transparent substrate (11) is preferable. The optically transparent substrate (11) may be made of, for example, glass or a polymer material having a certain degree of optical transparency. The polymer material may comprise polycarbonate (PC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), triacetyl cellulose (TAC), cycloolefin, polyarylate, polyacrylate, polyethylene naphthalate, polybutylene terephthalate or polyamide. However, the polymer material is not necessarily limited thereto. An opaque substrate (11) may be made of sapphire, silicon single crystal. However, the material of the substrate (11) is not limited to the aforementioned materials and various other materials can be utilized in consideration of the conditions of the target analyte, the fabrication process, and the like.
The thin film layer (13) is a layer formed on at least one of the both surfaces of the substrate (11) and is formed by dispersedly disposing conductive nanoparticles or nanostructures (14) that cause LSPR. The thin film layer (13) may be formed on only one surface (see
On the other hand, the conductive nanoparticles or nanostructures (14) are immobilized (see
The sensing unit (10) formed by disposing the thin film layer (13) on the substrate (11) is immersed in the target sample (3). At this time, the thin film layer (13) is also immersed in the target sample (3) as the substrate (11) is immersed from its free end in the target sample (3). Here, the free end of the substrate (11) refers to the opposite end of one end of the substrate (11) connected to the gripping unit (20) to be described later. When the thin film layer (13) is immersed in the target sample (3), a target analyte in the target sample (3) binds to the thin film layer (13).
At this time, a detection substance that specifically binds with the target analyte may be immobilized on the thin film layer (13) in order for the thin film layer (13) to bind with the target analyte. The detection substance may be, for example, a low molecular weight compound, an antigen, an antibody, a protein, a peptide, a DNA, an RNA, a PNA, an enzyme, an enzyme substrate, a hormone receptor, and a synthetic reagent having a functional group. However, the aforementioned detection substances are just exemplary ones and thus the detection substance is not necessarily limited thereto. The detection substance may comprise any known substances, including combinations of such substances, that combine with the target analyte. The detection substance is immobilized on the thin film layer (13), i.e., conductive nanoparticles or nanostructures (14), or on the binder (15); and specifically binds to the target analyte, thereby binding the target analyte to the thin film layer (13). However, the detection substance is not necessarily immobilized on the thin layer (13).
On the other hand, the sensing unit (10) may be one or more. When the sensing units (10) are plural, they are spaced apart from each other by a predetermined distance and arranged side by side. In this case, the sensing units (10) are arranged such that one surface of the substrate (11) is opposed to one surface or the other surface of another substrate (11). One or more of the sensing units (10) are connected to and fixed to the gripping unit (20).
Here, the gripping unit (20) is a unit held by a user and is connected to one end of the substrate (11). Thus, the user can hold the gripping unit (20) and immerse the sensing unit (10) into the cuvette (1) in which the target sample (3) is accommodated. Here, the cuvette (1) is a container to accommodate the target sample (3) for the spectroscopic analysis of a target sample (3). However, the target sample (3) is not necessarily accommodated in the cuvette (1) when the sensing unit (10) is immersed. Nevertheless, in the general sample analysis process, the target sample (3) is prepared in the cuvette (1) and the spectroscopic analysis is performed in a state in which the sensing unit (10) is immersed in the cuvette (1). Therefore, hereinafter, it is assumed that the target sample (3) is accommodated in the cuvette (1).
According to the aforementioned descriptions, the biosensor according to the present embodiment has a structure in which the thin film layer (13) having the conductive nanoparticles or nanostructures (14) dispersedly disposed on one surface of the substrate (11) is formed. The thin film layer (13) causes LSPR which can be utilized for identifying the specific target analyte and determining the concentration of the target analyte in a medium. The biosensor allows a label-free detection of the target analyte.
Meanwhile, a biosensor according to the present embodiment may further comprise a cap (30). Here, the cap (30) is configured to be removably inserted into the cuvette (1) and to close or seal the open inlet of the cuvette (1). The cap (30) is disposed under the gripping unit (20) and connects the gripping unit (20) and the substrate (11). The cap (30) is held in contact with the inner surface of the cuvette (1) and fix the substrate (11) so that the sensing unit (10) does not move in the cuvette (1).
Here, the sizes of the cuvette (1) are not all uniform. Therefore, depending on the size of the cuvette (1), a gap may be created between the outer surface of the cap (30) and the inner surface of the cuvette (1). Due to the gap, the cap (30) remains unfixed to the cuvette (1), making it difficult to accurately analyze the target sample (3). To avoid this problem, the biosensor may further comprise a fixing unit (40) to fix or secure the sensing unit (10) regardless of the size of the cuvette (1).
The fixing unit (40) is arranged on the outer surface of the cap (30). With this arrangement, the original position or shape of the fixing unit (40) is changed to create resilience when the cap (30) is inserted into the cuvette (1). The fixing unit (40) is brought into close contact with the inner circumferential surface of the cuvette (1) by the resilience. The sensing unit (10) connected to the cap (30) is fixed or secured in the cuvette (1) when the fixing unit (40) disposed on the cap (30) is in close contact with the cuvette (1).
Specifically, the fixing unit (40) may be an elastic unit that is deformed while being pressed by the inner surface of the cuvette (1) and comes into close contact with the inner surface of the cuvette (1) by elasticity when the cap (30) is inserted into the cuvette (1). The fixing unit (40) may use the inherent elasticity of the elastic material such as rubber or the like, or may use the properties of a unit such as a spring. However, the fixing unit (40) does not necessarily have to use the elasticity of the elastic material or the unit, but can be implemented through a predetermined structure, which will be described in detail below (see
The fixing unit (40) may extend from the outer surface of the cap (30) and may be bent in a predetermined direction. For example, the fixing unit (40) may extend outward from the outer surface of the cap (30) and may be bent in parallel to the outer surface of the cap (30) to form an inverted L shape. The outwardly protruding protrusion formed at one end of the fixing unit (40) is pressurized against the inner surface of the cuvette (1), and as a result, the fixing unit (40) can be brought into close contact with the inner surface of the cuvette (1) by tension. At this time, since the fixing unit (40) is moved toward the cap (30) when pressured, a portion of the outer surface of the cap (30) opposite to the fixing unit (40) may be recessed.
Alternatively, the fixing unit (40) may extend from the inner surface of the recessed portion of the cap (30) and the protrusion may be formed outward from the outer surface of the cap (30) to have an “L” shape.
Consequently, the fixing unit (40) may extend from the outer surface of the cap (30) and be freely modified into various structures so long as it can be brought into close contact with the inner surface of the cuvette (1) by tension when the cap (30) is inserted into the cuvette (1).
Meanwhile, in a biosensor according to the present embodiment, the substrate (11) may have a narrow portion (12). Here, the narrow portion (12) is a portion where the width of the substrate (11) is relatively narrowed at a predetermined height with respect to the other end (free end) of the substrate (11), as compared with other portions.
In order to analyze the target sample (3), the cuvette (1) is irradiated with light from the outside of the cuvette (1) in a state in which the sensing unit (10) is inserted. When the sensing unit (10) is inserted into the cuvette (1), a capillary force is created in the gap between the sensing unit (10) and the inner surface of the cuvette (1) or in the gap between the immediately arranged sensing units (10) in parallel and that leads to a rise of the target sample (3) (see
Specifically, the narrow portion (12) can be formed through the formation of the ascend prevention groove (17) recessed from the side surface of the substrate (11) concavely. Since the ascend prevention groove (17) is recessed by a predetermined depth from one side surface to the other side surface of the substrate (11), the width of the substrate (11), i.e. the distance between the both side surfaces of the substrate (11), at the position where the ascend prevention groove (17) is formed is small. The ascend prevention groove (17) may be formed only on one side surface of the substrate (11), but may be formed on both side surfaces of the substrate (11). In the case where the ascend prevention grooves (17) are formed on the both side surfaces of the substrate (11), they may be formed so as to face each other, but are not necessarily limited thereto. The ascend prevention grooves (17) may be staggered in a zigzag pattern. The ascend prevention grooves (17) may be formed in plurality along the side surfaces of the substrate (11) at a predetermined distance in the lengthwise direction.
The ascend prevention groove (17) may be formed to be rounded in shape but it is not necessarily formed in such a shape. The ascend prevention groove (17) may be recessed in any shape so long as the width of the substrate (11) is narrowed. At this time, since there is no particular limitation on the width of the ascend prevention groove (17), i.e. the distance perpendicular to the depth of the ascend prevention groove (17), the width of the ascend prevention groove (17) may range from a predetermined height of the substrate (11) to one end thereof.
Specifically, the adaptor (60) is formed in a block shape having a predetermined height and has an engaging groove (61) recessed or perforated from the outer surface. The adaptor (60) and the cuvette (1) are combined by inserting the bottom surface of the cuvette (1) into the engaging groove (61). Here, the adaptor (60) can be fabricated to have a different height and combined with the cuvette (1) to adjust the height of the cuvette (1).
The biosensor according to the present embodiment is configured to analyze target sample (3) by irradiating the cuvette (1) with light in a state in which the sensing unit (10) is immersed in the target sample (3), i.e. in a state where the sensing unit (10) is inserted into the cuvette (1). At this time, the analysis of the target sample (3) comprises protein assay, immunoassay, kinetic analysis, and small molecule detection. Conventionally the protein assay, immunoassay, kinetic analysis, and small molecule detection were performed separately using a separate device. However, the biosensor according to the present embodiment makes it possible to perform all the aforementioned analyses.
Hereinafter, a sample analysis method using the biosensor according to the present invention will be described.
As illustrated in
A sample analysis method using the biosensor according to the present embodiment comprises a step of preparing a biosensor (S100), a step of immobilizing a detection substance (S200), and a step of immersing the sensing unit in a target sample (S300). Here, the sample analysis method using the biosensor according to the present embodiment uses the above-described biosensor according to the present invention, so that redundant contents will be omitted or simply described.
According to the sample analysis method using the biosensor according to the present embodiment, any one of the above-described biosensors is prepared (S100), and the sensing unit of the biosensor is immersed in a detection sample containing a detection substance specifically binding to the target analyte to immobilize the detection substance on the thin film layer (S200). At this time, the detection substance may be prepared in a cuvette, and then the sensing unit of the biosensor may be immersed in the cuvette. When the biosensor is inserted into the cuvette and the sensing unit is immobilized with the detection substance, the biosensor inserted in the cuvette can be arranged in a spectroscopic analyzer to measure the absorbance. However, the measurement of absorbance here does not necessarily have to be performed.
When the detection substance is immobilized on the sensing unit, the sensing unit is inserted into the cuvette containing the target analyte solution to immerse the sensing unit in the target analyte (S300). At this time, the detection substance of the sensing unit binds to the target analyte. For example, when the detection substance is an antibody and the target analyte is an antigen, an antigen-antibody reaction is induced.
On the other hand, after the detection substance is immobilized on the sensing unit (S200), the sensing unit of the biosensor can be immersed in the rinsing solution before being immersed in the target analyte solution (S300). The detection substance is immobilized to the sensing unit via, for example, a physical or electrostatic reaction, thereby the immersion of the sensing unit in the rinsing solution removes the bound substance in an unintended manner. At this time, the rinse solution is prepared in a cuvette, and the sensing unit of the biosensor can be inserted into the cuvette to be immersed. When the detection substance is removed, the biosensor inserted in the cuvette can be arranged in a spectroscopic analyzer to measure the absorbance. However, this absorbance measurement is not an essential step to be performed.
After the sensing unit is immersed in the target analyte solution, the target sample can be analyzed by arranging the biosensor in a spectroscopic analyzer while inserting it into the cuvette and then measuring the absorbance. At this time, it is preferable to pre-heat the spectroscopic analyzer before the biosensor is arranged, and to arrange the biosensor in the spectroscopic analyzer as soon as the sensing unit is immersed in the target analyte solution. However, it is not necessary to pre-heat the spectroscopic analyzer in advance.
The results of analyzing the absorbance difference according to the concentration of the enzyme, MMP-9 (Matrix Metalloproteinase) in the urine through these steps can be verified in
Although the present invention has been described herein with reference to the specific embodiments, these embodiments do not serve to limit the invention and are set forth for illustrative purposes. It will be apparent to those skilled in the art that modifications and improvements can be made without departing from the spirit and scope of the invention.
Such simple modifications and improvements of the various embodiments disclosed herein are within the scope of the present invention, and the specific scope of the present invention will be additionally defined by the appended claims.
According to the present invention, it is possible to quantitatively detect a sample by generating an LSPR phenomenon, and to easily induce the reaction between biological samples or between biological and non-biological samples without a separate sample pretreatment process. Therefore, an industrial applicability of the biosensor is recognized.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2016-0060161 | May 2016 | KR | national |
This application is a continuation of PCT Application No. PCT/KR2017/004546, filed on Apr. 28, 2017, which claims priority to Korean Patent Application No. KR 10-2016-0060161, filed on May 17, 2016. Each of the above applications is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2017/004546 | Apr 2017 | US |
| Child | 16194089 | US |
