KR100991563B1 - Surface plasmon resonance sensor chip, method for manufacturing the same, surface plasmon resonance sensor system, and method for detecting analyzed material with surface plasmon resonance sensor system - Google Patents

Abstract

Disclosed are a surface plasmon resonance sensor chip, a manufacturing method thereof, a surface plasmon resonance sensor system, and a method for detecting an analyte using the same. Surface plasmon resonance sensor chip is a substrate; A metal thin film formed on the substrate; And a first dielectric layer formed on the metal thin film.

Description

SURFACE PLASMON RESONANCE SENSOR CHIP, METHOD FOR MANUFACTURING THE SAME, SURFACE PLASMON RESONANCE SENSOR SYSTEM, AND METHOD FOR DETECTING ANALYZED MATERIAL WITH SURFACE PLASMON RESONANCE SENSOR SYSTEM}

The present invention relates to a technique using a surface plasmon resonance phenomenon, and more particularly to a surface plasmon resonance sensor chip, a method of manufacturing the same, a surface plasmon resonance sensor system and a method for detecting analyte using the surface plasmon resonance sensor system.

When two media having different refractive indices exist, when incident light enters the low refractive index medium from the high refractive index medium, some of the incident light is reflected at the interface of the two media, and the rest is refracted. However, the incident light is all reflected above a certain angle of incidence.

If the incident light is p-polarized light, the high refractive index medium is a dielectric material, and the low refractive index medium is a metal thin film, the wave vector parallel to the metal thin film is the wave vector of the surface plasmon. The energy density of the incident light is absorbed by the metal thin film. This phenomenon is called Surface Plasmon Resonance (SPR).

The surface plasmon resonance phenomenon is applied to various industrial fields, in particular, the surface plasmon resonance sensor system having a surface plasmon resonance sensor chip is used as a means for detecting analyte in the biotechnology field.

The surface plasmon resonance sensor system is a kind of biosensor system, and has advantages such as specific reactivity and the possibility of general analysis on various types of analytes. In particular, since the surface plasmon resonance sensor system directly measures the mass change of the surface of the surface plasmon resonance sensor chip, it is possible to analyze the binding of antigen-antibody, ligand-receptor, etc. in real time, and label the analyte. The advantage is that labeling is unnecessary. In addition, the surface plasmon resonance sensor system can be measured while maintaining the intrinsic properties of the analyte without changing the activity or inherent properties of the analyte. In addition, since the surface plasmon resonance sensor system does not require the pretreatment of the analyte, the measurement can be performed within a short time, and the analysis procedure is considerably simpler than that of the conventional immunoassay method.

However, when measuring the low molecular weight or a small amount of analyte by the surface plasmon resonance sensor system, the surface plasmon resonance sensor system only measures the mass change of the surface of the surface plasmon resonance sensor chip, its sensitivity is low have. For this reason, conventionally, a separate signal amplification technique is required to measure a low molecular weight or a trace amount of analyte.

The present invention provides a surface plasmon resonance sensor chip, a method for manufacturing the same, and a surface plasmon resonance sensor system including the same, which can improve sensitivity to an analyte.

The present invention also provides a surface plasmon resonance sensor chip, a method of manufacturing the same, and a surface plasmon resonance sensor system including the same, which can shorten the manufacturing time and reduce the manufacturing cost.

In addition, the present invention provides a method for detecting an analyte using a surface plasmon resonance sensor system having improved sensitivity to an analyte.

Further objects to be solved by the present invention are not limited to the above-mentioned objects, and other objects not mentioned will be clearly understood by those skilled in the art from the following description. Could be.

Surface plasmon resonance sensor chip according to an aspect of the present invention is a substrate; A metal thin film formed on the substrate; And a first dielectric layer formed on the metal thin film.

Here, the surface plasmon resonance sensor chip according to an aspect of the present invention is formed on the first dielectric layer, it may further include a metal nanoparticle layer made of metal nanoparticles.

Here, the surface plasmon resonance sensor chip according to an aspect of the present invention may further include a second dielectric layer formed on the metal nanoparticle layer.

On the other hand, the manufacturing method of the surface plasmon resonance sensor chip according to an aspect of the present invention comprises the steps of preparing a substrate; Forming a metal thin film on the substrate; And forming a first dielectric layer on the metal thin film.

Here, the method of manufacturing a surface plasmon resonance sensor chip according to an aspect of the present invention may further include forming a metal nanoparticle layer made of metal nanoparticles on the first dielectric layer.

Here, the method of manufacturing the surface plasmon resonance sensor chip according to an aspect of the present invention may further include forming a second dielectric layer on the metal nanoparticle layer.

On the other hand, the surface plasmon resonance sensor system according to an aspect of the present invention includes a substrate, a metal thin film formed on the substrate and a first dielectric layer formed on the metal thin film, the surface plasmon resonance sensor chip for fixing the material to be analyzed ; A prism attached to one surface of the substrate on which the metal thin film and the first dielectric layer are not formed on both surfaces of the substrate; A light source generating incident light incident on the surface plasmon resonance sensor chip through the prism; And a light receiving unit configured to detect reflected light reflected by the surface plasmon resonance sensor chip among the incident light.

On the other hand, the method for detecting analyte using the surface plasmon resonance sensor system according to an aspect of the present invention comprises a surface plasmon resonance sensor chip comprising a substrate, a metal thin film formed on the substrate and a first dielectric layer formed on the metal thin film Preparing a surface plasmon resonance sensor system comprising; Activating the surface plasmon resonance sensor chip; Immobilizing a binding material that can be specifically bound to the analyte on the surface plasmon resonance sensor chip; Reacting the binding material and the analyte; And injecting light into the surface plasmon resonance sensor chip to measure a change in the surface plasmon resonance wavelength.

According to the present invention, the sensitivity of the analyte can be greatly improved, and through this, it is possible to detect not only the analyte that can be detected but also a low molecular weight or a trace analyte. In addition, a separate signal amplification technique is not required, and the manufacturing time of the surface plasmon resonance sensor chip and the surface plasmon resonance sensor system can be shortened, and the manufacturing cost can be reduced.

Hereinafter, a surface plasmon resonance sensor chip, a method of manufacturing the same, a surface plasmon resonance sensor system, and an analyte detection method using the same will be described in detail with reference to the accompanying drawings. Like reference numerals refer to like elements throughout.

In the present specification, when a component is described as being formed "on" another component, the case where there is another component between the said one component and the said other component is not excluded. That is, the certain component may be formed in direct contact with the other component, or another component may be interposed between the certain component and the other component.

In addition, in the present specification, "analyze material" refers to a material that can be analyzed using the surface plasmon resonance sensor system (hereinafter, referred to as "sensor system") according to the present invention, and in particular, These include low molecular weight or trace amounts of substances, as well as substances that could be analyzed. Here, the low molecular weight substance refers to a substance having a molecular weight of 10 to 1000, such as ethanol, drug and toxin substance, and the trace substance refers to a substance having a concentration of 1 pg / ml to 10 ng / ml.

In addition, in the present specification, "binding material" refers to a material which is fixed on a surface plasmon resonance sensor chip according to the present invention (hereinafter referred to as a "sensor chip") and which can be specifically combined with the analyte. . For example, when the analyte is an antigen, the binding agent may be an antibody to the antigen and vice versa. As another example, when the analyte is a ligand, the binding agent may be a receptor that accepts the ligand and vice versa. Herein, the antigen encompasses proteins, cells, bacteria, viruses, protozoa, parasites, serum components, red blood cells, and the like, and the present invention is not limited by the specific types of antigens.

The sensor chip according to the present invention is for fixing a material to be analyzed, and includes at least one dielectric layer, or at least one dielectric layer and a metal nanoparticle layer, wherein the outermost of the dielectric layer and the metal nanoparticle layer Existing components are used to fix the analyte. First, a sensor chip including one dielectric layer will be described in detail with reference to FIG. 1.

1 is a view schematically showing the configuration of an example of a surface plasmon resonance sensor chip according to the present invention.

The sensor chip 100 illustrated in FIG. 1 includes a substrate 110, a metal thin film 120 formed on the substrate 110, and a first dielectric layer 130 formed on the metal thin film 120. In addition, the sensor chip 100 may further include another metal thin film (not shown) formed between the substrate 110 and the metal thin film 120 as necessary, in which case the metal thin film has a multilayer structure. do.

The substrate 110 may be a transparent medium, for example, a transparent glass substrate. However, the present invention is not limited thereto, and may include a silicon (Si) substrate according to the function and feature of the sensor chip 100; Alternatively, a transparent oxide substrate such as titanium oxide (TiO ₂ ), tantalum oxide (Ta ₂ O ₅ ), or aluminum oxide (Al ₂ O ₅ ) may be used. The substrate 110 is preferably made of a material having the same or similar refractive index as the prism provided in the sensor system.

The metal thin film 120 reflects incident light incident from a light source provided in the sensor system, or causes surface plasmon resonance. The material and thickness of the metal thin film 120 changes surface plasmon resonance conditions. It acts as a variable that can be made.

The metal thin film 120 may be formed of gold (Au), silver (Ag), copper (Cu), aluminum (Al), alloys thereof, or the like, which may easily emit electrons by an external stimulus and have a negative dielectric constant. It can form in combination of 2 or more type. The silver (Ag) may exhibit the sharpest SPR resonance peak but may result in denaturation. The gold (Au) not only has excellent surface stability and biocompatibility, but also has a property of low denaturation. For this reason, when the metal thin film 120 is formed of the gold (Au), it may exhibit better characteristics.

The metal thin film 120 may be, for example, formed to a thickness of 30 to 60 nm, preferably 40 to 50 nm. If the thickness of the metal thin film 120 is less than 30nm, surface plasmon resonance hardly occurs. On the other hand, if the thickness exceeds 60nm, surface plasmon resonance occurs, but the signal is weak.

In some cases, the metal thin film 120 may have a low adhesive strength with the substrate 110. In order to prevent this, another metal thin film (not shown) having a thickness of 1 to 5 nm may be formed between the metal thin film 120 and the substrate 110, for example, chromium (Cr) or titanium (Ti). have.

The first dielectric layer 130 is for fixing the material to be analyzed, and may be formed of, for example, silica (SiO ₂ ). The first dielectric layer 130 formed of silica, that is, the silica layer is characterized in that it is very stable under severe conditions, such as when there is a continuous flow of fluid. In addition, the roughness (roughness) is a very important factor in the SPR measurement, when the silica layer is introduced to the surface or the lower portion of the metal nanoparticles, it is possible to improve the surface stability because it prevents the adhesion of foreign matters or aggregation of the particles. In addition, when the silica layer is formed using, for example, a compound having a silane group and a thiol group, not only the surface of the silica layer having various functional groups can be formed, but also the silica layer can be formed very simply. The first dielectric layer 130 may be formed directly on the metal thin film 120, or may be formed after introducing a first self-assembled monolayer (SAM) onto the metal thin film 120. Can be. This will be described in detail in the method of manufacturing the sensor chip 100 to be described later. Meanwhile, the case in which the first dielectric layer 130 is formed of silica has been described as an example, but the present invention is not limited thereto. For example, the first dielectric layer may include at least one inorganic material selected from the group consisting of aluminum oxide (Al ₂ O ₃ ), hafnium oxide / hafnium silicate, perovskite, and porous silica; Or aromatic thermosetting resin, polyvinyl phenol (PVP), polyvinylidenfluoride (PVDF), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate ( Polyethylenenaphthalate (PEN), Polycarbonate (PC), Polyacrylate (PAR), Polyethersulphone (PES), Polyimide (PI), Parylene (Parylene) selected from the group consisting of It may be formed of one or more organic materials, and the inorganic and organic materials may be used in combination.

The thickness of the first dielectric layer 130 may be 2 to 8 nm. If the thickness of the first dielectric layer 130 is less than 2 nm, the thickness is so thin that, for example, it may not be possible to cover all of the gold surface, while if the thickness of the first dielectric layer 130 exceeds 8 nm, There is a problem that a lot of the wavelength shift (wavelength shift) occurs so that the sample measurement range can be reduced later.

The sensor chip 100 having the above-described configuration can greatly improve the sensitivity of the analyte, thereby detecting not only the analyte that can be detected but also a low molecular weight or a small amount of the analyte. have.

2 is a view schematically showing the configuration of another example of the surface plasmon resonance sensor chip according to the present invention. The sensor chip illustrated in FIG. 2 is the same as that of FIG. 1 except for including the metal nanoparticle layer, and thus description thereof will not be repeated, and only the features thereof will be described.

The sensor chip 200 illustrated in FIG. 2 includes a substrate 210, a metal thin film 220 formed on the substrate 210, a first dielectric layer 230 formed on the metal thin film 220, and the first dielectric layer. Metal nanoparticle layer 240 formed on 230 is included.

The metal nanoparticle layer 240 is for fixing the material to be analyzed, and refers to a layer made of metal nanoparticles 242. The metal nanoparticle layer 240 may be formed of gold (Au), silver (Ag), copper (Cu), aluminum (Al), alloy nanoparticles thereof, or the like, alone or in combination of two or more thereof. Among them, the metal nanoparticle layer 240 may be formed of the same material as the metal thin film 220. The metal nanoparticle layer 240 may be formed after introducing a second self-assembled monolayer film on the first dielectric layer 230, which may serve as a binder on the first dielectric layer 230. This will be described in detail in the manufacturing method of the sensor chip 200 which will be described later.

If the metal nanoparticles 242 are gold nanoparticles, for example, they have the following characteristics. That is, the gold nanoparticles have a high refractive index and can easily adjust the average particle diameter. In addition, the gold nanoparticles can easily overcome the conventional limitations of phase equilibrium and kinetics due to their large surface area, and can form active complexes with abundant biological materials. In addition, since localized surface plasmon resonance (LSPR) occurs in the gold nanoparticles themselves, the localized surface plasmon resonance may be coupled with surface plasmon resonance occurring on the surface of the metal thin film 220. As a result, the surface plasmon resonance signal is amplified, so that the sensitivity of the sensor chip 200 can be greatly improved. In addition, the gold nanoparticles and the metal thin film 220 are spaced apart by a predetermined distance, that is, the thickness of the first dielectric layer 230 by the first dielectric layer 230, thereby amplifying the SPR sensitivity. Features due to the gold nanoparticles do not only occur in the gold nanoparticles, but may also occur in other metal nanoparticles 242.

The average particle diameter of the metal nanoparticles 242 may be 3 to 20 nm. If the average particle diameter of the metal nanoparticles 242 is less than 3 nm, it is difficult to manufacture the particles in reality, and it is difficult to distinguish the effects of the SPR influence and the surface roughness, whereas the wavelength exceeds 20 nm. There is a problem that a lot of (wavelength shift) occurs.

In the above, the case where the metal nanoparticle layer 240 is formed on the first dielectric layer 230 has been described as an example, but the present invention is not limited thereto. For example, a metal thin film may be formed in place of the metal nanoparticle layer 240.

The sensor chip 200 having the above-described configuration can greatly improve the sensitivity of the analyte to be analyzed, thereby detecting not only the analyte that can be detected but also a low molecular weight or a small amount of the analyte. have.

On the other hand, Figure 3 is a view schematically showing the configuration of another example of the surface plasmon resonance sensor chip according to the present invention. 3 is identical to FIG. 2 except that the sensor chip illustrated in FIG. 3 includes a second dielectric layer, and thus descriptions thereof will not be repeated and only features thereof will be described.

The sensor chip 300 illustrated in FIG. 3 includes a substrate 310, a metal thin film 320 formed on the substrate 310, a first dielectric layer 330 formed on the metal thin film 320, and the first dielectric layer. The metal nanoparticle layer 340 formed on the 330 and the second dielectric layer 350 formed on the metal nanoparticle layer 340 are included.

The second dielectric layer 350 is to fix the material to be analyzed and may be formed of the same material as the first dielectric layer 330, for example, silica (SiO ₂ ). In this case, the second dielectric layer 350 and the first dielectric layer 330 may appear as if they are integrally formed. For this reason, the second dielectric layer 350 may exhibit characteristics of the first dielectric layer 330. The second dielectric layer 350 may be formed directly on the metal nanoparticle layer 340, or may be formed after introducing a third self-assembled monolayer film on the metal nanoparticle layer 340. This will be described in detail in the method of manufacturing the sensor chip 300 described later.

The thickness of the second dielectric layer 350 may be 4 to 8 nm. When the thickness of the second dielectric layer 350 is less than 4 nm, there is a possibility that the metal nanoparticle layer 340 may not be sufficiently covered in some cases, whereas when the thickness of the second dielectric layer 350 exceeds 8 nm, the wavelength change There is a problem that a lot of (wavelength shift) occurs. The thickness of the second dielectric layer 350 may be greater than the thickness of the metal nanoparticle layer 340. In this case, the second dielectric layer 350 may be formed to cover the metal nanoparticle layer 340. Alternatively, the thickness of the second dielectric layer 350 may be smaller than the thickness of the metal nanoparticle layer 340, in which case a portion of the metal nanoparticle layer 340 may be exposed to the outside.

The sensor chip 300 having the above-described configuration can greatly improve the sensitivity of the analyte, thereby detecting not only the analyte that can be detected but also a low molecular weight or a small amount of the analyte. have.

On the other hand, Figure 4 is a schematic view showing a process for manufacturing an example of the surface plasmon resonance sensor chip according to the present invention, it shows a process for manufacturing the sensor chip shown in FIG.

Referring to FIG. 4, the method of manufacturing the sensor chip 100 may include preparing a substrate 110, forming a metal thin film 120 on the substrate 110, and forming a metal thin film 120 on the metal thin film 120. Forming a dielectric layer 130. Hereinafter, each step will be described in detail.

First, as shown in FIG. 4A, a substrate 110 having a clean surface is prepared. Then, as shown in FIG. 4B, for example, on the substrate 110. Gold (Au), silver (Ag), copper (Cu), aluminum (Al), alloys thereof, and the like, alone or in combination of two or more, forms the metal thin film 120 with a thickness of 30 to 60 nm. . Physical vapor deposition (PVD), chemical vapor deposition (CVD), thermal evaporation, and sputtering methods for forming the metal thin film 120 It is possible to use the method of, in addition to the known thin film formation technology can be used. Meanwhile, another metal thin film (not shown) may be formed between the substrate 110 and the metal thin film 120 as necessary.

Next, as shown in FIG. 4C, a first self-assembled monolayer film 125 is formed on the metal thin film 120. The first self-assembled monolayer film 125 may serve as a binder for fixing a material capable of forming the first dielectric layer 130 on the metal thin film 120. Since the material for forming the first self-assembled monolayer film 125 may be combined with the metal thin film 120 and the first dielectric layer 130, the material is not particularly limited, but the metal thin film 120 and the first film may be formed. This may be determined in consideration of the characteristics and the material of the dielectric layer 130. For example, when the metal thin film 120 is a gold thin film, the gold thin film may be immersed in a (3-mercaptopropyl) -trimethoxysilane (MPS) solution. Then, the first self-assembled monolayer film 125 may be formed by coordinating the thiol functional group of the (3-mermethopropyl) -trimethoxysilane with the gold thin film. The silane functional group of propyl) -trimethoxysilane may be used as a precursor for introducing the first dielectric layer 130. Instead of the MPS solution, 3-mercaptopropyl (dimethoxy) methylsilane may be used.

Next, as shown in FIG. 4D, the substrate 110 on which the first self-assembled monolayer film 125 is formed is immersed in a solution for forming the first dielectric layer 130. 1 dielectric layer 130 is formed. When the first dielectric layer 130 is formed of silica, a material capable of introducing the silica may include, for example, sodium silicate, tetraethyl orthosilicate (TEOS), and tetrameth. And a methoxysilane solution, and these may be used alone or in combination of two or more thereof. In addition, other materials may be used to introduce the silica.

If the immersion time is long, the thickness of the first dielectric layer 130 may be increased. Therefore, the processing time may be adjusted to adjust the thickness of the first dielectric layer 130, for example, 5 to 60 minutes. In this case, when the processing time exceeds a predetermined time, for example, 60 minutes, the thickness of the first dielectric layer 130 may converge to a predetermined thickness, for example, a thickness of 8 nm.

In the above, the method of forming the first dielectric layer 130 by introducing the first self-assembled monolayer film 125 has been described, but the present invention is not limited thereto. For example, a method of directly forming the first dielectric layer 130 on the metal thin film 120 may be performed without introducing the first self-assembled monolayer film 125.

Meanwhile, FIG. 5 is a view schematically showing a process for manufacturing another example of the surface plasmon resonance sensor chip according to the present invention, and shows a process for manufacturing the sensor chip shown in FIG. 2. 5 (a) to 5 (d) are the same as the manufacturing process shown in FIG. 4, the overlapping description is omitted and only the features thereof will be described.

Referring to FIG. 5, the method of manufacturing the sensor chip 200 may include preparing a substrate 210, forming a metal thin film 220 on the substrate 210, and forming a metal thin film 220 on the metal thin film 220. Forming a dielectric layer 230, and forming a metal nanoparticle layer 240 made of metal nanoparticles 242 on the first dielectric layer 230. Hereinafter, each step will be described in detail.

First, as shown in FIGS. 5A to 5D, the metal thin film 220 and the first dielectric layer 230 are sequentially formed on the substrate 210. Here, reference numeral 225 denotes a first self-assembled tomography film.

Next, as shown in FIG. 5E, a second self-assembled monolayer film 235 is formed on the first dielectric layer 230. The second self-assembled monolayer film 235 may serve as a binder for fixing the metal nanoparticles 242 on the first dielectric layer 230. The material for forming the second self-assembled monolayer film 235 is not particularly limited since it may be combined with the first dielectric layer 230 and the metal nanoparticles 242, but the first dielectric layer 230 is not limited thereto. ) And the characteristics and materials of the metal nanoparticles 242 may be determined in various ways. For example, when the metal nanoparticles 242 are charged, a functional group capable of inducing electrostatic interaction between the second self-assembled monolayer film 235 and the metal nanoparticles 242 may be used. The second dielectric layer 230 may be formed by treating a material including the first dielectric layer 230. As a specific example, when the gold nanoparticles have a negative charge, the second self-assembled monolayer film 235 using a 3-aminopropyl-trimethoxysilane (APS) solution containing an amine group having a positive charge Can be formed. Here, 3-aminopropyl-triethoxysilane solution, aminopropylmethyldimethoxysilane solution, aminopropylmethyldiethoxysilane solution, etc. may be used instead of the APS solution. have.

Next, as shown in (f) of FIG. 5, the metal nanoparticle layer by immersing the substrate 210 on which the second self-assembled monolayer film 235 is formed in a solution containing the metal nanoparticles 242. 240 is formed. The metal nanoparticles 242 may be charged to electrostatically bond with the second self-assembled monolayer film 235. This will be described taking the case of using gold nanoparticles as the metal nanoparticles 242 as an example. When the gold nanoparticles are prepared by reducing, for example, hydrogen tetrachloride (hydrogen tetrachloroaurate) with sodium citrate, since the citrate ions surround the gold nanoparticles, the gold Nanoparticles can carry negative charges. In this case, the second self-assembled monolayer film 235 having the amine group and the gold nanoparticles may have an electrostatic bond.

Meanwhile, FIG. 6 is a view schematically showing a process for manufacturing another example of the surface plasmon resonance sensor chip according to the present invention, and shows a process for manufacturing the sensor chip shown in FIG. 3. 6 (a) to 6 (f) are the same as the manufacturing process shown in FIG. 5, the overlapping description is omitted and only the features thereof will be described.

Referring to FIG. 6, the method of manufacturing the sensor chip 300 may include preparing a substrate 310, forming a metal thin film 320 on the substrate 310, and forming a metal thin film 320 on the metal thin film 320. Forming a dielectric layer 330, forming a metal nanoparticle layer 340 made of metal nanoparticles 342 on the first dielectric layer 330, and a second layer on the metal nanoparticle layer 340. Forming a dielectric layer 350. Hereinafter, each step will be described in detail.

First, as shown in FIGS. 6A to 6F, the metal thin film 320, the first dielectric layer 330, and the metal nanoparticle layer 340 are sequentially formed on the substrate 310. Here, reference numerals 325 and 335 denote the first self-assembled tomography film and the second self-assembled tomography film, respectively.

Next, as shown in (g) of FIG. 6, after forming the third self-assembled monolayer film 345 on the metal nanoparticle layer 340, as shown in (h) of FIG. 6, The second dielectric layer 350 may be formed by immersing the substrate 310 on which the third self-assembled monolayer film 345 is formed in a solution for forming the second dielectric layer 350. Here, the method of forming the third self-assembled monolayer film 345 is the same as or similar to the detailed description with reference to FIG. 4C, that is, the method of forming the first self-assembled monolayer film 125. Since the method of forming the second dielectric layer 350 is the same as or similar to that of the detailed description with reference to FIG. 4D, that is, the method of forming the first dielectric layer 130, a detailed description thereof will be omitted below.

On the other hand, Figure 7 is a view schematically showing the configuration of an example of the surface plasmon resonance sensor system according to the present invention. 7 shows what is shown in FIG. 1 as a sensor chip. However, the sensor system according to the present invention is not limited thereto, and may include the sensor chip illustrated in FIG. 2 or 3. On the other hand, since the sensor chip is the same as described above, overlapping description is omitted and only the features thereof will be described.

The sensor system 400 illustrated in FIG. 7 includes a sensor chip 100, a prism 410, a light source 420, and a light receiver 430.

The sensor chip 100 is for fixing an analysis target material, and includes a substrate 110, a metal thin film 120 formed on the substrate 110, and a first dielectric layer 130 formed on the metal thin film 120. It includes.

The prism 410 may be attached on one surface of the substrate 110 in which the metal thin film 120 and the first dielectric layer 130 are not formed on both surfaces of the substrate 110. The prism 410 may be formed of barium crown (BK4) glass or borosilicate crown (BK7) glass, but is not limited thereto, and may be formed as known in the art. The shape of the prism 410 may be any one of triangular, hemispherical, parallelogram, inverted trapezoidal or semi-cylindrical.

The light source 420 generates incident light incident to the sensor chip 100 through the prism 410, wherein light generated from the light source 420 is collected through an optical system and then incident to the prism 410. Or are incident on the prism 410 in parallel. The light source 420 may be a TM or p-polarized monochromatic light source that generates light having a short wavelength or multiple wavelengths, a white light source having a multi-wavelength band, a tungsten-halogen lamp (QTH lamp), a laser diode (LD), a light emitting diode. (LED) etc. can be used.

The light receiving unit 430 may detect the reflected light reflected by the sensor chip 100 among the incident light. That is, the light receiving unit 430 may quantitatively measure a change in wavelength due to resonance absorption of the surface plasmon, for example, a color change or an intensity change. To this end, the light receiving unit 430 includes a silicon photodiode (Si-PD), a charge coupled device (CCD) camera, a video camera or a projection film capable of shaping a two-dimensional plane, or as a near-field microscope. Optical microscopes, near field scanning microscopes.

When detecting the analysis target material by using the sensor system 400 according to the present invention having the above configuration, when the analysis target material is fixed on the sensor chip 100, the light generated from the light source 420 Is incident at a predetermined angle with respect to the substrate 110 through the prism 410, the light reflected by the sensor chip 100 is detected by the light receiving unit 430. At this time, when the wave vector component parallel to the surface of the sensor chip 100 coincides with the wave vector of the surface plasmon generated along the interface between the sensor chip 100 and the material to be analyzed, the energy of the incident light is mostly at the surface plasmon. The incident angle of the incident light at this time is called a resonance angle θ. The surface plasmon is exponentially reduced in both directions at the interface between the sensor chip 100 and the analyte, and the surface plasmon resonance condition is changed according to the thickness, refractive index, and concentration of the analyte, and the light receiving unit 430 Through the surface plasmon resonance condition change can be measured.

The sensor system 400 having the above-described configuration can greatly improve the sensitivity of the analyte, thereby detecting not only the analyte that can be detected but also a low molecular weight or a small amount of the analyte. have. In addition, a separate signal amplification technique may not be required.

8 is a view for explaining a method of detecting a material to be analyzed by using an example of the surface plasmon resonance sensor system according to the present invention. 8 shows what is shown in FIG. 1 as a sensor chip. However, the method for detecting analyte to be analyzed is not limited thereto, and a sensor system including the one illustrated in FIG. 2 or 3 may be used as the sensor chip.

Referring to FIG. 8, the method for detecting analyte using the sensor system 400 may include preparing a sensor system 400 including a sensor chip 100, activating the sensor chip 100, and the sensor. Immobilizing a binding material 510 that can be specifically bound to the analyte 500 on the chip 100, reacting the binding material 510 and the analyte 500, and the sensor Injecting light into the chip 100 to measure a change in the surface plasmon resonance wavelength. Hereinafter, each step will be described in detail.

First, after preparing the sensor system 400, to activate the sensor chip 100, the activation may vary depending on the structure of the sensor chip 100. For example, when the sensor chip 100 shown in FIG. 1 is used as the sensor chip, the first dielectric layer 130 may be activated by treating with a silane compound having an amine group. As another example, when the sensor chip 200 shown in FIG. 2 is used as the sensor chip, the metal nanoparticle layer 240 may be activated by treating with a thiol compound having an amine group. As another example, when the sensor chip 300 shown in FIG. 3 is used as the sensor chip 300, the second dielectric layer 350 may be treated with a silane compound having an amine group to be activated. Although it does not specifically limit as a silane compound which has the said amine group, For example, 3-aminopropyl- trimethoxysilane (APS) can be used. Although it does not specifically limit as a thiol compound which has the said amine group, For example, cysteamine can be used.

Next, the binding material 510 that can be specifically combined with the analyte 500 is immobilized on the sensor chip 100, wherein the binding material 510 is changed according to the analyte 500. Can be. For example, when the analyte 500 is an antigen, the binding agent 510 may be an antibody against the antigen. In addition, when the analyte 500 is a ligand, the binding agent 510 may be a receptor that accepts the ligand. On the other hand, if necessary, the active site in which the binding material 510 is not fixed to the sensor chip 100 is analyzed by using a material 520 that does not specifically bind to the analysis material 500 ( 500) may not be combined.

Next, the analyte 500 is introduced on the sensor chip 100 to specifically react the binding material 510 and the analyte 500.

Next, incident light generated from the light source 420 is incident on the substrate 110, and the reflected light is sensed using the light receiver 430, thereby measuring a change in wavelength due to resonance absorption of the surface plasmon. .

According to the method for detecting analyte using the sensor system 400 described above, the sensitivity of the analyte 500 is greatly improved, so that not only the analyte 500 that can be detected but also low molecular weight or trace amount The analyte 500 may be detected. In addition, a separate signal amplification technique may not be required.

Hereinafter, the present invention will be described in more detail through Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples and Comparative Examples.

[Example]

1. Fabrication of Surface Plasmon Resonance Sensor Chip

Example 1

A 5 nm thick titanium thin film and a 45 nm thick gold thin film were sequentially deposited on a glass substrate by thermal evaporation or sputtering to form a metal thin film having a two-layer structure. Subsequently, the gold thin film was treated with a 0.1M (3-mermethopropyl) -triethoxysilane solution to form a first self-assembled monolayer film. Subsequently, the substrate was immersed in an aqueous 1% sodium silicate solution for 5 minutes to prepare a sensor chip having the structure of FIG. 1.

Example 2

A sensor chip having the structure of FIG. 1 was manufactured in the same manner as in Example 1, except that the substrate was treated with an aqueous 1% sodium silicate solution for 10 minutes.

Example 3

A sensor chip having the structure of FIG. 1 was manufactured through the same process as in Example 1, except that the substrate was treated with an aqueous 1% sodium silicate solution for 15 minutes.

Example 4

A sensor chip having the structure of FIG. 1 was manufactured through the same process as in Example 1, except that the substrate was treated with an aqueous 1% sodium silicate solution for 20 minutes.

Example 5

A sensor chip having the structure of FIG. 1 was manufactured through the same process as in Example 1, except that the substrate was treated with an aqueous 1% sodium silicate solution for 30 minutes.

Example 6

A sensor chip having the structure of FIG. 1 was manufactured through the same process as in Example 1, except that the substrate was treated with an aqueous 1% sodium silicate solution for 60 minutes.

Example 7

A second self-assembled monolayer film was formed on the first dielectric layer of the sensor chip manufactured in Example 1 using 3-aminopropyl-trimethoxysilane aqueous solution. Subsequently, the substrate was immersed in an aqueous solution containing gold nanoparticles having an average particle diameter of 5 nm and manufactured by citrate reduction, thereby manufacturing a sensor chip having the structure of FIG. 2.

Example 8

The gold nanoparticle layer of the sensor chip prepared in Example 7 was treated with 0.1M (3-merctopropyl) -triethoxysilane solution to form a second self-assembled monolayer film. Subsequently, the substrate was immersed in an aqueous 1% sodium silicate solution for 20 minutes to prepare a sensor chip having the structure of FIG. 3.

Comparative Example 1

A sensor chip was manufactured by sequentially depositing a 5 nm thick titanium thin film and a 45 nm thick gold thin film on a glass substrate using a sputtering method.

2. Evaluation of sensor chip characteristics

In each of the sensor chips manufactured through Examples 1 to 6, the thickness change of the silica layer with the sodium silicate aqueous solution treatment time was measured by atomic force microscopy (AFM), and the results are shown in FIG. 9. In addition, in order to examine the effect of the sodium silicate aqueous solution treatment time on the surface plasmon resonance intensity, the surface plasmon resonance intensity according to the wavelength was measured using the sensor chip manufactured in each of Examples 1 to 6, and the result 10 is shown.

9, the thickness of the silica layer increased from about 2 nm to about 8 nm as the treatment time of the sodium silicate solution increased for 5 to 60 minutes, and the thickness of the silica layer converged to about 8 nm in about 60 minutes. Could know.

Referring to FIG. 10, for 5 to 60 minutes, the change in the resonance wavelength increased from about 2 nm to about 22 nm as the sodium silicate aqueous solution treatment time increased, and the change in the resonance wavelength was changed to about 22 nm in about 60 minutes. I could see that it converged. Here, the resonant wavelength means a portion having the lowest intensity in the surface plasmon resonance curve, and is used hereinafter with the same meaning.

Next, the surface plasmon resonance intensity according to the wavelength was measured using the sensor chip through Examples 1, 7, 8 and Comparative Example 1, respectively, and the results are shown in FIGS. 11 to 13, respectively.

Referring to FIG. 11, for Comparative Example 1, Example 1 showed a change in resonance wavelength of approximately 5 nm, while Example 7 showed a change in resonance wavelength of approximately 33 nm, as shown in FIG. Example 8 showed a change in resonance wavelength of approximately 40 nm, as shown in FIG. 13. As described above, it can be analyzed that the reason why the variation of the resonance wavelength increases in the order of Examples 1, 7, and 8 is that the mass of the sensor chip increases in the order of Examples 1, 7, and 8.

3. Fabrication of Surface Plasmon Resonance Sensor System

Example 9

The sensor system was manufactured by attaching a prism to the lower part of the sensor chip manufactured in Example 1 and configuring a light source and a light receiver. At this time, BK7 was used as a prism, a white light source was used as a light source, and a photo spectrometer was used as a light receiving unit.

Example 10

A sensor system was manufactured in the same manner as in Example 9, except that the sensor chip manufactured in Example 7 was used.

Example 11

A sensor system was manufactured in the same manner as in Example 9, except that the sensor chip manufactured in Example 8 was used.

Comparative Example 2

A sensor system was manufactured in the same manner as in Example 9, except that the sensor chip manufactured in Comparative Example 1 was used.

4. Detection of material to be analyzed using sensor system

Example 12

The sensor chip of the sensor system manufactured in Example 9 was activated by treatment with 0.1 M APS. Subsequently, 50 mM NHS / 200 mM EDC and 1 mM prostate-specific antibody (anti-PSA) were mixed on a PBS buffer, and then processed on a sensor chip to fix an anti-PSA as a binding material to the sensor chip. It was. Afterwards, the anti-PSA-immobilized active site on the sensor chip was inhibited by non-specific binding using bovine serum albumin (BSA). At this time, as a result of treatment with immunoglobulin G (Imgunoglobulin G: IgG), which is a material that does not specifically bind to anti-PSA, it was confirmed that the non-specific binding was not induced because the resonance wavelength did not change.

Next, PSA, an analyte, was treated in a sensor chip at concentrations of 0.1, 1, 10, and 100 ng / ml, and the change in the resonance wavelength was measured. The results are shown in FIG.

Example 13

The analyte was detected in the same manner as in Example 12, except that 0.1 M of cysteamine was used in the activation step as well as using the sensor system prepared in Example 10. At this time, PSA, an analyte, was treated in the sensor chip at concentrations of 0.1, 1, 10, and 100 ng / ml, and the change in the resonance wavelength was measured. The results are shown in FIG.

Example 14

Except that the sensor system manufactured in Example 11 was used, the analyte was detected in the same manner as in Example 12. At this time, PSA, an analyte, was treated in the sensor chip at concentrations of 0.1, 1, 10, and 100 ng / ml, and the change in the resonance wavelength was measured. The results are shown in FIG.

Comparative Example 3

Except for using the sensor system prepared in Comparative Example 2, the analysis target material was detected through the same process as in Example 12. At this time, PSA, an analyte, was treated in the sensor chip at concentrations of 0.1, 1, 10, and 100 ng / ml, and the change in the resonance wavelength was measured. The results are shown in FIG.

5. Evaluation of detection of analyte using sensor system

Prostate cancer diagnostic kits using the principles of conventional immunochromatography methods or commercially available are disadvantages in that the error rate is large because the classification of the cancer determination concentration is not clear. Conventionally, Enzyme-Linked ImmunSorbent Assay (ELISA), which uses an antigen-antibody reaction in the blood as a fluorescence method, is known to obtain the most accurate value. And there is a disadvantage that the portability and mobility of the equipment is difficult. In addition, the enzyme-linked immunosorbent method has the disadvantage that the accuracy is lowered because it diagnoses both cases of prostate cancer and enlarged prostate.

On the other hand, in general, prostate cancer diagnostic markers require specific responses of PSA and anti-PSA. In the case of PSA, when it is below 4ng / ml, it is diagnosed as prostate cancer when it is normal and above. In the conventional surface plasmon sensor system, ELISA, and fluorescence labeling, the antigen-antibody reaction alone is used to detect PSA protein at 4ng / ml level. It cannot be analyzed. For this reason, if the surface plasmon resonance signal is not effectively amplified, the detection and quantification of the protein cannot be made significant.

Therefore, in order to compare the sensitivity of the conventional general sensor system with the sensor system of the present invention, detection of prostate cancer diagnostic marker was attempted as described above.

Fig. 14 shows the difference between the measured value of the resonance wavelength after Anti-PSA immobilization and BSA treatment and the measured value of the resonance wavelength after PSA treatment. In the case of Comparative Example 3, even in the case of PSA at a concentration of 100 ng / ml, it was not detected, whereas in Examples 12 to 14, all were detected in the PSA at a concentration of 1 ng / ml. Through this, it could be confirmed that the sensor system manufactured through Examples 9 to 11 increased sensitivity by at least several hundred times than the sensor system manufactured through Comparative Example 2. In particular, the sensor system prepared in Example 10 was able to detect PSA at a concentration of 0.1 ng / ml while showing a change in a resonance wavelength of about 3 nm. In addition, the sensor system prepared in Example 10 showed approximately 2.5 times more sensitivity than the sensor system prepared in Example 9 or 11 even at a concentration of 100 ng / ml.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains can realize that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. I can understand that.

Therefore, it should be understood that the above-described embodiments are provided so that those skilled in the art can fully understand the scope of the present invention. Therefore, it should be understood that the embodiments are to be considered in all respects as illustrative and not restrictive, The invention is only defined by the scope of the claims.

1 is a view schematically showing the configuration of an example of a surface plasmon resonance sensor chip according to the present invention.

2 is a view schematically showing the configuration of another example of the surface plasmon resonance sensor chip according to the present invention.

3 is a view schematically showing the configuration of another example of the surface plasmon resonance sensor chip according to the present invention.

4 is a view schematically showing a process for manufacturing an example of the surface plasmon resonance sensor chip according to the present invention.

5 is a view schematically showing a process for manufacturing another example of the surface plasmon resonance sensor chip according to the present invention.

6 is a view schematically showing a process for manufacturing another example of the surface plasmon resonance sensor chip according to the present invention.

7 is a view schematically showing the configuration of an example of a surface plasmon resonance sensor system according to the present invention.

FIG. 8 is a diagram schematically illustrating a method of detecting an analyte by using an example of a surface plasmon resonance sensor system according to the present invention.

9 is a graph showing the change in the thickness of the silica layer according to the treatment time of sodium silicate aqueous solution.

10 is a graph showing surface plasmon resonance intensity curves according to treatment time of sodium silicate aqueous solution.

FIG. 11 is a graph showing surface plasmon intensity curves according to the surface plasmon resonance sensor chips of Example 1 and Comparative Example 1. FIG.

12 is a graph showing the surface plasmon intensity curve according to the surface plasmon resonance sensor chip of Example 7 and Comparative Example 1.

FIG. 13 is a graph showing surface plasmon intensity curves according to the surface plasmon resonance sensor chips of Example 8 and Comparative Example 1. FIG.

14 is a graph showing resonance wavelength change according to the concentration of PSA in Examples 12 to 14 and Comparative Example 3.

{Description of symbols for main parts of the drawing}

100, 200, 300: Surface Plasmon Resonance Sensor Chip

110, 210, 310: substrate 120, 220, 320: metal thin film

130, 230, and 330: first dielectric layers 240 and 340: metal nanoparticle layers

242 and 342: Metal nanoparticle 350: Second dielectric layer

400: surface plasmon resonance sensor system

410: prism 420: light source

430: light receiver 500: analyte

510: binding substance

Claims (21)

Board; A metal thin film formed on the substrate; A first dielectric layer formed on the metal thin film; And A metal nanoparticle layer formed on the first dielectric layer, the metal nanoparticle layer Surface plasmon resonance sensor chip comprising a. delete The method of claim 1, A second dielectric layer formed on the metal nanoparticle layer Surface plasmon resonance sensor chip further comprising. The method of claim 1, The metal thin film is a surface plasmon resonance sensor chip, characterized in that formed of at least one selected from the group consisting of gold (Au), silver (Ag), copper (Cu), aluminum (Al), or alloys thereof. The method of claim 1, The first dielectric layer is at least one inorganic material selected from the group consisting of silica (SiO ₂ ), aluminum oxide (Al ₂ O ₃ ), hafnium oxide / hafnium silicate, and perovskite; Or aromatic thermosetting resins, polyvinyl phenol (PVP), polyvinylidenfluoride (PVDF), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate ( Polyethylenenaphthalate (PEN), Polycarbonate (PC), Polyacrylate (PAR), Polyethersulphone (PES), Polyimide (PI), Parylene (Parylene) selected from the group consisting of Surface plasmon resonance sensor chip characterized in that formed of at least one organic material. The method of claim 1, Surface plasmon resonance sensor chip, characterized in that the thickness of the first dielectric layer is 2 to 8nm. The method of claim 1, Surface plasmon resonance, characterized in that the metal nanoparticles of the metal nanoparticle layer is formed of at least one selected from the group consisting of gold (Au), silver (Ag), copper (Cu), aluminum (Al), or alloy nanoparticles thereof. Sensor chip. The method of claim 1, Surface plasmon resonance sensor chip, characterized in that the average particle diameter of the metal nanoparticles of the metal nanoparticle layer is 3 to 20nm. The method of claim 3, The second dielectric layer is a surface plasmon resonance sensor chip, characterized in that formed of the same material as the first dielectric layer. The method of claim 3, The thickness of the second dielectric layer is a surface plasmon resonance sensor chip, characterized in that 4 to 8nm. Preparing a substrate; Forming a metal thin film on the substrate; Forming a first dielectric layer on the metal thin film; And Forming a metal nanoparticle layer made of metal nanoparticles on the first dielectric layer Method of manufacturing a surface plasmon resonance sensor chip comprising a. delete The method of claim 11, Forming a second dielectric layer on the metal nanoparticle layer Method of manufacturing a surface plasmon resonance sensor chip further comprising. The method of claim 11, The forming of the metal thin film may include any one of physical vapor deposition (PVD), chemical vapor deposition (CVD), thermal evaporation, and sputtering. Method of producing a surface plasmon resonance sensor chip, characterized in that the step of using the method. The method of claim 11, wherein forming the first dielectric layer comprises: Forming a first self-assembled monolayer (SAM) on the metal thin film; And Immersing the substrate on which the first self-assembled monolayer film is formed in a solution for forming the first dielectric layer Method of manufacturing a surface plasmon resonance sensor chip comprising a. The method of claim 11, wherein the forming of the metal nanoparticle layer, Forming a second self-assembled monolayer film on the first dielectric layer; And Immersing the substrate on which the second self-assembled monolayer film is formed in a solution containing the metal nanoparticles. Method of manufacturing a surface plasmon resonance sensor chip comprising a. The method of claim 13, wherein forming the second dielectric layer comprises: Forming a third self-assembled monolayer film on the metal nanoparticle layer; And Immersing the substrate on which the third self-assembled monolayer film is formed in a solution for forming the second dielectric layer Method of manufacturing a surface plasmon resonance sensor chip comprising a. A surface plasmon resonance sensor chip for holding a substrate, a metal thin film formed on the substrate, a first dielectric layer formed on the metal thin film, and a metal nanoparticle layer made of metal nanoparticles on the first dielectric layer, ; A prism attached to one surface of the substrate on which the metal thin film and the first dielectric layer are not formed on both surfaces of the substrate; A light source generating incident light incident on the surface plasmon resonance sensor chip through the prism; And A light receiving unit that detects the reflected light reflected by the surface plasmon resonance sensor chip among the incident light Surface plasmon resonance sensor system comprising a. Surface plasmon resonance sensor system comprising a surface plasmon resonance sensor chip comprising a substrate, a metal thin film formed on the substrate, a first dielectric layer formed on the metal thin film and a metal nanoparticle layer made of metal nanoparticles on the first dielectric layer. Preparing a; Activating the surface plasmon resonance sensor chip; Immobilizing a binding material that can be specifically bound to the analyte on the surface plasmon resonance sensor chip; Reacting the binding material and the analyte; And Measuring a change in the surface plasmon resonance wavelength by injecting light into the surface plasmon resonance sensor chip; An analyte detection method using a surface plasmon resonance sensor system comprising a. The method of claim 19, The analyte is an antigen, and the binding agent is an antibody to the antigen characterized in that the surface plasmon resonance sensor system characterized in that the antibody. The method of claim 19, The analyte is a ligand, and the binding material is an analyte detection method using a surface plasmon resonance sensor system, characterized in that the receptor for receiving the ligand.

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