KR20060083112A - Biochip platform having dielectric particle layer structures and photoanalyser using the same - Google Patents

Abstract

The present invention relates to a biochip platform for biochemical analysis of a sample, and more particularly, to a chip platform having a dielectric particle layer for biochemical analysis of a sample such as DNA or protein, and an optical analysis device having the same. It is about.

The biochip platform according to the present invention forms a regular particle layer using spherical dielectric particles on a substrate, and thus has excellent wavelength separation ability of the signal due to its regularity, and its surface area is widened so that the fluorescent signal for the target sample can be improved. It can be seen that the amplification is greatly improved. The bioplatform according to the present invention is particularly useful for an optical analysis device for analyzing a biochemical sample due to its high economic efficiency due to its easy manufacturing process and its high efficiency.

Description

Biochip platform having dielectric particle layer and optical analysis device having same {Biochip platform having dielectric particle layer structures and photoanalyser using the same}

1A is a schematic diagram showing a cross section of a dielectric material comprising a surface treatment material and a bioprobe in accordance with the present invention.

1B is a schematic diagram illustrating a DNA hybridization reaction according to the present invention.

Figure 2a is a process chart showing the gravity precipitation method which is a method of forming a dielectric particle layer according to the present invention.

Figure 2b is a photograph showing a process diagram according to the evaporation (evaporation (vertical dip coating)) method of forming a dielectric particle layer according to the present invention.

3 is a SEM photograph showing a dielectric particle layer according to the present invention having an average diameter of about 575 nm.

4A is a SEM photograph showing the surface (top) of the biochip platform prepared in Example 5. FIG.

4B is a SEM photograph showing a cross section (bottom) of the biochip platform manufactured in Example 5. FIG.

Figure 5 is a schematic diagram showing a fluorescence measurement method using a chip platform according to the prior art.

6A is a schematic diagram illustrating a fluorescence measurement method using a chip platform according to the present invention in which a light source is incident on a bottom surface of a substrate.

6B is a schematic diagram illustrating a fluorescence measurement method using a chip platform according to the present invention with a light source incident on top of a substrate.

7 is a graph showing photoluminescence results of visible light regions for chip platforms according to Example 1 and Comparative Example 1. FIG.

8 is a graph showing photoluminescence results for the presence of surface treatment materials in the chip platform according to Example 1 and Example 2. FIG.

9 is a graph showing the reflection peak position and the fluorescence intensity for the biochip platform obtained in Examples 5-7.

10 is a graph showing fluorescence intensity for each wavelength of the biochip platform obtained in Example 7 and Comparative Example 2.

11 is a spectrum showing the fluorescence intensity of the particle layer interface in the biochip platform obtained in Example 7.

The present invention relates to a biochip platform for biochemical analysis of a sample, and more particularly, to a chip platform having a dielectric particle layer for biochemical analysis of a sample such as DNA or protein, and an optical analysis device having the same. It is about.

In analyzing biochemical samples, various assays using two-dimensional arrays are known, such as ELISA analysis, and fluorescence methods are widely used in these various assays.

Conventional fluorometry devices include a light source such as a laser for inducing fluorescence in a sample, and a sample to which a phosphor labeled marker is added. When the excitation light is irradiated to the sample, the fluorescence emitting material present in the sample emits fluorescence, and the fluorescence is monitored by a photodetector. In general, at this time, a method of guiding the reflected light from the sample to the sensor using the optical fiber waveguide is used. However, the optical fiber waveguide depends solely on the internal reflection, thereby limiting the incident light only within the optical fiber core.

As described above, in the conventional fluorescence measurement technique, most of the fluorescence does not reach the photodetector, so that the fluorescence that cannot be collected and is lost is not small. This loss of fluorescence has resulted in an undesirably increased minimum detectable limit, and has been difficult to desensitize when high sensitivity experiments are required. In many cases, the signal-to-noise ratio was not sufficient, which was often insufficient to prevent signal loss.

Among the prior art, a technique using an evanescent wave is also known. It uses laser light that is trapped in a very thin layer and actually forms a so-called Ibernesent field that extends shortly out of the physical sensor. This field can interact with molecules attached to the surface of the sensor. This instantaneous excitation or interaction is limited to the area very close to the waveguide. Ibernesent fields are spatially localized and do not transmit their stored energy to other areas.

In order to solve the problems of the conventional fluorescence measuring method as described above, a lot of efforts have been made to increase the signal-to-noise ratio, reduce the use of the optical measuring device, and develop a fluorescence measuring method with a simple manufacturing process and measuring method.

U. S. Patent No. 6,437, 345 to Zeptosens discloses a detection element using a diffraction grating and an Ibernesent field. The device according to the above patent can selectively excite only the surface response matrix (hundreds of nm region), so that the signal-to-noise ratio is relatively high, while the process of fabricating the submicron pattern is very complicated, and the incidence using the diffraction grating is precise. There is a problem that a mechanical structure is required. In addition, since the diffraction grating and the Evernetcent field are used, the efficiency of the light source is reduced, so that a high sensitivity detection equipment is required.

In addition, according to the method using the porous silicon and chemiluminescence provided by Infineon, the surface area of the porous silicon can be increased to increase the concentration of the reaction material, and because the chemiluminescence is used, no light source is required, and the light source interferes with fluorescence guidance. There is no advantage in that the signal-to-noise ratio is high, but the micron fabrication process of porous silicon is very complicated, and the reaction is complicated and expensive due to the use of chemiluminescence, and high sensitivity detection equipment is required.

In addition, US Patent No. 6,454,924 to Zyomyx discloses a method using micro poles and fluorescence, which can increase contrast, but the process of fabricating micro poles is complicated, Dedicated equipment is required for the surface reaction, and there is a disadvantage that a high sensitivity detection equipment is required.

The present invention is to overcome the limitations of the conventional fluorescence measuring device as described above, to develop a fluorescence measuring device having a simple process, low cost, and high sensitivity.

SUMMARY OF THE INVENTION The present invention has been made in an effort to provide a biochip platform having excellent separation and amplification efficiency of a fluorescence signal and a simple manufacturing process and fluorescence measurement.

Another technical problem to be achieved by the present invention is to provide a fluorescence analysis device having the biochip platform.

The present invention to achieve the above technical problem,

Board; And it provides a biochip platform having a dielectric particle layer formed on the substrate.

According to an embodiment of the present invention, the dielectric particle layer has a photonic crystal structure.

According to one embodiment of the present invention, the photonic crystal structure has a face centered cube structure.

According to one embodiment of the invention, the probe is attached to the surface of the dielectric particles forming the dielectric particle layer.

According to the exemplary embodiment of the present invention, the labeling material included in the target material binding to the probe generates light of 300 nm (UV, VIS) to 1500 nm (near IR) by the light source.

According to the exemplary embodiment of the present invention, the light generated from the labeling material corresponds to an energy band gap region of the photonic crystal.

According to one embodiment of the invention, the energy bandgap of the photonic crystal may be determined by the particle size or refractive index of the dielectric particles constituting the photonic crystal.

According to one embodiment of the invention, the probe may be formed on the surface of the dielectric particles via a surface treatment material.

According to one embodiment of the present invention, oligonucleotide or protein may be used as the probe.

According to one embodiment of the present invention, the dielectric material forming the dielectric particle layer is silica, ZnSe, CdS, polystyrene, PMMA, or Fe ₃ O ₄ .

According to one embodiment of the present invention, the dielectric material forming the dielectric particle layer has an average particle diameter of 100nm to 1000nm, the shape is preferably spherical.

According to one embodiment of the invention, the thickness of the dielectric particle layer is in the range of 100nm to 100 microns.

According to one embodiment of the present invention, the dielectric particle layer may be formed by gravity precipitation or evaporation (vertical dip coating).

According to an embodiment of the present invention, the substrate may use a transparent substrate.

According to one embodiment of the invention, the transparent substrate is a glass containing a TiO ₂ , SiN ₃ , or ITO film, or Si.

The present invention to achieve the above other technical problem,

A light source for generating excitation light for sample excitation;

The above-described biochip platform; And

An optical analyzer comprising a detector for detecting detection light emitted from a sample.

According to one embodiment of the invention, the light generated from the light source is incident on the dielectric particle layer of the biochip platform, or the bottom surface of the substrate.

Hereinafter, the present invention will be described in more detail.

The present invention provides a biochip platform having a substrate and a spherical dielectric particle layer formed on the substrate.

Such a platform improves the weak sensitivity which is a problem with the conventional platforms, thereby providing a more improved sensitivity. That is, when fluorescence is measured by forming a layer of spherical dielectric particles on a transparent substrate and attaching a sample to be analyzed on the surface thereof, more samples can be attached due to an increase in the surface area due to the dielectric particle layer. Separation and amplification of the fluorescence signal is made more efficient.

In particular, when the fluorescent signal generated from the sample is absorbed or scattered in the dielectric particle layer, the fluorescent signal may be lost. In the present invention, a photonic crystal structure is used as the dielectric particle layer to prevent this. In particular, the photonic crystal composed of SiO ₂ has a function of transmitting and / or reflecting light in a predetermined wavelength region without absorbing it, and thus transmitting and / or reflecting it as it is without absorbing the fluorescence signal to minimize loss of the fluorescence signal It becomes possible. That is, in the case of the photonic crystal, since an energy band gap exists in a predetermined wavelength region, when the fluorescent signal corresponds to such an energy band gap region, internal reflection efficiency increases, resulting in a decrease in amplification efficiency of the fluorescent signal.

As such a photonic crystal structure, various structures may exist, and for example, a face centered cubic structure (FCC), a diamond, a inverse opal structure, and the like may be used. Of these, the face-centered cubic structure is more preferable because it has the highest packing density (0.740) in terms of geometry, and the surface volume ratio (2.22 / r, r is sphere radius).

The bioprobe is formed on the particle surface of the dielectric particle layer, which may have a photonic crystal structure as described above, to bind to the target material. These bio probes are attached to the surface of the spherical dielectric particles, and the final target material is fixed to fluoresce to analyze the sample. In this case, the generated fluorescence is generated by the labeling material included in the target material, and it is preferable to have a wavelength range of 300 nm to 1500 nm to facilitate detection around a region containing visible light. In particular, when the generated fluorescence corresponds to the energy band gap of the dielectric particle layer having the photonic crystal structure, the internal reflection efficiency may be increased, and thus the amplification efficiency may be reduced. The energy bandgap of the photonic crystal may be determined by the particle size of the dielectric particles constituting the photonic crystal. As the particle size or the effective refractive index increases, the energy bandgap may specifically reflect light having a larger wavelength.

On the other hand, the bioprobe attached to the surface of the dielectric particles forming the dielectric particle layer is preferably an oligonucleotide or protein, and more specifically, DNA, RNA, PNA, aptamers, antibodies, antigens, haptens, etc. Include.

The bioprobe as described above may be attached directly on the surface of the dielectric particles, but as shown in FIG. 1A, after treating the surface of the dielectric particles with a surface treatment material and attaching the same with a linker in terms of efficiency, More preferred. As such a surface treatment substance, substances, such as a silane type, an epoxy type, a carboxyl type, an amine type, and an aldehyde type, are possible. More specifically, aminopropyltriethoxysilane (APTES), glycidoxypropyltrimethoxysilane (GPTS), triethoxysilane undecanoic acid (TETU), polylysine (poly (lysine) ), 4-trimethoxysilylbenzaldehyde, and the like. In particular, after binding the probe DNA on the surface of the surface-treated particles as shown in Figure 1b, using a hybridization reaction to specifically bind the target DNA, it is also used as a method of detecting by the labeling material It is possible.

The dielectric material for forming the dielectric particle layer may be a material that forms a periodic array and may be easily attached to the bioprobe as described above, for example, silica, ZnSe, CdS, polystyrene, PMMA, Fe ₃ O ₄ and the like are preferable. These dielectric materials can use the thing whose dielectric constant is 1-16, The shape is preferable spherical. In particular, the average particle size is preferably 100 to 1000 nm. If the average particle diameter is more than 1000 nm, the area for attaching the bioprobe to the surface cannot be sufficiently provided, and it is difficult to make a colloidal, and the particle size is smaller than 100 nm. Since the size of is so small that the pores become too small, it is not preferable because unwanted substances can be trapped on the pores and inhibit the fluorescent signal.

Such dielectric materials are spherically stacked on a substrate to form a three-dimensional laminate (or photonic crystal), and the thickness of the laminate is preferably 100 nm to 100 microns. If the thickness of the laminate is less than 100 nm, the surface area decreases, so that a sufficient amount of sample cannot be attached. Therefore, the separation and amplification of the fluorescence signal is not easy. If the thickness exceeds 100 microns, the light is incident from the light source. There is a problem that it is difficult for a light to sufficiently reach the surface of the particles existing inside the particle layer and thus it is difficult to achieve sufficient amplification efficiency.

When the dielectric particle layer is composed of colloidal spherical particles, it can form a face-centered cube (FCC) structure, such a face-centered cube structure in which each particle is evenly arranged periodically to form a compact structure As a thermodynamically stable structure. In addition, sufficient space is formed between the adjacent particles, it is also a structure that can provide a space for attaching the bio probe through this space. By using the pore structure, the fluid can be moved using a channel having a size of 5 nm to 500 nm, and thus, a passage through which a bio probe can be attached to the surface of many particles can be provided.

As the substrate on which the dielectric particle layer is formed, any conventionally known substrate can be used without particular limitation. However, when the light source is incident from the rear surface, it is preferable to use a transparent substrate so that the light source can be easily incident from the rear surface. For example, TiO ₂ , SiN ₃ , glass containing an ITO film, Si, or the like can be used.

As the method for forming the dielectric particle layer on the substrate, various methods known in the art can be used. For example, gravity sedimentation, solvent evaporation, and capillary force method ), A spin coating method and the like can be used.

Gravity precipitation has the advantage that the colloidal solution containing particles is left for a long time as shown in FIG. The capillary force method uses a capillary force by placing a substrate vertically, and the spin coating method can form a particle layer by adjusting the spin speed. Gravity precipitation method of these is difficult to control the thickness, but as described above has the advantage that the process is easy to form a particle layer simply. In the case of forming the dielectric particle layer by such a gravity precipitation method, the surface smoothness may be formed to be somewhat uneven, so that the light source is incident from the back surface of the substrate. In this case, the transparent substrate as described above is used. It is desirable to. On the other hand, the capillary force method or the spin coating method is somewhat difficult to manufacture, but has the advantage that the thickness can be adjusted arbitrarily.

More specifically, in the capillary force method, dielectric particles having a predetermined size are dispersed in distilled water or alcohol, and then the substrate is immersed in the dispersed sol and dried. In the spin coating method, a pattern is formed on a substrate using photoresist, followed by hydrofluoric acid etching, and then, when the photoresist is removed, a jaw is present. When the sol is dropped while rotating the substrate where the jaw is present, dielectric particles are blocked on the substrate and the particles are accumulated.

Gravity precipitation is a method of preparing a colloidal solution containing dielectric particles, and then adding it to a substrate placed in a container and leaving it there for a long time to form a particle layer on the substrate sitting down by gravity. More specifically, when the dielectric particles having a colloidal diameter of 400 to 700 nm are kept at the minimum of external vibration, the particles gradually sink and form a particle layer from the bottom surface by gravity and thermal fluctuations. In this case, it is also possible to use a commercially available dielectric particle or to obtain a relatively uniform spherical particle by the Stober method. In this case, the particles may be fcc or hexagonal close-packed (fcc) or hcp (hexagonal close-packed) structures in which spherical dielectric particles are in close contact with each other in a planar triangular shape. have. The solvent in the spaces between the spherical dielectric particles is then slowly evaporated to form a so-called "opal" structure. It is also possible to sinter the opal structure at high temperature to create a neck between the spherical particles in order to later penetrate other materials into the sphere gap.

The solvent evaporation method is a method for which the manufacturing process is easy and the thickness control of the dielectric particle layer is easy. As shown in FIG. 2B, in the solvent evaporation method, dielectric particles are formed by a method of suspending, dispersing, colloid, and dissolving in a solvent, and then, the substrate is added thereto, followed by evaporation of the solvent to deposit dielectric particles formed in the solvent on the substrate. This is how it is accumulated. In this case, when the appropriate solvent and evaporation conditions are selected, it is possible to appropriately control the thickness of the dielectric particle layer formed on the substrate. This thickness control of the dielectric particle layer is important because it can affect the reflection wavelength by the formula of wavelength = 2 X refractive index X thickness, which in turn affects the amplification efficiency.

As a solvent usable in such a solvent evaporation method, common organic solvents having high volatility, such as alcohols and benzenes, may be used, but are not particularly limited. In addition, the form in which the dielectric particles are formed may vary depending on the conditions under which the solvent evaporates, that is, the temperature or the pressure. As solvent evaporation conditions that can be generally used, it is preferable to evaporate the solvent at a temperature of 10 to 40 ° C. under normal pressure or vacuum.

As described above, after the dielectric particle layer is formed on the substrate, the sintering treatment may be further performed at an appropriate temperature range in order to more stabilize their bonding. Such sintering treatment enables stronger bonding of the particles, and enables formation of a stable photonic crystal structure. Such sintering treatment is preferably carried out for 1 to 10 hours in the temperature range of 400 to 800 ℃, is not particularly limited to these, those skilled in the art selection of conditions for such sintering treatment is the solvent used, the particle layer to be formed It is considered easy depending on the thickness and the like.

When the selective sintering treatment is completed as described above, the surface of the dielectric particles is coated with a surface treatment material. Such a process may be selectively performed in the same manner as the sintering treatment. Can be determined. That is, in the case of using a probe that is easily bonded on the surface of the dielectric particles as a sample, the process of forming the surface treatment material may be unnecessary, but in the case of using a material that is difficult to bond on the surface of the dielectric particles as a probe By forming a surface treatment material on the surface of the dielectric particles as a linker, the binding of the probe can be further expanded to expect amplification of the fluorescence signal.

As the surface treatment material formed on the surface of the dielectric particles, any material having a functional group bondable to a probe can be used without limitation as long as the material can be bound on the surface of the dielectric particles. May be a physical bond or a chemical bond. In this case, as the surface treatment material that can be used, both the dielectric particles and a linker material capable of self-assembly may be used. For example, a material such as silane, epoxy, carboxyl, amine, aldehyde, etc. may be used. As the example, the compound etc. which were mentioned above can be used.

After the dielectric particle layer is formed on the substrate as described above, a probe may be attached thereto. As such a probe, oligonucleotides or proteins as described above may be used, and as the method of attaching these to the surface of the dielectric particles, a conventionally known self-assembled monolayer coating method, dip coating method, Methods such as spin coating, LB coating, and printing can be used. After the bioprobe is attached, a target substance that selectively reacts with them is added as a sample. The target substance is a substance to be analyzed, and substances which will selectively react with the bioprobe, and preferably include a component that is excited in response to an excitation light source, that is, a labeling substance. As such an excitation component, fluorescence is preferable.

The present invention provides an optical analysis device having a biochip platform according to the present invention as described above. The optical analysis apparatus according to the present invention includes a light source for generating excitation light for exciting a sample, the biochip platform, and a detector for detecting a detection beam emitted from the sample, and includes an optical waveguide and a lens. It may further include.

Such an optical analysis device detects a detection beam such as fluorescence generated by the excitation light incident on the biochip platform. In this case, as shown in FIG. 6B, the direction of incidence of the light may be incident to the upper layer of the substrate on which the dielectric particle layer is formed. However, the surface is somewhat inhomogeneous to generate a detection beam having a uniform direction and intensity. In this difficult case, as shown in Fig. 6A, light can be incident on the back surface of the substrate on which the dielectric layer is not formed to analyze the sample. In this case, after light is incident on the uniform particle layer formed on the rear surface, each sample is excited to generate a detection beam having a more uniform intensity and shape.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1

After preparing a colloidal solution in which silica (SiO ₂ ) having a diameter in the range of about 200 to 300 nm was dispersed in water, it was added into a vessel in which a size 1 inch by 3 inch glass substrate was placed. It was allowed to stand for 48 hours to slowly sink them by gravity and thermal fluctuations to produce a platform on which an average 10 micron thick silica particle layer having a surface centered cubic structure was formed on a glass substrate, which was then subjected to GAPS (3- Aminopropyltrimethoxysilane) was self-assembled to prepare a biochip platform. The surface photograph in which the silica particle layer was formed is shown in FIG.

Example 2

A colloidal solution prepared by dispersing SiO ₂ particles, dielectric particles having a diameter of 200 nm to 300 nm in water, was added to a vessel in which a size 1 inch by 3 inch glass substrate was placed. This was allowed to stand for 48 hours to gradually sink them by gravity and thermal fluctuations to prepare a biochip platform in which a 10 micron-thick silica particle layer having a surface-centered cubic structure was formed on a glass substrate.

Examples 3 and 4

GAPS (3-aminopropyltrimethoxysilane) was deposited on the surface of the silica particles as a surface treatment material on the biochip platform obtained in Examples 1 and 2 by deposition. Alexa Fluoro 532-Labled Human IG Antibody, which reacts selectively with this, was then covalently bound in a wet method to bind to the surface.

Example 5

After preparing a colloidal solution in which silica (SiO ₂ ) having a diameter of 210 nm was dispersed in isopropyl alcohol, it was added to a container in which a size 1 inch by 3 inch glass substrate was placed. The solvent was evaporated at room temperature for 24 hours to prepare a platform on which a 5 micron-thick silica particle layer having a face-centered cubic structure was formed on a glass substrate, and then sintered at a temperature of 600 ° C. for 4 hours to strengthen the bonding between the particles. . The surface and the cross-sectional photograph in which the silica particle layer was formed are shown in FIGS. 4A and 4B, respectively.

In order to treat the surface of the silica particles, an ethanol solution of 5% aminopropyl triethoxy silane was used. After coating for 1 hour at ambient temperature, the ethanol solution was sufficiently washed with ethanol, and then washed at about 110 ° C. The incubation process was carried out for about 1 hour. After sufficiently washing with ethanol, it was spin dried. Subsequently, a solution containing a mixture of PEG buffer (pH 10, 0.025 M) and formamide in 80 M amination-probe DNA, 9.0 mM NaHCO ₃ in a 1: 1: 2 ratio was prepared. The platform was patched and probe DNA solution injected into the patch. The immobilization reaction was carried out at room temperature for 2 hours or more while keeping humidity constant. Rinse with lx SSC buffer solution for 5 minutes and dry with distilled water. The Cy ₃ -labeled complementary target DNA solution and the 2X hybridization reaction buffer were mixed at a 1: 1 volume ratio, and hybridization was performed using a patch while maintaining a constant humidity like a probe DNA immobilization reaction. After washing for 5 minutes with 3X SSC solution, it was washed again with 1X SSC solution for 5 minutes and dried. The structure of the amination-probe DNA is 5'-NH ₂ -CCTCCTCCCCCCTGTCAGCA-3 '. Under optimized hybridization conditions, when 10 nM was used, the ratio of distinction of the sequence (3'-CAA GAC AAG AGA ACA-5 ') which did not coincide with the perfectly matched (3'-GGAGGAGGGGGGACAGTCGT-5') was 20. .

Example 6

Except that the diameter of the silica particles is 255nm was carried out the same process as in Example 5 to prepare a biochip platform, the same DNA hybridization process was carried out.

Example 7

Except that the diameter of the silica particles 295nm was carried out the same process as in Example 5 to prepare a biochip platform, the same DNA hybridization process was carried out.

Comparative Example 1

The fluorescent sample (registered name Alexa Fluoro 532) was formed on a glass substrate having a size of 1 inch by 3 inches in the thickness of a monolayer on the surface treated material GAPS.

Comparative Example 2

Biochip platforms were prepared by self-assembling GAPS (3-aminopropyltrimethoxysilane) as a surface treatment material on glass substrates having a size of 1 inch by 3 inches. Subsequently, DNA having a sequence of 5'-NH2-CCTCCTCCCCCCTGTCAGCA-3 'as a probe was bound to the surface treatment material. Cy3 labeled target DNA that selectively reacts with the probe DNA was then covalently bound by the spotting method to bind the probe. Cy3-labeled 3'-GGAGGAGGGGGGACAGTCGT-5 'was used as the target DNA. Unreacted target DNA or target DNA trapped in the cavity was removed by washing.

Experimental Example

Fluorescence was analyzed on the biochip platforms obtained in Comparative Examples 1 and 3 and 4 using an optical analyzer equipped with a light source and a detector. 5 shows a fluorescence measurement method for the biochip platform according to Comparative Example 1, Figure 6a shows a fluorescence measurement method for the biochip platform according to Examples 3 and 4. Figure 6b shows a fluorescence measurement method for the biochip platform according to Example 5.

Experimental measurement method, the hybridization reaction result for the photonic crystal was measured using the fluorescence 2D scan area intensity, spectrum and reflectance. By using a confocal fluorescence scanner (Axon Instrument, Genepix 4000B), the tolerance of Cy3 fluorescence intensity by 523 nm excitation light at 5 m spatial resolution was obtained using a bandpass filter (575DF35; AXON Instrument, 550 nm to 600 nm transmission filter). 10 nm). Fluorescence spectra according to colloid size were measured using a monochromator (Oriel) and cooled CCD (Oriel), and the number of layers and reflectance were measured using a microscope reflector (KMAC, SpectraThick 2000) and a fiber-coupled reflector.

The results of using the optical analyzer equipped with the biochip platform obtained in Example 1 and Comparative Example 1 are shown in FIG. As can be seen in FIG. 7, when the biochip platform according to the present invention is used, the separation and amplification of the fluorescence signal is greatly improved, so that photoluminescence of a target sample is shown. In the case of Comparative Example 1, the signal is represented. Almost could not be confirmed.

The photoluminescence measurement results of Examples 1 and 2 are shown in FIG. 8, and bioprobes are more efficiently attached than the case of Example 1 using a surface treatment material, thereby showing much better photoluminescence. It can be seen.

In addition, the results of analyzing the absorption peak position for the Examples 5 to 7 using a reflectance measuring device is shown in FIG. As can be seen from Figure 9, it can be seen that as the size of the silica particles used changed (210, 255, and 295 nm, respectively), the reflection peak positions also shifted to -462 nm, -560 nm, and -646 nm, respectively.

10 shows the results of measuring the fluorescence intensity of the platform after DNA hybridization obtained in Example 7 and Comparative Example 2. FIG. In Example 7, in which the dielectric particle layer was formed, the amplification efficiency of about 100 times was obtained as compared with Comparative Example 2 in which the dielectric particle layer was not formed. Accordingly, it can be seen that the dielectric particle layer according to the present invention dramatically improves the amplification efficiency of the fluorescent signal due to the surface area increasing effect.

FIG. 11 is a spectrum showing the fluorescence intensity on the interface on which the dielectric particle layer is formed according to Example 7, and shows that the fluorescence intensity is further increased as the thickness of the dielectric particle layer according to the present invention increases. This is because the surface area increases as the thickness of the dielectric particle layer increases, and the fluorescence intensity becomes stronger as the content of the probe bound to the particle layer further increases.

As described above, the biochip platform according to the present invention forms a regular particle layer using spherical dielectric particles on a substrate, and thus the wavelength separation ability of the signal is excellent due to its regularity, and the surface area thereof is widened to provide a desired sample. It can be seen that the amplification of the fluorescence signal is significantly improved. The bioplatform according to the present invention is particularly useful for an optical analysis device for analyzing a biochemical sample due to its high economic efficiency due to its easy manufacturing process and its high efficiency.

Claims (15)

Board; A dielectric particle layer formed on the substrate; And a probe attached to a surface of the dielectric particles forming the dielectric particle layer. The biochip platform of claim 1, wherein the dielectric particle layer has a photonic crystal structure. The biochip platform according to claim 2, wherein the photonic crystal structure is a face centered cube structure. The biochip platform according to claim 1, wherein the labeling material included in the target material binding to the probe generates light of 300 nm to 1500 nm by a light source. The biochip platform of claim 4, wherein the light generated from the label corresponds to an energy band gap region of the photonic crystal. The biochip platform according to claim 5, wherein the energy band gap of the photonic crystal is determined by the particle size or refractive index of the dielectric particles constituting the photonic crystal. The biochip platform of claim 1, wherein the probe is an oligonucleotide or protein. The biochip platform of claim 1, wherein the dielectric material forming the dielectric particle layer is silica, ZnSe, CdS, polystyrene, PMMA, or Fe ₃ O ₄ . The biochip platform according to claim 1, wherein the dielectric material forming the dielectric particle layer has a spherical particle diameter of 100 nm to 1000 nm. The biochip platform of claim 1, wherein the dielectric particle layer has a thickness of 200 nm to 100 microns. The biochip platform according to claim 1, wherein the dielectric particle layer is formed by gravity precipitation or evaporation. The biochip platform according to claim 1, wherein the substrate is a transparent substrate. The biochip platform according to claim 12, wherein the transparent substrate is glass including Si, TiO ₂ , SiN ₃ , or ITO film, or Si. A light source for generating excitation light for sample excitation; The biochip platform according to any one of claims 1 to 13; And And a detector for detecting the detection light emitted from the sample. The optical analysis device of claim 14, wherein the light generated from the light source is incident on the dielectric particle layer of the biochip platform or the bottom surface of the substrate.

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