KR100724314B1 - Ball shaped photonic crystal attaching with bioreceptor, preparation method thereof and using method thereof - Google Patents

Abstract

The present invention provides a photonic crystal sphere comprising a plurality of spherical pores uniformly to form a spherical diffraction grating and formed of a sphere filled with a medium around the pores, the photonic crystal sphere having a biosensing material fixed to the surface of the pore; A photonic crystal sphere formed of a spherical body in which a plurality of spherical medium portions are uniformly aggregated to form a spherical diffraction grating and filled with pores around the medium portion, the photonic crystal sphere having a biosensing material fixed to the surface of the medium portion; Its preparation method; And it provides a biochip or a biosensor comprising the photonic crystal sphere.

Biochip, biosensor, label-free, optical diffraction, photonic crystal, photonic crystal sphere

Description

Photonic crystals immobilized with biological sensing materials and methods for manufacturing and using the same {BALL SHAPED PHOTONIC CRYSTAL ATTACHING WITH BIORECEPTOR, PREPARATION METHOD THEREOF AND USING METHOD THEREOF}

1 is an electron micrograph of a photonic crystal sphere which is a core material of the present invention, FIG. 1A is a photonic crystal sphere having an inverse opal structure, and FIG. 1B is a photonic crystal sphere having an opal structure.

FIG. 2 is a photograph showing that materials having a regular diffraction grating reflect color and have a plane diffraction grating at a measurement angle (left: -30 degrees, right: +30 degrees). The reflection spectrum changes accordingly.

FIG. 3 is a photograph showing that materials having a regular diffraction grating reflect light and have a color. The photonic crystal sphere having the spherical diffraction grating (the photonic crystal sphere having the opal structure of FIG. 1B) is constant regardless of the measurement angle. It shows reflected light (lower left side: -15 degrees, lower middle: 0 degrees, lower right: +15).

Figure 4a and 4b is a schematic diagram showing an example of the application of the photonic crystal can be biologically analyzed according to the present invention, Figure 4a is an example of applying the opal crystal photonic sphere, Figure 4b is an example of applying the opal crystal photonic sphere .

Figure 5a is a schematic diagram showing the process of obtaining a spherical colloidal photonic crystal using an electro-hydraulic spraying device.                 

5B is a schematic diagram of an electrohydraulic spraying device.

FIG. 5C is a schematic diagram illustrating a method of manufacturing a photonic crystal sphere having an inverse opal structure using a colloidal templating method.

6 illustrates chemical linker molecules capable of covalently binding three types of surface-activated chemicals (amines, aldehydes, nickel) and correspondingly various types of biosensors to photonic crystals.

7 is a result obtained by using a UV-VIS spectrometer the change in color when performing a biological analysis according to the present invention.

The present invention relates to a photonic crystal sphere having a biosensing material usable in a biochip or a biosensor, a manufacturing method thereof, and a method for detecting a target biomolecule using the same, a biochip or a biosensor including the photonic crystal sphere.

Biochip is a high-density microarraying of various types of bioreceptors on the surface of a solid support of unit area, and according to the biosensing materials attached to the surface, DNA chips, protein chips, cell chips, neurons It can be divided into various kinds of chips such as chips.

The biosensor is composed of a biosensing substance and a signal transducer, and can selectively detect a substance to be analyzed.                         

Biosensors that can be used in biochips or biosensors include enzymes, antibodies, antigens, lectins, hormones, receptors and the like that can selectively react and bind with specific materials.

On the other hand, a method for detecting a target biomolecule that reacts or binds with a biosensor on a biochip or biosensor is a method using a fluorescent material, a method using a vibration of cantilever, a method using surface plasmon resonance (SPR) There are a variety of methods, such as using a quartz crystal microbalance, electrochemical methods. Currently, the most commonly used methods are fluorescence and electrochemical methods. However, both of these methods attach specific types of labels to biomolecules and detect the signal.

Recently, interest in methods for detecting biomolecules without attaching markers is increasing. Representative examples include SPR, ellipsometry, and reflectance interference spectroscopy.

Among them, the reflectance interference spectroscopy method is to create an interfering lattice structure and observe the change in the interference spectrum as the biomolecule is attached to the lattice surface. It is known that the sensitivity can be obtained up to about 10 fM, and the materials forming the lattice structure are various such as polymer hydrogel, silica, and titania.

As such, the grating structure using the interference of light has a lot of conceptual similarities with the photonic crystal. Photonic crystal refers to a material in which dielectrics are arranged periodically.                         

In general, materials with a crystalline structure have a periodic potential due to the regular arrangement of atoms or molecules that make up the material, which affects the movement of electrons. An important phenomenon that arises is the formation of a band gap. This concept applies to photons as well, with the dielectric acting as a potential for the photons. In this case, as in the case of the former, a band gap is formed, which is distinguished from the band gap of the electron, and is called a photonic band gap. The term band gap is applied to a particular photonic material because its components are expected to function like optical analogs in semiconductors. Just as electrons with some energy cannot move through a semiconductor because of an energy band gap, light of any frequency cannot spread into photonic band-gap crystals because of the optical frequency (or wavelength) band gap. In semiconductors, the gap is due to the electronic structure of the material, or in the case of photonic crystals, because of the crystal structure that strongly scatters certain wavelengths.

The material with this photonic crystal structure can eventually be colored without dyes by Bragg diffraction in terms of crystal lattice. Conventional optical diffraction detection techniques form two-dimensional optical diffraction gratings on substrate materials by deposition or embossing. In this case, the color may appear in various colors depending on the viewing angle (see FIG. 2) because the spacing of the lattice varies according to the incident angle of light in the general two-dimensional photonic crystal structure.

The present invention uses a photonic ball in which a biosensing material is fixed to a spherical diffraction grating instead of a two-dimensional diffraction grating in order to display the same color at all angles in a detection technique using an optical diffraction without a marker. I would like to.

Accordingly, it is an object of the present invention to provide a photonic crystal sphere in which a biosensing material is fixed to a spherical diffraction grating and a method of manufacturing the same.

Another object of the present invention is to provide a method for analyzing the binding between a biosensing material immobilized on a spherical diffraction grating on a photonic crystal sphere and a target molecule, and a biochip or biosensor using the same.

The present invention provides a photonic crystal sphere comprising a plurality of spherical pores uniformly to form a spherical diffraction grating and formed of a sphere filled with a medium around the pores, the photonic crystal sphere having a biosensing material fixed to the surface of the pore; Its preparation method; And it provides a biochip or a biosensor comprising the photonic crystal sphere.

In addition, the present invention provides a photonic crystal sphere formed of a spherical body in which a plurality of spherical medium portions are uniformly aggregated to form a spherical diffraction grating, and a periphery of the medium portion is filled with pores, wherein the photonic crystal is fixed to the surface of the medium portion. phrase; Its preparation method; And it provides a biochip or a biosensor comprising the photonic crystal sphere.

Furthermore, the present invention is a first step of applying a sample containing a target material to analyze whether or not bound to the bio-sensing material is fixed on the photonic crystal sphere; and a target material specifically bound to the bio-sensing material It provides a method for analyzing the binding between the target material and the biosensors immobilized on the photonic crystal sphere, comprising a second step of detecting.

Hereinafter, the present invention will be described in detail.

1. Types of photonic crystal spheres having spherical diffraction gratings and methods for their preparation .

According to the present invention, a photonic crystal sphere composed of a spherical diffraction grating to which a biosensing substance can be fixed includes (1) a spherical sphere having a plurality of spherical pores uniformly formed to form a spherical diffraction grating and filled with a medium around the pores. Photonic crystal sphere (inverse opal structure, see FIG. 4A) formed of a sieve, and (2) photonic crystal sphere formed of a spherical body in which a plurality of spherical medium portions are uniformly agglomerated to form a spherical diffraction grating, and the periphery of the medium is filled with pores (opal) Structure, see FIG. 4B).

At this time, in the opal crystal photonic sphere, the regularly formed medium portion serves as a lattice, and in the opal photonic crystal sphere, the regularly formed spherical pores serve as a lattice.

The difference between the refractive index of the pores and the medium is sufficient to be able to reflect due to optical diffraction.

In the case of a three-dimensional inverse opal structure, when the refractive index difference is more than 2.8, light in all directions can be scattered under the same conditions.

The material capable of forming the medium portion is preferably selected from metal or nonmetallic materials that can be produced by the sol-gel process, and non-limiting examples include silicon oxide (silica), titanium oxide (titania), aluminum oxide, zirconium oxide, and silicon nano. Particles, Cd-Se nanoparticles, gold particles, polymeric hydrogels or mixtures thereof. It is preferable to use particles such as titanium oxide or gold to give a sufficient change in the UV-VIS spectrum to observe the color change with the naked eye.

Tetramethylorthosilicate (TMOS), Tetraethylorthosilicate (TEOS), Methyltrimethoxysilane (MTrMOS), Ethyltriethoxysilane (ETrEOS), Tetramethoxysilicate (TMS), Methyltrimethoxysilicate (MTMS) The medium portion can be formed from one or more silicate derivatives selected from among 3-aminopropyltrimethoxysilicates. In addition to the silicon, it is possible to form a medium part from sol precursors such as zirconia, aluminum, and titanium, which have better mechanical properties and higher refractive index than silicon, and a material for medium parts in which nanoparticles of all metals, metal oxides, and ceramic materials are dispersed. Can be used

Filling the pores of the photonic crystal sphere of the present invention may be air (n = 1.0), and may be a solvent in which the photonic crystal sphere is dispersed. In the case of solvents, in principle, all solvents having a difference in refractive index of 2.8 or more are possible. Preference is given to solvents which can enable biochemical reactions in which the photonic crystal spheres of the present invention can be applied. Non-limiting examples include water, alcohols, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexane, toluene, ethers, oils and the like.

An example of a method of manufacturing the opal crystal photonic sphere (FIG. 4A) and the opal photonic crystal sphere (FIG. 4B) is described in Korean Patent Laid-Open No. 10-2004-0028360, which forms a part of the present specification.

Looking at the manufacturing method in detail as follows.

In the method of manufacturing the opal crystal photonic sphere (FIG. 4B), an AC power is applied between the metal capillary containing the suspension and the ground electrode to induce droplets of uniform size, and the solvent is removed from the droplets to form colloidal crystals. (See FIG. 5A).

The method of manufacturing a photonic crystal sphere having a reverse opal structure (FIG. 4A) is to prepare a porous structure using a colloidal crystal as a template. An AC power source is applied between a metal capillary and a ground electrode containing a suspension in which polymer and inorganic particles are mixed and dispersed. To induce droplets of uniform size, to remove the solvent from the droplets to form colloidal crystals, and to sinter the colloidal crystals (preferably at a temperature of 100 to 1500 ° C.) (FIG. 5a).

5A and 5B show an example of an electrohydraulic spraying apparatus used to make the colloidal crystals. The electro-hydraulic spraying device includes a metal capillary tube (1) and a ground electrode (2), and is connected to an alternating current power source (3) between the metal capillary tube and the ground electrode to change the periodic electric field induced from the alternating current source. Accordingly, droplets are generated at the end of the capillary in association with frequency by electrical stress acting on the surface, and spherical droplets of uniform size can be induced.

In FIG. 5A, the suspension penetrates into the capillary tube to apply AC power to induce droplets of uniform size, and when the solvent is evaporated from the droplets, the self-assembly process first concentrates into a low-energy face-centered cubic structure. The process of spherical colloidal photonic crystal is shown. Also shown is a spherical polymer-inorganic colloidal crystal in which inorganic particles are filled in a gap of the polymer colloidal crystal obtained by driving an electrohydrodynamic spraying apparatus in a suspension in which polymer and inorganic particles are mixed and dispersed. At this time, the spherical porous structure is obtained by sintering the polymer-inorganic colloidal crystals.

Particles contained in the suspension are substances capable of crystallization through self-assembly by evaporation of the solvent and need not be limited to a particular kind as long as the crystals are industrially useful. Examples of preferred particles include latex type polymers, and non-limiting examples thereof include polystyrene, poly (methyl methacrylate), polymethylacrylate, polyethylacrylate, polyvinylchloride, polyacrylonitrile, polyisoprene, Polycarbonate, polyvinyl alcohol, polyester, polyamide, and the like.

The solvent contained in the suspension is capable of self-assembly and is preferably selected from materials larger than the effective density of the selected particles. Non-limiting examples of such solvents include water, methanol, ethanol, ethylene glycol, glycerol, perfluorodecalin, perfluoromethyldecalin, perfluorononane, perfluoroisoacid, perfluorocyclohexane, perfluoro1,2-dimethyl Cyclohexane, perfluoro2-methyl2-pentene, perfluorokerosene and the like.

As shown in FIG. 5B, the resulting droplets are contained in a lower bath containing oils such as glycerol, ethylene glycol, silicone oil, fluorinated silicone oil, mineral oil and the like so as not to mix with the droplets in the aqueous solution. It is more effective when used with highly hydrophobic oils and surfactants in which the droplets can remain stable in the oil.

In one example, the latex-type polystyrene particles in the droplets produced in the electro-hydraulic spraying device have a structure in which a spherical colloidal structure is formed and the gap is filled with sol-gel silica particles. When the spherical polystyrene-silica structure is sintered at 500 degrees Celsius, the polystyrene is deteriorated to obtain a porous structure (see FIG. 5C).

2. Manufacturing method of photonic crystal in which a biosensing substance is fixed to a spherical diffraction grating .

Methods of immobilizing the biosensing material on the surface of the medium in the photonic crystal include physical adsorption (i.e. no chemical linker), chemical bond (i.e. chemical linker), electrochemical bond, electrostatic bond, hydrophobic bond and hydrophilicity. There is a bond. Double chemical bonding is the most stable method of fixing the biosensing material to the photonic crystal.

On the other hand, chemical binding includes those linked through the following functional groups: amine groups, aldehyde groups, nickel groups, acid groups, alkanes, alkenes, alkynes, aromatic groups, alcohol groups, ether groups, ketones Groups, ester groups, amide groups, amino acid groups, nitro groups, nitrile groups, carbohydrate groups, thiol groups, organic phosphate groups, lipid groups, phospholipid groups, steroid groups.

In addition, the photonic crystal surface having these functional groups can be used to fix several different types of chemical linkers to the photonic crystal (see FIG. 6). For example, amine surfaces can be used to link several types of linker molecules, and aldehyde surfaces can be used to link proteins directly without linkers. An example of a method of immobilizing a biosensor through a linker is described in FIG. 6.

In general, the biosensing material may be fixed using a metallic hydroxyl group of the medium part.

For example, silica constituting the medium portion of the photonic crystal can be modified with silanol groups having an active treatment on the surface thereof. The silanol groups on the surface can be easily reacted with silane derivatives such as 3-aminopropyltriethoxysilane (APTS) and 3-glycidoxypropyltrimethoxysilane (GPTS) to form reactive functional groups (in the case of APTS, In the case of an epoxy group), a linker can be introduced using this reactive functional group. The linker minimizes the steric hindrance effect on the silica surface and provides a linkage of physical or chemical bonds, and biological materials such as receptors or ligands can be effectively immobilized on the photonic crystal via this linker.

3. Application of photonic crystal sphere in which a biomaterial is fixed to a spherical diffraction grating .

The present invention is a first step of applying a sample containing a target material to analyze whether or not bound to the bio-sensing material fixed on the photonic crystal sphere; and detecting the target material specifically bound to the bio-sensing material It provides a method for analyzing the binding between the target material and the biosensor immobilized on the photonic crystal sphere, comprising a second step.

The photonic ball of the present invention is characterized in that the diffraction grating formed by the pore portion or the medium portion has a spherical three-dimensional structure, and the photonic crystal sphere itself is also spherical, and the biomolecule is fixed on the surface of the photonic crystal sphere. It is characteristic. Photonic crystals are crystal structures that strongly scatter specific wavelengths, i) bare photonic crystals (photonic crystals without biomaterials immobilized), ii) photonic crystals with biomaterials immobilized, and iii) target molecules are biosensitive. Photonic crystals bonded to the material have a shift in the reflected wavelength (see FIG. 7). That is, the size and refractive index of the diffraction grating are changed by the biochemical reaction on the surface of the photonic crystal, and the UV-VIS spectrum can measure the change.

Therefore, such photonic crystal spheres can be used in biochips or biosensors, and can detect binding between the biosensors immobilized on the photonic crystal spheres and the target molecules by using optical diffraction without a marker.

Further, according to the present invention, the photonic crystal sphere in which the biomaterial is fixed to the spherical diffraction grating uses a spherical diffraction grating, and since the photonic crystal sphere itself is spherical, the reflection spectrum is constant according to the viewing angle, so that the biosensing substance fixed to the photonic crystal sphere is fixed. Binding between the target material and the target material becomes possible. Therefore, the present invention can be applied to the analysis of biochemicals in the form of spherical photonic crystal spheres that do not require a marker. In addition, attaching the colloidal gold, silver, or latex polymer material to the target material may maximize the shift of the reflected wavelengths, thereby making the visual identification more apparent.

Photonic crystal spheres in which a biosensing material such as a ligand is prepared may be used in a dispersed form in a solution or may be used as a pattern in a substrate material or microfluidic chip. Therefore, the photonic crystal sphere of the present invention is used in biochips or biosensors that have a flat substrate or microfluidic channel and are used for various purposes such as medical, environmental, food diagnostics, high efficiency screening (HTS), and enzyme activity measurement. The reaction results of biomolecules can be confirmed without labeling.

According to the present invention, a biochip or a biosensor to which a photonic crystal device in which a biosensing material is immobilized is applied, for example, by patterning a solution containing a photonic crystal device on which a biosensing material is fixed to a flat substrate or a substrate in which microfluidic channels are present. I can make it.

While conventional optical diffraction detection techniques form optical diffraction gratings on a substrate material by deposition or embossing, photonic crystal spheres in which a biomaterial is fixed to a spherical diffraction grating according to the present invention have optical diffraction gratings. Since the particles are prepared and used in advance, the convenience of use is increased.

In addition, the photonic crystal sphere in which the biomaterial is fixed to the spherical diffraction grating according to the present invention has the advantage of the biochemical analysis technique of round particles. For example, spherical particles are often used as solid support in the field of diagnostics and high-efficiency screening. They have several advantages over general microtiter-well based methods. Since the surface area can be increased or adjusted and the particles are fluid, it is possible to shorten the time of biomolecule analysis and to increase the activity of the biomolecule. In addition, since the particles are surface-treated in a batch process or biomolecules are attached in advance in a state where the particles are dispersed in the suspension, an error generated when each is attached to the micro-titer plate can be minimized. Finally, it is possible to attach multiple markers to each particle to analyze multiple target molecules at once.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

[ Example ]

Example  One: Photonic crystal  Chemical formula

Silica photonic crystal spheres (bare photonic crystal spheres) prepared according to the method described in Korean Patent Laid-Open No. 10-2004-0028360 for 10 minutes in a piranha solution (H ₂ SO ₄ : H ₂ O ₂ (1/1)). It was left to stand and acid treated. Washed with distilled water and methanol and dried in vacuo (30 ℃) for 1 hour.

The acid treated photonic crystal was reacted at 30 ° C. in a 5% (v / v) 3-aminopropyltriethoxysilane (APTS) / chloroform solution for 30 minutes, and then washed with chloroform and methanol to have a surface amino group. Photonic crystal spheres (APTS-treated photonic crystal spheres) were prepared.

The prepared photonic crystal was 5mM FMOC-aminocaproxane (Fmoc-ACA), 5mM benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (Benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP)), 5 mM 1-hydroxybenzotriazole (HOBt), 10 mM diisopropylethylamine (DIEA) / DMF solution for 2 hours at 30 ° C. (Fmoc-ACA treated photonic crystal) Sphere formation). After several washings with DMF, the FMOC group was removed by treatment with 20% (v / v) piperidine / DMF solution at 30 ° C. for 3 hours. Washed with DMF and methanol and dried in vacuo to prepare photonic crystal spheres (FMOC-depleted photonic crystal spheres) having free amino alkyl chains on the surface. The free amino alkyl chain acts as a linker to enhance the surface access of the biomaterial.

Example  2: Photonic crystal ball  Detection of Biomaterials Used

Biotin immobilized on the photonic crystal was used to selectively detect streptavidin present in the assay sample. The biotin-streptavidin response is a cognitive response of representative biomaterials.

Photonic crystals linked with free amino alkyl chains according to Example 1 were reacted for 2 hours at 30 ° C. in 5 mM biotin, 5 mM BOP, 5 mM HOBt, 10 mM DIEA / DMF solution to fix the biotin (biotin-fixed photonic sphere) .

Microwell plates were pretreated with 2% (w / v) bovine serum albumin (BSA) to prevent nonspecific adsorption of the protein. Biotin-photonic crystals dried in vacuo were charged to a microwell plate, and then the assay sample containing 10 μg / mL streptavidin was dropped. The plate was allowed to stand for 20 minutes, then washed with PBS containing 0.1% (v / v) tween 20 as a cleaning agent and dried.

The UV-VIS spectrometer was used to measure the reflection spectra of each crystal sphere before and after the detection, and the variation of the maximum reflection peak was measured. The results are shown in FIG. 7.

While the invention has been described in detail above with reference to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the invention, and such modifications and variations fall within the scope of the appended claims. It is also natural.

 Since the photonic crystal device of the present invention always emits the same color according to the viewing angle, in the case of the rapid test that needs to be observed with the naked eye, uncertainty may be eliminated by ordinary people, not experts. In addition, compared to the conventional method using a colloidal gold or latex beads, it is possible to omit the labeling process, it is possible to manufacture a much simpler biochip or biosensor. In particular, it is more effective when introduced to a so-called lab-on-a-chip using microfluidics than conventional strip-type diagnostic kits or microarray-type biochips.

Claims (9)

A photonic crystal sphere comprising a plurality of spherical pores uniformly so as to form a spherical diffraction grating, and formed of a spherical body filled with a medium around the pores, wherein the biosensing material is fixed to the surface of the pore. A photonic crystal sphere formed of a spherical body in which a plurality of spherical medium portions are uniformly aggregated so as to form a spherical diffraction grating, and the periphery of the medium portion is filled with pores, wherein the biosensing material is fixed to the surface of the medium portion. The photonic crystal device according to claim 1 or 2, wherein there is a difference in refractive index between the pore portion and the medium portion so that reflection is possible due to the optical diffraction. The photonic crystal sphere according to claim 3, wherein the refractive index difference is 2.8 or more. The material of claim 1 or 2, wherein the medium portion is at least one selected from the group consisting of silicon oxide (silica), titanium oxide (titania), aluminum oxide, zirconium oxide, silicon nanoparticles, Cd-Se nanoparticles, and gold. Photonic crystal sphere characterized in that formed. The photonic crystal device according to claim 1 or 2, wherein the biosensing material is immobilized on the surface of the medium through physical adsorption, chemical bonding, electrochemical bonding, electrostatic bonding, hydrophobic bonding, or hydrophilic bonding. A first step of applying a sample comprising a target material to be analyzed whether or not bound to the bio-sensing material immobilized on the photonic crystal of claim 1 or claim 2; and the target material specifically bound to the bio-sensing material And a second step of detecting the binding between the biosensing material immobilized on the photonic crystal sphere and the target material. The method of claim 7, wherein the target material is a target material to which a colloidal gold, silver, or latex polymer material is attached. A biochip or a biosensor comprising the photonic crystal sphere of claim 1.

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