KR20100077567A - Biomolecular microarrays having enhanced fluorescene and method for preparing the same - Google Patents

Abstract

PURPOSE: A biomolecule array with improved fluorescence signal and a method for manufacturing the same are provided to obtain biomolecule array with repeat and uniform fluorescence signal. CONSTITUTION: A method for manufacturing a biomolecule array with improved fluorescence signal comprises: a step of forming an uniform metal nanopattern on a substrate; a step of forming organic and/or inorganic nanostructure thin film on the uniform metal nanopattern; and a step of arraying fluorescence-labeled biomolecules on the organic and/or inorganic nanostructure thin film or labeling fluorescence molecule.

Description

Biomolecule array with improved fluorescence signal and its manufacturing method {Biomolecular Microarrays Having Enhanced Fluorescene and Method for Preparing the Same}

The present invention relates to a biomolecule array with improved fluorescence signal and a method of manufacturing the same. The present invention relates to a biomolecule array with improved fluorescence signal obtained by introducing fluorescence showing excitation and divergence wavelengths, and a method of manufacturing the same.

Fluorescence has been used as the most dominant and critical detection method in biotechnology, cell imaging, medical diagnostics and drug development. In particular, since fluorescence has been used in most immunoassays, it is an important key to enable discovery of new drugs or diagnosis of new diseases. In polymerase chain reaction (PCR) and DNA chips for DNA enhancement and identification, fluorescence plays an important role in signal detection, which determines the mapping of genes and the detection of specific genes.

     The reason why the fluorescence can achieve various applications is that the fluorescence sensitivity reaches ~ fM and the signal is highly selected because it is excited and diverged only at a specific wavelength according to various fluorescence types. However, due to the limitation of the quantum yield due to the indirect method of labeling, the superposition due to the fluorescence of the sample itself, the stability of the fluorescence signal, and the need for high sensitivity signal detection using very small amounts, recent bio systems using fluorescence Two new approaches are being explored. First, a method of detecting signals by substituting other materials than fluorescence, and second, stably improving signals of fluorescence by using other structural materials.

The first approach is to detect signals using non-fluorescence, labeling materials such as metal nanoparticles, isotopes, quantum dots, or other non-labeling methods using only surface plasmon resonance. In the case of detecting a signal using a non-labeled method using only surface plasmon resonance, the ratio of noise to signal is increased by increasing or decreasing the energy while using a conventional method using fluorescence.

Experimental results on the enhancement of metal nanostructures and fluorescence have been published around the Lakowiz Group at the University of Maryland and attempts have been made to apply them to biodetection systems. In accordance with the existing application of fluorescence, studies have been reported for the use of high-luminescence protein fluorescent probes, detection of DNA hybridization, enhancement of two-photon or multiphoton absorption signals, and immunoassay signals. However, the metal nanostructures used were obtained by reducing metal salts to form metal dots of non-uniform size on the surface. Due to such non-uniformity, there is a difficulty in accurate augmentation or reproducibility of signals, and there are no researches on obtaining signals of large biomaterials such as array-based sensors or cells. As the size of the nanostructures formed on the surface along with the nonuniformity of the size is not uniform, it is also difficult to realize industrial microdevices (Sabanayagam, C. R, Nucleic Acids Res., 35 : e13, 2007).

Attempts to develop precisely shaped nanostructures include the introduction of quantum dots on nanopatterns fabricated by E-beam lithography (Pompa, P. P, Nature Nanotechnology 1 : 126, 2006) and the individual MEF effects of triangular nanoparticles. This study (Chen, Y, Nano letter 7 : 690, 2007) has not been studied on the exact function and association as a biodetector. Therefore, it is necessary to study the improvement of the quantitative signal according to the fluorescent substance labeled reactant because the biomaterial is fixed on the nanostructure having the correct pattern.

The inventors have developed a method for manufacturing various micropatterns using a soft PDMS and a hard PDMS composite mold, as disclosed in Patent Application No. 2007-0073233. The patent application is a disadvantage in that when the pattern is formed by using a flexible PDMS (polydimethylsiloxane) mold in the prior art, distortion or deformation phenomenon occurs when the pattern is down from several microns to nanometer scale due to the soft physical properties of the flexible PDMS In order to compensate for this problem, a soft PDMS and a hard PDMS composite mold were developed to manufacture even nanometer-scale patterns, and a technique capable of manufacturing various patterns by controlling the shape and size of the patterns as well as manufacturing the fine patterns. .

Accordingly, the present inventors have made efforts to develop a biomolecule array with improved fluorescence signal, and thus, by using the method of Patent Application No. 2007-0073233, a uniform metal nanopattern for generating plasmon resonance wavelengths in the visible light region is produced. Forming a multilayer polymer electrolyte nanostructure thin film using a layer-by-layer (LBL) assembly method on the metal nanopattern, and forming a multi-layer polyelectrolyte nanostructure thin film as a fluorescent molecule showing an excitation and diverging wavelength in a region proximate or overlapping with the plasmon resonance wavelength. As a result of the array of labeled biomolecules, it was confirmed that the fluorescence signal was improved by overlapping the plasmon resonance wavelength and the excitation / diffusion wavelength of the fluorescent molecule, thereby completing the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a biomolecule array and a method of manufacturing the same, which are formed on a uniform metal nanopattern and have an improved fluorescent signal.

In order to achieve the above object, the present invention comprises the steps of (a) forming a uniform metal nanopattern on the substrate; (b) forming an organic and / or inorganic nanostructured thin film on the uniform metal nanopattern; And (c) arraying biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructured thin film, or arranging biomolecules on the thin film and then labeling fluorescent molecules. Provides a method for producing an improved biomolecule array.

The present invention also provides an array of biomolecules with enhanced fluorescence signal prepared by the above method, wherein biomolecules labeled with fluorescent molecules are immobilized on organic and / or inorganic nanostructured thin films formed on uniform metal nanopatterns. do.

The invention also comprises the steps of (a) forming a uniform metal nanopattern on a substrate; (b) forming an initial thin film on the uniform metal nanopattern; (c) forming an organic and / or inorganic nanostructured thin film on the initial thin film; And (d) arraying biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructured thin film, or arraying biomolecules on the thin film and then labeling fluorescent molecules. Provided is a method of manufacturing a biomolecule array with improved signal.

The present invention also provides an array of biomolecules with enhanced fluorescence signal, wherein the biomolecules labeled with fluorescent molecules are immobilized on an initial thin film and an organic and / or inorganic nanostructured thin film prepared by the above method and formed on a uniform metal nanopattern. To provide.

According to the present invention, a biomolecule array in which the fluorescence signal is improved can be manufactured by the interaction of the plasmon resonance wavelength in the visible light region and the fluorescence excitation and divergence wavelength generated in the uniform metal pattern, and the uniformity of the metal nanopattern can be produced. By doing so, it is possible to obtain an array of biomolecules in which the enhanced fluorescence signal is repeatedly and constantly displayed.

The term 'uniform' used in the uniform metal nanopattern of the present invention means that a pattern having the same size (eg, the same horizontal, vertical and height) and shape in the metal nanopattern is formed at a constant aspect ratio on the substrate. Point. As a specific example, if a gold nanopattern having a dot (cylindrical model) shape having a diameter of 100 nm and a height of 35 nm is formed on a substrate with an aspect ratio of 250 nm, it may be referred to as a uniform gold nanopattern.

As used herein, the term 'initial thin film' refers to a thin film that is formed on a substrate before the multilayer polymer electrolyte nanostructure thin film is formed, and is intended to improve the bonding force between the substrate and the multilayer polymer electrolyte nanostructure thin film.

The present invention in one aspect, (a) forming a uniform metal nanopattern on the substrate; (b) forming an organic and / or inorganic nanostructured thin film on the uniform metal nanopattern; And (c) arraying biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructured thin film, or arraying biomolecules on the thin film and then labeling fluorescent molecules. A method of making an improved biomolecule array is disclosed.

In another aspect, the present invention, (a) forming a uniform metal nanopattern on a substrate; (b) forming an initial thin film on the uniform metal nanopattern; (c) forming an organic and / or inorganic nanostructured thin film on the initial thin film; And (d) arraying biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructured thin film, or arraying biomolecules on the thin film and then labeling fluorescent molecules. A method of making an improved biomolecule array is disclosed.

The method of manufacturing the fluorescence-enhanced biomolecule array according to the present invention finds a uniform metal nanopattern representing a plasmon resonance wavelength in the visible light region, and induces an interaction between the plasmon resonance wavelength and the excitation and divergence wavelength of the fluorescent material. It is characterized in that the fluorescence is enhanced and the interaction between the metal nanopattern and the fluorescence is made efficiently by forming an organic and / or inorganic nano thin film structure having an appropriate thickness, thereby producing a biomolecule array with enhanced fluorescence. .

The substrate used in the present invention may be a glass substrate, a silicon substrate and the like, but may be used without limitation as long as the substrate used in the art to manufacture a biomolecule array.

In the present invention, as a method of forming a metal nanopattern on a substrate, a 'method of forming various fine patterns using a polymer mold' disclosed in the inventor's patent application No. 2007-0073233 is used.

That is, in the present invention, the step (a) is performed by (i) spin coating a mixed solution of monomers, a catalyst and a curing agent to form a hard PDMS (polydimethylsiloxane) in a silicon mold having an embossed pattern, and then Injecting a mixed solution of a monomer and a curing agent to form and then curing to form a flexible PDMS and a rigid PDMS composite mold; (ii) forming a metal thin film on the substrate on which the nanopattern is to be formed; (iii) contacting the soft PDMS obtained in step (i) and the hard PDMS composite mold on the metal thin film of step (ii) followed by heat treatment; (iv) separating the flexible PDMS and the rigid PDMS composite mold of step (iii) to form a nanopattern of the metal thin film; And (v) etching the nanopattern of the metal thin film formed in step (iv) to form a uniform metal nanopattern having a desired shape and size. .

In this case, a silicon mold having an embossed nanopattern serving as a template may be prepared by an electron beam process or photolithography, and capillary lithography may be used to form a flexible PDMS and a rigid PDMS composite mold from the silicon mold. Duplicate

Thereafter, the flexible PDMS and the rigid PDMS composite mold are contacted on a substrate coated with a metal material having a thickness of 25 nm, and then thermally reacted at a temperature above the glass transition temperature.

The heat treatment causes the metal material on the substrate to behave like a liquid to form a metal nanopattern on the substrate by filling the empty spaces of the soft PDMS and the hard PDMS composite mold by capillary action.

After the nanopattern is formed, the size of the nanopattern may be adjusted by removing the remaining layer on the substrate (abutting portions of the soft PDMS and the hard PDMS composite mold) by using a reactive ion etching process, and controlling the etching time. That is, it can be formed by adjusting a uniform metal nanopattern representing the plasmon resonance wavelength of the visible light region used in the present invention.

The method of forming the micropattern disclosed in the patent application No. 2007-0073233 is difficult to manufacture a nanometer-scale fine pattern due to the soft physical properties of the flexible PDMS when the pattern is manufactured using only the conventional flexible PDMS mold To improve the shortcomings, flexible PDMS and rigid PDMS composite molds can be manufactured and controlled down to nanometer scale patterns.

Therefore, in the present invention, by using the method of Patent Application No. 2007-0073233, a uniform metal nanopattern exhibiting a plasmon resonance wavelength in the visible light region was manufactured, and applied to biomolecules, thereby producing a biomolecule array with improved fluorescence signal. .

In the present invention, the uniform metal nanopattern formed by the method may be characterized in that the minimum length of the width and length of the pattern is 1-20 nm, respectively, the minimum length of 1-40 nm of the height, the uniform The shape of the pattern in the metal nanopattern may be selected from the group consisting of dots, triangles and squares. When the shape of the pattern is a dot, the width and length of the pattern mean diameter. Preferably, the uniform metal nanopattern may be characterized in that the width and length of the pattern, respectively 20 ~ 200nm, the height is 30 ~ 40nm.

At this time, when the horizontal and vertical minimum lengths of the pattern in the uniform metal nanopattern are less than 1 nm or more than 20 m, respectively, there is a problem in that the plasmon resonance wavelength of the region capable of improving the fluorescence signal cannot be emitted.

In the present invention, the uniform metal nanopattern formed by the method may be characterized in that the minimum height of the pattern is 1 ~ 40nm, the shape of the pattern is a dot (dot) shape, the height of the metal nanopattern If the minimum length of is less than 1 nm, the absorption of light is lowered and consequently the fluorescence signal enhancement efficiency is lowered. When it exceeds 40 nm, the fluorescence signal enhancement efficiency is also lowered. This lowers the problem that the pattern formed by the microarray is easily spread.

Plasmon resonances of uniform metal nanopatterns have a narrow full width at half maximum (FWHM) compared to plasmon resonances of non-uniform metal nanopatterns and exhibit relatively high light absorption at the maximum absorption wavelength. At this time, the plasmon resonance of the uniform metal nanopattern is changed more sensitively depending on the reacting material or environment, and thus, the plasmon resonance is more sensitively reacted with fluorescence, thereby improving the fluorescence signal enhancement efficiency.

On the other hand, in order to improve the fluorescence signal in the present invention, there must be an overlap of the wavelength region of the plasmon resonance of the uniform metal nanopattern and the excitation / diffusion wavelength region of the fluorescence, and the overlap occurs to directly improve the fluorescence signal. In order to be able to confirm, both the wavelength region of the plasmon resonance of the uniform metal nanopattern and the excitation / diffusion wavelength region of the fluorescence should be in the visible region.

That is, in the present invention, the wavelength of the plasmon resonance of the uniform metal nanopattern may be characterized in that the visible light region of 380 ~ 800nm.

In addition, the wavelength range of the plasmon resonance of the uniform metal nanopattern and the fluorescence excitation and emission wavelength region in which the overlapping chip is characterized in that 380 ~ 800nm.

That is, the fluorescent molecules labeled on the biomolecules in the present invention may be characterized by an excitation and divergence wavelength of 380 ~ 800nm, the fluorescent molecules are Cyanine-based fluorescent molecules, Rodamine-based fluorescent molecules, Alexa-based fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) fluorescent molecules and Texas Red fluorescent molecules may be selected from the group consisting of.

As a result, in the biomolecule array according to the present invention, first, a uniform metal nanopattern should be formed on a substrate in order to exhibit a fluorescence signal enhancement effect, and second, a plasmon resonance wavelength generated in the uniform metal nanopattern Fluorescence having an excitation and divergence wavelength exhibiting an overlapping phenomenon is required, wherein both the plasmon resonance wavelength and the excitation and divergence wavelength of the fluorescence are controlled in the visible light region.

The uniformity of the metal nanopattern plays a major role in improving the fluorescence signal of the biomolecule array according to the present invention. In other words, when fluorescence is introduced into a metal nanopattern in a manner of fixing fluorescence-labeled biomolecules, the excitation and divergent energy of fluorescence induces plasmons on the metal surface, and then the emanation energy of plasmon and fluorescence divergence energy are The overlapping divergence energy is generated, and as a result, it is observed that the fluorescence signal is improved by improving the energy originally possessed by the fluorescence.

In other words, the plasmon resonance is larger when the metal nanopattern is more uniform than the non-uniformity, and the uniform metal nanopattern and thorn also generate plasmon resonance showing the wavelength of the visible light region as described above. The fluorescence of the excitation and divergence wavelengths in the light ray region overlaps, and the improved fluorescence signal can be confirmed.

In the present invention, the metal of step (a) may be selected from the group consisting of gold, silver, platinum, copper and aluminum. As a material for forming the pattern of step (a), any material is theoretically possible, but in order to exhibit fluorescence enhancement effect, it is preferable to form a pattern using a metal having a high scattering degree as described above.

In the present invention, the organic and / or inorganic nanostructured thin film is composed of a multi-layered polymer electrolyte nanostructured thin film, a self-assembled nanostructured thin film, a nanostructured thin film of metal oxide (Metal Oxide) and a nanostructured thin film of nitrate (nitrate) It may be characterized in that it is selected from the group.

In the present invention, the organic and / or inorganic nanostructured thin film is selected by a method selected from the group consisting of a layer-by-layer (LBL) assembly method, thermal deposition, plasma deposition, chemical vapor deposition, and self-assembled monolayer molecular deposition. It may be characterized in that it is formed.

In general, the LBL granulation method refers to a fabrication method in which a thin film lamination method using a charging phenomenon is alternately absorbed and charged with positive and negative charges. In the present invention, the LBL assembly method may form an organic and / or inorganic nanostructure thin film having a thickness capable of efficiently exhibiting an effect of improving fluorescence by interacting the metal nanopattern with fluorescence labeled on the biomolecule. .

In the present invention, the multilayer polyelectrolyte nanostructure thin film may be characterized in that an anionic polymer electrolyte nanostructure thin film and a cationic polyelectrolyte nanostructure thin film are laminated and stacked. The multi-layered polymer electrolyte nanostructure thin film allows the biomolecules having charges opposite to those of the uppermost thin film to be immobilized efficiently.

In the present invention, the polymer of the anionic polyelectrolyte is PSS (poly (sodium 4-styrenesulfonate), PAA (poly (acrylic acid)), Poly-L-glutamate and PSSA (poly (styrene sulfonic acid)) group It may be characterized in that, the cationic polymer electrolyte polymer is selected from the group consisting of PAH (poly (allylamine hydrochloride)), PANi (Polyaniline), PEI (Polyethyleneimine) and PDADMA (poly (diallyldimethylammonium)) It may be characterized by.

In the present invention, the thickness of the organic and / or inorganic nanostructured thin film may be characterized in that 1nm ~ 10㎛. When the thickness of the thin film is 1 nm to 10 μm, the interaction between the surface plasmon resonance and the fluorescence of the uniform metal nanopattern is maximized, thereby improving the fluorescence signal. When the thickness of the thin film is less than 1 nm, an extinction reaction of the fluorescence signal occurs, and when the thickness is greater than 10 μm, the effect of improving the fluorescence signal is reduced.

The thickness of the organic and / or inorganic nanostructure thin film is a thickness at which the interaction between the metal nanopattern and the fluorescence is performed to improve the fluorescence signal, and a charge opposite to the charge on the top of the organic and / or inorganic nanostructure thin film. Efficiently fixing the biomolecules to ensure that the biomolecules are effectively fixed on the large-area metal nanopattern and react with the molecules that interact with the biomolecules.

In the present invention, before forming the organic and / or inorganic nanostructured thin film may further comprise the step of forming an initial thin film by coating a uniform metal nanopattern, the initial thin film is polyethyleneimine (poly (ethyleneimene), PEI) thin film or an organic nano thin film having an amine (amine) as a terminal group may be characterized.

The initial thin film is to maintain the bonding by continuously increasing the bonding efficiency between the metal pattern formed on the substrate and the organic and / or inorganic nanostructure thin film, the PEI thin film is cationic and has excellent surface adsorption effect, and the amine The organic nano thin film having an end group also has excellent bonding strength with the anionic nanostructure thin film.

In the present invention, the material for forming the organic nano thin film having the amine as a terminal group is (4-aminophenyl) trimethoxysilane [(4-aminophenyl) trimethoxysilane], (3-aminopropyl) triethoxysilane [ (3-aminopropyl) triethoxysilane], (3-aminopropyl) diethoxymethylsilane [(3-aminopropyl) diethoxymethylsilane], (3-aminopropyl) ethoxydimethylsilane [(3-aminopropyl) ethoxydimethylsilane], 11-amino- It may be characterized in that it is selected from the group consisting of 1-undecanethiol (11-amino-1-undecanethiol) and cysteamine (cysteamine).

In the present invention, the biomolecule may be selected from the group consisting of DNA, RNA, PNA, enzymes, proteins, peptides, ligands, antigens and antibodies. Since the biomolecule is combined with the organic and / or inorganic nanostructured thin film, the biomolecule is charged opposite to the thin film on the top of the organic and / or inorganic nanostructured thin film.

In still another aspect, the present invention provides a biomolecule with an enhanced fluorescent signal, wherein a biomolecule labeled with a fluorescent molecule is immobilized on an organic and / or inorganic nanostructure thin film prepared by the above method and formed on a uniform metal nanopattern. Relates to an array.

In another aspect, the present invention provides a fluorescent signal in which a biomolecule labeled with a fluorescent molecule is immobilized on an initial thin film and an organic and / or inorganic nanostructure thin film prepared by the above method and formed on a uniform metal nanopattern. An improved biomolecule array is provided.

In the present invention, the size of the pattern in the uniform metal nanopattern may be characterized in that the minimum length of the horizontal and vertical are each 1 ~ 20nm, the minimum length of the height is 1 ~ 40nm, the uniform metal nano The shape of the pattern in the pattern may be selected from the group consisting of dots, triangles and squares. Preferably, the uniform metal nanopattern may be characterized in that the width and length of the pattern, respectively 20 ~ 200nm, the height is 30 ~ 40nm.

In the present invention, the wavelength of the plasmon resonance generated in the uniform metal nanopattern may be characterized in that the wavelength of 380 ~ 800nm, the wavelength of excitation and emission of the fluorescent molecules may be characterized in that 380 ~ 800nm. .

In the present invention, the fluorescent molecules in the group consisting of Cyanine-based fluorescent molecules, Rodamine-based fluorescent molecules, Alexa-based fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) fluorescent molecules and Texas Red fluorescent molecules It may be characterized by being selected.

In the present invention, the organic and / or inorganic nanostructured thin film of the biomolecule array is a multi-layered polymer electrolyte nanostructured thin film, self-assembled nanostructured thin film, nanostructured thin film of metal oxide (Metal Oxide) and nitrate nano It may be characterized in that it is selected from the group consisting of a structural thin film.

In the present invention, the multilayer polymer electrolyte nanostructured thin film may be characterized in that the anionic polymer electrolyte nanostructured thin film and the cationic polymer electrolyte nanostructured thin film are laminated with each other.

In the present invention, the polymer of the anionic polyelectrolyte is PSS (poly (sodium 4-styrenesulfonate), PAA (poly (acrylic acid)), Poly-L-glutamate and PSSA (poly (styrene sulfonic acid)) group The cationic polymer electrolyte polymer may be selected from the group consisting of poly (allylamine hydrochloride) (PAH), polyaniline (PANi), polyethyleneimine (PEI), and PDADMA (poly (diallyldimethylammonium)). It may be characterized by being selected.

In the present invention, the thickness of the organic and / or inorganic nanostructured thin film may be characterized in that 1nm ~ 10㎛.

In the present invention, the biomolecule may be selected from the group consisting of DNA, RNA, PNA, enzymes, proteins, peptides, ligands, antigens and antibodies.

In the present invention, the initial thin film may be characterized in that the polyimethylene (poly (ethyleneimene), PEI) thin film or an organic nano thin film having an amine as an end group, the organic nano having the amine as a terminal group Substances forming the thin film are (4-aminophenyl) trimethoxysilane, (3-aminopropyl) triethoxysilane, and (3-aminopropyl). Diethoxymethylsilane [(3-aminopropyl) diethoxymethylsilane], (3-aminopropyl) ethoxydimethylsilane [(3-aminopropyl) ethoxydimethylsilane], 11-amino-1-undecanethiol And it may be characterized in that it is selected from the group consisting of cysteamine (cysteamine).

The biomolecule array exhibits a fluorescence signal enhancement effect due to the interaction of uniform metal nanopatterns contained therein with fluorescence labeled on the biomolecules, and the fluorescence signal enhancement effect is repeated and due to the uniformity of the metal nanopatterns. Indicates homeostasis.

After all, from an industrial point of view, if mass production of substrates is possible using all the systems for efficiently manufacturing nanopatterns, the biomolecule array can efficiently utilize the various advantages of fluorescence while utilizing the existing fluorescence detection system. It can be applied to the detection system and is expected to contribute to the detection of ultra fine molecules, selective fluorescent signal enhancement, single biomolecule research, bioimaging improvement, and cost reduction of bioimaging equipment.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

1 illustrates a manufacturing process of a biomolecule array having an improved fluorescent signal formed on a uniform metal nanopattern according to the present invention.

The capillary lithography method is used to repeatedly replicate the variously scaled nanopatterns on the substrate, or the nanopatterning method is used to form large-area metal nanopatterns (FIG. 1A). In this case, the unique surface plasmon resonance wavelength of the metal nanopattern is present in the visible light region.

A 10 nm thick multilayered polymer electrolyte nanostructure thin film was formed using the LBL assembly method (FIG. 1B).

By preparing a biomolecule array having a polarity opposite to that of the final surface of the multilayer polymer electrolyte nanostructure thin film and labeled with fluorescent molecules, the biomolecule array is finally formed on a uniform metal nanopattern to improve the fluorescent signal ( (C) of FIG. 1).

2 illustrates an embodiment of a process of forming a PEI thin film as an initial thin film on a substrate, and then forming a multilayer polymer electrolyte nanostructure thin film.

The capillary lithography method is used to repeatedly replicate the various sized nanopatterns on the substrate, or the nanopatterning method is used to form large area metal nanopatterns. In this case, the unique surface plasmon resonance wavelength of the metal nanopattern is present in the visible light region.

In order to bond the polymer electrolyte thin film on the metal nanopattern, cationic PEI was coated to form a PEI thin film as an initial thin film layer (FIG. 2 (a)).

An anionic polymer electrolyte nanostructure thin film is formed on the cationic PEI thin film and bonded (FIG. 2 (b)).

The cationic polyelectrolyte nanostructure thin film and the anionic polyelectrolyte nanostructure thin film are laminated successively on the anionic polyelectrolyte thin film to form a multi-layer polyelectrolyte nanostructure thin film having a total thickness of 5 to 10 nm. c)).

FIG. 3 illustrates an embodiment of a process of forming a multi-layer polyelectrolyte nanostructure thin film after forming an aminosilane thin film as an initial thin film on a substrate, or after sequential treatment of amino silane and amine alkynthiol.

The capillary lithography method is used to repeatedly replicate the various sized nanopatterns on the substrate, or the nanopatterning method is used to form large area metal nanopatterns. In this case, the unique surface plasmon resonance wavelength of the metal nanopattern is present in the visible light region.

In order to bond the polymer electrolyte thin film on the metal nanopattern, cationic PEI was coated to form a PEI thin film as an initial thin film layer (FIG. 3 (a)).

An anionic polyelectrolyte nanostructure thin film is formed on the cationic PEI thin film and bonded (FIG. 3 (b)).

The cationic polyelectrolyte nanostructure thin film and the anionic polyelectrolyte nanostructure thin film are successively stacked on the anionic polyelectrolyte thin film to form a multilayer polyelectrolyte nanostructure thin film having a total thickness of 5 to 10 nm (FIG. 3 ( c)).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of a biomolecule array comprising a multilayer polymer electrolyte nanostructure thin film on a metal pattern

1-1. Metal nanopattern formation

Using the method shown in Patent Application No. 2007-73233, a metal nanopattern was formed as follows.

(a) trimethylsiloxy-terminated vinylmethylsiloxane-dimethylsiloxane copolymers, methylhydrosiloxane-dimethylsiloxane monomers, a catalyst Pt-divinyltetramethylsiloxane, and a curing agent 2,4,6,8 for the production of rigid PDMS (polydimethylsiloxane) in an embossed silicon mold Sylgard 184 A (DOW corning, US) and Sylgard 184 B (DOW corning, US) (a mixture of vinyl-terminated PDMS) for spin coating the mixed solution of -tetramethyl tetravinylcyclotetrasiloxane, followed by curing A solution containing trimethylsiloxyterminated poly (methylhydro-siloxane) polymers was injected to cure onto the flexible PDMS mold, and then cured to form a flexible PDMS and a rigid PDMS composite mold.

(b) A 35 nm thick gold thin film was formed on the glass substrate using an e-beam evaporator (Moohan Co. Ltd, Korea). At this time, in order to improve adhesion between the metal thin film and the surface, a Cr thin film having a thickness of 2 nm was formed using the same equipment, and then a gold thin film was formed.

(c) A 0.5 wt% polystyrene (molecular weight: 20,000) solution dissolved in toluene was spin-coated at 3000 rpm for 45 seconds on a gold thin film-deposited substrate.

(d) The flexible PDMS obtained in step (a) and the hard PDMS composite mold were contacted on the gold thin film of step (c), and then heat-treated at 135 ° C. for 30 minutes.

(e) The flexible PDMS and the rigid PDMS composite mold of step (d) were separated and a pattern of the polymer thin film was formed.

(f) A uniform gold nanopattern having a dot shape having a diameter of 100 nm and a height of 35 nm was formed by vertically etching along the shape of the polymer thin film pattern formed in step (d).

For etching, first, remove the residual polymer layer under the condition of O2: CF4 = 40 sccm: 60 sccm, 20 mTorr, 80 W using reactive ion etching equipment, and then ion milling equipment). By etching for 2 minutes using the final uniform gold nanopattern was obtained.

1-2. Forming a multilayer polymer electrolyte nanostructure thin film

The initial thin film was immersed in a solution of (3-aminopropyl) triethoxysilane in toluene at 2 wt% for 2 hours, washed sequentially with toluen, aceton and methanol, and left in a 110 ° C oven for 1 hour. As an example, aminosilane having a thickness of 2-3 nm was deposited in a monolayer.

3 mg / ml of poly (sodium 4-styrenesulfonate) (PSS) (molecular weight 70 kDa) (pH 6.5) solution was spin-coated at 3300 rpm for 15 seconds, and then spin-coated under the same conditions using distilled water to react with the unreacted surface. The material was removed. Next, 3 mg / ml of poly (allylamine hydrochloride) (PAH) (molecular weight 70 kDa) (pH 6.5) aqueous solution was spin-coated under the same conditions, followed by spin coating for washing with distilled water.

When PSS and distilled water spin coating and PAH and distilled water spin coating were performed as one process, the process was repeated four times to reach a thickness of 8 nm, thereby forming a multilayer polymer electrolyte nanostructure thin film.

1-3. Arraying biomolecules

As an experimental group, a single strand of one of the DNA pairs forming a double bond was immobilized on the surface of the multilayered polyelectrolyte nanostructure thin film formed in 1-2. At this time, the micro arrayer was used to form a DNA dot array having a uniform dot shape and 50 μM concentration on the surface.

In addition, as a control, a DNA array having a sequence inconsistent with a double bond pair was formed on the surface of the multilayer polymer electrolyte nanostructure thin film formed in 1-2.

Then, the unreacted surface was inactivated by reacting each of the experimental and control groups with 5 × Denhardt's solution (containing 0.1 mg / mL each of Ficoll, poly (vinylpyrrolidone), and bovine serum album) for 30 minutes.

Subsequently, Cy5 fluorescence was labeled on the DNA microarrays of the terminal and the control group, and the DNA molecules having complementary binding sequences with the DNA immobilized on the surface were dropped and left for 14 hours (14 hours). The final fluorescence signal was observed in the washed state.

As a result, as shown in Figure 4, the perfect fluorescent signal was detected in the DNA having the complementary binding sequence of the experimental group, the fluorescent signal was not detected at all in the DNA having no complementary binding sequence as the control group.

That is, from the above observations, first, the polymer electrolyte nanostructure thin film formed by laminating the polymer electrolyte by LBL while fixing the biomolecules and inducing the mutual coupling reaction is not separated from the surface of the substrate and maintains a stable system. Second, in the selective screening system of biomolecules by LBL, when the inactive treatment of the surface where the biomolecules are not immobilized properly, it was confirmed that the selective response was clear while minimizing the non-selective response.

Example 2 Preparation of Biomolecule Arrays Comprising an Initial Thin Film and a Multilayer Polymer Electrolyte Nanostructure Thin Film on a Metal Pattern

1-1. Metal nanopattern formation and initial thin film formation

As an experimental group, a dot-shaped pattern having a diameter of 150 nm and a height of 35 nm was formed on a glass substrate having a width of 2 cm and a length of 2 cm, and the vertical and horizontal axis spacings between the patterns were 250 nm and 2.7 mm and 2.7 mm, respectively. Phosphorus gold nanopattern was produced. The surface plasmon resonance wavelength of the gold nanopattern was initially 625 nm.

In this case, the glass substrate, except for the region where the gold nanopattern is formed on the glass substrate, that is, the glass substrate region as a control.

1-2. Forming a multilayer polymer electrolyte nanostructure thin film

A glass substrate on which the test group and the control group were formed at the same time was coated with 3W% PEI solution at 5000 rpm for 15 seconds to form an initial thin film.

Thereafter, a 3 mg / ml poly (sodium 4-styrenesulfonate) (PSS) (molecular weight 70 kDa) (pH 6.5) aqueous solution was spin-coated at 3300 rpm for 15 seconds, followed by spin coating under the same conditions using distilled water. Unreacted material was removed. Next, 3 mg / ml of poly (allylamine hydrochloride) (PAH) (molecular weight 70 kDa) (pH 6.5) aqueous solution was spin-coated under the same conditions, followed by spin coating for washing with distilled water.

When PSS and distilled water spin coating and PAH and distilled water spin coating were performed as one process, the process was repeated four times to reach a thickness of 8 nm, thereby forming a multilayer polymer electrolyte nanostructure thin film.

1-3. Arraying biomolecules

A single strand of one of the DNA pairs forming a double bond was immobilized on the surface of the multilayer polyelectrolyte nanostructure thin film formed in 1-2. At this time, the micro arrayer was used to form a DNA dot array having a uniform dot shape and 50 μM concentration on the surface.

Thereafter, the unreacted surface was inactivated by reacting with 5 × Denhardt's solution (containing 0.1 mg / mL each of Ficoll, poly (vinylpyrrolidone), and bovine serum album) for 30 minutes.

Subsequently, Cy5 fluorescence was labeled on the DNA microarrays of each of the terminal and the control group of the experiment group, and the DNA molecules having complementary binding sequences with the DNA immobilized on the surface were dropped and left for 14 hours, after which the unbound molecules were washed. The final fluorescence signal was observed.

As a result, as shown in FIG. 5, it was confirmed visually that the improvement of the fluorescence signal occurred in the DNA microarray formed on the gold nanopattern of the experimental group compared to the DNA microarray formed on the glass nanopattern as the control group.

In addition, when the concentration of DNA used to form the DNA microarray is 0 nM, 1 nM, 10 nM and 100 nM, respectively, the DNA microarray formed on the gold nanopattern, which is the experimental group, compared to the DNA microarray formed on the glass nanopattern at all concentrations. It was confirmed visually that the improvement of the fluorescence signal occurred at, and the average improvement was 5 times.

As a result, the fluorescence signal enhancement result of the biomolecule array formed from the metal nanopattern is higher than that of the fluorescence signal enhancement of the biomolecule array formed from the nonmetallic nanopattern, and formed from the metal nanopattern regardless of the concentration of the biomolecule used. It was confirmed that the effect of improving the fluorescence signal of the biomolecule array was great.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the manufacturing process of the biomolecule array with improved fluorescence signal according to the present invention.

2 illustrates an embodiment of a process of forming a PEI thin film as an initial thin film on a substrate, and then forming a multilayer polymer electrolyte nanostructure thin film.

FIG. 3 illustrates an embodiment of a process of forming a multi-layer polyelectrolyte nanostructure thin film after forming an aminosilane thin film as an initial thin film on a substrate, or after amine alkynthiol treatment on the amino silane thin film.

FIG. 4 shows a case in which fluorescently labeled cross-linking molecules are combined as biomolecules in a biomolecule array with improved fluorescence signal according to the present invention (a) and a molecule not having a fluorescent-labeled cross-linking (b) The fluorescence signal observation result is shown.

FIG. 5 shows the results of fluorescence signal observation in the case where a metal nanopattern is formed on a substrate as a biomolecule (a) and a nonmetallic nanopattern is formed on a substrate (b) in a biomolecule array with improved fluorescence signal according to the present invention. will be.

Claims (31)

A method of making a biomolecule array with enhanced fluorescence signal, comprising the following steps: (a) forming a uniform metal nanopattern on the substrate; (b) forming an organic and / or inorganic nanostructured thin film on the uniform metal nanopattern; And (c) arraying biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructured thin film, or arraying biomolecules on the thin film and then labeling fluorescent molecules. A method of making a biomolecule array with enhanced fluorescence signal, comprising the following steps: (a) forming a uniform metal nanopattern on the substrate; (b) forming an initial thin film on the uniform metal nanopattern; (c) forming an organic and / or inorganic nanostructured thin film on the initial thin film; And (d) arranging biomolecules labeled with fluorescent molecules on the organic and / or inorganic nanostructure thin film, or arranging biomolecules on the thin film and then labeling fluorescent molecules. The method of claim 1 or 2, wherein step (a) is performed by a method comprising the following steps: (i) spin coating a mixed solution of monomers, a catalyst and a curing agent to form a rigid PDMS (polydimethylsiloxane) into a silicon mold having an embossed pattern, and then injecting a solution containing a monomer and a curing agent to form a flexible PDMS; Curing to form a flexible PDMS and a rigid PDMS composite mold; (ii) forming a metal thin film on the substrate on which the nanopattern is to be formed; (iii) contacting the soft PDMS obtained in step (i) and the hard PDMS composite mold on the metal thin film of step (ii) followed by heat treatment; (iv) separating the flexible PDMS and the rigid PDMS composite mold of step (iii) to form a nanopattern of the metal thin film; And (v) etching the nanopatterns of the metal thin film formed in step (iv) to form uniform metal nanopatterns of desired shape and size. The method of claim 1 or 2, wherein the size of the pattern in the uniform metal nanopattern has a minimum length of 1-20 nm, and a minimum length of 1-40 nm, respectively. The method of claim 1 or 2, wherein the wavelength of the plasmon resonance generated in the uniform metal nanopattern is 380 ~ 800nm. The method according to claim 1 or 2, wherein an excitation and divergence wavelength of said fluorescent molecule is 380-800 nm. The method of claim 6, wherein the fluorescent molecule is a group consisting of Cyanine-based fluorescent molecules, Rodamine-based fluorescent molecules, Alexa-based fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) fluorescent molecules and Texas Red fluorescent molecules It is selected from. The method of claim 1 or 2, wherein the metal of step (a) is selected from the group consisting of gold, silver, platinum, copper and aluminum. The organic and / or inorganic nanostructured thin film of claim 1 or 2, wherein the organic and / or inorganic nanostructured thin film is formed of a multilayer polymer electrolyte nanostructured thin film, a self-assembled nanostructured thin film, a nanostructured thin film of metal oxide, and nitrate. And a nanostructured thin film. The method according to claim 1 or 2, wherein the organic and / or inorganic nanostructured thin film is composed of a layer-by-layer (LBL) assembly method, thermal deposition, plasma deposition, chemical vapor deposition, and self-assembled monolayer molecular deposition. Formed by the method selected. The method of claim 9, wherein the multilayer polymer electrolyte nanostructure thin film is an anionic polymer electrolyte nanostructure thin film and a cationic polyelectrolyte nanostructure thin film. The method of claim 11, wherein the polymer of the anionic polyelectrolyte is composed of poly (sodium 4-styrenesulfonate), polyacrylic acid (PAA), poly-L-glutamate and poly (styrene sulfonic acid) (PSSA) Characterized in that it is selected from the group. The method of claim 11, wherein the polymer of the cationic polyelectrolyte is selected from the group consisting of poly (allylamine hydrochloride (PAH), polyaniline (PANi), polyethyleneimine (PEI), and poly (diallyldimethylammonium) PDADMA. Way. The method of claim 1, wherein the organic and / or inorganic nanostructured thin film has a thickness of about 1 nm to about 10 μm. The method of claim 1 or 2, wherein the biomolecule is selected from the group consisting of DNA, RNA, PNA, enzymes, proteins, peptides, ligands, antigens and antibodies. The method of claim 2, wherein the initial thin film is a poly (ethyleneimene) film or an organic nano thin film having an amine (amine) as a terminal group. 17. The method of claim 16, wherein the material for forming the organic nano thin film having an amine as a terminal group is (4-aminophenyl) trimethoxysilane], (3-aminopropyl) triethoxysilane [(3-aminopropyl) triethoxysilane], (3-aminopropyl) diethoxymethylsilane [(3-aminopropyl) diethoxymethylsilane], (3-aminopropyl) ethoxydimethylsilane [(3-aminopropyl) ethoxydimethylsilane], 11-amino -1-undecanethiol (11-amino-1-undecanethiol) and cysteamine (cysteamine) characterized in that the method selected from the group consisting of. A biomolecule array with improved fluorescence signal, wherein the biomolecules labeled with fluorescent molecules are immobilized on the organic and / or inorganic nanostructure thin films prepared by the method of claim 1 and formed on a uniform metal nanopattern. A biomolecule array with improved fluorescence signal prepared by the method of claim 2, wherein the biomolecules labeled with fluorescent molecules are immobilized on the initial thin film and the organic and / or inorganic nanostructure thin film formed on a uniform metal nanopattern. 20. The biomolecule of claim 18 or 19, wherein the size of the pattern in the uniform metal nanopattern has a minimum length of 1-20 nm and a minimum length of 1-40 nm, respectively. Array. The biomolecule array according to claim 18 or 19, wherein the wavelength of plasmon resonance occurring in the uniform metal nanopattern is 380-800 nm. The biomolecule array according to claim 18 or 19, wherein an excitation and divergence wavelength of the fluorescent molecule is 380-800 nm. 20. The fluorescent molecule according to claim 18 or 19, wherein the fluorescent molecules are Cyanine-based fluorescent molecules, Rodamine-based fluorescent molecules, Alexa-based fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) fluorescent molecules, and Texas Red fluorescence Biomolecule array, characterized in that selected from the group consisting of molecules. 20. The method according to claim 18 or 19, wherein the organic and / or inorganic nanostructured thin film is a multilayer polymer electrolyte nanostructured thin film, a self-assembled nanostructured thin film, a nanostructured thin film of metal oxide (Metal Oxide) and nitrate (nitrate) of the Biomolecule array, characterized in that selected from the group consisting of nanostructured thin film. 25. The biomolecule array of claim 24, wherein the multilayer polyelectrolyte nanostructure thin film is formed by crossing an anionic polyelectrolyte nanostructure thin film and a cationic polyelectrolyte nanostructure thin film. 27. The method of claim 25, wherein the polymer of the anionic polyelectrolyte is composed of poly (sodium 4-styrenesulfonate), polyacrylic acid (PAA), poly-L-glutamate and poly (styrene sulfonic acid) (PSSA) Biomolecule array, characterized in that selected from the group. The method of claim 25, wherein the polymer of the cationic polyelectrolyte is selected from the group consisting of poly (allylamine hydrochloride) (PAH), polyaniline (PANi), polyethyleneimine (PEI) and PDADMA (poly (diallyldimethylammonium)) Biomolecule array. The biomolecule array of claim 25, wherein the organic and / or inorganic nanostructured thin film has a thickness of 1 nm to 10 μm. 20. The biomolecule array of claim 18 or 19, wherein the biomolecule is selected from the group consisting of DNA, RNA, PNA, enzymes, proteins, peptides, ligands, antigens and antibodies. 20. The biomolecule array according to claim 18 or 19, wherein the initial thin film is an organic nano thin film having a polyimethylene (PEI) thin film or an amine terminal group. 31. The method of claim 30, wherein the material forming the organic nano thin film having the amine as a terminal group is (4-aminophenyl) trimethoxysilane], (3-aminopropyl) triethoxysilane [(3-aminopropyl) triethoxysilane], (3-aminopropyl) diethoxymethylsilane [(3-aminopropyl) diethoxymethylsilane], (3-aminopropyl) ethoxydimethylsilane [(3-aminopropyl) ethoxydimethylsilane], 11- Bio-molecule array, characterized in that selected from the group consisting of amino-1-undecanethiol (11-amino-1-undecanethiol) and cysteamine (cysteamine).

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