KR20070072269A - Three-dimensional nanostructures with active enzymes by surface templated layer-by-layer assembly and method for building the same - Google Patents

Abstract

A three-dimensional micro/nano-structure is provided to be able to increase the sensitivity of a biochip without non-specific absorption by having an active enzyme, thereby increasing the reliability of a bio-sensor. The three-dimensional nano-structure includes a functional enzyme which is controllably prepared by layer-by-layer assembling of surface templates patterned by using the micro-contact printing manner. The method comprises the steps of: (a) printing an SAM strip as a resistant coating agent on a substrate having a cold surface; (b) filling an un-stamped area with an SAM which is made of biotin-terminated thiol; (c) assembling avidin on the biotinylated SAM area; and (d) binding a biotin-HRP to the avidin, wherein the steps (c) and (d) are repeated.

Description

THREE-DIMENSIONAL NANOSTRUCTURES WITH ACTIVE ENZYMES BY SURFACE TEMPLATED LAYER-BY-LAYER ASSEMBLY AND METHOD FOR BUILDING THE SAME}

1 is a cross-sectional view schematically showing a process for forming a three-dimensional structure with an active enzyme according to the present invention.

2 shows the thickness of avidin / biotin-HRP membrane according to the number of assembly cycles.

3 shows an atomic force microscope image of avidin / biotin-HRP three-dimensional structure according to the number of assembly cycles.

FIG. 4 is a graph showing absorbance changes over different time intervals at 571 nm for 1, 5 and 9 bilayer samples.

     <Description of the symbols for the main parts of the drawings>

10: substrate 20: PDMS stamp

30: EG ₆ OH strip

 The present invention relates to a method for manufacturing a biochip, and more particularly, to a method for forming a three-dimensional nanostructure with an active enzyme by interlayer assembly with a surface template.

Recently, as the structure of the human genome is almost revealed, interest in functional research constituting the human body is rapidly increasing. As such, the overall field of research that defines the function of genes is defined as functional genomics, which is a study of the entire group of proteins in genomics and cells that use genes as experimental materials. It is divided into proteomics that identify functions, and bioinformatics that wants both fields in common. Until recently, most of the molecular biology approaches to study the function of genes or cells have been focused on the regulation of expression of a single gene or mRNA expressed by the gene, but nowadays, it is changing to a protein body.

A core technology that can be used essentially for the protein body research is a protein chip (Gavin MacBeath and Stuart L. Schreiber, Science, 289: 1760 ~ 1763, 2000). A protein chip is an automated device system that analyzes protein binding simultaneously by immobilizing dozens or thousands of proteins on a small substrate. This is similar to a microarray DNA chip that detects gene expression or mutations by immobilizing dozens or thousands of genes on a substrate and simultaneously quantitating changes in genes through complementary binding between genes. Yasuda, H., Lamage, CE, J. A ppl.Polym.Sci. 17: 201, 1973; Muguruma, H., Karube, I., Trends Anal.Chem. 18: 62, 1999; Nalanishi, K., Muguruma, H., Karube, I., Anal.Chem. 68: 1695, 1996; Miyachi, H., Hiratsuka, A., Ikebukuro, K., Yano, K., Muguruma, H., Karube, I., Biotechnol. Bioengin. 69: 323, 2000). However, the protein chip can analyze the following parts not found by the DNA chip.

First, information on protein-protein interactions can be obtained. Intracellular signaling or regulation occurs in the form of protein-protein interactions that can be analyzed using protein chips.

Second, from higher organisms, including humans, to yeast, proteins undergo post-translational modifications regardless of genes, which provide information about secondary modifications such as phosphorylation and oxidation. Can be.

Third, problems occurring after mRNA generation can be detected. Many diseases result from problems such as post-transcriptional regulation, protein production, and protein localization, which are difficult to obtain from DNA chips that detect mRNA.

Fourth, since mRNA may not be expressed quantitatively between proteins, protein chips can overcome the limitations of DNA chips, which in some cases cannot provide accurate information about proteins.

Fifth, the difference in the nucleotide sequence of the gene identified by the DNA chip means a direct disease or does not show a difference in phenotype. Moreover, mutations to amino acids with similar properties usually do not cause disease or phenotypic differences because they retain the protein's intrinsic properties. However, protein chips can be used to identify proteins that show direct differences in disease and phenotype.

The use of protein chips enables the generation of data on protein-protein interactions, which can save time and money when developing new drugs. For example, it can dramatically reduce the time it takes to develop therapeutic drugs using current genes. Can be. In addition, the protein chip is not only industrial demand, but also potential demand, such as disease diagnosis, environmental monitoring and hazard screening.

For protein chips, the core technology is protein immobilization technology that binds proteins to the surface of the chip. Protein immobilization techniques are divided into three categories according to their characteristics. The first method is to fix a specific protein on the surface of the sensor chip using carboxymethyl-dextran (CM), the most widely used method of which amine (amine) bond is the most common Thiol bonds or avidin-biotin bonds are also used to bind acidic proteins, DNA, etc. The second is to bind a group of proteins with a number of identical properties. The third method is to treat the surface of the sensor chip, so that the third method is to bind a large number of unspecified proteins, such as polylysine or calix crown.

However, in immobilizing the protein, which is the core technology of the sensor chip on the substrate, nonspecific surface immobilization, loss of biological activity of the protein due to immobilization on a solid surface, relatively low surface concentration of the active substance, surface deviation There is a technical difficulty, such as the disappearance of the analysis signal due to this, the solution is urgent situation.

The most necessary technique for immobilizing proteins on a substrate is reliable patterning techniques for making nanometer-sized devices. In addition, the precise attachment of functional biomolecules to surfaces from micro- to nano-scales in relation to protein immobilization technology is of great importance for array-type high-efficiency screening technologies, new biosensors, bioelectronics, basic research in skin engineering and cell biotechnology. Do.

Such protein fixation techniques include inkjet printing, microfluidic, micro-contact printing (μCP), electron beam lithography and scanning probe technology.

As an optical method, photolithography, which has been used to date and will be used as a major technology until the near future, has a limitation of about 100 nm in terms of resolution. This is due to the interference problem of light in the size of less than 100nm, and since the mid 90s, much efforts have been made to replace the exposure process. Unless there is an economic problem, optical lithography methods with resolutions of 100 nm or less have already been developed. For example, electron-beam lithography, X-ray lithography, scanning probe lithography. ) Is an example of such a method. However, the optical pattern method has the disadvantage of using expensive light sources and using a lot of equipment. In addition, the light blocking material used in this manner is non-environmentally friendly and requires the introduction of other technologies.

As a prior art, US Patent No. 6,485,984, issued on Nov. 26, 2002, Calixcrown derivative, a preparation method thereof, and a self-assembled monomolecular film of a Calixcrown derivative prepared using the same and a self-assembled monomolecular film of Calixcrown derivative (Calixcrown derivatives, a process for the preparation particular, a self-assembled monolayer of the calixcrown derivatives prepared by using the same and a process for immobilizing a protein monolayer by using the self-assembled monolayer of the calixcrown derivatives) disclose a method of immobilizing proteins on a substrate using a self-assembled monolayer. However, the prior art did not selectively fix the protein in the desired position, the process was complicated and the pattern formation was not clear.

In addition, conventionally, photosensitive polyethylene glycol is patterned by capillary force lithography, then covered with PDMS coating, cured by irradiation with ultraviolet rays, and then etched with fluorocarbon plasma, and then the protein is immobilized at a selective position. Technology has been disclosed (Microstructures of polyethylene glycol by molding and dewetting, KY Suh, R. Langer, Appl. Phys. Lett., Vol. 83, No. 8, 1668, 2003). However, since the above technique is required to irradiate ultraviolet rays, it is difficult to provide a substrate of a biosensor at low cost because it is complicated and the patterning speed is low.

U.S. Patent Application No. 20030062334, filed on April 3, 2003, Method for forming a micro-pattern on a substrate by using capillary force, discloses a method of making polymer patterns in various ways using capillary lithography. . However, in the prior art, it is difficult to pattern a thin polymer film because all of them are made of liquid phase, and since the residue is generated after the capillary lithography process, it is difficult to use the prior art for forming a biomaterial pattern.

Biochips formed by the prior art have a problem that can not be used in applications such as high-sensitivity sensor because it has only one biomolecule layer. In addition, since non-specific adsorption still occurs in the conventional biochip, the biosensor has a problem of low reliability. In addition, despite extensive research in the field of layer-by-layer assembly with biomolecules, multilayer 3D micro / nano structures with active enzymes have not yet been realized.

Accordingly, the present invention has been made to solve the problems of the prior art, by implementing a multi-layer 3D micro / nano structure with an active enzyme to increase the sensitivity of the biochip and prevent the non-specific adsorption occurs, the reliability of the biosensor It aims at raising.

The present invention for achieving this object is manufactured to be controllable by layer-by-layer assembly on a surface template patterned using a micro-contact printing method. Achieved by three-dimensional (3D) nanostructures with enzymes.

Nonspecific interactions with proteins can cause nonspecific adsorption on the resist background. Therefore, in the present invention, in order to obtain a clearly formed multilayer structure, non-specific adsorption was minimized. Here, we use a self-assembled monolayer (SAM) terminated with hexa (ethylene glycol) groups known to reduce non-specific biomolecule adsorption as a resistive coating. In the present invention, an average 2.3 biotin-labeled horseradish peroxidase (Biotin-HRP) was selected as a model enzyme, and avidin was selected as a biocompatible linker, with each avidin being two on each protein side. This is because it has four biotin binding sites. It also takes advantage of the significant strong biospecific binding between avidin and biotin (dissociation constant, K _d ^{˜10 −15} M).

 Hereinafter, the present invention will be described with reference to the accompanying drawings.

1 is a cross-sectional view schematically showing a process for forming a three-dimensional structure with an active enzyme according to the present invention. In this embodiment, micro-contact printing (μCP) technology is used to pattern the substrate. In addition, in this embodiment, a micron size SAM strip 30 of EG ₆ OH (11-mercaptoundecylhexa- (ethylene glycol) alcohol) is printed on the substrate 10 having a flat gold surface (FIG. 1C). And 1d). The unstamped region 40 is then filled with a SAM of biotin terminated thiol (FIG. 1E).

Avidin is then assembled on the biotinylated SAM region using two of the biotin binding sites (FIG. 1F). And leaves two additional biotin binding sites for binding biotin-HRP.

Biotin-HRP then binds to the two biotin binding sites (FIG. 1G). This binding forms the outermost layer of biotin at the outermost side, where the second avidin layer can be bound.

In this embodiment the PDMS stamp has 2 μm strips separated by 2 μm gaps. The PDMS stamp is soaked with 2 mM (EG) ₆ OH solution in ethanol. The stamp is then blown dry with nitrogen and conformal contact with the gold plated substrate, maintaining this contact for about 20-30 seconds. After printing, the gold substrate is incubated with the biotin-disulfide solution for about 1 hour to fill the areas that are not stamped with the biotin terminated SAM. The substrate is cleaned with ethanol, methanol and milliQ water to remove unbound biotin-disulfide and template avidin / biotin-HRP interlayer assembly.

The substrate is incubated in 0.1 mg / ml avidin solution for about 0.5 hours, washed with PBS and then soaked in 0.2 mg / ml Biotin-HRP solution for about 0.5 hours. This is the first protein bilayer, abbreviated as (avidin / biotin-HRP) ₁ . Repeat the above steps to form additional bilayers.

As such, selective LBL assembly by avidin and biotin-HRP is repeated to grow a three-dimensional enzyme structure. Since the enzyme is assembled on top of the biocompatible protein (avidin) layer, the immobilized enzyme must maintain catalytic function. 3D enzyme nanostructures (including multilayer active enzymes) generally have higher overall catalytic activity than conventional immobilized enzyme arrays that contain only one layer of enzyme.

<Example>

In order to confirm the performance of the three-dimensional structure manufactured according to the present invention, it was carried out as follows. Multilayer avidin / biotin-HRP membranes were assembled on a gold coated silicon surface (˜200 nm thick gold with ˜10 nm chromium adhesion layer) and the thickness change was measured according to the number of assembly cycles using ellipsometry. The gold surface is coated with SAM of biotin terminated thiols by incubating in a biotin-disulfide solution (1 mM in ethanol) for about 1.5 hours and thoroughly cleaned with ethanol, methanol and milliQ water before use in assembly did. The first avidin layer is assembled by incubating the surface with avidin solution (0.1 mg / mL in PBS, 10 mM phosphate, 150 mM NaCl, 2 mM NaN ₃ , pH 7.4) for about 30 minutes. After washing with PBS, the surface is incubated with Biotin-HRP (0.2 mg / mL in PBS) for 30 minutes and washed with PBS.

2 shows the thickness of avidin / biotin-HRP membrane according to the number of assembly cycles. It can be seen from FIG. 1 that the protein membrane thickness increases with increasing number of assembly cycles, indicating that the enzyme membrane is correctly interlaminar assembled after the first bilayer. The average thickness of each avidin / biotin-HRP bilayer is ˜1.44 nm, which is considerably thinner than the bond thickness of the tightly packed HRP and avidin layers (about 8-10 nm).

3 shows an atomic force microscope image showing avidin / biotin-HRP three-dimensional structure according to the number of assembly cycles. An enzyme was assembled on the three-dimensional structure according to the present invention, and the enzyme pattern according to the LBL assembly process was observed. FIG. 3A shows an image of a SAM template patterned with micro-contact printing (μCP), which separates the 2 μm EG ₆ OH SAM strip into a biotin-thiol strip on a flat thin gold coated surface. EG ₆ OH strips are ˜2.4 nm higher than biotin-thiol strips. This means that there is a difference in thickness between the two SAMs.

After the avidin / biotin-HRP bilayer is assembled on the template, the topographic structure between the protein and the EG ₆ OH strip is reversed (FIG. 3B). The protein strip is now ˜1.2 nm higher than the EG ₆ OH strip because the protein is assembled on the biotin strip.

Despite the use of EG ₆ OH SAM, one of the most efficient resist coatings for biomolecules, some non-specific adsorption was observed on the resist strips. Most nonspecific adsorbed proteins in the present invention were removed by treating the surface with a surfactant (0.1% Tween 20 in PBS) for about 1.5 hours. This treatment increased the height difference between the two strips to ˜3.2 nm (FIG. 3C). By washing with a surfactant between each assembly step, the multilayered avidin / enzyme of the three-dimensional structure can be readily prepared on the patterned surface by LBL assembly.

3D shows an image of the patterned surface after assembly of nine avidin / biotin-HRP bilayers. The height of the enzyme strip is ˜12 nm from the resist background. This corresponds to a total protein layer thickness of 14.4 nm. This shows little non-specific adsorption on EG ₆ OH, indicating that three-dimensional enzyme structures can be accurately formed by interlayer assembly on functional pattern regions.

Assays for adsorption were used to determine the activity of immobilized enzymes. Absorbance changes for 1, 5 and 9 bilayer samples were measured at different time intervals at 571 nm (FIG. 4). In the presence of H ₂ O ₂ , HRP turns a colorless substrate (Amplex red) into a colored product (resorufin) with maximum absorbance at 571 nm. All three samples were catalytically active and the overall activity increased with the number of enzyme layers. The increase rate of activity according to the enzyme layer is not linear, and the activities of the 5 bilayers and 9 bilayers are about -25% and -40% higher than those of the 1 bilayer.

As described above, precisely multilayered three-dimensional enzyme structures can be successfully produced by using a micro-contact printing patterned SAM template to induce avidin / biotin-HRP interlayer assembly according to the present invention. The assembled enzyme maintains catalytic activity, so that the overall activity increases with the number of enzyme layers.

What has been described above has described one embodiment according to the present invention, and the present invention is not limited to the above-described embodiment, and as claimed in the following claims, without departing from the gist of the present invention, the field to which the present invention pertains. It will be said that the scope of the present invention to the extent that those skilled in the art can change.

The present invention implements a multi-layered three-dimensional micro / nano structure with an active enzyme to increase the sensitivity of the biochip and prevent non-specific adsorption from occurring, thereby increasing the reliability of the biosensor.

Claims (7)

Three-dimensional (3D) nanostructures with functional enzymes made to be controllable by interlayer assembly on patterned surface templates using micro-contact printing. a) printing a SAM strip as a resistive coating on a substrate having a gold surface; b) filling an unstamped region with a SAM of thiol terminated with biotin; c) assembling avidin on the biotinylated SAM region; d) binding biotin-HRP with said avidin; And Repeating the steps c) and d). The method of claim 2, further comprising treating the surface with a surfactant after performing any one of steps b) to d). a) soaking the PDMS stamp with a 2 mM (EG) ₆ OH solution in ethanol; b) contacting the stamp with a gold plated substrate for about 20-30 seconds; c) incubating the gold substrate with a biotin-disulfide solution for 1 hour to fill the areas that are not stamped with the biotin terminated SAM; d) cleaning the substrate with ethanol, methanol and milliQ water to remove unbound biotin-disulfide; e) template avidin / biotin-HRP interlayer assembly; f) incubating the substrate in 0.1 mg / ml avidin solution for about 0.5 hours; g) soaking the substrate in a 0.2 mg / ml biotin-HRP solution for about 0.5 hours; And h) repeating steps f) and g) to form additional bilayers. The method of claim 4, wherein the PDMS stamp has a 2 μm strip separated by a 2 μm gap. 5. The method of claim 4, further comprising cleaning the substrate with PBS after step f). Three-dimensional nanostructures prepared by the method according to any one of claims 2 to 6.

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