KR20080082388A - Quantum dot-based biochip and method for assaying hydrolytic proteins using the same - Google Patents

Abstract

A quantum dot-based biochip and a method for searching a hydrolyzed protein using the same are provided to measure the activity of a hydrolyzed protein from a small amount of a solution rapidly using energy transfer. A quantum dot-based biochip comprises: a support consisting of glass or silica; a self-assembled mono-layer membrane on the surface of the support; a quantum dot mono-layer membrane formed on the self-assembled mono-layer membrane by coating the surface of a quantum dot with a bio-molecule specifically bonded to biotin; and a binding layer of a substrate-energy receptor formed on the quantum-dot mono-layer membrane by specific binding between a biotin at a substrate end and the biomolecule exposed on the quantum dot mono-layer. A method for measuring the activity of a hydrolyzed protein regarding a substrate comprises the steps of: (a) reacting the biochip with the hydrolyzed protein; (b) after washing the reaction completed biochip, measuring luminescence of the quantum dot; and (c) comparing luminescence of a quantum dot of the biochip which is not reacted with the hydrolyzed protein with the luminescence of the step(b) to identify the hydrolytic activity of the protein regarding the substrate. Further, a concentration ratio of the biochip and the hydrolyzed protein is 1:1 to 1:100(mol/mol).

Description

Quantum dot-based biochip and method for assaying hydrolytic proteins using the same

1 is a schematic diagram showing a method for measuring the activity of hydrolyzed protein using a quantum dot-based biochip according to the present invention.

Figure 2 is a spectrum of the emission inhibition capacity of the quantum dot according to the concentration of gold nanoparticles and fluorescent material as the energy acceptor in aqueous solution.

3 is according to the present invention The spectrum of luminescence inhibition of quantum dots according to the concentration of gold nanoparticles and fluorescent materials used as energy acceptors in quantum dot-based biochips.

Figure 4 is a hydrolytic protein activity spectrum using different quantum dots and gold nanoparticles in aqueous solution.

5 is an electron micrograph confirming the activity of the hydrolyzed protein using quantum dot-gold nanoparticles in aqueous solution.

Figure 6 is a fluorescence analysis image and optical micrographs measuring the activity of the hydrolyzed protein using a quantum dot-based biochip and gold nanoparticles according to the present invention.

Figure 7 is a fluorescence analysis image of the hydrolysis protein and its inhibitory effect using a quantum dot-based biochip and gold nanoparticles according to the present invention.

The present invention is a quantum dot-based biochip and The present invention relates to a method for analyzing a hydrolyzed protein using the same, and more particularly, in a biochip and a biochip in which a quantum dot which is an energy donor is combined with a substrate-energy receptor, The present invention relates to a method for rapidly measuring a small amount of hydrolyzed protein activity on various substrates using energy transfer between quantum dots and energy receptors.

Hydrolyzed proteins (or enzymes) are generic terms for proteins that hydrolyze proteins, peptides, DNA, RNA, sugars, lipids, etc. In the cell, signal transduction, proliferation, inflammation, and self-killing It is an important biomolecule that performs both forward and reverse functions with respect to apoptosis. Therefore, measuring the activity of various hydrolytic proteins or searching for and screening inhibitors for these proteins is of great interest in the field of diagnosis and treatment of specific diseases along with basic research for the identification of life phenomena.

In general, the method of measuring the activity of the hydrolyzed protein is measured by the high performance liquid chromatography (Bjurlin, MS Bloomer, S. and the product produced by the hydrolyzed protein using a synthesized substrate) Nelson, CJ Biotechnol. Lett . 2002, 24, 191), and methods of measurement by thin-layer electrophoresis (Kotler, M. Katz, RA Danho, W. Leis, J. Skalka, AM Proc) Natl.Acad.Sci. USA 1988. 85, 4185). However, this method is not only complicated and time-consuming, but also difficult to quantitate substrates. Because of this problem, the method of using a fluorescent material to measure the activity of the hydrolyzed protein is mainly utilized. For example, in the case of proteases, fluorogenic peptides are used as substrates (Harris, JL Backes, BJ Leonetti, F. Mahrus, S. Ellman, JA and Craik, CS Proc. Natl Acad. Sci. USA 2000, 97, 7754), a method of using fluorescence resonance energy transfer (FRET) by attaching a pair of fluorescent materials or a pair of fluorescent materials-quencher to a peptide substrate. Widely used (Matayoshi, ED Wang, GT Krafft, GA Erickson, J. Science, 1990, 247, 954; US Publication No. 20050014160). In particular, the method by FRET is known to be able to more accurately measure the activity of the hydrolyzed protein because it depends largely on the distance between the pair of donors and acceptors. FRET is a long-wavelength fluorescence of a receptor because the donor, which is a fluorescent substance, absorbs light energy and emits it as fluorescence instead of emitting it as fluorescence. This is the principle of emission. If the acceptor and the donor are at a distance of 10 nm or more, or the absorption wavelength of the acceptor does not overlap with the emission wavelength region of the donor, excitation of the donor does not lead to emission of the acceptor because resonance does not occur. . However, the method of measuring hydrolyzed protein using fluorescence resonance energy transfer is difficult to measure for a long time because the two fluorescent materials can be rapidly attenuated by the initial external light energy, and the appropriate donor-receptor pair is extremely limited. As it is used, it is impossible to measure several kinds of hydrolyzed proteins simultaneously with one light source.

Recently, quantum dots are emerging as alternative materials that can solve such problems. That is, since the damping effect by the external light energy is small and has a high quantum yield, the measurement sensitivity can be improved. In addition, since the emission wavelength region of quantum dots having different sizes can be distinguished by one common absorption wavelength, it can be used for simultaneous analysis. In terms of energy transfer, a quantum dot as a donor can combine with a large number of receptors by introducing a surface functional group, which greatly increases the efficiency of FRET. Indeed, FRET between quantum dots and fluorescent materials has been used to measure protease activity (Medintz, IL Clapp, AR Brunel, FM Tiefenbrunn, T. Uyeda, HT Chang, EL Deschamps, JR Dawson, PE Mattoussi, H. Nat. Mater. 2006, 5, 581), as well as complementary DNA hybridization (Zhang, CY Yeh, HC Kuroki, MT Wang, TH Nat. Mater. 2005, 4, 826), protein analysis (Clapp, AR Medintz, IL Mauro) , JM Fisher, BR Bawendi, MG Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301). On the other hand, unlike the dipole-dipole energy transfer phenomenon in FRET, surface energy transfer between the dipole-electrons generated between the fluorescent material and the metal nanoparticles (SET) Has been reported that the distance between donor and acceptor can occur within 20 nm (Yun, CS Javier, A. Jennings, T. Fisher, M. Hira, S. Peterson, S. Hopkins, B. Reich, NO Strouse, GF J. Am. Chem. Soc. 2005, 127, 3115). In a manner similar to this surface energy transfer phenomenon, fluorescent and gold nanoparticles can be used to measure the activity of DNA hydrolyzed proteins (Ray, RC Fortner, A. Darbha, GK J. Phys. Chem B. 2006, 110, 20745), an example of analyzing the interaction of biomaterials based on energy transfer between quantum dots and gold nanoparticles (Oh, E. Lee, D. Kim, Y.-P. Cha, SY Oh, D.-B) Kang, HA Kim, J. Kim, H.-S. Angew. Chem. Int. Ed. 2006, 45, 7959; KR Application No. 10-2006-12213). However, the energy transfer phenomena by FRET or SET between the fluorescent material-fluorescent material, the quantum dot-fluorescent material, the fluorescent material-metal nanoparticle, and the quantum dot-metal nanoparticles exemplified above are all performed in an aqueous solution. As a result, nanoparticles (quantum dots and metal nanoparticles) are easily aggregated, making it difficult to measure. In addition, a large amount of nanoparticles are required in a reaction solution of at least several tens of microliters to several mL or more in a single measurement.

In order to solve this problem, as an example of measuring the activity of the hydrolyzed protein in the chip, there is a report measuring the activity of proteases using a fluorogenic peptide chip (Salisbury, CM Maly, DJ Ellman) , JA J. Am. Chem. Soc. 2002. 124, 14868) It is not applicable to the method of energy transfer using quantum dots, which may result in poor stability and difficulty in applying various fluorescent materials. In some cases, energy transfer between quantum dots and gold nanoparticles was used for complementary DNA binding on the chip surface (Dyadyusha, L. Yin, H. Jaiswal, S. Brown, T. Baumberg, JJ Booy, FP Melvin, T. Chem. Commun. 2005, 25, 3201) This is not a generally applicable method for measuring the activity of hydrolyzed proteins and is limited in the amplification of energy transfer due to the limited number of receptors that react with the donor (responding to 1: 1). It is difficult to expect effective quantitative analysis. To date, no quantum dots have been immobilized on the chip surface to measure the activity of hydrolyzed proteins based on energy transfer phenomena including FRET or SET.

The present invention is to solve the problems of the prior art as described above, by using the energy transfer phenomenon can quickly measure the activity of the hydrolyzed protein even with a small amount of solution based on a quantum dot-based biochip and fast It is an object to provide a search method.

Still another object of the present invention is to provide a method for simultaneously analyzing the activity of various types of hydrolyzed proteins using various quantum dots.

It is another object of the present invention to provide a method for rapidly selecting an inhibitor of a hydrolyzed protein and selecting a new drug candidate that inhibits protein activity in a very high speed.

The quantum dot-based biochip of the present invention for achieving the above object is (A) a support made of glass or silica; (B) a self-assembled monolayer film formed on the surface of the support; (C) a quantum dot monolayer film formed on the self-assembled monolayer membrane by the surface of the quantum dot is coated with a biomolecule that specifically binds to biotin, by combining the terminal of the self-assembled monolayer film and the biomolecule functional group on the surface of the quantum dot; And (D) biotin is bonded to one end of the substrate and the energy receptor is bound to the other end of the substrate to the hydrolyzed protein, and the biotin at the end of the substrate is specifically bound to the biomolecules exposed on the quantum dot monolayer. Characterized in that consisting of; binding layer of the substrate-energy receptor formed on the monolayer film.

The support is preferably made of glass or silica, and when using a metallic substrate such as gold, silver, aluminum, or titanium, the fluorescence of the quantum dots may be seriously inhibited, and thus, the support may not be used.

The self-assembled monolayers (SAMs) are regularly aligned organic molecular thin films coated on the surface of the support, which are alkanoic acid that forms an ionic bond with the support or organosilicon that forms pure covalent bonds. ) Compounds can be used. Specific examples of the compound forming the self-assembled monolayer include compounds such as alkylchlorosilanes, alkylalkoxysilanes, and alkylaminosilanes as n-alkanoic acid and alkyl silane, wherein the number of carbons in each compound is preferably 3-25.

Typically, the compounds that make up the self-assembled monolayer are composed of a reactor of the head that binds to the substrate, a long alkane chain of the body that enables regular molecular membrane formation, and a terminal functional group that determines the function of the molecular membrane. As the functional group of the terminal portion, the simplest functional group may be an alkyl group, for example, an amine, thiol, carboxyl group, aldehyde, epoxy, maleimide, NHS (N-hydroxyl succinimide), or a mixture of two or more of biotin. Preference is given to using in form. When selecting these functional groups, care should be taken when using a functional group that emits fluorescence in the region (420-460 nm) where the quantum dots are excited, since the light emission signal of the quantum dots may be severely inhibited.

The self-assembled monolayer film is prepared by preparing the compound in a concentration range of usually 2 mM to 10 mM and then immersing the support in a solution. At this time, it is preferable to first remove the oxide layer on the surface of the support, and in the process of forming the self-assembled monolayer film, it is preferable to react under nitrogen atmosphere to block the oxidation reaction by oxygen during the reaction. The oxide layer on the support can be removed by treating the support with a Pirana solution of sulfuric acid / hydrogen peroxide for 5-10 minutes. The reaction process of the compound for the self-assembled monolayer film is preferably carried out at room temperature for 2 to 24 hours, but even if the reaction time exceeds 24 hours, there is no problem in forming the self-assembled monolayer film. In order to induce a faster reaction, the reaction may be performed at 30 to 40 ° C. for only 2 hours.

Glass substrates with commercially available self-assembled monolayer films, such as NHS-hydrogel (N-hydroxyl succinimide hydrogel) -treated glass substrates (Nexterion ^™ Slide H, Schott, Germany), may be used.

In an embodiment of the present invention, the Strepptavidin-coated quantum dot (CdSe / ZnS) -based quantum dot (CdSe / ZnS) is a semiconductor nanoparticle coated with a biomolecule-coated quantum dot (streptavidin). QD-SA, Invitrogen) was used. When QD-SA is used, firstly, it is easy to attach quantum dots to the surface because streptavidin is a protein, and secondly, a large number of substrate-energy receptors are strongly bound to the surface of quantum dots by streptavidin-biotin binding. The non-specific binding can be reduced by the washing process. Third, the streptavidin-biotin binding allows the substrate to be spaced on the quantum dots, thereby providing a space for the hydrolyzed protein to effectively access and degrade the substrate. Although there is (FIG. 1), it is obvious that the quantum dot of the present invention is not limited thereto. As biomolecules for coating the quantum dot surface, avidin, neutravidin, or captavidin may be used in addition to streptavidin.

The monolayer film of the quantum dots is formed by combining the quantum dots with a monolayer film on the surface after construction of the self-assembled monolayer film. Coupling of the self-assembled monolayer film and the quantum dots is achieved by covalent bonding of the end groups of the self-assembled monolayer film and the surface functional period of the biomolecules coated on the quantum dots. For the construction of a quantum dot monolayer film, a solution of a quantum dot is formed by dropping or spraying on a surface of a self-assembled monolayer film on a support.

The monolayer film of the quantum dot may be formed in the entire region on the self-assembled monolayer film, but it is preferable to limit the amount of the quantum dot to a small amount in some regions because commercialization of the quantum dot is expensive. When the monolayer film of the quantum dots is formed to be limited to a portion of the self-assembled monolayer film , the fixation of the quantum dots may be performed by pipetting a small amount of the quantum dot solution on the surface of the self-assembled monolayer film or by microarray method. The reaction is usually carried out by washing the surface with distilled water after the reaction for 30 minutes to 2 hours at room temperature, but is not limited thereto.

The quantum dots (QD-SA) are already commercialized in various sizes (Invitrogen or Evident Technologies) and it is preferable to use quantum dots having a maximum emission wavelength in the range of 500 to 900 nm.

The quantum dot is attached Unresponsive to quantum dots around the support The presence of self-assembled monolayers can lead to nonspecific adsorption between the self-assembled monolayers and the substrate in subsequent reactions. In other words, if functional groups such as aldehyde, epoxy, NHS, and biotin constituting the terminal portion of the self-assembled monolayer film are exposed without binding to the quantum dots, the adsorption of the substrate may occur, thereby filling the space around the quantum dots to form nonspecific binding. It is desirable to prevent. Examples of the nonspecific binding inhibitor include ethylene glycol-based compounds such as ethylene glycol amine, ethylene glycol thiol, NHS (N-hydroxysuccinimide) -ethylene glycol, maleimide-ethylene glycol, polyethylene glycol amine, polyethylene glycol thiol, (NHS One or two selected from the group consisting of polyethylene glycol-based compounds such as) -polyethylene glycol, maleimide-polyethylene glycol, or polymers based on carbohydrate monomer binding as a biological compound, protein disulfide isomerase, and bovine serum albumin (BSA) It is preferable to be filled with the above compound. Filling around the quantum dots is made by spraying, dipping or flowing a solution of the compound after forming a monolayer film of the quantum dots. Depending on the reactivity and solubility of the compound, the solvent, pH, buffer solution, etc. may be determined differently, but a buffer solution having a pH range of 5 to 9 is more suitable than an organic solvent within a range that does not affect quantum dots attached to the surface. It is preferable to maintain the concentration of 0.1 mM ~ 10mM.

The energy acceptor may be any of those capable of suppressing light emission of the quantum dots by energy transition from the quantum dots when the energy receptor is in close proximity to the quantum dots. In this specification, energy transfer is used to refer to fluorescence resonance energy transfer (FRET) and surface energy transfer (SET) in a comprehensive sense.

In the present embodiment, only the gold nanoparticles (AuNP) which are widely used for the adhesion of Cy ^TM- 5 and the surface functional group and the simple size adjustment as the energy acceptor are not limited thereto. Fluorescent dyes such as TAMRA (carboxytetramethylrhodamine), Alexa ^TM and Cy ^TM , nanoparticles such as gold, platinum and silver or fluorescent quenchers such as QSY ^TM , BHQ ^TM and DABCYL ^TM can be used. have.

The energy receptor is attached to the other end of the substrate of the hydrolyzed protein with biotin bonded at one end, and thus substrate-energy on the monolayer membrane of the quantum dot by specific binding of the biotin attached to the substrate and the biomolecule coated on the quantum dot. The binding layer of the receptor is formed.

In the step of the present invention, only the peptide is illustrated as a substrate of the hydrolyzed protein, but is not limited thereto, and any substrate can be used as long as it can attach biotin to one end and the other end can bind to the energy receptor. . For example, the peptide CRPLALWRSK-biotin used in this example is biotin attached by C-terminal lysine and specifically with monomolecular maleimide gold nanoparticles (monomaleimide nanogold ^ㄾ , Nanoprobe) by N-terminal cysteine. It is possible to combine. In addition, instead of gold nanoparticles, a fluorescent dye, Cy ^TM- 5, can be attached directly to the N-terminal end of cysteine-free arginine (R) by amide bonds. As such a substrate, one can introduce a biotin group which is bonded to a biomolecule on the surface of a quantum dot and the other end can be a thiol (-SH), an amine group (NH ₂ ), a carboxyl group (-COOH), or a hydroxy group which can bind to an energy receptor. As having a hydroxyl group (-OH), Examples include peptides and DNA, RNA, PNA, aptamers, sugars, lipids.

The length of the substrate should be less than or equal to the predetermined length depending on the type and form of the energy acceptor bound for effective energy transfer. In other words, when using gold nanoparticles as energy acceptors, it is appropriate that the theoretical length of the substrate is 1-15 nm or less in consideration of the radius of the quantum dot (7.5 to 10 nm). It is appropriate to be 1-5 nm or less, but the use of shorter substrates within the exemplified distance conditions can be expected to be effective energy transfer. However, even if the substrate is too short and the substrate is long, if the position of the hydrolysis in the substrate is located on the quantum dot side, the hydrolyzed protein is difficult to access and the reaction efficiency may be low, so this should be considered sufficiently. When the peptide is used as a substrate, it is preferable that the peptide sequence is 20 or less when the gold nanoparticles are used as the energy receptor, and the peptide sequence is 10 or less when the fluorescent material is used as the energy receptor. If the length of the peptide is larger than the number, the distance between the quantum dot and the receptor may be increased, so that an effective energy transfer may not occur. However, if the peptide contains a sequence known to form an α-helix structure (eg, achiral alpha-amino isobutyric residue (Aib)) or forms a secondary or tertiary structure from the above-mentioned number of peptide sequences, Can be reduced so that more peptide sequences can be used.

For the construction of the substrate-energy receptor binding layer, a solution of the energy receptor with the substrate bound thereto is sprayed or dropped onto the surface of the quantum dot monolayer. The reaction is usually carried out by washing the surface with distilled water after the reaction for 30 minutes to 2 hours at room temperature, but is not limited thereto.

The quantum dot-based biochip according to the present invention may be constituted by forming the quantum dot monolayer film and the substrate-energy receptor binding layer stepwise on the support on which the self-assembled monolayer film is formed as described above. It may also be prepared by combining with a self-assembled monolayer film to form a quantum dot monolayer film and a substrate-energy receptor binding layer at the same time. In this case, the quantum dot and the substrate-energy receptor are combined by specific binding of the biomolecule coated on the quantum dot and the biotin bonded to one end of the substrate as in the stepwise formation of the quantum dot monolayer and the substrate-energy receptor binding layer.

In the quantum dot-based biochip according to the present invention, the quantum dot monolayer is composed of a mixture of two to five kinds of quantum dots, and the substrate-energy receptor binding layer is composed of different substrate-energy receptors specifically attached to different quantum dots. Can be. Theoretically, five or more kinds of quantum dots can be used. However, considering the combination of the maximum light emission wavelength and the energy acceptor, the number is preferably five or less. In this case, when using a fluorescent material may be used different types depending on the substrate, when using a fluorescent suppressor or metal nanoparticles can be used only one type.

The quantum dot-based biochip according to the present invention having the plurality of quantum dot-substrate-energy receptor combinations may have different types of quantum dots and substrate-energy receptors after binding different substrate-energy receptors to different kinds of quantum dots. The mixture is prepared by fixing quantum dots to a support on which a self-assembled monolayer film is formed.

Another aspect of the present invention relates to a method for measuring the activity of a hydrolyzed protein on a substrate using a quantum dot-based biochip according to the present invention. The hydrolysis protein screening method may include (A) reacting a quantum dot-based biochip with a hydrolyzed protein to measure activity; (B) measuring the luminescence of the quantum dots after washing the quantum dot-based biochip is completed; And (C) comparing the light emission of the quantum dot-based biochip not reacted with the hydrolyzed protein and the change in light emission of the quantum dots measured in step (B) to confirm the hydrolytic activity of the substrate of the protein. It is done.

In the method of screening the hydrolyzed protein for the substrate, the luminescent signal is reduced by energy transfer between the quantum dot and the adjacent energy receptor under conditions in which the quantum dot, which is an energy donor, and the energy receptor are interposed between the substrate, but are hydrolyzed to the substrate. When a specific portion of the substrate is cleaved by an active protein, quantum dots and energy receptors are separated, and thus the light emitting signal of the donor is recovered again because no energy transfer occurs (FIG. 1). That is, the photoluminescence (PL) recovery after protein treatment is observed by comparing and measuring the luminescence ability of the surface of the chip before and after the treatment with the protein to verify the hydrolytic activity on the substrate. It can be seen that it has a hydrolytic activity for.

As the hydrolyzed protein, a protease, a peptidase, a nucleosidase, a deoxynuclease, a phosphatase, a glucosidase, and a galactose It is characterized in that one or more hydrolyzed proteins selected from the group consisting of a galase (galactosidase), lipase (lipase), esterase (esterase). In the embodiment of the present invention, only the protease matrix metalloproteases (MMP-7) is illustrated, but by changing only the sequence of the peptide used as a substrate, other proteases (eg, collagenase, caspase, thrombin, HIV protease, cathepsins, etc.) Naturally, activity can be measured.

Hereinafter, the screening method of the hydrolyzed protein of the present invention will be described in detail using the quantum dot-based biochip and the hydrolyzed protein used in the examples.

As a substrate, the peptide RPLALWRSK-biotin, with cysteine attached to the N-terminus and biotin attached to the C-terminus, was used, which reacts specifically with matrix metalloproteinase-7 in the protease. Is a peptide substrate.

Before measuring the activity of the protein substrate using the quantum dot-based biochip according to the present invention, the aqueous solution using QD605-SA and Cy5-RPLALWRSK-biotin (pep-Cy5) or AuNP-CRPALWRSK-biotin (pep-AuNP) The decrease rate of the light emission signal due to the energy transition of the quantum dots was measured.

QD-SA There are 5 to 10 SAs around one quantum dot, and 2 to 4 effective binding sites exist on one SA that is randomly immobilized on the quantum dot surface. Therefore, the number of binding sites that can be attached to biotin can be inferred to about 10 to 40, and the number of exposed SAs can be reduced on the chip surface, so there are less than 40 predictable biotin binding sites. For this reason, it is possible to appropriately control the donor: receptor ratio by changing the concentration of Pep-AuNP of the receptor to be reacted with the concentration of QD-SA attached to the surface.

As a result of assaying the donor quenching due to energy transfer in aqueous solution, as the relative amount of the energy acceptor quantum dots increases, the luminous ability of the donor QD-SA could be effectively suppressed. In the case of Pep-AuNP, when the concentration of quantum dot: pep-AuNP is 1:40, the intensity of the luminescence signal is saturated even when the concentration of energy acceptor is higher because the quantum dot: pep-AuNP concentration is 1:40. It did not decrease any more. When the donor and the receptor are present in a ratio of 1:40, pep-AuNP exhibits up to 82.1% of luminescence inhibition and Pep-Cy5 up to 55.5% of luminescence suppression, compared to using fluorescent material (Cy ^TM- 5) as an energy acceptor. When the particles were used, excellent luminescence inhibition was shown.

Based on the principle of the general fluorescence resonance energy transfer phenomenon (FRET), the rate of decrease in luminescence signal ( Q _E ) at both receptors The differences were compared more clearly. First, considering the number of receptors bound to the donor, Q _E by FRET can be expressed by Equation 1.

(Formula 1)

That is, Q _E are donors for the fluorescence intensity (F _D) when the donor exist - means that the fluorescence intensity of the donor, the larger the Q _E as a reducing ratio of the receptor present upon the fluorescence intensity (F _DA) reduced by the acceptor. In addition, this value is the F ㆆ rster distance ( R ₀ , the distance at which the donor and the receptor will be close to 1/2 of the maximum Q _E when the donor and the receptor react in close contact with each other without peptide) and the number of receptors bound to the donor. ( n ) and the actual distance (r) between donor receptors spaced by the peptide. R ₀ can be calculated by the optical properties of the donor-receptor and can be expressed by Equation 2.

(Formula 2)

κ ² refers to the dipole-dipole position between donor receptors and has a value of about 2/3 in this example. n _D is the refractive index of the aqueous solution (refractive index) has a value of 1.4 in this embodiment. NA is Avogadro's number (Avogadro number), Q _D in the case of QD605 used in this example as the quantum efficiency of the donor (quantum yield) Q _D was about 0.55 (available measurements from Invitrogen). J (λ) is the value obtained by integrating the size of the region where the light emission spectrum of the donor overlaps with the absorption spectrum of the receptor. 4.53 ㅧ 10 ^-15 cm ³ M ^-1 for QD605-AuNP, 8.19 ㅧ 10 for QD605-Cy5 ^It was measured ^-15 cm ³ M ^-1 (result not shown). Therefore, R ₀ values of QD605-AuNP and QD605-Cy5 were calculated to be 5.8 nm and 6.4 nm, respectively.

The actual donor-receptor distance r is derivable from Equation 1 and can be calculated from Equation 3 from known measurements ( R _0, Q _E and n ).

(Formula 3)

As can be seen in FIG. 2, Q _E is 0.82 and n is about 40 for QD605-AuNP, and Q _E is 0.55 and n for about 40 for QD605-Cy5, so the actual donor-receptor measured by Equation 3 The distance r was about 8.3 nm for QD605-AuNP and about 11.4 nm for QD605-Cy5.

The radius of QD-SA is 7.5 ~ 10nm (provided by Invitrogen) and the theoretical maximum distance of peptide can be expected as 3.8nm (distance between amino acid and amino acid (3.8 Å) ㅧ amino acid number) The r _theory can be estimated as about 11.3 to 13.8 nm (QD-AuNP) and 10.9 to 13.4 nm (QD-Cy5). Therefore, when comparing the measured distance r by the FRET and the expected distance r _theory , it was found that the QD605-Cy5 was in good agreement, but the QD605-Au did not follow the general FRET equation because the r _theory and r value did not match. Did. In the case of QD605-Au, the r value was measured to be smaller than the r _theory value, and it was predicted that the luminous signal was suppressed by energy transfer more effectively than the mechanism by FRET. This is illustrated in Table 1.

These results indicate that AuNPs can effectively cause surface energy transfer (SET) at extended distances (up to 20 nm) than FRET, and have been reported to have excellent quenching ability (Yun, CS). et al. J. Am. Chem. Soc. 2005, 127, 3115). Therefore, from the above results, the distance from the donor core to the receptor used in the present invention is approximately 11-13 nm, whereas the QD-AuNP is a light-emitting signal even if the distance is more than 10 nm by the peptide than QD-Cy5. It can be seen that the quantum dot-gold nanoparticles can be more effectively used to measure the protease activity than the quantum dot-fluorescent material.

In the case of QD-Cy5 following the FRET mechanism, FRET does not occur at a distance exceeding 10 nm in general, but the emission signal of the donor is not reduced, but in the case of QD-Cy5, the separated distance ( r = 11.4 nm) is 2 there was a light emission signal (about 40) up to 55% can be suppressed, because a range within the large amount of R ₀ (R ₀ 2 ?? 12.8 nm), receptor (Cy -5 ^TM) bound to the donor (QD) . This simply does not happen when the donor and the receptor are combined only 1: 1, which is an advantage that only quantum dots can have.

Although not mentioned in detail in the present embodiment, when nanoparticles are used as energy acceptors, Q _E may be changed due to energy transfer phenomena depending on the diameter size. Quantum dots can be used by selecting an appropriate size in the range of 1-100 nm, but as the particle size is larger, the accessibility by the hydrolyzed protein may be limited and the recovery of the luminescent signal may be reduced.

As a result of confirming luminescence suppression of the quantum dot by the energy acceptor on the chip (FIG. 3), as in the aqueous solution, as the amount of Pep-AuNP which is the energy acceptor increased, the luminous ability of the donor QD-SA was effectively suppressed. However, unlike in the aqueous solution, even in the case of quantum dot: pep-AuNP = 1: 40, the emission signal of the quantum dot was not saturated, and the emission signal of the quantum dot was continuously decreased even at a concentration ratio of 1: 100, thereby suppressing the emission of the quantum dot by up to 90%.

The results indicate that, unlike in aqueous solutions, the greater the concentration of energy receptors reacting to the surface, the greater the amount of nonspecific binding on the surface, and this non-specific adsorption during the drying process results in energy between the donor and the acceptor. It is presumably because it greatly affects the transmission. Therefore, in order to prevent such nonspecific binding, it is not recommended to react the energy receptor at high concentration, and it is preferable to determine the concentration ratio of donor: energy receptor within the range of 1: 1 to 100 (mol / mol). Although no specific data was described in the Examples, when the ratio of the energy receptor to the donor was 1: 100, the recovery of the luminescence signal was not as high as about 32% due to nonspecific adsorption during the treatment of the hydrolyzed protein (results) Not shown). However, if the concentration ratio of the energy acceptor to the donor on the chip surface is maintained 1:30 ~ 1:50 (mol / mol), it is more preferable to measure the activity of the hydrolyzed protein is not a problem. However, the optimum concentration ratio may vary according to various conditions, in particular, the length of the substrate is preferably 1: 1-100.

Likewise, its effectiveness was confirmed in aqueous solution before assaying the hydrolytic activity of the protein substrate on the chip. For effective comparison, the luminescence signal recovery by MMP-2 without hydrolysis activity on the substrate was measured together with MMP-7 having hydrolysis activity on the substrate. MMP-7 specifically hydrolyzes the binding between alanine and leucine in the peptide sequence to restore the donor's luminescence signal.

As a result of calculating the PL recovery of the donor (quantum dot) emission signal according to Equation 4, MMP-7 was recovered to about 50% of the initial emission signal but did not increase any more. In contrast, the light emission signal of the quantum dots hardly increased by MMP-2.

(Formula 4)

I _DA : Luminescence signal size when donor and receptor are combined

I _D : Luminescence signal size after hydrolysis protein treatment when there is only a donor without acceptor

I _E : Luminescence signal size after hydrolysis protein treatment when donor and receptor are bound

100% luminescence recovery means that all peptide substrates bound to the donor by the hydrolytic protein are completely hydrolyzed, so that the recipient is not near the donor. Basically, all peptides bound to the donor are not expected to exhibit 100% efficiency because the hydrolyzed protein cannot fully access all peptides due to the steric hindrance with adjacent peptides. However, depending on the type and size of the hydrolyzed protein, the specificity of the substrate, the location of the specific site to be hydrolyzed, the distance between the donor and the receptor, the size of the donor, and the ability of the receptor to inhibit the luminous signal, the degree of recovery of the luminous signal of the quantum dot may be different. Therefore, it should always be judged by comparing the recovery degree of the light emission signal under the same measurement conditions.

Although not mentioned in the present embodiment, the luminescent signal of the quantum dot was not recovered at all under the condition of only the buffer solution without the protease enzyme (10 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ pH 7.4). The reaction time of the (MMP-7) was used as an appropriate time during the protease reaction because the degree of recovery of the luminescence signal was saturated when the reaction time was performed at 37 ° C. for 1 hour or more. In addition, the recovery rate of the luminescence signal increased according to the concentration of MMP-7, but was saturated at 100 nM of MMP-7 concentration and showed only about 6% PL recovery at the minimum concentration of 1 nM (result not shown). The above embodiment is limited only to the protease MMP-7, and may be differently applied depending on the composition and reaction time of the buffer used according to the type of hydrolyzed protein.

When measuring the activity of the hydrolyzed protein using a quantum dot-based biochip according to the present invention, The reaction between the substrate and the hydrolyzed protein on the chip is performed by dropping a solution of the protein to be measured onto a quantum dot-based biochip. The reaction is usually carried out by washing the surface with distilled water after the reaction for 30 minutes to 3 hours at 30 ~ 37 ℃, the reaction temperature or time is preferably controlled according to the characteristics of the hydrolyzed protein and the substrate, and the no. In particular, Quantum dot based biochip with protein solution In the case of dropping the reaction, it is preferable to cover the chip surface with a chamber made of a thin (1-5 mm) silicon film in order to prevent the protein solution from flowing. In the case of using a chamber having a plurality of wells, the hydrolysis activity of various proteins on a substrate is simultaneously measured on a quantum dot-based biochip by measuring the recovery rate of the luminescence signal of each well by reacting a different protein solution for each well. It is possible to do

As a result of measuring the hydrolytic activity of the protein using the quantum dot-based biochip according to the present invention, the luminescence signal of the quantum dot was recovered to about 60%, and silver staining As a result, it was confirmed that the gold nanoparticles attached to the quantum dots fell off in the sample where the luminescence signal was recovered, indicating that the quantum dots and the gold nanoparticles were separated by hydrolysis of the substrate. As a result, it was confirmed that the quantum dot-based biochip according to the present invention can effectively select the hydrolyzed protein for the substrate.

In addition, although not described in the embodiment, when the hydrolyzed protein is selected using the quantum dot-based biochip of the present invention, as the concentration of the same hydrolyzed protein increases, the emission recovery signal of the quantum dot generated by the decomposition of the substrate increases, so that hydrolysis is performed. It can be effectively used for quantitative analysis to check protein concentration. That is, a standard curve is prepared for the degree of recovery of the luminescence signal of the quantum dots according to the concentration of the hydrolyzed protein, and then the degree of recovery of the luminescence signal of the unknown protein is measured and compared with the standard curve. Concentration can be quantified.

In addition, in the case of using the quantum dot-based biochip of the present invention having a quantum dot monolayer composed of a plurality of quantum dot-substrate-energy receptor combinations and a substrate-energy receptor binding layer, the hydrolyzed protein is formed by reaction with a hydrolyzed protein. Since only the luminescent signal of the quantum dots bound to the active substrate is recovered and increased, it is possible to analyze the hydrolytic activity of the proteins for various substrates at once.

As another aspect of the present invention, (A) the Reacting the quantum dot-based biochip with the hydrolyzed protein; (B) reacting the quantum dot-based biochip with a mixture of a hydrolyzed protein and a hydrolysis inhibitor; (C) measuring luminescence of quantum dots after washing the quantum dot-based biochips of (A) and (B), respectively, in which the reaction is completed; And (D) measuring the change in luminescence of the quantum dots of the chips (A) and (B) measured in step (C) to confirm the inhibitory ability of the inhibitor against the hydrolyzed protein. Provided are methods for screening inhibitors for.

The screening method of the inhibitor for the hydrolyzed protein is that the hydrolysis does not occur when there is an inhibitor specific to the hydrolyzed protein, so that the light emission signal of the quantum dot is not recovered. The characteristics of the enzyme used in the screening method of the inhibitor of the hydrolyzed protein are the same as described in the above screening method of the hydrolyzed protein.

As an example of the inhibitor selection, for proteases that selectively hydrolyze peptides Inhibitors that inhibit the activity of hydrolyzed proteins may be screened as new drug candidates by mixing with various kinds of synthetic compounds, natural products, polypeptides, hormones, and the like, which are candidates for inhibitors.

In the present invention, the energy receptor bound to the substrate is limited to a method of selecting a hydrolyzed protein by treating a hydrolyzed protein or a mixture of a hydrolyzed protein and an inhibitor on a chip coupled with a quantum dot. It is also possible to select a hydrolyzed protein or inhibitor by first reacting with a hydrolyzed protein or a mixture of a hydrolyzed protein and an inhibitor before binding to the quantum dots, then attaching to the quantum dots and measuring the degree of emission of the quantum dots.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example 1 Preparation of Peptide-Gold Nanoparticles (pep-AuNP) with Biotin Attached

6 nmol of Monomaleimide Nanogold ^ㄾ (Nanoprobe), a 1.4 nm gold nanoparticle composed of monomolecular maleimide, was dissolved in 100 μL of dimethylsulfoxide (DMSO), followed by adding 900 μL of distilled water to vortex for 5 minutes. I shook it. Peptide CRPLALWRSK-biotin (Peptron) with cysteine and biotin attached at the N- and C-terminus, respectively, at a concentration of 50 nmol / mL, which is 5-10 times (30-60 nmol) of gold nanoparticle concentration in 1 mL of distilled water. After dissolving, the mixture was mixed with 6 nmo / 1mL of the solution in which the gold nanoparticles were dissolved, and left at 4 ° C. for 16-18 hours. When the reaction was accompanied by stirring, the dissolved oxygen in the reaction solution was increased so that it could be inhibited in the maleimide reaction. Since maleimide can specifically covalently bind to the thiol group of the cysteine located at the N-terminal of the peptide, it is possible to bind gold nanoparticles 1: 1 with the peptide.

To remove peptides that did not participate in the binding, centrifuge at 10,000 g for 30 to 40 minutes using microcentricon (microcentricon YM-10, 10 kDa cut-off), and add about 500 μL of distilled water to the same centrifugation conditions. Washed 2-3 times in.

Since the gold nanoparticles are about 15kDa and the peptide is approximately 1.5kDa, only micropeptides can be used to easily filter the peptides. After filtration, only peptide-bound gold nanoparticles were present on the microcentricon surface. Gold nanoparticles AuNP-CRPLALWRSK-biotin (pep-AuNP) incorporating the peptide remaining on the surface of the microcentric silicon was diluted in distilled water or a buffer solution to be used in the next experiment. The concentration of pep-AuNP in the diluted solution was measured using the extinction coefficient of gold nanoparticles. The absorption coefficient of gold nanoparticles at 420 nm is 1.1 ㅧ 10 ⁵ M ^-1 cm ^-1, so that the absorbance of the diluted solution is measured by UV-spectrum (UV-2550, Shimadzu), and then the absorbance is divided by the absorption coefficient. The concentration was calculated. Since gold nanoparticles and peptides were bound 1: 1, the concentration of gold nanoparticles was the same as that of pep-AuNP.

Example 2 Measurement of Luminescence of Donor According to Receptor Concentration in Solution

In order to compare the quantum dot quenching phenomenon due to the energy transfer of the gold nanoparticles or the fluorescent material with the quantum dots, the quantum dot-gold nanoparticles and the quantum dot-fluorescein (dye) The luminescence intensity was compared and analyzed according to the concentration ratio of gold nanoparticles or fluorescent material.

The QD605-SA (Invitrogen) having a total diameter of 15-20 nm and 5-10 streptavidin (SA) per surface of quantum dots was used as an energy donor. QD605-SA emits light at 605 nm.

As the energy acceptor, the gold nanoparticles are pep-AuNP prepared in Example 1, and the fluorescent material is Cy5-RPLALWRSK-biotin (AnyGen) to which Cy ^TM- 5 is attached instead of the N-terminal cysteine in the peptide of Example 1 ( pep-Cy5) was used. The concentration of Pep-Cy5 was quantified using an extinction coefficient of 2.5 ^TM 10 ⁵ M ^-1 cm ^-1 of Cy ^TM -5 at 649 nm.

The energy donor solution was prepared by diluting QD605-SA to a concentration of 20 nM with distilled water to make 1 mL. Pep-AuNP or Pep-Cy5 as an energy acceptor was diluted in distilled water so as to have concentrations of 20, 50, 100, 200, 400, 600, 800, and 1000 nM, respectively, and 50 μL each was prepared. After mixing the same volume of the QD605-SA solution in 50μL of the energy receptor solution of each concentration and reacted for 30 minutes at room temperature. 100 μL of the final reaction solution in the 96-well plate was divided into each well, and the fluorescence intensity of QD605-SA was measured through a fluorescence plate reader (plate reader M-200, TECAN, Austria GmBH), and is shown in FIG. 2. In FIG. 2, QD represents QD605-SA, Au represents Pep-AuNP, and Cy5 represents Pep-Cy5.

As can be seen in FIG. 2, as the concentration ratio of the receptor to the donor QD605-SA increases, the fluorescence intensity of the donor decreases, in particular, Pep-AuNP (FIG. 2 a)) is higher than Pep-Cy5 (FIG. 2 b)). The light emission signal of the donor was effectively suppressed. In the case of Pep-AuNP, the emission signal was saturated at a concentration ratio (mol / mol) of 40 times higher than that of the donor, and although not illustrated in the drawing, the signal strength of the donor was higher even at 100 Au / QD where the concentration of Pep-AuNP was higher. It did not decrease over. Comparing the quenching efficiency ( Q _E ) of the luminescence signal through three repeated experiments, Pep-AuNP was up to 82.1% (average) when the donor and acceptor were present in a concentration ratio of 1:40. Error 7.3%), Pep-Cy5 showed up to 55.5% (mean error 4.9%) Q _E (FIG. 2 c)).

Example 3 Measurement of Luminescence of Donor According to Receptor Concentration on Chip Surface

1) Fixation of quantum dots as energy donors on chip surface

QD605-SA used in Example 2 was diluted to a final 10 nM in 2% BSA-added buffer (50 mM borate buffer, pH 8.5), and then 20 μL was added to a 96-well plate to remove bubbles. Centrifugation was performed for 1 minute at 500 rpm.

As a substrate for attaching the quantum dots, a glass substrate treated with NHS-hydrogel (N-hydroxyl succinimide hydrogel) (Nexterion ^™ Slide H, Schott, Germany) was used. The QD605-SA solution was spotted at a designated location on the substrate using an automated microarray (Robotic Microarray System, Microsys, Cartesian Techonologies, USA) and a spotting pin (CMP3 spotting pin, Telechem International, Sunnyvale, USA). The amount of QD605-SA solution corresponding to one spot was about 1nL, and the size of one spot was about 150 ~ 200μm in diameter, and a total of four spots (2x2) were spotted at 4 mm intervals. After leaving the substrate for about 1 hour in a chamber maintained at room temperature and 70% humidity, a buffer solution containing 2% BSA (50 mM borate buffer, pH) to block NHS groups remaining in or around the spot. 8.5) was pipetted onto each spot consisting of 2 × 2 onto each of the spots with a pipette of about 3 μL and then left at room temperature for 1 hour while storing the glass substrate to prevent drying. Thereafter, the glass substrate was washed three times with distilled water and then slowly dried with nitrogen gas.

2) Combination of Energy Receptor and Quantum Dots on Chip

Pep-AuNP or Pep-Cy5 prepared in Example 1 was bound as an energy acceptor to the quantum dots attached to the surface of the chip prepared in 1). In other words, Pep-AuNP or Pep-Cy5 aqueous solution was prepared in the spot area of QD605-SA composed of 2 ㅧ 2 on the substrate by the method described in Examples 1 and 2, and each 3 μL was dropped by pipette and sealed to prevent drying. In a state left for 30 minutes at room temperature. In this way, Pep-AuNP according to the concentration was reacted by dropping the same on the QD605-SA spot for each concentration of 100, 200, 300, 400, 1000 nM. Thereafter, the reaction solution was removed, the substrate was washed three times with distilled water, and then slowly dried with nitrogen gas. Pep-Cy5 was also combined with quantum dots attached to the chip surface in the same manner as Pep-AuNP.

3) Measurement of emission intensity of donor on chip

The energy intensity of the quantum dots combined with Pep-AuNP or Pep-Cy5 attached on the chip prepared in step 2) is based on a spot (QD control) in the absence of an energy acceptor. QD: acceptor = 1:10, 1:20, 1:30, 1:40, 1: 100, mol / mol) as measured by the emission image of the donor appeared. Luminescent image is then analyzed using a fluorescent scanning slides chip scanner ^{(ArrayWoRx e, Applied Precision, USA} ) , which was shown in Figure 3 to that picture a). At this time, the fluorescence filter was measured using a combination of absorbance 460nm / emission 605nm (Chroma).

The fluorescence image obtained by scanning is median and standard deviation of signal strength for four (2 ㅧ 2) spots reacted under the same conditions using array analysis software (GenePix Pro 4.0, Axon). were measured, each measured value was calculated by the formula _E Q 2 using the Excel (Microsoft excel 2003) (b in FIG. 3)).

In the same manner as in Example 1, it was observed that Pep-AuNP inhibited luminescence of quantum dots more effectively than Pep-Cy5, but unlike aqueous solution, luminescence of quantum dots was suppressed up to 90% as the concentration of the receptor increased. That is, when gold nanoparticles are used as the acceptor, the decrease in the donor's emission signal is not saturated even at a concentration ratio of 1:40 (donor: receptor, mol / mol), and the emission signal of the donor continues up to 1: 100. Decreased.

From the experimental results, it was possible to effectively analyze the energy transfer by the quantum dots and the receptor on the chip surface, and it was confirmed that the use of gold nanoparticles as the receptor can effectively suppress the emission of quantum dots than the fluorescent material.

Example 4 Determination of Hydrolytic Activity of Proteins in Solution

1) Measurement of luminescence of quantum dots

In this example, it was tested whether the activity of the hydrolyzed protein could be effectively measured in the solution using only Pep-AuNPs having confirmed excellent luminescence inhibitory ability as a receptor. In addition, in order to simultaneously check whether Pep-AuNP has the same effect on different quantum dots, the emission intensity was measured by using QD525-SA (Invitrogen) having the property of emitting light at 525 nm with QD605-SA as an energy donor. 4 is shown.

Quantum dots QD525-SA and QD605-SA were dissolved in a buffer solution (10 mM HEPES buffer, pH 7.4) at a concentration of 20 nM, and 50 μL of each was injected into four eppendorf tubes for each quantum dot. (1) To each tube of QD525-SA and QD605-SA, add 50 μL of Pep-AuNP at 1 μM concentration diluted with buffer solution (10 mM HEPES buffer, pH 7.4), and mix for 30 minutes at room temperature. It was left to react. Thereafter, in order to induce the reaction of the hydrolyzed protein, the matrix metalloprotease-7 (MMP-7, Calbiochem) was dissolved in a buffer solution (10 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ pH 7.4), and 0.5 μL was added to each tube so that the final concentration of MMP-7 was 100 nM, followed by reaction at 37 ° C. for 1 hour, and the emission of quantum dots was measured (+ MMP-7). (2) In another tube of QD525-SA and QD605-SA, add 50 μL of Pep-AuNP at 1 μM concentration diluted with buffer solution (10 mM HEPES buffer, pH 7.4) and mix for 30 minutes at room temperature. The same was left. Thereafter, MMP-7-free buffer solution (10 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ pH 7.4) was added to each of the tubes by 0.5 μL and reacted equally at 37 ° C. for 1 hour and inhibited by Pep-AuNP. It was used to measure the light emission of the quantum dots (QD-AuNP). (3) Substrate-receptor-free control, in which each tube of QD525-SA and QD605-SA was further diluted with 50 μL of the buffer solution, and then added with MMP-7 (10 mM HEPES, 150). mM NaCl, 5 mM CaCl ₂ pH 7.4) were added to each tube at 0.5 μL (final MMP-7 concentration of 100 nM) and reacted at 37 ° C. for 1 hour. (QD only). (4) A sample was prepared in the same manner as in (1) except that MMP-2 (Sigma) was used instead of MMP-7, and the emission of quantum dots was measured (+ MMP-2).

As shown in FIG. 4, both QD525-SA and QD605-SA were effectively quenched by Pep-AuNP and specifically recovered by the MMP-7 proteases. From this, the specific reaction of the hydrolyzed protein was confirmed that the peptide connected between the quantum dot and the gold nanoparticles is hydrolyzed to recover the light emission signal of the quantum dot.

2) Assay by field emission transmission electron microscope

In order to more specifically prove the hydrolysis by the protein of 1), the protease using a field emission transmission electron microscope (FE-TEM, model name: Tecnai F30 S-Twin, FEI, Netherlands) Forms of quantum dots and gold nanoparticles before and after the reaction are shown in FIG. 5. Samples containing 1.4 nm gold nanoparticles were measured after adsorption on an ultrafine grid supported by holey carbon (Ted Pella). QD605-SA and Pep-AuNP conjugated samples treated with protease MMP-7 were filtered through a microcentricon (microcentricon YM-50, 50 kDa cut-off) at 10,000 g for 15 minutes to discard the cutout and discard the quantum dots. Only the supernatant containing the adsorbed on the grid was measured.

As can be seen in Figures a) and b) and enlarged c) and d), the gold nanoparticles bound to the surface of the quantum dots were significantly reduced after treatment with the hydrolyzed protein. From this, the peptide was specifically hydrolyzed (cleavage) by the activity of the hydrolyzed protein to break the bond between the quantum dot and the gold nanoparticles, it can be seen that the light emission signal of the quantum dot is restored.

Example 5 Determination of Hydrolytic Activity and Inhibitory Effect of Proteins on Biochip Surfaces

1) Recovery of luminescence signal by substrate specificity

The activity of the hydrolyzed protein was measured using energy transfer of quantum dots and receptors on the chip surface identified in Example 3.

In the same manner as in Example 3, (1) a chip having QD605-SA attached thereto and (2) a chip having Pep-AuNP bonded at a concentration ratio (mol / mol) of 1:50 to QD605-SA attached to the chip surface; (3) The emission image at 605 nm of the chip treated with MMP-7 on the chip of (2) was measured and shown in Fig. 6A). Meanwhile, MMP-7 specifically cleaves between alanine, A) and leucine (Leucine, L) in the sequence of peptides by hydrolysis, thus preparing Pep _RGD- AuNP for comparison with nonspecific peptide substrates. After preparing three chips identical to Pep-AuNP, the emission images were measured and shown in b) of FIG. 6 to test the specificity of the hydrolyzed protein. Pep _RGD- AuNP was prepared in the same manner as in Example 1 except that CALNNRGDK-biotin (Pep _RGD , Peptron, Co. Ltd.) was used as the peptide. From the fluorescence image for each spot was measured in the average signal intensity and standard deviation using array analysis software (GenePix Pro 4.0, Axon) is shown in Figure 6 c).

A specific method of treating MMP-7 on a chip in which quantum dots and gold nanoparticles are combined is as follows. MMP-7 (Calbiochem) was dissolved at a concentration of 100 nM immediately before reacting with a buffer solution (10 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ pH 7.4), and then dropped on each spot and reacted . To prevent the MMP-7 solution from flowing, place a multiwell silicon chamber (1 mm high by 3 mm in diameter, Simga) spaced so that 2 x 2 spots enter a single well on a glass substrate with quantum dots and gold nanoparticles. After cover, 10 μL of MMP-7 solution was dropped per spot. Thereafter, the glass substrate was placed in a Petri dish and reacted at 37 ° C. for 1 hour while being sealed.

As shown in FIG. 6, Pep-AuNP effectively inhibited the luminescence signal after binding to the quantum dot (a2) and after the treatment of the hydrolase MMP-7, the luminescence signal of the quantum dot was recovered to ~ 60%. (a3). On the other hand, Pep _RGD -AuNP effectively suppressed the luminescence signal after combining with the quantum dot (b2), but the signal recovery was relatively weak after the protease treatment (b3).

In order to prove this in more detail, silver staining was performed to confirm whether gold nanoparticles were actually cleaved and lost by the protease reaction. The dyeing method is a method of identifying the presence of gold nanoparticles because the color is colored in proportion to the amount of gold nanoparticles. The silver dye was reacted by dropping silver salt solution and initiator solution into 1: 1 (v / v) and dropping 3 μL onto the spotting part of the chip surface. Immediately after the reaction for 1-2 minutes, the resultant was washed with distilled water and observed with an optical microscope. The photograph was shown beside each fluorescent image of FIG. 6. As shown in the optical micrograph of FIG. 6, it was confirmed that the dyes were darkly stained at the portion where Pep-AuNP or Pep _RGD -AuNP was coupled (a2, b2), and only gold nanoparticles were treated by PMP-AuNP by MMP-7 treatment. It can be seen that the decrease of dyeing is reduced by the decrease of (a3, b3). From the above results, it can be seen that MMP-7 reacts specifically with the substrate and contributes to signal recovery of quantum dots.

2) Measurement of protease activity according to the size of quantum dots

In order to test whether gold nanoparticles can be effectively used for quantum dots of various sizes on the chip surface, the proteases activity was also measured in QD525-SA together with QD605-SA in the same manner as 1) and shown in FIG. 7. As can be seen in FIG. 7, not only the QD605-SA but also the QD525-SA fixed to the chip surface effectively recovered the light emission signal by the MMP-7. NHDDPC (N-Hydroxy-1,3-di- (4-methoxybenzenefulfonyl) -5,5-dimethyl- [1,3] -piperazine-2-carboxamide, MMP Inhibior II, Calbiochem) Diluted with MMP-7 in a buffer solution (10 mM HEPES, pH 7.4) at a concentration of about 100 nM, the quantum dots did not recover due to the disruption of peptides in both types of quantum dots (a4). And b4).

By the experiment, the activity of the hydrolyzed protein can be measured using the energy transfer between the various quantum dots and gold nanoparticles on the chip surface, it can be seen that it is very effective in identifying the inhibitor.

As described above, the quantum dot based According to the biochip, it is possible to perform ultra-fast mass analysis of the activity of hydrolyzed proteins using energy transfer of quantum dots on the chip surface.

In addition, the use of gold nanoparticles as energy acceptors enables measurement by energy transfer between donor and acceptor at distances of more than 10 nm, and can be applied to quantum dots of different sizes, allowing simultaneous analysis of various hydrolyzed proteins or various substrates. It is possible. In particular, it is expected that the present invention can be effectively used not only for the purpose of mass diagnosis of hydrolyzed proteins known as disease-related markers in blood, but also for ultra-fast screening of related inhibitors.

<110> Korea Advanced Institute of Science and Technology <120> Quantim dot-based biochip and method for assying hydrolytic          protein using the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Substrate for hydrolytic protein <400> 1 Cys Arg Pro Leu Ala Leu Trp Arg Ser Lys   1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Substrate for hydrolytic protein <400> 2 Arg Pro Leu Ala Leu Trp Arg Ser Lys   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Substrate for hydrolytic protein <400> 3 Cys Ala Leu Asn Asn Arg Gly Asp Lys   1 5  

Claims (16)

(A) a support made of glass or silica; (B) a self-assembled monolayer film formed on the surface of the support; (C) a quantum dot monolayer film formed on the self-assembled monolayer membrane by the surface of the quantum dot is coated with a biomolecule that specifically binds to biotin, by combining the terminal of the self-assembled monolayer film and the biomolecule functional group on the surface of the quantum dot; And (D) Biotin is bonded at one end of the substrate to the hydrolyzed protein and energy receptor is bound at the other end, and the quantum dot monolayer is formed by specific binding of the biotin at the end of the substrate to the biomolecules exposed on the quantum dot monolayer. A binding layer of substrate-energy receptor formed on the membrane; Quantum dot-based biochip comprising a. The method of claim 1, The end group of the self-assembled monolayer film is any one or two or more functional groups selected from the group consisting of aldehyde (aldehyde), epoxy (epoxy), NHS (N-hydroxyl succinimide) and biotin (biotin) . The method of claim 1, The biomolecule is a quantum dot-based biochip, characterized in that one or more biomolecules selected from the group consisting of streptavidin, avidin, avidin, neutravidin and captavidin. The method of claim 1, The quantum dot is a quantum dot-based biochip, characterized in that the maximum emission wavelength region of 525nm ~ 800nm. The method of claim 1, The space between the quantum dots in the monolayer film of the quantum dots is ethylene glycol amine, ethylene glycol thiol, NHS (N-hydroxysuccinimide)-ethylene glycol, maleimide-ethylene glycol, polyethylene glycol amine, polyethylene glycol thiol, (NHS)-polyethylene glycol, A quantum dot-based biochip characterized by being filled with one or more compounds selected from the group consisting of maleimide-polyethylene glycol, a polymer based on carbohydrate monomer binding, and a protein disulfide isomerase and bovine serum albumin. The method of claim 1, The energy acceptor is a quantum dot-based biochip, characterized in that the metal nanoparticles, fluorescent (fluorescent dye) or fluorescent quencher (fluorescent quencher). The method of claim 6, The metal nanoparticles are quantum dot-based biochip, characterized in that the gold nanoparticles, silver nanoparticles or platinum nanoparticles of 1 ~ 100nm size. The method of claim 1, The substrate is a quantum dot-based biochip, characterized in that one or more substrates selected from the group consisting of peptides, DNA, RNA, PNA, aptamers, sugars and lipids. The method of claim 8, When the energy acceptor is a metal nanoparticle, the length of the substrate is 1 to 15 nm, If the energy receptor is a fluorescent material, the length of the substrate is a quantum dot-based biochip, characterized in that 1 ~ 5nm. The method of claim 1, The quantum dot monolayer consists of a mixture of 2 to 5 kinds of quantum dots having different maximum emission wavelengths, The substrate-energy receptor binding layer is a quantum dot-based biochip, characterized in that made of a mixture of substrate-energy receptors specifically bound to the various quantum dots constituting the quantum dot monolayer. (A) reacting the quantum dot-based biochip according to claim 1 with a hydrolyzed protein to measure activity; (B) measuring the luminescence of the quantum dots after washing the quantum dot-based biochip is completed; And (C) confirming the hydrolytic activity of the protein substrate by comparing the luminescence of the quantum dots of the quantum dot-based biochip not reacted with the hydrolyzed protein and the change in the luminescence of the quantum dots measured in step (B); Method for measuring the activity of a hydrolyzed protein on a substrate comprising a. The method of claim 10, In the quantum dot-based biochip, the concentration ratio of the quantum dot and the energy acceptor is 1: 1 to 1: 100 (mol / mol). Method for measuring the activity of a hydrolyzed protein on a substrate, characterized in that. The method of claim 10, The hydrolyzed protein may be protease, peptidase, nucleosidase, deoxynuclease, DNase, phosphatase, glucosidase, galacto A method for measuring the activity of a hydrolyzed protein against a substrate, characterized in that one or more hydrolyzed proteins selected from the group consisting of galactosidase, lipase and esterase. (A) reacting the quantum dot-based biochip and the hydrolyzed protein according to claim 1; (B) measuring the luminescence of the quantum dots after washing the quantum dot-based biochip is completed; And (C) quantifying the protein concentration by comparing the degree of luminescence of the quantum dots measured in step (B) with a standard curve according to the hydrolyzed protein concentration; Method for quantifying the concentration of hydrolyzed protein for a substrate comprising a. (A) reacting a mixture of the quantum dot-based biochip according to claim 10 and the hydrolyzed protein to measure the activity; (B) measuring the luminescence of each quantum dot after washing the quantum dot-based biochip is completed; And (C) confirming the hydrolysis activity of each substrate of the protein by measuring the change in the emission of each quantum dot of the quantum dot-based biochip not reacted with the hydrolyzed protein and the emission of each quantum dot measured in step (B) ; Method for measuring the activity of a hydrolyzed protein on a substrate comprising a. (A) reacting the quantum dot-based biochip according to claim 1 or 11 with the hydrolyzed protein; (B) reacting the quantum dot-based biochip according to claim 1 or 11 with a mixture of hydrolyzed protein and hydrolysis inhibitor; (C) measuring luminescence of quantum dots after washing the quantum dot-based biochips of (A) and (B), respectively, in which the reaction is completed; And (D) measuring the change in luminescence of the quantum dots of the chips (A) and (B) measured in step (C) to confirm the inhibitory ability of the inhibitor against the hydrolyzed protein; Method for screening an inhibitor for a hydrolyzed protein comprising a.

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