KR20120127129A - Hydrophilic/hydrophobic patterned biochips for on chip MALDI-TOF MS - Google Patents

Abstract

PURPOSE: A patterned biochip of hydrophilicity/hydrophobitiy and a method for manufacturing the same are provided to enable mass spectrometry of target proteins with high reliability and to be used in SPR imaging analysis. CONSTITUTION: A method for preparing a patterned biochip of hydrophilicity/hydrophobicity comprises: a step of forming a metal grid pattern on a substrate; a step of forming a self-assembled monolayer containing thiol on the metal surface; and a step of attaching hydrophobic molecules on the substrate. The metal is Ni, Cu, Zn, Au, or alloy thereof. The self-assembled monolayer is formed by adding thiol-mixed polyethylene glycol(PEG). The thiolp-mixed PEG carboxyl-terminated hexa (ethylene glycol) undecane thiol, hydroxyl group-terminated tri(ethylene glycol)undecane thiol, or a mixture thereof. The hydrophobic molecule is perfluoroalkyl silane.

Description

Hydrophilic / hydrophobic patterned biochips for on chip MALDI-TOF MS}

The present invention relates to a hydrophilic / hydrophobic patterned biochip, a method for manufacturing the same, and a method for mass spectrometry / detection using the same.

In general, a mass spectrometer is an analyzer that measures the mass of a compound, and is designed to determine the molecular weight of the compound by measuring mass-to-charge (m / z) after charging and ionizing the analyte. . As the ionization method of the compound, an electron ionization method using an electron beam, a method of colliding atoms at high speed, a method using a laser, a method of spraying a sample into an electric field, and the like are known.

On the other hand, as a method for mass spectrometry of biochemical substances having large molecular weight such as proteins and nucleic acids, MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectroscopy) using laser, Mass spectrometer using this has been developed and widely commercialized and used. Mass spectrometry using MALDI-TOF is performed as follows. A small amount of matrix solution is added to the metal plate to dry, and the sample solution to be analyzed is dropped and dried. When the laser is irradiated where the matrix and the crystallized sample are located, the sample is desorbed / ionized with the help of the matrix. Usually an electric field is placed between the metal plate on which the sample is located and the mass spectrometer, so that the ionized sample is moved towards the sensor by the potential difference of the electric field. In this case, when the amount of charge of the sample is known, the mass of the sample may be analyzed using a variable such as the time of arrival at the sensor. As in the principle of mass spectrometry, when analyzing a synthetic polymer sample by MALDI mass spectrometry, an organic acid matrix is mixed with the analytical sample in order to assist with desorption and ionization by a laser. If the organic acid matrix is not used together, decomposition of the polymer sample occurs because the polymer sample absorbs the laser directly. As such, various organic acid matrices are used in MALDI-TOF mass spectrometry to prevent the sample itself from degrading. In addition, MALDI-TOF is used in a wide range of areas because of its ease of use and rapid analysis of many samples, and analysis using a mass spectrometry technique such as MALDI-TOF after enrichment of a target protein with an antibody. The method provides a variety of information about the target protein that is not available in conventional immunoassay, showing potential for new diagnostic techniques. However, until now, there is a lack of research on MALDI mass spectrometry biochip itself that can detect only desired protein from a sample mixed with multiple proteins and accurately mass spectrometry, and when several proteins are mixed such as human serum, Interference of many background protein-related peaks reduces the accuracy of the sample. In particular, in on-chip analysis, in which a step of capturing a target protein using an antibody and mass spectrometry is performed on one chip, it is considered to be a more reliable protein mass spectrometry by guaranteeing a simplified analysis method. Development of biochips capable of performing mass spectrometry on a large number of proteins using chips has been insufficient.

On the other hand, Surface Plasmon Resonance (hereinafter referred to as 'SPR') sensor technology is a biosensor and biochip measuring method using a phenomenon that changes the signal when a biomaterial such as protein is bound to the sensor surface. (Nice, EC and Catimel, B., BioEssays, 1999, 21, 339-352). Surface plasmons are quantized vibrations of free electrons that propagate along a conductor surface, such as a metal surface. Such surface plasmons are excited by incident light incident on the metal thin film at an angle above a critical angle of the dielectric medium through a dielectric medium such as a prism and cause resonance at a constant angle. The angle of incidence, ie the resonance angle, at which this SPR occurs is sensitive to the change in refractive index of the material in proximity to the metal thin film. SPR sensors use these properties to measure the thickness of a sample that is close to a metal thin film, that is, a quantitative analysis of a sample, qualitative analysis, and a thin film from a change in refractive index of a sample. The biggest advantage of SPR biosensor is that it is a label-free sensor and it has been applied to various biotech related research fields including proteomics, genetic research, medical diagnosis and new drug development, food processing and environmental science. It is usefully used up to (Ince and Narayanaswamy, Anal. Chim. Acta, 569: 1-20, 2006; Lee, et al., Langmuir, 22: 5241-5250, 2006).

Accordingly, the present inventors fabricated a biochip patterned with hydrophilicity / hydrophobicity, and applied a sample in which multiple proteins were mixed to perform on-chip immuno-MALDI mass spectrometry. By confirming that the analysis can be performed, the present invention has been completed.

The object of the present invention can be applied not only to MALDI-TOF, but also to SPR imaging system, and a hydrophilic / hydrophobic patterned biochip capable of reliable mass spectrometry of various target proteins from a mixture of multiple proteins and a method of manufacturing the same. To provide.

Still another object of the present invention is to provide a method for detecting and analyzing a target substance using the biochip.

The present invention to solve the above problems is (1) forming a metal lattice pattern on the substrate; (2) forming a self-assembled monomolecular layer mixed with thiol on the metal surface; And (3) attaching hydrophobic molecules to the substrate.

As used herein, the term "biochip" refers to a high density of biomaterials such as DNA, proteins, cells, etc. attached to a predetermined position on various kinds of solid surfaces including polymer substrates such as surface-modified glass, silicon, polypropylene, etc. .

The substrate may be selected from the group consisting of glass, silicon, polypropylene, nylon, plastic and metal, but is not limited thereto. Preferably it is a glass substrate.

In the above production method, the metal may be selected from the group consisting of Ni, Cu, Zn, Au and alloys thereof, but is not limited thereto. Preferably the metal is Au.

In the present invention, a metal thin film may be formed by depositing the metal, in which the deposition is performed by an electron beam evaporator, a chemical vapor deposition (CVD), a physical vapor deposition (PVD), a combined droplet chemical vapor deposition machine, a simple vapor deposition machine, or the like. It may be carried out by, but any deposition method commonly used in the art can be used without limitation. One of the features of the biochip of the present invention is that gold is deposited in a lattice pattern. In one embodiment of the present invention, a shadow mask was used to pattern the metal on the glass substrate, and 5 x 4 metal spots per 1 mm diameter were included (Example 1).

In the manufacturing method of the present invention, step (2) is a step of forming a self-assembled monolayer by adding polyethylene glycol (PEG) mixed with a thiol.

In the present invention, the term "self-assembly monolayer" when the substrate is placed in a molecular solution, the molecule forms a self-aligned arrangement on the surface of the substrate by the interaction between the molecules and the interaction between the molecules A thin film composed of one molecular layer.

Through step (2), in the biochip patterned with gold, a thiol-PEG linker may be attached onto the gold thin film. The linker is not limited in kind as long as it is a hydrophilic molecule, but is preferably hexa (ethylene glycol) undecane thiol having a carboxyl group at the terminal, and a hydroxyl group at the terminal. Tri (ethylene glycol) undecane thiol or mixtures thereof. In one embodiment of the present invention, the ethanol solution mixed with 0.1 mM HS-PEG-COOH and 0.9 mM HS-PEG-OH is reacted for 16 hours, the self-assembled monolayer (self) -assembled monolayer (SAM) was allowed to form (Example 2).

In the manufacturing method of the present invention, step (3) attaches hydrophobic molecules to the surface of the substrate other than the gold thin film, thereby adding hydrophobicity around the gold thin film to which the hydrophilic molecules are bound. The kind of the hydrophobic molecule is not limited, but a silane material including the hydrophobic molecule is preferable, and more preferably, a perfluoroalkylsilane. In one embodiment of the present invention, 2% perfluoroalkylsilane (FM1010) was reacted with a chip to add hydrophobicity to the substrate surface (Example 2).

In addition, the present invention comprises the steps of (1) forming a metal grid pattern on the substrate; (2) attaching hydrophobic molecules to the substrate; And (3) forming a protein G layer on the metal surface by adding cysteine-tagged protein G to form a hydrophilic / hydrophobic patterned biochip. to provide.

The substrate may be selected from the group consisting of glass, silicon, polypropylene, nylon, plastic and metal, but is not limited thereto. Preferably it is a glass substrate.

In the above production method, the metal may be selected from the group consisting of Ni, Cu, Zn, Au and alloys thereof, but is not limited thereto. Preferably the metal is Au.

The biochip consists of forming a metal lattice pattern on a substrate, first attaching hydrophobic molecules to the substrate, and then attaching cysteine tagged protein G on the metal surface.

Cysteine tagged protein G, well known as antibody binding protein, forms a gold thin film surface protein layer and can bind well with antibodies.

The biochip manufacturing method of the present invention enables all reactions of on-chip immuno-MALDI assays of various stages, such as antibody immobilization, antigen reaction, on chip proteolysis and MALDI matrix treatment, to be carried out at a constant condition, and thus from a mixture of proteins. Biochips capable of SPRi analysis can be prepared for reliable mass spectrometry of various target proteins and for quantitative analysis of antibody-antigen responses.

In addition, the present invention is characterized in that in the lattice patterned biochip with gold, a hydrophilic molecule or cysteine tagged protein G is bonded to the gold surface, and hydrophobic molecules are bonded to the substrate other than the gold surface. A biochip patterned hydrophilic / hydrophobic.

The biochip of the present invention can be manufactured by the above-described manufacturing method. In the biochip of the present invention, hydrophilic molecules are bonded to a gold thin film, and hydrophobic molecules are bonded to a surface of a substrate around the gold thin film, so that antibodies may be specifically fixed to hydrophilic molecules of the gold thin film. Target proteins can be easily detected. In addition, the biochip of the present invention can be used for MALDI-TOF mass spectrometry. The biochip of the present invention enables all reactions of on-chip immuno-MALDI assays of various stages, such as on chip proteolysis and MALDI matrix treatment, to be performed under certain conditions, thereby allowing masses of only proteins of interest in a mixture of multiple proteins. There is an advantage that can be measured accurately. In particular, in one embodiment of the present invention it was confirmed that the detection and mass spectrometry of the target protein in the serum of a human is mixed with multiple proteins. In addition, the biochip can be directly used as a substrate of the SPR imaging system prior to mass spectrometry, thereby enabling quantitative analysis of antibody-antigen reactions. Combining the MALDI-TOF-MS and SPR imaging systems enables quantitation of antibody-antigen binding without the use of internal standards prior to MS analysis. In an embodiment of the present invention, after the SPR imaging analysis using the hydrophilic / hydrophobic patterned biochip of the present invention, MALDI-TOF MS analysis can be performed by directly applying a MALDI matrix to a spot.

In addition, the present invention comprises the steps of: (1) immobilizing the antibody of the target material to the protein G tagged with the hydrophilic molecule or cysteine of the biochip patterned hydrophilic / hydrophobic; And (2) reacting a sample containing a target material to the biochip, the method of detecting a target material using the biochip or a mass spectrometry method.

As used herein, the term "target substance" is a substance which is intended to detect whether it is present in a sample using a biochip, and the type of target substance is not limited as a substance commonly used in the art, but preferably PNA. (peptide nucleic acid), polypeptide, protein. Examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

As used herein, the term "sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine containing the target substance to be detected.

In the detection method, using the biochip of step (2), SPR imaging analysis, MALDI-TOF (Matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry, or MALDI-TOF after SPR imaging analysis The method may further comprise performing mass spectrometry.

In one embodiment of the present invention, after immobilizing various types of antibodies to protein G tagged with hydrophilic molecules or cysteines of a biochip, antigens specific for the antibodies were reacted with the antibodies. In addition, SPR imaging analysis or MALDI-TOF-MS analysis was performed using the biochip. As a result, target antigens were successfully detected and accurate mass information was obtained. In addition, in the patterned gold biochip system of the present invention, the application of antigen-bound, nitrogen-dried chips directly to the SPR imaging system showed that antibody fixation and capture of specific antigens were accurately represented in the SPR imaging data. . After SPR imaging experiments, the analyzed biochips were separated in an SPR imaging analyzer. Then, MALDI-MS analysis was performed by applying the MALDI matrix to the antigen including the gold spot, so that SPR imaging analysis and MALDI-MS could be performed with one biochip. Therefore, the biochip of the present invention has a certain reaction condition in on chip MALDI-TOF MS analysis due to its hydrophilicity / hydrophobic nature, so that accurate mass analysis is possible, and various biomaterials can be immobilized using gold thin films.

As described above, when performing on chip immuno-MALDI-TOF mass spectrometry using the hydrophilic / hydrophobic patterned biochip of the present invention, only a desired protein is accurately detected and analyzed even in a mixture containing a plurality of proteins. can do. In addition, the hydrophilicity / hydrophobicity of the surface of the biochip was maintained during the on-chip immuno-MALDI-MS, which was composed of several complex steps, allowing each step of the on-chip immuno-MALDI-MS to be carried out under constant conditions. In addition, it is possible to simplify the accurate mass spectrometry of various target proteins from a mixture of multiple proteins, and can also be used for SPR imaging analysis.

FIG. 1 is a diagram illustrating a method of manufacturing a biochip patterned with hydrophilicity / hydrophobicity and on chip MALDI-MS analysis (A). In addition, photographs of the chip patterned with gold (left picture), the immobilized antibody on the surface of the chip patterned with hydrophilicity / hydrophobicity (middle picture), and the chip (right picture) with MALDI matrix applied before modification were applied. It is shown (B).
FIG. 2 shows MALDI mass spectral results of angiotensin II (A) and cytochrome C (B) using chips of the present invention (left) and known MALDI sample plates (right).
3 is a result of MALDI mass spectrometry after immobilizing a human IgG antibody on a thiol-mixed SAM or a Cys3-protein G layer.
FIG. 4 shows MALDI of antigens captured from multiple protein mixtures after immobilization of antibodies against β2-microglobulin (A), IL6 (B), CRP (C), PSA (D), human IgG (E) on a chip The mass spectrometry results are shown. The peaks corresponded to the molecular weight of the target antigen within the various charge states.
Figure 5 shows the proteins contained in human serum samples after immobilization of antibodies against β2-microglobulin (A), IL6 (B), CRP (C), PSA (D), human IgG (E) on the chip The result of MALDI mass spectrometry of the antigen captured from this is shown.
FIG. 6 shows the results of MALDI mass spectrometry of antigens captured from multiple protein mixtures after forming a protein G layer on a gold thin film, immobilizing antibodies on the protein G layer. The antibodies used are antibodies against β2-microglobulin (A), IL6 (B), CRP (C), PSA (D), human IgG (E).
FIG. 7 shows the results of MALDI mass spectrometry of CRP captured through the antibody, using an antibody against human IgG (E) as an anti-CRP antibody (AD) or control. In addition, as a result of performing MALDI mass spectrometry with different concentrations of CRP antigen, the mass peak increased in a concentration-dependent manner.
8 shows SPR images of antigen-antibodies bound to thiol mixed SAMs. Ab: antibody, Ag: antigen.
Figure 9 shows the SPR image of the antigen-antibody bound on the Cys3-protein G layer. Ab: antibody, Ag: antigen.
10 shows the mass spectrometry results of CRA (A), PSA (B), and IL6 (C) after SPR imaging analysis.
11 shows the results of MALDI mass spectrometry after digestion with PASfmf trypsin captured by PSA antibody. (A) is the case where PSA antibody is used, (B) is the case where no antibody is used, (C) is the case where CRP antibody is used as a control. Trypsin autolysis peak is indicated by 'T' and PSA decomposition peak is indicated by '*'.
12 shows the results of MALDI mass analysis of trypsin digest of PSA.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Material Preparation

Carboxyl-terminated hexa (ethylene glycol) undecane thiol (SH-PEG-COOH) having a carboxyl group attached to the terminal and a tri (ethylene glycol) undecane thiol having a hydroxyl group bonded to the terminal tri (ethylene glycol) undecane thiol, SH-PEG-OH) was purchased from Nanoscience Instrument.

N - (3- dimethylaminopropyl) - N '- ethylcarbodiimide (N - (3-Dimethylaminopropyl) - N' -ethylcarbodiimide, EDC), N - hydroxy-succinimide (N -hydroxysuccinimide, NHS), ammonium Ammonium acetate, hexane, β2-microglobulin and MALDI matrix α cyano-4-hydroxy cinnamic acid (CHCA) and 2 -(4-hydroxy-phenylazo) benzoic acid (2- (4-hydroxy-phenylazo) benzoic acid (HABA)) was purchased from Sigma-Aldrich.

Anti-human kallikrein 3 / PSA antibodies, anti-human interleukin 6 antibodies, recombinant human kallikrein 3 / PSA and recombinant human interleukin 6 antibodies were purchased from R & D Systems (Minneapolis, MN). Anti-β2-microglobulin antibodies were purchased from Abchem (UK) and anti-CRP antibodies and recombinant human CRP were purchased from Calbiochem. Amine PEG was purchased from SunBio, Inc. Perfluoroalkylsilane (FM1010) was purchased from Nanocrystal CO., LTD.

Example 2 Preparation of Hydrophilic / Hydrophilic Patterned Gold Thin Film Biochip

A hydrophilic / hydrophobic patterned gold thin film array (diameter 1 mm) can be formed on a glass chip patterned with gold through a two-step chemical reaction, as shown in FIG. 1.

First, gold was evaporated through a shadow mask to form a gold pattern on the glass substrate. The patterned gold thin film chip produced had a size of 2.2 x 2.2 cm and contained 5 x 4 gold spots per 1 mm diameter. The patterned gold thin film chip was washed with concentrated piranha solution mixed with 70% (v / v) sulfuric acid and 30% (v / v) hydrogen peroxide and washed with deionized water. The chip was reacted with an ethanol solution containing 0.1 mM HS-PEG-COOH and 0.9 mM HS-PEG-OH for 16 hours to prepare a self-assembled monolayer (SAM) containing thiol on the chip's gold surface. This was allowed to form. Next, the chip was washed with ethanol and dried with nitrogen gas. In order to add hydrophobicity to the glass substrate surface, the chip was reacted with hexane in which 2% perfluoroalkylsilane (FM1010) was dissolved for 30 minutes, and the chip was washed with hexane and ethanol and dried with nitrogen gas. Chips prepared by this method can be stored at 4 ° C. or activated for antibody immobilization.

In order to activate the carboxyl group of the SAM mixed with thiol, a solution of 0.1 M EDC and 0.4 M NHS dissolved in distilled water was poured for 15 minutes, washed with water, and dried with nitrogen gas.

In addition, cysteine-tagged protein G (Cys3-protein G) was used to form a protein G layer on the gold surface of the chip. Prior to immobilizing Cys3-protein G on the gold surface, hexane dissolved in 2% perfluoroalkylsilane (FM1010) and the chip were reacted for 30 minutes to add hydrophobicity of the glass substrate. After washing the chip with hexane and ethanol, and drying with nitrogen gas, 0.5 μL of PBS solution dissolved in 0.1 mg / mL Cys3-protein G was added to the gold surface of the chip and allowed to react for 1 hour. The chip was washed with PBS with 0.1% tween 20. In the same manner as described above to prepare a chip in which the Cys3-protein G is fixed on the gold surface, it was used for antibody immobilization.

Example  3: antibody / antigen assay

For antibody immobilization, antibodies dissolved in PBS (0.5 μL, 0.1 mg / mL) were added over gold spots of hydrophilic / hydrophobic patterned chips and allowed to react for 90 minutes.

Next, the cells were washed with PBST (10 mM phosphate-saline buffer, 0.1% tween20), and the chip was immersed in 1 mg / mL BSA blocking solution (in PBST) for 30 minutes, thereby binding to nonspecifically bound antibody and PBST. Was removed. Prior to application of the BSA blocking solution, the NHS ester bound chip and 1 mg / mL amine PEG (in PBS) were reacted for 30 minutes to remove unreacted NHS ester groups. Next, antigen (0.5 μL, 10-100 μg / mL) was added to each spot and reacted for 2 hours. The chip was washed with PBST and PBS, then rinsed with 10 mM ammonium acetate (pH 7.5) and dried with nitrogen.

Example  4: SPR Imaging  analysis

SPR imaging analysis was performed by known methods using a house-customized SPR imaging system. Nonspecific human IgG, β2-microglobulin (11.6 KDa), human interleukin 6 (IL6, 20 KDa), C-reactive protein (CRP, 23 KDa) on the gold spot of the biochip Antibodies to prostate-specific antigen (PSA, 30 KDa) were attached.

Specifically, anti-CRP antibody (0.5 μL, 0.1 mg / mL), anti-PSA antibody (0.5 μL, 0.1 mg / mL), nonspecific human IgG (0.5 μL, 0.1 mg / mL, β2-microglobulin (0.5 μL) , 0.1 mg / mL), Or anti-IL6 antibody (0.5 μL, 0.1 mg / mL) was immobilized on the gold spot. Antigen (0.5 μL, 0.1 mg / mL) was applied to each gold spot and reacted for 1 hour. Specifically, a protein mixed solution containing four antigens (β2-microglobulin, IL6, CRP, PSA; each antigen contained 20 μg / mL) and 0.1 mg / mL BSA were applied to the gold spot to which the antibody was attached. Finally, as described above, the chips were washed, rinsed and dried.

Example  5: trypsin digestion ( On - chip tryptic digestion )

To decompose the protein captured by the biochip into trypsin, a 0.5 μL trypsin solution in 10 mM ammonium acetate (0.01 mg / mL, pH8.0) on each gold surface of the sampled biochip. Reacted. After digesting overnight at 37 ° C., the αcyano-4-hydroxy cinnamic acid (CHCA) matrix solution (0.5 μL, 10 mg / mL) in 50% ACN / 0.1% TFA was added to react and evaporated.

Example  6: MALDI - TOF  Mass spectrometry

AXIMA-CFR plus with pulsed nitrogen laser operating at 337 nm MALDI-TOF-MS analysis was performed using a MALDI-TOF mass spectrometer (Shimadzu / Kratos, Manchester, UK).

MALDI matrix solution with 10 mg / mL CHCA or 2- (4-hydroxy-phenylazo) benzoic acid (2- (4-hydroxy-phenylazo) benzoic acid (HABA)) 50% acetonitrile / 0.1% trifluoroacetic acid It was prepared by dissolving in (trifluoroacetic acid).

Carbon tape was used to fix the patterned biochip onto the MALDI sample substrate.

Spectra for proteins (linear mode) and peptides (reflectron mode) were obtained in positive-ion mode. Linear mode was measured using a mixture of cytochrome C and bovine serum albumin, and reflectron mode was angiotensin II ([M + H] ⁺ _Average = 1046.54) and ACTH fragment 1839 ([M + H). ] ⁺ _Average = 2467.19).

The protein mass obtained by performing MALDI with the hydrophilic / hydrophobic patterned biochip of the present invention was compared with the case using a known MALDI sample substrate. As a result, mass analysis of peptides (angiotensin II, 1046.54 Da and cytochrome C, 12361.9 Da), as well as model proteins, was carried out using known sample substrates and chips of the present invention. Similar signal intensity (mv) was shown in Fig. 2.

Example  7: using gold thin film chip with carboxyl group SPRI  And / or MALDI - MS  Perform analysis

In order to detect the target antigen by SPRI and / or MALDI-MS analysis, various kinds of antibodies were immobilized on the surface of the gold thin film chip to which the carboxyl group was attached. One of the five antibodies below was attached to each spot of the patterned gold thin film biochip.

Attached antibodies: nonspecific human IgG, β2-microglobulin (β2-microglobulin (11.6 KDa)), human interleukin 6 (IL6, 20 KDa), C-reactive protein (CRP, 23 KDa), prostate Antibodies to prostate-specific antigen (PSA, 30 KDa)

Only MALDI mass spectrometry of human IgG immobilized on the chip showed several charge states of the antibody (FIG. 3).

A protein mixed solution containing 4 antigens (β2-microglobulin, IL6, CRP, PSA each containing 20 μg / mL) and 0.1 mg / mL BSA were applied to the gold spot to which the antibodies were attached. After washing, the surface was dried with nitrogen gas and then treated with MALDI matrix to perform immuno-MALDI-TOF MS analysis of the captured antigen.

The mass spectrum of each antibody spot showed specific antigen peaks corresponding to the molecular weight of the target antigen within various charge states (FIG. 4). In addition, although peaks of serum components were observed nonspecifically in the mass range of 6000-9000 m / z, On chip immuno-MALDI MS analysis of several proteins was successful for protein mixtures in human serum samples (FIG. 5).

Example  8: Cys3 - protein  Using chip with G MALDI - MS  Perform analysis

Cysteine-tagged protein G, well known as antibody binding protein, formed a gold thin film surface protein layer and bound well with the antibody.

The protein G layer in the present invention has been successfully applied for the preparation of hydrophilic / hydrophobic antibody arrays.

In this case, as described in Example 2, the glass surface of the chip was reacted with perfluoroalkylsilane, and then protein G was attached to the remaining gold spot.

As in Example 7, the antibody was bound, the antigen was reacted, and on chip MALDI-TOF-MS analysis was performed.

Target antigens were successfully detected via on chip MALDI-TOF-MS for each protein G (FIG. 6).

Example  9: SPR Imaging  System and MALDI - TOF - MS Combination of

Following mass spectrometry performed in Example 8, concentration dependent MS analysis of CRP antigen was also performed using an on chip MALDI-MS system. Increasing the concentration of CRP antigen added from 1 μg / mL to 100 μg / mL resulted in an increased mass peak (FIG. 7).

Accurate quantitation of analytes usually requires the use of internal standards with careful experimental design. Combining the MALDI-TOF-MS with the SPR imaging system allows for quantitative analysis of antibody-antigen binding without the use of internal standards prior to MS analysis. In the patterned gold biochip system of the present invention, the antigen bound and nitrogen dried chips were directly applied to the SPR imaging system.

Antibody immobilization and capture of specific antigens, either covalently or with the help of protein G, was shown exactly in SPR imaging data (FIGS. 8, 9).

FIG. 9 is an SPR image showing antigen antigens bound to a thiol mixed SAM, and FIG. 10 is an SPR image showing antigen antigens bound to a Cys3-protein G layer.

After SPR imaging experiments, the analyzed biochips were separated in an SPR imaging analyzer. The MALDI matrix was then applied to the antigen containing the gold spots to perform MALDI-MS analysis (FIG. 10).

Example  10: on - chip  Protein breakdown and MALDI - MS  analysis

Anti-PSA antibodies were used to capture PSA in gold spots according to the protein assay described above. Trypsin solution (0.5 μL, 10 μg / mL) was dropped onto the gold spot where PSA was captured.

The hydrophilic / hydrophobic nature of the biochip enabled consistent degradation conditions to be applied to all gold spots. Without going through a wash step, the MALDI matrix for the peptide was applied directly to the spot and analyzed by MALDI-TOF MS.

MALD mass spectrometry of the trypsin digest of PSA captured by the antibody is shown in FIG. 11. PAS degraded peptide peaks were clearly distinguished from degradation peaks of antibodies and trypsin. In addition, ions were identified at m / z 1409.92, 1875.78 and 2233.46, which coincided with the peaks obtained from trypsin digest of PSA (FIG. 12).

Claims (14)

(1) forming a metal lattice pattern on the substrate;
(2) forming a self-assembled monolayer of thiol mixed on the metal surface; And
(3) attaching hydrophobic molecules to the substrate
Method of producing a biochip patterned to hydrophilic / hydrophobic comprising a.
The method of claim 1, wherein the substrate is selected from the group consisting of glass, silicon, polypropylene, nylon, plastic, and metal.
The method of claim 1, wherein the metal is selected from the group consisting of Ni, Cu, Zn, Au, and alloys thereof.
The method of claim 1, wherein in step (2), polyethylene glycol (PEG) mixed with thiol is added to form a self-assembled monomolecular layer.
The polyethylene glycol (PEG) in which the thiol is mixed is hexa (ethylene glycol) undecane thiol having a carboxyl group bonded to the terminal, and a hydroxyl group at the terminal. Process for producing a combined tri (ethylene glycol) undecane thiol or a mixture thereof.
The method of claim 1, wherein the hydrophobic molecule of step (3) is characterized in that the perfluoroalkylsilane (perfluoroalkylsilane).
(1) forming a metal lattice pattern on the substrate;
(2) attaching hydrophobic molecules to the substrate; And
(3) adding protein G tagged with cysteine to form a protein G layer on the metal surface
Method of producing a biochip patterned to hydrophilic / hydrophobic comprising a.
8. The method of claim 7, wherein the hydrophobic molecule of step (2) is a perfluoroalkylsilane.
8. The method of claim 7, wherein said substrate is selected from the group consisting of glass, silicon, polypropylene, nylon, plastic, and metal.
The method of claim 7, wherein the metal is selected from the group consisting of Ni, Cu, Zn, Au and alloys thereof.
A biochip patterned with hydrophilicity / hydrophobicity prepared by the method of any one of claims 1 to 10.
(1) immobilizing the antibody of the target substance to protein G tagged with the hydrophilic molecule or cysteine of the biochip of claim 11; And
(2) reacting a sample containing a target substance to the biochip;
Including, the detection method of the target substance using the biochip.
The method according to claim 12, wherein the biochip of step (2) is used for SPR imaging analysis, MALDI-TOF (Matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry, or MALDI- after SPR imaging analysis. The method for detecting a target substance, characterized in that it further comprises the step of performing TOF mass spectrometry.
The method of claim 12, wherein the sample is obtained from serum, tissue, cells, saliva, cerebrospinal fluid, or urine.

Images