KR100561187B1 - Metallic chloride compound coating plate for protein chips and their manufacturing process - Google Patents

Abstract

In the present invention, a metal chloride compound for protein chips prepared by spin coating a metal chloride compound having a distilled water: metal chloride ratio of 20: 1 to 5: 1 using a metal chloride compound containing NiCl₂, CaCl₂ and FeCl₂ on a glass substrate. It relates to a coating plate. In another aspect, the present invention comprises the steps of preparing a metal chloride compound coating plate by spin-coating with a metal chloride compound solution of distilled water: metal chloride ratio of 20: 1 to 5: 1 on the glass substrate; And attaching a FITC-conjugated protein or a FITC-conjugated protein with a His-tag attached thereto at the same time.

Glass substrate, distilled water, metal chloride compound, nickel chloride, spin coating, protein chip, nickel chloride coating plate, FITC-conjugated protein, His-tag protein

Description

Metallic chloride compound coating plate for protein chips and their manufacturing process             

Figure 1 (a) shows the SEM analysis results of 100,000 times the glass substrate deposited for 20 seconds at 4000 rpm by spin coating method according to the NiCl2 concentration change.

Figure 1 (b) shows the results 200,000 times of the glass substrate deposited for 20 seconds at 4000 rpm by the spin coating method according to the NiCl2 concentration change.

Figure 2 (a) shows the results of the SEM analysis of 100,000 times according to the rpm change of the sample deposited with distilled water / NiCl2 weight ratio of 8: 1.

Figure 2 (b) shows the SEM results of 200,000 times according to the change in the rpm of the sample of distilled water / NiCl 2 weight ratio deposited 8: 1.

Figure 3 shows the XRD pattern according to the change in distilled water / NiCl₂weight ratio.

Figure 4 shows the surface characteristics of the sample as the AFM results according to the change of NiCl2 concentration.

5 shows the protein adhesion tendency on the substrate according to the NiCl 2 concentration change.

Figure 6 shows the trend according to the rpm change of the sample of distilled water / NiCl2 weight ratio of 8: 1.

Figure 7 shows the tendency of protein adhesion on the substrate according to the coating time change of the sample of distilled water / NiCl2 weight ratio of 8: 1.

Figure 8 shows the tendency of protein adhesion on the substrate according to the drying time of the sample of distilled water / NiCl2 weight ratio of 8: 1.

Figure 9 shows the tendency of protein adhesion on the substrate according to the drying temperature of the sample of distilled water / NiCl2 weight ratio of 8: 1.

The present invention relates to a metal chloride compound coating plate for protein chips spin-coated a thin film of a metal chloride compound containing NiCl 2, CaCl 2, FeCl 2 on a glass substrate and a method of manufacturing the same. More specifically, the present invention relates to a metal chloride coating plate for protein chips prepared by spin coating a metal chloride compound solution having a distilled water: metal chloride ratio of 20: 1 to 5: 1 on a glass substrate. In another aspect, the present invention comprises the steps of preparing a metal chloride compound coating plate by spin-coating with a metal chloride compound solution of distilled water: metal chloride ratio of 20: 1 to 5: 1 on the glass substrate; And attaching a FITC (Fluorescein isothiocyanate) -conjugated protein or a FITC-conjugated and simultaneously His-tag protein onto the plate.

Protein chips are protein arrays in which hundreds to thousands of different kinds of proteins are immobilized on glass substrates. Therefore, since the protein chip is capable of simultaneous analysis of immobilized proteins, it can be used for protein discovery, drug screening and disease diagnosis with specific physiological activities (Fung, ET, Thulasiraman, V., Weinberger, SR, and Dalmasso, E. (2001) Protein biochips for differential profiling Current Opinion in Biotechnology 12:. 65-69 and MacBeath, G. and Schreiber, S. ( 2000) Printing proteins as microarrays for high-throughput function determination Science 289:. 1760 -1763). Protein chips are highly valuable due to their potential for use in human disease diagnosis and treatment, while manufacturing various protein chips suitable for their purpose is required depending on the purpose of use.

The most basic technique of protein chip manufacturing is substrate pretreatment technology for attaching protein onto glass substrate. The pretreatment of the substrate should be made in accordance with the method of protein attachment (MacBeath, G. and Schreiber, S. (2000) as microarrays for high-throughput function determination Science 289:. 1760-1763, Nicolau, DV, Taguchi, H., Taniguchi, H., and Yoshikawa, S. (1998) Micron-sized protein patterning on diazonaphthoquinone / novolak thin polymeric films Langmuir 14: 1927-1936 and Bashir, R., Gomez, R., Sarikaya, A., Ladisch, MR, Sturgis, J., and Robinson, JP (2001) Adsorption of avidin on microfabricated surfaces for protein biochip applications. Biotechnol. Bioeng. 73: 324-328). Glass substrates reported to date are amine groups (Nicolau, DV, Taguchi, H., Taniguchi, H., and Yoshikawa, S. (1998) Micron-sized protein patterning on diazonaphthoquinone / novolak thin polymeric films. Langmuir 14: 1927- 1936) and aldehyde groups (MacBeath, G. and Schreiber, S. (2000) Printing proteins as microarrays for high-throughput function determination; see Science 289: 1760-1763). The protein was attached to the protein by direct attachment of the protein or by using a crosslinker. However, this method of attachment results in the loss of its function when the protein functional domain is attached to the glass substrate because any portion of the protein exposed surface is unspecifically attached to the substrate.

The present invention is intended to bind a protein with a His-tag on the substrate to specifically bind to the metal ion of the metal chloride compound including NiCl₂, CaCl₂, FeCl₂ in order to prevent any portion of the protein surface attached to the substrate In order to prevent the adhesion of a protein's functional domain to a glass substrate, a protein that binds to the surface of the substrate is genetically attached to a desired target protein so as not to lose the function of the target protein. .

The present invention relates to a metal chloride coating plate for protein chip spin coating a thin film formed of at least one metal chloride compound selected from the group consisting of NiCl 2, CaCl 2 and FeCl 2 on a glass substrate, and a method for manufacturing the same. More specifically, the present invention provides a solution of a metal chloride compound having a distilled water: metal chloride compound ratio of 20: 1 to 5: 1 using at least one metal chloride compound selected from the group consisting of NiCl2, CaCl2 and FeCl2 on a glass substrate. It relates to a metal chloride coating plate for protein chips prepared by spin coating. In addition, the present invention by spin-coating a metal chloride compound solution of distilled water: metal chloride compound ratio of 20: 1-5: 1 using at least one metal chloride compound selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂ on a glass substrate Preparing a metal chloride coating plate; And attaching a FITC-conjugated protein or a FITC-conjugated and his-tag protein at the same time on the plate.

 The present invention is to prepare a thin film formed of any one or more metal chloride compound selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂ on the glass substrate by the Sol-Gel method and then coating the metal chloride compound coating for protein chips by examining the physical properties and protein adhesion characteristics Relates to the application of the plate. According to the present invention, after adjusting the weight ratio of distilled water and a metal chloride compound using any one or more metal chloride compounds selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂, Sol is prepared and spin coated on a glass substrate to form NiCl₂, CaCl₂, FeCl₂. A metal chloride compound thin film was prepared, and the prepared thin film was analyzed by SEM (Scanning Electron Microscope) analysis (LEO 1530), XRD (X-ray diffraction) analysis (Scintag XDS 2000) and AFM (Atomic Forces Microscope) analysis (DI Nanoscope 3A) and protein adhesion test were performed. As a result of SEM analysis, the particle size was measured to be about 10 nm, and the increase in the concentration of metal chloride compound and rpm including appropriate NiCl₂, CaCl₂ and FeCl₂ decreased the incidence of cracks and improved protein adhesion. The results of XRD and AFM showed that the crystals were grown to the (101) plane, and the main peak was improved and the crystallinity increased as the concentration of metal chloride compounds including NiCl₂, CaCl₂ and FeCl₂ increased. there was. When the metal chloride compound thin films containing NiCl₂, CaCl₂ and FeCl₂ were naturally dried, the protein adhesion ability was greatly decreased, but the protein adhesion characteristics were improved at the drying temperature above 40 ℃.

In order to manufacture a plate coated with at least one metal chloride compound selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂ in the present invention, NiCl₂, CaCl₂, The metal chloride compound containing FeCl₂ and distilled water were weighed in a weight ratio of 20: 1 to 5: 1 using an electronic balance to remove surface impurities on the substrate before coating the prepared sample on a glass substrate. Ultrasonic washing with ethyl alcohol and distilled water for 5 minutes and then dried. The glass substrate thus washed is mounted on a spin coater, and then a solution of a metal chloride compound including NiCl₂, CaCl₂, and FeCl₂, prepared in a sol state, is applied thereon, at various times of 1000 rpm to 4000 rpm. Spin-coating and then dried on a hot plate according to the temperature change. XRD analysis (Scintag XDS 2000) was performed to analyze the crystallization characteristics of each sample according to composition and rpm, and SEM analysis (LEO 1530) was performed to observe grain formation and microstructure of the samples. AFM (DI Nanoscope 3A) was used to investigate the surface condition and surface roughness characteristics of the sample coated.

FITC-conjugate on a glass substrate coated with a metal chloride compound containing NiCl₂, CaCl₂, FeCl₂ in order to attach the protein to a plate formed of any one or more metal chloride compounds selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂ in the present invention Protein or FITC-conjugated protein with His-tag at the same time for 10 minutes to 1 hour at a concentration of 10 ㎍ / ml-1mg / ml, washed three times with PBS buffer for 5 minutes and dried Analysis was by fluorescence image analyser. The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 1 Preparation of NiCl 2 Coated Plates

In order to coat NiCl₂ on a glass substrate, NiCl₂5g and distilled water were weighed in a weight ratio of 20: 1 to 5: 1 using an electronic balance. Before coating the prepared sample on a glass substrate, it was ultrasonically washed for 5 minutes using 100 ml of 99.9% ethyl alcohol to remove surface impurities on the substrate and dried. After the glass substrate thus washed was mounted on a spin coater, NiCl₂ solution prepared in a sol state was applied to 0.8cc per plate and spin-coated at various times of 1000 rpm to 4000 rpm. It was dried on a hot plate according to the temperature change.

XRD analysis (Scintag XDS 2000) was performed to analyze the crystallization characteristics of each sample according to the composition and rpm, and SEM analysis (LEO 1530) was performed to observe grain formation and microstructure of the samples. AFM (DI Nanoscope 3A) was used to investigate the surface condition and surface roughness characteristics of the sample coated.

Example 2 FITC-Conjugated Protein Attachment to NiCl2 Coated Plates

The FITC-conjugated protein was reacted at a concentration of 10 µg / ml-1mg / ml for 10 minutes to 1 hour at room temperature on a nickel-coated glass substrate, washed three times with PBS buffer for 5 minutes, dried, and then fluorescence image analyser. Analyzed.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 3 Protein Attachment with His-tag While FITC-Conjugated with NiCl2 Coated Plate

The FITC-conjugated, His-tag-containing protein was reacted at 10 µg / ml to 1mg / ml at room temperature for 10 minutes to 1 hour at room temperature on a nickel-coated glass substrate, and then three times for 5 minutes with PBS buffer. After washing, drying and analyzing by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 4 Preparation of CaCl 2 Coated Plates

Example 1 was carried out in the same manner as in Example 1 except for using CaCl 2 instead of using NiCl 2.

Example 5 FITC-Conjugated Protein Attachment to CaCl2 Coated Plates

The FITC-conjugated protein was reacted at a concentration of 10 µg / ml to 1 mg / ml at room temperature for 10 minutes to 1 hour on a CaCl₂-coated glass substrate, washed three times with PBS buffer for 5 minutes, dried, and then fluorescence image analyser. Analyzed.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 6 CaCl2 Coated Plate FITC-Conjugated and His-tag Attached Protein

The FITC-conjugated, His-tag-containing protein was reacted at 10 µg / ml-1mg / ml at room temperature for 10 minutes-1 hour at room temperature for 3 minutes with PBS buffer. After washing, drying and analyzing by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 7 Preparation of FeCl 2 Coated Plates

Example 1 was carried out in the same manner as in Example 1 except for using FeCl₂ instead of using NiCl₂.

Example 8 FITC-Conjugated Protein Attachment to FeCl2 Coated Plates

FITC-conjugated protein on FeCl₂-coated glass substrate was reacted for 10 minutes to 1 hour at 10 µg / ml ~ 1mg / ml at room temperature, washed three times with PBS buffer for 5 minutes and dried, and then fluorescence image analyser Analyzed.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 9 Protein Attachment with His-tag While FITC-Conjugated with FeCl2 Coated Plate

The FITC-conjugated, His-tag-containing protein was reacted at 10 µg / ml to 1mg / ml at room temperature for 10 minutes to 1 hour at room temperature for 3 minutes with PBS buffer. After washing, drying and analyzing by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 10 Preparation of a Plate Coated with a Mixed Compound of NiCl2 and FeCl2

Instead of using NiCl 2 in Example 1 was carried out in the same manner as in Example 1 except for using a mixed compound of NiCl 2 and FeCl 2 mixed 1: 1.

Example 11 FITC-Conjugated Protein Attachment to a Plate Coated with a Mixture of NiCl2 and FeCl2

FITC-conjugated protein was reacted on a glass substrate coated with a mixed compound of NiCl₂ and FeCl₂ at a concentration of 10 µg / ml-1mg / ml for 10 minutes to 1 hour at room temperature, washed three times with PBS buffer for 5 minutes and dried. After analysis by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 12: A plate coated with a mixed compound of NiCl 2 and FeCl 2 FITC-conjugated and His-tag attached protein

The FITC-conjugated, His-tag-containing protein was reacted at room temperature for 10 minutes to 1 hour at a concentration of 10 µg / ml to 1mg / ml on a glass substrate coated with a mixed compound of NiCl₂ and FeCl₂ PBS buffer solution. After washing three times for 5 minutes to dry and analyzed by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 13 Preparation of a Plate Coated with a Mixture of NiCl2, CaCl2, and FeCl2

Instead of using NiCl 2 in Example 1 was carried out in the same manner as in Example 1 except for using a mixed compound of NiCl 2, CaCl 2 and FeCl 2 in a 1: 1: 1 mixture.

Example 14 FITC-Conjugated Protein Adhesion to a Plate Coated with a Mixture of NiCl 2, CaCl 2 and FeCl 2

FITC-conjugated protein was reacted on a glass substrate coated with a mixed compound of NiCl₂, CaCl₂ and FeCl₂ at a concentration of 10 µg / ml-1mg / ml for 10 minutes to 1 hour at room temperature and washed three times with PBS buffer for 5 minutes. After drying by fluorescence image analyzer was analyzed.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

Example 15 A plate coated with a mixed compound of NiCl 2, CaCl 2 and FeCl 2 FITC-conjugated and His-tag attached protein at the same time

After FITC-conjugated and His-tag-attached protein on a glass substrate coated with a mixture of NiCl₂, CaCl₂ and FeCl₂, the reaction was allowed to react at room temperature for 10 minutes to 1 hour at a concentration of 10 µg / ml to 1mg / ml. After washing three times with PBS buffer for 5 minutes, dried and analyzed by fluorescence image analyser.

The FITC-conjugated protein immobilized on the glass substrate was measured by fixing the excitation wavelength 490 nm and emission wavelength 670 nm in a Bio-Imaging Analyzer System (Bas; Fuji Photo Film Co).

FIG. 1 (a) shows the results of SEM analysis of 100,000 times the glass substrate deposited at 4000 rpm for 20 seconds by the spin coating method according to the NiCl2 concentration change, and FIG. 1 (b) shows the spin coating method according to the NiCl2 concentration change. It shows 200,000 times the result of the glass substrate deposited at 4000 rpm for 20 seconds. As can be seen from the overall SEM results, the surface is relatively uniformly coated with very small bead-shaped particles. The particle size was measured at about 10 nm. In the case of 20: 1 weight ratio of distilled water / NiCl₂, the degree of crack that weakens the crystallinity was clearly observed, and the increase of NiCl₂ concentration greatly reduced the degree of crack. The uniformity of was greatly improved. The results of the SEM measurements were also consistent with the results of 100,000 and 200,000 times observed at different locations. The SEM results were in close agreement with the results of FIG. 5, and the protein adhesion ability was improved by increasing the concentration of NiCl₂ from 20: 1 to 8: 1 by weight ratio of distilled water / NiCl₂.

FIG. 2 (a) shows a SEM analysis result of 100,000 times according to the change of rpm of the sample deposited with distilled water / NiCl₂weight ratio of 8: 1, and FIG. 2 (b) shows the sample deposited with distilled water / NiCl₂weight ratio of 8: 1. SEM results of 200,000 times according to the change of rpm are shown. In FIG. 2, it can be observed that very small spherical particles are deposited relatively uniformly as in FIG. 1. As shown in FIG. 2 (a), particles are relatively well bonded at 3000 rpm to maintain uniformity, but the degree of cracking is increased at 1000 rpm. This fact can be observed to coincide with the result of 200,000 times of FIG. 2 (b), and it can be seen that rpm affects particle cracking.

Figure 3 shows the XRD pattern according to the change in distilled water / NiCl 2 weight ratio. In the case of 20: 1, a weak peak was observed near 31 °, but the peak gradually improved with increasing NiCl2 concentration. Although several peaks were expected due to the nature of NiCl₂, the crystals grown on the (101) plane seemed to be dominant in this experiment.

Figure 4 shows the surface characteristics of the sample by the AFM results according to the change in NiCl2 concentration. In FIG. 4 (a), it is determined that the surface roughness characteristics are improved as the spacing of the grains becomes denser according to the polycrystalline form in which the crystals seem to be spread somewhat or the NiCl 2 concentration increases.

5 shows the protein adhesion tendency on the substrate according to the NiCl 2 concentration change. Distilled water / NiCl 2 weight ratio of about 8: 1 protein adhesion tendency is greatly improved.

Figure 6 shows the tendency of protein adhesion on the nickel substrate according to the rpm change of the sample of distilled water / NiCl 2 weight ratio of 8: 1. As can be seen in Figure 6 it can be seen that the adhesion ability of the protein is gradually improved at more than 2000 rpm, as seen in the SEM analysis results it can be seen that the adhesion capacity is greatly increased at 1000 ~ 4000 rpm.

Figure 7 shows the tendency of protein adhesion on the substrate according to the coating time change of the sample of distilled water / NiCl2 weight ratio of 8: 1. It shows almost similar adhesion tendency in the coating time range of more than 5 seconds, which is considered that the NiCl2 thin film does not depend very much on the coating time.

Figure 8 shows the tendency of protein adhesion on the substrate according to the drying time of the sample of distilled water / NiCl2 weight ratio of 8: 1. As shown in the drawing, the adhesion capacity did not change significantly in the drying time of 3 minutes or more.

9 is a protein on the substrate according to the drying temperature of the sample of distilled water / NiCl 2 weight ratio of 8: 1. Shows the tendency of attachment. In the case of naturally dried samples, the protein adhesion ability was greatly decreased, and the adhesion ability was gradually improved at the drying temperature over 40 ℃.

Coating the thin film of one or more metal chloride compounds selected from the group consisting of NiCl₂, CaCl₂ and FeCl₂ on the glass substrate by the Sol-Gel method and attempting SEM analysis through various conditions changes, the results of NiCl₂, CaCl₂, FeCl₂ for the protein chip of the present invention The metal chloride coating plate containing was able to observe crystals of the metal chlorides containing NiCl₂, CaCl₂ and FeCl₂ in the form of fine rounds, and the increase of the concentration of the metal chloride compound including the appropriate NiCl₂, CaCl₂ and FeCl₂ and the rpm increase were observed. Reduction resulted in improved crystallinity. This is almost consistent with the results of the protein adhesion test. From the SEM results, the particle size was determined to be about 10 nm.

From the XRD and AFM results, the crystallinity tends to be improved by increasing the concentration of metal chloride compounds containing appropriate NiCl₂, CaCl₂ and FeCl₂. Changes in coating time and drying time did not significantly affect the properties of the metal chloride thin films including NiCl₂, CaCl₂ and FeCl₂. Similar results were obtained in the deposition time of more than 5 seconds and the drying time of more than 3 minutes. In the case of naturally dried NiCl₂, CaCl₂ and FeCl₂ thin metal chloride compound thin films, the adhesion of protein decreased significantly, but the adhesion of protein increased significantly at the drying temperature above 40 ℃.

As described above, any one or more metal chloride compound coated substrates selected from the group consisting of NiCl2, CaCl2 and FeCl2 for manufacturing the protein chip of the present invention has a simple manufacturing method and is cheaper than other substrate manufacturing methods. Since the functional damage of the His-tagged target protein attached on the substrate is much lower than that of other protein attachment techniques, it is suitable as a substrate for a protein chip, and the FITC-conjugated protein or FITC-conjugated and His-tag on the plate at the same time Since the attached protein can be used for protein chip by simultaneous attachment of immobilized proteins, it is a very useful invention in biotechnology and medicine industry such as protein search, new drug screening and disease diagnosis with specific physiological activity.

Claims (7)

In the protein chip, a coating plate and a fluorescein isothiocyanate (FITC) on the coating plate, characterized in that the spin coating with a solution of distilled water and nickel chloride (NiCl ₂ ) in a ratio of 20: 1 to 5: 1 on the glass substrate -A protein chip characterized in that the conjugated and at the same time attach the protein attached to His-tag. delete delete delete The protein chip according to claim 1, wherein the spin speed is coated at 1000 to 4000 rpm during spin coating. According to claim 1, Protein chip, characterized in that the drying temperature in the drying step after spin coating in the range of 40 ~ 80 ℃. delete

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