KR101324199B1 - Protein binding method using mixed self assembled monolayers and protein chip using thereof - Google Patents

Protein binding method using mixed self assembled monolayers and protein chip using thereof Download PDF

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KR101324199B1
KR101324199B1 KR1020100091053A KR20100091053A KR101324199B1 KR 101324199 B1 KR101324199 B1 KR 101324199B1 KR 1020100091053 A KR1020100091053 A KR 1020100091053A KR 20100091053 A KR20100091053 A KR 20100091053A KR 101324199 B1 KR101324199 B1 KR 101324199B1
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self
assembled monolayer
assembled
membrane
protein
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KR1020100091053A
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Korean (ko)
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KR20120029144A (en
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양민양
김종수
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한국과학기술원
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Abstract

The present invention relates to a method for binding a protein using a mixed self-assembled monomolecular membrane and a protein chip using the method, wherein the method for reducing unwanted adsorption and increasing desired adsorption of a protein using a mixed self-assembled monomolecular membrane; It relates to the production of protein chips using the same. Forming a self-assembled monolayer film 200 mixed with the glass substrate 100 for this purpose (S110); Removing the self-assembled monomolecular film 200 to be removed from the self-assembled monomolecular film 200 formed on the glass substrate 100 (S120); Forming a new self-assembled monomolecular film 300 at a position where the self-assembled monomolecular film 200 is removed (S130); And a method of binding the protein 400 to the new self-assembled monomolecular membrane 300 (S140).

Description

Protein binding method using mixed self assembled monolayers and protein chip using the method

The present invention relates to a method of binding a protein using a mixed self-assembled monomolecular membrane and a protein chip using the method. More particularly, the use of a self-assembled monomolecular membrane is mixed to reduce unwanted adsorption of proteins and to achieve desired adsorption. The present invention relates to a method of increasing and manufacturing a protein chip using the same.

In the case of a conventional micro array biochip, in the process of forming a micro array pattern, a amine (Amine), an aldehyde (Aldehyde), an epoxy coating, etc. are applied to the entire glass substrate, and then a micro array device (for example, a contact pin, Ink-jet, Electrospray) was used to pick up protein or cell samples one by one to form an array pattern.

After making an array pattern in this way, the polymerization reaction is performed one by one on one spot of the array, or the reaction reagent is raised by using glass to examine the desired reaction. However, this process problem requires a lot of time required to put the desired samples one by one, and since the front coating is used to cause the next reaction, there has been a problem that an unwanted reaction occurs next to the array spot. In addition, there is a problem in that the blocking (blocking) process must be additionally performed to eliminate the reaction of the unwanted portion. Conventionally, a hydrophilic surface treatment was used to reduce unwanted binding. In this case, it is difficult to selectively raise the protein solution because the surface energy of the place where the protein is adsorbed and the place where the protein is not adsorbed is similar.

Therefore, in the technical field to which the present invention belongs, it has been required to develop a protein binding method and a protein chip using a self-assembled monomolecular membrane which reduces the adsorption of unwanted portions and increases the adsorption of desired portions while requiring a short time.

Therefore, the present invention was created to solve the above problems, by using the difference in the surface energy of the self-assembled monolayer membrane to reduce the adsorption of the unwanted site and increase the adsorption of the desired site to bind the protein only to the desired site Its purpose is to make it possible.

However, the objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

An object of the present invention described above, the step of forming a self-assembled monomolecular film 200 mixed in the glass substrate 100 (S110); Removing the self-assembled monomolecular film 200 to be removed from the self-assembled monomolecular film 200 formed on the glass substrate 100 (S120); Forming a new self-assembled monomolecular film 300 at a position where the self-assembled monomolecular film 200 is removed (S130); And it can be achieved by providing a protein binding method using a mixed self-assembled monolayer membrane, characterized in that the step of binding the protein 400 to the new self-assembled monolayer membrane (300) (S140).

In addition, after the step (S130) of forming a new self-assembled monomolecular membrane (300), further comprising the step of cross-linking (S135) to further fix the protein 400 is characterized in that it is chemically bonded.

In addition, the self-assembled monomolecular film 200 may be formed on the entire surface of the glass substrate 100.

In addition, the mixed self-assembled monolayer membrane 200 is characterized in that the hydrophilic self-assembled monolayer membrane 210 and the hydrophobic self-assembled monolayer membrane 220.

In addition, the hydrophobic self-assembled monolayer membrane 220 is characterized by having a CH 3 functional group or a CF 3 functional group.

In addition, the hydrophilic self-assembled monolayer film 210 is characterized in that it has an OH functional group.

In addition, the hydrophilic self-assembled monomolecular film 210 may be formed more than the hydrophobic self-assembled monomolecular film 220.

In addition, the hydrophobic self-assembled monolayer membrane 220 and the protein 400 is characterized in that it is coupled to form a constant contact angle.

In addition, the new self-assembled monolayer membrane 300 is characterized in that the hydrophilic self-assembled monolayer membrane 210.

In addition, the hydrophilic self-assembled monolayer membrane 210 is characterized in that it has an NH 2 functional group or a COOH functional group.

In addition, the self-assembled monolayer film 200 may be removed using any one of plasma, UV laser, and UV lamp.

In addition, the combination of the self-assembled monolayer film 200 and the glass substrate 100 or the combination of the new self-assembled monolayer film 300 and the glass substrate 100 uses a silane-based self-assembled monolayer film. It is characterized by.

In addition, the glass substrate 100 is characterized in that provided by replacing the gold substrate (100).

In addition, the combination of the self-assembled monomolecular film 200 and the glass substrate 100 or the combination of the new self-assembled monomolecular film 300 and the glass substrate 100 may use a thiol-based self-assembled monomolecular film. It features.

On the other hand, the object of the present invention can be achieved by pore the protein chip using the mixed self-assembled monomolecular membrane prepared by any one of claims 1 to 12 as another category.

And, it can be achieved by providing a protein chip using a mixed self-assembled monomolecular membrane prepared by any one of claims 13 or 14.

According to the present invention as described above, it is convenient to fix the protein by binding the protein using the surface energy difference of the self-assembled monomolecular membrane, there is an effect that can produce a protein chip faster than using a micro array device .

Moreover, according to this invention, an array pattern has a uniform effect compared with using a micro array device.

In addition, according to the present invention, the protein is bound only to the desired site, thereby significantly reducing the amount of the sample.

In addition, according to the present invention, binding of unwanted sites can be reduced by using a self-assembled monomolecular membrane.

And according to this invention, the manufacturing cost of a protein chip can be reduced significantly.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate preferred embodiments of the invention and, together with the description, serve to further the understanding of the technical idea of the invention, It should not be construed as limited.
1 is a flow chart sequentially illustrating a method according to the invention,
2a to 2d are process state diagrams for each step according to the invention.

Hereinafter, a preferred embodiment of the present invention will be described with reference to the drawings. In addition, the embodiment described below does not unduly limit the content of the present invention described in the claims, and the entire structure described in this embodiment is not necessarily essential as the solution means of the present invention.

<Physical protein binding method using mixed self-assembled monomolecular membrane>

1 is a flow chart sequentially illustrating a method according to the present invention, and FIGS. 2A-2D are process state diagrams of each step according to the present invention.

As shown in FIG. 1, the protein binding method using the mixed self-assembled monomolecular membrane according to the present invention is performed including steps S110 to S140. Hereinafter, a protein binding method using a mixed self-assembled monolayer membrane according to the present invention will be described with reference to FIGS. 1 and 2.

First, as shown in FIG. 2A, a step of forming a self-assembled monolayer 200 mixed with the substrate 100 is performed (S110). In this case, the substrate 100 may form a self-assembled monolayer film 200 using a glass substrate or a gold substrate.

On the other hand, in the case of using a glass substrate, the combination of the self-assembled monomolecular film 200 and the substrate 100 or the combination of the new self-assembled monomolecular film 300 and the substrate 100 is Silane-based (SiCl). 3 -terminated) self-assembled monomolecular membranes can be used for binding.

In the case of using a gold substrate, the combination of the self-assembled monomolecular film 200 and the substrate 100 or the combination of the new self-assembled monomolecular film 300 and the substrate 100 is a thiol-based (SH-terminated) Self-assembled monomolecular membranes can be combined with each other.

Meanwhile, the self-assembled monomolecular film 200 is formed on the entire surface of the substrate 100, and the hydrophilic self-assembled monomolecular film 210 and the hydrophobic self-assembled monomolecular film 220 are mixed to form a front surface of the substrate 100. To form. At this time, the hydrophobic self-assembled monolayer membrane 220 is a CH 3 functional group or CF 3 It is preferable to have a functional group. The mixed self-assembled monolayer membrane is a mixture of hydrophilic or hydrophobic self-assembled monolayer membranes 210 and 220. In addition, the hydrophilic self-assembled monolayer film 210 preferably has an OH functional group.

Next, as shown in FIG. 2B, the self-assembled monomolecular film 200 desired to be removed from the self-assembled monomolecular film 200 formed on the glass substrate or the gold substrate is removed (S120). . The self-assembled monolayer film 200 to be removed may be removed using a plasma, UV laser, or UV lamp. Therefore, FIG. 2B illustrates a state in which a portion of the self-assembled monolayer film 200 is removed, and an empty space is formed to form a hydrophobic self-assembled monolayer film 220 to be described later. In this case, it is preferable that more hydrophilic self-assembled monolayer film 210 is formed than hydrophobic self-assembled monolayer film 220. This is because when the protein 400 binds to the self-assembled monolayer membrane 200, the protein 400 binds to the hydrophobic self-assembled monolayer membrane 220 at a desired position and does not bind to the remaining hydrophilic self-assembled monolayer membrane 210. to be. In other words, it is desirable to prevent the protein solution from forming on the protein chip. However, the above-described removal method will be omitted since it is obvious in the art.

Next, as shown in FIG. 2C, a step of forming a new self-assembled monomolecular film 300 is performed at the position where the self-assembled monomolecular film 200 is removed (S130). The new self-assembled monomolecular membrane 300 is meant to be a hydrophobic self-assembled monolayer membrane 220.

Finally, as shown in FIG. 2D, the step of binding the protein to the new self-assembled monolayer membrane 300 is performed (S140). In this case, when a large number of the hydrophilic self-assembled monolayers 210 are formed on the substrate 100, the hydrophobic self-assembled monolayers 220 may be distributed in small portions so that the protein solution does not form on the chip. That is, the protein 400 and the hydrophobic self-assembled monomolecular membrane 220 may be combined with each other to give a difference in contact angle between the self-assembled monomolecular membrane and the desired region.

Performing the steps S110 to S140 will be described the step of physically bonding the new self-assembled monolayer membrane 300 and protein 400. Hereinafter, the steps of chemically bonding the new self-assembled monolayer membrane 300 and the protein 400 will be described.

Mixed self-assembly Single molecule  Chemical Protein Binding Using Membrane>

Physically coupling the new self-assembled monolayer membrane 300 and protein 400 is similar to chemically binding. In the following, only the steps having a difference will be described.

As shown in Figures 2a to 2b, in the case of chemical bonding is performed step S110 to S140. However, after forming a new self-assembled monolayer membrane 300 (S130), it may be carried out further comprising the step of cross-linking (S135) to fix more of the protein 400 (S135). . At this time, the new self-assembled monomolecular membrane 300 is a hydrophilic self-assembled monomolecular membrane 210, and preferably has an NH 2 functional group or a COOH functional group.

Mixed self-assembly Single molecule  Protein Chips Using Membranes>

Protein chips using mixed self-assembled monolayer membranes can be made using the glass substrates described above or using gold substrates, and are produced by physical or chemical bonding. Such a protein chip is a biochip technology, in which high-density immobilization of related substances such as antibodies, receptors, nucleic acids, carbohydrates, and the like that can react with a specific protein. The ability to perform a series of protein analyses, ranging from protein isolation, quantification and functional analysis, to widespread use not only in the existing biotechnology industry but also in the field of protomixes that ultimately extend disease identification from the genetic to the protein level. Can be.

< Variation example >

As another embodiment of the present invention, in FIG. 2A, the ratio of the hydrophilic self-assembled monomolecular membrane 210 is absolutely high, but the ratio of the hydrophilic self-assembled monomolecular membrane 210 and the hydrophobic self-assembled monomolecular membrane 220 is shown. Can be combined in many ways. In addition, a certain ratio is not limited to one embodiment of the present invention, and one embodiment of the present invention is implemented by any ratio.

Although the present invention has been described with reference to the embodiment thereof, the present invention is not limited thereto, and various modifications and applications are possible. In other words, those skilled in the art can easily understand that many variations are possible without departing from the gist of the present invention.

100: substrate
200: self-assembled monomolecular membrane
210: hydrophilic self-assembled monolayer membrane
220: hydrophobic self-assembled monomolecular membrane
300: new self-assembled monolayer membrane
400: Protein

Claims (16)

Forming a self-assembled monomolecular film 200 in which the hydrophilic self-assembled monomolecular film 210 and the hydrophobic self-assembled monomolecular film 220 are mixed on the glass substrate 100 (S110);
Selectively removing the self-assembled monomolecular film 200 to be removed from the self-assembled monomolecular film 200 formed on the glass substrate 100 (S120);
Forming a new self-assembled monomolecular film 300 at a position where the self-assembled monomolecular film 200 is removed (S130);
Cross linking to fix the protein 400 more (S135); And
Comprising the step of binding the protein 400 to the new self-assembled monolayer membrane (S140); including,
The new self-assembled monolayer membrane (300) is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that the hydrophilic self-assembled monolayer membrane (210). delete The method of claim 1,
The self-assembled monolayer membrane 200 is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that formed on the entire surface of the glass substrate (100). delete The method of claim 1,
The hydrophobic self-assembled monolayer membrane (220) is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that it has a CH3 functional group or a CF3 functional group. The method of claim 1,
The hydrophilic self-assembled monolayer membrane 210 is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that it has an OH functional group. The method of claim 1,
The hydrophilic self-assembled monomolecular membrane 210 is formed more than the hydrophobic self-assembled monolayer membrane, characterized in that the protein binding method using a mixed self-assembled monolayer membrane. The method of claim 7, wherein
The hydrophobic self-assembled monolayer membrane (220) and the protein (400) is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that coupled to form a constant contact angle.
delete The method of claim 1,
The hydrophilic self-assembled monolayer membrane 210 is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that it has an NH 2 functional group or a COOH functional group. The method of claim 1,
Removing the self-assembled monolayer film 200 is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that the removal using any one of the plasma, UV laser, and UV lamp. The method of claim 1,
The combination of the self-assembled monolayer film 200 and the glass substrate 100 or the combination of the new self-assembled monolayer film 300 and the glass substrate 100 may use a silane-based self-assembled monolayer film. Protein binding method using a mixed self-assembled monolayer membrane characterized in that. The method of claim 1,
The glass substrate 100 is a protein binding method using a mixed self-assembled monolayer membrane, characterized in that provided by replacing with a gold substrate (100).
The method of claim 13,
The combination of the self-assembled monolayer film 200 and the glass substrate 100 or the combination of the new self-assembled monolayer film 300 and the glass substrate 100 uses a thiol-based self-assembled monolayer film. Protein binding method using a mixed self-assembled monolayer membrane.
A protein chip using a mixed self-assembled monomolecular membrane prepared by the bonding method according to any one of claims 1, 3, 5 to 8, 10 to 12. A protein chip using a mixed self-assembled monomolecular membrane prepared by the binding method according to any one of claims 13 and 14.
KR1020100091053A 2010-09-16 2010-09-16 Protein binding method using mixed self assembled monolayers and protein chip using thereof KR101324199B1 (en)

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Citations (4)

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KR20030088782A (en) * 2002-05-15 2003-11-20 삼성전자주식회사 A process for producing array plate for a biomolecule comprising a hydrophilic region and a hydrophobic region
US20050002985A1 (en) 2001-10-30 2005-01-06 Heal Richard David Albert Device with recessed tracks for forming a cellular network
US20050074898A1 (en) * 2002-07-31 2005-04-07 Caliper Technologies Corp. High density reagent array preparation methods
KR20110095503A (en) * 2010-02-19 2011-08-25 한국과학기술원 Bio chip and manufacturing method by using surface energy difference, methed for forming micro array pattern and method for dispensing sample

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050002985A1 (en) 2001-10-30 2005-01-06 Heal Richard David Albert Device with recessed tracks for forming a cellular network
KR20030088782A (en) * 2002-05-15 2003-11-20 삼성전자주식회사 A process for producing array plate for a biomolecule comprising a hydrophilic region and a hydrophobic region
US20050074898A1 (en) * 2002-07-31 2005-04-07 Caliper Technologies Corp. High density reagent array preparation methods
KR20110095503A (en) * 2010-02-19 2011-08-25 한국과학기술원 Bio chip and manufacturing method by using surface energy difference, methed for forming micro array pattern and method for dispensing sample

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