KR20230138692A - Ferrocene-based acrylate copolymer multilayer structure with efficient antifouling and electrochemical redox reaction properties and preparation method thereof - Google Patents

Abstract

The present invention relates to a ferrocene-based acrylate copolymer multilayer film structure with improved antifouling and electrochemical redox reaction properties and a method for manufacturing the same. More specifically, the present invention relates to 2-dimethyl aminoethyl methacrylate (DAMA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) ) and a second layer containing methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). It relates to a multilayer film structure including a bilayer made of and a method of manufacturing the same by a layer by layer coating method.

Description

Ferrocene-based acrylate copolymer multilayer structure with efficient antifouling and electrochemical redox reaction properties and preparation method thereof}

The present invention relates to a ferrocene-based acrylate copolymer multilayer film structure with improved antifouling and electrochemical redox reaction properties and a method for manufacturing the same. More specifically, the present invention relates to 2-dimethyl aminoethyl methacrylate (DAMA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) ) and a second layer containing methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). It relates to a multilayer film structure including a bilayer made of and a method of manufacturing the same by a layer by layer coating method.

Electrochemical biosensors have recently been in the spotlight in the biosensor market as sensors that are familiar to consumers due to their smaller device size and cost savings compared to optical sensors. Electrochemical biosensors basically have an electrode made of metal formed inside a channel, and a biological material that can specifically react with the substance to be detected is immobilized on a metal plate. In order to immobilize organic biological materials on the inorganic metal surface, treatment with chemicals such as self-assembled monolayer (SAM) is required.

However, it is known that by forming a self-assembled monolayer on the electrode, the electrically active material cannot reach the electrode surface due to the self-assembled monolayer and the electrochemical signal is reduced through non-specific binding with the self-assembled monolayer (K.L. Prime & G. M. Whitesides, J. Am. Chem. Soc. 1993, 115, 10714-10721; W. Senaratne, L. Andruzzi & C. K. Ober, Biomacromolecules. 2005, 6, 2427-244; S. Ko, B. Kim, S.S. Jo, S.Y. Oh, J.K. Park, BiosensBioelectron. 2007, 23, 51-59.). In addition, immobilization of biomaterials to the electrode surface not only acts as a factor that impedes the flow of the electrode or reduces the intensity of the signal through non-specific binding to the electrically active material, but also causes the biomaterial itself to act as a factor that impedes the flow of the electrode. It can bind non-specifically to the electrode surface without any surface treatment, thereby inhibiting the recognition of electrically active materials. As a result, interference with the movement of electrically active materials by self-assembled monolayers and biomaterials reduces the sensitivity of electrochemical biosensors.

In order to restore the reduced sensitivity of electrochemical sensors, methods have been implemented to control electrode fouling caused by non-specific binding of biological materials by coating the surface of the electrode with conductive or non-conductive, natural, ionic polymers, etc. As an example, Republic of Korea Patent No. 10-2067689 discloses a ferrocene-based functional polymer that has bioreactive functionality for binding biomolecules, anti-biofouling functionality, and can control redox active groups. However, by using a layer by layer coating method, a multilayer membrane structure containing a bilayer made of each copolymer is used to fundamentally prevent electrode contamination due to non-specific binding of biomaterials and improve the sensitivity of the sensor. There is still no known way to increase it.

Accordingly, the present inventors made diligent efforts to develop a multilayer film structure with excellent antifouling and electrochemical redox reaction properties, and as a result, the ITO substrate was made of 2-dimethyl aminoethyl methacrylate (DAMA) and ferrocenyl methyl methacrylate. A first layer containing acrylate (Ferrocenyl methyl methacrylate, FMMA) and polyethylene glycol methacrylate (PEG), and methacrylic acid (MAA), Ferrocenyl methyl methacrylate, When coated with a multilayer film containing a bilayer consisting of FMMA) and a second layer containing polyethylene glycol methacrylate (PEG), the adsorption of biomaterials is significantly reduced compared to the control group (Bare ITO). In addition, after confirming that the electrical signal increased, the present invention was completed.

The present invention includes 2-dimethyl aminoethyl methacrylate (DAMA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). A double layer consisting of a first layer and a second layer containing methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) The purpose is to provide a multilayer film structure including a bilayer.

The present invention also provides a) a substrate phase comprising 2-dimethyl aminoethyl methacrylate (DAMA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate. , coating with a first solution containing PEG), and

b) The substrate coated with the first solution is coated with methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). The purpose is to provide a method for manufacturing a multilayer film structure including the step of coating with a second solution.

A schematic diagram of the ferrocene-based acrylate copolymer multilayer film structure with improved antifouling and electrochemical redox reaction properties according to the present invention and its manufacturing method is shown in Figure 1.

The ferrocene-based acrylate copolymer multilayer film structure according to the present invention can not only significantly reduce the adsorption of biomaterials, but also increase the electrical signal. Therefore, it is expected that it will be possible to fundamentally prevent sensor electrode contamination and increase sensitivity.

Figure 1 shows a schematic diagram of a ferrocene-based acrylate copolymer multilayer film structure with improved antifouling and electrochemical redox reaction characteristics and a method for manufacturing the same according to the present invention.
Figure 2 shows the results of ¹ H-NMR analysis of the FP acrylate copolymer prepared according to Example 1.
Figure 3 shows the composition, molecular weight, and dispersion degree of the FP acrylate copolymer prepared according to Example 1 as confirmed through size exclusion chromatography (SEC, GPC).
Figure 4 shows the contact angle of the FP acrylate copolymer laminated on the ITO substrate of the multilayer thin film prepared according to Example 2.
Figure 5 shows the thickness, surface charge, and surface roughness of the multilayer thin film prepared according to Example 2.
Figure 6 shows the anti-biofouling effect of the multilayer thin film prepared according to the present invention according to Experimental Example 1.
Figure 7 shows the electrochemical effect of the multilayer thin film prepared according to the present invention according to Experimental Example 2.
Figure 8 shows the electrochemical effect of the multilayer thin film prepared with (FP2)10 according to Experimental Example 2.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms, but the present embodiments only serve to ensure that the disclosure of the present invention is complete and are within the scope of common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims. Hereinafter, the multilayer film structure and its manufacturing method according to the present invention will be described in detail.

According to the first implementation example,

The present invention relates to (A) 2-dimethyl aminoethyl methacrylate (DAMA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) A first layer comprising, and (B) methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). The purpose is to provide a multilayer film structure including a bilayer consisting of a second layer.

In the multilayer film structure according to the present invention, the molar ratio of DAMA:PEG:FMMA in the first layer is 10 to 1.25:2.5 to 1.5:1, and the molar ratio of MAA:PEG:FMMA in the second layer is 10 to 1.25: It is characterized by 2.5 ~ 1.5: 1. Preferably, the molar ratio of DAMA:PEG:FMMA is 2.5:1.5:1, and the molar ratio of MAA:PEG:FMMA is 2.5:1.5:1.

In the multilayer film structure according to the present invention, the multilayer film structure is characterized in that it includes 1 to 10 double layers consisting of a first layer and a second layer. Preferably, the multilayer film structure may include 2 to 5 double layers. More preferably, the multilayer structure includes two double layers.

In the multilayer film structure according to the present invention, the multilayer film structure is used in an electrochemical sensor.

According to the second implementation,

The present invention seeks to provide a method for manufacturing a multilayer film structure, which method includes:

a) The substrate was coated with 2-dimethyl aminoethyl methacrylate (DAMA), Ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). coating with a first solution comprising, and

b) The substrate coated with the first solution is coated with methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). Characterized by comprising the step of coating with a second solution.

In the method for manufacturing a multilayer film structure according to the present invention, the molar ratio of DAMA:PEG:FMMA in the first solution is 10 to 1.25:2.5 to 1.5:1, and the molar ratio of MAA:PEG:FMMA in the second solution is 10. It is characterized by ~1.25:2.5 ~1.5:1. Preferably, the molar ratio of DAMA:PEG:FMMA is 2.5:1.5:1, and the molar ratio of MAA:PEG:FMMA is 2.5:1.5:1.

In the method of manufacturing a multilayer film structure according to the present invention, the substrate is an ITO (indium tin oxide) substrate treated with oxygen plasma.

In the method for manufacturing a multilayer film structure according to the present invention, the method is characterized in that steps (a) and (b) are repeated 2 to 10 times. Preferably, the method is characterized by repeating steps (a) and (b) 2 to 5 times. More preferably, the method is characterized by repeating steps (a) and (b) twice.

In the method of manufacturing a multilayer film structure according to the present invention, the step (a') of washing and drying the coated substrate is further included after step (a).

In the method of manufacturing a multilayer film structure according to the present invention, the step (b') of washing and drying the coated substrate is further included after step (b).

Hereinafter, various embodiments are presented to aid understanding of the invention. The following examples are provided to make the invention easier to understand, and the scope of protection of the invention is not limited to the following examples.

<Substance>

500g/mol) 및 2-디메틸 아미노에틸 메타크릴레이트(DAMA, 99%, Merck)를 저해인자 제거 컬럼(inhibitor removal column)을 통과시킨 후 랜덤 공중합체(CPF 및 APF 시리즈)를 제조하는데 사용하였다. 2.2'-아조비즈(2-메틸프로피오니트릴)(AIBN, 99%, Junsei)는 메탄올(MeOH, 99.8%, Daejung)에서 재결정화시킨 후 사용하였다. 테트라히드로푸란(THF, 99.9%, Merck), 탈이온수(DIW, HyClone), 인산완충생리식염수(PBS, HyClone), 디메틸 설폭사이드(DMSO-d₆, 99.9%, Merck)를 추가 정제 없이 용매로 사용하였다. ITO-코팅 유리는 U.I.D. (한국, 면저항 = 15 ± 3 Ω/sq)에서 구매하였다. 소 혈청 알부민(BSA, biotechnology grade, Bioshop) 및 알부민-플루오레세인 이소티오시아네이트 컨쥬게이트(Albumin-FITC, Merck)를 사용하여 특정 농도의 PBS에서 용해시킨 후 단백질 흡착 특성을 특성화하였다.Ferrocenyl methyl methacrylate (FMMA, 95%, Aaron Pharmatech Ltd.), methacrylic acid (MAA, 99%, Merck), polyethylene glycol methacrylate (PEG, 99%, Merck, Mn)

500 g/mol) and 2-dimethyl aminoethyl methacrylate (DAMA, 99%, Merck) were used to prepare random copolymers (CPF and APF series) after passing through an inhibitor removal column. 2.2'-Azobeads (2-methylpropionitrile) (AIBN, 99%, Junsei) were used after recrystallization in methanol (MeOH, 99.8%, Daejung). Tetrahydrofuran (THF, 99.9%, Merck), deionized water (DIW, HyClone), phosphate buffered saline (PBS, HyClone), and dimethyl sulfoxide (DMSO-d ₆ , 99.9%, Merck) were used as solvents without further purification. used. ITO-coated glass was purchased from UID (Korea, sheet resistance = 15 ± 3 Ω/sq). Bovine serum albumin (BSA, biotechnology grade, Bioshop) and albumin-fluorescein isothiocyanate conjugate (Albumin-FITC, Merck) were used to characterize protein adsorption properties after dissolution in PBS at specific concentrations.

<Example>

Example 1. Preparation of ferrocene-based copolymer

1-1. Preparation of FP acrylate copolymer

Methacrylic acid (MAA) or 2-dimethyl aminoethyl methacrylate (DAMA) as the first monomer, ferrocenyl methyl methacrylate (FMMA) as the second monomer, and polyethylene glycol methacrylate (PEG) as the third monomer. FP acrylate copolymer was prepared using free radical polymerization. First, MAA, DAMA, and PEGMA were passed through an inhibitor removal column to remove monomeric inhibitors. The CFP and AFP series were defined based on the mole of ferrocene terminal monomer and were prepared according to the contents shown in Table 1 below.

FPs M.A.A. DAMA PEGMA FMMA CFP1 5mmol 3mmol 0.5mmol CFP2 5mmol 3mmol 2mmol CFP3 5mmol 3mmol 4mmol AFP1 5mmol 3mmol 0.5mmol AFP2 5mmol 3mmol 2mmol AFP3 5mmol 3mmol 4mmol

Specifically, MAA (5 mmol) or DAMA (5 mmol), FMMA (0.5 mmol), PEGMA (3 mmol), and AIBN (0.12 mmol) were each dissolved in 14 mL THF, the reaction mixture was sealed, and then degassed with an Ar gas stream for 10 min. Then, it was maintained at 70°C for 24 hours using a preheated oil bath. After copolymerization, the obtained polymer solution (20% concentration) was rapidly cooled and stored at 4°C.

1-2. Characterization of FP acrylate copolymer

The product was precipitated three times with an excess of hexane, dried overnight in a vacuum oven at room temperature, and the composition, molecular weight, and dispersion of the copolymer were determined through nuclear magnetic resonance ( ^1H -NMR) and size exclusion chromatography (SEC, GPC). The degree was confirmed. Number-average molecular weight (Mn) and dispersion (ÐM) were measured using GPC (Agilent 1200S/Agilent 1200S) equipped with three columns (Agilent PLgel MIXED-C*2, Agilent PLgel MIXED-E*1, Agilent Technologies) and a refractive index (RI) detector. Measurements were made using miniDAWN TREOS; Agilent Technologies). As an elution solvent, THF (HPLC grade, Merck) was used at a rate of 1 mL per minute at 30°C. Calibration was made with polystyrene narrow standards (PS narrow standards) (6.30×10 ² to 5.08×10 ⁵ g/mol).

As a result, as confirmed by ¹ H-NMR using DMSO-d ₆ as a solvent, it was confirmed that the copolymer was successfully synthesized according to the monomer feed (FIG. 2): δ = 4.8 (br, 2H, CO ₂ - CH ₂ of FMMA), 4.4 - 4.1 (br, 9H of FMMA), 4.1 (br, 2H; CO ₂ -CH ₂ of PEGMA), 3.79 - 3.58 (br, 30H; PEGMA), 1.82 - 1.90 (C-CH ₃ of MAA), 2.33 (N-CH ₃ of DAMA), 3.7 - 3.3 (br, 20H), 2.7 - 2.5 (br, 18H), 2.0 - 1.7 (br, 15H), 1.1 - 0.8 (br, 17H) . In addition, as the content of FMMA in the copolymer increased, the molecular weight and dispersion were found to significantly increase (Figure 3).

Example 2. Preparation of multilayer thin film using FP acrylate copolymer

2-1. Manufacture of multilayer thin film by layer by layer coating method

Multilayer thin films containing 1, 3, 5, and 10 FP1 bilayers using AFP1 and CFP1 copolymers, FP2 bilayers using AFP2 and CFP2 copolymers, and 1, 3, 5, and 10 FP3 bilayers using AFP3 and CFP3 copolymers were prepared, respectively.

First, an ITO substrate with a size of 1.5 × 1.5 (cm) was prepared, and then the surface of the substrate was treated with oxygen plasma to create a negative charge on the surface for 5 minutes. After completely removing the THF solvent of the CFP and AFP polymer solution prepared in Example 1 in a vacuum oven, the polymer was dissolved in DIW and the concentration of the new coating solution was adjusted to 1.0 mg/mL (0.1%). Next, the negatively charged ITO substrate surface was dip coated in the AFP coating solution for 10 minutes to give it a (+) charge, washed three times with DIW, and dried in the air. The AFP-coated ITO substrate was immersed in the CFP coating solution for 10 minutes to give it a negative charge, then rinsed with DIW three times and dried in the same manner to stack one bilayer (FP1). The same coating process as above was repeated to stack 3 bilayer (FP3), 5 bilayer (FP5), and 10 bilayer (FP10) thin films, respectively. To check whether the FP acrylate copolymers were well coated on the ITO substrate, the contact angle (WCA, θ) was measured in water, and a bare ITO substrate (contact angle: 66.8°) was used as a control.

As a result, as the content of FMMA in the FP acrylate copolymer increased, the value of the contact angle increased for the same number of bilayers. Also, even if coated with the same polymer, the content of FMMA increased as the number of bilayers increased, so the value of the contact angle increased significantly. It was found that it does. Meanwhile, it was confirmed that the contact angle value did not show a significant difference starting from 5 bilayers (Figure 4).

2-2. Confirmation of properties of multilayer thin film manufactured by layer coating method

(1) Surface element analysis of multilayer thin films

The surface elements of the multilayer thin film produced by the layer coating method were confirmed using XPS.

Atomic% C (1s) N (1s) O(1s) In (3d5) Sn (3d5) Fe (2p3) Bare ITO 44.19 0.45 38.23 15.17 1.96 - (FP2)1 46.04 0.84 40.89 10.83 1.40 - (FP2)3 74.82 1.21 23.62 0.14 - 0.21 (FP2)5 74.57 0.99 24.12 - - 0.31 (FP2)10 72.13 1.08 26.54 - - 0.25

As a result, as can be seen from Table 2 above, the main peaks of the bare ITO substrate were 3d (445 or 452 eV), Sn3d (487 or 495 eV), and O1 (530 eV), and the bare ITO substrate was It was confirmed that C1s (293 eV) and N1s (408 eV) peaks appeared at 285 eV due to small organic contaminants that were not removed during the cleaning process. Meanwhile, in the case of the multilayer thin film according to the present invention, C1s gradually increases from (FP2)1 to (FP2)10 to 46.0-72.1 atomic%, N1s to 0.8-1.1 atomic%, and Fe2p to 0-0.3 atomic%. In particular, as the number of layers of the FP2 bilayer on the ITO substrate increases, the In3d and Sn3d peaks disappear, and the FP acrylate copolymer is stably coated on the ITO substrate by the layer-by-layer coating method, effectively It was confirmed that surface modification had occurred.

(2) Thickness, surface charge and surface roughness analysis of multilayer thin films

The thickness, surface charge, and surface roughness of the multilayer thin film manufactured by the layer coating method were confirmed using DLS (ELS-Z2, Otsuka Electronics Co.) equipment. As a result, the thickness increased depending on the number of bilayer stacks, and the surface polarity was found to change by performing (+) polar coating and (-) polar coating, respectively. Additionally, it was confirmed that the surface roughness decreased as the number of coating layers increased (Figure 4).

<Experimental example>

Experimental Example 1. Analysis of anti-biofouling effect of multilayer thin film manufactured by layer coating method

BSA and albumin-FITC were selected to evaluate the degree of adsorption of unspecified proteins on bare ITO substrates and ITO substrates coated with (FP2)10. First, drop a 10 mg/mL BSA solution on the surface of a 1.5 The substrates were washed several times with PBS and DIW and completely dried at room temperature. Surface samples were observed using a field emission scanning electron microscope (FE-SEM; MiRa3, TESCAN, Czech Republic), and all samples for SEM analysis were prepared after air drying and platinum coating. (FP2)10 To evaluate the effective antibiofouling effect of the coated ITO substrate, 0.5 mg/mL albumin-FITC solution was dropped at 5 μm intervals on the PDMS mold patterned in the form of dots and pre-dried for 10 seconds. I ordered it. Each surface of the bare ITO substrate and the (FP2)10 coated ITO substrate was coated with a constant load using a PDMS mold to which albumin-FITC was applied. After adsorbing the fluorescent protein for 1 hour at room temperature in the dark, all specimens were soaked in PBS and DIW. After washing several times, it was dried naturally. The degree of albumin-FITC absorption on the substrate surface was analyzed using a fluorescence microscope (Eclipse NI-U; Nikon, Tokyo, Japan) equipped with a lens optimized for green fluorescent dye and a general filter.

As a result, BSA and albumin-FITC were detected in the case of the bare ITO substrate, whereas BSA and albumin-FITC were hardly confirmed in the case of the ITO substrate coated with (FP2)10, indicating that the FP acrylate copolymer according to the present invention The excellent anti-biofouling effect of was proven (Figure 6).

Experimental Example 2. Analysis of electrochemical effect of multilayer thin film prepared by layer coating method

The electrochemical effects of the bare ITO substrate and the ITO substrate coated with FP were measured at a scan rate of 100 mV s ^-1 in 0.1 M PBS as a supporting electrolyte using cyclic voltammetry (CV).

As a result, in the case of layered coating of FP polymer, the efficacy was found to increase as the number of layers increased (Figure 7). In particular, when coated with (FP2)10 polymer, a large signal was generated depending on the scan rate. Reproducibility was shown to be excellent as the square root value was close to 1 (Figure 8). Therefore, when the redox level, coating stability, and reproducibility were comprehensively checked when current was applied, coating using FP2 polymer was confirmed to be the most stable.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

(A) Containing 2-dimethyl aminoethyl methacrylate (DAMA), Ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) first floor, and
(B) Bilayer consisting of a second layer containing methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG) ) A multilayer film structure comprising a.
According to paragraph 1,
The molar ratio of DAMA: PEG: FMMA in the first layer is 10 to 1.25: 2.5 to 1.5: 1, and the molar ratio of MAA: PEG: FMMA in the second layer is 10 to 1.25: 2.5 to 1.5: 1. A multilayer film structure.
According to paragraph 2,
The molar ratio of DAMA:PEG:FMMA is 2.5:1.5:1, and the molar ratio of MAA:PEG:FMMA is 2.5:1.5:1.
According to paragraph 1,
The multilayer film structure is characterized in that it includes 1 to 10 double layers consisting of a first layer and a second layer.
According to paragraph 1,
The multilayer film structure is characterized in that it is used in an electrochemical sensor.
a) The substrate was coated with 2-dimethyl aminoethyl methacrylate (DAMA), Ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). coating with a first solution comprising, and
b) The substrate coated with the first solution is coated with methacrylic acid (MAA), ferrocenyl methyl methacrylate (FMMA), and polyethylene glycol methacrylate (PEG). A method of manufacturing a multilayer film structure, comprising the step of coating with a second solution.
According to clause 6,
The molar ratio of DAMA: PEG: FMMA in the first solution is 10 to 1.25: 2.5 to 1.5: 1, and the molar ratio of MAA: PEG: FMMA in the second solution is 10 to 1.25: 2.5 to 1.5: 1. A method of manufacturing a multilayer film structure.
In clause 7,
The molar ratio of DAMA:PEG:FMMA is 2.5:1.5:1, and the molar ratio of MAA:PEG:FMMA is 2.5:1.5:1.
In clause 7,
A method of manufacturing a multilayer film structure, wherein the substrate is an ITO (indium tin oxide) substrate treated with oxygen plasma.
In clause 7,
The method is characterized in that steps (a) and (b) are repeated 2 to 10 times.