학술논문
항균 펩타이드를 이용한 병원성 대장균 검출 전기화학 바이오센서 개발 / Development of an antimicrobial peptide based electrochemical biosensor for the detection of Escherichia coli O157:H7
Document Type
Dissertation/ Thesis
Author
Source
Subject
Language
English
Abstract
식중독은 선진국과 개발도상국 모두에서 두드러지게 나타나는 전 세계적인 공중보건 문제이다. 특히 E. coli O157:H7은 소량으로도 급성 감염을 일으킬 수 있는 식품 매게 병원체로, 이에 대한 예방을 위해서 항균제 개발과 함께 민감하고 신속한 검출 방법의 확립이 필수적이다. 기존의 E. coli O157:H7 검출 방법으로는 집락 형성 단위 측정, 효소 결합 면역흡착 분석, 중합효소 연쇄반응 등이 있지만, 이러한 방법들은 검출 시간이 오래 걸리고, 환경에 영향을 많이 받아 불안정하며, 전문가의 숙련된 조작 및 고가의 장비가 필요하다는 한계가 있다. 또한, 검출을 위해 사용되는 대표적인 생체 인식 요소인 항체, 효소는 열악한 환경에서의 안정성과 낮은 재현성 등 다양한 한계점을 가지고 있다. 본 연구에서는 이러한 한계를 극복하여, E. coli O157:H7을 신속하고 민감하게 분석할 수 있는 항균 펩타이드 기반 전기화학 바이오센서를 개발하였다. 구체적으로, 펩타이드 공학을 통해 magainin 1과 clavanin A에서 유래한 세 가지 항균 펩타이드를 도출하였고, 항균 활성 분석을 통해 E. coli O157:H7에 대해 가장 높은 항균 활성을 보인 AMP 3을 생체 인식 요소로 선정하였다. 항균 활성은 박테리아 성장 시간별 탁도 측정과 최소 억제 농도 (MIC) 측정을 통해 확인하였고, 시각적으로는 공초점 레이저 주사 현미경 (CLSM)과 주사전자현미경 (FE-SEM)을 통해 확인하였다. 민감하고 선택적인 검출을 위해 금 전극 (Au)에 친수성 탄닌산 (TA)-폴리에틸렌이민 (PEI) 하이드로겔을 개질하고, 자외선 가시광선 분광법, X선 광전자 분광법 (XPS), 주사전자현미경 (FE-SEM), 물 접촉각 (WCA), 원자간력 현미경 (AFM), 차동 펄스 전압전류법 (DPV)을 사용하여 특성화 실험을 진행했다. 이후, AMP 3을 커플링 화학법을 활용하여 하이드로겔에 고정화하여 AMP 기반 전기화학 센서를 개발했다. 개발된 Au/TA-PEI/AMP 3 바이오 센서의 전기 화학적 성능은 순환 전압전류법 (CV)과 차동 펄스 전압전류법 (DPV)을 통해 모니터링하였다. 그 결과, 최적의 반응 조건에서 3.4 CFU/mL의 검출 한계와 101-105 CFU/mL의 선형 검출 범위를 보였다. 또한, 바이오센서를 활용하여 수돗물 및 우유 시료에서 E. coli O157:H7을 정량적으로 검출한 결과, 89.1~122.0%의 우수한 회수율을 달성하였다. 하지만, 현장에서의 실제 적용을 위해 MXene 및 그래핀과 같은 나노 소재를 접목하여 전도성을 향상시키고, 특이성을 높이기 위해 항균 펩타이드의 추가적인 설계가 필요하다. 전반적으로, 개발된 AMP 기반 바이오센서는 E. coli O157:H7 검출에 있어 우수한 민감도와 재현성을 보였으며, 이는 공중 보건 및 안전에 위협이 되는 박테리아를 보다 민감하고 효과적으로 검출하는 데 기여할 것으로 기대된다.
Foodborne diseases are a worldwide public health problem affecting both developed and underdeveloped countries. Escherichia coli O157:H7, which can cause acute infections even in extremely small numbers, is the most common pathogen responsible for foodborne diseases. Both the development of antimicrobial agents and establishment of a sensitive and rapid detection method are essential to prevent foodborne diseases. Previously used methods for detecting E. coli O157:H7 include colony-forming unit (CFU) assays, enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), however, the use of these methods is limited by long detection times, instability, and the need for skilled operators and expensive equipment. In addition, antibodies and enzymes, the typical bioreceptors used for detection, suffer from poor stability in harsh environments, the need for sample preparation before analysis, and low reproducibility. In this study, we developed an antimicrobial peptide (AMP)-based electrochemical biosensor for the rapid and sensitive detection of E. coli O157:H7 to overcome these limitations. Specifically, three AMPs were derived from magainin 1 and clavanin A through peptide engineering. Subsequently, AMP 3, which showed the highest antimicrobial activity against E. coli O157:H7 in antibacterial activity assays, was screened as a potential candidate bioreceptor. The antimicrobial activity was determined by turbidimetric measurements and minimum inhibitory concentration (MIC), and visually by confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FE-SEM). To achieve selective and sensitive detection, a hydrophilic tannic acid (TA)-polyethyleneimine (PEI) hydrogel was modified on a gold electrode (Au), and characterization experiments were performed using UV-visible spectroscopy, X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FE-SEM), water contact angle (WCA), and atomic force microscopy (AFM). Subsequently, AMP 3 was immobilized into the hydrogel using coupling chemistry to develop an AMP-based electrochemical sensor. The electrochemical performance of the Au/TA-PEI/AMP 3-based electrochemical biosensor was monitored using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results showed a detection limit of 3.4 CFU/mL and a linear detection range of 101-105 CFU/mL. Furthermore, the biosensor was used to detect E. coli O157:H7 quantitatively in tap water and milk samples, with excellent recovery rates ranging from 89.1 to 122.0%. Overall, because the developed AMP-based biosensor exhibits a good sensitivity and reproducibility in the detection of E. coli O157:H7, the method is supposed to be a potential tool for detecting bacteria that pose a threat to safety and public health.
Foodborne diseases are a worldwide public health problem affecting both developed and underdeveloped countries. Escherichia coli O157:H7, which can cause acute infections even in extremely small numbers, is the most common pathogen responsible for foodborne diseases. Both the development of antimicrobial agents and establishment of a sensitive and rapid detection method are essential to prevent foodborne diseases. Previously used methods for detecting E. coli O157:H7 include colony-forming unit (CFU) assays, enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), however, the use of these methods is limited by long detection times, instability, and the need for skilled operators and expensive equipment. In addition, antibodies and enzymes, the typical bioreceptors used for detection, suffer from poor stability in harsh environments, the need for sample preparation before analysis, and low reproducibility. In this study, we developed an antimicrobial peptide (AMP)-based electrochemical biosensor for the rapid and sensitive detection of E. coli O157:H7 to overcome these limitations. Specifically, three AMPs were derived from magainin 1 and clavanin A through peptide engineering. Subsequently, AMP 3, which showed the highest antimicrobial activity against E. coli O157:H7 in antibacterial activity assays, was screened as a potential candidate bioreceptor. The antimicrobial activity was determined by turbidimetric measurements and minimum inhibitory concentration (MIC), and visually by confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FE-SEM). To achieve selective and sensitive detection, a hydrophilic tannic acid (TA)-polyethyleneimine (PEI) hydrogel was modified on a gold electrode (Au), and characterization experiments were performed using UV-visible spectroscopy, X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FE-SEM), water contact angle (WCA), and atomic force microscopy (AFM). Subsequently, AMP 3 was immobilized into the hydrogel using coupling chemistry to develop an AMP-based electrochemical sensor. The electrochemical performance of the Au/TA-PEI/AMP 3-based electrochemical biosensor was monitored using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results showed a detection limit of 3.4 CFU/mL and a linear detection range of 101-105 CFU/mL. Furthermore, the biosensor was used to detect E. coli O157:H7 quantitatively in tap water and milk samples, with excellent recovery rates ranging from 89.1 to 122.0%. Overall, because the developed AMP-based biosensor exhibits a good sensitivity and reproducibility in the detection of E. coli O157:H7, the method is supposed to be a potential tool for detecting bacteria that pose a threat to safety and public health.