초록 ▼

○ 연구결과
본 연구진은 농·축산품에 함유된 항생제를 현장에서 검사할 수 있는 검출시스템을 개발하였다. 소고기, 돼지고기, 닭고기 및 달걀, 우유등에 함유된 Tetracycline 계열(Tetracycline, Chlorotetracycline,Oxytetracycline, Doxytetracycline), Chloramphenicol, Sulfadimethoxine 등을 검출할 수 있는 센서와 계측기 및 시료전처리 키트를 개발하였는데, 의료기기 진단 센서로 널리 이용되는 바이오센서를 항생제 측정 시스템에도 적용하였다. 대량

○ 연구결과
본 연구진은 농·축산품에 함유된 항생제를 현장에서 검사할 수 있는 검출시스템을 개발하였다. 소고기, 돼지고기, 닭고기 및 달걀, 우유등에 함유된 Tetracycline 계열(Tetracycline, Chlorotetracycline,Oxytetracycline, Doxytetracycline), Chloramphenicol, Sulfadimethoxine 등을 검출할 수 있는 센서와 계측기 및 시료전처리 키트를 개발하였는데, 의료기기 진단 센서로 널리 이용되는 바이오센서를 항생제 측정 시스템에도 적용하였다. 대량 생산형에 맞춘 전기화학 분석용 Lab-on-a-film 타입 칩과형광 분석용 래피드 테스트 칩이 개발 완료되었으며 이 칩을 측정할 수 있는 계측기 또한 시제품화하였다. 소고기, 돼지고기, 닭고기 등의 시료에서 항생제를 추출하기 위해 실험실이 아닌 현장에서도 사용할 수 있는 시료 전처리키트도 개발 완료되었으며, 간이전처리 키트와 모세관 전기영동(capillary electrophoresis)을 이용한 microfluidic 칩이 일체화 되어 항생제 검출이 가능한 칩 또한 개발 완료되었다. 칩을 제작하는데 필요한 원료로는 항체, 효소 및 형광물질 conjugate가 주로 사용되었지만 cellulose-dsDNA로도 검출 가능함을 확인하였고, 압타머 개발을 통해 항체 대체 물질로의 적용 가능성을 확인함으로써 대량 생산 적용 시 항체 성능의 변화로 칩 자체의 성능 변화를 줄일 수 있는 가능성을 확인하였다.

Abstract ▼

Project by i-SENS, Inc.
Antibiotics are widely used in stock farm animals for prevention and control of infectious disease by bacteria. Widespread use of antibiotics in animal husbandry, however, greatly increase the risk in human health due to the residual antibiotics left in meats. European Uni

Project by i-SENS, Inc.
Antibiotics are widely used in stock farm animals for prevention and control of infectious disease by bacteria. Widespread use of antibiotics in animal husbandry, however, greatly increase the risk in human health due to the residual antibiotics left in meats. European Union and several developed countries around the world have set ever tightened acceptable maximum residue limits (MRLs) for various antibiotics to minimize the health risk by increased antimicrobial resistance or the emergence of methicillin-resistant Staphylococcus aureus (MRSA). The new trend in tightening regulations demands accurate and fast analytical techniques with high specificity, sensitivity and simplicity for on-site analysis.
A novel microfluidic electrochemical enzyme immunoassay chip for the sensitive detection of tetracycline (TC), chloramphenical (CAP) and sulfadimethoxine (SDM) are described in detail in this report. In this work, lab-on-a-film (LOAF) chips with microfluidic channels were formed using photolithography and wet etching of dry photoresist film laminated on the poly-ethylene terephthalate (PET) substrate. The electrochemical detection system was made by printing carbon paste electrode on the substrate, immobilizing the antibody (Ab) for antibiotics on the carbon working electrode, and dispensing and drying alkaline phosphatase enzyme (ALP)-antigen (Ag) conjugate and the substrate on the path of microfluidic channel, respectively. The sample applied to the LOAF flows through the two divided fluidic channels; sample in one path carries the ALP-Ag conjugate on its way to the electrode (sample flow) and sample in another path (wash flow) subsequently carries the substrate for electrochemical enzymatic reaction while washes off the unreacted ALP-Ag conjugate from the electrode. This system determines the concentration of antibiotics through competitive immunoassay and a chronoamperometric means. The electrochemical detection could be made within 1 minute and total assay time was about 10 minutes. The LOAF could detect residual antibiotics in meats in MRL range with sufficiently high sensitivity. This LOAF chip-based method can be used to develop "easy-to-use" on-site antibiotic determination kit in food samples.
Codevelopment project by Hankyong University team
The present work describes the development of a simple and rapid sample extraction method and the design of sampling kit for determination of antibiotics (tetracycline, sulfadimethoxine and chloramphenicol) in meats including beef, pork and chicken. A simplified method described here includes the eliminations of homogenization, centrifugation and evaporation steps replaced by filter, syringe, and connector, respectively. The simplified method is validated by comparing the recovery rate of conventional method using high-performance liquid chromatography. Covariance(CV) values for all the examined tetracycline, sulfadimethoxine and chloramphenicol were lower than 5.0 % and all values of accuracy for tetracycline, sulfadimethoxine and chloramphenicol were within ±10%. Tetracycline, sulfadimethoxine and chloramphenicol were recovered by 70 to 120% from all kind of matrices and standard deviations of this recovery tests were below 10%. The recoveries of tetracycline and sulfadimethoxine were 70 to 120% similar to results from the previous methods.
Simple sample extraction kit that can be used in the field without much training was designed and applied for the quantitative determination of antibiotics in meats using the LOAF chip.
Codevelopment project by Pusan University/POSTECH team
Electrochemical sensing system, a new aptamer synthesis and spectral analysis method for measuring antibiotics were developed. Electrochemical sensing system was composed of the pretreatment appatus, electrochemical biosensor, immunosensor and multi channel microfluidic chip. We also developed SELEX systems using magnetic bead and affinity chromatography for in vitro selection of small molecules as well as large molecules. Through these systems, we searched new aptamers for kanamycin.
Codevelopment project by Hallym University team
The advantages of DDI (DNA-Directed Immobilization) method are combined with unique properties of 9G DNA membrane enabled quantitative detection of free SDM with high sensitivity and reproducibility in few minutes. A model system comprising SDM-DNA conjugate (sulfadimethoxine) and secondary Cy5-modified SDM antibody is firstly mixed in solution phase which contains free SDM then loaded on the 9G DNA Membrane to demonstrate proof-of-concept. The detection of free SDM in water is relied on competing with SDM-DNA conjugate,which plays the role as bridge between secondary Cy5-modified (SDM) antibody with oligonucleotide probe on 9G membrane, quantitative data are obtained by recording the optical responses of the test zone with a portable strip reader. In conventional method, SDM was immobilized by using protein-SDM conjugate (such as BSA), it exposed some disadvantages due to unstable properties of protein. However DNA surfaces are robust and stable, and also easy to be fabricated compared to protein surfaces. DNA-directed immobilization also allows
us to avoid long-term storage and harsh incubation and spotting processes of unstable proteins on various assay surfaces. Furthermore, 9G DNA membrane in which oligonucleotide probes are immobilized on self-assembled monolayer of supramolecular modified glass fiber (9G membrane) by recognizing consecutive 9Guanine bases of probe sequence forms vertical space between probes that enabled DNA target to hybridize with excellent efficiency(80% within 10min). The responds of introduced biosensor (SDM Membrane Test Kit)showed linear over the range of 1-1000ng/ml(SDM concentration) with a detection limit of 0.1ng/ml. By comparing with previous reports or similar products the sensor shows great promise of application for point-of-care or in-field detection of biotic molecules as well as proteins.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 8
• CONTENTS ... 11
• 목차 ... 12
• 제 1 장 연구개발과제의 개요 ... 14
• 제 1 절 연구개발의 최종 목표 ... 14
• 제 2 절 연구개발의 필요성 ... 14
• 1. 항생제 남용의 심각성 ... 14
• 2. 휴대형 농·축산품 잔류 항생제 검출 시스템의 필요성 ... 15
• 제 2 장 국내외 기술개발 현황 ... 17
• 제 1 절 국내외 기술개발 현황 ... 17
• 1. 바이오칩(Biochip) ... 17
• 2. 바이오멤스(Bio-MEMS; Bio Micro Electro Mechanical System) ... 18
• 제 2 절 개발 핵심 기술의 국내외 기술과의 차별성 ... 20
• 1. Lab-on-a-film 칩 ... 20
• 2. 전기영동을 이용한 마이크로플루이딕 칩 ... 21
• 3. 면역크로마토그래피 ... 21
• 4. 압타머 ... 22
• 제 3 장 연구개발 수행 내용 및 결과 ... 23
• 제 1 절 연구개발 수행 내용 및 결과 ... 23
• 1. 세부연구 (주)아이센스 ... 23
• 2. 협동연구 한경대학교 ... 36
• 3. 협동기관 부산대학교 ... 56
• 4. 협동연구 한림대학교 ... 75
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 95
• 제 1 절 연구개발 목표의 달성도 ... 95
• 1. 세부연구 (주)아이센스 ... 95
• 2. 협동연구 한경대학교 ... 96
• 3. 협동연구 부산대학교 ... 97
• 4. 협동연구 한림대학교 ... 99
• 제 2 절 관련분야에의 기여도 ... 99
• 1. 세부연구 (주)아이센스 ... 99
• 2. 협동연구 한경대학교 ... 100
• 3. 협동연구 부산대학교 ... 100
• 4. 협동연구 한림대학교 ... 100
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 101
• 제 1 절 연구 개발 성과 ... 101
• 1. 논문게재 성과 ... 101
• 2. 특허 성과 ... 101
• 3. 인력 지원 성과 ... 102
• 제 2 절 성과 활용 계획 ... 102
• 1. 실용화·산업화 계획 ... 102
• 2. 특허, 논문 등 지식재산권 확보계획 등 ... 103
• 3. 추가연구, 타연구에 활용 계획 등 ... 103
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 104
• 제 7 장 참고문헌 ... 108
• 끝페이지 ... 108