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난치성 질환 유전자 치료제 개발 사업단에서는 암 그리고 선천성 유전질환 등의 난치성질환 극복을 위하여 다음의 과제를 진행하여 그 결과를 보고하고자 한다. 1. 종양 선택적 아데노바이러스를 이용한 두경부암 치료용 신약 개발종양 선택적 아데노바이러스 (DWP418)는 암세포에만 활성화되어 있는 효소인 텔로머라제 (Telomerase)가 활성화되어 있는 세포에서만 복제가 이루어지며, 그 결과 암세포만을선택적으로 살상하는 효과를 가져 주변 정상세포에는 독성이 없는 표적 지향적 암치료제이다. 또한 항종양 효과를 증대시킬 수 있는 릴렉신 (Relaxin) 유전자를 DWP418 내에 탑재하여, 바이러스의 암 조직내 침투력 및 확산기능을 향상시켜 효과적인 암세포 파괴를 가능케하는 새로운 항암 유전자치료제이다. DWP418의 약효를 확인하기 위해 뇌종양과 간암, 자궁암, 폐암, 두경부암에 걸린 쥐의 종양부위에 주사한 결과, 60일 이후 모든 암에서 90% 이상의 암세포가 사멸하는 것을 관찰할 수 있었다. 전임상 독성시험을 통해 실험동물에서 중대한 부작용은 없는 것을 확인하였다. 이러한 결과를 바탕으로 식약청 승인을 받아 기존 치료에 불응하는 고형암 환자를 대상으로 연세 암센터에서 임상 1상을 진행 중이다. 2. 종양 및 종양내피세포 선택적 암 유전자치료제 개발항종양혈관생성 활성이 있는 LK8 유전자를 발현하는 새롭고 우수한 종양선택적 살상아데노바이러스를 제작하여, 증진된 항암효과를 갖춘 항암유전자치료제를 개발하고 있다. 피하 비소세포폐암과 동소이식 비소세포폐암 동물모델에서 통계적으로 유의하고 우수한 항종양 효과를 확인하였으며, LK8 유전자의 동시 발현에 따른 증진된 항암효과를 확인하였다. 현재 생산 공정의 확립과 scale-up 하였으며 앞으로 전임상 시험 진행예정이다. 3. TRAIL 발현 아데노바이러스를 이용한 암유전자 치료제 개발 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)은 강력한 항암 후보 물질이다. 뇌암을 타겟으로하여 secretable timeric TRAIL(stTRAIL)을 발현하는 denovirus를 개발하였으며, 동물 모델에서 항암 억제 효과가 탁월함을 보았다. 단독으로 뿐만 아니라 항암제나 방사선 치료와 병용 투여 시에 그 효과가 증가하였다. 이런 결과를 바탕으로 하여 뇌암에 대한 1상 임상시험을 시작할 예정이다. 4. 선천성 질환 유전자 치료제 개발면역 결핍질환인 만성육아종 질환 환자의 혈액줄기세포에 정상 유전자를 도입하여 환자에게 다시 이식함으로써 면역기능을 회복시키는 유전자 치료제 Retro-CGD® (VM106)를 개발하였다. 현재 서울대학교 병원에서 1/2상 임상시험을 진행 중이다. 안전성을 확인하기 위하여 환자의 체내 복제가능 레트로바이러스 존재 확인 시험 및 정상단백질에 대한 항체 존재 확인 시험, 레트로바이러스 벡터 삽입양상 확인 시험을 진행하고 있고 현재까지 치료제와 관련된 이상반응은 관찰되지 않았다. 또한 만성육아종 질환 환자의 유전자 분석 및 진단을 위한 분자생물학적 기법을 확립하고 이 기법을 이용하여 만성육아종질환 환자를 대상으로 유전자 분석을 실시하여 질환과 관련된 주요 유전자 돌연변이에 대한 국내 database를 구축하였다.

단백질 산업은 유용 단백질을 동정, 대량 생산하여 생물의약, 생물화학, 바이오 식품, 농약 등 다양한 분야에 응용하는 산업으로 포스트게놈 시대의 초고속 성장 핵심 동력으로 글로벌 연구프로젝트의 표적이 되고 있으며, 특히 최근, 식물세포와 형질전환 식물체를 이용한 고부가가치 단백질 생산기술 개발은 생명공학 BT분야의 새로운 주연구 분야로 관심이 집중되고 있고 최근에 많은 관련 생명공학 기업들과 연구자들이 “molecular farming” 분야에 관심을 집중시키고 있다. 식물체 또는 식물세포 이용 기술이 보다 유리한 점은 낮은 생산비로 식물세포 배양 시 동물세포에 비해 단백질 생산 비용이 매우 낮으며, 식물체 경작 시 단위면적당 높은 수확량과 생산 규모의 확대가 용이하며, 환경 친화적인 특성으로 식물유래 단백질에 대한 높은 선호도로 대중적 인지도가 높으며 동물성 바이러스, 세균, 독소 및 프리온 등으로부터 안전하고 유전적으로도 안전성이 높고 순수 분리□정제가 용이하여 생산성 향상과 비용 절감의 효과를 얻을 수 있다. 이와 같은 무한한 가능성과 잠재성으로 향후 전망이 밝으며, “그린의약품”이 2020년 한국의 유망산업으로 선정되어 개발 사업이 본격적으로 진행되고 있다. 본 사업의 목표는 식물 세포주를 이용하여 저비용□고부가가치 단백질 생산체계를 확립하여 국내 단백질 제품의 수요를 충족시켜 수입대체 효과를 얻는 것이며, 특히 고부가가치의 의약품 중에서도 식물세포에서 성공적으로 발현될 수 있는 후보물질에 초점을 맞추었다. 또한 생산성이 향상된 단백질 제품의 수출을 확대하고 차세대 그린의약품의 생산 및 기반기술을 확보하여 신규기능 단백질의 대량생산 체계를 구축하는 것이다. 사업의 성과를 간단하게 요약하면 벼세포주로부터 생산된 관절염 치료제 CTLA4-Ig와 암환자 구내염 치료제 hGM-CSF의 발현에 성공하였고, 산업용 효소인 Trypsin의 발현 또한 성공하였다. 의약품으로서의 사업화에 필요한 전임상 자료인 효능 및 독성 평가를 실시하였으며, 제품화를 위한 제제화 연구를 수행하였다. 또한, CTLA4-Ig의 당구조 변형 기술을 활용하여 약물 동태 면에서 효능이 증가된 새로운 제품을 생산하는데 성공하였다. 전임상 독성평가는 식약청의 고시에 따라 GLP 인증기관을 통해 수행하였으며, 지속적인 효능모델에 서의 검증을 통하여 사업화에 필요한 자료를 축적하였다. hGM-CSF는 지속적인 독성시험과 함께 제제화를 위한 기초자료 및 개발 조건을 확립하였고 당 분석을 통한 단백질의 특성을 분석하여 상용화를 위한 연구를 순조롭게 진행하고 있다.

석유의 대체원료로 사용될 수 있는 식물성 바이오오일은 콩, 옥수수, 카놀라 등의 다양한 식물로부터 대량 생산된다. 바이오 오일은 탄수화물이나 셀룰로오즈 바이오매스에 비하여 그 화학적 구조와 물성이 기존의 석유와 매우 유사하여 비교적 간단한 반응에 의하여 에스테르 화합물, 지방알콜, 지방아민, 글리세롤 계통의 많은 화학물질 등으로 전환이 가능하다. 바이오오일을 사용하여 기존 석유화학공정을 통해 생산하는 화학소재를 대체할 경우 기존 우리 산업 구조의 취약점인 과도한 석유의존성과 화석연료 남용에 따른 환경오염의 문제점을 해결할 수 있다. ‘바이오오일 유래 친환경 화학소재 생산기술개발’(총괄과제)는 2006 년 11 월 차세대신기술개발사업으로 선정되어 2013 년까지 총 3 단계 7 년 예정으로 진행되고 있다. 본 과제는 BT 기반 친환경 지방산 알킬에스테르생산기술 개발(세부 1 과제; 생산기술연구원, 서울대, 수원대, 한일과학산업㈜, ㈜나라엔텍), 글리세롤의 생물 전환을 통한 3-hydroxypropionic acid 의 생산기술 개발(세부 2 과제; 부산대, 서울대, CTC 바이오㈜), 글리세롤 및 유도체의 생물 전환을 통한 광경화형 아크릴계 유도체 생산 기술 개발(세부 3 과제; 태광산업㈜, 화학연구원), 바이오나노촉매를 이용한 글리세롤카보네이트/유도체 합성 공정 개발(세부 4 과제; GS 칼텍스㈜, 광운대), 바이오 오일 유래 친환경 윤활유 첨가제 및 연료유 첨가제 개발(세부 5 과제: 화학연구원, 이맥솔루션㈜, 에코솔루션㈜) 등, 총 5 개의 세부과제로 구성되어 있고 기업 7 개, 대학 4 개, 연구소 2 개 등 총 13 개 기관이 참여하고 있다. 본 발표에서는 본 과제의 목표와 구성, 그리고 지난 2 년간 얻어진 중요 연구결과를 요약하여 설명한다.

5개 세부 과제의 결과를 요약하면 다음과 같다. 제 1 세부과제는 감도 바이오표면 분석을 위하여 단채널/다채널 SPR/E 시스템을 제작하였다. 실험결과 타원계측 퓨리에상수의 표준편차는 7×10-5 이하, SPR 측정감도 1 RU 이하의 높은 측정 감도를 얻었으며, geldanamycin과 같은 저분자 물질의 측정에도 SPR/E 시스템이 용이하게 활용될 수 있음을 확인하였다. 타원계측법을 이용한 자기조립박막층의 분석을 위하여 MUA SAM의 다층 박막층을 제조하여 두께분석을 통해 생체계면의 SPR/E 분석의 기반기술을 확립하였다. 제 2 세부과제는 “All-optical nanosurgery workstation” 제작 및 운용 기술 개발 및 단일세포 수준 물리적 특성측정기술 및 fs-laser 초미세 공정/수술 장치 활용기술에 중점을 두고 사업을 수행하였다. 특히 돼지 안구에 대한 나노 수술기법 및 수술 장치를 체계적으로 개발하였다. 제 3 세부과제는 기본적 개념이 검증된 나노갭 소자의 제작 기술을 개선하고 이를 이용한 항원-항체 검출 범위의 확대를 추진하였으며 나노갭 소자 제작 기술의 최적화, 갭 소자 집적 기술의 개선, 생체시료에서의 직접 검출, 센서의 응용 범위 확장, 정량 검지를 위한 측정 루틴의 확립, 단백질 고정의 효율성 제고 및 시료전 처리 기술 개선 등을 함하여 추진함으로써 각 요소기술을 완성하였다. 제 4 세부과제는 CARS 바이오현미경/내시경 기술 실용화에 요구되는 핵심기술을 확보하기 위해 2차년도 연구개발목표로 설정한 CARS 현미경의 분자화학종 선택성 향상를 위한 멀티플렉스 분광측정 기술과 내시경 광학표면의 생체 단백질 흡착억제 표면개질 기술 개발이 목표한 수준으로 차질 없이 수행되었다. 제 5 세부과제는 덴드론 박막이 단분자 간의 상호작용력 측정을 보장해 줄 수 있음을 증명하였고, 이를 이용해 다양한 단분자를 탐침의 정점에 도입할 수 있다는 가능성을 보였다. 또한 실험 목적 및 실험에 이용하고자 하는 생분자의 크기에 따라 적합한 덴드론 군을 선택하여 실제 생체 시료에서 단분자 간 상호작용력을 측정할 수 있었다. 결론적으로 레이저, 나노탐침, 나소자를 이용한 단일세포 생체 조직 이미징, 수술, 고감도 센서, 등의 나노바이오 융합 연구를 수행하여 바이오의료 분야의 측정분석, 의료진단 수술 분야의 원천기술를 개발하였다.

표적지향성 유전자치료제는 원하는 조직 또는 세포 등 치료대상 부위에서 선택적으로 유전자를 발현시켜 치료효능과 안전성이 진일보된 차세대 유전자치료제로써, 기존 유전자치료제의 큰 단점인 국소 투여의 제한점을 극복할 수 있을 뿐 아니라, 치료효율은 향상시키는 반면에 정상세포에 미치는 부작용은 최소화시킬 수 있는 장점이 있다. 본 연구과제는 질환의 분자생물학적 특성을 밝힘으로서 질환 특이적인 치료 표적을 선정하고 이에 해당하는 표적 특이적 리간드가 접합된 표적지향성 유전자치료제를 개발하고자 한다. 표적지향성 유전자치료제 개발을 위한 핵심기술요소는 나노복합체, 바이러스복합체, 표적 리간드, siRNA로서, 질환별 치료기술의 성격이 높아 기술목적에 부합하여 특화된 제품개발이 가능하고, 또한 원천기술의 확보가 용이하여 기술 확장 효과와 독점적인 기술우위를 점할 수 있는 잇점이 있다. 본 과제의 최종 목표는 병소 조직 특이적 유전자 전달, 발현 조절기술, 표적화 부여기술을 활용한 표적지향성 유전자치료제를 개발하는 것으로서, 다음과 같은 4개의 세부과제로 구성되어 있다: ①생분해성 폴리머젤을 이용한 지속형 표적지향성 유전자치료제 개발 ②특이 항체 부착 리표솜을 이용한 혈액암 표적지향성 유전자치료제 개발 ③Nanocomplex와 종양 선택적 살상 바이러스를 이용한 전신투여형 유방암 표적지향성 유전자치료제 개발 ④표적지향성 전달체를 이용한 siRNA 암 유전자치료제 개발. 본 과제의 1단계 개발목표로서 나노복합체, 바이러스, siRNA를 이용한 표적지향적 유전자 전달 또는 발현 조절 핵심기술을 개발하고 동물 질환모델을 이용한 치료효과를 검증하고자 하며, 2단계에서는 암 치료용 표적지향성 유전자치료제 후보 도출을 통하여 표적지향성 유전자치료제 대량생산 공정을 개발하고 전임상시험에 진입하고자 하며, 3단계에서는 약동력학 분석과 동물독성 시험을 완료하고, 4종 이상 임상시험을 위한 IND 신청 및 완료와 전임상/임상시료를 생산하고자 한다. 현재까지 1단계 1차년의 연구를 진행하였으며, 그 결과 ①유전자 전달용 생분해성 폴리머젤을 제작 및 특성 평가. 유전자- 폴리머 복합체(polymeric complex)의 형성능 확인 및 물리적 특성 분석을 완료. ② 백혈병/림프종 특이적 JL1 항체의 생산. JL1 항체 유전자 기반 minibody형 항체변이체의 제작. 백혈병/림프종 동물 모델에서 면역리포솜의 유전자 전달 효능 연구를 진행하였으며, ③ 나노복합체를 이용하여 아데노 바이러스를 포획하는 기술을 확립하고 암치료 효율이 증대된 종양 선택적 살상 아데노바이러스를 개발하였으며, ④ 고효율의 항암 siRNA 억제제를 개발하고, siRNA-PEG에 표적지향성 리간드를 부착하는 기술을 확립하였다. 표적지향성 유전자치료제 분야는 기존 치료법의 한계를 극복할 수 있는 미래의학의 첨단 분야로 신약 개발의 영역을 넓힐 수 있고 새로운 시장을 형성시킬 수 있는 blue ocean 분야이다. 따라서 본과제가 성공적으로 수행된다면, 고부가가치를 가진 신약 분야를 개척할 수 있으며 제품 및 기술의 수출을 통해 첨단 바이오산업 강국으로 도약할 수 있는 기반을 제공할 수 있을 것으로 생각된다.

성체줄기세포는 치료제로 쓰기에는 충분한 양만큼의 분화와 증식이 되지 않는 등 문제점이 대두되었으나 최근 수많은 연구로 성체줄기세포의 증식에 관한 해결책이 보고되고 있어 실용화에 큰 기대를 모으고 있다. 특히 골수의 MAPC 혹은 피부의 SKP 등은 거의 전분화능에 가까운 분화 잠재력을 나타내고 있어 면역학적으로 전혀 문제가 없는 자가 성체 줄기세포의 임상적 활용가능성은 무한하다고 볼 수 있다. 하지만 세포 자체만으로는 치료의 안정성이 보장되지 못하며, 그 기능 면에서도 조직과 비교하여 상당부분 떨어지는 문제점으로 손상된 생체조직의 복원을 위해서는 적절한 지지체에서 줄기세포를 배양하여 실제의 장기와 유사한 기능을 가진 인공 조직을 체내에 이식하는 형태의 조직치료제가 요구되고 있다. 현대 의학에 있어 세포 및 조직학에 대한 재료공학의 접목 즉 조직공학은 세포 및 조직치료제의 개발에 큰 계기를 부여하고 있다. 사고나 노화로 인해 손상된 조직과 기관의 기능을 회복시키거나 유지 향상 시키는 재생의학의 분야인 조직공학은 생물학적인 대체물의 발전과 더불어 공학과 생명과학의 원리를 기초로 하여 형성된 응용학문으로 인공기질(artificial matrix)의 개념이 함축되어 있다. 인공기 질의 개념은 세포가 조직으로 형성되기 위해서는 지지체의 역할을 하는 세포외기질(ECM)이필요하며 이를 인공적으로 만들었을 때도 같은 역할을 할 것이라는 이론에서 기인하였다. 이러한 이론적 배경을 바탕으로 살아있는 조직과 장기의 모양으로 만들어진 고분자지지체로 인체의 손상된 부분을 대체하거나 재생되도록 도와주는 매개체인 이른바 조직치료제에 대한 연구를 착수 하게 되었다. 본 연구는 질환별로 최적 상태로 적용하기 위한 지지체의 개발을 기반으로 개발 성공시 의료계와 바이오산업에 지대한 파급효과가 예상됨과 동시에 국내 의료기술 수준을 상향시킬 수 있는 3가지 질환 군 - 허혈성 심질환, 허혈성 뇌 망막질환, 골 질환 - 에 대한 조직공학제제의 개발을 최종 목표로 한다. 2008년 지식경제부의 차세대 신기술사업의 수혜를 받아 지난해 9월부터 7년간 3단계 과정으로 진행되고 있는 본 연구는 가톨릭대학교 의과대학 기초의학교실과 임상의학교실 연구진, 서울대학교 의과대학 연구진을 비롯하여 KIST, KRICT, KITECH 등의 정부출연 연구소의 전문가 등이 대거 참여하여 공동연구를 진행 중에 있다.

Single stranded DNA aptamers that bind to tetracycline (TET) with high affinity and specificity were identified by selection from an oligonucleotide library of 1015molecules using modified SELEX, which was employing tosylactivated magnetic beads. The binding affinities of seven aptamers are in nanomolar range. These DNA aptamers are highly selective for TET as much as distinguishable of only single hydroxyl group on 5 or 6 carbon atoms in other tetracycline group. TET binding DNA aptamer, No.20 (Kd=63.6nM) was used for the electrochemical detection of TET. To fabricate electrochemical aptasensor, No.20 DNA aptamer was immobilized on screen-printed gold electrode chip on which all of three working, reference and counter electrode were integrated. After treatment of sample containing chemicals, cyclic voltammetry and squire wave voltammetry were analyzed. In result, the electrochemical aptasensor showed a good sensitivity in a range of 1-10000nM and the selectivity of this aptasensor for TET detection was also verified with other antibiotics of tetracyclines' group.

We report highly enhanced performance of the organophosphorus hydrolase-based biosensor for determination of organophosphate pesticides by using Mesoporous Carbons(CMK) as a anodic layer onto GE(glassy carbon electrode). The CMK/GE layer exhibits not only a greatly improved amperometric response, but also promoted electron transfer of the enzymatically generated phenolic compounds(p-nitrophenol) in comparison with a carbon nanotube(CNT)-modified electrode. The well-ordered nanopore and high specific surface area of Mesoporous carbon(CMK) especially give them the benefits to sensitivity and linear range in a low nanomolar range of paraoxon, which make them suitable for application in real-time biosensors. Under the optimal experimental conditions, the biosensor performance shows the detection limits of 0.12μM(36ppb) and sensitivity of 250nA/μM for paraoxon.

Cell cycle-dependent protein kinases (CDKs) regulates the cells entry into mitosis during the transition from G2 to M phase, by phosphorylating a group of key proteins including RNA polymerase II and histone H1. A recombinant substrate for the enzyme was expressed as a recombinant protein from a plasmid pMFS-cdc2 in E. coli, and it was purified. In this study, we present a strategy for the detection of a phosphorylation of a substrate catalysed by cdc2 protein kinase by using a radioisotope detection technique in microarrays. We conclude that the use of Mep45-fused substrate in developing a protein biochip for the detection of the activity of cdc2 protein kinase by using a RI detection technique is highly economical and can be used for both high throughput screening and studying protein-protein interactions. Keywords: Cdc2 protein kinase, Gene cloning, Microarrays, Radioisotope detection

Basic biological and medical research continues to provide the building blocks necessary to develop solutions to human health problems and to preserve and repair the natural environment. In this study, we investigated the feasibility of applying a RI detection technique to a protein kinase C assay for the development of a diagnostic biochip. Using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, a Mep45-fused substrate was cloned and purified for use as substrate to detect the activity of protein kinase C. The resultant MFS-PKC protein was used as a substrate for a protein kinase C assay and the signal intensity of the kinase assay system was examined with varying concentrations of the kinase. The results are found to be promising and can be applied when using a RI detection technique for developing a highly sensitive biochip to determine the enzyme activity for a disease diagnosis and subsequent new drug development. Keywords: Protein kinase C, Protein-fused substrate, Microarrays, Radioisotope

Non-crosslinked, monodisperse polystyrene spheres were synthesized using an emulsifier-free emulsion polymerization Technique. Ordered 3D arrays of Hydrogel inverse opals were fabricated via capillary-attraction-induced methods by using nanoparticles of polystyrene beads as sacrificial templates. The inverse opaline samples obtained by gravity sedimentation method. Films obtained by Uv expose. The sample was immersed in dimethylbenzene for completely removing polystyrene spheres to from inverse opal hydrogels(IOHPEG). The interpenetrating polymer networks(IPNs) were formed by synthesis of a crosslinked poly(acrylic acid)(PAA) network within PEG hydrogel in order to reduce decrease of mechanical properties of PAA/PEG IPNs and PAA possesses carboxyl groups that could serve as sites for immobilization of various protein. Materials achievable with this technique. These structures could potentially find applications as Biosensor via fill biomaterials like a protein or glucose in the air sphere.

A panel of seven different biosensing cells, each constructed using a promoter that is responsive to DNA damage, was used for detecting and classifying genotoxicity. Four of these recombinant bioluminescent bacteria were newly developed for this study using the promoters for the alkA,sulA, sbmC, and recN genes from E. coli. The responses of these recombinant bioluminescent bacterial strains were successfully characterized and classified by using certain classes of genotoxic chemicals, such as mitomycin C, 1-methyl-1-nitroso-N-methylguanidine, nalidixic acid and 4-nitroquinoline N-oxide. The responses obtained from these cells were compared with each other and the differential effects among the strains in response to the different types of genotoxic agents have been successfully characterized. These results demonstrate that these seven bioluminescent bacteria can be used to elucidate and classify the genotoxic impacts of samples or chemical solutions and that together they may be utilized in the pre-screening steps of new drugs or to characterize unknown or newly synthesized chemicals, the toxicity of food samples and environment samples.

PDMS is well known as one of the best biomaterials due to its stability and biocompatibility. To apply PDMS to cell based biochip, proper surface modifcations such as collagen and gelatin are required to adhere cell on its surface. In this study, the nano-scaled irregular patterns on the PDMS surface was developed to fabricate cell based biochip without chemical and biological treatment. PDMS surface was modified by formation of irregular nano patterns using surface deformation technology. Hydrophobicity of modified PDMS surface was sustained with basic PDMS surface. To measure adhesion properties and viability of Human Mammary Epithelial Cells(HMEC) on the surface proposed here, AFM and confocal microscopy were utilized and their results were compared with that from commercial culture dish. It implies modified PDMS surface can be directly applied to cell based biochip without even any reagents. Acknowledgments: This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10755160002-07N5516-00210).

The volume reduction process is required to increase the sensitivity for the detection of pathogenic bacteria in environmental sample that has relative large volume, considering microchip format. With the verification of multi-processes (concentration/amplification/detection) in single chamber to detect pathogenic bacteria in large volume sample, the optimization of chamber in chip was accomplished to enhance its operating stability and detection capability. Chamber was designed for fast concentration by high speed sample loading. Glass beads was enlarged to 100 μm to prevent the its loss under high pressure by high speed of sample loading. Consequently, the sample loading was carried out with 9ml/min. RNA was concentrated by its adsorption on glass bead under pH5 and 50mM of acetate buffer. RNA concentraed on glass bead was desorbed under amplification solution (pH8.9, Nucleic Acid Sequence Based Amplification mixture) and was amplified for 90 min. The proposed PDMS microchip can be measured E.coli in the concentration of 1 c.f.u/ml with 10ml of sample. Acknowledgments : This subject is supported by Ministry of Environment of the Republic of Korea as "The Eco-Technopia 21 project" and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-D00099)

The aim of this study is to evaluate the sensitivity and specificity of the DNA chip developed in previous study by comparing conventional clinical diagnostic methods and to verify the sensitivity and simplicity of the DNA chip according to clinical DNA sources. Two hundred twenty seven participants volunteered for this test. Diagnosis was first performed by a slit-lamp eye examination, which is a common medical diagnostic method, and then each DNA sample from peripheral blood was analyzed by DNA chip and sequencing simultaneously. All the tests were performed independently as blind tests. The evaluation of slit lamp examination and DNA chip was finally done by comparing the DNA sequencing results. Seven heterozygous GCD II patients were selected to examine how DNA chip is sensitive and which samples are applicable except for blood. DNA were extracted from their buccal epithelial cells, hair roots, epidermis stuck on skin patch sticker and blood. A total of 28DNA samples were collected and sent for further analysis by DNA chip and sequencing. DNA sequencing showed that of the 227 participants, 124 are GCD II patients (54.6%) and 103 are normal healthy individuals - non GCD II (45.4%). The sensitivity and specificity of DNA chip were both 100%, while the sensitivity and specificity of slit lamp method were 99.19% and 100%, respectively. When DNA samples from different clinical sources of 7 patients were tested usingDNA chip and sequencing simultaneously, only DNA chip was able to give the results directly in all types of samples, but DNA sequencing was only successful using DNA from blood. It was firmly concluded that DNA chip allows rapid and successful diagnosis of GCD II, which is better than conventional clinical method and it also allows simple and easy detection with various types of clinical samples better than DNA sequencing. [This work was supported by Medigenes Co., Ltd. and by the Brain Korea 21 Project. Further support through the LG Chemicals Chair Professorship is appreciated.]

The evaluation of cell disruption efficiency is important in monitoring durability and diverse cellular physiologies of host cells and improving recovery yield of intracellular products. Based on our previously suggested pH-controlled fluorescence resonance energy transfer (FRET) system, we herein demonstrated that easy and effective estimation of the degree of cell disruption under certain stress environments is possible using a simple measurement of changes in fluorescence spectra of two fused fluorescent proteins, because its unique linear correlation was established with cell disruption efficiency from the conventional colony counting method.

To detect bacterial pathogens, 16s rRNA was analyzed using immunochromatographic test strip with a surface-modified gold nanoparticle. The 16s rRNA has been used as a genetic marker for identifying organism, and can be analyzed directly without PCR amplification due to relatively high copy number [1]. Immunochromatographic strip test can be performed easily without sophisticated skills and instruments. It takes a little time to identify the result with naked eyes. Also, this method require small sample volumes so, more economic. For easy visual judging, surface-modified reddish gold nanoparticle was used. In this study, detection of bacterial 16s rRNA using peptide nucleic acid (PNA) probes was developed. The backbone of PNA is neutral, we carried out effective hybridization between PNA and RNA by decreasing the electrostatic repulsion [2]. To visualize the interaction between PNA and 16s rRNA, surface of gold nanoparticle was designed as a cationic nanoparticles to interact with 16s rRNA selectively. Using our method, identification of bacterial pathogens was possible.

This study presented simple surface modification for the immobilization of biomolecules such as proteins and cells in order to integrated with microfluidic system. Firstly, the fabrication of thin film composed of glycidyl methacrylate(GMA) that amine-attractive epoxide monomer was fabricated with photopolymerization. The orthogonal surface using simple micromolding in capillaries (MIMIC) was obtained. One region included polyethylene glycol (PEG) microstructures that prevent nonspecific binding of proteins and cells. The other region was the epoxide surface that promoted protein and cell immobilization. The orthogonal difference in surface functionality between the epoxide region and background PEG show strong possibility of simple patterning of biomolecules. It will be useful platform technology for the constrcution of organic material patterning , protein array and cell.

The carbon nanotube(CNT)-OPH biosensor was prepared onto the CNT plate and the magnetically separable polyaniline nanofiber(PAMP)-CNT-OPH sensor was prepared onto PAMP coated CNT plate. The OPH was immobilized by glutaraldehyde(GA) cross-linker methods. The detection of pesticides in buffer solution was implemented on the CNT and CNT-PAMP-sensor using CV method. The giving voltage range was from-1.2V to 0.5V. This system can detect from 1 to 500uM for paraoxon and can simply detect the concentration of organophosphate pesticides(OPs) from vegetables.

Nanoimprint lithography (NIL) is one of the most promising technologies to fabricate nanopattern on solid substrate because of its several advantages such as resolution, reliability, and process speed, compared to conventional lithography. By using NIL, nano-scale to micro-scale nanostructures was fabricated on large scale silicon on insulator (SOI) plate at mild condition. The fabricated nanowire patterns were characterized by FE-SEM. An antibody was immobilized on the fabricated nanowire pattern, which has affinity for target molecule of interest. And then target molecules were detected by analyzing the change of nanowire conductance using semiconductor parameter analyzer. In this study, the proposed NIL technique can be useful as a method for the fabrication of nanoscale biosensor. Acknowledgments: This research was supported by the Core Environmental Technology Development Project for Next Generation funded by the Ministry of Environment of Korea, and by Nuclear R&D program through the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. M20706010003-08M0601-00310)

In this study, we have developed shear horizontal (SH) surface acoustic wave (SAW) sensors for the detection of immunoglobulin G (IgG) [1] on the gold coated delay line of SH-SAW devices. The sensor consists of twin SAW delay lines operating at 100MHz, one reference channel and one sensing channel, fabricated with Au/Ti on 36YX LiTaO3piezoelectricsubstrate.The delay line was exposed to a solution of gold nanocatalyst-anti-mouse IgG(G-anti MIgG) conjugate with in PBS solution. Target mouse IgG solution was injected onto the delay line. As the result of the experiment, we could uniformly immobilize anti-MIgG(mouse IgG) conjugate on the surface of gold. When displaying results of immobilization on the surface of gold using G-anti MIgG conjugate and blocking buffer in frequency shift, G-anti MIgG conjugate showed frequency shift of 75.1 kHz in the initial frequency, and blocking buffer showed frequency shift of 215.7 kHz. When various concentration of MIgG was added in 100MHz type, the sensor showed 46.3, 127.45, 161.21 and 262.39 kHz frequency shift at 25, 50, 75 and 100 μg MIgG concentration compare with phosphate buffered saline (PBS) solution. The result shows that is primarily detected the frequency shift due to the change in mass loading, which results from interactions between the prove and the target layer.

The kinetic parameters for Salmonella typhimurium binding with the immunoassays were evaluated quantitatively. The lower limit of detection for the cell was also investigated in the immunoassays using a surface plasmon resonance biosensor. The lower limit of detection for the cell was improved to 101 CFU/ml by application of the sandwich assay. The association rate constants and affinities of the cell by the sandwich assay were above twice as large as those by the direct assay. The improvement of the lower limit of detection by the sandwich assay was derived from the enhancement of binding attraction between the cell and a detection antibody.

We demonstrated the Human IL5 detection of the antibody microarry immunoassay as low-concentration level on surface plasmon resonance imaging (SPRI) and ellipsometry imaging (EI). In order to increase the sensitivity, alkaline-phosphatase was used for enzyme-catalyzed precipitation reaction with nitro blue tetrazolium(NBT) and 5-bromo-4-chloro-3-indolyl phophate p-toluidine(BCIP) as substrates. Enzyme-catalyzed signal enhancement has been used widely for detecting the low concentration of target molecule and increasing the sensitivity on biochip/sensor technology1). In this study, human IL5 was ranged from 1 pg/ml to 100 pg/ml. There can be a useful method as application for high-throughput screening (HTS) on diagnosis disease or drug screening very usefully.

It is very critical to develop the surface plasmon resonance (SPR) detection in order to achieve an avian influenza (AI) virus sensor. This work is specifically focused on the development of biosensor system to obtain recombinant proteins with the gold-specific affinity. The strategy is based on the principle that the gold binding polypeptide (GBP) as a fusion partner specifically binds onto the gold surface for immobilizing proteins. The AI virus surface antigen was employed as a model protein for the specific immobilization of GBP-fusion protein. The fusion proteins were successfully immobilized on the gold surface and examined by SPR and SPR imaging analysis. We also analyzed the immobilization efficiency of intracellular protein on the micro-patterned gold surface. Finally, the GBP-fusion method immobilized proteins onto the gold surface without surface modification and in a bioactive form, which is suitable for studying interactions of protein-protein, and other biomolecules [This work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through IITA].

Identification of the chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health problems. In response to the need for more efficient methods to identify potential developmental neurotoxicants, we designed a cell based chip to detect the environmental chemical effects on neurite outgrowth in PC-12 cell, which is a cancer cell line derived from a pheochromocytoma of the rat adrenal medulla. Cell based chip were fabricated by binding synthetic oligopeptide (RGD) onto a gold electrode. PC- 12 cells were immobilized on the gold electrode. Induction of the neurite cell to grow was observed by the neurite growth factor (NGF). Electrochemical behavior of the effect of the environmental chemical (aldrin) on the neurite outgrowth was measured using a cyclic voltammogram. Voltammetric current peak behavior shows that as the concentration of aldrin increases, the measured peak current decreases. This indicates a decrease in cell viability. The cell chip used in this study can be applied to detect the inhibitory effects of a large number of environmental toxins on neurite outgrowth.

When one makes a decision based on a quantified value, one must know the precision of the value, the LOD and the ROQ. According to the guideline for the validation of the ICH, the precision of an analytical value by ELISA can be described from the standard deviation or related standard deviation (RSD) by repeated experiments at various concentrations of the analyte. We developed a mathematical model for predicting precision without performing repeated experiments using ELISA. When the model is applied to ELISA, the RSD of measurements is expressed as the error parameters and the calibration curve. The optimum conditions for ELISA, such as the reaction time, the concentration of the antibodies, and the concentration of the enzyme-conjugated antigen (antibody) have been determined by trial and error. In this study, to reduce the number of optimization experiments, the initial screening of the candidate antibodies and the determination of the optimal reaction conditions has often been carried out on the basis of the kinetic properties against the antigen of interest. The LOD and ROQ were determined from a model describing the calibration curve and precision profile of the ELISA.

In recent years, the cell-based assay has been used for the detection of drugs, toxicants, and other cell functional characterization. For these cell-based assays, there is an increasing demand for more rapid and simple procedures for studying the cell viability and the effect of drugs on cell behavior. Immobilization of cell is very important process in the fabrication of cell chip since the performance is highly affected by the cells that are immobilized on substrate. HeLa cells have been used to study the effect of anticancer drugs on cells. In order to to enhance adhesion of HeLa Cells on surface, we fabricated nanoconcaved surface using nanoporous alumina. Uniform nanoconcaved patterns were revealed on surface of aluminum after the removal of nanoporous alumina formed during anodization. Au was deposited on nanoconcaved surface by sputtering. HeLa cells were immobilized on the Au nanoconcaved surface. The results imply that gold-coated nanoconcaved surface can be utilized as nanobioplatform to construct biochip such as cell chip. Acknowledgement: This research was supported by the Nano/Bio science & Technology Program (M10536090001-05N3609-00110) of the Ministry of Education, Science and Technology (MEST), by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST)(2006-05374) and by the Core Environmental Technology Development Project for Next Generation funded by the Ministry of Environment of Korea.

The cationic lipid or surfactant encapsulation is known as one of the most effective and straightforward strategies because of the persistent quantum yield and no additional steps for conjugation with peptides or antibodies. In spite of the successful fluorescent cellular labelings with those ligands, in some ways, there are still limitations remained such as not clear fluorescent images of cellular morphology, low exclusion size for encapsulating QDs, and relatively complex composition of the system. There have been reports of solublilzation of nanocrsytals including quantum dots, gold nanoparticles and nanorods by CTAT micelle. Not yet is success of CTAB QD reported as intracellular labels. Therefore, herein we represent capability of CTAB QD as cellular labels particularly for fluorescent morphological distinct images.

A recent adaptation of genetic algorithm, NSGA-II-JG, was applied systematically to each key to be completed for developing an optimal batch chromatographic process for 1,3-propanediol purification. The genetic algorithm was used first in the task of determining intrinsic parameters such as adsorption isotherms and mass-transfer coefficient. At this study, the genetic algorithm plays a part in searching the values of intrinsic parameters that lead to the minimum difference between the experimental and the model predicted elution profiles. The genetic algorithm was applied to optimize a series of operating parameters, which include flow rate, feed loading volume, starting and ending times of product collection, and the time of gap between adjacent two feed injections. Such optimization, which aims for the highest productivity under the constraints of product purity and pressure drop, was repeated by varying adsorbent particle size. The optimization results confirmed that the productivity for small particles was limited by the pressure drop whereas the productivity for large particles was limited by the mass-transfer efficiency. In consequence, the optimal particle size for the maximum productivity falls on the boundary between the two limiting regions. The results of this study indicate that the application of genetic algorithm is effective in each of a batch chromatographic process development, which covers from parameters estimation to process optimization.