이 작품은 한국 연구 재단 (NRF) 보조금 (2013R1A2A2A05004314, 2012R1A1A2043747), 한국 정부에 의해 투자 한국의 의료 기술 R &amp; D 프로젝트, 보건 복지부 (A121994)와 IBS-R020-D1의 정부에서 보조금에 의해 지원되었다.

주 : 혈액 건강한 사람에서 도출하고, 혈액 수집 튜브에 수집 하였다. 서면 동의서는 모든 자원 봉사자들로부터 얻은 것입니다. 이산화 티탄 NF 매트 1. 제작 <ol><li> 전구체 용액 (22)의 제조 <ol><li> 에탄올 (99.9 %, 3 ml) 및 빙초산 (3 mL)을 혼합하여 티타늄 테트라 이소 프로 폭 사이드 (TTIP) 1.5 g을 용해시키고, 자기 교반기에서 30 분 동안 RT에서의 수용액 (25 ° C)을 혼합한다. </li><li> 에탄올 (99.9 %)의 3.64 g의 폴리 비닐 피 롤리 돈 (PVP)의 11 중량 %를 용해시키고, 핫 플레이트, 자기 교반기를 사용하여 1 시간 동안 65 ° C에서 상기 용액을 교반한다. </li><li> 결합 및 핫 플레이트 자석 교반기 4 시간 동안 65 ° C에서 두 솔루션을 저어. </li></ol></li><li> 전기 방사 <ol><li> 200 μm의 내측 diame의 스테인리스 바늘에 연결된 주사기로 제조 된 용액을로드터. 플런저를 제거한 후, 주사기에 직접 용액을 붓는다. </li><li> 높은 DC 전압 (15 kV로)에 접지 된 기판 상에 배치 된 실리콘 웨이퍼 상에 10 분 동안 0.3 ml / hr의 유량 (2cm X 2cm)에 항상 용액을 소개한다. 전기 방사 중에 니들 10cm에 접지 기판 사이의 거리를 설정한다. </li></ol></li><li> NF 개의 매트의 하소 <ol><li> 노에서 NF 매트를 놓고 고진공 조건 (5 × 10-5 토르)하에 3 ℃ / 분으로 램핑 속도로 500 ℃까지 온도를 증가시킨다. </li><li> 3 시간 동안 500 ° C의 온도를 유지한다. 노의 RT (25 °의 C)로 샘플의 온도를 냉각. </li></ol></li></ol> 원심 미세 유체 디스크에 티오 2. 통합이 NF 매트 <ol><li> 디스크의 제조 <ol><li> 3 차원 설계 소프트웨어 (예를 들면, 솔을 사용하여idWorks 또는) 유사한 디스크의 각 층의 챔버, 채널 또는 밸브를 묘사한다. 코드 생성 소프트웨어 (예를 들어, Edgecam 또는 유사)를 사용하여 컴퓨터 수치 제어 (CNC) 코드 설계 파일을 변환합니다. 상기 명령에 따라 소프트웨어를 사용 설명서 23,24 참고.이 장치에 사용되는 일반적인 채널 치수 X 0.3 mm (폭 X 깊이) 1.0 mm이다. </li><li> (CNC 밀링 머신의 운영 소프트웨어를 열고 예., DeskCNC 또는 이와 유사한 것) 및 운영 소프트웨어 CNC 코드를로드하고 순서대로 다음을 클릭합니다 : &quot;AUTO&quot;-&gt; &quot;G CODE OPEN을&quot;-&gt; 생성 된 CNC 코드를 선택합니다. </li><li> CNC에 밀링 머신의 PC 판을 부착 : 디스크 층에 대한 상위 계층과 5mm의 PC 판을위한 1mm의 PC 판을 사용합니다. &quot;시작&quot;버튼을 클릭하여 디스크의 각 층을 절단하는 CNC 밀링 머신을 실행합니다. </li></ol></li><li> 디스크 상에 이산화 티탄 NF 매트의 전송<ol><li> 실리콘 기판 40 μL (트리 데카 -1,1,2,2- tetrahydrooctyl)에서 30 분 동안 진공 하에서 진공 챔버 -1- 트리클로로 실란을 함유하는 작은 페트리 접시를 놓는다. 플루오로 실란이 증발하여 기판에 도포 얻는다. </li><li> 진공 챔버에서 탈기 한 비율 10 : 경화제로 폴리 디메틸 실록산 (PDMS) 예비 중합체를 혼합한다. 스핀 코트를 60 초 동안 650 rpm으로 실리콘 기판 상에 PDMS. </li><li> 10 분 점착성 상태를 달성하기 위해 65 ° C의 오븐에서 실리콘 기판 상에 코팅 된 PDMS 큐어. 주 : 경화 시간은 환경 조건에 따라 변화 될 수 있고, 접착력은 레오 미터를 사용하여 점착성 시험에 의해 시험 될 수있다. </li><li> 트위터를 사용하여 PDMS 코팅 된 실리콘에 전기 방사 및 소성에 의해 제조 된 이산화 티탄 NF 매트를 전송합니다. 그런 다음, 수동 (유리 슬라이드, 예.) 평면 객체에 전송 된 이산화 티탄 NF 매트에 균일 한 압력을 적용하고 개체를 제거합니다. 놓다4 시간 동안 65 ℃에서 또는 1 시간 동안 80 ° C의 오븐에서 샘플이 완전히 PDMS 층을 경화한다. </li><li> 20 μL를 PDMS / 경화제 혼합물과 디스크에 코트 결합 반응 챔버 (10 : 1), 접착제 층 역할을하는 디스크에 이산화 티탄 NF 매트를 부착합니다. </li><li> PDMS의 층에 이산화 티탄 NF 매트에 에탄올 (99.9 %)의 50 μL를 분배 6 mm 직경의 펀치 구멍과 NF 매트를 절단하고, 20 μL로 코팅 된 결합 반응 챔버 이산화 티탄 NF 매트를 절단 전송 이전 단계에서 PDMS의. 참고 :이 단계에서, 에탄올 Si 기판에서 절단 이산화 티탄 NF 매트를 분리하는 데 도움이 될 것입니다. </li><li> 1 시간 완전 경화 PDMS를 4 시간 동안 80 ° C에서 65 ° C의 오븐에서 이산화 티탄 NF 매트 내장형 디스크 부화. </li></ol></li><li> 디스크상의 이산화 티탄 NF 매트의 표면 개질 <ol><li> 피에탄올 (3- 글리시 독시) methyldiethoxy 실란 (GPDES)의 repare 1 % v / V를 용액 (99.9 %). </li><li> 140 W, 180 초 동안 50 sccm의 산소 흐름에 산소 플라즈마와 이산화 티탄 NF 매트 통합 된 디스크를 취급합니다. 각각의 나노 섬유 매트에 GPDES 용액 100 μl를 분배하고 2 시간 동안 RT (25 °에 C)에 품어. </li><li> 세척 병을 사용하여 에탄올 (99.9 %)을 분배하여 기판 간단히 세척 한 후 디스크를 반전하고 깨끗한 대해 그것을 닦아 블로 팅에 의해 완전히 에탄올을 제거합니다. 1 시간 동안 80 ℃에서 경화. 상술 한 바와 같이 물리적으로 흡착 바운드 GPDES 분자를 제거하고, 동일한 방식으로 에탄올 (99.9 %)로 두 번 세척 하였다. </li><li> 건식 진공 상태에서, 질소 기류로 바람을 불어 건조. 주 : 디스크를 사용할 때까지 실온 (25 ℃)에서 밀폐 용기에 저장 될 수있다. </li></ol></li><li> 표면에 항체의 고정화 <ol><li> 포획 항체 200 μg의 / ㎖의 용액 (마우스 모노클로 날 항 만들기인간 hsCRP 또는 인산염 완충 식염수 (PBS) 완충액 (pH 7.4)로 항체를 희석하여 단일 클론 마우스 항 cTnI는). 마이크로 피펫을 사용하여 디스크의 각 NF 매트 상 용액 5 μL를 분배. 항체에 대한 자세한 내용은 자료의 목록을 참조하십시오. </li><li> 가습 실에서 디스크를 유지하고 4 시간 동안 37 ° C에서 품어. 0.1 % BSA-PBS 버퍼와 NF 매트 코팅 된 항체를 씻으십시오. 마이크로 피펫을 사용하여 세척 완충액 100 ㎕와 챔버를 채우기, 흡입 또는 경사에 의해 버퍼를 제거합니다. 마지막으로, 디스크를 반전하고 깨끗한 닦아에 대해 그것을 가릴 다음 디스크를 조립한다. </li></ol></li><li> 디스크 어셈블리 <ol><li> 캐드 프로그램 (예 AutoCAD의 또는 유사한)를 사용하여 양면 접착 테이프, 디스크의 디자인을 그린다. 커팅 플로터에 CAD 설계를 넣습니다. </li><li> 커팅 플로터를 사용하여 양면 접착 테이프를 잘라. 이중 접착 테이프의 한 보호 층을 벗겨과 내가 첨부디스크 층의 상단에 t. 다른 보호 층을 벗겨 디스크 상에 상부 층을 연결합니다. </li><li> 프레스 기계에 미리 조립 된 디스크를로드하고 정확하게 각 밸브, 채널 및 챔버를 연결하는 각 층에 정렬 마크를 사용하여 상단 / 접착제 / 디스크 층을 맞 춥니 다. 프레스 기계를 사용하여 균일 한 압력을 적용합니다. </li></ol></li></ol> 3. 면역 <ol><li> 마이크로 피펫을 이용하여 1 % BSA-PBS 완충액 챔버를 채우고, 1 시간 동안 37 ° C에서 디스크를 배양한다. 마이크로 피펫을 사용하여 흡인하여 버퍼를 제거합니다. 미 반응 사이트를 차단하고, 디스크에 단백질의 비특이적 흡착을 감소시키기 위해이 단계를 수행한다. </li><li> 충전 및 마이크로 피펫을 사용하여 실을 흡입하여 0.1 % BSA-PBS로 두 번 세척 하였다. 참고 :이 단계에서 디스크를 사용할 때까지 4 ° C에 저장할 수 있습니다. </li><li> micropipett을 사용하여 디스크에 CRP 검출을위한 항원 아군 전체 혈액 또는 CRP 무 혈청로드 10 μL이자형. 교정 그래프를 만들기 위해, 100ng의 / ㎖ 1 pg / ml이다에서 CRP 농도를 사용한다; cTnI는 10 pg / ml 인 내지 100 ng / ml이되게 준비 하였다. 참고 μM의 범위이다 전혈에서 CRP의 높은 수준으로 인해 CRP 무 혈청 장치의 낮은 검출 한계를 보여주기 위해 사용되었다. </li><li> 적혈구를 분리하는 60 초 동안 3600 RPM (XG 391)에서 디스크를 스핀. </li><li> 개방 밸브 레이저 조사에 의해 1 번, 그리고 전사 3 초 동안 2400 rpm으로 디스크를 회전시켜 HRP가 결합 된 항체를 검출 8 μL를 포함하는 챔버에 뜨는 플라즈마 4 μL. 주 :. 밸브 활성화 및 디스크 동작의 시각화를위한 일반적인 프로세스는 이전의 보고서에 상세히 설명 21 </li><li> 단백질 검출 항체의 결합을 5 초 동안 혼합 모드 (15 Hz에서의 -1, 15 °)를 적용합니다. </li><li> 오픈 밸브 # 2, 결합 반응 챔버 6 단계에서 제조 된 혼합물을 트랜스퍼 (2,400 RPM3 초). 이어서, 혼합물을 이산화 티탄 연맹에 결합하는 항체 사이의 면역 반응을 달성하기 위해 20 분 동안 혼합 모드 (60 Hz에서의 -1,2- °)을 적용한다. 상기 반응 개방 밸브 # 3 후 폐 챔버 (2400 RPM, 10 초)에 잔류 혼합물을 옮긴다. </li><li> 밸브 # 4를 열고 디스크 (2400 rpm으로 4 초)을 회전시켜 결합 반응 챔버의 세정 완충액 (600 μL)를 옮긴다. 이어서 챔버 내의 이산화 티탄 연맹 씻어 120 초 동안 혼합 모드 (30 Hz에서의 -1, 30 °)를 적용한다. </li><li> 단계 3.8의 동일한 조건에 따라 두 번 더 이산화 티탄 연맹을 세척하고, 20 초 동안 2,400 rpm에서 디스크를 회전시켜 완전히 잔류 세척 버퍼를 제거합니다. 그리고, 밸브 # 5를 닫는다. </li><li> 화학 발광 반응을위한 밸브 # 6 및 방사를 개방함으로써 결합 반응 챔버에 화학 발광 기질 용액을 전송 한 후 1 분 동안 혼합 모드 (30 Hz에서의 -1,2- °)를 적용이후 10 초 동안 2,400 rpm에서 디스크. </li><li> 밸브를 개방 # 7, 10 초 동안 2400 rpm으로 디스크를 회전시켜 상기 검출 챔버로 반응 기질 용액을 전송. </li><li> 광전자 증 배관 (PMT) 모듈을 갖춘 검출 시스템을 사용하여 반응 기질 용액에서 화학 발광 신호를 측정한다. 주 : 스텝 모터, 현미경 성분 변환 단계, 및 상업적으로 입수 한 PMT : 검출 계는 다음과 같은 부품을 사용하여 맞춤형 기계이다. 현미경 부품, 스텝 모터 장치를 유지하도록 조립되고, 모터는 디스크를 회전시킬 수있다. 검출은 현미경 부에 고정 된 PMT에 의해 수행되고, 이는 상기 장치의 검출 챔버 위에 배치된다. 스텝 모터를 조작함으로써, 검출 챔버 바로 PMT의 검출 영역 아래에 정렬된다. </li></ol>

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	<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+Ultrasensitive+Protein+Detection.">Nanomaterials for Ultrasensitive Protein Detection.</a> Adv. Mater. 25 (28), 3802-3819 (2013).</li><li>Hu, W., Li, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterial-based+advanced+immunoassays.">Nanomaterial-based advanced immunoassays.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 3 (2), 119-133 (2011).</li><li>Yang-Kyu, C., Chang-Hoon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon+Nanowire+Biosensor+for+Cancer+Markers.">Silicon Nanowire Biosensor for Cancer Markers.</a> Biosensors and Cancer. , 164-183 (2012).</li><li>Baltazar, R., Vistas, C. R., Ferreira, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+Applications+Using+Nanoparticles.">Biosensing Applications Using Nanoparticles.</a> Nanocomposite Particles for Bio-Applications. , 265-282 (2011).</li><li>Roy, P., Berger, S., Schmuki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO2+Nanotubes:+Synthesis+and+Applications.">TiO2 Nanotubes: Synthesis and Applications.</a> Angew. Chem. Int. Ed. 50 (13), 2904-2939 (2011).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+Nanofibrous+Membranes:+A+Novel+Solid+Substrate+for+Microfluidic+Immunoassays+for+HIV.">Electrospun Nanofibrous Membranes: A Novel Solid Substrate for Microfluidic Immunoassays for HIV.</a> Adv. Mater. 20 (24), 4770-4775 (2008).</li><li>Chantasirichot, S., Ishihara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+phospholipid+polymer+substrate+for+enhanced+performance+in+immunoassay+system.">Electrospun phospholipid polymer substrate for enhanced performance in immunoassay system.</a> Biosens. Bioelectron. 38 (1), 209-214 (2012).</li><li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+TiO2+Nanofiber-Based+Cell+Capture+Assay+for+Detecting+Circulating+Tumor+Cells+from+Colorectal+and+Gastric+Cancer+Patients.">Electrospun TiO2 Nanofiber-Based Cell Capture Assay for Detecting Circulating Tumor Cells from Colorectal and Gastric Cancer Patients.</a> Adv. Mater. 24 (20), 2756-2760 (2012).</li><li>Ali, M. A., Mondal, K., Singh, C., Dhar Malhotra, B., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-epidermal+growth+factor+receptor+conjugated+mesoporous+zinc+oxide+nanofibers+for+breast+cancer+diagnostics.">Anti-epidermal growth factor receptor conjugated mesoporous zinc oxide nanofibers for breast cancer diagnostics.</a> Nanoscale. 7 (16), 7234-7245 (2015).</li><li>Mondal, K., Ali, M. A., Agrawal, V. V., Malhotra, B. D., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Biofunctionalized+Mesoporous+Electrospun+TiO2+Nanofiber+Based+Interface+for+Biosensing.">Highly Sensitive Biofunctionalized Mesoporous Electrospun TiO2 Nanofiber Based Interface for Biosensing.</a> ACS Appl. Mater. Interfaces. 6 (4), 2516-2527 (2014).</li><li>Tu, W., Dong, Y., Lei, J., Ju, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Potential+Photoelectrochemical+Biosensing+Using+Porphyrin-Functionalized+TiO2+Nanoparticles.">Low-Potential Photoelectrochemical Biosensing Using Porphyrin-Functionalized TiO2 Nanoparticles.</a> Anal. Chem. 82 (20), 8711-8716 (2010).</li><li>Liu, S., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coadsorption+of+Horseradish+Peroxidase+with+Thionine+on+TiO2+Nanotubes+for+Biosensing.">Coadsorption of Horseradish Peroxidase with Thionine on TiO2 Nanotubes for Biosensing.</a> Langmuir. 21 (18), 8409-8413 (2005).</li><li>Portan, D. V., Kroustalli, A. A., Deligianni, D. D., Papanicolaou, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biocompatibility+between+TiO2+nanotubes+layer+and+human+osteoblasts.">On the biocompatibility between TiO2 nanotubes layer and human osteoblasts.</a> J.Biomed.Mater.Res. Part A. 100 (10), 2546-2553 (2012).</li><li>Dettin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+surface+modification+of+titanium+oxide+with+different+adhesive+peptides:+Surface+characterization+and+osteoblast-like+cell+adhesion.">Covalent surface modification of titanium oxide with different adhesive peptides: Surface characterization and osteoblast-like cell adhesion.</a> J. Biomed. Mater. Res. Part A. 90 (1), 35-45 (2009).</li><li>Kim, W. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Protein+Immobilization+Efficiency+on+a+TiO2+Surface+Modified+with+a+Hydroxyl+Functional+Group.">Enhanced Protein Immobilization Efficiency on a TiO2 Surface Modified with a Hydroxyl Functional Group.</a> Langmuir. 25 (19), 11692-11697 (2009).</li><li>Son, K. J., Ahn, S. H., Kim, J. H., Koh, W. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+Copolymer-Templated+Mesoporous+TiO2+Films+Micropatterned+with+Poly(ethylene+glycol)+Hydrogel:+Novel+Platform+for+Highly+Sensitive+Protein+Microarrays.">Graft Copolymer-Templated Mesoporous TiO2 Films Micropatterned with Poly(ethylene glycol) Hydrogel: Novel Platform for Highly Sensitive Protein Microarrays.</a> ACS Appl. Mater. Interfaces. 3 (2), 573-581 (2011).</li><li>Kar, P., Pandey, A., Greer, J. J., Shankar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+sensitivity+assays+for+human+cardiac+troponin+I+using+TiO2+nanotube+arrays.">Ultrahigh sensitivity assays for human cardiac troponin I using TiO2 nanotube arrays.</a> Lab Chip. 12 (4), 821-828 (2012).</li><li>Agarwal, S., Wendorff, J. H., Greiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrospinning+technique+for+biomedical+applications.">Use of electrospinning technique for biomedical applications.</a> Polymer. 49 (26), 5603-5621 (2008).</li><li>Ding, B., Wang, M., Wang, X., Yu, J., Sun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanomaterials+for+ultrasensitive+sensors.">Electrospun nanomaterials for ultrasensitive sensors.</a> Mater. Today. 13 (11), 16-27 (2010).</li><li>Liu, Y., Yang, D., Yu, T., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+electrospun+nanofibrous+PVDF+membranes+into+a+microfluidic+chip+assembled+by+PDMS+and+scotch+tape+for+immunoassays.">Incorporation of electrospun nanofibrous PVDF membranes into a microfluidic chip assembled by PDMS and scotch tape for immunoassays.</a> ELECTROPHORESIS. 30 (18), 3269-3275 (2009).</li><li>Lee, W. S., Sunkara, V., Han, J. -. R., Park, Y. -. S., Cho, Y. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+TiO2+nanofiber+integrated+lab-on-a-disc+for+ultrasensitive+protein+detection+from+whole+blood.">Electrospun TiO2 nanofiber integrated lab-on-a-disc for ultrasensitive protein detection from whole blood.</a> Lab Chip. 15 (2), 478-485 (2015).</li><li>Li, D., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Titania+Nanofibers+by+Electrospinning.">Fabrication of Titania Nanofibers by Electrospinning.</a> Nano Lett. 3 (4), 555-560 (2003).</li><li>Lombard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SolidWorks 2013 BIBLE. , (2013).</li><li>Tickoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EdgeCAM 11.0 for Manufacturers. , (2007).</li><li>Zhu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+adhesion+of+interconnected+TiO2+nanofiber+network+on+conductive+substrate+and+its+application+in+polymer+photovoltaic+devices.">Improved adhesion of interconnected TiO2 nanofiber network on conductive substrate and its application in polymer photovoltaic devices.</a> Appl. Phys. Lett. 93 (1), 013102 (2008).</li><li>Song, M. Y., Ahn, Y. R., Jo, S. M., Kim, D. Y., Ahn, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO2+single-crystalline+nanorod+electrode+for+quasi-solid-state+dye-sensitized+solar+cells.">TiO2 single-crystalline nanorod electrode for quasi-solid-state dye-sensitized solar cells.</a> Appl. Phys. Lett. 87 (11), 113113 (2005).</li><li>Katsuhiro, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+processed+nanofibrous+TiO2+membranes+for+photovoltaic+applications.">Electrospinning processed nanofibrous TiO2 membranes for photovoltaic applications.</a> Nanotechnology. 17 (4), 1026-1031 (2006).</li></ol>

The authors have nothing to disclose.

이산화 티탄 NF 통합 된 디스크에 대한 분석은 혈액의 매우 낮은 볼륨에 존재하는 낮은 풍부한 단백질의 고감도 검출을위한 신속하고 저렴하고 편리한 기술이다. 이 기술은 적은 양의 샘플 (10 μL)를 사용하는 장점을 가지며, 동시에 다수의 샘플 분석을위한 의무이다. 이것은 다중 면역 장치로서 큰 잠재력을 제공한다. 상기 장치는 통상적 인 ELISA를에 필요한 혈장 분리, 같은 시료의 전처리...

질병 진단을위한 여러 플랫폼 1,2- 예컨대 나노 와이어, 나노 입자 (3), (4) 나노 튜브 (5)와 나노 파이버 (NFS) 6-8과 같은 나노 재료에 기초하여 개발되었다. 이 나노 물질은 독특한 물리 화학적 특성으로 인해 매우 민감한 생물 검정을위한 새로운 기술의 디자인 적으로도 뛰어난 전망을 제공합니다. 예를 들어, 메조 포러스 산화 아연 나노 유방암 바이오 마커의 펨토 몰 민감한 검출을 위해 사용되었다. 구 최근 화학적 안정성을 고려하여 생체 시료 애플리케이션 10 탐구되었다 티탄 (TiO2), 이산화에 기초하여 나노 물질을 11 무시할 단백질 변성 12 및 또한 생체 적합성.도 13은 이산화 티탄의 표면에 수산기 화학적 변형 및 생체 분자의 공유 결합을 용이하게한다. 14,15 무늬 이산화 티탄 생N 막 16 이산화 티탄 (17) 표면적이 증가하여 표적 단백질의 검출 감도를 향상시키는 데에 이용 된 나노 튜브; 그러나, 제조 공정이 다소 복잡하고 값 비싼 장비를 필요로한다. 한편, 전기 방사 NFS가 간단하고 저렴한 제조 공정뿐만 아니라 그들의 높은 표면적 주목 받고; 18,19 아직 상기 전기 방사 이산화 티탄 NF 매트 취약한 느슨한 특성 다루기가 어렵고 미세 유체 장치와 통합 할 수 있습니다. 6,20을 따라서, 이산화 티탄 NF 매트는 거의, 생체 시료 응용 프로그램에서 거친 세척 조건을 필요로 특히 활용되지 않았다. 본 연구에서는 이러한 한계를 극복하기 위해, 우리는 얇은 폴리 디메틸 실록산 (PDMS) 접착제 층을 이용하여 임의의 피 처리 기판의 표면에 매트 NF는 전기 방사를 전송하는 새로운 기술을 개발 하였다. 작고에hermore, 우리는 성공적으로 폴리 카보네이트 (PC)로 이루어진 원심 미세 유체 소자로 전기 방사 티오 NF는 매트 (2)의 적분을 보여왔다. 이 장치를 사용하여, C 반응성 단백질의 민감한 높은 완전 자동화 된, 통합 검색 (CRP)뿐만 아니라 심장 트로포 닌 I (cTnI는)는 전체 혈액의 10 μL에서 30 분 이내에 달성 하였다. (21)로 인해 결합에 는 NFS와 원심 플랫폼의 특성의 장점은 분석은 검출 하한 100 ng를 / ㎖ (0.8 ~ PM) 1 pg / ml 인 (~ 8 FM)에서 크기의 여섯 주문의 넓은 동적 범위를 전시 CRP (37) PG / ㎖ (~ 오후 1시 5분)의 검출 한계 100ng의 / ㎖ (~ 4 ㎚) 10 pg / ml 인 (0.4 ~ PM)의 동적 범위의 0.8 pg / ml이다 (~ 6 FM)의 cTnI는합니다. 이러한 검출 한계는 300 ~되고, 20 배, 대응하는 종래의 ELISA 결과와 비교하여 낮은. 이 기술은 적절한 항체 모든 표적 단백질의 검출에 적용될 수있다. 전반적으로,이 장치 공동예를 들면, 전혈을 10 μl를 그것은 생물학적 샘플의 극소량에서조차 좋은 감도로 표적 단백질의 드문 양을 검출 할 수 있기 때문에 ULD은 체외 진단 및 생화학 적 분석에 크게 기여한다. 우리는이 연구에서 ELISA를 사용하여 혈청 단백질 검출을 증명하지만, 미세 유체 소자와 전기 방사의 NFS 전사 통합 기술은 더 광범위하게 높은 검출 감도를위한 큰 표면적을 필요로하는 다른 생화학 적 반응에 적용될 수있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Si wafer</td>
			<td>LG SILTRON</td>
			<td>Polished Wafer, test grade</td>
			<td>Dia. (mm) = 150, orientation = &#60;100&#62;, dopant = boron, RES(Ohm-cm) = 1 - 30, thickness (&#956;m) = 650 - 700</td>
		</tr>
		<tr>
			<td>Polycarbonate (PC)&#160;</td>
			<td>Daedong Plastic</td>
			<td>PCS#6900</td>
			<td>Thickness (mm) = 1 and 5&#160;</td>
		</tr>
		<tr>
			<td>Titanium tetraisopropoxide, 98%,</td>
			<td>Sigma-Aldrich</td>
			<td>205273</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone, Mw = 1,300,000</td>
			<td>Sigma-Aldrich</td>
			<td>437190</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS and curing agent</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>GPDES</td>
			<td>Gelest Inc</td>
			<td>SIG5832.0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J T Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FE-SEM</td>
			<td>FEI</td>
			<td>Nova NanoSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray photoelectron spectroscopy</td>
			<td>ThermoFisher</td>
			<td>K-alpha</td>
			<td></td>
		</tr>
		<tr>
			<td>3D modeling machine</td>
			<td>M&#38;I CNC Lab, Korea</td>
			<td></td>
			<td>CNC milling machine</td>
		</tr>
		<tr>
			<td>Wax-dispensing machine</td>
			<td>Hanra Precision Eng. Co. Ltd., Korea</td>
			<td></td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>Double-sided adhesive tape</td>
			<td>FLEXcon, USA</td>
			<td>DFM 200 clear 150 POLY H-9 V-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting plotter</td>
			<td>Graphtec Corporation, Japan</td>
			<td>Graphtec CE3000-60 MK2</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>MIDAS</td>
			<td>SPIN-3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Furnace (calcination)</td>
			<td>R. D. WEBB COMPANY</td>
			<td>WEBB 99</td>
			<td></td>
		</tr>
		<tr>
			<td>Rheometer (Tack test)</td>
			<td>Thermo Scientific</td>
			<td>Haake MARS&#160;III - ORM Package</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma system</td>
			<td>FEMTO</td>
			<td>CUTE</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse antihuman hsCRP</td>
			<td>Hytest Ltd., Finland</td>
			<td>4C28</td>
			<td>(clone # C5)</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-cTnI</td>
			<td>Hytest Ltd., Finland</td>
			<td>4T21</td>
			<td>(clone # 19C7)</td>
		</tr>
		<tr>
			<td>HRP conjugated goat polyclonal anti-hsCRP</td>
			<td>Abcam plc., MA</td>
			<td>ab19175</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated mouse monoclonal anti-cTnI</td>
			<td>Abcam plc., MA</td>
			<td>ab24460</td>
			<td>(clone # 16A11)</td>
		</tr>
		<tr>
			<td>hsCRP</td>
			<td>Abcam plc., MA</td>
			<td>ab111647</td>
			<td></td>
		</tr>
		<tr>
			<td>cTnI</td>
			<td>Fitzgerald, MA</td>
			<td>30-AT43</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Amresco Inc</td>
			<td>E404</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>BD vacutainer</td>
			<td>367844</td>
			<td>K2 EDTA 7.2 mg plus blood 
			collection tubes</td>
		</tr>
		<tr>
			<td>SuperSignal ELISA femto</td>
			<td>Invitrogen</td>
			<td>37074</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular multilabel plate reader</td>
			<td>Perkin Elmer</td>
			<td>Envision 2104</td>
			<td></td>
		</tr>
		<tr>
			<td>Disc operating machine</td>
			<td>Hanra Precision Eng. Co. Ltd., Korea</td>
			<td></td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>Photomultiplier tube (PMT)</td>
			<td>Hamamatsu Photonics</td>
			<td>H1189-210</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>AutoDesk</td>
			<td>Version 2012</td>
			<td>Design software</td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software&#160;</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2013</td>
			<td>3D Design software,</td>
		</tr>
		<tr>
			<td>Edgecam</td>
			<td>Vero software</td>
			<td>version 2009.01.06928</td>
			<td>Code generating software</td>
		</tr>
		<tr>
			<td>DeskCNC</td>
			<td>Carken Co.</td>
			<td>version 2.0.2.18</td>
			<td>CNC milling machine software</td>
		</tr>
	</tbody>
</table>

fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

이 프로토콜, 높은 감도를 제조 하였다으로 전체 혈액에서 단백질 검출을위한 완전 자동화 된 원심 미세 유체 장치를 사용. 이산화 티탄 NF 매트는 전기 방사 및 소성 공정에 의해 제조 하였다. 이러한 유량, 전압 및 회전 시간 등의 조건을 전기 방사 원하는 직경, 형태 및 두께는 NFS를 제조하기 위해 최적화되었다. 조건이 최적화되지 않은 경우는 NFS 형성의 질이 저...

Watch this Scientific Journal Video about Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood at JoVE.com

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

전혈에서 고감도 단백질 검출을위한 완전 자동화 된 원심 미세 유체 장치

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

fully-automated-centrifugal-microfluidic-device-for-ultrasensitive

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.4K 조회수.  School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, Republic of Korea. This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 µL of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

비디오: Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

종양 Microenvironment를 재현을위한 마이크로 유체 장치<em&gt; 체외에서</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

연구

생체공학

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

microfluidic 비말 플랫폼 형광 검출 방법

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

단백질 접기 유학을위한 Microfluidic 믹서

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

여러 고유 긴장 유학을위한 마이크로 유체 장치

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

처리되지 않은 전체 혈액에서 호중구 주 화성을 측정하기위한 미세 유체 플랫폼

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

마이크로 기둥 미세 유체 장치에서 바이오 필름 보리 형성을위한 프로토콜

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

적혈구 집계 동적 조사를위한 미세 유체 제어 환경

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

마이크로 스케일 슐 리렌 기술에 의해 미세 유체 소자 믹싱 비균질성 분석

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

판막 다층 미세 유체 장치에 대한 여러 단계의 변수 높이 포토 리소그래피

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...