이 연구는 건강의 국립 연구소 (NIH) 보조금 R01 EB008268에 의해 지원되었다.

1. 칩 제조 12 <ol><li> Zeonex 690R 알약에서 두 액자 만들기 : 5 분에 198 ° C에서 가열 언론에 금속 플레이트, 예열의 중심에 균일하게 8~9g Zeonex 알약을 배포 한 다음 다른 5 분에 2,500 PSI로 천천히 압력을 적용 할 수 있습니다. 이 단계를 완료하려면, 우리는 카버 핫 프레스를 사용했습니다. </li><li> 에폭시 몰드와 상패의 마이크로 유체 채널을 양각. 금형 제조에 대한 자세한 내용은 다른 곳에서 12 일 (그림 2B의 채널 디자인) 설명되어 있습니다 : 에폭시 몰드에 한 플라크를 넣어 한 후 157에있는 온수 보도에 앞서 열을 가하다 그 ° 10 분에 C와 다른 10 분 동안 천천히 1,000 PSI에 압력을 적용 . 손으로 금형에서 플라크를 제거하거나 아래로 냉각하기 전에 집게를 사용합니다. (참고 :이 단계에서 열 장갑을 착용하십시오.) </li><li> 입구, 폐기물 포트, 그리고 마이크로 유체 채널의 출구에서 양각 칩에 구멍을 뚫는다. 우리는 1.25 mm의 diam를 사용세 구멍을 만들기 위해 드릴 비트를 eter. </li><li> 본드 다른 플라크 (상단)과 양각 칩은 :; 또 다른 350 PSI에 한 다음 Enter 키를 누릅니다 10 분 동안 131 ° C에서 가열 언론에 공기 건조하고 예열을하고 IPA, RNAse 거리에 있으며 순서에 탈 이온수로 칩을 씻으십시오 10 분. </li><li> 입구, 폐기물 및 별도 JB 용접 에폭시를 사용하여 콘센트 포트에 Nanoports를 첨부, 제조업체의 지시 사항에 따라 15-24 시간에 대한 치료. </li><li> 100 μl nuclease 무료 물 다음에 50 μl RNAse 멀리있는 SPE 채널을 씻어. </li><li> 접목 솔루션 (3 % w / v를 benzophenone과 메틸 메타 크릴 레이트) 4 μl와 SPE 채널을로드 한 다음 365 나노 미터 UV 파장과 2,000 MJ / cm 2 이하 UV 오븐에 10 분 동안 잠복기로 메타 크릴 레이트를 crosslink. 진공과 잔류 접목 솔루션을 제거합니다. 우리는 벽 진공을 사용했습니다. 최상의 결과를 얻으려면 신선한 솔루션을 사용해야합니다. 그것은 일반적으로 수용하지만,를 저장할최대 일주일 4 ° C에서 준비한 솔루션입니다. </li><li> 16 % 에틸렌 dimethacrylate, 24% 부틸 메타 크릴 레이트, 42 % 1-dodecanol, 18 %의 cyclohexanol, 1 % 2 - 디메틸 아미노 -4 - (메틸 phenylamino) 페놀을 추가 SPE 열 솔루션을 (모든 V / V %) 확인 사진 기자로. </li><li> SPE 열 솔루션 (1:1 혼합물을하는)와 같은 실리카 마이크로 솔루션의 동일한 볼륨을 가지고, 완전히 ° C 하룻밤 진공 오븐에 70에서 건조. 폐쇄 뚜껑과 함께 튜브에 활용하여 펠릿을 해봐. 건조 실리카 microsphere 튜브와 혼합 할 소용돌이에 SPE 열 솔루션을 추가합니다. </li><li> 동일한 채널에 SPE 솔루션 4 μl를로드 한 다음 2.5 분 동안 UV 조사하여 crosslink. 그런 다음 위에 칩을 뒤집어 다른 2.5 분에 비추다. polymerized의 SPE 항목은 불투명 한 흰색 고체해야합니다. </li><li> 초과 반응물을 멀리 씻어하기 위해 100 % 메탄올 500 μl와 채널을 씻으십시오. 이 페인트는 내부 폴리머 PO를 제거주위 조건에서 건조 할 때 rogenic 용매는이를 SPE 칼럼의 열린 기공 구조를 만드는 두 가지 방법이 있습니다. </li><li> 열 전도성 테이프 칩의 맨 아래에 두 개의 박막 히터를 부착합니다. 히터의 측면은 변성 및 (그림 1 참조) 별도로 어닐링을위한 다양한 채널의 가장자리를 정렬해야합니다. </li><li> 죽은 통해 칩에 장소 다섯 열전쌍은 각 칩의 열린 쪽 채널을 (그림 2B 참조) 종료했습니다. 열전대는 언론 운동을 통해이 채널에있어. 테이프는 더 열전대의 위치를​​ 수정하는 데 사용되었다. </li></ol> 2. 고체 단계 추출 방법 <ol><li> DEPC - 처리 및 autoclaved 물 (deionized 또는 Millipore-필터링)을 70 % 에탄올의 96 μl를 준비합니다. </li><li> 25X RNA의 추가 4 μl 한 관에 70 % 에탄올의 96 μl를 확보하고, 별도의 관 100 % 에탄올과 같은 혼합물을 중복. </li><li> 300 준비다음을 혼합하여 채널 버퍼의 μl : 6 M 구아니딘의 thiocyanate (GuSCN), 2 - 프로 파놀의 150 μl, nuclease 무료 물의 63 μl, 그리고 보안 25X RNA 12 μl의 75 μl. </li><li> 6 M GuSCN, 2 - 프로 파놀 200 μl 및 보안 12 μl 25X RNA의 100 μl : 혼합하여 용해 버퍼의 312 μl를 준비합니다. </li><li> RNA 보안을 활성화하려면 10-20 분을위한 60 ° C에서 70 %의 에탄올을 가열, 100 % 에탄올, 채널 버퍼와 용해 버퍼. </li><li> 마이크로 유체 채널을 평형하려면, 0.8 ML / 시간의 유량에서 SPE 채널을 통해 채널 버퍼를 실행합니다. </li><li> 물 욕조에서 37 ° C에서 VTM에 테스트 샘플을 빠른 해동하고, 가능한 한 빨리이 해동 될 때 물통에서 즉시 샘플을 제거합니다. </li><li> 10 분에 13,000 rpm으로 샘플을 원심 분리기 및 용해 버퍼 300 μl로 표면에 뜨는 100 μl를 전송합니다. 1 μg / μl 캐리어 RNA의 6 μl를 추가하여 혼합을 완료합니다. </li><li> 혼합 할 수 소용돌이, 그리고에 대한 스핀 후5 초는 Luer - 무사히 다녀 오게 한 CC의 주사기로 전체 lysate를로드합니다. </li><li> 0.8의 유량에서 SPE 채널을 통해 lysate를 실행 ML / 시간. </li><li> 1 ML / 시간 100 μl of100 %의 에탄올에 이어 70 %의 에탄올 100 μl와 SPE 채널을 씻으십시오. (내보냈되지 않습니다 주사기 팁에 50 μl 죽은 볼륨이 있으므로 실제로는 50 μl의 채널을 통해 진행됩니다.) </li><li> 0.5 ML 마크의 빈 주사기를 위치 1에서 건조 할 채널을 통해 공기를 밀어 ML / 시간.주세요 </li><li> 0.5에서 바인딩 된 핵산을 elute ML / 시간 및 폐기물 포트에서 nanoport에서 13.5 μl를 수집하는 채널을 통해 nuclease - 무료 물 67.5 μl를 실행합니다. (유체 손실이 주사기 팁과 SPE 채널에 54 μl 죽은 볼륨 때문입니다.) </li></ol> 3. RT-PCR <ol><li> Qiagen OneStep RT-PCR 키트의 시약을 사용하여 공기 건조 단계 (2.12) 동안 RT-PCR 마스터 믹스의 36.5 μl를 준비합니다. </li><li> RT-PC를로드R은 쓰레기 포트에서 nanoport의 시약 및 RNA와 혼합 다음 50 μl RT-PCR 반응을 얻을 수 : nuclease 무료 물, 4 μl RT-PCR 효소 혼합, 10 μl Q 솔루션, 10 μl1X 하나에 13.5 μl 용출 RNA를 단계 버퍼, 2 μl 25 MM MgCl 2, 1 μl 50 앞으로 μM 프라이머 5&#39;-GAC 미쳤 (다고하더라도) TCC TGT CAC CTC TGA C-3 &#39;, 1 μl 50 μM 역 프라이머 5&#39;-AGG GCA TTY TGG ACA AAK CGT CTA-3 &#39;, 2 μl dNTP, 0.75 μl 1.0 % w / V BSA, 0.73 μl PEG8000과 5.02 μl nuclease 무료 물. </li><li> 부드러운 진공으로 RT 채널 (우리가 벽 진공을 사용)에 RT-PCR 혼합물을로드하고 폐쇄 피팅으로 Nanoport을 봉쇄. </li><li> 히터 1-35 V를 적용 할 수 있습니다. 히터 1 50 ° C.에 equilibrates 후 30 분 동안 RT 채널에 시약 보관 95에서 히터 하나 equilibrates 후 15 분 ° C.에 대한 RT 채널에 시약 보관 </li><li> 히터 1과 히터 2-50 V 27 V를 적용, 60 &amp; 드에 3 분 정도 또는 히터 1 equilibrates 때까지 기다리g, C 및 히터 2 95에 equilibrates ° C. </li><li> 0.5 μl / 분의 PCR 채널에 시약을 밀어 넣습니다. 시약은 굴곡 채널을 통해 유동하는 것은 약 20 분 정도 소요됩니다. </li><li> 샘플 굴곡 채널의 끝 부분에 낸다으로 적절하게 크기 pipettor 및 팁을 사용하여 전원 콘센트에서 PCR 제품을 수집합니다. </li></ol> 4. PCR 제품 검색 <ol><li> 우리는 제품을 감지 할 수있는 애질런트 높은 감도 DNA 검사를 사용합니다. 이 테스트는 냉장고에서 테스트하기 전에 30 분 애질런트 높은 감도 DNA 키트를 꺼내 실행하십시오. </li><li> Bioanalyzer 전원을 켜고 2100 전문가 소프트웨어를 엽니 다. </li><li> 테스트에 PCR 제품의 1 μl를로드하고 애질런트 프로토콜 (따라 <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent의 %의 20Manual.pdf을</a> ). </li><li> 분석하고 데이터를 기록합니다. </li></ol>

<ol>
	<li>Nicholls, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+Influenza:+The+Inside+Story.">Pandemic Influenza: The Inside Story.</a> PLoS Biol. 4, (2006).</li><li>Wenzel, J. 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=Analytical+performance+determination+and+clinical+validation+of+the+novel+roche+realtime+ready+influenza+A/H1N1+detection+set.">Analytical performance determination and clinical validation of the novel roche realtime ready influenza A/H1N1 detection set.</a> Journal of Clinical Microbiology. 48, 3088-3094 (2010).</li><li>Chan, K. H., 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=Analytical+sensitivity+of+rapid+influenza+antigen+detection+tests+for+swine-origin+influenza+virus+(H1N1.">Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1.</a> Journal of Clinical Virology: the. 45, 205-207 (2009).</li><li>Ginocchio, C. C., 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=Evaluation+of+multiple+test+methods+for+the+detection+of+the+novel+2009+influenza+A+(H1N1)+during+the+New+York+City+outbreak.">Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak.</a> Journal of. 45, 191-195 (2009).</li><li>Aeron, C. H., 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=Performance+of+influenza+rapid+point-of-care+tests+in+the+detection+of+swine+lineage+A(H1N1)+influenza+viruses.">Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses.</a> Influenza and Other Respiratory Viruses. 3, 171-176 (2009).</li><li>Takahashi, H., Otsuka, Y., Patterson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+for+influenza+and+other+respiratory+viruses:+determining+performance+specifications+based+on+clinical+setting.">Diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting.</a> Journal of Infection and Chemotherapy. 16, 155-161 (2010).</li><li>Selvaraju, S. B., Selvarangan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Three+Influenza+A+and+B+Real-Time+Reverse+Transcription-PCR+Assays+and+a+New+2009+H1N1+Assay+for+Detection+of+Influenza+Viruses.">Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses.</a> Journal of Clinical Microbiology. 48, 3870-3875 (2009).</li><li>Northrup, M. A., Ching, M. T., White, R. M., Watson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transducer'93, seventh international conference on solid state sensors and actuators. , 924-926 (1993).</li><li>Bhattacharyya, A., Klapperich, 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=Thermoplastic+microfluidic+device+for+on-chip+purification+of+nucleic+acids+for+disposable+diagnostics.">Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.</a> Analytical Chemistry. 78, 788-792 (2006).</li><li>Cao, Q., Kim, M. -. C., Klapperich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+microfluidic+chip+for+continuous-flow+polymerase+chain+reaction:+Simulations+and+experiments.">Plastic microfluidic chip for continuous-flow polymerase chain reaction: Simulations and experiments.</a> Biotechnology Journal. 6, 177-184 (1002).</li><li>Cao, Q., 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=Microfluidic+Chip+for+Molecular+Amplification+of+Influenza+A+RNA+in+Human+Respiratory+Specimens.">Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens.</a> PLoS ONE. 7, e33176 (2012).</li><li>R&#229;dstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Characterization+of+PCR-Inhibitory+Components+in+Blood+Cells.">Purification and Characterization of PCR-Inhibitory Components in Blood Cells.</a> J. Clin. Microbiol. 39, 485-493 (2001).</li><li>Boddinghaus, B., Wichelhaus, T. A., Brade, V., Bittner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+PCR+Inhibitors+by+Silica+Membranes:+Evaluating+the+Amplicor+Mycobacterium+tuberculosis+Kit.">Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor Mycobacterium tuberculosis Kit.</a> J. Clin. Microbiol. 39, 3750-3752 (2001).</li><li>Andreasen, 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=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50, S6-S9 (2010).</li></ol>

저자는 경쟁 재정 이익을 선언 없습니다.

진단 방법은 높은 특이성과 낮은 검출 한계가있는 환자 ​​표본에서 인플루엔자 RNA를 증폭 할 수있는 통합 마이크로 플라스틱 칩의 능력을 보여 여기에 제시된 13 우리는 케어 테스트의 잠재적 지점이 칩을 설계 :. (A) 온도와 유체 컨트롤이 간단했다, (b)는 칩은 낮은 비용과 사출 성형을 사용하여 높은 처리량 제조에 적합, 그리고 (c) 칩은 따라서 표본 교차 오염의 우려를 줄이고, 종이 및...

수백만 명의 죽음은 20 세기 1 세 인플루엔자 전염병 동안보고되었습니다. 또한, 가장 최근의 인플루엔자 유행은 2009 년에 세계 보건기구 (WHO) 2로 선언되었고, 같은 2010년 8월 1일의, 18,449 사망자는 WHO 3보고되었다. 이 유행은 다시 전염성 질병의 높은 부담, 및 빠른 질병 확인, 적절한 공중 보건 대응하고 효과적인 치료 4 활성화 인플루엔자의 신속하고 정확한 탐지의 필요성을 보여 주었다. 널리 독감을 진단에 사용되는 여러 가지 방법이 있습니다, 이들은 빠른 immunoassays 직접 형광 항원 검사 (DFA) 및 바이러스 성 문화 방법이 포함되어 있습니다. 다른 두 방법은 노동 집약적 인 9 소비 시간이있는 동안 급속한 immunoassays은 극적으로, 감도 5-8가 부족합니다. 분자 테스트는 짧은 턴 - 주위에 시간, 높은 sensit 등의 여러 장점을 제공합니다ivity, 높은 특이성. 여러 상업 기관은 전염병에 대한 빠른 분자 테스트 (또한 핵산 검사 또는 NATs이라고도 함)으로 작업되었으며, 몇은 파이프 라인에서 인플루엔자 assays 있습니다. 그러나 대부분은 오프 칩 샘플 준비가 필요합니다. 분자 검사 검정 카트리지 나 모듈에 샘플 준비를 통합 면제 임상 실험실 개선 개정 (CLIA)의 없음. 랩 - 온 - 어 - 칩 기술은 포인트의 케어 테스트 장치의 개발에 중요한 역할을한다. 1993 10 첫 번째 PCR 칩의 도입 후, 많은 노력 핵산 테스트 칩을 개발에 투입되었습니다. 그러나, 이들의 몇은 하류 증폭과 통합 원유 샘플 준비되어 있습니다. 우리는 이전에 플라스틱 마이크로 칩 11으로 견고한 위상 추출 컬럼 (SPE)의 소형화을 입증하고 개발 한연속 흐름 PCR 칩 12 ment 및 최적화. 여기, 우리는 임상 진단을위한 단일 칩에 RT와 PCR 단계로 SPE를 통합하고 환자 nasopharyngeal (NP) 면봉과 aspirates에서 핵산을 증폭하기위한 능력을 평가하는 이전 작업을 확장합니다.

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 1 dodecanol </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 443816-500G </td></tr><tr><td> 2,2-Dimethoxy-2-phenylacetophenone </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 196118-50G </td></tr><tr><td> 2100 Bioanalyzer </td><td> 애질런트 테크놀로지스, 산타 클라라, CA </td><td> G2943CA </td></tr><tr><td> 2 - 프로 파놀 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 19,516 </td></tr><tr><td> Benzophenone </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 239852-50G </td></tr><tr><td> BSA </td><td> 온도 피셔 과학, pittsburge, PA </td><td> A7979-50ML </td></tr><tr><td> 부틸 메타 크릴 레이트 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 235865-100 ML </td></tr><tr><td> 캐리어 RNA </TD&gt; <td> Qiagen, 발렌시아, CA </td><td> 1017647 </td></tr><tr><td> Cyclohexanol </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 105899-1L </td></tr><tr><td> 에탄올 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> E7023 </td></tr><tr><td> 에틸렌 dimethacrylate </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 335861 </td></tr><tr><td> 에틸렌 글리콜 dimethacrylate </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 335681-100ML </td></tr><tr><td> 유리 주사기 250 μl </td><td> 해밀턴, 리노, NV </td><td> 81,127 </td></tr><tr><td> 구아니딘의 thiocyanate </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 50,981 </td></tr><tr><td> 높은 감도 DNA 키트 </td><td> 애질런트 테크놀로지스, 산타 클라라, CA </td><td> 5067-4626 </td></tr><tr><td> 핫 프레스 </td><td> 카버 와바 시가에 </td><td> 4386 </td></tr><tr><td> JB 용접 Epoxies </td><td> Mcmaster-CARR, 허스트, IL </td><td> 7605A11 </td></tr><tr><td> Luer - 무사히 다녀 오게 주사기 </td><td> BD-의료, 프랭클린 호수, NJ </td><td> 309628 </td></tr><tr><td> 염화 마그네슘 </td><td> 온도 피셔 과학, pittsburge, PA </td><td> AB-0359 </td></tr><tr><td> 메타놀 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 494437 </td></tr><tr><td> 메틸 메타 크릴 레이트 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch 과학 </td><td> N-333-01 </td></tr><tr><td> Nanoport 피팅 </td><td> Upchurch 과학 </td><td> F-120x </td></tr><tr><td> Nuclease 무료 물 </td><td> 온도 피셔 과학, pittsburge, PA </td><td> PR-P1193 </td></tr><tr><td> OneStep RT-PCR 키트 </td><td> Qiagen, 발렌시아, CA </td><td> 210210 </td></tr><tr><td> PEG8000 </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 41,009 </td></tr&gt; <tr><td> 전원 공급 장치 </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> 멀리 RNAse </td><td> 시그마 - 알드리치, 세인트 루이스, MO </td><td> 83931-250ML </td></tr><tr><td> RNASecure </td><td> 응용 Biosystems, 포스터 시티, CA </td><td> AM7005 </td></tr><tr><td> 실리카 마이크로 </td><td> Polysciences, 링톤, PA </td><td> 24324-15 </td></tr><tr><td> 주사기 펌프 </td><td> 하버드 장치, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> 열 전도성 테이프 </td><td> Mcmaster - 카, 허스트, IL </td><td> 6838A11 </td></tr><tr><td> 열전대 </td><td> 오메가 공학, 스탬 포드, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> 박막 히터 </td><td> Minco, 미니애폴리스, MN </td><td> HK5166R529L12A </td></tr><tr><td> 자외선 Crosslinker </td><td> UPV, Upland, CA </td><td> CL-1000 </td></tr><tr><td> Zeonex </td> &lt;TD&gt; Zeon 화학 물질, 루이빌, KY </td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

빠르고 효과적인 진단은 효과적인 환자 관리 및 치료를함으로써 전염병을 제어하는​​ 데 중요한 역할을한다. 여기, 우리는 환자 nasopharyngeal (NP) 면봉과 aspirates의 인플루엔자 바이러스를 증폭 할 수있는 능력을 통합 마이크로 열가소성 칩을 제시한다. 환자 샘플을로드하면, 마이크로 유체 장치는 순차적으로, 온칩 세포 용해, RNA 정화 및 고체 위상 추출 (SPE), 역 전사 (RT) 및 RT-PCR 챔버에서 중합 효소 연쇄 반응 (PCR)에 농도 단계를 수행 각각. 엔드 포인트 검출은 오프 칩 Bioanalyzer을 (애질런트 테크놀로지스, 산타 클라라, CA)를 사용하여 수행됩니다. 이 박막 히터는 RT와 PCR 챔버의 열원으로 사용하는 동안 주변 기기를 들어, 우리는 시약 및 샘플을 늘리기 위해 하나의 주사기 펌프를 사용했습니다. 이 칩은 fabricati을 줄이기 위해 높은 처리량 제조를위한 단일 레이어와 적합하도록 설계되어시간과 비용 있습니다. 마이크로 칩은 관심의 새로운 병원균을 감지하는 데 필요한 시약 디자인의 변화 만 제한 바이러스와 박테리아의 다양한 분석 할 수있는 플랫폼을 제공합니다.

<html> 전형적인 결과는 인플루엔자에 감염된 nasopharyngeal 세척 표본에 대한 그림 3에 표시됩니다. 각 환자 표본에서 인플루엔자 바이러스의 서로 다른 양의로 인해, PCR 제품의 최종 농도는 달라질 수 있습니다. 좋은 결과는 낮은 노이즈, 두 명확 래더 봉우리 (35 10,380 BP)과 긍정적 인 샘플에 대한 설계 제품 사이즈 (107 BP)에서 하나의 제품 피크가 있어야합니다. 제품 피크는 이론적으로 부?...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

통합 마이크로 열가소성 칩은 분자 진단으로 사용하기 위해 개발되었습니다. 이 칩은 핵산 추출, 역 transcriptase 및 PCR을 수행합니다. 제조 및 칩을 실행하기위한 방법은 설명되어 있습니다.

마이크로 유체 칩 제작 및 인플루엔자을 감지하는 방법

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

Research

JoVE Journal

Bioengineering

14.8K 조회수.  Boston University. 통합 마이크로 열가소성 칩은 분자 진단으로 사용하기 위해 개발되었습니다. 이 칩은 핵산 추출, 역 transcriptase 및 PCR을 수행합니다. 제조 및 칩을 실행하기위한 방법은 설명되어 있습니다.

비디오: Microfluidic Chip Fabrication and Method to Detect Influenza

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 ...

연구

생체공학

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

단일 세포의 다목적 화학 분석을위한 미세 유체 칩

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

미리 설계된 크기와 모양의 중합체 및 바이오 하이브리드 섬유의 미세 유체 제조

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMS 샘플 소개를위한 미세 유체 칩

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

혈관 유래 미세 유체 네트워크의 이미지 유도, 레이저 기반의 제조

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

면역 측정법에 대한 3 차원 종이 기반 미세 유체 장치의 제작

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

드라이 필름 포토 레지스트 기반 전기 화학 미세 바이오 센서 플랫폼: 장치 제작, 온 칩 시험 준비 및 시스템 운영

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

제조 및 통합된 전극 Transendothelial 전기 저항을 직접 측정 하는 기관-온-칩 시스템의 유효성 검사

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Stretchable, 듀얼 채널, 미세 기관 칩의 확장 가능한 제작

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...