Sandia는 미국이 계약 DE-AC04-94AL85000 하에서 에너지의 국립 핵 보안 관리학과를 명시에 대한 Sandia 공사, 록히드 마틴 사에 의해 운영 multiprogram 연구소입니다. 저자는 Sandia 국립 연구소 &#39;연구실에서 기금을 인정하고 연구 개발 (LDRD) 프로그램을 감독.

1. Microneedle 제작 <ol><li> 입체 모델링 소프트웨어 Solidworks (다쏘 Systemes SA, Velizy, 프랑스)를 이용하여 피라미드 모양의 중공 microneedle 배열을 설계 (그림 1). 3-5 </li><li> Magics RP 13 소프트웨어 (Materialise NV, Leuven, 벨기에)를 사용 microneedle 배열에 대한 지원 구조를 디자인합니다. 지원 구조는 수지가 제조하는 동안 장치에서 드레인 수 있으며 microneedles이 구축되어있는 기반을 제공합니다. 예를 들어, 지원 구조는 그림 1에 표시됩니다. </li><li> 연계 지원 및 microneedle 배열 파일은 제조 공정을 제어 Perfactory RP 소프트웨어 (EnvisionTEC 게엠베하, Gladbeck, 독일)에 업로드됩니다. 이 소프트웨어 패키지 내에서 가공되는 microneedle 배열의 개수를 선택하고 제조 접시 장치의 위치를​​ 결정합니다. </li><li> Perfactory 신속한 PR 180 MW에서 자외선 모드에서 보정을 실행합니다ototyping 제조 시스템과 에너지의 편차를 확인은 ± 2 MW 이내입니다. </li><li> 제조가 완료되면 15 분 동안 이소프로판올 년 기​​초판에서 microneedle 배열을 제거하고 개발합니다. 압축 공기가있는 배열을 건조하고 완전한 중합을 보장하기 위해 Otoflash Postcuring 시스템 (EnvisionTEC 게엠베하, Gladbeck, 독일)이 50 초 동안 실온에서 microneedles을 치료. </li><li> 현미경을 통해 microneedle 제조의 유효성을 검사하고 각 microneedle 구멍이 투명하고 탁 트인 있는지 확인합니다. 완벽하게 조립 microneedles는 그림 2에 표시됩니다. </li></ol> 2. 탄소 붙여넣기 전극 배열의 제작 <ol><li> 구멍을 잘라내어였다 플랫 플렉시블 케이블 (21039-0249)에서 기본 개별적으로 주소 지정 연결하는 구리선을 폭로 60 W 모델 6.75 CO 2 래스터 / 벡터 레이저 시스템 (유니버셜 레이저 시스템즈, 스코 츠 데일, AZ)를 사용 상용 소스 (모에서 얻은렉스 커넥터 공단, Lisle, IL) (그림 3 (A와 B)). 제대로 레이저 박리 접시에 그들을 정렬하기 위해 지그에 플랫 플렉시블 케이블을 놓습니다. 유연한 케이블의 절연 부분에서 500 μm의 직경 충치를 만드는 rastering 접근법을 사용하십시오. 절제를위한 패턴은 CorelDraw에서 만든 (코렐, 오타와, 온타리오)와 레이저 시스템으로 보내집니다. </li><li> 40 PSI의 에어 브러쉬 그 스프레이를 아세톤으로 바뀌었 플랫 플렉시블 케이블을 제거합니다. 이소프로판올 및 탈이온수로 rinsing하여 청소 완료. 더 단열 필름 노출된 구리 스트립 위에 남아 없다는 것을 현미경으로 확인합니다. </li><li> 다음 단계는 탄소 붙여 넣습 포장에 대한 들고 충치를 만드는 것입니다. Melinex 테이프는 (압력에 민감한 아크릴 접착제와 함께 단일 면에 코팅 0.002 &quot;두께) 전극 ablated 전극 스트립여 지향 스트립, 그리고 적절한 연결을 보장하기 위해 2 분을 3,000 PSI에 압축과 같은 패턴으로 ablated 있습니다.에이 이 CASE는 캐비티 직경이 750 μm의입니다. </li><li> Melinex 테이프의 추가 레이어 (0.004 &quot;압력에 민감한 아크릴 접착제와 양면에 코팅 두께)은 이후 싱글 양면 접착 테이프와 같은 패턴으로 ablated되고 탄소 페이스트 전극 배열로 결합 microneedle 배열에 정렬 후 사용합니다 . </li></ol> 3. 기능성 탄소 붙여 넣습의 합성과 전극 안의 공간의 포장 <ol><li> 포도당 민감한 탄소 페이스트는 이전의 제조법을 근거로하고 균일한 혼합물을 얻을 때까지 포도당 산화 효소와 폴리 (ethylenimine)의 2.2 밀리그램의 10 밀리그램을 혼합하여 얻어진다. 16이 혼합되어, 탄소 분말에 로듐의 60 밀리그램 (최대 5% 로딩)가 추가됩니다. 40 MG의 광유가 추가되고 이후에 혼합됩니다. 붙여 넣습가 준비 후 최대 1 주일 정도 사용되며 붙여 넣습는 ° C에서 사용할 때까지 4에 저장됩니다. </li><li> 산도에 민감한 탄소 페이스트가 혼합 30% (W / W) 광유 70 % (W / W) 흑연 P에 의해 얻어진다owder. 섹션 3.4에서 설명한대로 전극 캐비티에 붙여넣 싸. 0.5 M의 인산 10 밀리미터 패스트 블루 RR diazonium 소금 (4 benzoylamino-2 ,5-dimethoxybenzenediazonium 염화 헤미 (염화 아연) 소금)의 솔루션입니다. 17 확인을위한 포장 페이스트 전극을 통해이 솔루션은 20 μl 드롭 놓으십시오 30 분 자발적 빠른 블루 홍보 diazonium 소금을 chemisorb합니다. 버퍼 또는 탈이온수 사용하지 않을에서의 탈이온수 및 매장과 린스. </li><li> 락테이트 민감한 탄소 페이스트는 이전의 제조법을 근거로하고 있으며 탄소 분말과 락트 산 산화 효소 2.5 밀리그램에 로듐 2.5 밀리그램을 혼합하여 얻은 것입니다, 다섯 회전 동안 sonication 5 분, vortexing 5 분 사이에 교대. 18 </li><li> 준비된 플랫 플렉시블 케이블로 수정된 붙여 넣습의 포장이 전극 충치 통해 각각 붙여 넣습을 적용하여 수행됩니다. 흙손과 팩을 t으로 플라스틱의 얇은 조각을 (예, 플라스틱 무게는 보트의 가장자리) 사용매끄러운 표면을 얻을 때까지 그는 붙여 넣습니다. 과잉 붙여넣기가 제거되기 전까지 두 번째 깨끗한 무게 보트로 반복합니다. 탈이온수로 씻으십시오. 개략도 탄소 붙여 넣습 포장, 충치를 만드는 레이저 절제를 보여주는, 그리고 microneedle 통합은 (섹션 2와 3에서 설명) 그림 3에서 제공됩니다. </li></ol> 4. 감지 및 센서 교정 <ol><li> 락테이트 감지 M 인산 버퍼 (산도 = 7.5). 그림 4 (가) 락트 산의 검출을위한 electrocatalytic 반응의 구조도를 포함 -0.15 V에서 센서의 chronoamperometric 응답을 측정하고 0.1에 15 초 후 전류를 기록하여 수행됩니다 . </li><li> 포도당 감지 -0.05 V에서 센서의 chronoamperometric 응답을 측정하고 0.1 M 인산 완충액 (산도 7.0)에서 15 초 후 전류를 기록하여 유사한 방법으로 수행됩니다. 그림 4 (B)를위한 electrocatalytic 반응의 구조도를 포함하고 디포도당의 etection. </li><li> 산도는 100 MV / s의 -0.7 V에서 0.8 V로 주기적 voltammetric 스캔을 실행하고 산화 피크 잠재력의 위치를​​ 기록하여 감시하고 있습니다. 산도 검출을위한 산화 환원 반응의 개략도는 그림 5에 표시됩니다. </li><li> 포도당과 젖산 센서 보정 곡선은 각 analyte의 연속 첨가에 의해 만들 어질 수 있으며, 섹션 5.1 및 5.2에 설명된대로 chronoamperometric 측정은 각 analyte 추가 후에하게됩니다. 또는 고정 잠재 chronoamperometric 성능은 현재의 안정화에 대한 각 analyte 추가 사이에 충분한 시간을 (~ 10-1백초) 허용하는 동안 교반하에 만들 수 있습니다. </li><li> 산도 교정 곡선은 1.0 산도 단위 단위로 5 ~ 8로 알려진 산도 값 일련의 이상 산화 피크 잠재력의 위치를​​ 측정하고 섹션 5.3에 설명된대로 순환의 voltammograms을 기록하여 만들 수 있습니다. </li></ol> 5. 대표 재sults 수정된 탄소 붙여넣으-가득한 microneedles과 정지 솔루션 chronoamperometric 커브 (예 : 포도당 감지이나 락테이트 감지)를 획득하면, 현재는 즉시 해당 검색 잠재​​력 신청에 따라 줄어 듭니다. 그것은 결국 정상 상태 값에 가옥됩니다. 대표적인 결과는 그림 6에 표시되며이 결과는 락테이트 microneedle에서 락테이트 및 녹음 2 MM 첨가로부터 얻은 것입니다. 솔루션은 간단히 각 락테이트 또한 이후 흔들해야합니다. 15초 후 현재는 젖산의 농도를 증가시 상승, 현재 응답 후 알 수없는 솔루션에 젖산의 농도를 결정하는 데 사용할 수 있습니다. 또한, 지속적인 모니터링이 증가 포도당 농도 (그림 5)와 솔루션을 시연으로 흔들 솔루션 (또는 흐르는 용액에서)에서 사용할 수 있습니다. 다시시 전류의 증가는 T를 증가그 포도당의 농도는 알 수없는 솔루션 포도당 응답을 표준화하는 데 사용할 수 있습니다. 충분한 시간이 솔루션은 안정 수 있도록하기 위해 각 스파이크 후에 허용하여야합니다. 0.1 M 인산 완충액의 산도에 민감한 microneedle의 순환 voltammograms는 5 그림 6에서 8 1에서 pH의 단위 증가에 대한 네 가지 산도 솔루션을 통해 표시됩니다. 증가 산도와 산화 피크 잠재력 교대는, 이러한 현상은 산도 가치의 지표로 사용됩니다. <img alt="그림 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> 그림 1. Solidworks (A) 및 지원 구조 (B)를 보여줍니다 인쇄 화면의 생성 microneedle 배열의 STL 파일의 이미지. <img alt="그림 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> 그림 2. microneedle 배열 (A)이 배열 내에 단일 microneedle (B)의 전자 micrographs를 스캔. 그림 3. 플랫 유연한 케이블 어셈블리의 도식. 참여 단계, 플랫 플렉시블 케이블 () 수정 (B) 패턴 동그라미를 흡열, 탄소 페이스트 (C)로 가득뿐만 아니라 두 번째 ablated Melinex 레이어를 추가하고이 교미하는 초기 ablated Melinex 레이어를 추가 포함 microneedle 어레이 (D)가. <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 누르십시오</a> . <img alt="그림 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> 그림 4. 0.1 M 인산 완충 (산도 = 7.5)에서 -0.15 V에서 15초 chronoamperometric 스캔과 락트 산에 민감한 페이스트의 보정. 락트 산의 2 밀리미터 외에도 현재 해당 각각 증가. <img alt="그림 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> 그림 5. </s응답의 각 감소는 포도당의 2 밀리미터 이외에 해당 -0.05 V.에서 chronoamperometric 스캔을 실행 포도당에 민감한 페이스트의&gt; 보정을 tron​​g. 0.1 M 인산 버퍼 용액 (산도 = 7.0) 보정 기간 동안 흔들되었다. 외부 자세 / AgCl과 PT 참조 및 카운터 전극이 연구에 사용되었다. <img alt="그림 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> 산도에 민감한 탄소 그림 6. 순환의 voltammogram (이력서) 1 산도 단위 증가 (물오리 = 산도 8.0, 녹색 = 산도 7.0, 보라색 = 산도 6.0, 적색 = 산도 5.0)에 산도 5-8 이상 0.1 M 인산 완충액에 붙여 넣습니다. 다섯째 이력서는 자세 / AgCl 참조 및 PT 와이어 카운터 전극 비해 분석에 사용되었다.

<ol>
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C., Xiao, X., Brozik, S. M., Wang, J., Polsky, R., Narayan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microneedle-based+biosensor+array+for+characterization+of+metabolic+acidosis.">Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.</a> Talanta. 88, 739-742 (2012).</li><li>Rofstad, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment-induced+cancer+metastasis.">Microenvironment-induced cancer metastasis.</a> Int. J. Radiat. Biol. 76, 589-605 (2000).</li><li>Vander Heiden, M. G., Cantley, L. C., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Warburg+effect:+the+metabolic+requirements+of+cell+proliferation.">Understanding the Warburg effect: the metabolic requirements of cell proliferation.</a> Science. 324, 1029-1033 (2009).</li><li>Warburg, O., Wind, F., Negelein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+tumors+in+the+body.">The metabolism of tumors in the body.</a> J. Gen. Physiol. 8, 519-530 (1927).</li><li>Goode, J. A., Chadwick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tumour+Microenvironment:+Causes+and+Consequences+of+Hypoxia+and+Acidity.">The Tumour Microenvironment: Causes and Consequences of Hypoxia and Acidity.</a> Novartis Foundation Symposium 240. , (2008).</li><li>Cardone, R. A., Casavola, V., Reshkin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+disturbed+pH+dynamics+and+the+Na+/H++exchanger+in+metastasis.">The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis.</a> Nature Rev. Cancer. 5, 786-795 (2005).</li><li>Robergs, R. A., Ghiasvand, F., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+exercise-induced+metabolic+acidosis.">Biochemistry of exercise-induced metabolic acidosis.</a> Am. J. Phys. 287, R502-R516 (2004).</li><li>Wang, J., Liu, J., Chen, L., Lu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+membrane-free,+mediator-free+glucose+biosensor.">Highly selective membrane-free, mediator-free glucose biosensor.</a> Anal. Chem. 66, 3600-3603 (1994).</li><li>Makos, M. A., Omiatek, D. M., Ewing, A. G., Heien, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+voltammetric+carbon-fiber+microelectrode+pH+sensor.">Development and characterization of a voltammetric carbon-fiber microelectrode pH sensor.</a> Langmuir. 26, 10386-10391 (2010).</li><li>Wang, J., Chen, Q., Pedrero, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+biosensing+of+lactate+at+lactate+oxidase+containing+rhodium-dispersed+carbon+paste+electrodes.">Highly selective biosensing of lactate at lactate oxidase containing rhodium-dispersed carbon paste electrodes.</a> Anal. Chem. Acta. 304, 41-46 (1995).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

이 microneedle 기반 센서의 설계의 여러 측면은 장치 제조 이전으로 간주되었다. 실시간 감지 기능이 센서를 사용하기 위해서는 센서의 응답 시간은 낮은해야하며이 프로토콜에서는, 각 시험 센서 십오초 아래의 응답 시간을 전시. 이 프로토콜에서 사용되는 붙여 넣습 또한 전극 반응을 방해할 수 electroactive biomolecules를 포함하는 생체내 환경에서 내 자신의 선택도에 대한 선정되었습니다. 구...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 플랫 플렉시블 케이블 </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;사이드 양면 테이프 </td><td> Melinex </td><td></td></tr><tr><td> 0.004 &quot;두 번 양면 테이프 </td><td> Melinex </td><td></td></tr><tr><td> 락트 산 산화 효소 </td><td> 시그마 </td><td> L0638 </td></tr><tr><td> 포도당 산화 효소 </td><td> 시그마 </td><td> G7141 </td></tr><tr><td> 탄소에 로듐 </td><td> 시그마 </td><td> 206,164 </td></tr><tr><td> 흑연 분말 </td><td> 시그마 </td><td> 385,031,000 </td></tr><tr><td> 폴리 (ethylenimine) </td><td> Acros </td><td> 178,570,010 </td></tr><tr><td> 미네랄 오일 </td><td> 시그마 </td><td> M5904 </td></tr><tr><td> 포도당 </td><td> 시그마 &lt;/ TD&gt; <td> G8270 </td></tr><tr><td> 락테이트 </td><td> 시그마 </td><td> L1750 </td></tr><tr><td> 빠른 블루 RR 소금 </td><td> 시그마 </td><td> F0500 </td></tr><tr><td> E-쉘 300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> E-쉘 200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> AG / AgCl 참조 전극 </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> PT 와이어 </td><td> Basi </td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash Postcuring 시스템 </td><td> EnvisionTEC </td><td></td></tr><tr><td> 인산 </td><td> 어부 </td><td> A366-4 </td></tr><tr><td> 60W 모델 6.75 CO 2 래스터 / 벡터 레이저 시스템 </td><td> Univer샐 레이저 시스템 </td><td> PLS6.75 </td></tr><tr><td> CorelDraw </td><td> 코렐 </td><td></td></tr><tr><td> Solidworks </td><td> 다쏘 Systemes </td><td> 2009 </td></tr><tr><td> Magics RP13 </td><td> Materialise </td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

생물학 - 관련 분자의 신속한 분석을위한 최소한 침략적 다중 모니터링 시스템의 개발은 만성적인 신체 조건에서 그들의 즉각적인 생리적 상태의 손쉬운 평가를 겪는 개인을 제공할 수 있습니다. 또한, 그것은 복잡한 multifactorial 의료 조건의 분석을위한 연구 도구의 역할을 수 있습니다. 실현되는 등 multianalyte 센서에 대한 위해서는, 사용자에게 통증이나 손상없이 발생합니다 간질성 액체 샘플링, 최소한 침입 있어야하고, 분석은 신속한뿐만 아니라 선별해야합니다. 처음에는 통증이없는 약물 전달을 위해 개발, microneedles는 피부를 통해 백신과 pharmacologic 대리인 (예 : 인슐린)을 전달하기 위해 사용되었습니다. 1-2 이러한 장치는 중간 공간을 액세스할 수 있기 때문에, microelectrodes과 통합되어 microneedles는 transdermal로 사용할 수 전기 화학 센서. 포도당, 글루 탐 산염, 락트 산의 선택적 검출, Hydrogen의 과산화수소, 그리고 ascorbic 산성은 transducing 요소로 재직 탄소 섬유, 수정 탄소 붙여 넣습니다, 그리고 백금 코팅 폴리머 microneedles와 통합 microneedle-전극 장치를 이용하여 증명되었습니다. 3-7,8 이 microneedle 센서 기술은 여러 analytes의 현장과 동시 검출에 대한 소설과 정교한 분석 접근 방식을 사용할 수있다. 멀티플렉싱은 신속하고 최소한 침략적 방식으로 성격 달리 어려운 복잡한 microenvironments을 모니터링의 가능성을 제공합니다. 예를 들어,이 기술의 세포외 수준의 동시 모니터링 활용 수있다, 포도당, 젖산 및 산도, 9 중요한 신진 대사 질환 상태 7,10-14의 지표 (예, 암 증식) 및 운동 유발성 산증은 있습니다. 15

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

이 문서에서는 세부 멀티 플렉스 microneedle 기반 센서의 건설. 장치는 신속하고 선택적으로 여러 analytes의 현장 샘플링과 전기 화학 분석을 위해 개발되고있다. 우리는 임상 의학을 구상하고 생명 의학 연구는 이러한 microneedle 기반의 센서를 위해 사용합니다.

다중 Transdermal 전기 감지를위한 빈 Microneedle 기반 센서

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K 조회수.  University of North Carolina and North Carolina State University. 이 문서에서는 세부 멀티 플렉스 microneedle 기반 센서의 건설. 장치는 신속하고 선택적으로 여러 analytes의 현장 샘플링과 전기 화학 분석을 위해 개발되고있다. 우리는 임상 의학을 구상하고 생명 의학 연구는 이러한 microneedle 기반의 센서를 위해 사용합니다.

비디오: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Interferometric 반사율 이미징 센서 (IRIS)를 채용 Biomolecular 감지

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

연구

생체공학

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

마그네틱 족집게를 사용하여 다중 단일 분자 강제 Proteolysis 측정

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

전기 화학 센서를 사용하여 세균의 검출 및 식별

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

유기 전기 트랜지스터와 장벽 조직 혼란의 감지

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

라벨없는 DNA의 혼성화 분석 용 미세 유체 기반 전기 바이오칩

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

용매를 이용한 지질 이중층 (SALB) 방법에 의해 생체막 제작

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

에 대한 중공의 생물 반응<em&gt; 생체</em&gt; -처럼 포유류 조직 문화

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

마이크로 캡슐화 애플리케이션을위한 추출 공장 기반 캡슐

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

교차 reactivity 무료 및 정찰 병 무료 스냅 칩 샌드위치 Immunoassays 다중화

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Foodborne 병원 체를 검출 하는 전기 화학 DNA 바이오 센서의 개발

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...