이 작품은 국립 과학 재단 FIBR (NSF EF-0623664)와 IDBR (NSF DBI-0754570)에 의해 지원됩니다. 이 작품은 부분적으로 협동 협정 PHY 0,120,999하에 캘리포니아 대학, 데이비스에 의해 관리 Biophotonics, NSF 과학 기술 센터를위한 센터에서 관리 국립 과학 재단 FIBR 그랜트 0,623,664로부터 자금 지원에 의해 지원되었다. 리사 Lapidus, 박사의 연구 버로우즈 웰컴 기금에서 과학 인터페이스에서 경력 수상에 의해 부분적으로 지원됩니다.

<ol>
	<li>Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+and+mechanisms+in+protein+folding.">Fast kinetics and mechanisms in protein folding.</a> Annual Review of Biophysics and Biomolecular Structure. 29, 327-359 (2000).</li><li>Hertzog, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtomole+mixer+for+microsecond+kinetic+studies+of+protein+folding.">Femtomole mixer for microsecond kinetic studies of protein folding.</a> Analytical Chemistry. 76, 7169-7178 (2004).</li><li>Hertzog, D. E., Ivorra, B., Mohammadi, B., Bakajin, O., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+microfluidic+mixer+for+studying+protein+folding+kinetics.">Optimization of a microfluidic mixer for studying protein folding kinetics.</a> Analytical Chemistry. 78, 4299-4306 (2006).</li><li>Yao, S., Bakajin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+Mixing+Time+and+Mixing+Uniformity+in+Devices+Designed+for+Studies+of+Protein+Folding+Kinetics.">Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics.</a> Analytical Chemistry. 79, 5753-5759 (2007).</li><li>Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Hydrophobic+Collapse+and+Early+Folding+Steps+Observed+in+a+Microfluidic+Mixer.">Protein Hydrophobic Collapse and Early Folding Steps Observed in a Microfluidic Mixer.</a> Biophysical Journal. 93, 218-224 (2007).</li><li>Bilsel, O., Kayatekin, C., Wallace, L. A., Matthews, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchannel+solution+mixer+for+studying+microsecond+protein+folding+reactions.">A microchannel solution mixer for studying microsecond protein folding reactions.</a> Review of Scientific Instruments. 76, (2005).</li><li>Chan, C. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+protein+folding+kinetics+studied+by+ultrarapid+mixing.">Submillisecond protein folding kinetics studied by ultrarapid mixing.</a> PNAS. 94, 1779-1784 (1997).</li><li>Shastry, M. C. R., Luck, S. D., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous-flow+capillary+mixing+method+to+monitor+reactions+on+the+microsecond+time+scale.">A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.</a> Biophysical Journal. 74, 2714-2721 (1998).</li><li>Pollack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compactness+of+the+denatured+state+of+a+fast-folding+protein+measured+by+submillisecond+small-angle+x-ray+scattering.">Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 10115-10117 (1999).</li><li>Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+cytochrome+c+initiated+by+submillisecond+mixing.">Folding of cytochrome c initiated by submillisecond mixing.</a> Nature Structural Molecular Biology. 4, 44-50 (1997).</li><li>Knight, J. B., Vishwanath, A., Brody, J. P., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+focusing+on+a+silicon+chip:+Mixing+nanoliters+in+microseconds.">Hydrodynamic focusing on a silicon chip: Mixing nanoliters in microseconds.</a> Physical Review Letters. 80, 3863-3866 (1998).</li><li>DeCamp, S. J., Naganathan, A. N., Waldauer, S. A., Bakajin, O., Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Downhill+Folding+of+lambda-Repressor+in+a+Microfluidic+Mixer.">Direct Observation of Downhill Folding of lambda-Repressor in a Microfluidic Mixer.</a> Biophysical Journal. 97, 1772-1777 (2009).</li><li>Waldauer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+in+the+folding+landscape+of+protein+L.">Ruggedness in the folding landscape of protein L.</a> Hfsp Journal. 2, 388-395 (2008).</li><li>Shastry, M. C. R., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+barrier-limited+protein+folding+kinetics+on+the+microsecond+time+scale.">Evidence for barrier-limited protein folding kinetics on the microsecond time scale.</a> Nature Structural Biology. 5, 385-392 (1998).</li><li>Uzawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+and+search+dynamics+of+apomyoglobin+folding+revealed+by+submillisecond+observations+of+alpha-helical+content+and+compactness.">Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 1171-1176 (2004).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Multiple+Folding+Pathways+for+the+Villin+Headpiece+Subdomain.">Evidence of Multiple Folding Pathways for the Villin Headpiece Subdomain.</a> The Journal of Physical Chemistry B. 115, 12632-12637 (2011).</li></ol>

우리는 공개 할게 없다.

그것이 긴 biomolecules의 분자 프로세스 picoseconds에서 초에 이르기까지 다양한 시간 단위를 통해 발생하는 인식 되었기 때문에 급속한 믹싱은 수년 동안 단백질 폴딩의 필드에 대한 개발 목표왔다. 기존 정지 - 흐름 믹서는 주로 난류에 의해 제한되는 1-5 MS의 죽은 시간이 있습니다. 30-300 μs의 혼합 시간과 지속적인 난류 유동 믹서는 그룹의 소수에 의해 지난 15 년간 개발된지만 일반적으로 다음과 믹싱 시간을 달성하기 위해 높은 유량을 필요로하므로 샘플 6-8를 많이 사용되었습니다. 이 프로토콜은 층류 정권에 신속하게 희석를 달성하기위한 microfluidic 혼합 칩의 사용을 설명합니다. 가이 정권 내에서 작동으로 여러 장점이 있지만 기본 하나 하나가 혼합 반응을 수정할 수있는 높은 정밀도와 전체 믹싱 과정을 시뮬레이션할 수있는 능력입니다. 간단한 geom 다른 그룹에 의해 개발된 T-믹서etries는 주로 채널 9-10의 크기에 의해 제한되었다 100-200 μs의 혼합 시대를 증명하고있다. 10 μm의 나이트로 채널의 크기를 축소함으로써 ~ 10 μs 11 ~ 혼합 시간 단축. 야오와 Bakajin은 기하학의 작은 변화가 혼합 시간 4의 상당한 개선으로 이어질 수 있다고했다. 그러나 그 작품은 혼합의 가속도도뿐만 아니라 느린 단계로 이어질 수있다 보여주었다. 이 작품은 12-13에 사용되는 디자인 denaturant 농도의 느린 지수 붕괴를 최소화하고 또한 DRIE 에칭의 기능이 더욱 재현할 수 있도록 참조 4의 두 최고의 디자인 사이의 타협이다. 혼합 시간의 정의를 통해 믹싱 ultrarapid의 분야에서 어떤 논쟁이 일어났습니다. 그것은 &quot;혼합 시간&quot;과 &quot;죽은 시간&quot;의 차이를 인식하는 것이 중요합니다. 죽은 시간 동안 measuremen 시간을 난류 믹서에서 정의됩니다T는 만들 수 없으며 실제로는 실제 혼합 시간보다 훨씬 더 오래있을 수 있습니다. 그것은 일반적으로 다양한 농도에서 의사 최초 주문 bimolecular 반응 (예 : N bromosuccinate 의한 트립토판 형광 담금질 등)의 여러 측정함으로써 결정됩니다. 관찰된 지수 붕괴는 t = 0의 것으로 추정 하나의 값으로 수렴하기 위해 장착하고 그 시점과 첫 번째 측정 시점 사이의 시간은 죽은 시간이다됩니다. 도 죽은 시간에만 믹싱 시간이 없다 있도록 판상 흐름 믹서의 경우 성능은 믹싱 과정에서 만들 수 있습니다. 혼합 시간은 단순히 충분한 균일에 도달할 때까지 서로 다른 두 솔루션을 결합하는 시간입니다. 우리는 이전에 10분의 90 시간, unmixed 가치의 10 % ~ 90 % 감소한하기 denaturant의 농도에 대한 시간이 될 혼합 시간을 정의했습니다. 그러나, 혼합 곡선의 형태는 작은 지수 구성 요소를 갖는 부패의 꼬리와 완벽하게 대칭이 아닙니다. 또한, 단백질 폴딩이있다denaturant 농도에서 높은 비선형 의존도 그 접는가 자주 denaturant가 두 배 이상 감소되는 경우에도 개시하게 할수 있습니다. 따라서 우리는 최근 80 % denaturant 농도를 감소시키는 시간으로 혼합 시간을 정의했습니다. 물론 다른 정의는 실험의 요구에 따라 사용할 수 있습니다. 단백질 접힘 연구를 위해이 믹서의 사용은 지속적으로 놀라운 결과를 발표했다. 오랫동안 지속적인 흐름 믹서로 여러 개의 접는 단계와 초기 결과를 가지고 연구되어 시토크롬 C와 apomyoglobin의 개고 것은 ~ 100 μs timescale 10,14-15에서 발생하는 붕괴를 보여주었다. 이 믹서와의 측정합니다 (느린 단계 다음 믹서의 혼합 시간 내에, 매우 빠르고 가능성이 불특정, 붕괴합니다 (TRP 형광 스펙트럼의 변화로 측정)이 timescale에 최소한 2 단계가있을 보여준 ) 총 TRP 방출의 담금질에 의해 측정된 그 가능성 F입니다기본 구조 5 irst 형성. 우리는 또한이 단백질은 2 주 폴더에 13이라는 종래의 해석을 overturning, 단백질 L의 지하 도메인에 50 μs 과정을 관찰했습니다. 이러한 급속한 믹서의 장점은 일반적 nanosecond T-점프 악기에 의한 measureable 있었 빠른 접는 단백질의 폴딩을 알아내기 수있다는 것입니다. T-점프는 일반적으로 개고 / 단백질의 녹는 온도 근처에서 휴식을 펼쳐 따라서 전체 인구의 접는을 따르고 절대 관찰이 필요합니다. 이 믹서를 통해 우리는 TRP 총 방출 및 스펙트럼 시프트 모두와 γ-억제의 폴딩 (λ 6-86)를 방문한 적이 있으며 단백질 이전 T에 액세스할 수 없다는 강한 접는 조건 하에서 개고 아무 장애가 없다는 증거를 발견했습니다 측정에게 12 - 점프. 마지막으로 우리는, T 중 하나를 villin의 투구 HP-35 접는을 측정했습니다그는 빠른 폴더는 아직 측정 및 혼합 후 측정 접는 비율이 동일한 조건 16 세 미만 T-점프로 측정하는 것보다 ~ 5 배 느립니다 것으로 나타났습니다. 이것은 접는 속도론이 분야의 주요 가정을 overturning, 시작 조건에 따라하는 것이 좋습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> AZ P4110 포토 레지스트 </td><td> AZ 전자 재료 </td><td></td><td></td></tr><tr><td> AZ 400 K 개발자 </td><td> AZ 전자 재료 </td><td></td><td></td></tr><tr><td> 베이커 푸에르 토리코 2,000 포토 레지스트 스트리퍼 </td><td> Avantor 실적 재료 </td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBond 기술 </td><td> AquaBond 55 </td><td></td></tr><tr><td> 500 μm의 표지 웨이퍼 </td><td> 센서 초등학교 서비스 </td><td></td><td> O : 100mm ± 0.5 mm 두께 : 0.5 mm ± 0.025 mm 표준 오차 (명시하지 않는 한) 표면 마침 : SI: 4-8A, SII : 8-12A 아파트 : 하나는 기본 SEMI-STD; 하나 스크 라이브 깨끗하고 광택이 양측 </td></tr><tr><td> 170 μm의 표지 웨이퍼 </td><td> 센서 초등학교 서비스 </td><td> 7980 2G 웨이퍼 </td><td> O : 100mm ± 0.5 mm 두께 : 0.17 mm ± 0.025 mm 표준 오차 (명시하지 않는 한) 표면 마침 : SI : 4-8A, SII : 8-12A 아파트 : 하나는 기본 SEMI-STD; 하나 스크 라이브 깨끗하고 광택이 양측 </td></tr><tr><td> 산화물에 대한 깊은 반응성 이온 식각 </td><td> ULVAC </td><td> ULVAC NL-6000 </td><td></td></tr><tr><td> 아르곤 이온 레이저 </td><td> 캠브리지 레이저 실험실 </td><td> Lexel 95 SHG </td><td> λ = 258 nm의 </td></tr><tr><td> 아르곤 이온 레이저 </td><td> Melles Griot </td><td></td><td> λ = 488 nm의 </td></tr><tr><td> 현미경 </td><td> 올림포스 산 </td><td> IX-51</td><td></td></tr><tr><td> 현미경 목표 </td><td> 토르 실험실 </td><td> OFR LMU-40X-UVB 0.5 NA </td><td> λ = 258 nm의 </td></tr><tr><td> 현미경 목표 </td><td> 올림포스 산 </td><td> UPLSAPO 60XW 1.2 NA </td><td> λ = 488 nm의 </td></tr><tr><td> Nanopositioner </td><td> 매드 시티 실험실 </td><td> 나노 LP100 </td><td></td></tr><tr><td> 동력 번역 스테이지 </td><td> Semprex 사 </td><td> KL-시리즈 12-6436 </td><td></td></tr><tr><td> GaAsP 광자 카운터 </td><td> 하마 마츠 </td><td> H7421-40 </td><td></td></tr><tr><td> Monochrometer </td><td> Horiba 인 스트 루먼트 INC </td><td> MicroHR </td><td></td></tr><tr><td> CCD 카메라 </td><td> Andor 기술 </td><td> iDus 420A-BU </td><td></td></tr><tr><td> 구아니딘 염산염 (GuHCl) </td><td> 시그마 - 올드 리치 </td><tD&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

그 기본 형태로 단백질 폴드는 생물학과 인간의 건강 아직은 저조한 이​​해 깊은 관련성이있는하는 과정입니다. 이것에 대한 한 가지 이유는 폴딩이 단백질의 1에 따라 나노 초에서 초 이상으로, timescales 다양한 통해 이루어지는 것입니다. 기존 정지 - 흐름 믹서는 접는 속도론의 측정은 약 1 MS에서 시작 허용했습니다. 우리는 최근 ~ 8 μs 2 denaturant는 ~ 100 배 dilutes microfluidic 믹서를 개발했습니다. 정지 - 흐름 믹서와 달리,이 믹서는 난기류가 발생하지 않습니다하는 층류 정권에서 운영하고 있습니다. 난류의 부재 3-4 실험하는 훌륭한 합의와 믹서 내의 모든 흐름의 정확한 숫자 시뮬레이션이 가능합니다. 층류는 ≤ 100을 다시 레이놀즈 번호에 대해 이루어진다. 수성 솔루션의 경우,이 마이크론 스케일의 형상을 필요로합니다. 우리는 실리콘 또는 융합된 sili로, 하드 기판을 사용CA, (그림 1 참조) 채널 5-10 μm의 넓은 및 10 μm의 깊이 있도록. 작은 크기는, 혼합 지역으로 입구 크기 1 μm의 순서에 있습니다. 이 칩은 광학 액세스 얇은 유리 또는 융합된 실리카 coverslip으로 날인합니다. 대표적인 총 선형 유속은 ~ 1 M / s의 매우 저조한 재 ~ 10이지만 단백질 소비가 전용입니다 ~ 0.5 NL / s 또는 1.8 μL / 시간.에게 단백질 농도는 검출 방법에 따라 다릅니다 : 트립토판 형광의 경우 전형적인 농도는 100 μm의 (1 TRP / 단백질의 경우)이며 전형적인 농도가 ~ 100 nm의입니다 안달을 위해. 접는 과정은 6 M에서 0.06 M 구아니딘 염산염으로 denaturant의 급속한 희석에 의해 시작됩니다. 높은 denaturant의 단백질은 중앙 채널 아래로 흐르고 denaturant합니다 (그림 2 참조) ~ 100 배 빠르게 이동하지 않고 버퍼에 의해 혼합 영역에서 양쪽에 만난됩니다. 이 기하학은 좁은으로 단백질 흐름의 급격한 수축을 초래제트 ~ 100 nm의 폭. 무거운 단백질 분자의 확산이 많이 느린 한 MS의 diffusing 미만 1 μm의 상태에서 빛을 denaturant 분자의 보급은 매우 빠른 속도이다. 단백질 주위 denaturant의 효과적인 농도를 감소 denaturant과 단백질 스트림에서 denaturant의 급속한 희석에있는 단백질 결과의 지속적인 확산의 차이,. 단백질 제트는 스캐닝 공촛점 현미경 5를 사용하여 관찰할 수있다 개고 동안 단백질의 관찰 채널 및 형광 아래로 일정한 속도로 흐른다.

Watch this Scientific Journal Video about Microfluidic Mixers for Studying Protein Folding at JoVE.com

Microfluidic Mixers for Studying Protein Folding

이 작품에서 우리는 ~ 8 μs 두 솔루션을 혼합 수 microfluidic 믹서의 제조 및 사용을 설명합니다. 또한 자외선 형광 및 형광 공명 에너지 전달을 (무서워)를 사용 spectroscopic 감지 이러한 믹서의 사용을 보여줍니다.

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 ...

microfluidic-mixers-for-studying-protein-folding

Research

JoVE Journal

Bioengineering

15.0K 조회수.  Michigan State University. 이 작품에서 우리는 ~ 8 μs 두 솔루션을 혼합 수 microfluidic 믹서의 제조 및 사용을 설명합니다. 또한 자외선 형광 및 형광 공명 에너지 전달을 (무서워)를 사용 spectroscopic 감지 이러한 믹서의 사용을 보여줍니다.

비디오: Microfluidic Mixers for Studying Protein Folding

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

단일 입자 Microfluidic 기반 유체 트랩

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

연구

생체공학

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

막 단백질 폴딩의 열역학은 형광 분광학에 의해 측정

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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 ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

마이크로 유체 플랫폼을 사용하여 높은 처리량 단백질 표현 생성기

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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 ...

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 ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

공부 단백질 기능 및 항체의 간섭과 3 차원 재건에 의해 변경 단백질 발현의 역할

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

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.

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.

전혈에서 고감도 단백질 검출을위한 완전 자동화 된 원심 미세 유체 장치

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

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

단일 또는 공존 화학 / 전기 / 전단 응력 자극에서 세포 응답 유학을위한 미세 유체 장치 설계

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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 ...