이 연구 작업은 원자 수준의 촉매 디자인, 미국 에너지 부, 과학, 보너스 번호 DE-SC0001058에서 기초 에너지 과학 국의 재정 지원 에너지 프론티어 연구 센터의 센터의 한 부분으로 지원하고 또한 이사회에 의해 지원됩니다 부여 보너스 번호 LEQSF (2009-14) EFRC-MATCH와 LEDSF-EPS (2012)-OPT-IN-15에서 리전트. MRCAT 작업은 에너지 부와 MRCAT 회원 기관에 의해 지원된다. ANL에서 고급 광자 소스의 사용은 계약 번호 DE-AC02-06CH11357에서 미국 에너지 부, 과학, 기초 에너지 과학의 사무실의 사무실에 의해 지원됩니다. JTM에 대한 재정 지원은 아톰 효율적인 화학 변환에 대한 연구소 (IACT), 미국 에너지 부에 의해 투자 에너지 프론티어 연구 센터, 과학의 사무실, 기초 에너지 과학의 사무실의 일부로 제공되었다.

Millifluidics 세트 - 최대 : 220mm (L) × 2mm (W) X 0.15 mm (H)의 크기와 뱀 채널이 Microplumbers Microsciences LLC에서 (폴리 에스테르 테레 프탈레이트 폴리머로 제작) millifluidic 칩을 구입하실 수 있습니다. 펌프에 칩을 연결하는 0.25 mm의 ID의 크기, OD 1 / 16로 FEP 튜브를 사용합니다. 두 개의 서로 다른 실험을 위해 두 개의 서로 다른 펌프를 사용합니다. 첫 번째 실험 (구리 나노 입자)와 두 번째 실험 (금 나노 입자)의 millifluidic 장치를 사용하여 P-펌프. , 채널 내에서 가스 버블의 문제점을 최소화하기 위해 새로 제조에 NaBH 4 용액을 기체 기포가 용액으로부터 벗어날 수 있도록 칩에 펌핑하기 전에 10-15 분간 ~ 동안 정치 개방 하였다. 이 단계는 실험의 모든 하였다. 1. 매우 작은 구리 나노 클러스터 (UCNCs)의 합성 <ol><li> 필요한 화학 물질 : 구리를 얻습니다 (II) 니트라TE 수화물, 수소화 붕소 나트륨, 수산화 나트륨 펠렛 및 O-[2 - (3 - mercaptopropionylamino) 에틸]-O&#39;-methylpolyethylene 글리콜 (MW = 5000) [MPEG]을 더 정제하지 않고 모든 화학 물질을 사용한다. 실험 nanopure 물 (18.2 MΩ-cm)를 사용합니다. </li><li> 사용 P-펌프 실험 질소 압력 하에서 규제. 해당 흐름 속도 (㎖ / 시간)와 연관시키는 실험 전에 다른 압력에서 용매로 물을 펌프를 테스트합니다. 실험을 시작하기 전에 탈 이온수로 millifluidic 반응기 튜브를 씻어. </li><li> 구리의 174 밀리그램 (0.95 밀리몰)을 녹이고, (II) 질산 및 O-[2 - (3 - mercaptopropionylamino) 에틸] 610 밀리그램 (0.122 mmol)을에-O&#39;-methylpolyethylene의 nanopure 물 28 ㎖의에 글리콜, 계속 그 하나의 입력 채널에 연결하기 바이알 </li><li> 다른 바이알에 다른 수소화 붕소 나트륨 111 mg을 용액 (2.93 mmol) 및 102 mg의 (2.78 mmol)을 28 ㎖ 중의 수산화 나트륨 (약 pH 13)을 유지하고 그것을 연결다른 입력 채널. </li><li> 다른 흐름 속도 (아래)에서 millifluidic 반응기 내에서 동시에 솔루션 흐름과 유리 병에있는 출구에서 결과 UCNCs를 수집합니다. 질소를 용액을 제거하고, 질소하에 저장한다. </li><li> 의 합성 상온에서 50 밀리바 (6.81 ㎖ / 시간), 100 밀리바 (14.31 ㎖ / 시간), 200 밀리바 (32.7 ㎖ / 시간) 300 밀리바 (51.4 밀리리터 / 시간)의 다른 일정 압력에서 펌프를 작동 다른 흐름 속도로 UCNCs. </li></ol> 합성 절차가 P-펌프가 millifluidic 셋업을 사용하여 설명되었지만, 그것은 또한 Millifluidica로부터 휴대용 millifluidic 장치를 사용하여 수행 될 수있다. 2. 시간은 금 나노 입자의 형성에 현장 운동 연구에서 해결 <ol><li> 필요한 화학 물질 : 염화 금산 (. HAuCl 4 3H 2 O) 메소 -2,3 - dimercaptosuccinic 산 (DMSA)와 소듐 보로 하이드 라이드 &amp; # 받기(160)와, 추가의 정제없이 모든 화학 물질을 사용한다. 실험 nanopure 물 (18.2 MΩ-cm)를 사용합니다. </li><li> 완전 자동화, 맥동 무료 주사기 펌프는 칩 내의 액체 흐름, 높은 정밀도를 사용합니다. 필요한 유량을 최적화하기 위해 실험 전에 다른 흐름 속도로 용매로 물을 펌프를 테스트합니다. </li><li> (I) HAuCl 4의 표준 용액을 준비합니다. 3H 2 O (10 밀리몰, 118.2 mg/30 ml)에 nanopure 수산화 나트륨 (산도 12)의 50 밀리그램 (II) DMSA (20 밀리몰, 109.2 mg/30 ml)을 물. </li><li> 자동 펌프를 이용하여 10 ㎖ / hr의 일정한 유량에서 millifluidic 칩에 두 개의 별도의 주사기를 통해 두 용액을 피드. </li><li> 커플 XYZ 방향으로의 이동에 대한 액세스를 가지며 해법은 칩을 통해 펌핑 하였다로서 칩에 다른 시간대에 XAS 데이터를 수집 금속 스테이지를 사용 싱크로트론 빔 라인에 millifluidic 칩. </li></ol> &lt;동안EM&gt; 시츄 분석 절차는 P-펌프가 millifluidic 셋업을 사용하여 입증되었고, 또한 휴대용 millifluidic 장치를 사용하여 수행 될 수있다. 3. 연속 흐름 금 촉매 이 절차는 핸드 헬드 장치를 사용 millifluidic 입증되었다. <ol><li> 필수 화학 물질 : 염화 금산 (. HAuCl 4 3H 2 O), 메소 -2,3 - dimercaptosuccinic 산 (DMSA), 수소화 붕소 나트륨, 4 - 니트로 페놀, 4 - 아미노 페놀을 구하여이를 추가적인 정제없이 모든 화학 물질을 사용한다. 실험 nanopure 물 (18.2 MΩ-cm)를 사용합니다. </li><li> 촉매 제조 :. HAuCl 4의 표준 용액을 준비 3H 2 O Nanopure (10 밀리몰, 118.2 mg/30 ml)에 DMSA (20 밀리몰, 109.2 mg/30 ㎖)의 NaBH 4 (10 밀리몰, 11.34 mg/30 ml)을 물. </li><li> 두 개의 유리 병에 HAuCl 4 DMSA 솔루션 10 ㎖를 각각 가지고 일을 흐름45 분 동안 12 ㎖ / hr의 일정한 유속으로 소형 millifluidic 장치를 이용하여 칩 내의 EM. </li><li> 오에 오 (I)를 줄이기 위해 15 분 동안 12 ㎖ / 시간의 흐름 속도로 칩 내에서 10 밀리몰의 NaBH 4 흐름 (0). </li><li> 마지막으로, 촉매 반응 실험을 수행하기 전에 동일한 유량에서 30 분 동안 nanopure 물과 칩을 씻어. </li><li> 촉매 반응 : 아래로 금 촉매 (상기에서 제조) 내에서 4 - 아미노 페놀 (4-AP)에 4 - 니트로 페놀의 화학적 전환 반응 (환원) (4-NP)을 수행은 millifluidic 채널을 입혔다. </li><li> 4 nitrophenolate 이온 (4-NPI)를 형성하기 위해 0.65 몰의 NaBH 4 용액을 3.3 ㎖로 4-NP의 9 × 10 -5 몰 용액 15 ㎖를 혼합한다. </li><li> 촉매 활성을 평가하기 위해 5 ㎖ / hr의 일정한 유량에서 칩 내에 증착 금 촉매를 통해 얻어진 용액을 합격. 250-50의 파장 범위 내에서 수집 된 제품의 UV-VIS 스펙트럼을 분석0 nm의 4-NP의 전환을 확인합니다. </li><li> 4 - NPI의 검량선을 얻음으로써, 반응의 촉매 활성을 추정한다. 검량선 다른 표준 농도 4 NPI의 실험적 관찰 흡수 강도 (I을) 플롯에 의해 획득 될 수있다. UV-VIS 흡수 커브 (399 nm에서)의 피크 높이가 흡수 강도 (I)의 값을 나타내고, 비어 램버트의 법칙에 따라, 피크 높이 값의 변화가 그 농도에 대응하는 변화를 보여줄 것이다. 따라서, 검량선으로부터 반응물의 초기 및 최종 농도에 차이를 발견함에 따라 촉매 활성을 추정한다. 피크 높이가 1 단위 (도 6) 인 경우 예를 들어, (캘리브레이션 플롯 기준) 90 %의 촉매 전환에 상당한다. </li></ol>

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Rev. , (2013).</li><li>Shahbazali, E., Hessel, V., No&#235;l, T., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallic+nanoparticles+made+in+flow+and+their+catalytic+applications+in+organic+synthesis.">Metallic nanoparticles made in flow and their catalytic applications in organic synthesis.</a> Nanotech. Rev. , (2013).</li></ol>

CSSR 쿠마를 제외한 모든 저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다. C. SSR 쿠마 회사 Millifluidica LLC의 설립자이다.

-O&#39;-methylpolyethylene 글리콜 (MW = 5000) [MPEG] - UCNCs는 중합체 캐핑 에이전트 O-[(3 - Mercaptopropionylamino) 에틸 2]의 존재하에 수소화 붕소 나트륨과 질산 구리의 환원 반응에 의해 형성 하였다. 반응은 6.8 ㎖ / 시간, 36.3 ㎖ / 시간, 32.7 ㎖ / 시간 및 형성 UCNCs에 흐름 비율의 효과를 연구하기 위해 51.4 ㎖ / 시간으로 다른 흐름 속도로 millifluidic 칩 반응기 내에서 수행 하였다. 위의 흐름 속도에 대한 각각의 체?...

화학 합성 실험실 - 온 - 칩 (LOC) 장치가 증가 질량과 열전달, 뛰어난 반응 제어, 높은 처리량 및 안전한 작업 환경 (1)의 측면에서 상당한 이점을 증명하고있다. 이러한 장치는 광범위 칩 기반의 유체와 nonchip 기반 유체 소자로 분류 될 수있다. 칩 기반의 유체 사이에서, 마이크로 유체 잘 조사하고, 주제는 문학 2-5에서 잘 덮여. Nonchip 기반 LOC 시스템은 관형 반응기 (6)를 사용합니다. 통상적으로, 마이크로 유체 시스템은 정확한 컨트롤과 기하학적 서브 밀리미터 규모로 제한하는 유체의 조작에 사용됩니다. 우리는 최근 (폭 또는 깊이 또는 채널의 양쪽 모두는 크기가 최소 mm이다) 7-9 밀리미터 스케일 채널에서 유체의 조작을 위해 사용될 수있다 칩 기반 millifluidics의 개념을 도입 하였다. 또한, millifluidic 칩은 WHI를 제조하기가 비교적 쉽다르 흐름 속도 및 시약의 조작을 제어하기위한 비슷한 방법을 제공합니다. 이 칩은 좁은 크기 분포를 갖는 나노 입자의 제어 합성의 스케일 업에 대한 가능성을 제공함으로써, 작은 체류 시간을 만들고, 높은 흐름 속도로 작동 할 수 있습니다. 예를 들어, 우리는 최근 초소형 구리 나노 클러스터의 합성을 시연하고 현장 X-선 흡수 분광법에 사용뿐만 아니라 TEM을 특징으로했다. 구리 나노 클러스터 (7)의 안정된 콜로이드의 형성에 매우 효율적 두자리 PEG 화 안정 화제입니다 MPEG의 사용과 함께 millifluidic 채널 내에서 작은 체류 시간을 얻을 수있는 능력. 화학 물질과 나노 물질의 합성뿐만 아니라, millifluidics 때문에 프로브 영역에서 높은 양 및 농도, 더 일반화하고 효율적인 시간 분해 동역학 연구도 achie이다 합성 플랫폼에 제공 할마이크로 유체 시스템 7,10보다 노이즈 비율로 신호가 잘 보이는 군. 우리는 5 밀리 초 (11)와 같은 작은 시간 해상도와 동일계 XAS에서 사용한 용액으로부터 금 나노 구조물의 성장 시간이 해결 분석을위한 예로서 millifluidic 칩의 사용을 보여준다. 또한, 촉매 응용 프로그램에 대한 최신 개발 된 마이크로 반응기의 대부분은 실리콘 (12, 13)를 기준으로합니다. 생성 된 작은 볼륨뿐만 아니라 고가의 제조는 대규모 제조되지는 않습니다. 나노 촉매와 채널 코팅하는 두 가지 일반적인 방법 - 화학적 및 물리적 종종 실리콘 코팅 절차 칭함 보그 14,15 현재이다. 고가의 마이크로 제조 외에도 채널의 막힘은 마이크로 반응기의 촉매가 대규모 생산에 부적합하게 될 수있다. 마이크로 반응기는 마이크로 연속 관류 공정의 earli 이종 촉매에 사용되었지만16-18 어, 치수를 제어 할 수있는 능력, 지속적인 흐름 채널에 내장 된 금 나노 촉매의 형태는 이전에 탐험되지 않았습니다. 우리는 최근의 Au 촉매와 millifluidic 채널을 코팅하는 기술을 개발 한 나노 형태와 치수 (그림 5) (11)를 제어하는 데, 산업적으로 중요한 화학 반응의 촉매 작용을 수행하기위한. 예를 들어 우리는 millifluidic 채널 내에서 코팅 된 나노 금 촉매로 4 - 아미노 페놀에 4 - 니트로 페놀의 변환을 증명하고있다. 단일 millifluidic 반응 칩이 50 ~ 60 ㎖ / 시간, 7 높은 처리량 및 화학 물질의 제어 합성의 흐름 속도를 생성 할 수 있음을 감안하면 어느 연속 흐름 작업 또는 병렬 처리를 통해 가능하다. millifluidics 이상으로 설명 몇 가지 예제와 함께 제공하는 가능성을 활용하기 위해, 우리는 또한 보여 사용자 친화적 인이식하고있다 millifluidic 장치는 millifluidic 칩, 매니 폴드, 유동 제어기, 펌프 및 전기적 연결과 같은 모든 필요한 구성 요소가 통합. 이러한 millifluidic 장치는,도 7에 도시 된 바와 같이, 지금의 회사 Millifluidica LLC (에서 구할 <a href="http://www.millifluidica.com" target="_blank">www.millifluidica.com</a> ). 원고는 나노 물질, 반응 메커니즘과 연속 유동 촉매의 시간 분해 분석 제어 합성에 대해, 후술하는 바와 같이, 핸드 헬드 장치를 사용 millifluidic 프로토콜을 제공한다.

<table><tbody><tr><td>Copper (II) nitrate hydrate</td><td>Sigma-Aldrich</td><td>13778-31-9</td><td>99.999% pure</td></tr><tr><td>O-[2-(3-mercaptopropionylamino)ethyl]-O&amp;prime;-methylpolyethylene glycol</td><td>Sigma-Aldrich</td><td>401916-61-8</td><td>MW=5,000</td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt;3H&lt;sub&gt;2&lt;/sub&gt;O (Chloroauric acid)</td><td>Sigma-Aldrich</td><td>27988-77-8</td><td>99.999% pure</td></tr><tr><td>meso-2,3-dimercaptosuccinic acid (DMSA)</td><td>Sigma-Aldrich</td><td>304-55-2</td><td>~98% pure</td></tr><tr><td>4-Nitrophenol</td><td>Sigma-Aldrich</td><td>100-02-7</td><td>spectrophotometric grade</td></tr><tr><td>4-Aminophenol</td><td>Sigma-Aldrich</td><td>123-30-8</td><td>&gt;99% pure (HPLC grade)</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>16940-66-2</td><td>98% pure</td></tr><tr><td>Sodium hydroxide pellets</td><td>Sigma-Aldrich</td><td>1310-73-2</td><td>99.99% pure</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Millifluidic Chips</td><td>Microplumbers Microsciences LLC</td><td>SDC-01</td><td>Made from polyester terephthalate polymer</td></tr><tr><td>Pressure Pump</td><td>Mitos P-Pump, Dolomite</td><td>3200016</td><td></td></tr><tr><td>Automated Syringe Pump</td><td>Cetoni Automation and Microsystems, GmbH</td><td></td><td>Syringe pump neMESYS</td></tr><tr><td>UV-3600 UV-VIS-NIR Spectrophotometer</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Hand-held Millifluidic Device</td><td>Millifluidica</td><td>SCMD-1008</td><td>&lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr></tbody></table>

millifluidics for chemical synthesis and time-resolved mechanistic studies

화학 합성 시간이 해결 기계론의 연구에 대한 millifluidic 장치를 이용하는 절차는 세 가지 예를 복용에 의해 설명되어 있습니다. 먼저, 초소형 구리 나노 클러스터의 합성은 설명한다. 두 번째 예는 현장 X-선 흡수 분광법에 사용하는 금 나노 입자의 형성을 분석하여 화학 반응의 시간이 해결 된 역학 조사에 대한 자신의 유틸리티를 제공합니다. 마지막 예제는 나노 구조 촉매로 코팅 millifluidic 채널 내부 반응의 연속적인 흐름의 촉매 작용을 보여줍니다.

잘 분산 및 좁은 크기 분포를 갖는 균일 한 크기의 구리 나노 클러스터는 millifluidic 칩 설정 (도 1A)를 사용하여 수득 하였다. 합성을 위해 사용되는 상이한 흐름 속도는 클러스터의 크기에 유의 한 영향을 미치지 않았다. 그럼에도 불구하고, 유속의 증가와 함께, 크기 분포의 축소에 관찰 가능한 개선이있다. 유용한 좁은 크기 분포를 갖는 UCNCs은 32.7 ㎖ / hr의 유량으로 얻었다. 32.7 ㎖ / 시간의 유량?...

Watch this Scientific Journal Video about Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies at JoVE.com

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Millifluidic 장치는 나노 물질, 반응 메커니즘과 지속적인 흐름 촉매의 시간 분해 분석의 제어 합성에 이용된다.

화학 합성 및 시간 - 해결 기계론의 연구 Millifluidics

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

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11.1K 조회수.  Louisiana State University.  Millifluidic 장치는 나노 물질, 반응 메커니즘과 지속적인 흐름 촉매의 시간 분해 분석의 제어 합성에 이용된다. 

비디오: Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

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

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

연속 흐름 처리를 사용하여 수행 반응의 실시간 모니터링 : 예를 들어 3 Acetylcoumarin의 제조

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

화학

Microfluidic Mixers for Studying Protein Folding

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

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

단백질 접기 유학을위한 Microfluidic 믹서

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

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

현장에서 크리스탈 x-선 회절 및 현장에서 직렬 결정학에 대 한 동적 빛 산란에 대 한 미세 칩

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

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 ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

콜 로이드 반도체 나노의 체계적인 연구를 위한 모듈형 미세 기술

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

공학

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

단일 DNA 분자에서 대규모 병렬 단백질 합성을 위한 펨토리터 물방울 배열

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

단일 분자 연구를 위한 이중 광학 핀셋 및 미세 유체 공학 사용

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

생화학

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

장기간 무세포 유전자 발현의 전도를 위한 다층 미세유체 플랫폼

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

고처리량 미세 유체 장치를 사용하여 동적 환경 조건의 생성

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...