이 작품은 양적 생명 과학의 캘리포니아 공과 대학 (QB3), 로저스 가족 재단, 혁신적인 생물 의학 연구, BASF에서 보조금에 대한 UCSF / 샌들러 재단 프로그램에서 브리징 격차 상, 그리고 NSF를 통해로부터 연구 상 수상에 의해 지원되었다 학부 조기 경력 개발 (경력) 프로그램 (DBI-1253293).

1. SU8 마스터 제작 <ol><li> AutoCAD 소프트웨어를 사용하여 두 개의 층을 제조하기위한 미세 유체 구조를 디자인하고 10 μm의 해상도로 회로 기판 필름 공급 업체에서 인쇄 된 디자인을 가지고있다. 장치 설계의 세부 사항은 첨부 된 기준 (11)에 부여하고, 채널 형상은도 1에 도시되어있다. 각 층은 층 (12)의 제조에서의 기능을 배치 할 수 있도록 정렬 마크를 포함한다. </li><li> 스핀 코터에 미리 청소 3 인치 직경의 실리콘 웨이퍼를 놓고 척에 부착하는 진공를 켜. 50 ㎛의 막 두께를 제공하고, 2,000 rpm에서 30 초, 500 rpm에서 20 초간 스핀 웨이퍼의 중심에 SU8-3035 1 ㎖를 적용. </li><li> 웨이퍼를 제거하고, 30 분 동안 135 ° C 열판에 굽는다. 웨이퍼는 다음 단계로 이동하기 전에 실온으로 냉각되도록. </li><li> 1 차 레이어 마스크로 코팅 된 웨이퍼 (그림 2A 노출 </stronG&gt;) 90 초 동안 LED nm의 평행 190 mW의 365 미만. 노출 후, 다음 단계를 진행하기 전에, 1 분 동안 실온에 다음 시원한를 135 ° C 형 열판에 웨이퍼를 놓습니다. </li><li> 스핀 코터에 웨이퍼를 놓고 척에 부착하는 진공의 전원을 켭니다. 135 ㎛의 두께의 부가 층을 제공 결과, 1,375 rpm에서 30 초, 500 rpm에서 20 초간 스핀 웨이퍼의 중심에 SU8-2050 1 ㎖를 적용. </li><li> 다음 단계로 이동하기 전에 30 분 동안 135 ° C의 열판에 RT에 다음 멋진 웨이퍼와 빵을 제거합니다. </li><li> 1.3 패턴 형상에 2 차 레이어 마스크 (그림 2B)를 맞추고 3 분 동안 LED nm의 평행 190 mW의 365에 코팅 된 웨이퍼를 노출. 노출 후, 다음 단계를 진행하기 전에, 1 분 동안 135 ° C의 열판에 RT에 다음 시원한 놓습니다. </li><li> 30 분 동안 프로필렌 글리콜 모노 메틸 에테르 아세테이트 교반 조에서 침지하여 마스크를 개발한다. 웨이퍼를 세척1 분 동안 135 ° C의 열판에 이소프로판올 및 빵에. PDMS 성형 100mm 페트리 접시에서 개발 마스터를 놓습니다. </li></ol> 2. PDMS 소자 제조 <ol><li> 플라스틱 컵에 경화제 5 g과 실리콘계의 50g을 조합하여 1 PDMS : 10을 준비한다. 볶음 스틱 장착 된 회전 도구를 사용하여 내용을 섞는다. 모든 기포가 제거되고 30 분 동안, 또는 건조기 내부까지 혼합물을 탈기. </li><li> 마스터 이상 3mm의 두께를주고 더 탈 가스에 대한 데시 케이 터에 다시 배치 할 PDMS를 붓고. 일단 모든 기포를 제거하고, 2 시간 동안 60 ℃에서 구워 장치. </li><li> 패턴면을 위로하여 깨끗한 표면에 메스과 장소를 사용하여 금형에서 장치를 잘라. 마스터 2 (그림 3a)에서 석사 (1)를 분리하기 위해 면도날로 절반에 PDMS 몰드를 잘라. 마스터 (1)에 의해 임프린트 된 50㎛의 유체 취급을 포함하는 형상 부재에, 0.7와 유체 유입구 및 출구를 펀치5mm 생검 펀치. </li><li> 플라즈마는 300 W 플라즈마 청소기에 60 초 동안 1 mbar에서의 O 2 플라즈마에 장치를 취급합니다. 일시적 PDMS-PDMS 본딩을 지연 및 윤활제 역할을 DI 물 방울 PDMS의 unpunched 조각의 표면을 적시고. 마스터 2면에 스테레오 현미경, 장소 마스터 1을 통해보기 및 기계식 잠금 장치가 달성 될 때까지 상대적으로 표면을 밀어 동안 때 오목 프레임과 그림 3A 메이트에서 돌출 프레임. </li><li> 60 ° C의 오븐에서 장치를 놓고 물과 완전한 결합을 증발시켜 60 ℃에서 이틀 동안 조립 된 장치 (도 3b)을 굽는다. </li></ol> 시약 3. 준비 <ol><li> 내부 단계의 증류수로 1 ML의 주사기를 채우십시오. </li><li> 1 중량으로 HFE 7500 불소계 오일 1 ML의 주사기를 채우십시오. 중간 단계에 대한 % 생체 적합성 계면 활성제 계면 활성제 (13). </li><li> 10 중량으로 10 ML의 주사기를 채우십시오. % 폴리에틸렌1 중량 % 함유하는 수용액에서는 E 글리콜 (PEG). % 트윈 20과 1 중량. 연속 상 % 나트륨 도데 실 설페이트. </li></ol> 4. 시스템 준비 <ol><li> &lt;100 마이크로 초 셔터 속도를 낼 수있는 디지털 카메라와 결합 거꾸로 현미경의 무대에서 마이크로 유체 칩을 놓습니다. </li><li> 주사기 펌프에 모든 주사기를 탑재하고 27 G 바늘을 연결합니다. 바늘 ~ PE-2 튜브의 30cm 길이를 연결하고 해당 장치에 구멍을 천공으로 느슨한 끝을 삽입합니다. </li><li> 장치의 출력 포트에 PE-2의 10cm 길이를 삽입하고 폐기물 수집 용기에서 다른 쪽 끝을 배치합니다. </li><li> 프라임 튜브 세그먼트에서 유체의 속도까지 높은 속도로 시린지 펌프 (000 μL / 분)를 실행하여 장치는 장치의 흡입구에 도달한다. </li></ol> 5. 에멀젼 생성 <ol><li> 50 μm의 × 50 μm의 구멍과를 포함하는 지역에 현미경 초점하류 출구 채널. </li><li> 연속 상 250 μL / 내부 단계의 시간, 100 μL / 중간 단계의 시간, 700 μL / hr의 유량으로 이중 에멀젼 발생기에 유체를 제공하고 평균 10 분을 기다려야 주사기 펌프를 설정합니다. </li><li> 각각 250 μL / 시간에서 100 μL / 시간 내측 및 중간 단계의 유량을 유지한다. 1,050 μL / 시간 외측 위상의 유량을 설정한다. 이중 에멀젼 생성이 유동 조건의 설정에 따라 안정을 위해 3 ~ 5 분을 기다립니다. </li><li> 수동 이미지 분석을 통해 오프라인으로 처리를위한 30 Hz에서 영상 5 초를 취득. </li><li> 표 1에 주어진 유량과 5.3 및 5.4를 반복한다. 내측 및 중간 단계의 유량은 일정하게 유지되고 반송파 위상 유량 주사기 펌프의 설정을 조절함으로써 변화된다. </li></ol>

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The authors have nothing to disclose.

여기에 설명 된 이중 에멀젼 발생 형상은 유리 모세관 장치 (8)의 물리를 모방하도록 설계된다. 이들에 정렬 원통형 유리 모세관 균일 이중 에멀젼 방울에 전단되는 삼상 동축 제트을 만드는 데 사용됩니다. 우리 3D PDMS 소자의 기능은 전체 높이가 320 μm의 아르 반송파 위상 채널과 50 μm의 높이로 형성 작은 ​​제작 기능의 중앙 정렬에 의존한다. 마스크가 정확히 정렬되지 않는 경우에 50 ?...

<html> 이중 에멀젼은 인해 산업, 의약품에서 사용 가능성, 및 생물학적 응용 1 중간, 비혼 화성 유체 층에 의해 담체 상으로부터 분리 액적로 구성하고, 특히 중요하다. 어떤 경우에는, 이중 에멀젼의 코어에 고 부가가치 화합물을 캡슐화 할 수있는 능력은 보호 물질과 제어 방식으로 방출 될 수있다. 예를 들어, 약물이 외부 캐리어 유체 (2)에 적합하지 않은 조건에서 용해도 캡슐화 될 수있다. 또한, 중간 유층 약물, 화장품 및 영양 3의 캡슐화 및 전송을위한 캡슐 템플릿으로 사용될 수있다. 그들은 서브 나노 리터 실험의 대규모 수가 다음, 실시 감지 (FACS) 악기 4,5 정렬 형광 활성화 된 셀을 사용하여 정렬 할 수 있기 때문에 생물학, 이중 에멀젼은 또한 높은 처리량 검사에 유용하다. ENT는 &quot;&gt; 원하는 성능 특성을 갖는 이중 에멀젼의 디자인은 이중 에멀젼 크기, 조성 및 균일도의 정밀한 제어를 필요로한다. 이러한 막 유화 같은 벌크 유화 공정은, 업계에서 사용되지만, 생성 된 에멀젼은 높은 다 분산되어 보이는 기능적 특성 (1)의 다양한. 액적 미세 유체 분야는 자연적으로 신중하게 제어 구성 6 단 분산 에멀젼의 생성을 적합하다. 미세 유체 이중 에멀젼 생성이 초점을 두 가지 주요 전략을 순차적 드롭 결정과 유리 모세관 흐름 달성되었다. 두 번 에멀젼 수 공정을 두 단계의 방울을 사용 평면 PDMS 장치에서 생성 될 수있다. 먼저, 수성 유 에멀젼이 유 중수 드롭하게 소수성 채널 벽을 가진 디바이스의 영역이있다. 다음을 사용하여 생성되며, 에멀젼 일 수있다 흐르는 물에 기름에 적합한 친수성 ​​벽 드롭 만드는 영역에 재 주입드롭 만들기 (4). 그러나, PMD를 친수성 표면 처리는 추가적인 제조 공정을 필요로하며 종종 임시 7이다. 이중 에멀젼을 형성하기 위해 대부분의 제어 및 반복 가능한 방법이 동축 흐름을 중심으로하고, 기술은 세 단계를 포함하는 동심 제트 단 분산 액적 (8)을 생성하기 위해 작은 오리피스를 통해 전단된다 유리 모세관 마이크로 유체를 사용 개척했다. 이 기술은 각상의 유속의 함수 인 이중 에멀젼의 정확한 크기 및 구성으로, 채널 치수보다 훨씬 작은 방울의 생산을 허용한다. 액적 크기 및 채널과 상기 보호 외피 흐름 사이의 큰 차이는, 채널 벽과 접촉 불필요한 표면 처리 렌더링로부터 방울을 방지한다. 그러나, 유리 장치는주의 조립 및 씰링과 함께, 테이퍼 모세관 팁 사용자 정의 제조를 필요로한다. 이전 연구자들은 3D 소프트 리소그래피를 사용했다그래피로 물리학을 중심으로 흐름을 사용하여 이중 에멀젼을 생성하지만, 이러한 장치는 직경&gt; 150 μm의 9, 10, 일반적으로 외과로 분류 개체보다 큰 크기의 약 주문 에멀젼을 생산했다. 매력적인 대안은 PDMS 소프트 리소그래피 제조의 용이성에 초점을 맞추고 유리 모세관 동축 흐름의 강력한 기능과 작은 방울 생성을 포함한다. 본 논문에서는 50 μm의 에멀젼 ≤ 생산 초점 동축 흐름을 사용하여 완전히 3D 소프트 리소그래피 (11)를 사용하여 구성되어 이중 에멀젼 발생기를 설명합니다. 우리는 디바이스 뽑아 유리 모세관 노즐 에멀젼 형성 공정을 근사하는 작은 전단 채널 (도 1)를 포함하는 장치를 제조 폴더 형 접근법을 사용한다. 더 중요하게, 이러한 장치는 특별한 표면 처리를 필요로하지 않고, 모든 중합체 구조는 간단하고 반복적 인 제조 SC드립니다중복 된 장치들의 많은 수 alable. 여기서는 더블 에멀젼 발생기의 설계, 제조 및 테스트를 개요. 더블 에멀젼 세대는 14 μm의의 물방울 직경에 이르기까지 강력하고 반복적으로 표시됩니다. 제조의 용이성과 기능의 결합은이 장치 새로운 더블 에멀젼 응용 프로그램의 개발을위한 매력적인 옵션이 있습니다.

<table><tbody><tr><td>Photomasks</td><td>CadArt Servcies</td><td></td><td></td></tr><tr><td>3&quot; silicon wafers, P type, virgin test grade</td><td>University Wafers</td><td>447</td><td></td></tr><tr><td>SU-8 3035</td><td>Microchem</td><td>Y311074</td><td></td></tr><tr><td>SU-8 2050</td><td>Microchem</td><td>Y111072</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Krayden</td><td>4019862</td><td></td></tr><tr><td>1 ml syringes</td><td>BD</td><td>309628</td><td></td></tr><tr><td>10 ml syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>27 gaugue needles</td><td>BD</td><td>305109</td><td></td></tr><tr><td>PE 2 polyethylene tubing</td><td>Scientific Commodities, Inc.</td><td>B31695-PE/2</td><td></td></tr><tr><td>Novec 7500</td><td>Fisher Scientific</td><td>98-0212-2928-5</td><td>Commonly knowns as HFE 7500</td></tr><tr><td>Biocompatable surfactant</td><td>Ran Biotechnologies</td><td>008-FluoroSurfactant</td><td></td></tr><tr><td>35,000 MW PEG</td><td>Sigma Aldrich</td><td>1546660</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1369</td><td></td></tr><tr><td>Sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma Aldrich</td><td>L3771</td><td></td></tr></tbody></table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

이중 에멀젼 발생기 3D PDMS 제조를 (도 1a)를 사용하여 만든 공동 축류 집속 장치로 구성된다. 지오메트리는 삼상 동축 제트의 형성 물 / 오일 / 물 이중 에멀젼 (도 1b,도 1c)의 형성을 허용 광장 × 50 μm의 오리피스 50㎛의로 전단 될 수있다. 내부 수상과 유상 중간 10 ㎛, 채널 치수에 함께 접합하게된다 × 50 μm의 (도 1D,</...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

폴리 디메틸 실록산 (PDMS) 공동 축류 초점 장치를 사용하여 이중 에멀젼 생성

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Research

JoVE Journal

Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

16.0K 조회수.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

비디오: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Microfluidic 칩은 탄성 Microvalve의 배열을 제어

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

연구

생물학

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

콜로이드 입자의 자기 조립 과정을 통해 PDMS 미세 유체 칩의 하위 50 Nm의 나노 유체 접합 만들기

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

공학

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

마이크로 소용돌이를 이용한 순차 분자 전달을위한 Electroporator을

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

생체공학

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

크기 제어 폴 리 (에틸렌 글리콜) 빛 방울 장치를 통해 세미 3 차원 흐름 초점 미세의 세대

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

폐 선포 항공 호흡 Biomimetically의 미세 유체 모델

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

방울과 에멀젼을 생성하는 유리 기반 장치

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

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 ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

편광 TIRF 현미경에 의해 감시 미세 유동 흐름 전지에 닿는 지원 이중층 단일 테오의 SNARE 매개 퓨전

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

신경과학

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 ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

구형 에멀젼 방울의 기본 유사 분열 스핀들의 재구성

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...