This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. 포토 마스크 제작 <ol><li> 석영 탱크에 침수 120 ℃에서 20 분 동안 : 피라냐 솔루션 4 &quot;유리 웨이퍼 (1 비율 H 2 2 SO 4 / H 2 O 2)를 청소합니다. </li><li> 크롬 / 금 층 (CR 30 nm의 Au로 / 1 ㎛)의 전자 빔 증발기 (7) (도 1a)를 사용하여 유리 웨이퍼 상에 층을 증착. <ol><li> 전자 빔 증발기에 웨이퍼를 놓습니다. 진공은 5 × 10-6 토르에 도달하면, 고전압 소스 (10 kV로)를 켜. 모니터 제어 패널의 두께를 제어한다. </li><li> 전자빔 총을 사용하여 30 초, (웨이퍼상의 증착을 방지하기 위하여), 셔터 &quot;OFF&quot;를 유지하기위한 각 물질 사전 세정. </li></ol></li><li> 유리의 깊은 습식 식각을위한 CR / 금 포토 레지스트 마스크 층을 생성합니다. <ol><li> 스핀 코터 분무 시스템을 사용하여 3,000 rpm에서 30 초 동안 용액을 5 ml의 회전에 의해 2 ㎛의 두께의 포토 레지스트를 적용한다.1.5 분 동안 100 ℃에서 핫 플레이트상에서 프리 베이크 포토 레지스트. </li><li> 노출 및 경질 핫 플레이트상에서 30 분 동안 120 ℃에서 레지스트를 굽는다. 또한, 소수성 표면과 금속층 포토 레지스트의 강한 접착력을 생성하는 것이 중요하다. 패턴은 포토 레지스트를 통해 CR 또는 Au 에칭을 이용하여 CR / 금 층은 8,9 마스크. </li></ol></li><li> 비 - 패턴 화 유리 표면의 보호를 위해 일시적으로 더미 실리콘 웨이퍼에 유리 웨이퍼를 접합. 9 <ol><li> 110 ℃에서 핫 플레이트 상에 유리 웨이퍼를 배치하고 (웨이퍼의 전체 표면을 왁스로 피복되는 방식으로) 유리 웨이퍼의 반대측을 용융 왁스. </li><li> 글래스 웨이퍼와 접촉 더미 실리콘 웨이퍼를 놓고 과잉 왁스를 제거하기 위해 누른다. 왁스의 유출을 방지하기 위하여, 핫 플레이트상에서 청정실 티슈 페이퍼를 배치했다. </li></ol></li><li> 최적화 된 불화 수소산을 이용하여 렌즈의 등방성 에칭을 수행한다 (49 %의 v / v)의. 8.5 분간 마그네틱 교반기 (10)의 HCl의 존재는 생성 된 렌즈의 양호한 표면 품질을 달성하는데 중요하다 : (1 (10)의 체적 비율)와 염산 (37 % v / v)의 용액. <ol><li> 에칭 속도가 7 μm의 / 분인지 확인; 에칭 용액 200 ml의 총 부피를 사용. 플라스틱 용기에 에칭을 수행하고,이 처리 단계에 대한 안전 조치를 취할. </li><li> 세척 및 실온에서 추가로 건조 탈 (DI) 물에 웨이퍼를 청소합니다. </li></ol></li><li> 처리 종료 후, 더미 실리콘 웨이퍼로부터 유리 웨이퍼를 분리하여 15 초 동안 100 ℃에서 핫 플레이트를 사용하여 왁스를 워밍업. 왁스가이 온도에서 용융 된 바와 같이, 더미 실리콘 웨이퍼로부터 유리 웨이퍼를 분리. </li><li> 초음파 탱크에서 80 ℃에서의 용매로서 N 메틸 -2- 피 롤리 돈을 사용하여 1 시간 동안 초음파를 사용하여 렌즈의 가장자리에 남아있는 왁스, 포토 레지스트 및 돌출 CR / 금 층을 제거한다.</li><li> 포토 마스크 (11)에 제작 된 마이크로 렌즈의 PDMS 몰드 복제본을 생성합니다. </li><li> 각각 주 사형 전자 현미경을 이용하여 실체 현미경 레플리카 포토 마스크 치수 (길이 및 폭)과 마이크로 렌즈 PDMS 몰드 (깊이 및 직경)를 특성화. 12-14 </li></ol> 2. 미네소타 샤프트 제작 <ol><li> 유리의 어느 측면에 장착 된 유리 슬라이드를 사용하여 0.9 cm × 2.5 cm의 구멍을 만듭니다. 양측에 적층 된 유리 슬라이드의 개수는 스페이서의 두께 (도 1b)로 알려진 공동의 높이를 결정한다. </li><li> 상 / 2- 하이드 록시 -2- 메틸 프로피 오페 w (HMP) w, 0.5 %의 폴리 (에틸렌 글리콜) 디 아크릴 레이트 (PEGDA, MW = 258 다)를 함유하는 예비 중합체 용액의 얇은 층을 도포하여 유리 슬라이드의 각 층을 확보 2 초 고강도 자외선 설정 (UV) 광의 조사 하였다 유리 슬라이드. </li><li> photoma의 위치를SK 공동의 내부 대향 CR / Au로 코팅 된 표면이 (이전에 제조 된). 공동 벽의 측면이 포토 마스크에 포함 된 렌즈를 모호하지 있는지 확인하십시오. </li><li> CR / Au로 코팅 된 표면이 보이는 기포없이 용액에 접촉 할 때까지 예비 중합체 용액 공동을 채운다. </li><li> 3백20부터 5백까지 nm의 UV 필터 범위의 UV 경화 스테이션을 사용하여 UV 소스로부터 3.5 cm의 거리를 1 초 동안 원하는 강도의 높은 강도의 자외선을 조사 설정. 자외선 프로브 시준 어댑터를 사용하십시오. </li><li> 복사계를 사용하여 UV 광의 세기를 측정. </li><li> 자외선 노출에 따라, 이동 노드의 배열과 포토 마스크를 제거합니다. 재사용을 위해 원래의 용기에 다시 그 과정에서 중합되지 않은 초과 프레 폴리머 용액을 붓고. </li><li> 제조업체의 지시에 따라 실체 현미경을 이용하여 이동 노드의 길이와 직경을 정량화 팁. </li></ol><p클래스 = &quot;jove_title&quot;&gt; 3. 미네소타 지지층 제작 <ol><li> 도 1c에 도시 된 바와 같이, 핀셋으로, 24- 웰 플레이트의 웰에 부착 포토 마스크를 MN들 (이전에 제조 된) 놓는다. </li><li> 바늘이 원하는 높이로 빠져들 때까지 우물에 프레 폴리머 용액의 - (400 μL 300) 지정된 볼륨을 추가합니다. 이 볼륨 생성 백킹 층의 두께를 결정한다. </li><li> 10.5 cm 떨어진 1 초의 시간 동안 UV 소스로부터 고강도 UV 광 (15.1 W / ㎝ 2)과 설정을 조사한다. </li><li> 날카로운 칼날을 사용하여 포토 마스크에서 MN 배열의 받침 층을 분리합니다. </li><li> 제조업체의 지시에 따라 실체 현미경을 사용하여 백킹 층의 MN 길이, 팁 직경과베이스 직경을 정량화. </li></ol>

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The authors declare no conflict of Interest.

MN들 어레이의 제조를 위해 상술 프로토콜 ~ 1cm 2의 MN 어레이를 제작하기 위해 제시되었다. 어레이는 대형 캐비티를 만들어 더 큰 포토 마스크를 이용하여 확장 할 수있다. 증가 된 공동 크기는 양측에 스페이서 사이의 폭을 증가시킴으로써 생성 될 수있다. 프로토콜 MN 어레이를 제조하는 단계는 각각 중요하지만, 가장 중요한 단계는 다음과 같다 : 포토 마스크의 설치 위치, 프레 폴리머 ?...

경피 약물 전달은 특히 거의 독점적으로 피하 주사에 의해 관리되는 생체 분자에 대한, 약물 투여에 대한 매력적인 대안 접근 방식을 제공합니다. 그러나, 피부, 특히 상부층 (각질층)는, 인체에 유입 외인성 분자 방지 강력한 장벽이다. 최근 미네소타 장치는 피부를 통해 약물을 전달하는 도구를 가능하게 등장했다. MN 장치는 약물 분자의 흐름이 개선 환자 순응도 및 편의 1-3와 원하는 생리 활성을 달성 할 수 있도록 각질층 내부에 임시 구멍을 만든다. 다양한 제조 방법은 중합체의 MN (4)를 제조하기 위해 채택되었다. 그러나, 그들은 일반적으로 MN의 어레이를 제조하기 위해 긴 시간 및 / 또는 고온을 필요로하는 복잡하고 여러 단계의 공정을 포함한다. (4)를, 단일 단계 성형 공정없이 사용 제조 공정을 단순화포토 마스크는 최근 5,6- 개발되었다. 그러나,이 방법으로, 메커니즘은 포토 리소그래피에 포함 된 자외선 (UV) 빛의 경로를 수정하는 위치에 없기 때문에 이동 노드가, 무딘 바늘 끝을했다 제작. 본 연구에서는, 포토 마스크에 내장 마이크로 렌즈가 이동 노드의 형상을 정의하는 것이 제안되어왔다. 프로토콜은 포토 마스크 임베디드 마이크로 렌즈로 구성되고이어서 포토 마스크를 이용 첨예가보고와 제조를 MN을 제조.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td>
			<td>SIGMA&#160;</td>
			<td>475629-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxy-2-methyl-propiophenone (HMP)</td>
			<td>SIGMA&#160;</td>
			<td>405655-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type 1, FITC conjugate&#160;</td>
			<td>SIGMA&#160;</td>
			<td>C4361</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing station&#160;&#160;&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>OmniCure S200-XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Adaptor&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>EXFO 810-00042</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ 1500 stereomicroscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dillon GL-500 digital force gauge&#160;</td>
			<td>Dillon, USA</td>
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			<td>Nikon, Japan</td>
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polymeric microneedle array fabrication by photolithography

이 원고는 포토 리소그래피에 의해 고분자 미세 바늘 (MN) 어레이의 제조에 대해 설명합니다. 이는 임베디드 마이크로 렌즈로 이루어진 포토 마스크를 이용하여 간단한 몰드없는 공정을 포함한다. 임베디드 마이크로 렌즈 형상 MN (선명도)에 영향을 미치는 것으로 밝혀졌다. 제작 된 두 개의 서로 다른 길이 (1336 μm의 ± 193 μm의 μm의 ± 171 μm의 957)와, μm의 13.7 ± μm의 μm의 8.4 ± 41.5 μm의 사이에 71.6까지 팁 직경 강력한 미네소타 배열. 이 MN 배열은 피부를 통해 저분자 및 고분자 치료제의 전달에 잠재적 인 응용 프로그램을 제공 할 수있다.

이동 노드의 구조는 크게 포토 마스크 및 임베디드 마이크로 렌즈의 특성에 의해 영향을받을 수있다. 굴절 정도 MN 형상 (도 2A)에 영향을 UV 광선의 전달 경로에 영향을 미친다. 각각의 마이크로 렌즈는 350 μm의 지름을 갖는 것으로하여, 130 ㎛의 볼록 표면을 평탄화하고, 62.3 μm의 깊이 (도 2B-D). 피타고라스 정리를 사용하여, 제 1면의 곡률 반경은 272.89 ㎛,이었다. 초점 길이?...

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

포토 리소그래피에 의해 고분자 미세 바늘 어레이 제작

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

polymeric-microneedle-array-fabrication-by-photolithography

Research

JoVE Journal

Bioengineering

11.9K 조회수.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

비디오: Polymeric Microneedle Array Fabrication by Photolithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

동적 마스크 프로젝션 석판술를 사용하여 신경 문화 시스템의 Micropatterned Hydrogels의 제작

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

연구

생체공학

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanoprecipitation 기법을 통해 디블록 고분자 Nanoparticles의 제형

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

다중 Transdermal 전기 감지를위한 빈 Microneedle 기반 센서

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

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

고분자 유전자 배달에 대한 평가는 Nanoparticle 추적 분석 및 높은 처리량 유동 세포 계측법에 의해 나노 입자

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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

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

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

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

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

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

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

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

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

용매를 이용한 지질 이중층 (SALB) 방법에 의해 생체막 제작

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

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

바이오 메디컬 응용 프로그램에 대한 초 소수성 고분자 재료를 제조

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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 ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

멀티플렉스 인공 세포 MicroEnvironment 배열의 제작

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.