이 작품은 보조금의 원조 교육부, 문화, 스포츠, 일본의 과학 기술, 교토 Technoscience 센터 및 연구 개발에 대한 미타니 재단 젊은 과학자 (16K17956)에 의해 지원되었다.

1. 악기 <ol><li> 편광 장치 <ol><li> 편광 소자를 구성하는, 할로겐 램프, 도광판, 편광판, 시료 스테이지, 광학 레일로드 스탠드, 디지털 일안 리플렉스 카메라 (갖는 광원을 제공하는 편광에 대한도 1C와 자재 목록 참조 장치 부). </li></ol></li></ol> 생체 고분자의 용액의 제조 (2) <ol><li> 다당류 솔루션 <ol><li> 이상 ~ 12 시간 동안 80 ℃에서 교반하여 순수한 물 (100 mL) 중 sacran 7 (0.5 g)을 녹인다. 용해 중에, 증발을 방지하기 위해 플라스틱 포장으로 용기 커버. 동일하게 크 산탄 검의 수용액을 준비한다. </li><li> 0.5 중량 % 수용액을 얻었다 ~ 25 ℃에서 용액을 냉각. </li><li> 불순물 (48,400 XG, 4 ℃, 1 시간, 3 팀을 제거하는 용액을 원심 sacranES). </li></ol></li><li> MT 솔루션 <ol><li> 브리튼 - 로빈슨 완충액에서 0.5 중량 %의 튜 불린 용액 (1 ml)에 준비 (80 mm의 N, N &#39;비스은 피페 라진 (2- 에탄 설 폰산) (PIPES), 1 mM의 에틸렌 글리콜 비스 (β 아미노 에틸 에테르) - N, N, N &#39;, N&#39;-tetraacetic 아세트산 (EGTA); 얼음 8 5 밀리미터의 MgCl 2, pH를 6.8). </li><li> 0.5 중량 % 튜 불린 용액 (50 μL) 및 사용 구아노 신 - 5 &#39;- [(α, β) -methyleno] 포스페이트 (GpCpp) (5 μL)는 GpCpp 함유 튜 불린 용액 (50 μL)을 제조 하였다. 안정한 MT 핵을 얻기 위해 3 시간 동안 37 ℃에서 인큐베이션. 참고 : GpCpp의 역할은 MT 형성을 지원하기 위해 완전히 튜 불린에 MT의 해중합을 억제하는 것입니다. </li><li> 안정한 0.5 중량 % MT 용액을 수득 1 일 ~ 25 ° C에서 0.5 중량 % 튜 불린 용액 (950 μL) 및 GpCpp 튜 불린 - 함유 용액 (50 μL)를 섞는다. </li></ol></li><li> DNA 솔루션<ol><li> (1mM의 EDTA 10 개 mM 트리스, pH를 8.0) 트리스 EDTA 완충액에서 0.5 중량 %의 DNA 용액 (1 ml)로 준비한다. </li></ol></li><li> 건조 실험을 25 ℃에서 0.5 중량 %의 시료 생체 고분자 용액을 유지한다. </li></ol> 교차 편광 3. 건조 실험 및 관찰 <ol><li> 한 측면에서 개방 셀 솔루션 (도 1a) <ol><li> 실리콘 낱장 1mm의 두께를 적절한 형상으로 (자재 목록 참조) (5-15 mm의 1mm의 내부 크기 및 ~ 20mm;도 1a). <ol><li> ~ 20mm 두 비 개질 된 유리 슬라이드 (76mm × 1 mm × 26 mm)을 5-15 mm 1 mm의 내 치수를 갖는 실리콘으로 이루어지는 스페이서의 한쪽 개방 셀을 조립하고. 누출에서 시료 용액을 유지하기 위해 사전에 더블 클립 셀의 양면을 수정합니다. </li></ol></li><li> 천천히 ~ 1 mm의 기공 크기를 피펫을 사용하여 0.5 중량 %의 생체 고분자 용액 (100-300 μL)을 각각 추가~ 25 ℃에서 각 셀에 TE 팁. 주사기 바늘을 사용하여 세포로부터 기포를 제거한다. </li><li> 증발 대기압하에 60 ℃에서 공기 순환 오븐에서 함께 세포로; 증착 방향이 중력의 반대이다. </li></ol></li><li> 교차 편광하에 관찰 (도 1B-1C) <ol><li> 광역 (80mm X 80mm) 위에 평평한면 광원으로는 100 W 할로겐 램프를 통해 직선 가시 광선을 제공한다. 홀더 (도 1C)를 사용하여 45 ° 및 135 °에 편광판을 조정한다. </li><li> 광학 레일로드 스탠드 (도 1C)를 사용하여, 광원, 편광기, 샘플 스테이지, 카메라의 위치를 고정한다. 두 편광판 사이에 시료 스테이지를 배치 (편광판 사이의 거리는 5cm ~이어야한다). 집중 할 수 있도록 샘플 단계부터 ~ 20cm를 카메라를 놓습니다. </li><li> 주어진 시간에, 폴라 사이의 단계 3.1.3에서 샘플을 배치XZ 평면에 평행 한 단에 rizers 검은 커튼 장치를 커버; 실제 장치가도 1c에 도시되어있다. </li><li> 표준 줌 렌즈와 디지털 일안 리플렉스 카메라를 사용하여 선형 교차 편광판을 통해 샘플을 촬영 (재료 목록 참조). 컴퓨터 소프트웨어 (자료 목록을 참조)을 사용하여, 같은 초점 거리와 같은 카메라 설정을 제어 할 수 있습니다. </li></ol></li><li> 투과광 강도의 시공간적 분석 (도 1C) <ol><li> 건조 공정의 순서 배 양성 파라미터의 변화를 평가하기 위해 24 시간 동안 시간당 사진을 수집한다. </li><li> (예 ImageJ에) 화상 처리 프로그램을 사용하여 그레이 값과 Z 방향의 중심선을 따라 투과광의 강도를 측정한다. </li><li> 상부 개방 측으로부터의 거리의 함수로서 그레이 값의 그래프를 그린다. </li></ol></li><li> 교차 편광 조명 관리에서 현미경 관찰t (도 1D) <ol><li> , 방법을 확인 CCD 카메라 (9)가 장착 된 편광 현미경 현미경 관찰을합니다. 빛의 경로에 일차 위상차 판을 보관하십시오. PC에 소프트웨어를 사용하여 사진에 대한 조건을 제어합니다. </li></ol></li></ol>

<ol>
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저자가 공개하는 게 없다.

이 때문에 투과광 강도가 너무 낮있는 카메라 샘플에 초점을 때로는 어려웠다. 이러한 경우, 무대에서 확장 된 투명 플라스틱 필름을 배치하는 것은 초점을 준비하는 데 도움이. 관측 가능한 해상도의 한계는 ~,이 경우에는 10 μm의 카메라 렌즈에 의존했다. 샘플의 두께, ΔY, 상기 관찰 ~ 제한이 경우 10mm 램프의 최대 광 강도에 의존했다. 도 1b에 도시 된 장치의 장?...

생체 고분자의 강성, 막대 형상의 구조체에 초점을 동적 부드러운 물질은 다당류 생물막 행렬 1, 세포 골격 단백질이 이루어지는 &quot;활성 겔&quot;을 원하는 형상 (3)의 &quot;DNA 접기」등, 다양한 용도로 사용되고있다. 구조적 특성을 명확히하기 위해, 많은 전략들이 이러한 투과형 전자 현미경, 주사 전자 현미경, 원자력 현미경, 및 공 초점 형광 현미경으로, 탐구되었다. 이러한 방법은 대부분 건조 또는 정적 상태에서 수행되기 때문에 그러나, 실제 생활 시스템에서 볼 수 있듯이, 거시적 규모의 동적 행동을 설명하기 어렵다. 최근에는 성공적으로 편광 된 광 (4)를 통해 수성 공기 LC 인터페이스 생체 고분자의 동적 거동을 관찰 하였다. 배향 구조의 시각화 동안 고분자 물질을 건조하는 동안용액의 시간적 변화는 불안정한 공기 LC 인터페이스 생체 고분자의자가 통합을 나타냈다. 여기서는 편광 수단을 이용하여, 공기 계면에서 LC LC 생체 고분자 용액의 건조를위한 프로토콜을 기술한다. 5,6 건조 고려하지 않는다는 LC상의 다른 분석 달리, 건조 공정 동안 LC 역학은 일측 개방 셀 내의 유체의 상 측면도의 배 양성 위해 파라미터를 평가하여 현재 조사했다 . 셀 증착의 조합 제어 증발 방향 육안 모니터링 허용 편광 수단의 사용. 또한,이 방법의 효과를 입증하기 위해, 분자량, 농도 등의 영향을받은 흡착 마이크로 도메인의 결정 구조에 집중하여 건조 기록을 확인할 수 있었다 건조 프로다당류, 미세 소관 (MTS), 및 DNA 등의 딱딱한 막대 모양 기본 생체 고분자의 세 스는 조사 하였다. 그들이 megamolecular 무게와 계층 거대 분자의 전형적인 예이며, 자신의 분자간 상호 작용 LC 상태를 형성 할 수 있도록하기 때문에 우리는이 생체 고분자를 선택했다.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">sacran</td>
			<td style="width:151px;">Green Science Materials Inc., Japan</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">From Aphanothece sacrum. 
			Mw = 1.9 &#215; 107 g mol-1</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">xanthan gum</td>
			<td style="width:151px;">Taiyo Kagaku Co., Japan</td>
			<td style="width:131px;">Neosoft XC</td>
			<td style="width:163px;">From Xanthomonas campestris. 
			Mw = 4.7 &#215; 106 g mol-1</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">tubulin</td>
			<td style="width:151px;">Cytoskeleton, Inc., USA</td>
			<td style="width:131px;">T240</td>
			<td style="width:163px;">From porcine brain.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">GpCpp</td>
			<td style="width:151px;">Jena Bioscience, Germany</td>
			<td style="width:131px;">NU405L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">piperazine-N,N&#8242;-bis(2-ethanesulfonic acid)</td>
			<td style="width:151px;">Sigma-Aldrichi, Co. LLC.</td>
			<td style="width:131px;">P6757-500G</td>
			<td style="width:163px;">PIPES</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:172px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:151px;">Dojindo Molecular Technologies, Inc.</td>
			<td style="width:131px;">342-01314</td>
			<td style="width:163px;">EGTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">MgCl2-6H2O</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">135-15055</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">KOH</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">162-21813</td>
			<td style="width:163px;">pellet</td>
		</tr>
		<tr height="21">
			<td height="58" style="height:58px;width:172px;">DNA</td>
			<td rowspan="2" style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td rowspan="2" style="width:131px;">D1626</td>
			<td rowspan="2" style="width:163px;">From salmon testes. 
			Mw = 1.3 &#215; 106 Da (~2000 bp)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Tris-EDTA buffer solution</td>
			<td style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td style="width:131px;">T9285-100ML</td>
			<td style="width:163px;">10 mM Tris, pH 8.0, with 1 mM EDTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">slide glass</td>
			<td style="width:151px;">Matsunami Glass Ind., Ltd., Japan</td>
			<td style="width:131px;">S1111</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">silicon rubber sheet</td>
			<td style="width:151px;">Asone Co.</td>
			<td style="width:131px;">6-611-32</td>
			<td style="width:163px;">Thickness: 1 mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">centrifuge</td>
			<td style="width:151px;">Beckman-Coulter, Inc., USA</td>
			<td style="width:131px;">Avanti J-25</td>
			<td style="width:163px;">equipped with a JA-20 rotor</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light source</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">LS-LHA</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light guide</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">GF7.2-1-L1500R-M80 (AAAR-015M)</td>
			<td style="width:163px;">80 mm &#215; 80 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">halogen lamp</td>
			<td style="width:151px;">Ushio Inc., Japan</td>
			<td style="width:131px;">JCR 15V150WBN</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarizer</td>
			<td style="width:151px;">Luceo, Co.,Ltd.</td>
			<td style="width:131px;">POLAX-42S</td>
			<td style="width:163px;">40 x 80 x 2t (45&#730;)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">KMH-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample stage</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">TARW-25503L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">SHA-25RO</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">rod</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">ROU-12-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-6-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-60</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-130</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">carrier</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CAA-25LS</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">camera holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CMH-2</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">medium optical rail</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">OBA-500SH</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L25 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L50 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">multiband</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">80&#966;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">V plate</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">V plate 60S</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">plate holder</td>
			<td style="width:151px;">Viexen, Co.,Ltd.</td>
			<td style="width:131px;">plate holder SX</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">EOS Kiss X7i&#160;</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">8594B001</td>
			<td style="width:163px;">with a standard zoom lens,&#160; EFP 18-55 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">EOS Utility</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">PC</td>
			<td style="width:151px;">Microsoft</td>
			<td style="width:131px;">Surface</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarization microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">BX51</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">first order retardation plate</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">U-TP530</td>
			<td style="width:163px;">&#955; = 530 nm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">CCD camera</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">DP80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">cellSens Standard</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Java-based image processing program</td>
			<td style="width:151px;">the National Institutes of Health</td>
			<td style="width:131px;">ImageJ</td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

methods for the self-integration of megamolecular biopolymers on the drying air-lc interface

물을 사용 살아있는 유기체는 항상 환경에서 건조하는 경향이 있습니다. 관다발 물을 이동 및 피부 층에 물 보습의 경우에서 볼 수 있듯이 이들의 활동은 생체 고분자 기반의 마이크로 및 매크로 구조에 의해 구동된다. 본 연구에서는 건조 생체 고분자로 이루어지는 수계 결정 (LC) 용액의 효과를 평가하기위한 방법을 개발 하였다. LC의 바이오 폴리머가 megamolecular 무게를 가지고, 우리는 다당류, 세포 골격 단백질과 DNA를 연구하기로 결정했습니다. 편광하에 건조시 생체 고분자 용액의 관찰 불안정한 공기 LC 인터페이스부터 milliscale자가 통합을 보여준다. 수성 LC 생체 고분자 용액의 역학은 일측 개방 셀로부터 물을 증발에 의해 모니터링 될 수있다. 교차 편광을 이용하여 촬영 된 영상을 분석함으로써, 배 양성 위해 파라미터의 시공간적 변화를 인식 할 수있다. 이방법은 인공 다양한 분야에서 물질뿐만 아니라 자연의 살아있는 조직뿐만 아니라의 특성 유용 할 수 있습니다. 우리는 생물 의학 및 환경 분야에서의 부드러운 소재에 대한 평가 방법을 제공 할 것입니다 믿습니다.

<html> 도 1에 도시 된 바와 같은 장치를 사용하여 미소 영역에서 자기 통합 에어 LC 인터페이스 평가 건조 (도 2A)에 macrodomain한다. 건조 실험의 처음 입증 된 바와 같이, megamolecular 다당류 sacran (M w = 1.9 × 10 7g 몰 -1) 및 크 산탄 검 (4.7 × 106 그램 몰 -1) 두 종류, 비교 하였다. 도 2b는 교차 편광?...

Watch this Scientific Journal Video about Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface at JoVE.com

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

공기 액정 성 인터페이스 megamolecular 생체 고분자의 건조 - 유도 된자가 통합을위한 방법이 여기에 제공된다. 이 방법은 생체 고분자의 거시적 인 잠재력을 이해뿐만 아니라, 생물 의학, 환경 분야의 부드러운 소재에 대한 평가 방법으로뿐만 아니라 도움이 될 것입니다.

건조 공기-LC 인터페이스에 Megamolecular 생체 고분자의 자체 통합을위한 방법

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

methods-for-self-integration-megamolecular-biopolymers-on-drying-air

Research

JoVE Journal

Bioengineering

6.0K 조회수.  Japan Advanced Institute of Science and Technology.  공기 액정 성 인터페이스 megamolecular 생체 고분자의 건조 - 유도 된자가 통합을위한 방법이 여기에 제공된다. 이 방법은 생체 고분자의 거시적 인 잠재력을 이해뿐만 아니라, 생물 의학, 환경 분야의 부드러운 소재에 대한 평가 방법으로뿐만 아니라 도움이 될 것입니다. 

비디오: Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Biofabrication와 바이오 전자 인터페이스 브리지

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

연구

생체공학

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

글라이딩 Assays를 사용 Biopolymers의 굽힘 강성 측정

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

포토 리소그래피 정의 파릴 렌-C에 대한 세포 패터닝 : 그런가<sub&gt; 2</sub&gt; 기판

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

분자 얽힘과 생체 고분자의 Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

바이오 필름 및 서식지의 이질성의 공동 개발을 특성화하기위한 방법

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip : 단백질 - 포스 포이 노시 티드 상호 작용에 대한 라벨없는 연구

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

셀 루 로스 Nanofiber 단방향 동결에 의해 준비 Microhoneycomb Monoliths 기반 Sols: 메서드 및 확장

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

성인 쥐의 복합 재생 말초 신경 인터페이스 (C-RPNI)의 제조

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.