우리는 MAB 장치의 와이어 본딩을위한 자금 지원 (보조금 번호 103498 및 103692), 퍼듀 대학교는 Birck Nantechnology 센터 게일 록우드에 대한 NASA 우주 생물학 과학 기술 장비 개발 (ASTID) 프로그램을 감사 좋아하고, 준 진형 공원에 대한 것 흐름 셀 챔버의 CAD 드로잉입니다.

1. 폴리 (4 - 스티렌 설포 네이트 나트륨) (PEDOT : PSS) - 이온 H +와 CO에 대한 Electropolymerization 솔루션 3 2 폴리 (3,4 - 에틸렌)의 준비 <ol><li> 완전히 (약 10 초) 분산 될 때까지 10 ㎖ 탈 이온수 (DI) 물과 소용돌이에 - 70 밀리그램 폴리 (4 - 스티렌 설포 네이트 나트륨) (나 + PSS)를 추가합니다. </li><li> 용액이 완전히 혼합 될 때까지 1.1 솔루션 소용돌이 10.7 ㎕의 3,4 - ethlyenedioxythiophene (EDOT)를 추가합니다. </li></ol> 2. 칼슘 황산염 (PEDOT : 카소 4) 칼슘 2 Electropolymerization 솔루션 + 이온 폴리 (3,4 - 에틸렌)의 준비 <ol><li> 10 ㎖ DI 물과 소용돌이에 136 밀리그램의 칼슘 황산염 CASO (4) 추가, 솔루션은 완전히 분산 유백색 표시되지 않습니다. </li><li> 완전히 혼합 될 때까지 2.1와 소용돌이의 솔루션에 10.7 μL EDOT를 추가합니다. </li></ol> 3. PEDOT 기반의 Electropolymerization전도성 고분자 <ol><li> 생 분석 시스템 주식 회사 (BASI) C3 셀 스탠드 (그림 3)과 EC 엡실론 텐쇼 / galvanostat는 electropolymerization에 대한 전기 화학적 셀을 형성하는 데 사용됩니다. 용존 산소를 제거하는 20 분 동안 전기 세포와 질소 거품 PSS electropolymerization 솔루션 : EDOT를 놓습니다. </li><li> 이제 전기 화학 셀의 상대 전극 위치에서 백금 거즈 클립. 그런 백금 거즈에 직면 작동 전극과 전기 화학 셀의 작동 전극 위치에서 MAB 클립. PSS electropolymerization 솔루션 : 만 원형 전극 PEDOT에 빠져들되도록 MAB 깊이를 조정합니다. 사각 전기 접촉 패드를 가진 솔루션의 접촉을 피하십시오. </li><li> BASI 포화은 / 염화은 (자세 / AgCl을) 전기 화학 셀의 참조 전극의 위치에 전극을 배치합니다. 기준 전극이 작동하고 counte 사이에 있지 않은지 확인R 전극. </li><li> PEDOT의 경우 : PSS 증착 : 거품 전기 20 분간 세포와 0V에서 단일주기 voltammogram을 실행하는 EC 엡실론 텐쇼 / galvanostat을 사용 - 20의 스캔 속도와 1.1V ± 100 μA의 규모 MV / 초. </li><li> 에 대한 PEDOT : 카소 4 증착 : 거품 20 분 동안 전기 화학 셀, 30 분 nA의 814에 chronopotentiometry를 실행하는 EC 엡실론 텐쇼 / galvanostat를 사용합니다. </li></ol> 4. K 3 철의 PEDOT 계 고분자 복합체의 순환 전압 전류계 (CN) 6 <ol><li> 3.1-3.3 위의 단계를 수행합니다. </li><li> ± 10 μA의 규모 MV / 초 (25, 50, 75, 100, L25, 150, 175, 200)의 스캔 속도를 변화와 -653 MV에서 853 mV에서 하나의 순환 전압 전류를 실행하는 EC 엡실론 텐쇼 / galvanostat을 사용하여 . </li></ol> 5. 표면 기능화 프로토콜 <ol><li> 3 단계로 관심의 이온에 대한 특정 예금 전도성 고분자 결합. </li><li> 6 단계로 이온 선택 막을 적용합니다. </li></ol> 6. 이온 선택 막 적용 <ol><li> 진공 회 척을 중심 MAB. </li><li> MAB 및 런타임의 중앙 위에 보증금 100 μL 막. </li><li> 5 초 램프 아래로 30 초 동안 1,500 rpm으로 스핀 코터와 스핀 코트 이온 선택 막. </li><li> 30 분 동안 스핀 코팅 MAB를 진공 청소기로 청소 20 분 동안 70 ° C 오븐에서 칩을 굽는다. </li></ol> 7. 이온 선택 막 - pH와 탄산 PEDOT-PSS 전도성 고분자 접합 (CO 3 2)의 보정 <ol><li> 조건에 10 μM의 중탄산 나트륨 (NaHCO3를) 및 조류 미디어 5 MM의 칼륨 염화물 (KCl을)에서 하룻밤 MAB. </li><li> 마이크로 유체 유동 세포 칩 홀더에 MAB를 삽입합니다. </li><li> 초기의 pH 값 또는 농도 (CO 3 2 예, pH가 4 또는 10 μM -)와 5ml를 시험 용액을 주입. 온것을 제거흐름 세포 칩 홀더에서 준한. </li><li> 흐름 세포 전기기구 상 유동 세포 칩 홀더를 놓습니다. </li><li> EC 엡실론 소프트웨어를 열고 개방 회로 전위 (OP) 모드로 들어갑니다. 300 분, ± 1V에 전압 스케일, 10 kHz로 컷오프 주파수에 시간을 설정하고, 값마다 2 초를 기록합니다. </li><li> MAB는 보정 과정을 계속하기 전에 (플랫 행을 찾습니다) 안정 수 있습니다. </li><li> MAB가 안정되면, 시험 용액으로하여 흐름 세포를 세척하고 보정 할 다음 농도를 주입 (산도 5 또는 3 CO 2를 25 μM -). 더 거품이 흐름 세포를 입력 할 수 없습니다 있는지 확인합니다. pH가 6, 7, 8, 9 또는 CO 3 2 단계 7.5 및 7.6를 반복 - 50의 농도, 75, 100, 250, 500, 750, 1000 μM. </li><li> 최종 농도가 실행 된 후, 질소 공기와 MAB 건조를 제거합니다. </li><li> MAB 다시 다음 사용 때까지 신선한 조절 용액에 넣습니다. </li></ol>2 카소 4 전도성 고분자 접합 <ol><li> 조건 0.1 M 염화칼슘과 10 μM에 NaNO 3 7 ML에서 하룻밤 MAB. </li><li> 7.10-7.2과 같은 단계를 수행합니다. 단계 8.3, 0.01 mM의 염화칼슘의 초기 농도와 탄산 테스트 솔루션을 대체합니다. 시험 용액 0.05의 농도, 0.1, 0.5, 1, 10 MM에 대해이 단계를 반복합니다. </li></ol>

<ol>
	<li>Migdalski, J., Bas, B., Blaz, T., Golimowski, J., Lewenstam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Miniaturized+and+Integrated+Galvanic+Cell+for+the+Potentiometric+Measurement+of+Ions+in+Biological+Liquids.">A Miniaturized and Integrated Galvanic Cell for the Potentiometric Measurement of Ions in Biological Liquids.</a> J. Solid State Electrochem. 13, 149-155 (2009).</li><li>Buehler, M. G., Kounaves, S. P., Martin, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Water-quality+Monitor+with+Ion-selective-electrodes.">Designing a Water-quality Monitor with Ion-selective-electrodes.</a> 1, 331-338 (2001).</li><li>Adamchuk, V. I., Lund, E. D., Sethuramasamyraja, B., Morgan, M. T., Doberman, A., Marx, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Soil+Chemical+Properties+on-the-go+using+Ion-selective-electrodes.">Direct Measurement of Soil Chemical Properties on-the-go using Ion-selective-electrodes.</a> Journal Computers and Electronics in Agriculture. 48 (3), 272-294 (2005).</li><li>Oel&#946;ner, W., Hermann, S., Kaden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Sensors+and+Sensor+Module+for+Studying+Biological+Systems+in+Space+Vehicles.">Electrochemical Sensors and Sensor Module for Studying Biological Systems in Space Vehicles.</a> Aerospace Science and Technology. 1, 291-296 (1997).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polymer-based+Solid-state+Ion-selective+Electrodes.">Conducting Polymer-based Solid-state Ion-selective Electrodes.</a> Electroanalysis. 18 (1), 7-18 (2006).</li><li>Buck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Selective Electrodes in Analytical Chemistry. , (1980).</li><li>Nam, H., Cha, G. 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Chem. 72, 4694-4699 (2000).</li><li>Song, F., Ha, J., Park, B., Kwak, T. H., Kim, I. T., Nam, H., Cha, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-solid-state+Carbonate+Selective+Electrode+based+on+a+Molecular+Tweezer-type+Neutral+Carrier+with+Solvent-soluble+Conducting+Polymer+Solid+Contact.">All-solid-state Carbonate Selective Electrode based on a Molecular Tweezer-type Neutral Carrier with Solvent-soluble Conducting Polymer Solid Contact.</a> Talanta. 57, 263-270 (2002).</li></ol>

우리는 공개 아무것도 없어.

MAB 바이오 칩 백금 전극, 측정 전기 신호로 관심의 이온 농도를 transduces하는의 조합에 PEDOT 기반의 CP 공액 전달 층 꼭대기 ISM에서 건설 ASSISEs으로 구성되어 있습니다. 안정된 전극 전위는 CP 층과 ISM 층 모두에 의해 정의됩니다. 두 레이어는 MAB 및 측정 전기 신호의 품질 (소음, 드리프트)의 작동 수명을 결정합니다. PEDOT 모두의 이온 및 전자 성질 (산화 된 형태)로 전달 레이어 ?...

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the items</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>3,4-Ethylenedioxythiophene</td>
			<td>Sigma-Aldrich</td>
			<td>483028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly(sodium 4-styrenesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>243051</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EC epsilon galvanostat/potentiostat</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>e2P</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Saturated Ag/AgCl reference electrode</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>MF-2052</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pt gauze</td>
			<td>Alfa Aesar</td>
			<td>10283</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium nitrate</td>
			<td>J.T. Baker</td>
			<td>3190-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td>Mallinckrodt/ Macron</td>
			<td>7412-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S-7127</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>237132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C3 cell stand</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>EF-1085</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flow-cell chip holder</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames</td>
		 </tr>
		 <tr>
			<td>Flow-cell electrical fixture</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Specific reagents and equipment.</td>
		 </tr>
		</tbody>
 </table>

multi-analyte biochip (mab) based on all-solid-state ion-selective electrodes (assise) for physiological research

환경, 생물 의학, 농업, 생물, 그리고 우주 비행 연구 실험실 - 온 - 칩 (LOC) 응용 프로그램은 복잡한 생물학적 매체 1-4 장기간 보관을 견딜 수있는 이온 선택성 전극 (ISE)가 필요합니다. 모든 고체 이온 선택성 전극 (ASSISE)는 앞서 언급 한 응용 프로그램에 특히 매력적이다. 일괄 처리를 허용, 쉬운 건축, 낮은 유지 보수 및 (잠재력)의 소형화 : 전극은 다음과 같은 유리한 특성을 가지고 있어야합니다. 미세하게 ASSISE은 H +, 캘리포니아 2 +, 및 CO 3 2 정량화하기위한 것 - ​​이온이 건설되었다. 그것은 고귀한 금속 전극층 (즉, 백금), 전달 계층 및 이온 선택 막 (ISM) 층으로 구성되어 있습니다. 전달 계층 함수는 측정 전기 신호로 이온 선택 막 농도 의존적​​ 화학 잠재력을 형질 도입합니다. 티그는 ASSISE의 수명은 층 / 멤브레인 인터페이스 5-7 전도성의 잠재력을 유지에 의존 찾을 수 있습니다. ASSISE 작동 수명을 연장함으로써 계면 층에서 안정 가능성을 유지하기 위해, 우리는 (자세 / AgCl을)은 / 염화은 대신 전도성 고분자 (CP) 폴리 (3,4 - 에틸렌) (PEDOT) 7-9 활용 변환기 레이어. 우리는 우리가 다중 분석 바이오칩 (MAB) (그림 1)라는 실험실 - 온 - 칩 형식으로 ASSISE을 구축. (측정 범위 0.01 밀리미터 - 1 - MM) 및 CA 2 + (1 mm의 로그 선형 범위 0.01 ㎜) 테스트 솔루션의 보정은 MAB가 산도 (작동 범위 산도 4-9), CO 3 2를 모니터 할 수 있습니다 것을 보여 주었다. 산도의 MAB는 조류 중간에 거의 한 달 저장 한 후 가까운 따른다 Nernstian 경사 응답을 제공한다. 탄산 바이오칩은 기존의 이온 선택성 전극의 그것과 유사 전위차 프로필을 보여줍니다. 관심 회원ogical 측정 모델 시스템의 생물학적 활성, microalga 클로렐라의 vulgaris를 모니터링하기 위해 사용되었다. MAB 크기, 다양성의 장점을 전달하고, 분석 감지 기능 멀티 플렉스, 지구 또는 공간에 많은 제한 모니터링 상황이 적용하고. 바이오칩 설계 및 실험 방법 바이오 칩 차원에서 10 × 11mm이며, 작업 전극 (WES) 5 Ag / AgCl 기준 전극과 같은 기준 전극 (해상도)로 지정 9 ASSISEs 있습니다. 각 작업 전극 (WE) 직경 240 μm의과 동등 직경 480 μm의 수 있습니다 정규 표현식에서 1.4 mm 이격되어 있습니다. 이 전극은 0.5 mm × 0.5 mm의 차원과 전기 접촉 패드에 연결됩니다. 회로도는 그림 2에 표시됩니다. 순환 전압 전류 법 (CV)와 galvanostatic 증착 방법은 Bioanalytic를 사용하여 PEDOT 필름을 electropolymerize하는 데 사용됩니다알 시스템즈 (BASI) C3 셀 스탠드 (그림 3). PEDOT 필름의 카운터 이온이 관심의 분석 이온에 맞게 맞게 조정됩니다. 폴리 (스티렌) 카운터 이온 (PEDOT / PSS)와 PEDOT는 H +와 CO 3 2 활용 - 황산염 (카소 4와 같은 솔루션에 추가)와 하나가 칼슘 2 +에 활용되는 동안. PEDOT 코팅의 전기 화학적 특성은 우리는 산화 환원 활성 솔루션 (페리 시안화 칼륨 즉, 2 MM (K 3 철 (CN) 6))에 이력서를 사용하여 분석됩니다. CV 프로필을 기반으로, 랜들 - Sevcik 분석은 유효 표면적 10를 결정하는 데 사용되었다. 1,500 rpm으로 스핀 코팅 MAB 작업 전극 (WES)에 1 ~ 2 μm의 두께 이온 선택 막 (ISMS)를 캐스팅하는 데 사용됩니다. MAB는 조류 매체의 150 μL 볼륨 가득 미세 유체 흐름 셀 챔버에 포함되고 접촉 패드는 전기적으로 (그림 BASI 시스템에 연결되어URE 4). 클로렐라의 vulgaris의 광합성 활동은 주위의 빛과 어두운 조건에서 모니터링됩니다.

<html> PEDOT의 순환 voltammogram (CV) 결과의 예 : PSS와 해당 음극 피크 전류 (I P) 대 스캔 속도 (V 1 / 2)는 각각 그림의 5A 및 5B에 표시됩니다. PEDOT : 다양한 스캔 속도 및 음극 피크 전류에서 카소 4 표시되지 않습니다. PSS와 PEDOT : 랜들 - Sevcik 분석 10, 고체 접촉 PEDOT의 유효 표면적을 이용하여 이온 선택성 막 않고 카소 4는 각각...

Watch this Scientific Journal Video about Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research at JoVE.com

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

전도성 고분자 (CP) 트랜스 듀서에서 생성 모든 고체 이온 선택성 전극 (ASSISEs) 액체 매체의 기능적 수명이 몇 달을 제공합니다. 여기, 우리는 실험실 - 온 - 칩 형식 ASSISEs의 제조 및 교정 과정을 설명합니다. ASSISE 복잡한 생물학적 매체에서 장기간 보관 후 가까운 따른다 Nernstian 기울기 프로파일을 유지 한 것으로 설명된다.

생리 연구를위한 모든 고체 이온 선택성 전극 (ASSISE)를 기반으로 다중 분석 바이오칩 (MAB)

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

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Bioengineering

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

17.1K 조회수.  Purdue University. 전도성 고분자 (CP) 트랜스 듀서에서 생성 모든 고체 이온 선택성 전극 (ASSISEs) 액체 매체의 기능적 수명이 몇 달을 제공합니다. 여기, 우리는 실험실 - 온 - 칩 형식 ASSISEs의 제조 및 교정 과정을 설명합니다. ASSISE 복잡한 생물학적 매체에서 장기간 보관 후 가까운 따른다 Nernstian 기울기 프로파일을 유지 한 것으로 설명된다.

비디오: Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

의 주문형 정량 분석​​을위한 미세 유체 기반 Electrotaxis<em&gt; Caenorhabditis의 엘레</em&gt; &#39;로코

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

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Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

재생 연구를위한 고조파 나노 입자

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

라벨없는 DNA의 혼성화 분석 용 미세 유체 기반 전기 바이오칩

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

인간 아데노 바이러스 유형 5를 기반으로 대용량 아데노 바이러스 벡터의 클로닝 및 대규모 생산

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired 디자인을위한 프로토콜 : 접지 샘플러 성게 턱을 바탕으로

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

드라이 필름 포토 레지스트 기반 전기 화학 미세 바이오 센서 플랫폼: 장치 제작, 온 칩 시험 준비 및 시스템 운영

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

제조 및 통합된 전극 Transendothelial 전기 저항을 직접 측정 하는 기관-온-칩 시스템의 유효성 검사

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

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 ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

곡선 변환 기반 도구를 사용하여 피브릴라 콜라겐 조직을 정량화

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

Microtubule Plus-End Dynamics Visualization in Huntington&#039;s Disease Model based on Human Primary Skin Fibroblasts

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

인간 1차 피부 섬유아세포를 기반으로 한 헌팅턴 병 모델의 마이크로튜블러 플러스 엔드 다이내믹스 시각화

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...