The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

다양한 솔루션 및 단백질 샘플의 1 준비 <ol><li> 50 밀리미터의 NaH 2 PO 4, 50 mM의 염화나트륨을 함유하는 인산염 완충액 (PBS-G) 100 ㎖, pH를 6.8에서 10 % 글리세롤을 준비한다. </li><li> pH가 7.4에서 10 mM의 HEPES, 150 mM의 염화나트륨, 0.005 % 트윈 20을 함유하는 HEPES 완충 용액 250 ㎖를 준비한다. </li><li> PBS-G에서 0.2 밀리미터에 유당 (그림 1) 황산 빌딩 블록 1 (BB1) 유당 및 빌딩 블록이 (BB2)의 주식 솔루션을 준비합니다. </li><li> 1 μM에서 꽃대의 hypogaea 렉틴 (AHL) 500 nm의, 미오글로빈에서, 및 500 nm에서 라이소자임 : HEPES 단백질 용액 1 ㎖를 준비합니다. </li><li> 초순수에 1 % SDS 20 ㎖를 준비한다. </li></ol> CoCRR 배열의 2 준비 <ol><li> 75 % 산소 25 %, 아르곤, 0.6 밀리바, 전력 40 W. : 이러한 조건에서 3 분 동안 플라즈마 클리너와 함께 사용하기 전에 프리즘 48 시간의 금 표면을 청소 </li><li> 열한 순수하고 혼합 solutio 준비[BB1] / BB2와 BB1과의 NS ([BB1] + [BB2]) PBS-G 0.1 ㎜의 전체 BB 농도에서 10 %의 증가에 의해 0과 100 % 사이의 비율. </li><li> 입금 합계 44 점 (도 2)와 각 감속비 사통에서 비접촉 감시인을 이용한 프리즘면에 순수 및 이들 혼합 용액 8 NL 방울. NOTE : PBS-G에서 추가 10 % 글리세롤, 용매 증발 및 증착 후 BB 농도의 변화를 감소시키는 것이 매우 중요하다. </li><li> 초순수 1 ㎖를 함유하는 페트리 접시 안에 프리즘을 배치하고 금 표면에 BB2와 BB1의 자기 조립을위한 RT에서 O / N을 떠난다. 여기서,이 혼합의 SAM의 표면 평균 조성은 증착 혼합 용액의 조성 (도 3) (11)를 반영하는 것으로한다. </li><li> 초순수로 충분히 프리즘을 세척 한 후 아르곤의 흐름 아래를 건조. </li></ol> 3 단백질 감지 SPRI에 의해 <ol><li> incubato 설정R이있는 SPRI 장치는 단백질 감지하는 동안 온도 변화에 의해 유도 된 굴절률 변화를 방지하기 위해 25 ° C에 배치됩니다. </li><li> (PEEK) 컴퓨터 제어 시린지 펌프, 탈기 및 6 포트 중압 분사 밸브에 연결된 셀 흐름 (도 4) 10 μl를 폴리 에테르 에테르 케톤의 비 - 작용 린스 프리즘입니까. 갓 여과하고 버퍼 HEPES를 실행 탈기와 흐름 시스템에 입력합니다. </li><li> 린스 프리즘을 제거하고 유동 셀 CoCRR 어레이를 포함 프리즘을 삽입한다. 100 μL / min의 유량으로 실행 HEPES. CCD 카메라의 도움으로 신속하게 실행 버퍼를 전달하여 프리즘 표면에 존재하는 기포를 제거합니다. </li><li> 어레이의 잘 대조 화상과 SPRI 시스템의 통합 소프트웨어의 도움으로 기초하여 관심 영역에 대해 동일한 직경을 가진 원을 그림으로써, 각 자리에 대한 연구 영역을 정의한다. </li><li> 반영 플라즈몬 나타내는 곡선을, 추적모든 반점의 입사각의 함수에의 ivity 곡선. </li><li> 반사율 곡선의 가장 높은 슬로프에서 작업 각도 (운동 곡선이 기​​록됩니다되는 위치)를 선택합니다. 운동 측정을위한 선정 작업 각도를 해결하기 위해 스캐닝 미러를 돌립니다. </li><li> 모든 관광 명소에 대한 반사 신호가 안정적이고 일정하게 될 때까지 흐름 시스템을 통해 HEPES를 계속 실행됩니다. </li><li> 위치 &quot;부하&quot;에 주입 밸브 시료 루프 (500 μL)에 주사기와 1 ㎖의 AHL 솔루션을 주입한다. 그런 다음 &quot;주입&quot;위치로 주입 밸브를 넣어. 모든 단백질 주입에 사용 유속 100 μL / 분이었다. </li><li> 모든 지점에서 동시에 시간에 대한 반사율의 변화를 모니터링하여 운동 측정을 시작합니다. </li><li> 단백질 사출의 끝에서 8 분 동안 버퍼를 실행 어레이를 헹군다. 마지막으로, 그것을 다시 생성 한 ML 1 % SDS를 주입. </li><li> 다른 페이지에 대해 동일한 절차를 반복roteins. </li></ol> (4) 데이터 처리 및 분석 <ol><li> 유동 세포에 단백질 용액의 항목에 따라, 분자 결합이 발생하고 플라즈몬 커브의 시프트 및 반사율의 변화를 유도한다. 화상 획득 소프트웨어 sensorgrams (도 5B)을주고, SPR 이미지 5A 도표주는 스케일 레벨을 그레이 측정 광 강도 값을 변환하고, 시간에 대한 반사율의 변화를 생성한다. </li><li> [BB1] 대 각 단백질 주입 단부에 반사율 (R의 %)을 플롯 수학 컴퓨팅 소프트웨어를 사용하여 / ([BB1] + [BB2])는 각 샘플에 대한 2 차원 연속 진화 프로파일 (CEP)을 생성하는 비율 (도 5C). </li><li> [BB1] / ([BB1] + [BB2]) sensorgrams의 비율을 추가합니다 (그림 5B)는 각 단백질 (그림 5D)에 대한 3D 연속 진화 풍경 (CEL)을 생성합니다. </li></ol>

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The authors have no conflicts of interest to declare.

<html> 이 등의 건설을위한 가장 중요한 단계는 시스템의 재현성을 보장하기 위해 최선을 다하고 있습니다. 예를 들어, BB1과 BB2의 열한 순수 및 혼합 용액에 글리세롤의 10 %를 첨가, 사용 전에 표준화 된 절차와 프리즘의 금 표면을 세정하는 BB들은 프리즘의 금 표면에 자기 조립 중에 용매를 증발 제거하고, 각 [BB1] 여러 복제물 / ([BB1] + [BB2]) 비율 등을 증착하는 단계를 포함한다. SPRI 의?...

정확하고 빠른 단백질 감지 방법은 의료 진단 및 단백질 체학 (proteomics)에서 매우 중요합니다. 이러한 바이오칩 클래식 단백질 검출 배열은 &quot;자물쇠 및 열쇠&quot;인식의 원리에 기초하고 앱 타머, 항체 또는 모방 체 등의 특정 수용체​​를 필요로한다. 최근 몇 년 동안, 인간의 후각 및 맛보기에 의해 영감을 차동 감지 대안 1로 떠오르고있다. 이 전자 코 / 혀 (EN / ET) 접근법은 표적 분자에 매우 특정 또는 선택적으로 필요하지 않은 교차​​ 반응성 수용체 (CRRs)의 배열 분석 물질의 결합 미분에 기초하므로 번잡을 극복하도록 매우 선택적 수용체의 개발 방법. 그것은 그것의 식별을 가능하게 지문처럼 각각의 샘플에 대한 고유 한 패턴을 만들어 모든 수용체의 결합 반응이다. ELEC의 개발이 핵심 과제효과적인 단백질 검출 용 트로닉 코 / 혀는 구조적으로 유사한 분석 및 바인딩 이벤트에 적합한 전달 시스템을 구별 할 수있는 능력을 가지고 촬상 소자의 제조이다. 지금까지 연구는 어레이 개발이에 다양한 접근 방법이 신고되었습니다. 한 연구에서, 예를 들어 단백질의 스토리지 기반 식별은 다양한 아미노산 또는 단백질에 대한 별개의 청구 둘레 대한 친 화성, 소수성 코어를 갖는 차동 수용체를 제공하는 디 펩티드에 카르복실기를 결합하여 테트라 carboxyphenylporphyrin 유도체로부터 제조 CRRs를 사용하여 개발되었다 부여 차동 결합. 이 시스템을 사용하면, 다른 단백질과 단백질 혼합물은 분석 물질과의 상호 작용에 따라 3,4 수용체 형광 소광을 측정함으로써 확인되었다. 또 다른 연구에서, hexasubsti에 조합 방식으로 합성 트리 펩티드 및 보론 산 잔기를 함유하는 29 CRRs의 라이브러리환 된 벤젠 비계 표시 - 흡수 비색 검출 5,6로 단백질을 감지하기 위해 개발되었다. 같은 디자인으로, 각각의 수용체는 당 단백질에서 단백질의 차별화에 도움 펩타이드 팔에 분산에 따라 단백질, 및 붕산과 차동 결합 능력을 보여 주었다. 최근 음이온 형광 고분자 폴리 (p-페닐 렌) (PPE)가 결합 다른 양이온 작용 금 나노 입자로 구성된 배열을 감지하고 단백질 칠을 식별하기 위해 만들어졌습니다. 경쟁 단백질 고유 인식 패턴을 생성 단백질 분석 물질 간의 바인딩과 형광을 재생 PPE / 금 나노 입자 복합체를 켄칭. 이 연구에서, 기능화 된 나노 입자 - 단백질 상호 작용은 나노 단부 그룹의 물리 화학적 특성을 변화시킴으로써 조정 하였다. 또한,이 방법은 복잡하고 단백질이 풍부한 배지 등의 단백질 분석을 위해 효과적임을 나타내었다생리 학적으로 관련이 농도에서 인간 혈청으로서, 이와 질환 상태를 진단하기 위해 8 실제 시료 프로파일에서 ET의 전위를 도시. 매우 유망하지만,이 시스템은 몇 가지 본질적인 한계가있다. 그들은 설계 및 매우 복잡한 구조를 5 ~ 29 CRRs에서 합성이 필요합니다. 또한, 각각의 측정 다음 리셋 후각 시스템과 달리, 단백질은 샘플 당 센싱 어레이를 제조 요구한다. 마지막으로, 실시간 모니터링에 이벤트 바인딩은 매우 어렵습니다. 이러한 맥락에서, 조합 접근 방식은 서로 다른 물리 화학적 특성을 가진 간단하고 쉽게 접근 분자의 적은 수의 사용에 의해 제안되었다 (중성 음으로 하전 된 양전하 친수성, 소수성,,,, 등.) 빌딩 블록 (BBS) 9있다. 에 혼합물을 변화의 BB들은하고 제어 비율을 혼합 허용함으로써의 금 표면에 자기 조립 (self-assembly)프리즘, 결합 단백질에 적합한 특성을 갖춘 조합 표면의 배열을 만들었습니다. 특히,이 시스템의 자기 조립 단층 신속하고 효율적으로 생산 될 다양한 조합 교차 반응 수용체 (CoCRRs)를 가능하게 높은 분기하는 방식으로 표면 특성의 범위를 쉽게 튜닝 할 수 있습니다. 단백질이 검출 광 검출 시스템을 사용하여 수행되었으며, 플라즈몬 공명 영상 (SPRI)를 표면. 간략하게, LED에서 넓은 빔 단색 편광 프리즘의 표면 전체 CoCRR 어레이 영역을 조명한다. 고해상도 CCD 비디오 카메라는 CoCRR 어레이의 모든 지점에 걸쳐 실시간 차분 화상을 제공한다. 이것은 바인딩 이벤트 및 운동 프로세스 10에 대한 상세 정보를 제공 CoCRR 어레이의 표면에서 로컬 변경을 모두 포착. 한편, 이미징 소프트웨어의 도움으로, 스폿에 대응하는 SPR 이미지는 자동 versu 반사율의 변화로 변환sensorgrams 불리는 결합 곡선 운동의 시리즈를 생성하는 시간이야. 따라서, SPRI 바인딩 이벤트 라벨없는, 동기, 병렬 및 실시간 관찰을 허용한다. 또한, 수득 CoCRR 어레이는 단백질 분석을위한 재생 가능한 재사용이다. 이 프로토콜은 빌딩 블록으로서 만이 작은 분자를 사용하여 전자 혀의 구성을 설명하고 SPRI 연속 인식과 구매 패턴에 따라 공통 단백질 분석 용의 응용을 도시한다.

<table border="0" cellpadding="0" cellspacing="0" width="1511">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td style="width:799px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRi apparatus&#160;</td>
			<td>Horiba Scientific-GenOptics</td>
			<td style="width:225px;"></td>
			<td>SPRi apparatus is placed in a temperature regulated incubator at 25&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Memmert</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump&#160;</td>
			<td>Cavro scientific instruments</td>
			<td style="width:225px;">Cavro XLP 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Micro Elite Degasser&#160;</td>
			<td style="width:203px;">Alltech</td>
			<td style="width:225px;">AT590507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-port medium pressure injection valve</td>
			<td>Upchurch Scientific</td>
			<td style="width:225px;"></td>
			<td>The volume of injection loop used is 500 &#181;l.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Femto plasma cleaner (version 7)</td>
			<td>Diener Electronic</td>
			<td style="width:225px;"></td>
			<td>On-line degassing system with 2 channel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spotter</td>
			<td style="width:203px;">Siliflow</td>
			<td style="width:225px;"></td>
			<td>&#160;It is a non-contact piezoelectric spotter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SPRi-Biochip</td>
			<td>Horiba Scientific-GenOptics</td>
			<td align="right" style="width:225px;">36000067</td>
			<td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">erythrina cristagalli lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L5390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">arachis hypogaea lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L0881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">myoglobin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lysozyme</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#945;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#947;&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sulfated lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">G5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SDS</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">tween 20</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td align="right" style="width:225px;">274348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">HEPES</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium chloride</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S3014</td>
			<td></td>
		</tr>
	</tbody>
</table>

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

<html> 일반적인 단백질 분석 용 전자 혀의 능력을 검사하기 위해, 세 가지 단백질을 사용 하였다 : AHL, 미오글로빈 및 리소자임을. 도 6에 도시 된 바와 같이 각각의 단백질의 경우, 별개의 연속적인 진화 2D 프로파일, CEP는 ET에 의해 생성되었다. 또한, 각각의 단백질에 대해, 시간에 따라 연속 인식 패턴을 실시간으로 흡착 및 탈착 반응 속도를 모니터링 할 수 SPRI, ?...

Watch this Scientific Journal Video about Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis at JoVE.com

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

단백질 분석을위한 지속적인 인식 패턴을 생성하는 전자 혀

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

electronic-tongue-generating-continuous-recognition-patterns-for

Research

JoVE Journal

Bioengineering

7.8K 조회수.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

비디오: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

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

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

증식하는 연속 고해상도 현미경으로 관찰 신진 효모의 노화

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

세포 물질의 상호 작용 연구를위한 나노 기공 골드 패턴의 미세

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

양적, 피부가 상처 관리를위한 등각 피부와 같은 전자 시스템의 제조 및 특성

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

평면에 단일 분자 형광 현미경은 이중층 지원

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

단일 벽 탄소 나노 튜브에 바이오 모방 고분자와 공학 분자 인식

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

섬유에 유기 전자 장치를위한 간편하고 확장 가능한 제조 방법

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

최적화 된 설치 및 히스테리시스 측정 현장에서 자기 도메인 이미징에 대 한 프로토콜

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

시험관 내 마이크로 유기 전하 변조 필드 효과 트랜지스터 어레이에 의한 다단계 세포 분석

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...