저자는 SENSOTEM 및 NANOTIC 연구 프로젝트의 프레임 워크 내에서 벨기에의 왈 롱 지역 뿐만 아니라 국가 자금에 대 한의 과학 연구 (F.R.S.-F.N.R.S) 그들의 재정 지원에 대 한 인정합니다.

1. 단백질 샘플 준비
<ol><li>고 정화 하이브리드 단백질 BlaP-택시-Lys3로 우리의 이전 연구15에 보고 합니다. 50 m m 인산 버퍼 pH 7.4 다음 구성에서에서 단백질을 저장: 8 g의 NaCl, KCl의 0.2 g, 나2HPO4 의 1.44 g, KH2포4 증류수 800 mL에 용 해의 0.24 g 수정 전에 7.4 해결책의 pH 1 솔루션의 최종 볼륨을 조정 L. 필터 단백질 해결책 소독.</li>
	<li>암 탉 달걀 흰 lysozyme (HEWL) 재고 솔루션을 준비 합니다. 인산 염 버퍼 식 염 수 (PBS 단계 2.1.1 참조) 10 mL에 상업적으로 구입한 HEWL의 100 밀리 그램 (40000 단위/mg)을 분해. 0.22 μ m 휴즈와 필터를 사용 하 여 여과 하 여 단백질 해결책 소독.</li>
</ol>2. 바이오 센서 분석 실험
<ol><li>
솔루션 및 버퍼 준비
	<ol>
<li>8 g의 NaCl, KCl의 0.2 g, 나2HPO4, 1.44 g 및 KH2포4 증류수 800 ml에서의 0.24 g을 용 해 하 여 50 mM PBS를 준비 합니다. 1 나 필터를 소독 및 4 ° c.에 저장 솔루션의 볼륨을 조정 하기 전에 pH 7.4 1에 N HCl 또는 1 N NaOH 조정</li>
		<li>카 제인의 위에서 설명한 대로 준비 하는 PBS의 100 ml에서의 3 g을 용 해 하 여 채도/차단 솔루션을 준비 (2.1.1 단계 참조). 필터를 소독 및 4 ° c.에 저장</li>
		<li>카 제인의 위에서 설명한 대로 준비 하는 PBS의 100 ml에서의 1 g을 용 해 하 여 바인딩 솔루션 준비 (2.1.1 단계 참조). 필터를 소독 및 4 ° c.에 저장</li>
		<li>세척 솔루션 준비 (0.1% 트윈-PBS) 위에서 설명한 대로 준비 하는 PBS의 100 mL에 트윈 20 (100%)의 100 μ를 추가 하 여 (2.1.1 단계 참조). 4 ° c.에 게</li>
		<li>전극 준비 솔루션 준비 (1% 트라이 톤 X-100-PBS) 100 ml PBS 준비 위에서 설명한 대로의 트라이 톤 X-100 (100%)의 1 mL을 추가 하 여 (2.1.1 단계 참조). 4 ° c.에 게</li>
		<li>최종 볼륨을 증류수에 KCl의 26 g을 용 해 하 여 전극 재생 솔루션 (3.5 M KCl) 준비 100 mL. 살 균 필터와 4 ° c.에 게</li>
		<li>1 나 필터를 소독 및 4 ° c.에 저장의 최종 볼륨을 증류수에 NaCl의 0.29 g를 용 해 하 여 5 mM NaCl 솔루션을 준비 그런 다음, 5 mM NaCl 솔루션의 20 mL에 benzylpenicillin의 26.7 mg를 용 해 하 여 탐지 솔루션 (4 m m benzylpenicillin)를 준비 합니다. 필터를 소독 하 고-20 ° c.에 저장</li>
	</ol>
</li>
	<li>
센서 준비 및 재생 참고:는 폴리아닐린 코팅 센서 칩 개발 하 고 친절 하 게 박사 P. Bogaerts, 닥터 미 유 누스 교수부터 Y. Glupczynski의 (가톨릭 대학교의 루 베인 라-브-추 몽-Godinne)에 의해 제공 되었다. 폴리아닐린 전기 합 프로토콜 이러한 센서를 합성 하는 데 사용 되는 센서의 설명 그들의 이전 일17에 자세히 나와 있습니다. 간단히,이 시스템은 고전 인쇄 회로 기판 (PCB) 기술에 의해 제조 된 8 개의 개별 칩의 재사용 가능한 센서를 사용 합니다. 개별 칩 3 개의 전극으로 구성 된 라운드 명소. 상위 1은 작업 전극은 폴리아닐린에 전기 합성 했다. 중간에 한 참조 전극 이며 하단 전극 구성 카운터 전극. 둘 다, 참조 및 카운터 전극은 기능성 탄소 레이어 위에 단단한 Ag/AgCl 혼합을 사용 하 여.<ol>
<li>3 세척 전극의 전극 준비 솔루션의 300 µ L/잘을 포함 하는 96 잘 접시의 우물에는 팁을 찍기 여 수행 (1% 트라이 톤 X-100-PBS, 참조 단계 2.1.5.). 실 온에서 부드러운 혼합으로 2 분 동안 각 세척을 수행 합니다.</li>
		<li>실 온에서 부드러운 혼합으로 2 분 증류수의 300 µ L/잘을 포함 하는 96 잘 접시의 우물에는 팁을 찍기 하 여 전극 린스.</li>
		<li>포함 하는 재생 솔루션의 300 µ L/잘 96 잘 접시의 우물으로 팁을 찍기 여 전극을 다시 생성 (3.5 M KCl, 단계 2.1.6 참조) 4 ° C 또는 실 온에서 1 h에서 하룻밤.</li>
		<li>인산 염 버퍼 염 분의 300 µ L/잘을 포함 하는 96 잘 접시의 우물에는 팁을 찍기 하 여 전극의 3 세척을 수행 (2.1.1 단계를 참조 하십시오.). 실 온에서 부드러운 혼합으로 2 분 동안 각 세척을 수행 합니다.</li>
	</ol>
</li>
	<li>
센서에 수행 분석 결과 바인딩
	<ol>
<li>40 µ g/mL PBS에서 준비 하는 전극 표면에 HEWL의 15 µ L 드롭 입금 하 여 전극의 PANI (폴리아닐린) 표면 코트 HEWL. 4 ° C 또는 실 온에서 1 시간에서 밤새 품 어.</li>
		<li>인산 염 버퍼 식 염 수와 전극의 3 개의 세척을 수행 (2.1.1 단계 참조) 포함 하는 인산 염 버퍼 염 분의 300 µ L/잘 96 잘 접시의 우물으로 센서 칩의 전극 부분을 찍기로. 실 온에서 부드러운 혼합으로 2 분 동안 각 세척을 수행 합니다.</li>
		<li>50 µ L 방울 차단 솔루션을 추가 하 여 전극 포화 (단계 2.1.2 참조) 전극 표면에. 실 온에서 1 h에 품 어. 다음 단계 (단계 2.3.2 참조)는 이전에 설명 된 대로 세 번 세척.</li>
		<li>BlaP-택시-Lys3 솔루션 바인딩 솔루션에서 20 µ g/mL를 희석 (단계 2.1.3 참조) 15 µ L 방울 전극에 희석된이 솔루션을 적용. 실 온에서 10 분 동안 품 어. 항 원-nanobody 반응 후 세척 솔루션을 사용 하 여 이전 단계에서 세척을 3 번으로 설명 (2.1.4 단계 참조). 다음 한 번 PBS와 전극 린스 (2.1.1 단계 참조).</li>
		<li>검색, 디지털 멀티 미터에 구리 회로 부분을 통해 센서 칩을 연결 합니다. 그런 다음 50 µ L 방울 탐지 솔루션을 적용 하 여 센서 응답 시작 (단계 2.1.7 참조) 긍정적인 전극에 적용 (단계 2.1.7 참조) 부정적인 전극에 NaCl 5mm 솔루션의 50 µ L 드롭. 실 온에서 30 분 동안 품 어. 디지털 멀티 미터와는 전도성을 모니터링 합니다. 참고:는 멀티 미터 박사 P. Bogaerts, 닥터 미 유 누스 교수부터 Y. Glupczynski의 (가톨릭 대학교의 루 베인 라-브-추 몽-Godinne에 의해 제공 된. 이 potentiostat 컴퓨터 USB 포트를 통해 제어 하 고 동시에 센서의 8 개의 서로 다른 칩을 분석 합니다. 유 누스와 동료17 에 의해 만들어진 소프트웨어는 실시간 플롯 시간에 대 한 참조 및 샘플 전극의 전도도 차이의 측정을 나타내는 만듭니다.</li>
	</ol>
</li>
</ol>

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저자는 공개 없다.

이 작품에서는 캐리어 단백질으로 BlaP β-lactamase를 사용 하 여 nanobody를 functionalize 하는 방법을 소개 하 고 우리는 우리가 성공적으로 구현할 수 결과 하이브리드 단백질 potentiometric 센서 분석 결과 보여. 다른 바이오 센서 분석 실험에 비해 우리의 작업의 주요 혁신 측면은 전기 신호를 생성 하는 효소 활동을 항 체 부분의 공유 결합. 이 소위 단백질 삽입 기술 선물 장점과 한계가이 섹션의 주요 초?...

바이오 센서는 바이오 분자 상호 변환기1이라고 하는 물리적 또는 화학적 신호 장치를 결합 하는 분석 장치. 기록 된 신호 다음 해석 하 고 고정 및 무료 파트너 간의 상호 작용을 모니터링 변환할 수 있습니다. 바이오 센서의 대부분은 호르몬 등 다른 병원 체 마커2analytes를 검출 하는 항 체의 사용을 포함. 다른 센서 형식을 사용할 수 있습니다 하 고 질량, 자석, 광학 또는 전기 화학 바이오 센서를 포함 합니다. 후자는 가장 일반적으로 사용 되는 센서, 그리고 바인딩 이벤트를 전기 신호로 변환 하 여 작동. 공연 및 모든 항 체 기반 바이오 센서의 감도 기본적으로 두 개의 매개 변수에 강하게 의존: i)는 항 체와 ii)2신호 생성 하는 데 사용 하는 시스템의 속성의 품질.
항 체는 2 개의 무거운 사슬과 2 개의 가벼운 사슬 구성 높은 분자 질량 dimeric 단백질 (150-160 kDa). 빛과 무거운 사슬 사이 상호 작용은 주로 소수 성 상호 작용 보존된 이황화 결합에 의해 안정 된다. 각 체인에는 본질적으로 보완 결정 영역 (CDR1-2-3) 라는 3 하이퍼 영역을 통해 항 원와 상호 작용 하는 변수 도메인 포함 되어 있습니다. 필드, 낮은-비용 식 시스템 (예를 들면, 대장균) 전체 길이 항 체의 대규모 식에서에서 수많은 진보에도 불구 하 고 종종 불안정 하 고 집계 된 단백질의 생산에 지도 한다. 이 때문에 단일 체인 변수 조각3 (ScFvs ≈ 25 kDa)와 같은 다양 한 항 체 파편을 설계 되었습니다 있다. 그들은 각각 한 중 고 covalently는 합성 아미노산 서 열에 의해 연결 된 1 개의 경 쇄의 가변 도메인 구성 됩니다. 그러나,이 파편은 종종 가난한 안정성 표시 하 고 집계, 때문에 그들은 그들의 소수 성 영역 용4의 큰 부분을 노출 하는 경향이 있다. 이러한 맥락에서 단일 체인 camelid 항 체 단편, nanobodies 또는 VHHs, ScFvs에 대 한 우수한 대안을 것 같다. 이러한 도메인 camelid 단일 체인 항 체의 가변 도메인에 해당합니다. 기존의 항 체, 달리 camelid 항 체 경 쇄가 없는 고 두 무거운 체인5포함. 따라서, nanobodies는 가장 작은 단위체 항 체 파편 (12 kDa) 기존의 항 체6의 선호도와 항 원에 바인딩할 수 있습니다. 또한, 그들은 향상 된 안정성과 가용성 다른 전장 항 체 또는 항 체 파편에 비해 제시. 마지막으로, 그들의 작은 크기와 그들의 확장된 CDR3 루프 그들이 이상한 epitopes를 인식 하 고 효소 활성 사이트7,8바인딩할 수 있습니다. 요즘, 이러한 도메인 상당한 관심을 받고 있다 고 바이오 센서 기술에 결합 되었습니다. 예를 들어 황은 외. 탐지 및 정량화 인간 전립선 특정 항 원 (PSA)9의 nanobody 기반 바이오 센서를 개발 했습니다.
언급-위, 바이오 센서 분석에 중요 한 매개 변수는 전기 신호를 생성 하는 데 사용 하는 시스템의 효율성. 이러한 이유로, 효소 기반 전기 화학 바이오 센서 증가 관심을 받고 있다 고 건강 관리, 식품 안전, 환경 모니터링 등 다양 한 응용 프로그램에 대 한 널리 사용 되었습니다. 이 바이오 센서는 전기 신호를 생성 하는 효소에 의해 기판의 촉매 가수분해에 의존 합니다. 이러한 맥락에서 β-lactamases 더 구체적인, 더 과민 하 고 알칼리 성 인산 가수분해 효소 또는 양 고추냉이 과산화 효소10같은 많은 다른 효소 보다 실험적으로 구현 하기 더 쉬운 것 표시 했다. Β-lactamases 효소 그들을 hydrolyzing 하 여 β-락탐 항생제를 세균성 저항에 대 한 책임은 있습니다. 그들은 작은 크기의 단위체, 매우 안정, 효율적인, 그리고입니다. 또한, β-lactamases에 도메인/펩 티 드 삽입 생성 bi 기능 공상 단백질 단백질-리간드 상호작용을 연구 하는 효율적인 도구 표시 했다. 실제로, 최근 연구의 대상 항 원에 높은 선호도와 바인딩할 수 남아 공상 단백질에서 TEM1 β-lactamase 결과 항 체 변수 조각의 그 삽입을 나타났습니다. 흥미롭게도, 항 원 바인딩 TEM1 촉매 활동11,12의 allosteric 규칙 유도 표시 했다. 또한, 우리는 단백질 도메인 삽입 허용 루프에 균 licheniformis BlaP β-lactamase 단백질-리간드 상호작용13 모니터링 하는 기능 공상 단백질 생성 여러 연구에서 보였다 ,14. 우리는 최근 택시-Lys3, BlaP15이 허용 삽입 사이트에 명명 하는 nanobody를 삽입. 이 nanobody 암 탉 달걀 흰 lysozyme (HEWL)에 바인딩할 및16의 효소 활동 억제 하기 위해 표시 했다. 우리는 생성 된 하이브리드 단백질, BlaP-택시-Lys3, 라는 높은 특이성을 유지 / HEWL β-lactamase 활동 동안에 대 한 선호도 그대로 보였다. 그럼 우리가 성공적으로 β-lactamase 기술 하이브리드는 전기 화학 바이오 센서를 결합 하 여 생성 된 전기 신호 양을 했다 BlaP-택시-Lys3와 HEWL는 전극에 움직일 수 간의 상호 작용에 따라 다릅니다. 실제로, BlaP에 의해 β 락탐 항생제의 가수분해 양성자 방출 양적 전기 신호로 변환할 수 있는 유도 합니다. 이 조합은 전기 화학 바이오 센서와 하이브리드 β-lactamase 기술의 빠른, 민감한, 양적, 이며 생성 된 신호의 실시간 측정을 수 있습니다. 이 방법론은 여기 설명 되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="1471">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Reagents</td>
			<td style="width:215px;"></td>
			<td style="width:128px;"></td>
			<td style="width:615px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td>Sigma-Aldricht</td>
			<td>V000225&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td>Sigma-Aldricht</td>
			<td>1551128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldricht</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8211;HCl</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldricht</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>NIST2186II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Sigma-Aldricht</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alanine</td>
			<td>Sigma-Aldricht</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HClO4</td>
			<td>Fluka</td>
			<td>34288</td>
			<td>1M HClO4 solution, distributor : Sigma-Aldricht</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">casein hydrolysate</td>
			<td>Sigma-Aldricht</td>
			<td>22090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzylpenicillin sodium</td>
			<td>Sigma-Aldricht</td>
			<td>B0900000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">hen egg white lysozyme</td>
			<td>Roche</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">heptane</td>
			<td>Sigma-Aldricht</td>
			<td>246654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">methanol</td>
			<td>Sigma-Aldricht</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium hydroxide solution</td>
			<td>Sigma-Aldricht</td>
			<td>380539</td>
			<td>28% NH3&#160;in H2O, purified by double-distillation (concentrated?)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laboratory consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657165</td>
			<td>CELLSTAR 6-Well Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>WTW</td>
			<td>1AA110</td>
			<td>Lab pH meter inoLab pH 7110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vacuum and filtration system</td>
			<td>Nalgene</td>
			<td>NALG300-4100</td>
			<td>Filter holders with receiver, distributor : VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">potentiometric sensor chips</td>
			<td></td>
			<td></td>
			<td>manufactured by Yunus and colleagues (ref 16)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGSTAT30 Autolab</td>
			<td>Metrohm Autolab</td>
			<td></td>
			<td>discontinued, succesor Autolab PGSTAT302N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital multimeter, METRAHit 22M</td>
			<td>Gossen Metrawatt</td>
			<td></td>
			<td>discontinued, successor Metrahit Base</td>
		</tr>
	</tbody>
</table>

the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions

바이오 센서 병원 체 검출, 분자 진단, 환경 모니터링, 식품 안전 관리 등 다양 한 분야에서 점점 더 중요 하 고 구현 되 고 있다. 이러한 맥락에서 우리는 효율적인 기자 효소가 여러 단백질 단백질 상호 작용 연구로 β-lactamases를 사용. 또한, 강하게 펩 티 드 또는 구조적된 단백질/도메인의 삽입을 허용 하도록 그들의 능력 공상 단백질을 생성 하는이 효소의 사용을 권장 합니다. 최근 연구에서 우리 균 licheniformis BlaP β-lactamase에 단일 도메인 항 체 단편을 삽입. Nanobodies, 라고도 하는 이러한 작은 도메인 camelids에서 단일 체인 항 체의 항 원 묶는 도메인으로 정의 됩니다. 일반 이중 체인 항 체 처럼 그들은 높은 화력과 특이성 그들의 목표에 대 한 표시. 결과 공상 단백질 β-lactamase 활동을 유지 하면서 대상에 대 한 높은 선호도 전시. 이 nanobody 및 β-lactamase moieties 유지 기능 제안 합니다. 현재 작업에서 우리는 우리의 하이브리드 β-lactamase 시스템 바이오 센서 기술에 결합 하 여 상세한 프로토콜을 보고 합니다. 특정 바인딩 대상에 nanobody의 효소의 촉매 활동 발표 양성자의 conductimetric 측정 덕분에 검색할 수 있습니다.

설계 및 엔지니어링의 공상 단백질 BlaP-택시-Lys3
그림 1 균 licheniformis에서 BalP 클래스 A β-lactamase의 관대 한 루프에 택시 Lys3의 삽입을 나타냅니다. 삽입은 잔류물 Asp198와 Lys199 사이 수행 되었다. 트 롬 빈 분열 사이트 택시-Lys3의 각 측면에 도입 되었다. BlaP-택시-Lys3 공상 단백질 인코딩 구성 식 플라스 미드와 변형 세포 암 ?...

Watch this Scientific Journal Video about The Use of a ÃŽÂ²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

이 작품에서는, 우리는 하이브리드 β-lactamase 기술에 따라 conductimetric 바이오 센서를 사용 하 여 단백질 단백질 상호 작용을 공부 하는 새로운 방법을 보고 합니다. 이 메서드는 β-lactams의 가수분해 시 양성자의 출시에 의존합니다.

바이오 상호 작용을 검출 하는 β-lactamase 기반 Conductimetric 바이오 센서 분석 결과의 사용

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

the-use-lactamase-based-conductimetric-biosensor-assay-to-detect

Research

JoVE Journal

Bioengineering

8.9K 조회수.  University of Liège. 이 작품에서는, 우리는 하이브리드 β-lactamase 기술에 따라 conductimetric 바이오 센서를 사용 하 여 단백질 단백질 상호 작용을 공부 하는 새로운 방법을 보고 합니다. 이 메서드는 β-lactams의 가수분해 시 양성자의 출시에 의존합니다.

비디오: The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned 표면은 암세포와 Hyaluronic 산성 상호 작용을 연구하기 위해

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

연구

생체공학

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Interferometric 반사율 이미징 센서 (IRIS)를 채용 Biomolecular 감지

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

G-단백질 결합 수용체에 의해 칼륨 채널의 변조를 감지하기 위해 라벨이없는 광 바이오 센서의 사용

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

높은 처리량의 사용<em&gt; 체외</em&gt; 미세 유체 시스템 구강 다중 종의 바이오 필름을 개발하는

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

복 부 복 부 헤 르 니 아 수리 공부 쥐 모델의 사용

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

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 ...

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 ...