저자는 재정 지원에 대한 자신의 조기 연구원 상 프로그램을 통해 자연 과학 및 캐나다의 공학 연구 협의회, 캐나다 연구 의자 프로그램, 건강 연구의 캐나다 연구소, 그리고 연구와 혁신의 온타리오 교육부 감사드립니다.

1. 마스터 금형 제작을위한 포토 리소그래피 <ol><li> 30 초 후 30 초와 메탄올 아세톤 실리콘 웨이퍼 인치 3 목욕. 5 분 dH보다 2 0 물로 씻어. </li><li> N2 블로우 총으로 웨이퍼의 표면을 건조. 2 분 동안 140 ° C에서 뜨거운 접시에 웨이퍼를 가열한다. </li><li> 플라즈마 실리콘 웨이퍼 (1 분, 50 W)의 표면을 산화. </li><li> 3 ML SU-8 100 포토 레지스트 (; 1,750 rpm으로 40 초)을 스핀 코팅 웨이퍼의 표면. </li><li> 65 뜨거운 접시에 미리 구워 코팅 웨이퍼 ° C 10 분 후, 95 ° C 2 분 이상 온도를 진입로. 추가 1 시간이 설정을 유지합니다. </li><li> 원하는 채널 설계를 포함하는 포토 마스크를 맞 춥니 다. 550-600 UV 빛의 엠제이 / ㎠ (350-400 nm의)에 저항을 노출합니다. 포토 마스크는 AutoCAD에서 설계 및 고해상도 인쇄와 투명에 인쇄 할 수 있습니다. </li><li> T를 올렸에서 10 분 동안 1 분, 95 ° C, 65 ° C에서 뜨거운 접시에 웨이퍼 포스트 구워이전과 그 온도. </li><li> 15 분 SU-8 개발자 용액에 웨이퍼를 담가. 이소프로판올로 세척함으로써 개발 완료를 확인합니다. 백색의 침전이 나타나면, 계속 개발. 마스터 형은 그림 2A에 표시됩니다. </li></ol> 2. 마이크로 채널 제작 용 소프트 리소그래피 <ol><li> 3.5 ML PDMS 경화제와 함께 35 ML 폴리 디메틸 실록산 (PDMS) 탄성 중합체 기반을 섞는다. </li><li> 알루미늄 호일로 늘어선 페트리 접시에 제작 된 마스터 몰드 (모양이 위를 향하게)와 빈 실리콘 웨이퍼를 놓습니다. </li><li> 두 번째 접시에 마스터 금형 접시 15 ML에 20 ML PDMS의 프리폴리머를 붓는다. 부드럽게 일회용 나무 주걱으로 그들을 눌러 웨이퍼 아래에 공기 주머니를 제거합니다. </li><li> 두 접시를 커버하고 치료하는 일에 대해 따로 설정합니다. 또는 2, 빠른 치료를 위해, 진공 degasifier를 사용하여 PDMS에서 기포를 제거하고 80 뜨거운 접시에 요리를 남겨 ° C시간. </li><li> 웨이퍼에서 호일 PDMS 껍질을 제거합니다. </li><li> 채널의 양쪽 끝에서 유체 액세스 포트를 펀치 해리스 유니 코어 (2.5 mm)을 사용합니다. 비슷한 크기의 스트립에 채널과 빈 PDMS를 잘라. </li><li> 플라즈마 산화에 채널 빈 PDMS 스트립 유리 슬라이드 (75 × 25mm 2)로드 가능성 클린 룸에 위치해 있습니다. 40 W 전원에서 40 초 동안 산소 플라즈마에 노출. </li><li> 빈 스트립의 반대편에 채널 조각과 유리 슬라이드를 붙입니다. 결합을 완료하는 데 2​​ 시간 방치. </li><li> 120 핫 플레이트에 어셈블리를 배치 ° C. PDMS의 예비 중합체를 사용하여 구멍을 뚫은 저수지, 각 장기에 최소 6 in (내경 1 / 32 &quot;, 외경 3 / 32&quot;) 플라스틱 튜브를 연결합니다. 하나 또는 두 개의 튜브에 부착는 유체 플라스틱 커넥터 주사기 첨부를 허용하거나, 상업적으로 이용 가능한 피팅을 사용합니다. </li><li> PDMS는 치료하는 튜브를 확보 할 수 있습니다. EAC로 22 게이지 절연 구리 와이어의 3 &quot;길이를 삽입입구 관 및 채널과 PDMS 예비 중합체와 보안 사이의 시간 저수지. 완성 된 제품은 그림 2B에 표시됩니다. </li></ol> 3. Electrotaxis 실험 <ol><li> 모니터 (그림 1)에 연결 장착 된 카메라 현미경의 무대 (가급적 XY-이동)에 마이크로 놓습니다. </li><li> 의 마이크로 전극에 전원 공급 장치 또는 앰프의 출력 선을 연결합니다. 만 DC 신호가 필요한 경우 단순 DC 전원 공급 장치는 충분하지만, 함수 발생기에 연결된 앰프뿐만 아니라 펄스 DC 및 AC 신호의 응용 프로그램을 할 수 있습니다. </li><li> 일회용 주사기에의 마이크로 출력 튜브를 연결합니다. 잠수함 M9 생리 버퍼에있는 입구 관의 입과 부드럽게은 주사기 내부의 부압 (수동 또는 주사기 펌프를 사용)를 적용하여 채널에 액체를 열망. 입구와 출구 튜브 M9로 채워 둘 때, 주사기 F를 분리롬 관. 수압 구동 흐름을 방지하기 위해 같은 높이에 두 튜브를 수평. </li><li> 채널에 DC 전압을 적용하고 (R = V / I)이 (긴 50mm를위한 0.3 mm 폭 ~ 0.1 mm 깊이 마이크로) MΩ 0.6 주위에 그 저항을 확인합니다. </li><li> 채널의 무결성을 만족하는 경우, 채널에 희석 현탁액에서 웜을로드 위의 단계를 수행하십시오. </li><li> 주사기를 분리하고 수압 관 &#39;상대 높이를 조정하여 흐름을 조작 할 수 있습니다. 채널의 중심에있는 벌레를 놓고 같은 높이에 두 튜브가 평평하다이 방법을 사용합니다. </li><li> L3 단계 동물 용 4-12 V / cm의 L4S 4-10 V / cm, 2-4 V / 젊은 성인을위한 CM : 적절한 전압 전원 공급 장치를 설정합니다. 전기 신호를 활성화하고 현장에 순응하는 웜 사전 노출의 1 분을 허용합니다. 웜은 음극쪽으로 이동을 시작합니다. 분이 경과하면 녹음을 시작하려면 카메라를 사용합니다. </li><li> AC 및 펄스7, 8을 원하는대로 D DC 실험, 최대 반응 전계 신호의 이상과 주파수 및 듀티 사이클에서 채택 될 수 변조 할 수 있습니다. </li><li> 실험이 완료되면, 채널에서 모든 액체 (와 웜)를 제거, dH보다 2 0으로 헹굼 ° C 건조 125 핫 플레이트에 장치를 둡니다. </li><li> 수동으로 NIH ImageJ에 (사용 녹화 된 비디오에서 locomotory 데이터를 추출 <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) 또는 사용자 정의 MATLAB 기반의 웜 추적 소프트웨어입니다. </li></ol>

<ol>
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미세 electrotaxis 분석 기술은 미국과 캐나다, 미국에 특허 출원되어 있습니다.

처음으로 추앙와 동료 11,12의 dielectrophoretic 조작 작업에 GABEL 및 동료와 건물에 의해 기술 된 행동 현상을 활용, 우리의 마이크로 유체 기반 electrotaxis 분석은 운동을 같이 사용하여 웜에 신경 활동을 조사하기 쉽고 강력하고 중요한 방법을 제공합니다 출력. 운동 매개 변수의 분석은 서로 다른 유전자형 사이의 정량적 인 비교를 할 수 있습니다. 함께 마이크로 제조 및 전기 분야의 응용 프?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

선충 Caenorhabditis의 elegans의 때문에 질병 관련 유전자와 경로뿐만 아니라 재배의 용이성 자사의 보존 생물 의학 연구를위한 다양한 모델 생물이다. 여러 C. 엘레 질병 모델은 같은 도파민 (DA) 뉴런 1의 변성을 포함 파킨슨 병 (PD)와 같은 퇴행성 신경 질환 등이보고되었다. 도입 유전자와 신경 독성 화학 물질을 모두 신경 변성 및 신경 유전자와 화합물을 2,3에 대한 화면의 기초로 조사를 허용, DA의 신경 퇴화 및 웜의 필연적 인 움직임 결함을 유도하는 데 사용되었습니다. C. 같은 낮은 진핵 생물의 화면 엘레은 신경 신호에 영향을 미치는 화합물 유전자를 식별 할 수있는 효율적이고 경제적 인 방법을 제공합니다. 기존의 스크린은 일반적으로 수동으로 수행하고 육안 검사에 의해 채점, 결과적으로, 그들은 시간이 단점입니다uming과 인간의 오류가 발생하기 쉬운. 또한, 세포 수준 분석에 대부분의 초점은 운동을 무시하면서, 이는 운동 장애를위한 특히 중요한 매개 변수입니다. 우리는 C.를 제어하고 정량화 새로운 마이크로 유체 심사 시스템 (그림 1) 개발 elegans의 &#39;운동은 마이크로 내부 전기장 자극을 사용하여. 우리는 직류 (DC) 필드가 견고 음극쪽으로 운동 ( &quot;electrotaxis&quot;4) 주문형 유도 할 수있는 것으로 나타났습니다. 필드의 극성을 반전하면 웜이 빠르게뿐만 아니라 방향을 반전됩니다. 우리는 또한 도파민과 다른 감각 뉴런에 결함이 수영 응답 5을 변경 것으로 나타났습니다. 따라서 신경 세포의 신호 전달에 이상이 읽기 아웃으로 운동을 사용하여 확인할 수 있습니다. 운동 반응은 정확히 같은 속도, 몸을 구부리는 주파수와 반전 시간을 수영과 같은 매개 변수의 범위를 사용하여 정량화 할 수있다. 4에 비해 더 빠르게 이동합니다. 이러한 연구 결과는 우리가 수동적으로 연령과 표현형 (6)에 의해 벌레를 정렬하는 새로운 microfluidic 장치를 설계했다. 우리는 또한 펄스 DC에 웜의 반응을 테스트하고 전류 (AC) 전기 분야를 교류했다. C. 둘 다에있는 다양한 듀티 사이클 펄스 DC 필드 효과적으로 생성 electrotaxis 엘레과 사촌 C. 7 briggsae. 또 다른 실험에서, 1 Hz에서에서 3 kHz의 주파수 범위 대칭 AC 필드는 8 채널 내부의 벌레를 고정. 미세 환경에서 전기장의 구현은 electrotaxis 분석의 신속하고 자동 실행을 할 수 있습니다. 이 방법은 요소에 대한 높은 처리량 유전 및 화학 화면을 촉진하기 위해 약속신경 세포의 기능과 생존에 영향을 미치는.

웜 추적 소프트웨어에서 야생형 젊은 성인 선충의 electrotaxis 그 위치와 속도 출력의 대표적인 비디오 보충 비디오 1과 그림 3에 나와 있습니다. 동작 분석 소프트웨어 자체 필드 극성의 방향 및 극성 반전의 시간을 인식하지 않고 오히려이 정보는 소스 비디오에서 얻을 수 있어야합니다. 이 동영상의 오디오 또는 시각적 신호를 사용하거나 실험 조건 및 조작을 작성 할 수 있습니다. </p...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

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

에 주문형 운동을 유도하는 반자동 마이크로 전기 유체 방법<em&gt; Caenorhabditis의 엘레</em&gt;이 설명되어 있습니다. 이 방법은 미세 유체 채널 내부에 약한 전기 필드 ( &quot;electrotaxis&quot;)에 응답 벌레의 neurophysiologic 현상을 기반으로합니다. 미세 electrotaxis는 신경 세포 건강에 영향을 미치는 요인 화면으로 신속하고 민감한, 낮은 비용, 확장 성이 뛰어난 기술로 제공합니다.

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 ...

microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis

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Bioengineering

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

9.7K 조회수.  McMaster University. 에 주문형 운동을 유도하는 반자동 마이크로 전기 유체 방법 Caenorhabditis의 엘레이 설명되어 있습니다. 이 방법은 미세 유체 채널 내부에 약한 전기 필드 ( "electrotaxis")에 응답 벌레의 neurophysiologic 현상을 기반으로합니다. 미세 electrotaxis는 신경 세포 건강에 영향을 미치는 요인 화면으로 신속하고 민감한, 낮은 비용, 확장 성이 뛰어난 기술로 ?...

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

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Micropatterned 세포를 사용하여 약물의 영향 향상된 시각화 및 정량 분석

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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생체공학

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

단일 입자 Microfluidic 기반 유체 트랩

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

자유롭게 미생물 수영의 양적 운동 연구는 레이저 회절을 사용하여

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

정량은 SENP1 단백 분해 효소 반응 속도론 결정에 대해 (포스터 공명 에너지 전송) 분석 안달

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

아데 닐 레이트 사이 클라 제의 약물에 의한 과민성 : 작은 분자와 siRNA의 심사 응용 프로그램에 대한 분석 최적화 기술과 소형화

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

멀티 채널 듀얼 전기장 미세 유체 칩을 이용하여 폐암 세포의 Electrotaxis 연구

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

물방울 미세 유체에 대한 여러 가지 빛깔의 형광 검출 광학 섬유를 사용하여

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

주의 골 기능 재생성 된 대 퇴 골으로 이어지는 쥐 모델에 대 한 효율적이 고 재현 가능한 프로토콜

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

꼬마 선 충 에 경도 이미징을 위한 미세 플랫폼

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

의 주문형 정량 분석​​을위한 미세 유체 기반 Electrotaxis

의 주문형 정량 분석을위한 미세 유체 기반 Electrotaxis