microfluidic 채널 순환 폴리올레핀 (Zeonex ® 690R, Zeon 화학 주식 회사, 루이빌, KY)에 가공하고 있습니다. Zeonex는 다공성 모놀 형성에 사용되는 조명 감독 화학에 필수적인 어떤, 136 ° C의 유리 전이 온도 (T g)를 보유하고 투명 UV 있습니다. microchannels은 microfluidic 플랫폼의 부정 (반전) 기능을 가진 니코 electroformed 마스터 - 곰팡이와 마이크로 핫 엠보싱으로 가공하고 있습니다. 핫 엠보싱은 다음과 같이 이루어집니다 : <ol><li> 몰드 및 고분자 기판은 두 병렬 금속 platens 사이에 위치하고 있습니다. </li><li> platens는 Zeonex의 T g 이상 30 º C입니다 엠보싱 온도 (166 ° C)까지 가열합니다. </li><li> platens 그런 다음 약 5 분 함께 누르면되고 압력은 최대 250 PSI에서 관리하고 있습니다. </li><li> 몰드와 기판은 다음 1-2 분 냉각 허용 가열 platens에서 제거하고 수동으로 서로 구분됩니다. </li><li> 1.5 mm 직경의 웰스는 샘플 소개 및 수집 채널의 끝에 뚫고있다. </li><li> 양각 플라스틱 기판은 다음 열 동봉된 microfluidic 채널을 형성 Zeonex의 다른 조각와 결합됩니다. 양각 기판과 커버 조각은 UV - 오존 또는 씰링 효율을 향상시키고 동봉된 채널 구조 (Bhattacharyya 및 Klapperich, 2007)의 충실도를 보장하기 전에 열 본딩을 처리 플라즈마 수 있습니다. </li></ol> 플라스틱 microfluidic 채널에서 고체 상 추출 (SPE) 칼럼의 제작 고체 단계의 제작은 두 단계로 이루어집니다. 처음에는 가공 microchannels은 플라스틱 장치 SPE 열의 접착력을 향상시키기 위해 표면을 수정합니다. 표면 photopolymerization 통해 이식하는 것은 고분자 microchannels의 벽을 functionalize하는 데 사용됩니다. 다음과 같이 접목 과정은 다음과 같습니다 : <ol><li> microchannels은 수소 초록 photoinitiator됩니다 3 % benzophenone와 에틸렌 diacrylate (EDA) 및 메틸 메타 크릴 레이트 (MMA)의 1:1 혼합으로 가득합니다. EDA, MMA와 benzophenone는 시그마 - 알드리치, 세인트 루이스, MO에서 구입한 있습니다. </li><li> 칩은 자외선 노출기구 (CL - 1000 자외선 Crosslinker, UPV 주식 회사, 업랜드, CA)에서 254 nm의 UV 파장 200 MJ / cm 2의 에너지 10 분 후 UV - 조사입니다. 접목은 두번째 단계에서 모놀의 준비에 사용되는 중합 혼합물 내에 통합 얻을 표면 닿는 고분자 사슬에 H - 추출 중합 및 결과를 통해 발생합니다. </li><li> 초과 단량체는 진공 열망​​을 사용하여 메탄올 15 μL와 rinsing하여 채널에서 제거됩니다. </li></ol> photografting 과정 후, microporous 고분자 모놀은 microchannels 내에 형성되는 DNA 추출에 대한 모함 실리카는 입자. 다음과 같이 다공성 모놀리스는 원래의 장소에 준비가되어 있습니다 : <ol><li> 표면으로 바뀌었 채널 BuMA (15 % WT), EDMA (10 % WT), 1 - dodecanol (52.5 % WT), cyclohexanol (22.5 % WT), DMPAP 구성된 모놀 전구체 혼합물과 관련하여 (1 % WT로 채워집니다 단량체)와 0.7 μm의 실리카 입자 (사전 폴리머 혼합물의 전체 볼륨에 대하여 15 %)합니다. 실리카 microspheres는 Polysciences 주식 회사 (워링턴, PA)에서 구입한 및 단량체와 porogenic 용제는 시그마 - 알드리치, 세인트 루이스, MO에서 구입한 있습니다. </li><li> 이 칩은 중합을 시작 1.1 분 200 MJ / cm 2 UV crosslinker에서 UV로 조사하고, 초과 모노머는 진공 열망을 사용하여 메탄올 15 μL와 rinsing하여 채널에서 제거됩니다. </li><li> 유체 연결 (NanoPorts TM 연결 업처치 과학, 오크 하버, WA) 다음 microfluidic 채널의 도입 및 수집 우물에 첨부됩니다. </li><li> 다공성 모놀은 다음 100mL/hour의 유량에서 주사기 펌프를 사용하여 적어도 30 분 메탄올로 다시 세탁 있습니다. </li></ol> 플라스틱 microfluidic 채널에서 CNT 용해 모놀의 제작 CNT (탄소 나노튜브) 용해 모놀의 제작은 두 단계로 이루어집니다. 먼저, 가공 microchannels은 플라스틱 장치에서 SPE 열에 대한 것과 동일한 방식으로 표면에 수정 (이식할)입니다. photografting 과정 후, microporous 고분자 모놀은 다중 벽의 탄소 나노튜브를 임신되는 microchannel 내에 형성됩니다. 다음과 같이 다공성 모놀리스는 원래의 장소에 준비가되어 있습니다 : <ol><li> 다중 벽으로 둘러싸인 나노튜브는 2.27M의 농도에 cyclohexanol에 resuspended 있습니다. 이 resuspension는 50 %의 진폭에서 30 분 ultrasonicated 및 sonifier 세포 방해 (함께 50 %의 듀티 사이클이다 Sonifier S - 250초음파 브랜슨, 단베리, CT)에 의해 제조 D. sonication은 CNTs의 효율적이고 안정적​​인 0.5M 정지를 제공했습니다. CNTs는 Nanolabs (브라이튼, MA)에서 구입했다. </li><li> 표면 수정된 채널 이제 cyclohexanol의 솔루션 + CNTs를 사용 제외하고 유사한 모놀 전구체 혼합물로 채워지게된다. 모놀리스 혼합물은 BuMA (15 % WT), EDMA (10 % WT), 1 - dodecanol (52.5 % WT), 그리고 cyclohexanol + CNTs (22.5 %의 WT)로 구성되어 있습니다. 이 솔루션에 photoinitiator의 DMPAP는 (단량체에 대하여 WT 1.13 %)이 추가됩니다. 단량체, porogenic 솔벤트와 DMPAPA는 시그마 - 알드리치, 세인트 루이스, MO에서 구입한 있습니다. </li><li> 이 칩은 중합을 시작하는 데 0.9 분 120 MJ / cm 2 UV crosslinker의 UV와 함께 조사합니다. 장치는 crossliker에서 180도 뒤집힌 120 MJ / cm 2 또 다른 0.9 분 조사입니다. 초과 단량체는 진공 열망​​을 사용하여 메탄올 50 μL와 rinsing하여 채널에서 제거됩니다. </li><li> 유체 연결 (NanoPorts TM 연결 업처치 과학, 오크 하버, WA) 다음 microfluidic 채널의 도입 및 수집 우물에 첨부됩니다. </li></ol> CNT 용해 모놀리스와 함께 사용하기위한 세균 샘플 준비 박테리아 샘플에 대한 샘플 준비 채널을 막히는 것을 피하기 위해 샘플이 매우 너무 집중되지 않도록 600nm에서 광학 밀도 측정을 복용 포함됩니다. <ol><li> 미디어 박테리아와 600nm에서 OD 읽기를 가지고, 이것은 biophotometer (Eppendorf BioPhotometer, Eppendorf 과학 주식 회사, 베리, NY)으로 이루어집니다. OD의 수치가 범위 0.18-0.25 A. 이내에 가을까지 샘플을 희석 </li><li> 5 분 6,500 RPM에서 샘플을 원심과 뜨는을 가만히 따르다. </li></ol> 용해 <ol><li> GuSCN에 Resuspend 박테리아 * + 0.01 % SDS + 4% Proteinase K (0.8mg/ml). 소용돌이 간단히 (미디어에서 박테리아의 1ml 1ml GuSCN에 resuspended해야합니다.) Proteinase K는 세균의 DNA에 붙어 있으며 DNAases 및 RNAases을 억제하는 단백질을 denaturing와 원조에 대한 해결책에 추가되었습니다. proteinase K의 추가적인 장점은 또한 그람 음성 세균은 일반적으로 더 많은 단백질을 가지고 있지만, 그람 양성 세균 및 그람 음성 세균의 세포 벽 모두 단백질의 대량 (포함되기 때문에 바람직 세포 벽의 단백질을 무너 뜨와 에이즈는 것입니다 ). chaotropic 버퍼 또한 세제는 세포 벽을 방해하면서 단백질 denaturing와 proteinase K를 에이즈. Proteinase K와 guanidinium thiocyanate의 (GuSCN)은 Qiagen 주식 회사 (발렌시아, CA)에서 구입했다. SDS는 (나트륨 Dodecyl의 황산) 피어스 (록퍼드, IL)에서 구입했습니다. </li><li> 즉시 피크 튜브에 연결된 주사기에 샘플을로드합니다. 더 공기가 시스템에 없다는 것을 때문에 주사기 대기음. nanoport 및 채널에 튜브를 연결합니다. </li></ol> KDS100 주사기 펌프는 (KD 과학, 홀리스튼, MA 제조) 실험에 사용되며 사용되는 유량은 모든 단계에 450 μL / 시간가되었습니다. 450ul/hr에서 샘플 용해 채널을 흐름. <ol><li> 샘플을 수집하고 SPE를 통해 DNA 추출 (DNA 추출 프로토콜을 참조)을 참조하십시오. 참고 : 정지가 GuSCN 있기 때문에이 박테리아 샘플,로드 2 단계를 수행하지 않아도됩니다 *. </li></ol> DNA 추출 프로토콜 추출 절차는 3 단계로 구성되어 있습니다 : <ol><li> 로드합니다. </li><li> 워시 </li><li> Elute. </li></ol> KDS100 주사기 펌프는 (KD 과학, 홀리스튼, MA 제조) 실험에 사용되며 사용되는 유량은 300 μL / 모든 단계에 대한 시간이었습니다. 하중 <ol><li> 실험을 시작하기 전에 1-2 분 chaotropic 버퍼와 채널 (GuSCN * guanidinium thiocyanate) 조건. </li><li> 1시 1분 비율로 chaotropic 버퍼로 샘플을 섞는다. </li><li> 3 분위한 채널을 통해 흐름 샘플 혼합합니다. </li></ol> 빨래 <ol><li> 2 분 70 %의 에탄올로 채널을 씻으십시오. </li><li> 2 분 100 %의 에탄올로 채널을 씻으십시오. </li></ol> Elute <ol><li> 3 분위한 millipore 물에 추출한 DNA를 Elute. 정화의 15 μL, PCR - 준비 DNA를 수집합니다. </li></ol> * Qiagen 키트 (버퍼 RLT)에서 GuSCN가 사용되었습니다.

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

우리가 실험실에서, 우리는 성공적으로 직접 폴리머 / nanoparticle 합성 microscale 용해와 고체 상 추출 컬럼을 사용하여 인간의 혈액, 소변 및 대변의 microliter과 submicroliter 볼륨에서 핵산을 격리합니다. 회수 샘플 추가 정리없이 PCRable 아르 집중, 소량의 샘플입니다. 여기서 우리는 핫 엠보싱 및 열 실링을 사용하여 열가 소성 microfluidic 칩을를 조작하는 방법을 보여줍니다. 그렇다면, 우리는 합성 고체 위상 열을 형성 밀봉된 칩을 통해 현장 조명 감독 접목 표면 중합에 사용하는 방법을 보여줍니다. 우리는 접목과 세균 세포의 용해를위한 탄소 나노튜브 / 고분자 복합 열 중합 보여줍니다. 그렇다면 우리는 실리카 / 폴리머 복합 컬럼을 사용하여 칩 샘플로부터 핵산의 견고한 위상 추출 다음에 용해 과정을 보여줍니다. 첨부 프로토콜은 용해와 고체 상 추출 컬럼을 모두 만드는 방법에 대한 자세한 지침이 포함되어 있습니다.

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

여기 핫 엠보싱 및 열 실링을 사용하여 열가 소성 microfluidic 칩을를 조작하는 방법을 보여줍니다. 그럼 우리가 합성 고체 위상 열을 형성 밀봉된 칩을 통해 현장 조명 감독 접목 표면 중합에 사용하는 방법을 보여줍니다.

열가 소성 Microfluidic 채널 제작

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

fabrication-of-the-thermoplastic-microfluidic-channels

Nanyang Technological University

Research

JoVE Journal

Biology

13.3K 조회수.  Boston University. 여기 핫 엠보싱 및 열 실링을 사용하여 열가 소성 microfluidic 칩을를 조작하는 방법을 보여줍니다. 그럼 우리가 합성 고체 위상 열을 형성 밀봉된 칩을 통해 현장 조명 감독 접목 표면 중합에 사용하는 방법을 보여줍니다.

비디오: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

신경 소마와 Axons의 Compartmentalization위한 Microfluidic 장치의 제작

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

연구

생물학

PDMS Device Fabrication and Surface Modification

PDMS 장치 제작 및 표면 변경

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Microfluidic 장치의 저렴한 제작에 대한 PDMS의 비 플라즈마 본딩

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

일회용 진단을위한 Microfluidic 응용

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oocytes의 표현 이온 채널 패치 클램프 녹음

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Amperometric 전극 제작

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Myogenic 엔지니어링된 조직 구조의 제작

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic 프로브 : 운영 및 현지화된 표면 처리에 사용

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRK 채널의 변조기를 식별을위한 형광 검사 분석

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...