High-Efficiency Separation in Microfluidic Devices for High-Throughput Screening of Kinases

Author(s):  
Bahram Fathollahi ◽  
Matthew B. Kerby ◽  
Spencer Wu ◽  
Ring-Ling Chien
2019 ◽  
Vol 24 (6) ◽  
pp. 641-652
Author(s):  
Keisuke Kawasaki ◽  
Yoshiaki Suzuki ◽  
Hisao Yamamura ◽  
Yuji Imaizumi

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based “test cell” expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.


2015 ◽  
Vol 20 (8) ◽  
pp. 932-942 ◽  
Author(s):  
Christin Luft ◽  
Robin Ketteler

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.


2013 ◽  
Vol 19 (4) ◽  
pp. 483-496 ◽  
Author(s):  
Oliver J. Dressler ◽  
Richard M. Maceiczyk ◽  
Soo-Ik Chang ◽  
Andrew J. deMello

Over the past two decades, the application of microengineered systems in the chemical and biological sciences has transformed the way in which high-throughput experimentation is performed. The ability to fabricate complex microfluidic architectures has allowed scientists to create new experimental formats for processing ultra-small analytical volumes in short periods and with high efficiency. The development of such microfluidic systems has been driven by a range of fundamental features that accompany miniaturization. These include the ability to handle small sample volumes, ultra-low fabrication costs, reduced analysis times, enhanced operational flexibility, facile automation, and the ability to integrate functional components within complex analytical schemes. Herein we discuss the impact of microfluidics in the area of high-throughput screening and drug discovery and highlight some of the most pertinent studies in the recent literature.


2006 ◽  
Vol 950 ◽  
Author(s):  
Zhengshan Zhao ◽  
Gerardo A. Diaz-Quijada ◽  
Régis Peytavi ◽  
Éric LeBlanc ◽  
Johanne Frenette ◽  
...  

ABSTRACTMicroarrays have become one of the most convenient tools for high throughput screening and have catalyzed major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is a high interest in employing polymeric materials as a low cost alternative suitable for mass production. We present proof-of-concept DNA arrays on a plastic platform for the detection of four important respiratory pathogens: Influenza A virus, respiratory syncytial virus, human enterovirus, and human metapneumovirus.This was accomplished by amplifying the genetic material from the viruses and simultaneously labeling the amplicons with a fluorescent dye (Cy3) via a highly sensitive multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR). The resultant RT-PCR product was hybridized, without further purification, with an array of specific oligonucleotide probes (20 mers) that had been covalently bound to a plastic substrate. Results indicate a high signal to background ratio that is comparable to commercially available microarray glass slides. In addition, 5 minute hybridization on this plastic substrate has been demonstrated using a centrifugal microfluidic platform, paving the way to a rapid medical diagnostic device for point-of-care use that is based on a low-cost portable Micro-Total-Analysis-System (μ-TAS).


2021 ◽  
Author(s):  
Sascha Dehler ◽  
Lukas PM Kremer ◽  
Santiago Cerrizuela ◽  
Thomas Stiehl ◽  
Jonas Weinmann ◽  
...  

The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, AAV9_A2 and AAV1_P5, both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled labeling of 30-60% of the NSC compartment, which was validated by FACS analyses and single cell RNA sequencing. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high regional tropism towards the v-SVZ with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.


Author(s):  
Matthew Rienzo ◽  
Shaina J. Jackson ◽  
Lawrence K. Chao ◽  
Timothy Leaf ◽  
Thomas J. Schmidt ◽  
...  

2014 ◽  
Vol 86 (19) ◽  
pp. 9570-9575 ◽  
Author(s):  
Ryo Oyobiki ◽  
Taisuke Kato ◽  
Michinobu Katayama ◽  
Ai Sugitani ◽  
Takeshi Watanabe ◽  
...  

Lab on a Chip ◽  
2010 ◽  
Vol 10 (3) ◽  
pp. 341-348 ◽  
Author(s):  
Sarvesh Upadhyaya ◽  
P. Ravi Selvaganapathy

2005 ◽  
Vol 897 ◽  
Author(s):  
Gerardo Antonio Diaz-Quijada ◽  
Regis Peytavi ◽  
André Nantel ◽  
Emmanuel Roy ◽  
Michel G. Bergeron ◽  
...  

AbstractMicroarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is a high interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) and Zeonor™ 1060R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotide were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.


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