NDLI: High throughput screening using enzyme assay microarrays

Content Provider	IEEE Xplore Digital Library
Author	Gosalia, D.N. Diamond, S.L.
Copyright Year	2002
Description	Author affiliation: Dept. of Bioeng., Pennsylvania Univ., Philadelphia, PA, USA (Gosalia, D.N.)
Abstract	We report a slide based microarray platform for assaying multiple enzyme activities using fluorogenic substrates. The method enables us to achieve the microfluidic requirements for rapid reaction assembly and compartmentalization. We can thus determine enzymatic activities in individually controlled reaction environments containing cofactors, inhibitors and activators. Fluorogenic substrates in glycerol were arrayed onto glass slides with reaction volumes < 5 nL and feature sizes of <150 /spl mu/m. Our method allowed rapid multiple sample deliveries onto the slide (<3 nL/spot) with no cross contamination between array positions. It enabled us to detect the activation of the fibrinolytic and coagulation proteases namely, thrombin, plasmin, factor Xa, tPa and kallikrein in human plasma. Enzyme-substrate-inhibitor assays using ten caspases were also performed. With over 400 spots/cm/sup 2/, combinatorial substrate libraries with different proteases can now be rapidly profiled. An assay to detect the dose response of a thrombin inhibitor benzamidine was performed. The inhibitor was arrayed in replicates onto selected positions on the chip. After sequential subnanoliter delivery of the reaction components, the result from the array was analyzed. The expected dose response from benzamidine was seen. A CV of 5.26% was achieved for 232 positions on the array not spiked with the inhibitor. Thus, with potentially several thousand compounds per slide, using rapid sub-nanoliter delivery of components and standard equipment, the true potential of the method is in the field of high throughput screening.
Starting Page	1628
Ending Page	1629
File Size	169801
Page Count	2
File Format	PDF
ISBN	0780376129
ISSN	1094687X
DOI	10.1109/IEMBS.2002.1106572
Language	English
Publisher	Institute of Electrical and Electronics Engineers, Inc. (IEEE)
Publisher Date	2002-10-23
Publisher Place	USA
Access Restriction	Subscribed
Rights Holder	Institute of Electrical and Electronics Engineers, Inc. (IEEE)
Subject Keyword	Throughput Biochemistry Inhibitors Microfluidics Assembly Glass Contamination Coagulation Humans Plasmas
Content Type	Text
Resource Type	Article

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

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