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Nucleic Acids Research Advance Access originally published online on September 12, 2008
Nucleic Acids Research 2008 36(19):e129; doi:10.1093/nar/gkn564
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Nucleic Acids Research, 2008, Vol. 36, No. 19 e129
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics

Brent C. Satterfield1,2, Michael R. Caplan1,* and Jay A. A. West3

1Harrington Department of Bioengineering, Arizona State University Tempe, AZ, 2Cooperative Diagnostics, Greenwood, SC and 3Arcxis Biotechnologies, Pleasanton, CA, USA

*To whom correspondence should be addressed. Tel: +1 480 965 5144; Fax: +1 480 727 7624; Email: michael.caplan{at}asu.edu

Received December 28, 2007. Revised August 18, 2008. Accepted August 19, 2008.

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 µl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their ‘on–off’ signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1 x 104 improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.


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