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Proceedings Paper

Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays
Author(s): Arjang Hassibi; Thomas H. Lee; Ronald W. Davis; Nader Pourmand
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Paper Abstract

A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

Paper Details

Date Published: 18 July 2003
PDF: 11 pages
Proc. SPIE 4966, Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis, (18 July 2003); doi: 10.1117/12.478095
Show Author Affiliations
Arjang Hassibi, Stanford Univ. (United States)
Xagros Technologies (United States)
Thomas H. Lee, Stanford Univ. (United States)
Ronald W. Davis, Stanford Univ. (United States)
Nader Pourmand, Stanford Univ. (United States)
Xagros Technologies (United States)


Published in SPIE Proceedings Vol. 4966:
Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis
Dan V. Nicolau; Ramesh Raghavachari, Editor(s)

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