title: Bioluminescence regenerative cycle (BRC) system: theoretical considerations for nucleic acid quantification assays.
abstract: A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).
authors.affiliation: Center for Integrated Systems, Stanford University, Stanford, CA, USA.
authors.forename: Arjang
authors.initials: A
authors.lastname: Hassibi
authors.forename: Christopher
authors.initials: C
authors.lastname: Contag
authors.forename: Marcel O
authors.initials: MO
authors.lastname: Vlad
authors.forename: Maryam
authors.initials: M
authors.lastname: Hafezi
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Lee
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Nader
authors.initials: N
authors.lastname: Pourmand
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D011756:Diphosphates / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry*; D017785:Photons
chemical_list: D011756:Diphosphates; D009696:Nucleic Acids; D000255:Adenosine Triphosphate