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).

Figure 1

Bioluminescence regenerative cycle (BRC) with ATP-sulfurylase and firefly luciferase detecting the released pyrophosphate from nucleic acid polymerization.

Figure 2

(a) CCD camera system which simultaneously measures the light from reference buffer and target sample, and (b) PMT imaging system in luminometer.

Figure 3

Pyrophosphate generation from DNA polymerization and RNA reverse transcription.

Figure 4

Bioluminescence regenerative cycle block diagram.

Figure 5

Simulation results based on the kinetics of the enzymes from [9] and [17] comparing light intensity of ATP assay and BRC system (Luciferin=0.1mM, APS=0.1mM).

Figure 6

Photon intensity (photon/sec) measured by CCD imaging systems in a 96-well microtiter plate format from (a) 1 to 100 fmol of 40bp oligo-loop and (b) 10amol to 1fmol of 230bp PCR product (Maltose binding protein – MBP).

Figure 7

Relative luminescence units measured by luminometer. Results are independent but identical normalized 1fmol to 1amol dilution series (incorporated dNTPs) for (a) ATP (b) 40bp oligo-loop (c) 230bp PCR product (Maltose binding protein – MBP)