Bioluminescence Regenerative Cycle (BRC)

We have developed a novel bioluminescence method for quantifying the nucleic acid molecules and adenosine triphosphate (ATP). Our bioluminescence regenerative cycle (BRC) approach is based on hybridization of specific primer-target DNA that does not require any molecular modification or labeling but rather counts the inorganic pyrophosphate (PPi) molecules released during the DNA polymerization, which then is correlated to the DNA copy number. This technique implements a bioluminescence regenerative cycle (BRC), activated by the generated PPi molecules, which are proportional in quantity to the number of target molecules. The regenerative cycle consists of (i) ATP-sulfurylase enzyme that converts PPi to ATP by consuming APS (adenosine phosphosulfate), and (ii) firefly luciferase, which in the presence of luciferin, consumes ATP as an energy source to generate photons as a detectable signal, and again PPi as a byproduct.
We have demonstrated that the photon emission rate with PPi regeneration becomes steady and is a monotonic function of the introduced PPi. Due to this positive feedback, the total number of photons generated by the BRC can potentially be orders of magnitude higher than typical chemiluminescent processes. For very low concentrations of PPi (lower than 10-8M), the total generated photons, in a fixed time interval, is proportional to the number of PPi molecules, and thus proportional to the number of nucleic acid molecules present in the solution. Employing this proposed method, an additional enzymatic cascade is added to the standard BRC reaction: (i) Adenylate Kinase (AK) in the presence of APS substrate and (ii) Pyruvate Kinase (PK) in the presence of phosphoenolypyruvate (PEP). The additional enzymes serve to create two ATP molecules from a single ATP by substrate cycling. This process would exponentially increase the concentration of ATP molecules in the reaction buffer and thereby result in signal amplification rather than target amplification.