A fluorophore (1) is in close proximity to a FRET center (2) embedded in patent-pending nano-structured ceramic film MetaFluorex. The hairpin sequence (3) and a probe sequence (4) are covalently bound (5) to the surface. A quencher (6) is at the other end of the hairpin, keeping the fluorophore from fluorescing prior to hybridization with complementary ssDNA (7). In the presence of DNA polymerase the distances between quencher and fluorophore increase beyond the FRET radius (>10 nm).
The polymerase exonuclease activity eliminates the hairpin and the quencher goes into solution. Non-radiative electro-optical fluorescence energy transfer can now take place. Fluorescence decay lifetimes of the FRET centers are 1,000 longer than the fluorophore, enabling multiple fluorescence events to take place and over 1,000% fluorescence enhancement.
References
[2] The interview with Dr. Gary Procop, who heads COVID-19 testing at the Cleveland Clinic, has not been published or peer-reviewed. https://www.npr.org/sections/health-shots/2020/04/21/838794281/study-raises-questions-about-false-negatives-from-quick-covid-19-test
[3] (a) Arevalo-Rodriguez, A, et-al, False-negative results of initial RT-PCR assays for COVID-19: A systematic review, https://www.medrxiv.org/content/10.1101/2020.04.16.20066787v1 (b) Yang, Y. et-al, Evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections. MedRxiv, https://doi.org/10.1101/2020.02.11.20021493.
[4] West CP, Montori VM, Sampathkumar P, COVID-19 Testing: The Threat of False-Negative Results, Mayo Clinic Proceedings (2020), doi: https://doi.org/10.1016/j.mayocp.2020.04.004
[5] Nanopec Pending Patents: a) “Controlled Doping of Anodic Aluminum Oxide For Enhanced Fluorescence and Methods of Preparation”, USPTO 16799169, b) “Nano-porous anodic aluminum oxide membrane for healthcare and biotechnology”, USPTO 16442608, c) “Enhanced signal to noise ratios for PCR testing within a FRET doped nano-structured ceramic film”, USPTO 63010268
[6] (a) The University of Arizona Functional Genomics Core Lab, (b) Horiba Instruments USA, (c) Edinburgh Instruments, U.K.
[7] “FRET as the source of fluorescence enhancement in anodic aluminum oxide nano-structured films”, Blanco, M., Natt, S., Nanopec, April, 2020, to be submitted.
[8] Vogels, C.B.F et-al, “Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays”, March 30, 2020, medRxiv preprint doi: https://doi.org/10.1101/2020.03.30.20048108.
[9] X. Che, “Biodetection and biointerface based on Nanostructured Aluminum Oxide (NAO): From fluorescence enhancement to MS effect on single neural cells”, Graduate Theses and Dissertations, Iowa State University, 2018
[10] Grace Biolabs Oncyte microscope nitrocellulose slides: https://gracebio.com/products/microarray-tools/microarray-substrates/oncyte-film-slides/
[11] “Digital PCR (dPCR) and Real-time PCR (qPCR) Market”, Report Code: BT 3161, MarketsandMarkets Report, Published Date: May 2019