Fluorescence Enhanced COVID-19 Testing

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.

Fluorescence Resonance Energy Transfer (FRET) has received proper recognition (Nobel Prize in Chemistry, 2008) in combination with GFP.  Applied to all fluorophores FRET is becoming the gold standard for fluorescence bio-assays. Drastic reductions in  the number of wash steps and higher sensitivity for ELISA are just two major advantages. However, the difficulties in creating soluble FRET-tagged antibodies that avoid water quenching, protected by aromatic groups,  their high costs and development times, have limited their widespread use. Nanopec has developed ready to use "FRET doped nano-structured ceramics".  MetaFluorex is of general use and more cost-effective than comparable FRET technologies.  It is not tied to a specfic antibody, nor is limited to FISH applications. The video below show how MetaFluorex technology works.

References

[1] https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations#coronavirus2019

[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

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