Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

Kocheril, Philip A.; Lenz, Kiersten D.; Jacobsen, Daniel E.; Kubicek-Sutherland, Jessica Z.

doi:10.3389/fsens.2022.948466

Title: Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

Journal Article · Tue Oct 04 00:00:00 EDT 2022 · Frontiers in Sensors

DOI:https://doi.org/10.3389/fsens.2022.948466· OSTI ID:1891018

Kocheril, Philip A.; Lenz, Kiersten D.; Jacobsen, Daniel E.; Kubicek-Sutherland, Jessica Z.

Early detection of pathogens using nucleic acids in clinical samples often requires sensitivity at the single-copy level, which currently necessitates time-consuming and expensive nucleic acid amplification. Here, we describe 1) a redesigned flow cell in the shape of a trapezoid-subtracted geometric stadium, and 2) modified experimental procedures that allow for the measurement of sub-attomolar analytes in microliter quantities on a fluorescence-based waveguide biosensor. We verified our instrumental sensitivity with a 200-μL sample of a fluorescent streptavidin conjugate at 100 zM (100 zeptomolar, or 100·10 ⁻²¹ mol L ⁻¹ ) and theoretically explored the applicability of this modified sensing platform in a sandwich immunoassay format using a Langmuir adsorption model. We present assays that demonstrate specific detection of synthetic influenza A DNA (in buffer) and RNA (in saliva) oligonucleotides at the single-copy level (200 μL at 10 zM) using a fluorescent molecular beacon. Lastly, we demonstrate detection of isolated genomic influenza A RNA at a clinically relevant concentration. This work constitutes a sensitivity improvement of over twelve orders of magnitude compared to our previous nucleic acid detection work, illustrating the significant enhancements that can be gained with optimized experimental design.

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Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA); USDOE Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: 20190392 ER Strategic Investment Initiative; 89233218CNA000001; 20190392 ER

OSTI ID:: 1891018

Alternate ID(s):: OSTI ID: 1894850

Report Number(s):: LA-UR-22-24090; 948466

Journal Information:: Frontiers in Sensors, Journal Name: Frontiers in Sensors Vol. 3; ISSN 2673-5067

Publisher:: Frontiers Media SACopyright Statement

Country of Publication:: Switzerland

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
biological science
waveguide
fluorescence spectroscopy
nucleic acid
amplification-free
molecular beacon
langmuir adsorption
single molecule

Title: Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

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