Optical Biosensor Platforms Display Varying Sensitivity for the Direct Detection of Influenza RNA

Abstract

Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention’s (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC’s probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.

Total Internal Reflection of Two Lasers in a Single Planar Optical Waveguide

“…To verify the efficacy of this biosensor, we prepared a supported lipid bilayer with 1% biotinylated lipids on the surface of our waveguide and measured fluorescence from fluorescent conjugates of streptavidin; the remainder of our methods concern previously published procedures and can be found in the Supplementary Material. 4,19…”

“…18 Furthermore, multiplex biosensing allows for different classes of biomolecules to be simultaneously interrogated in a single sample, such as combining a fluorescent molecular beacon for the detection of a specific nucleic acid with a fluorescent antibody sandwich assay for the specific detection of an immunogenic glycolipid in a single assay. 19,20 Among current biosensing approaches (including interferometry, surface plasmon resonance, microwave sensing, electrochemical sensing, Bloch surface wave sensing, and more), fluorescence-based waveguide biosensors are popular for their relative ease of use, potential low cost, theoretically nonexistent background signal, and fast, low-noise detectors. 21,22 Fluorescence-based methods additionally are broadly applicable through fluorophore labeling, and they are generally sensitive and specific even in complex biofluid samples such as blood, urine, sputum, and cerebrospinal fluid.…”

“…[23][24][25] Our laboratory and others have previously reported waveguide-based optical biosensing assays for a variety of biologically relevant targets, including protein toxins, lipopolysaccharides, and viral nucleic acids. 4,19,20,26,27 Multiplex waveguide biosensing is often achieved by the use of a multichannel waveguide, enabling the performance of multiple assays with a single optical source. 4,28 However, due to the additional complexity and cost of manufacturing multichannel waveguides, we sought an alternative means of achieving multiplex biomarker detection that remained applicable to complex samples.…”

Portable Waveguide-Based Optical Biosensor

“…We prepared our flow cell using previously published procedures [30,[33][34][35][36]41,42]. A planar optical waveguide and a glass coverslip (3" × 1" glass microscope slide with two 1-mm holes drilled 1.5 cm from the center along the long axis of the coverslip; 48300-036, VWR International, Radnor, PA, USA) were cleaned for 5 min each in chloroform (319988, Millipore Sigma, St. Louis, MO, USA), ethanol (EX0276, Millipore Sigma), and ultrapure water (Direct-Q 3 UV-R, Millipore Sigma) by bath sonication (2510R-DTH Ultrasonic Cleaner, Branson Ultrasonics, Brookfield, CT, USA), dried under argon gas (Airgas, Radnor, PA, USA), and further cleaned by ultraviolet-ozone treatment (Model T10 × 10/OES, UVOCS Inc., Lansdale, PA, USA) for 40 min.…”

“…Our team at Los Alamos National Laboratory has previously reported a benchtop waveguide-based optical biosensor (WOB) that combines the spatial specificity of evanescent field sensing, the specificity of biotin-streptavidin binding, and the spectral sensitivity of a fluorescence detection platform [30]. This biosensor has been used to detect many different compounds of biochemical interest, including lipopolysaccharides, lipoteichoic acids, lipoarabinomannan, protein toxins, disease biomarkers, and viral nucleic acids [31][32][33][34][35][36]. In addition, we previously developed a highly portable biosensor, but that platform was designed to specifically detect cholera toxin and ricin [37,38].…”