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.

Keywords: RNA; biosensor; detection; diagnostics; flow cytometer; influenza; waveguide.

Figure 1

Direct detection of influenza RNA without amplification using a thermal cycler. (a) The lowest concentration detected by 100 nM of the IAV probe with exact match RNA at room temperature was 8 nM (p  <  0.05). Dotted line indicates background signal. (b) Mismatch tolerance of MB probes was determined using synthetic RNA with exact match sequence and a single mismatch (Table 1). All targets were detected above probe background noise (p < 0.0001 for match A, mismatch A, match B, mismatch B). (c) Hybridization kinetics at varying IAV MB probe concentrations (0–200 nM) was determined using 50 nM synthetic RNA exact match target with even the lowest 25 nM probe concentration sufficient to detect 50 nM RNA over background noise (p < 0.0001). Values plotted are mean ± standard deviation. Statistical significance was determined by Student’s t-test for (a–b) and by two-way ANOVA with Dunnett’s multiple comparisons test to determine individual variances for (c). RFU, relative fluorescence units.

Figure 2

Direct detection of RNA using a waveguide-based optical biosensor. (a) Schematic of functionalized waveguide surface using a phospholipid bilayer and streptavidin to capture a biotinylated molecular beacon probe where fluorescence is quenched until hybridization with influenza RNA occurs (not drawn to scale). The fluorophore is excited by the evanescent field emitted from total internal reflection of light coupled in the waveguide limiting detection in this system to surface-bound molecules. Q, quencher; F, fluorophore. Figure created with Biorender.com. (b) Measurement of 50 nM (light pink line) and 1 µM (teal line) IAV RNA from the detection of AF532-labeled FEVER MB probe as compared to the quenched probe alone in the absence of RNA target (black line) in 1X PBS. (c) 100 nM (dark pink line) and 1 µM (teal line) RNA was also detected directly in human saliva. RFU, relative fluorescent units.

Figure 3

Flow cytometry bead-based detection of influenza RNA. (a) Schematic of flow cytometry bead-based assay using streptavidin-coated polystyrene particles coated with biotinylated MB probes. In the absence of target RNA fluorescence is quenched, but when target is present the fluorophore is separated from the quencher and fluorescence resulting from excitation is measured in the FITC channel of the flow cytometer. Figure created with Biorender.com. (b) The flow cytometer was able to detect 1 nM IAV RNA with 200 nM MB. The fluorescent signal from MB-RNA hybridization was quantified by FITC counts derived from the mean of 20,000 events.

Figure 4

Viral RNA detection using exonuclease selection. (a) Schematic of the exonuclease RNA detection strategy. Probe bound with viral RNA is not digested by ExoI. After digestion, remaining probes are hybridized with complementary reporter probes containing a single fluorophore. Figure created with Biorender.com. (b) 500 pM RNA was detected using 10 nM probes. (c) 3 nM RNA was detected directly in human saliva. Values plotted are the mean ± standard deviation. Statistical significance of FITC counts with RNA present compared to no RNA control was determined by Student’s t-test (** p < 0.01, *** p < 0.001).