(K-427) Multiplex detection of SARS-CoV-2, influenza A, and B viruses with signal-amplified and programmable lateral flow assays

Introduction

Coronavirus disease 2019 (COVID-19) was declared a pandemic by the World Health Organization in March 2020 and continues to have a profound impact on our daily lives [1, 2]. Despite global efforts and vaccine development, the virus persists and evolves into new variants such as the highly contagious omicron subvariant [2-4]. Additionally, the asymptomatic or mild symptoms of COVID-19 cases, which can resemble common flu, prevent effective tracking and monitoring of COVID-19 infection [5, 6], emphasizing the urgent need for accessible and accurate self-testing methods that can distinguish between SARS-CoV-2 and influenza viruses. Lateral flow assays (LFAs) have emerged as a popular platform for self-testing due to their portability, affordability, ease of use, and rapid results [7, 8]. However, the inherent low sensitivity of commercial LFAs [8] compels invasive swabs such as nasopharyngeal or nasal swabs to obtain a concentrated sample of the target. This creates a sense of reluctance towards the assay, discourages frequent self-testing, and ultimately, defeats the primary goal of early detection to minimize the virus transmission.

In this study, we report an LFA with enhanced sensitivity for multiplex detection of SARS-CoV-2, influenza A, and B viruses in non-invasively acquired specimens (e.g., saliva). Our device automatically runs an internal autometallographic reaction to enhance the colorimetric signals through a built-in flow controller. Programmed by imprinting features on paper with different inks, our device can orchestrate the capillary flow of different reagents leaving optimal incubation times in between to optimally execute a chemical reaction.

Materials and Methods

Our assay comprised two LFA test strips, each targeting SARS-CoV-2 and influenza A & B viruses, along with a custom-programmed paper-based flow controller designed to execute an internal signal amplification reaction on the respective test lines (Figure 1a). The flow controller was designed as a multi-layered paper-based device, where adhesive polymer tapes were used for bonding the individual paper layers and laminating the entire device (Figure 1b). Each paper layer was engineered to carry specific reagents from their respective inlets to the test lines in LFA test strips at the desired time. The flow paths within the paper layers were created by imprinting distinct patterns with combinations of water-insoluble inks that differentially modified the adhesion between the wetted paper and polymer tape.

To automate the execution of the signal amplification reaction, the flow controller was physically programmed by painting with a palette of water-insoluble inks (Figure 1c). For tuning the timing of delivery for different reagents within the device, we optimized the operation of the flow controller with dye solutions instead of actual chemicals (Figure 1c). Through this optimization, we achieved the sequential delivery of the sample and signal amplification reagents to the test lines, performing the autometallographic reaction to enhance the colorimetric signals from the LFAs for higher sensitivity (Figure 1d). Our design allowed for a total assay time of 20 minutes, which is comparable to the conventional LFAs commonly used for COVID-19 testing.

Results and Discussion

To characterize our device, we processed saliva samples spiked with recombinant NP from SARS-CoV-2, influenza A, and B viruses. In testing these samples, we quantified the normalized intensity (NI) of the test line and attempted to determine the limit of detection (LoD) based on these measurements. We found that the signals with NI< 0.1 were not visible to the naked eye, but the signals with NI >0.1 consistently produced visible signals.

For SARAS-CoV-2, we found that the lowest concentration capable of producing a visible signal in the commercial LFA was 2 pg/ml versus the 0.08 pg/ml of our assay (Figure 2a). Notably, our assay produced a comparable signal level at 0.08 pg/ml to that of the commercial LFA at 2 pg/ml. This enhancement is remarkable as our assay's ability to detect SARS-CoV-2 at concentrations as low as 0.08 pg/ml can be considered comparable to molecular assays such as polymerase chain reaction; NP concentrations below 0.5 pg/ml have been associated with low virus concentrations, resulting high cycle threshold values ( >30) in N gene [10]. For influenza A virus, the commercial LFA only produced visible signals at 1 ng/ml and above in contrast with our assay’s LoD at 0.04 ng/ml (Figure 2b). As for influenza B, the lowest concentration that commercial LFA could reliably detect was 5 ng/ml versus 0.2 ng/ml from our assay (Figure 2c). In all of the experiments, there was no cross-talk observed between SARS-CoV-2, influenza A, and B test lines (Figure S1).

Collectively, these results demonstrated that our assay is capable of detecting SARS-CoV-2, influenza A, and B viruses at concentrations ~25X lower than the commercial assay due to the built-in chemical amplification of the colorimetric signal.

Conclusions

We have developed an LFA that can (1) differentiate between COVID-19 and common flu through a multiplexed test panel and (2) detect both SARS-CoV-2 and influenza viruses with a ~25X sensitivity enhancement compared to the conventional LFAs due to a built-in chemical amplification. This enhanced sensitivity of our device could help in reducing the false negative results that are commonplace when using the current COVID-19 self-tests.

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Acknowledgments

This work was supported by the start-up funds provided to A.F.S. by Georgia Institute of Technology, School of Electrical and Computer Engineering.