Portable Nanoplasmonic Sensor for Real-Time SARS-CoV-2 RNA Detection

IntroductionFast, accurate, and in-situ environmental detection of SARS-CoV-2, the virus which causes COVID-19, is currently an unmet challenge. The gold standard detection method, polymerase chain reaction (PCR) is highly sensitive, but requires centralized lab infrastructure, specialized training, and is time consuming (> 3 hrs. time to result) [1].Other approaches more conducive to real-time and portable sensing are in development, including electrochemical [2] and plasmonic sensors [3]. However, improvements are still needed in device portability and robustness. Here, we introduce a new nanoplasmonic sensor configuration for SARS-CoV-2 RNA detection, which leverages commercially available surface plasmon readout instrumentation [4] for portable, real-time operation.MethodsThe sensor is configured as a plasmonic nanoparticle on mirror (NPoM) system, formed by a gold nanoparticle attached to a gold mirror by a peptide nucleic acid (PNA) probe complementary to the target RNA sequence (Figure 1A).The PNA capture probes are conjugated to the gold nanoparticles and then attached to the gold mirror using a two-step process [5]. Simple amine-coupling is used to attach the probes to the particle first, and then onto the activated gold mirror. Particle quantification is used to determine conjugation efficacy. The PNA probes act as a spacer layer, separating the nanoparticles from the mirror by approximately 10 nm.Upon hybridization between the PNA and RNA, the nanoparticle is pulled to within approximately 7 nm of the mirror below. The electromagnetic coupling between the nanoparticle and mirror is strongly dependent on the distance between the two and is transduced with nanometer precision by a pronounced shift in the resonance dip (Figure 1B). A change in distance of 3 nm is predicted to yield a ~40 nm shift in resonance wavelength.The NPoM system can either be interrogated in reflection mode (Figure 1B inset), or by total internal reflection with oblique illumination from the back side of the gold mirror. The latter enables integration with commercially available hardware for a portable, robust detection platform.Results and ConclusionBaseline experimental SARS-CoV-2 RNA detection has been obtained using gold nanoparticles on a glass substrate (Figure 1C). Standardized positive control material including the entire RNA genome of SARS-CoV-2 was linearly diluted to clinically relevant viral loads, and spiked into buffer for testing. The nanoparticles were functionalized with PNA capture probes complementary to a CDC-approved PCR panel sequence, and, upon RNA hybridization, a change in local refractive index was transduced by a shift in the extinction spectra peak (Figure 1C). The limit of detection of this system approached clinically relevant RNA loads (100,000 copies/mL).The NPoM geometry is expected to improve sensitivity and on-chip/portable system readout, owing to the narrower resonance width (cf. Figure 1B and 1C top). It is envisioned that this integrated platform could also be applied more broadly to the detection of DNA/RNA of other pathogens of interest including other viruses and bacterial species.References1. A. Afzal, Molecular diagnostic technologies for COVID-19: Limitations and challenges, J. Adv. Res. 26 (2020) 149–159. doi: 10.1016/j.jare.2020.08.0022. S.D. Bukkitgar, N.P. Shetti, T.M. Aminabhavi, Electrochemical investigations for COVID-19 detection—A comparison with other viral detection methods, Chem. Eng. J. (2020) 127575. doi: 10.1016/j.cej.2020.1275753. G. Qui, Z. Gai, Y. Tao, J. Schmitt, G.A. Kullak-Ublick, J. Wang, Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection, ACS Nano 14(5) (2020) 5268-5277. doi: 10.1021/acsnano.0c024394. Affinite Instruments P4SPR System, https://affiniteinstruments.com/products/p4spr/5. A. Tadimety, Y. Zhang, K.M. Kready, T.J. Palinski, G.J. Tsongalis, J.X.J. Zhang, Design of peptide nucleic acid probes on plasmonic gold nanorods for detection of circulating tumor DNA point mutations, Biosens. Bioelectron. 130 (2019) 236-244. doi: 10.1016/j.bios.2019.01.045Figure 1