Rapid, sensitive and multiplexed detection of SARS-CoV-2 viral nucleic acids enabled by phase-based surface plasmon resonance of metallic gratings

A rapid, sensitive and easy-to-implement approach is proposed for the detection of pathogenic nucleic acids based on phase-based plasmonic spectroscopy of metallic gratings. The plasmonic sensors were fabricated using interference lithography and functionalized with single-stranded DNA probes to specific target SARS-CoV-2. The biosensor achieved the detection of 40 fM viral nucleic acids within 5 min; furthermore, a detection capability of 1 aM (0.6 copies/µL) was acquired when combining with the recombinase polymerase amplification. Additionally, the multiplexed sensing system was demonstrated to simultaneously detect three genomic sequences on a single sensor chip, thereby enhancing diagnostic accuracy and enabling high-throughput detection.


RAPID, SENSITIVE AND MULTIPLEXED DETECTION OF SARS-COV-2 VIRAL NUCLEIC ACIDS ENABLED BY PHASE-BASED SURFACE PLASMON RESONANCE OF METALLIC GRATINGS: SUPPLEMENTAL DOCUMENT
Materials and Reagents.Probes, complementary targets, non-complementary targets were purchased from Guangzhou IGE Biotechnology Co., Ltd (Guangzhou, China).6-mercapto-1hexanol (MCH) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China).PBS solution was purchased from Shanghai Titan Scientific Co., Ltd (Shanghai, China).DNase/RNase-Free, Sterile Water was purchased from Beyotime biotechnology Co. Ltd (Shanghai, China).The Polydimethylsiloxane (PDMS, 184 Silicone Elastomer Base) was purchased from Dow Inc (USA).None of the chemicals were further purified, and deionized water was used throughout the entire.
Equipment.To experimentally investigate the SPP sensor for nucleic acid detection, an angular spectral detection equipment is established in the laboratory, as illustrated in Figure S1.The broadband light source is coupled into the enclosed light path through a fiber and subsequently reflected by a SPP chip.Finally, the light signal containing detection information is coupled into spectroscopy via the enclosed light path.The angle of incident light is precisely controlled by four stepper motors, enabling an angle range from 10 to 60 degree.Simulation.Drawing upon the structural parameters ascertained through SEM in Figure 2(a), the one-dimensional grating structure is numerically investigated using CST Microwave Studio software to elucidate the performance of the SPP sensor.The electric field distributions of grating at absorption state is delineated in Figure S2, demonstrating that the resonance of proposed grating is a consequence of the SPPs.
Figure S2 In simulation, the electric field distributions of grating.
Phase measurements.Based on the phase measurements method, the SPP sensor information can express as a narrow peak on the background of relatively weak spectrum, especially when applied the phase shift rate in spectrum.The schematic diagram of SPP grating phase detection is depicted in Figure S3.Firstly, the input polarizer is oriented at 45° and the output polarizer is oriented at -45° with respect to the axis associated with P-polarization of the incident optical wave.The quarter-wave plate is oriented at 0°, 45° and 90° separately, and the spectral transmission is measured with spectroscope as R0, R 45 and R 90 .The phase information φ can be obtained by the formula as follow: ( ) ( ) ( ) Finally, the phase shift rate Δφ is obtained by differential calculus on φ.

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Figure S1 (a) The equipment for SPP based nucleic acid detection.(b) Picture of the microfluid-integrated biosensor chip.(c) Picture of the three-channel microfluid-integrated biosensor.
Figure S3 Schematic diagram of SPP grating phase detectionIn phase measurements, the reflective spectra obtained at the quarter-wave plate set at 0°, 45° and 90° separately are shown in FigureS4(a).Additionally, the phase calculated using the method outlined in FigureS3is displayed in FigureS4(b) and reveals a significant discontinuity.The detailed spectra of the differential phase, presented in FigureS4(c), exhibit a narrow FWHM of about 1.2nm.The variations in phase jump with the incident angle are presented in FigureS4(d), demonstrating an exact alignment with the positions of reflectance dips.