Direct Detection of Viral Infections from Swab Samples by Probe‐Gated Silica Nanoparticle‐Based Lateral Flow Assay

Abstract Point‐of‐care diagnosis is crucial to control the spreading of viral infections. Here, universal‐modifiable probe‐gated silica nanoparticles (SNPs) based lateral flow assay (LFA) is developed in the interest of the rapid and early detection of viral infections. The most superior advantage of the rapid assay is its utility in detecting various sides of the virus directly from the human swab samples and its adaptability to detect various types of viruses. For this purpose, a high concentration of fluorescein and rhodamine B as a reporting material was loaded into SNPs with excellent loading capacity and measured using standard curve, 4.19 μmol ⋅ g−1 and 1.23 μmol ⋅ g−1, respectively. As a model organism, severe acute respiratory syndrome coronavirus‐2 (CoV‐2) infections were selected by targeting its nonstructural (NSP9, NSP12) and envelope (E) genes as target sites of the virus. We showed that NSP12‐gated SNPs‐based LFA significantly outperformed detection of viral infection in 15 minutes from 0.73 pg ⋅ mL−1 synthetic viral solution and with a dilution of 1 : 103 of unprocessed human samples with an increasing test line intensity compared to steady state (n=12). Compared to the RT‐qPCR method, the sensitivity, specificity, and accuracy of NSP12‐gated SNPs were calculated as 100 %, 83 %, and 92 %, respectively. Finally, this modifiable nanoparticle system is a high‐performance sensing technique that could take advantage of upcoming point‐of‐care testing markets for viral infection detections.


Calculation of F/F0 and R/R0 during measurement
The probe-gated CoV-2 strips were measured before and after the analysis of the real samples.F0 refers to before TL intensity, and F refers to after TL measurement.R0 refers to before CL intensity, and R refers to after CL measurement.
The formula is that: ; If this ratio> 1.2, the result is positive, if not it is negative.

Figure S. 2 .
Figure S.2.Different probes-gated SNPs-based LFA responses against several combinations of synthetic sample solution; PBS as a negative control, Viral solution for TL, Viral solution for TL-CL, and CL solution.A) TL, B) CL analysis.NSP12-gated SNPs-based LFA strips generated higher signals than other groups, which we selected as an optimum design for viral infections.

Figure S. 4 .
Figure S.4.The probe-gated SNPs-based LFA strip responses for negative control, viral solution for TL-CL, non-infected and infected human swab samples.For negative samples, it is seen that TL and CL intensities are almost the same when viewed with the naked eye (rows 1-3).The situation is different for positive samples.Looking at TL and CL (rows 2-4), it is seen that the particle intensities have decreased.

Figure S. 5 .
Figure S.5.Test stability of viral solution (TL+CL) after measurement.The released sensing material accumulated at the absorbent pad after one week.

Figure S. 6 .
Figure S.6.pH optimization.A) Non-infected human swab samples, B) Infected human swab samples pH optimization between pH 9-6 since pH 7 gave an optimum fluorescence intensity in both TL and CL measurements.

Figure S. 7 .
Figure S.7.Human swab samples TL and CL signal responses for A) E-gated SNPs B) NSP9gated SNPs C) NSP12-gated SNPs based LFA.The before-after measurements of CL were almost the same for the three groups since the TL intensity difference was higher before-after measurements for positive samples.The intensity difference in the E gene-gated and NSP9gated SNPs-based LFA was not adequate for test meanings.In contrast, NSP12-gated SNPsbased LFA strips have higher intensity to separate the infected and non-infected groups.

Figure S. 8 .
Figure S.8.Human swab samples evaluation using cut-off values.A) F/F0 of E gene-gated SNPs-based LFA.B) R/R0 of NSP9-gated SNPs-based LFA.C) F/F0 of NSP9-gated SNPsbased LFA.D) R/R0 of NSP9-gated SNPs based LFA human swab samples measurement results.According to the results, sensitivity is 100%, specificity is 75% for E gene-gated SNPsbased LFA while sensitivity is 50%, and specificity is 75% for NSP9-gated SNPs-based LFA.The numbers' reliability is insufficient; therefore, these sequences could not be optimum for CoV-2 detection.

Table 2 .
qRT-PCR confirmed infected and non-infected human swab samples E gene-gated SNPs-based LFA F/F0 measurement results.