The Development of Aptamer-Based Gold Nanoparticle Lateral Flow Test Strips for the Detection of SARS-CoV-2 S Proteins on the Surface of Cold-Chain Food Packaging

Abstract

The COVID-19 pandemic over recent years has shown a great need for the rapid, low-cost, and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to realize the visual detection of wild-type SARS-CoV-2 spike proteins (SPs) and multiple variants. Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S proteins of 0.68 nM was acquired, and the actual detection recovery was 83.3% to 108.8% for real-world samples. This suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy, and a new method for the development of alternative antibody test strips for the detection of other viral targets.

Keywords: AuNPs; COVID-19; S protein; SARS-CoV-2; aptamer; lateral flow test strips.

Figure 1

TEM test results. (a) AuNPs; (b) AuNPs; (c) AuNP–aptamer.

Figure 2

The UV absorption spectra of AuNPs and AuNP–aptamer conjugation.

Figure 3

Effect of aptamer concentration on test strip results. (a) Physical diagram of coupling solution at different aptamer concentrations: from left to right, aptamer concentrations were 0.5 µM, 1 µM, 2 µM, 3 µM, 4 µM, and 5 µM. (b) Absorbance values of test strips measured by the immunochromatographic reader at different aptamer concentrations.

Figure 4

Optimization of test strip conditions. (a) Absorbance values of test strips measured by the immunochromatographic reader at different SA concentrations. (b) Absorbance values of test strips measured by the immunochromatographic reader at different aptamer concentrations at different ratios of SA to C- and T-line aptamers. (c) Absorbance values of test strips measured by the immunochromatographic reader with different NC membrane types. (d) Absorbance values of test strips measured by the immunochromatographic reader with different binding buffers.

Figure 4

Optimization of test strip conditions. (a) Absorbance values of test strips measured by the immunochromatographic reader at different SA concentrations. (b) Absorbance values of test strips measured by the immunochromatographic reader at different aptamer concentrations at different ratios of SA to C- and T-line aptamers. (c) Absorbance values of test strips measured by the immunochromatographic reader with different NC membrane types. (d) Absorbance values of test strips measured by the immunochromatographic reader with different binding buffers.

Figure 5

Molecular docking and aptamer-complementary chain element optimization. (a) DA20 docking site and its aptamer sequence. (b) DA66 docking site and its aptamer sequence. (c) Absorbance values of test strips measured by the immunochromatographic reader with different aptamer-complementary sequences with blank conditions. (d) Absorbance values of test strips measured by immunochromatography with different aptamer-complementary sequences with SP 500ppb conditions. (e) MOE molecular docking result.

Figure 6

Optimization of detection conditions. (a) T/C and inhibition ratio with different amounts of coupling solution added. (b) Test strip experiments with different coupling solution spiking amounts under blank conditions, from left to right: 1 µL, 2 µL, 3 µL, 4 µL, and 5 µL. (c) Test strip experiments with different coupling solution spiking amounts under 500ppb SP conditions, from left to right: 1 µL, 2 µL, 3 µL, 4 µL, and 5 µL. (d) T/C and inhibition rate change over time.

Figure 7

Quantitative detection of SP. (a) Standard curve. (b) Calibration curve and color development of test strips established at 15 min detection time, from left to right: 0 ng/mL, 100 ng/mL, 300 ng/mL, 600 ng/mL, 800 ng/mL, and 1000 ng/mL.

Figure 8

Results of a selectivity study of nucleic acid aptamer test strips for SP detection.

Figure 9

Stability evaluation result.

Figure 10

Schematic representation of SP detection via Apt-LFA. (a) Structure of LFA strip. (b) Negative result of Apt-LFA (without SP). (c) Positive result of Apt-LFA (with SP).