fM–aM Detection of the SARS-CoV-2 Antigen by Advanced Lateral Flow Immunoassay Based on Gold Nanospheres

Abstract

The SARS-CoV-2 global pandemic created an unprecedented need for rapid, sensitive, and inexpensive point-of-care (POC) diagnostic tests to treat and control the disease. Many POC SARS-CoV-2 lateral flow immunoassays (LFAs) have been developed and/or commercialized, but with only limited sensitivity (μM–fM). We created an advanced LFA based on gold nanospheres (GNSs) with comprehensive assay redesign for enhanced specific binding and thermal contrast amplification (TCA) on GNSs for signal amplification, which enabled fM–aM detection sensitivity for SARS-CoV-2 spike receptor-binding domain (RBD) proteins within 30 min. The advanced LFA can visually detect RBD proteins down to 3.6 and 28.6 aM in buffer and human nasopharyngeal wash, respectively. This is the first reported LFA achieving sensitivity comparable to that of the PCR (aM–zM) by visual reading, which was much more sensitive than traditional LFAs. We also developed a fast (<1 min) TCA reading algorithm, with results showing that this TCA could distinguish 26–32% visual false negatives for clinical commercial LFAs. When our advanced LFAs were applied with this TCA, the sensitivities were further improved by eightfold to 0.45 aM (in buffer) and 3.6 aM (in the human nasopharyngeal wash) with a semiquantitative readout. Our proposed advanced LFA with a TCA diagnostic platform can help control the current SARS-CoV-2 pandemic. Furthermore, the simplicity and speed with which this assay was assembled may also facilitate preparedness for future pandemics.

Fast and ultrafast thermal contrast amplification of gold nanoparticle-based immunoassays

“…As summarized in Fig. 1 e, faster reading can be achieved with the pulsed laser ultrafast TCA (seconds) than CW laser TCA either with discrete or continuous reading algorithms (1–15 min) as detailed in previous work 9 , 13 , 14 . Additionally, different temperature measurement products (IR camera vs. sensor) were used to fit with the lasers as summarized in Fig.…”

“…Laser wavelength: ; absorption cross section: . Laser irradiation Gold nanoparticles (W) increase (fold) References Types (nm) Intensity, ( ) Power/energy Geometry Diameter (nm) ( ) CW 532 3.2 25 mW Sphere 30 1.45E3 4.6E−9 1 9 , 13 , 15 60 1.11E4 3.6E−8 8 15 100 2.08E4 6.7E−8 14 12 , 15 120 2.35E4 7.5E−8 16 14 CW 1064 12.7 100 mW Silica-cored gold shell Core: 198 Shell: 242 1.01E4 1.3E−7 28 This work …”

“…A 1064 nm CW laser (Spectrum Stabilized Laser Module, Model #: I1064SR0300B, Innovative Photonic Solutions Inc.) was used, and the power reaching the substrates was 100 mW. Details of the two reading algorithms and other components were described elsewhere 13 , 14 . Briefly, in discrete reading, the scanning was made point-by-point at 0.125 mm distance.…”

“…To cut down the time of reading, various improvements in signal amplification methods have been investigated 5 – 8 . In the case of TCA, a fast reader was developed with a continuous-reading algorithm and a continuous-wave (CW) laser; it enabled < 1 min reading per test while maintaining a sensitivity improvement similar to that of a previous discrete but-slow reading (~ 15 min) format 14 . To achieve the speed limit, it is also interesting to explore the potential of ultrafast TCA reading using a ms pulsed laser which would enable a complete readout within seconds.…”

Current Advances in Paper-Based Biosensor Technologies for Rapid COVID-19 Diagnosis

“…With TCA, the gold nanoparticles captured in the test line were excited by laser irradiation and exhibited a substantial photothermal effect, enabling the detection of subvisual positives. They successfully detected SARS-CoV-2 receptor-binding domain (RBD) antigen as low as 28.6 aM in a human nasopharyngeal swab [ 93 ]. Selenium nanoparticles exhibit favorable biocompatibility and are readily conjugated with biological molecules without losing their activity [ 94 ].…”