Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay

Abstract

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

“…Besides, some modified immunological technologies have also shown their advantages in sensitivity. For example, with the employment of the antibody sandwich protocol for target antigen capture and an ultrabright fluorescence-labeled antibody for signal readout, the obtained method could sensitively detect SARS-CoV-2 and influenza A antigens from clinical samples ( Stambaugh et al, 2021 ). Moreover, some on-chip sample preparation systems could also remarkably promote the hybridization between the capture probes and target virus RNA and enhance the capture efficiency, e.g., through using metered air bubbles to stir up the magnetic beads, which will certainly improve the following detection sensitivity ( Du et al, 2017 ).…”

Comparative Study of In Situ Techniques to Enlarge Gold Nanoparticles for Highly Sensitive Lateral Flow Immunoassay of SARS-CoV-2

“…The expected further developments in the area of point-of-care detection of SARS-CoV-2 will be related to highly sensitive and multiplex assays. Multiplexing (i.e., detection with a single test SARS-CoV-2 and other respiratory viruses) will facilitate a more informative assay and distinguish COVID-19 and infections with similar symptoms [42]. Significant efforts have been made for the multiplexing of LFIA [43], and well-developed prototypes (i.e., LFIA with multiple test-zones [44]) can be easily created.…”

Performance Comparison of Flow-Through Optofluidic Biosensor Designs

“…Optofluidic biosensors utilizing fluorescing molecules to tag pathogens in a lab-on-achip platform are in development for clinical use [1]. These biosensors operate by passing biosample targets tagged with fluorescing markers through an intersecting exciting light.…”

“…The markers fluoresce in this light, and the fluorescence can be detected and measured by a photodetector as a signal for target presence. Targets detectable by this biosensor platform include antigens, cancer biomarkers, liposomes, nucleic acids, proteins, ribosomes, and virions [1][2][3][4][5][6].…”

“…Notably, this biosensor platform has been used to detect the H1N1 virus [7], various antibiotic-resistant bacterial genes [5], and the recent SARS-CoV-2 virus [1]. Meena et al demonstrated the detection of E. coli, E. aerogenes, K. pneumonia, KPC, and VIM with accuracies of 91.6%, 90.2%, 85%, 90%, and 90%, respectively; their tests yielded an average signal-to-noise ratio (SNR) of 39.…”