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.
Detection of molecular biomarkers with high specificity and sensitivity from biological samples requires both sophisticated sample preparation and subsequent analysis. These tasks are often carried out on separate platforms which increases required sample volumes and the risk of errors, sample loss, and contamination. Here, we present an optofluidic platform which combines an optical detection section with single nucleic acid strand sensitivity, and a sample processing unit capable of on-chip, specific extraction and labeling of nucleic acid and protein targets in complex biological matrices. First, on-chip labeling and detection of individual lambda DNA molecule down to concentrations of 8fM is demonstrated. Subsequently, we demonstrate the simultaneous capture, fluorescence tagging and detection of both Zika specific nucleic acid and NS-1 protein targets in both buffer and human serum. We show that the dual DNA and protein assay allows for successful differentiation and diagnosis of Zika against cross-reacting species like dengue.
The recent massive Zika virus (ZIKV) outbreak illustrates the need for rapid and specific diagnostic techniques. Detecting ZIKV in biological samples poses unique problems: antibody detection of ZIKV is insufficient due to cross-reactivity of Zika antibodies with other flaviviruses, and nucleic acid and protein biomarkers for ZIKV are detectable at different stages of infection. Here, we describe a new optofluidic approach for the parallel detection of different molecular biomarkers using multimode interference (MMI) waveguides. We report differentiated, multiplex detection of both ZIKV biomarker types using multi-spot excitation at two visible wavelengths with over 98% fidelity by combining several analysis techniques.
Nucleic acids and proteins are the two most important target types used in molecular diagnostics. In many instances, simultaneous sensitive and accurate detection of both biomarkers from the same sample would be desirable, but standard detection methods are highly optimized for one type and not cross-compatible. Here, we report the simultaneous multiplexed detection of SARS-CoV-2 RNAs and antigens with single molecule sensitivity. Both analytes are isolated and labeled using a single bead-based solid-phase extraction protocol, followed by fluorescence detection on a multi-channel optofluidic waveguide chip. Direct amplification-free detection of both biomarkers from nasopharyngeal swab samples is demonstrated with single molecule detection sensitivity, opening the door for ultrasensitive dual-target analysis in infectious disease diagnosis, oncology, and other applications.
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