Research Project
10196257
Abstract The rapid development of the COVID-19 pandemic reveals the shortcomings of current technologies for diagnosis. The limited availability, insufficient sensitivity and/or specificity of gene-based and antigen/antibody- based tests resulted in relatively high rates of false negative/positive test results, which further led to failure of patient quarantine and confusion among health authorities and the public. The fundamental limitations of current gene-based assays stem from their reliance upon amplification and detection of specific nucleic acid sequences within the viral genome. The current test requires labor-intensive, laboratory-based sample preparation protocols for virus lysis, extraction of genetic materials, purification of the isolated materials, thermal cycling for enzymatic amplification of viral nucleic acid sequences, and interpretation of complex results by professionals. We seek a new paradigm for rapid and direct pathogen detection, identification, and quantification in which the intact virions are directly recognized through their distinct surface epitope features, and the resultant fluorescent signal is immediately captured by a portable, smartphone-based fluorimeter. To achieve specific recognition of SARS- CoV-2 virions, we customized a designer DNA nanostructure (DDN)-based capture probe that harbors a macromolecular ?net? whose vertices precisely match the intra- and inter-spatial pattern of SARS-CoV-2 trimeric spike glycoprotein clusters, and integrates a net-shaped array of SARS-CoV-2 spike specific-targeting aptamers that are designed for maximum affinity and specificity when binding with spikes in a polyvalent and pattern- matching fashion. When exposed to a test sample, such as saliva or nasopharyngeal swab material in solution, the DNA rhombus-shaped ?virus nets? rapidly and selectively bind intact virions to trigger the release of fluorescence. We have successfully developed a smartphone-based instrument that can detect and quantify fluorescent signals in point-of-care (POC) settings. Thus, the fluorescent signal released from the virus net upon binding to SARS-CoV-2 can be readily detected by our smartphone-based fluorimeter in POC settings. We propose to combine DDN capture probes and a smartphone fluorimeter for the first time, to develop and demonstrate a rapid, room temperature, single-step, virus-specific, and ultrasensitive diagnostic assay for COVID-19 that can be performed immediately after sample collection at the point of care, and provide a result in < 5 minutes. Our aims include development of a COVID-19 assay in POC settings, and statistically robust characterization of its sensitivity, specificity, reproducibility and cost-effectiveness. Our study will conclude with a preliminary validation of the system using clinical specimens and direct comparison against a gold-standard laboratory PCR test.
