Engineering a Rigid Nucleic Acid Structure to Improve the Limit of Detection for Genetic Assays.

Detecting nucleic acids at ultralow concentrations is critical for research and clinical applications. Particle-based assays are commonly used to detect nucleic acids. However, DNA hybridization on particle surfaces is inefficient due to the instability of tethered sequences, which negatively influences the assay's detection sensitivity. Here, we report a method to stabilize sequences on particle surfaces using a double-stranded linker at the 5' end of the tethered sequence. We termed this method Rigid Double Stranded Genomic Linkers for Improved DNA Analysis (RIGID-DNA). Our method led to a 3- and 100-fold improvement of the assays' clinical and analytical sensitivity, respectively. Our approach can enhance the hybridization efficiency of particle-based assays without altering existing assay workflows. This approach can be adapted to other platforms and surfaces to enhance the detection sensitivity.

Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels.

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Surveilling and Tracking COVID-19 Patients Using a Portable Quantum Dot Smartphone Device.

The ability to rapidly diagnose, track, and disseminate information for SARS-CoV-2 is critical to minimize its spread. Here, we engineered a portable smartphone-based quantum barcode serological assay device for real-time surveillance of patients infected with SARS-CoV-2. Our device achieved a clinical sensitivity of 90% and specificity of 100% for SARS-CoV-2, as compared to 34% and 100%, respectively, for lateral flow assays in a head-to-head comparison. The lateral flow assay misdiagnosed ∼2 out of 3 SARS-CoV-2 positive patients. Our quantum dot barcode device has ∼3 times greater clinical sensitivity because it is ∼140 times more analytically sensitive than lateral flow assays. Our device can diagnose SARS-CoV-2 at different sampling dates and infectious severity. We developed a databasing app to provide instantaneous results to inform patients, physicians, and public health agencies. This assay and device enable real-time surveillance of SARS-CoV-2 seroprevalence and potential immunity.

Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

Mobile phone technology is a perfect companion for point-of-care diagnostics as they come equipped with advanced processors, high resolution cameras, and network connectivity. Despite several academic pursuits, only a few mobile phone diagnostics have been tested in the field, commercialized or achieved regulatory approval. This review will address the challenges associated with developing mobile diagnostics and suggest strategies to overcome them. We aim to provide a resource for researchers to accelerate the development of new diagnostics. Our Account includes an overview of published mobile phone diagnostics and highlights lessons learned from their approach to diagnostic development. Also, we have included recommendations from regulatory and public health agencies, such as the U.S. Food and Drug Administration and World Health Organization, to further guide researchers. We believe that the development of mobile phone point-of-care diagnostics takes place in four distinct steps: (1) Needs and Value Assessment, (2) Technology Development, (3) Preclinical Verification, and (4) Clinical Validation and Field Trials. During each step, we outline developmental strategies to help researchers avoid potential challenges. (1) Researchers commonly develop devices to maximize technical parameters such as sensitivity and time which do not necessarily translate to increased clinical impact. Researchers must focus on assessing specific diagnostic needs and the value which a potential device would offer. (2) Often, researchers claim they have developed devices for feasible implementation at the point-of-care, yet they rely on laboratory resources. Researchers must develop equipment-free devices which are agnostic to any mobile phone. (3) Another challenge researchers face is decreased performance during field evaluations relative to initial laboratory verification. Researchers must ensure that they simulate the field conditions during laboratory verification to achieve successful translation. (4) Finally, proper field testing of devices must be performed in conditions which match that of the final intended use. The future of mobile phone point-of-care diagnostic devices is bright and has the potential to radically change how patients are diagnosed. Before we reach this point, researchers must take a step backward and focus on the first-principles of basic research. The widespread adoption and rapid scaling of these devices can only be achieved once the fundamentals have been considered. The insights and strategies provided here will help researchers avoid pitfalls, streamline development and make better decisions during the development of new diagnostics. Further, we believe this Account can help push the field of mobile diagnostics toward increased productivity, leading to more approved devices and ultimately helping curb the burden of disease worldwide.

Diagnosing COVID-19: The Disease and Tools for Detection.

COVID-19 has spread globally since its discovery in Hubei province, China in December 2019. A combination of computed tomography imaging, whole genome sequencing, and electron microscopy were initially used to screen and identify SARS-CoV-2, the viral etiology of COVID-19. The aim of this review article is to inform the audience of diagnostic and surveillance technologies for SARS-CoV-2 and their performance characteristics. We describe point-of-care diagnostics that are on the horizon and encourage academics to advance their technologies beyond conception. Developing plug-and-play diagnostics to manage the SARS-CoV-2 outbreak would be useful in preventing future epidemics.