Biology subjects

Wilson, B. D.

Publications and source records attributed to Wilson, B. D..

5 recordsLinked to original sources

bioRxiv2021

A Metric for Quantifying the Resolution of Molecular Assays

Many assay developers focus on limit of detection (LOD) as a primary performance metric, and LOD is indeed useful for assays designed to determine the presence or absence of an analyte. However, LOD is less useful for continuous assays designed to discriminate between concentrations of an analyte--e.g., glucose monitoring in diabetes, where clinical care is guided by particular concentration ranges and thresholds. In such scenarios, it would be valuable to quantify discriminatory resolution--i.e., whether an assay can differentiate 1 pM from 10 pM-- but no such standardized metric currently exists. Here, we propose a useful solution, termed resolution of molecular concentration (RMC). RMC offers a simple means for characterizing quantitative resolution and quickly comparing the quantitative performance of assays. By raising awareness of the limitations of current metrics for evaluating assay performance, we hope to empower the molecular diagnostics community to evaluate their methods in a more application-appropriate manner.

bioengineering↗

bioRxiv2021

Accelerated electron transfer in nanostructured electrodes improves the sensitivity of electrochemical biosensors

Electrochemical biosensors hold the exciting potential to integrate molecular detection with signal processing and wireless communication in a miniaturized, low-cost system. However, as electrochemical biosensors are miniaturized to the micron scale, their detection sensitivity degrades precipitously, thereby greatly reducing their utility in the context of molecular diagnostic applications. Studies have reported that nanostructured electrodes can greatly improve electrochemical biosensor sensitivity, but the underlying mechanism remains poorly understood, thus making it difficult to fully exploit this phenomenon to improve biosensor performance. In this work, we propose and experimentally validate a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity. We show that this mechanism can be exploited to achieve up to 24-fold increase in signal and nearly four-fold lower limit-of-detection relative conventional planar electrodes. This accelerated electron transfer mechanism should prove broadly applicable for improving the performance of electrochemical biosensors.

bioengineering↗

bioRxiv2020

Re-evaluating the conventional wisdom about binding assays

Analytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Indeed, contemporary technologies such as single-molecule and digital measurements have challenged, or even rendered obsolete, core aspects of the conventional wisdom related to binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low KD and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays optimally suited for a given detection application.

bioengineering↗

bioRxiv2019

Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. In particular, aptamers have proven useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be readily engineered into a wide range of nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution (i.e., switching time) can only be achieved at the cost of lower sensitivity or high background. Here, we describe a general design strategy that decouples the thermodynamic and kinetic behavior of aptamer switches to achieve independent control of sensitivity and temporal resolution. We used this strategy to generate an array of aptamer switches with effective dissociation constants (KD) ranging from 10 M to 40 mM and binding kinetics ranging from 170 ms to 3 s--all generated from the same parent ATP aptamer. Our strategy is broadly applicable to other aptamers, enabling the efficient development of switches with characteristics suitable for diverse range of biotechnology applications.

bioengineering↗

bioRxiv2019

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the Oxford Nanopore Technologys (ONT) MinION platform, and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings. One Sentence SummaryWe introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform.

bioengineering↗