Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test.

Zackary A Zimmers,Frederick R Haselton,Alexander D Boyd,Nicholas M Adams,Hannah E Stepp

doi:10.3390/mi12101204

Zackary A Zimmers, Frederick R Haselton + Show 3 more

Open Access

https://doi.org/10.3390/mi12101204

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Journal: Micromachines	Publication Date: Sep 30, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: Vanderbilt University

Abstract

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

Highlights

Nucleic acids are among the most important biomarkers of disease, and a large variety of diagnostic nucleic acid tests (NATs) have been developed to detect their presence in diagnostic settings, including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification [1,2,3,4]
There have been several interesting demonstrations of non-enzymatic NATs that achieve increased sensitivity via exponential growth, including cascaded catalyzed hairpin assembly [11], branched or hyperbranched hybridization chain reaction [12,13], and dendritic amplification [14], but perhaps the most promising is the recent demonstration of a dumbbell DNA amplification scheme [15]
The autoPiLOT assay exhibited a limit of detection of 5 fM, and showed promising results when applied to the detection of S. mansoni DNA, demonstrating the potential for a sensitive, low-cost, point-of-care non-enzymatic NAT

Summary

Introduction

Nucleic acids are among the most important biomarkers of disease, and a large variety of diagnostic nucleic acid tests (NATs) have been developed to detect their presence in diagnostic settings, including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification [1,2,3,4] Very powerful, these tests rely on the use of enzymes, which poses several challenges for use at the point of care or in low-resource settings. Enzymes are typically the most expensive reagent in a NAT, and they require robust low-temperature storage conditions and labor-intensive sample preparation methods to achieve high sensitivity [5,6] In response to these challenges, non-enzymatic NATs have been developed which amplify a target-induced signal using only thermodynamically-driven DNA hybridization reactions.

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