Abstract
Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and therefore are not suitable for point-of-care testing. Here, we present a 3D microfluidic paper-based electrochemical NAAT. These devices use off-the-shelf gold plasma-coated threads to integrate electroanalytical readouts using ex situ self-assembled monolayer formation on the threads prior to assembling into the paper device. They further include a sandwich hybridization assay with sample incubation, rinsing, and detection steps all integrated using movable stacks of filter papers to allow time-sequenced reactions. The devices use glass fiber substrates for storing recombinase polymerase amplification reagents and conducting the isothermal amplification. We used the paper-based device for the detection of the toxic microalgae Ostreopsis cf. ovata. The NAAT, completed in 95 min, attained a limit of detection of 0.06 pM target synthetic DNA and was able to detect 1 ng/μL O. cf. ovata genomic DNA with negligible cross-reactivity from a closely related microalgae species. We think that the integration of thread electrodes within paper-based devices paves the way for digital one-time use NAATs and numerous other advanced electroanalytical paper- or textile-based devices.
Highlights
Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and are not suitable for point-of-care testing
A tailed primer consists of a single-stranded DNA sequence (“tail”) that is added to the primer using a 3-C alkyl chain spacer, which prevents elongation of the tail during amplification
Tailed primers and sandwich hybridization assay (SHA) have been reported for recombinase polymerase amplification (RPA),[32] and we chose this strategy for our device because of the following: (i) It avoids post-amplification steps prior to detection to generate single-stranded sequences able to hybridize to the surface-immobilized probe; and (ii) It is highly specific as it involves two hybridization events through capture and reporter probes
Summary
Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and are not suitable for point-of-care testing.
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