Issue 8, 2019

A nuclease protection ELISA assay for colorimetric and electrochemical detection of nucleic acids

Abstract

Early and accurate diagnosis is crucial to monitor infection outcomes and provide timely interventions. However, gold standard polymerase chain reaction assays (PCR) are labor-intensive and require expensive reagents and instrumentation. Nuclease protection has been used for decades to detect and quantify nucleic acid but has not yet been investigated as a diagnostic tool for infectious disease. In this work, we describe a nuclease protection enzyme-linked immunosorbent assay (NP-ELISA) for accurate and sensitive detection of nucleic acid. Briefly, binding of a nucleic acid target to an oligo probe protects it from digestion of un-hybridized nucleic acid by S1 nuclease. Following the workflow of an ELISA, a horseradish peroxidase (HRP)-conjugated antibody binds the probe and oxidizes its substrate to generate signal. The assay was validated with three HRP substrates for absorbance, chemiluminescence, and electrochemical readouts, demonstrating great versatility. Electrochemical detection with 3,3′,5,5′-tetramethylbenzidine (TMB) gave the highest assay sensitivity with a limit of detection of 3.72 × 103 molecules per mL. Furthermore, non-complementary targets did not generate a response, indicating a high degree of specificity. This proof of principle serves as a stepping stone towards developing miniaturized, multiplexed nuclease protection assays for point-of-care diagnosis.

Graphical abstract: A nuclease protection ELISA assay for colorimetric and electrochemical detection of nucleic acids

Supplementary files

Article information

Article type
Paper
Submitted
14 dec 2018
Accepted
23 jan 2019
First published
24 jan 2019

Anal. Methods, 2019,11, 1027-1034

Author version available

A nuclease protection ELISA assay for colorimetric and electrochemical detection of nucleic acids

J. E. Filer, R. B. Channon, C. S. Henry and B. J. Geiss, Anal. Methods, 2019, 11, 1027 DOI: 10.1039/C8AY02729C

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements