Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

Miriam Jauset-Rubio,Calum Mcneil,Markéta Svobodová,Abdulrahman O Alyoubi,Neil Keegan,Ciara K O’Sullivan,Teresa Mairal,Abdulaziz S Bashammakh,Mohammad S El-Shahawi

doi:10.1021/acs.analchem.6b03256

Abstract

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Highlights

L upin is a leguminous plant consumed extensively in the Mediterranean region due to its’ nutritional benefits, being an attractive alternative for gluten-free foodstuffs
The sandwich formats take advantage of platforms developed for nucleic acid lateral flow assays (NALFs), in which a biotinylated capture aptamer is immobilized on a streptavidin coated test line, and the target sandwiched between this aptamer and a gold nanoparticle (AuNP) labeled reporter aptamer
Independent of the concentration of β-conglutin, all biotinylated-SA-HRP aptamer was observed to bind to the immobilized complementary sequence indicating that any bound labeled aptamer was effectively displaced from β-conglutin

Summary

Introduction

L upin is a leguminous plant consumed extensively in the Mediterranean region due to its’ nutritional benefits, being an attractive alternative for gluten-free foodstuffs. The sandwich formats take advantage of platforms developed for nucleic acid lateral flow assays (NALFs), in which a biotinylated capture aptamer is immobilized on a streptavidin coated test line, and the target sandwiched between this aptamer and a gold nanoparticle (AuNP) labeled reporter aptamer. Competitive type lateral flow assays have been reported for ochratoxin[27] and the HCV core antigen,[28] where competition occurs between the AuNP labeled aptamer with an addended polyA tail, and a DNA probe complementary to the aptamer sequence immobilized on streptavidin coated test lines on the membrane. We developed a competitive based lateral flow aptamer assay, where β-conglutin immobilized on a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The two approaches are compared in terms of detection limits and assay time required

Methods

Results

Conclusion