Diffractometric Detection of Proteins Using Microbead-based Rolling Circle Amplification

Overview

Journal Anal Chem

Specialty Chemistry

Date 2009 Dec 2

PMID 19947589

Citations 11

Authors

Joonhyung Lee

Kutay Icoz

Ana Roberts

Andrew D Ellington

Cagri A Savran

Affiliations

Soon will be listed here.

Abstract

We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.

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