Characterization of Binding of Magnetic Nanoparticles to Rolling Circle Amplification Products by Turn-On Magnetic Assay.

Sobhan Sepehri,Teresa Zardán Gómez De La Torre,Björn Agnarsson,Dag Winkler,Maria Strømme,Jakob Blomgren,Justin F Schneiderman,Christer Johansson,Mats Nilsson,Alexei Kalaboukhov,Aldo Jesorka,Jan Albert

doi:10.3390/bios9030109

Abstract

The specific binding of oligonucleotide-tagged 100 nm magnetic nanoparticles (MNPs) to rolling circle products (RCPs) is investigated using our newly developed differential homogenous magnetic assay (DHMA). The DHMA measures ac magnetic susceptibility from a test and a control samples simultaneously and eliminates magnetic background signal. Therefore, the DHMA can reveal details of binding kinetics of magnetic nanoparticles at very low concentrations of RCPs. From the analysis of the imaginary part of the DHMA signal, we find that smaller MNPs in the particle ensemble bind first to the RCPs. When the RCP concentration increases, we observe the formation of agglomerates, which leads to lower number of MNPs per RCP at higher concentrations of RCPs. The results thus indicate that a full frequency range of ac susceptibility observation is necessary to detect low concentrations of target RCPs and a long amplification time is not required as it does not significantly increase the number of MNPs per RCP. The findings are critical for understanding the underlying microscopic binding process for improving the assay performance. They furthermore suggest DHMA is a powerful technique for dynamically characterizing the binding interactions between MNPs and biomolecules in fluid volumes.

Highlights

Rolling circle amplification (RCA) is a versatile tool with applications in nanobiotechnology, diagnostics and biodetection [1]
To investigate the binding kinetics, four different rolling circle amplification times of 10, 20, 40 and 60 min and two rolling circle products (RCPs) concentrations of 1.13 and 11.3 pM labelled with 250 μg/mL
The magnetic nanoparticles (MNPs) markers are incubated with RCPs for 20 min at 55 ◦ C and hybridization buffer is used for diluting the samples

Summary

Introduction

Rolling circle amplification (RCA) is a versatile tool with applications in nanobiotechnology, diagnostics and biodetection [1]. For detection of nucleic acids, the assay uses padlock probe ligation [3] for target recognition and RCA [4] to create a single strand concatemer, i.e., the rolling circle product (RCP). Numerous methods have been developed for sensing the magnetically labelled target analytes in a volume [13] and on a sensor surface and some of which are used for detection of magnetically labeled RCPs including: magneto-resistive sensors [14], superconducting quantum interference devices (SQUIDs) [9,15,16,17], induction coils [18,19], optomagnetic sensors [20,21], and ferromagnetic resonance sensors [22]. This size change appears as a shift in the susceptibility curve

Methods

Results

Conclusion