Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

Luka Vanjur,Marco Buscaglia,Francesco Damin,Giuliano Zanchetta,Laura Sola,Thomas Carzaniga,Marcella Chiari,Luca Casiraghi

doi:10.3390/polym13223897

Abstract

The physical–chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal’s amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M−1·s−1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

Highlights

Capturing DNA or RNA strands with specific sequences by surface-immobilized complementary probe strands is the basis of established DNA microarray technology [1] and many innovative DNA biosensors [2]
We studied the hybridization of 23-mer single-strand DNA in solution with complementary probe strands immobilized on the Reflective Phantom Interface (RPI) biosensor surface
The target strands were added in solution at increasing concentrations, and the kinetic binding curves were measured after each addition

Summary

Introduction

Capturing DNA or RNA strands with specific sequences by surface-immobilized complementary probe strands is the basis of established DNA microarray technology [1] and many innovative DNA biosensors [2]. In label-free biosensors, the signal originates directly from the presence of the target strands through the change of some physical properties of the interface hosting the probes, such as mass [5], electrical conductivity [6], or refractive index [7]. In all these cases, the detection performance relies on the molecular recognition process between the two complementary strands. Many studies have shown that nucleic acid hybridization with surface-immobilized probe strands provides different features than the same process occurring between strands freely diffusing in solution [8,9]. Despite such an accumulation of evidence, a comprehensive understanding of the phenomena affecting surface hybridization is still missing, and quantitative comparison between different surface treatments for covalent immobilization of probes and passivation remains challenging to achieve

Methods

Results

Conclusion