Abstract
We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current. A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5′ end (1 × TINA) or at both the 5′ and 3′ ends (2 × TINA) were compared. TINA modifications were found to provide a relative decrease of koff by a factor of 6-20 at temperatures from 57.5 °C to 60 °C. The values of kon were generally in the range between 0.5-2 × 105 M−1s−1 and showed lower values for the unmodified probe than for the TINA modified probes. The observations correlated well with measured melting temperatures of the DNA hybrids.
Highlights
DNA hybridization is a key element of the majority of bioassays targeting nucleic acids
Experiments were performed at fixed temperature where the magnetic nanoparticle (MNP)-labeled ss-DNA target was first incubated on a single chip with three nominally identical sensors functionalized with unmodified probes, probes modified with Twisted Intercalating Nucleic Acid (TINA) at the 5 ́ end (1 ×TINA) and probes modified at both the 5 ́ and 3 ́ ends (2 ×TINA) to study the hybridization and washed to study the denaturation
Results obtained vs. time at the other temperatures, including those with 2 ×TINA probes, are presented in the supplementary material Fig. S1
Summary
DNA hybridization is a key element of the majority of bioassays targeting nucleic acids. The PCR reaction starts with hybridization of a short primer sequence to the template DNA whereas the microarray recognition is based on hybridization of the target DNA to allele specific probes tethered to the surface of a microarray slide. In these applications, a high degree of hybridization translates into higher sensitivity. The functional advantages offered by ortho-TINA modified capture probes were studied in a homogeneous hybridization assay, where the use of ortho-TINA modified probes presented a 27-fold sensitivity increase at high stringency conditions while retaining specificity to single point mutations[6]. It is sensitive to variation in the buffer composition and temperature as well as unspecific binding
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