Analyzing Binding Specificity in a Microparticle-Based DNA Displacement Assay Using Multiharmonic QCM-D

Employing particles as a label is a common approach for signal amplification in various surface-based biosensors. However, the size dependency of adhesive forces can increase the likelihood of nonspecific interactions between the particles and surface. Hence, using microscale particles in surface-based sensors requires both developing surface chemistries with enhanced antifouling properties and precise methods for evaluating these properties. Here we employ a quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate the binding specificity of microparticles anchored multivalently to DNA-functionalized surfaces. We design a competitive particle displacement assay by implementing toehold-mediated strand displacement as an actuator in the microparticle-substrate interface, in which specifically anchored particles dissociate from the surface upon DNA displacement. We evaluate the efficiency of particle displacement in various modified surfaces by measuring the dissipation change (ΔD) following the addition of invader single-strand DNA. We further show that, prior to the particle displacement step, the specificity of particle binding can be inferred from comparing QCM-D harmonics in the particle binding step. Our results suggest that the frequency of zero crossing in the coupled-resonator model, f_ZC, can be used to characterize the specificity of particle binding. In combination, f_ZC in the particle binding step and ΔD in the particle displacement step can be considered synergic measures for evaluating the specificity of particle binding on DNA-coated surfaces.