Tracking the Brownian motion of DNA-functionalized magnetic nanoparticles for conformation analysis beyond the optical resolution limit

DNA-functionalized colloidal particles in the nano-and micrometer size range are versatile tools for engineering self-assembled structures and play a pivotal role in detecting various biological and non-biological substances. [1,2,3] Pioneering studies successfully demonstrated the controlled bottom-up synthesis of larger structures from DNA oligonucleotide-functionalized gold nanoparticles and nanocrystals. [4,5] Detection schemes are reported for specific DNA-sequences [6], metal ions [7], enzymes [8], and other analyte species [9] using nanoparticles or quantum dots hybridized with short DNA strands as probes. A steady interest can be noted in integrating magnetic nanoand microparticles for separation, fluid mixing, drug delivery, and hyperthermia by magnetic field-induced particle motion. [10,11,12,13,14,15,16] This technique also allows novel methods for analyte binding detection, like surface interaction-mediated traveling wave magnetophoresis [17], magnetic particle spectroscopy [18], and magnetic particle aggregation assays [19], aiding the development of point-of-care medical diagnostics. For DNA-functionalized magnetic particles, the concentration of attached DNA, also known as the grafting density, is of major importance for their physical properties. For high grafting densities, DNA strands typically form a rigid polymer brush around the particle, enabling binding of a specific target in every direction. [2] The DNA brush at high grafting densities significantly changes the particle's hydrodynamic radius and, therefore, impacts its dynamic response to an applied magnetic field when suspended in a liquid. [20] At low grafting densities, the conformation of particle-bound DNA strands is coiled, where single strands are wrapping around the particle. This effectively increases the probability of non-specific binding of DNA on the particle's surface as compared to the brushed state. [2,20] A mixture of coiled and brushed single-stranded DNA (ssDNA) attached to magnetic nanoparticles was found to yield the highest sensitivity in magnetic particle spectroscopy-based biosensing of target DNA sequences. [20] Experimental validation of the conformation of DNA bound to magnetic nano-or microparticle surfaces is, thus, essential for understanding their binding affinity toward specific targets. Atomic force microscopy [21] is a possible imaging technique, providing high spatial resolution, but typically yields a low throughput and is hard to apply in situ. The low throughput issue might be solved by using either magnetic particle spectroscopy [18,22,20] or dynamic light scattering (DLS). [23] The former, however, is only applicable for magnetic DNA-functionalized particles and solely probes the dynamic response of the particles to an alternating current (AC) magnetic field, which does not necessarily reflect the quasi static, undisturbed state of DNA conformation at the particle's surface. DLS is indeed useful to investigate the hydrodynamic radius of particles, which in turn gives information about the structure of particle-bound DNA, but is prone to overestimating the weight of larger particles or aggregates in the obtained size distribution, is highly dependent on the carrier liquid properties, and is in general a low-resolution technique. [23] Other methods to determine the hydrodynamic radius of DNA-functionalized particles include the capture inside an optical trap with low stiffness and subsequent analysis of the particle's Brownian motion. [24] Here as well, the low measurement throughput is not ideal, investigating only a single particle at a time. Finally, the free end of a particle-bound DNA strand can be attached to a container wall, and subsequent observation of the particle's restricted Brownian motion can be used to monitor changes in the conformation of the attached DNA strand. This is also known as tethered particle motion. [25,26] While delivering accurate results for the behavior of single strands, it does not cover the combined structure of many DNA strands immobilized on the particle's surface. It also struggles with low throughput, as only a single particle is observed per experiment. This work presents an efficient yet simple experimental approach for characterizing the structural conformation of short, double-stranded (ds) DNA bound to the surface of spherical magnetic nanoparticles (MNPs). The basic idea behind our study is sketched in Figure 1. By combining optical brightfield microscopy with a customized multiple particle tracking algorithm to observe the free two-dimensional Brownian motion of MNPs, we obtained data on the particles' diffusivity with high statistical significance. The resulting diffusion coefficient distributions are then connected to effective hydrodynamic diameters through the Stokes-Einstein relation. From the measured distribution of diffusivity, the effective hydrodynamic diameter distribution is reconstructed inversely. In parallel, we analyze the bare MNPs

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