Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.

Simultaneous Isolation of Circulating Nucleic Acids and EV-Associated Protein Biomarkers From Unprocessed Plasma Using an AC Electrokinetics-Based Platform.

The power of personalized medicine is based on a deep understanding of cellular and molecular processes underlying disease pathogenesis. Accurately characterizing and analyzing connections between these processes is dependent on our ability to access multiple classes of biomarkers (DNA, RNA, and proteins)-ideally, in a minimally processed state. Here, we characterize a biomarker isolation platform that enables simultaneous isolation and on-chip detection of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated proteins in unprocessed biological fluids using AC Electrokinetics (ACE). Human biofluid samples were flowed over the ACE microelectrode array (ACE chip) on the Verita platform while an electrical signal was applied, inducing a field that reversibly captured biomarkers onto the microelectrode array. Isolated cfDNA, EV-RNA, and EV-associated proteins were visualized directly on the chip using DNA and RNA specific dyes or antigen-specific, directly conjugated antibodies (CD63, TSG101, PD-L1, GPC-1), respectively. Isolated material was also eluted off the chip and analyzed downstream by multiple methods, including PCR, RT-PCR, next-generation sequencing (NGS), capillary electrophoresis, and nanoparticle size characterization. The detection workflow confirmed the capture of cfDNA, EV-RNA, and EV-associated proteins from human biofluids on the ACE chip. Tumor specific variants and the mRNAs of housekeeping gene PGK1 were detected in cfDNA and RNA isolated directly from chips in PCR, NGS, and RT-PCR assays, demonstrating that high-quality material can be isolated from donor samples using the isolation workflow. Detection of the luminal membrane protein TSG101 with antibodies depended on membrane permeabilization, consistent with the presence of vesicles on the chip. Protein, morphological, and size characterization revealed that these vesicles had the characteristics of EVs. The results demonstrated that unprocessed cfDNA, EV-RNA, and EV-associated proteins can be isolated and simultaneously fluorescently analyzed on the ACE chip. The compatibility with established downstream technologies may also allow the use of the platform as a sample preparation method for workflows that could benefit from access to unprocessed exosomal, genomic, and proteomic biomarkers.

Cancer Detection at your Fingertips: Smartphone-Enabled DNA Testing.

High molecular weight cell-free DNA (hmw cfDNA) found in biological fluid, such as blood, is a promising biomarker for cancer detection. Due to the abundance of background apoptotic cell-free DNA in blood, quantifying the native concentration of hmw cfDNA using existing methods is technically challenging, time consuming, and expensive. We have developed a novel technology which utilizes Alternating Current Electrokinetics (ACE) to isolate hmw cfDNA directly from blood. Furthermore, we integrated this technology into a handheld device which utilizes a smartphone for power, instruction transmission, optical detection, image processing, and data transmission. The detection of hmw cfDNA in blood plasma demonstrated the performance of the device. We are continuing development of this device as a future point of care in vitro diagnostic.

Dynamics of forced nucleosome unraveling and role of nonuniform histone-DNA interactions.

A coarse-grained model of the nucleosome is introduced to investigate the dynamics of force-induced unwrapping of DNA from histone octamers. In this model, the DNA is treated as a charged, discrete worm-like chain, and the octamer is treated as a rigid cylinder carrying a positively charged superhelical groove that accommodates 1.7 turns of DNA. The groove charges are parameterized to reproduce the nonuniform histone/DNA interaction free energy profile and the loading rate-dependent unwrapping forces, both obtained from single-molecule experiments. Brownian dynamics simulations of the model under constant loading conditions reveal that nucleosome unraveling occurs in three distinct stages. At small extensions, the flanking DNA exhibits rapid unwrapping-rewrapping (breathing) dynamics and the octamer flips ∼180° and moves toward the pulling axis. At intermediate extensions, the outer turn of DNA unwraps gradually and the octamer swivels about the taut linkers and flips a further ∼90° to orient its superhelical axis almost parallel to the pulling axis. At large extensions, a portion of the inner turn unwraps abruptly with a notable rip in the force-extension plot and a >90° flip of the octamer. The remaining inner turn unwraps reversibly to leave a small portion of DNA attached to the octamer despite extended pulling. Our simulations further reveal that the nonuniform histone/DNA interactions in canonical nucleosomes serve to: stabilize the inner turn against unraveling while enhancing the breathing dynamics of the nucleosome and prevent dissociation of the octamer from the DNA while facilitating its mobility along the DNA. Thus, the modulation of the histone/DNA interactions could constitute one possible mechanism for regulating the accessibility of the nucleosome-wound DNA sequences.

Twist propagation in dinucleosome arrays.

We present a Monte Carlo simulation study of the distribution and propagation of twist from one DNA linker to another for a two-nucleosome array subjected to externally applied twist. A mesoscopic model of the array that incorporates nucleosome geometry along with the bending and twisting mechanics of the linkers is employed and external twist is applied in stepwise increments to mimic quasistatic twisting of chromatin fibers. Simulation results reveal that the magnitude and sign of the imposed and induced twist on contiguous linkers depend strongly on their relative orientation. Remarkably, the relative direction of the induced and applied twist can become inverted for a subset of linker orientations-a phenomenon we refer to as "twist inversion". We characterize the twist inversion, as a function of relative linker orientation, in a phase diagram and explain its key features using a simple model based on the geometry of the nucleosome/linker complex. In addition to twist inversion, our simulations reveal "nucleosome flipping", whereby nucleosomes may undergo sudden flipping in response to applied twist, causing a rapid bending of the linker and a significant change in the overall twist and writhe of the array. Our findings shed light on the underlying mechanisms by which torsional stresses impact chromatin organization.