Super-resolution imaging of linearized chromatin in tunable nanochannels.

Nanofluidic linearization and optical mapping of naked DNA have been reported in the research literature, and implemented in commercial instruments. However, the resolution with which DNA features can be resolved is still inherently limited by both Brownian motion and diffraction-limited optics. Direct analysis of native chromatin is further hampered by difficulty in electrophoretic manipulation, which is routinely used for DNA analysis. This paper describes the development of a three-layer, tunable, nanochannel system that enables non-electrophoretic linearization and immobilization of native chromatin. Furthermore, through careful selection of self-blinking fluorescent dyes and the design of the nanochannel system, we achieve direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging of the linearized chromatin. As an initial demonstration, rDNA chromatin extracted from Tetrahymena is analyzed by multi-color imaging of total DNA, newly synthesized DNA, and newly synthesized histone H3. Our analysis reveals a relatively even distribution of newly synthesized H3 across two halves of the rDNA chromatin with palindromic symmetry, supporting dispersive nucleosome segregation. As a proof-of-concept study, our work achieves super-resolution imaging of native chromatin fibers linearized and immobilized in tunable nanochannels. It opens up a new avenue for collecting long-range and high-resolution epigenetic information as well as genetic information.

Systems for multiplexing homogeneous immunoassays.

High-throughput multiplex protein biomarker assays continue to gain significance in the fields of biomarker discovery and drug development, due to their economical use of not only the precious clinical biological samples but also expensive reagents. Among these platforms, homogeneous multiplex systems have potential for short assay run times and cost-effective reagent consumptions. However, these systems must overcome challenges of signal cross talk and biochemical cross-reactivity. Despite these obstacles, several homogeneous multiplex immunoassays have been demonstrated. These include fluorescent polarization, fluorescent resonance energy transfer with quantum dots or graphene, luminescent oxygen-channeling immunoassay coupled with aqueous two-phase systems and DNA proximity assays. The balance between speed/simplicity and high multiplexing and robustness of these homogeneous multiplex immunoassays are discussed in this review.