With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 - 8.88; PMMA/COC device range = 1.4 - 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O activated after device assembly with the dose of the UV/O affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.
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http://dx.doi.org/10.1016/j.chroma.2021.461892 | DOI Listing |
J Med Virol
December 2024
Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA.
RNA helicase DDX5 is a host restriction factor for hepatitis B virus (HBV) biosynthesis. Mass spectrometry (LC-MS/MS) identified significant DDX5-interacting partners, including interferon-inducible protein 16 (IFI16) and RBBP4/7, an auxiliary subunit of polycomb repressive complex 2 (PRC2). DDX5 co-eluted with IFI16, RBBP4/7, and core PRC2 subunits in size exclusion chromatography fractions derived from native nuclear extracts.
View Article and Find Full Text PDFEcotoxicol Environ Saf
December 2024
Acibadem Mehmet Ali Aydınlar University, Institute of Health Sciences, Department of Medical Biotechnology, Istanbul, Turkey; Acibadem Mehmet Ali Aydınlar University, School of Medicine, Department of Medical Microbiology, Istanbul, Turkey. Electronic address:
Background/purpose: Nanobacteria, known to date as self-replicating, nano-scale size organisms, smaller than bacteria. However, whether these are living organisms or agglomeration of biomolecules was one of the most controversial issues for many years. One of the reasons for debate is their lack of any genetic material.
View Article and Find Full Text PDFMolecules
November 2024
College of Chemistry, Zhengzhou University, Zhengzhou 450001, China.
The early diagnosis of tumorigenesis is crucial for clinical treatment, but the resolution and sensitivity of conventional short-wavelength biomarkers are not ideal because of the complicated interference in living tissue. Herein, a nicotinamide adenine dinucleotide (NAD)-responsive probe with deep-red emissive ratiometric fluorescence was synthetized as a promising target for energy metabolism patterns during tumorigenesis. Interestingly, the solvents HPO and 2,2'-dithiodibenzoic acid enhanced the red emission (640 and 680 nm) of o-phenylenediamine-based carbon dots (CDs), leading to the formation of a nanoscale graphite-like skeleton covered with -P=O, -CONH-, -COOH and -NH on their surfaces.
View Article and Find Full Text PDFElectrophoresis
November 2024
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan, China.
Chem Commun (Camb)
October 2024
The RNA Institute, University at Albany, State University of New York, New York, NY, USA.
In this work, we analyzed the electrophoretic behavior of a double crossover (DX) DNA motif in various counter ions. The influence of the type and concentration of counter ions on electrophoretic behavior is different for the DX motif compared to a duplex of the same molecular weight. Higher concentrations of divalent ions Mg and Ca in the gel and running buffer reduce the electrophoretic migration of the DX motif.
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