We synthesized reversible terminators with tethered inhibitors for next generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides and were used to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high quality sequence data without the need for target amplification and thus avoided concomitant biases.
A novel solid-state polymer electrolyte was constructed using layer-by-layer (LbL) polyelectrolyte assembly of linear poly(ethylenimine) (LPEI) and poly(acrylic acid) (PAA), combined with a plasticization step using oligoethylene glycol dicarboxylic acid (OEGDA). This composite film exhibits a relatively high ionic conductivity of 9.5 x 10(-5) S/cm at 25 degrees C and 22% relative humidity. Detailed characterization of the composite was undertaken using grazing-angle Fourier transform infrared (GA-FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and impedance spectroscopy. After immersing the LPEI/PAA films into OEGDA aqueous solutions, the films exhibited a swelling behavior and increased surface roughness indicative of porosity induced by reorganization of ionic interactions between LPEI and PAA in acidic solution. This internal porous structure allows inclusion of OEGDA within the multilayer and increased ionic conductivity under ambient conditions due to the combined effects of plasticization of the LbL matrix by atmospheric water as well as the added mobility of ions in molten OEGDA within the composite.
Stable multilayer thin films, assembled by a combination of layer-by-layer processing of polyelectrolytes and inorganic quantum dot monolayers prepared using the Langmuir-Schaefer deposition procedure, have been examined. Film formation and structure are confirmed using UV-vis absorption measurements and fluorescence spectroscopy. Growth is shown to be linear over several polyelectrolyte-quantum dot multilayers. Films consist of either (trioctylphosphine/trioctylphosphine oxide)-capped CdSe (4.1 nm ( 0.16 nm) or dodecanethiol-capped Au quantum dots (6.0 nm ( 0.9 nm) within polyelectrolyte bilayers of the well-studied (poly(allylamine hydrochloride)/poly(styrene sulfonate)) system. Electrostatic and hydrophobic interactions combine as the basis for assembly using this versatile new procedure.The introduction of layer-by-layer (LbL) assembly of polyelectrolytes from aqueous solution in the early 1990s enabled the formation of useful thin film architectures. 1-3 Subsequently, a variety of schemes for the formation of multilayer films were introduced, incorporating semiconductor or metal quantum dots (QDs), which utilized charged surface-passivating ligands on the QDs to facilitate aqueous LbL assembly. 4-8 While the advantages of LbL assembly, such as assembly from aqueous solution, and lack of a substrate shape constraint, are important when applied to mixed systems of polyelectrolytes and QDs, questions remain about the internal structure of the layers and the impact this internal structure may play in certain applications. Deposition of QDs directly from solution affords little control over many of the assembly parameters, such as the packing density of the QDs, and the amount of phase segregation between the QDs and the polyelectrolyte layers. The LbL assembly of polyelectrolytes produces films with highly interpenetrated layers. 9-12 This characteristic mixing of layers in an LbL film has been shown to translate closely to QD layers produced using the LbL approach. 5 In contrast, studies incorporating R-zirconium phosphate sheets 13 or clay sheets 14 in an LbL scheme present examples of preformed inorganic layers limiting the amount of interpenetration. In applications such as the production of organic light-emitting diodes (OLEDs), the precise deposition and controlled environment around the active layers (i.e., the QD layers) are of utmost importance. It has recently been shown that the amount of phase segregation and monolayer character of QD layers in QD-small molecule organic devices represents a major factor in device performance. 15 Formation of Langmuir films is a viable route to the formation of QD monolayers, given they are capped with hydrophobic ligands. These films are typically studied after their transfer to solid substrates using the LangmuirBlodgett (LB) technique. 16,17 Initial studies relied on traditional amphiphiles to form a monolayer on the subphase surface, either directly as capping groups stabilizing the QDs or onto which a layer of QDs could be synthesized or deposited. It was subs...
A new procedure to directly pattern a luminescent porous silicon substrate by dry‐removal soft lithography is outlined. The ability to transfer a removed microstructure of porous silicon to a free‐standing flexible polymer film is also demonstrated, and by removing strips in orthogonal directions, an array of PSi pillars is produced (see Figure).
Tumor antigen-driven selection may expand T cells having T cell receptors (TCRs) of shared antigen specificity but different amino acid or nucleotide sequence in a process known as TCR convergence. Substitution sequencing errors introduced by TCRβ (TCRB) repertoire sequencing may create artifacts resembling TCR convergence. Given the anticipated differences in substitution error rates across different next-generation sequencing platforms, the choice of platform could be consequential. To test this, we performed TCRB sequencing on the same peripheral blood mononuclear cells (PBMC) from individuals with cancer receiving anti-CTLA-4 or anti-PD-1 using an Illumina-based approach (Sequenta) and an Ion Torrent-based approach (Oncomine TCRB-LR). While both approaches found similar TCR diversity, clonality, and clonal overlap, we found that Illumina-based sequencing resulted in higher TCR convergence than with the Ion Torrent approach. To build upon this initial observation we conducted a systematic comparison of Illumina-based TCRB sequencing assays, including those employing molecular barcodes, with the Oncomine assay, revealing differences in the frequency of convergent events, purportedly artifactual rearrangements, and sensitivity of detection. Finally, we applied the Ion Torrent-based approach to evaluate clonality and convergence in a cohort of individuals receiving anti-CTLA-4 blockade for cancer. We found that clonality and convergence independently predicted response and could be combined to improve the accuracy of a logistic regression classifier. These results demonstrate the importance of the sequencing platform in assessing TCRB convergence.
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