Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer.

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

CO-Photolysis-Induced H-Atom Transfer from MnIO-H Bonds.

The formation of TEMPOH from a mixture of [Mn(CO)3(µ3-OH)]4 (1) and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) is shown to occur through a light-initiated CO photolysis from 1 (illumination at 300-375 nm). One hypothesis is that the loss of carbon monoxide (CO) causes significant O-H bond weakening to render proton-coupled electron transfer (PCET) to TEMPO favorable. For instance, the ground-state O-H bond dissociation free energy (BDFEO-H) of 1 (computed with density functional theory and estimated using effective BDFE reagents) is too high to transfer an H-atom to TEMPO. We also demonstrate that TEMPO and 1 interact in the dark through a hydrogen-bonded "precomplex" (1···TEMPO). We suggest that the PCET reaction that forms TEMPOH is the result of a H-atom-transfer reaction that occurs immediately after photolysis of a CO ligand(s).

Organically modified titania nanoparticles for sustained drug release applications.

In this paper, we report the synthesis, characterization of drug-doped organically modified titania nanoparticles, and their applications in sustained drug release. The drug-doped nanoparticles were synthesized in the hydrophobic core of oil-in-water microemulsion medium. Structural aspects obtained through TEM and FESEM depicted that organically modified titania nanoparticles are monodispersed with spherical morphology, with an average size of around 200 nm. Their polymorphic forms and porosity were determined using powder XRD and BET, respectively, which showed that they are present in the anatase form, with a surface area of 136.5 m(2)/g and pore-diameter of 5.23 nm. After synthesis and basic structural characterizations, optical properties were studied for both fluorophore and drug encapsulated nanoparticles. The results showed that though the optical properties of the fluorophore are partially diminished upon nanoencapsulation, it became more stable against chemical quenching. The nanoparticles showed pH-dependent drug release pattern. In vitro studies showed that the nanoparticles were efficiently uptaken by cells. Cell viability assay results showed that though the placebo nanoparticles are non-cytotoxic, the drug-doped nanoparticles show drug-induced toxicity. Therefore, such porous nanoparticles can be used in non-toxic drug delivery applications.