A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor.

Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABAA receptors (GABAAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11ß-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAAR PAM, to characterize steroid-binding sites in the Torpedo α2ßγδ nAChR in its native membrane environment. We found that F4N3Bzoxy-AP (IC50 = 31 µm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [3H]tenocyclidine to nAChRs in the desensitized state. At 300 µm, neither steroid inhibited binding of [3H]tetracaine, a closed-state selective channel blocker, or of [3H]acetylcholine. Photolabeling identified three distinct [3H]F4N3Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of ßVal-261 and δVal-269 (position M2-13'); 2) at the interface between the αM1 and αM4 helices, identified by photolabeling in αM1 (αCys-222/αLeu-223); and 3) at the lipid-protein interface involving γTrp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and αM1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroid-binding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists.

Ionization of covalent immobilized poly(4-vinylphenol) monolayers measured by ellipsometry, QCM and SPR.

Covalently immobilized poly(4-vinylphenol) (PVP) monolayer films were fabricated by spin coating PVP on perfluorophenyl azide (PFPA)-functionalized surface followed by UV irradiation. The pH-responsive behavior of these PVP ultrathin films was evaluated by ellipsometry, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). By monitoring the responses of these films to pH in situ, the ionization constant of the monolayer thin films was obtained. The apparent pKa value of these covalently immobilized PVP monolayers, 13.4 by SPR, was 3 units higher than that of the free polymer in aqueous solution.

Scalable antifouling reverse osmosis membranes utilizing perfluorophenyl azide photochemistry.

We present a method to produce anti-fouling reverse osmosis (RO) membranes that maintains the process and scalability of current RO membrane manufacturing. Utilizing perfluorophenyl azide (PFPA) photochemistry, commercial reverse osmosis membranes were dipped into an aqueous solution containing PFPA-terminated poly(ethyleneglycol) species and then exposed to ultraviolet light under ambient conditions, a process that can easily be adapted to a roll-to-roll process. Successful covalent modification of commercial reverse osmosis membranes was confirmed with attenuated total reflectance infrared spectroscopy and contact angle measurements. By employing X-ray photoelectron spectroscopy, it was determined that PFPAs undergo UV-generated nitrene addition and bind to the membrane through an aziridine linkage. After modification with the PFPA-PEG derivatives, the reverse osmosis membranes exhibit high fouling-resistance.

Photoconjugation of temperature- and pH-responsive polymer with silica nanoparticles for separation and enrichment of bacteria.

A new photoconjugation approach was developed to prepare nanoparticle-supported boronic acid polymer for effective separation and enrichment of bacteria. The photo-activated polymer immobilization was demonstrated by coupling an azide-modified copolymer of N-isopropylacrylamide and glycidyl methacrylate to a perfluorophenyl azide-modified silica surface. The thermoresponsive polymer was synthesized using reversible addition fragmentation chain transfer polymerization followed by conversion of the pendant epoxides into azide groups. The perfluorophenyl azide-modified silica nanoparticles were synthesized by an amidation reaction between amino-functionalized silica and pentafluorobenzoyl chloride, and a subsequent treatment with sodium azide. Bacteria-capturing boronic acid was conjugated to the silica-supported polymer chains via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. The particle size, morphology and organic content of the composite nanoparticles were characterized systematically. The capability of the nanocomposite to bind Gram-positive and Gram-negative bacteria was investigated. The nanocomposite exhibited high binding capacities for E. coli (13.4 × 107 CFU/mg) and S. epidermidis (7.66 × 107 CFU/mg) in phosphate buffered saline. The new photoconjugation strategy enables fast and straightforward grafting of functional polymers on surface, which opens many new opportunities for designing functional materials for bioseparation and biosensing.

Carbohydrate Functionalization of Few-Layer Graphene through Microwave-Assisted Reaction of Perfluorophenyl Azide.

The excellent physical and chemical properties of graphene make it an attractive nanomaterial and a component in high-performance nanocomposite materials. To prepare graphene-based nanocomposite materials, chemical functionalization is often necessary. Water-soluble ligands such as carbohydrates not only make the functionalized graphene compatible with aqueous media, but also introduce biorecognition, which is important for graphene to be used in biotechnology. In this study, we report the derivatization of few-layer graphene (FLG) with carbohydrates through microwave-assisted reaction of perfluorophenyl azide (PFPA). FLG was first treated with PFPA under microwave radiation. Subsequent conjugation with glycosyl amine gave carbohydrate-presenting FLG. Thermogravimetric analysis showed that microwave radiation gave a higher degree of functionalization compared to conventional heating, with higher weight losses for both PFPA and Man ligands. The carbohydrates (mannose and galactose) retained their bioactivity, as demonstrated by the lectin binding assays. Higher degree of binding toward lectins was obtained for the carbohydrate-functionalized FLG prepared by microwave radiation than the conventional heating.

Reducing Friction in the Eye: A Comparative Study of Lubrication by Surface-Anchored Synthetic and Natural Ocular Mucin Analogues.

Biomaterials used in the ocular environment should exhibit specific tribological behavior to avoid discomfort and stress-induced epithelial damage during blinking. In this study, two macromolecules that are commonly employed as ocular biomaterials, namely, poly(vinylpyrrolidone) (PVP) and hyaluronan (HA), are compared with two known model glycoproteins, namely bovine submaxillary mucin (BSM) and α1-acid glycoprotein (AGP), with regard to their nonfouling efficiency, wettability, and tribological properties when freely present in the lubricant, enabling spontaneous adsorption, and when chemisorbed under low contact pressures. Chemisorbed coatings were prepared by means of photochemically triggered nitrene insertion reactions. BSM and AGP provided boundary lubrication when spontaneously adsorbed in a hydrophobic contact with a coefficient of friction (CoF) of ∼0.03-0.04. PVP and HA were found to be excellent boundary lubricants when chemisorbed (CoF ≤ 0.01). Notably, high-molecular-weight PVP generated thick adlayers, typically around 14 nm, and was able to reduce the CoF below 0.005 when slid against a BSM-coated poly(dimethylsiloxane) pin in a tearlike fluid.

Carbohydrate conjugation through microwave-assisted functionalization of single-walled carbon nanotubes using perfluorophenyl azides.

Carbohydrate-functionalized single-walled carbon nanotubes (SWNTs) were synthesized using microwave-assisted reaction of perfluorophenyl azide with the nanotubes. The results showed that microwave radiation provides a rapid and effective means to covalently attach carbohydrates to SWNTs, producing carbohydrate-SWNT conjugates for biorecognition. The carbohydrate-functionalized SWNTs were furthermore shown to interact specifically with cognate carbohydrate-specific proteins (lectins), resulting in predicted recognition patterns. The carbohydrate-presenting SWNTs constitute a new platform for sensitive protein- or cell recognition, which pave the way for glycoconjugated carbon nanomaterials in biorecognition applications.

Delineating fibronectin bioadhesive micropatterns by photochemical immobilization of polystyrene and poly(vinylpyrrolidone).

Bioadhesive micropatterns, capable of laterally confining cells to a 2D lattice, have proven effective in simulating the in vivo tissue environment. They reveal fundamental aspects of the role of adhesion in cell mechanics, proliferation, and differentiation. Here we present an approach based on photochemistry for the fabrication of synthetic polymer micropatterns. Perfluorophenyl azide (PFPA), upon deep-UV exposure, forms a reactive nitrene capable of covalently linking to a molecule that is in close proximity. PFPA has been grafted onto a backbone of poly(allyl amine), which readily forms a self-assembled monolayer on silicon wafers or glass. A film of polystyrene was applied by spin-coating, and by laterally confining the UV exposure through a chromium-on-quartz photomask, monolayers of polymers could be immobilized in circular microdomains. Poly(vinylpyrrolidone) (PVP) was attached to the background to form a barrier to nonspecific protein adsorption and cell adhesion. Micropatterns were characterized with high-lateral-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS), which confirmed the formation of polystyrene domains within a PVP background. Fluorescence-microscopy adsorption assays with rhodamine-labeled bovine serum albumin demonstrated the nonfouling efficiency of PVP and, combined with TOF-SIMS, allowed for a comprehensive characterization of the pattern geometry. The applicability of the micropatterned platform in single-cell assays was tested by culturing two cell types, WM 239 melanoma cells and SaOs-2 osteoblasts, on micropatterned glass, either with or without backfilling of the patterns with fibronectin. It was demonstrated that the platform was efficient in confining cells to the fibronectin-backfilled micropatterns for at least 48 h. PVP is thus proposed as a viable, highly stable alternative to poly(ethylene glycol) for nonfouling applications. Due to the versatility of the nitrene-insertion reaction, the platform could be extended to other polymer pairs or proteins and the surface chemistry adapted to specific applications.

Development of a C(60)-fullerene bonded open-tubular capillary using a photo/thermal active agent for liquid chromatographic separations by π-π interactions.

This short communication describes a newly developed open-tubular capillary which was coated with C60-fullerene by a covalent bonding via a photo/thermal active agent. We utilized perfluorophenyl azide (PFPA) as an active agent, which can be used for the "photo click" coupling of the carbon materials. The inner wall of a fused silica capillary was treated with silane conjugated PFPA, and then C60-fullerene was chemically modified by a photoreaction or a thermal reaction. Through evaluations of the capillaries by liquid chromatography, the separation characteristics of three polycyclic aromatic hydrocarbons (PAHs) were confirmed in both capillaries. With comparison of the retention behavior to a commonly used C18 column, the prepared capillaries showed the specific separation ability based on the π-π stacking by C60-fullerene. The capillary prepared by the thermal reaction provided the base line separation of phenanthrene, triphenylene, and benz[a]pyrene within 3min at 18.8cm capillary length.