Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Effects of conductive hearing loss on temporal aspects of sound transmission through the ear.

Effects of conductive hearing loss on level and spectrum are well known. However, little is known about possible additional effects on temporal aspects of sound transmission. This study investigated effects of earplugs and middle ear effusions on amplitude and timing of cochlear microphonic (CM) responses in gerbils. Bilateral CM responses to pure tones (1-16 kHz) were monitored before and after (i). unilateral earplug insertion or (ii). injection of silicone oil, of various viscosities, into one middle ear. Earplugs produced flat hearing losses (mean 13 dB) and delayed CMs more at lower (mean 80 micros, 1-6 kHz) than at higher (20 micros, 8-16 kHz) frequencies. Effusions also produced flat hearing loss. On average, high viscosity effusions produced larger hearing losses (36 dB) than medium (25 dB) or low (20 dB) viscosity effusions. Low and medium viscosity effusions delayed responses to lower (mean 82 and 65 micros respectively, 1-6 kHz) more than to higher (mean 20 and 10 micros respectively, 8-16 kHz) frequencies. High viscosity effusions produced smaller delays across all frequencies (mean 31 micros, 1-16 kHz). In normal animals, CM responses were not delayed over a wide range of stimulus levels. Therefore, in addition to attenuation, conductive loss distorts acoustic temporal cues important for hearing.

Single-site beta-diiminate zinc catalysts for the ring-opening polymerization of beta-butyrolactone and beta-valerolactone to poly(3-hydroxyalkanoates).

Polymerization of beta-butyrolactone (BBL) and beta-valerolactone (BVL) using the zinc alkoxide initiator (BDI-1)ZnO(i)()Pr [(BDI-1) = 2-((2,6-diisopropylphenyl)amido)-4-((2,6-diisopropylphenyl)imino)-2-pentene] proceeds very rapidly under mild conditions to produce poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyvalerate) (PHV), respectively. For the monomer-to-initiator ratio 200:1, PHB number-average molecular weights (M(n)) are proportional to conversion throughout the reaction and polydispersity indices (PDIs) are narrow, consistent with a living polymerization. Higher monomer-to-initiator ratios can be used to achieve high molecular weight PHB (M(n) > 100 000). End-group analysis verifies that the polymerization of BBL follows a coordination-insertion mechanism, where complexes of the form (BDI-1)ZnOCH(Me)CH(2)CO(2)R are the active species. Variable temperature NMR experiments show that (BDI-1)ZnO(i)()Pr is monomeric in benzene-d(6) solution. In contrast, (BDI-2)ZnO(i)()Pr [(BDI-2) = 2-((2,6-diethylphenyl)amido)-4-((2,6-diethylphenyl)imino)-2-pentene] is a poor initiator at room temperature because it prefers to form a bis-mu-isopropoxide dimer in solution. According to kinetic studies, propagation by (BDI-1)ZnO(i)()Pr is first order in both monomer as well as (BDI-1)ZnO(i)()Pr concentration. These results lead us to propose a monometallic active species. Several results suggest that elimination side reactions are slowly catalyzed by zinc alkoxides, leading to degradation of the polymer.