High-throughput screening of transglutaminase activity using plasmonic fluorescent nanocomposites.

We describe a high-throughput screening (HTS) assay for transglutaminase (TG) enzyme activity using plasmonic fluorescent nanocomposites. We used TG to covalently crosslink 500 µM solution of 5'-biotinamidopentylamine (BP) to N,N'-dimethylcasein (DMC) which was adsorbed onto 384-well microplates. We then bound 0.2 - 2.0 × 10(11)/mL of 10 nm gold nanoparticles-streptavidin conjugate (10 nm AuNPs-SA) to BP via biotin-streptavidin interactions. Finally, J-aggregation of cyanine 1 (25 µM) or 2 (10 µM) upon the 10 nm AuNPs elicited absorption and fluorescence signaling of TG catalysis. The cyanines could be added sequentially to elicit green (590 nm) and red (700 nm) spectral responses from the same set of reactions. Catalysis was linear (r(2) > 0.98) up to 10 min within a linear dynamic range (LDR) of 0.1 - 5 µg/mL enzyme. The multi-wavelength interrogation offered fast results (< 5 min), sensitivity (limit of detection, LOD of 5 ng or 64 fmol TG) and intermediate precision (relative standard deviation, RSD of < 20% over 42 days). Plasmonic fluorescent nanocomposites offer new ways of interrogating biomolecules in HTS format.

Rational redesign of glucose oxidase for improved catalytic function and stability.

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.

Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.

The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure(®)) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-h period.

Antimicrobial peptide interactions with silica bead supported bilayers and E. coli: buforin II, magainin II, and arenicin.

Using the unique quantitative capabilities of hyperspectral confocal microscopy combined with multivariate curve resolution, a comparative approach was employed to gain a deeper understanding of the different types of interactions of antimicrobial peptides (AMPs) with biological membranes and cellular compartments. This approach allowed direct comparison of the dynamics and local effects of buforin II, magainin II, and arenicin with nanoporous silica bead supported bilayers and living E. coli. Correlating between experiments and comparing these responses have yielded several important discoveries for pursuing the underlying biophysics of bacteriocidal specificity and the connection between structure and function in various cellular environments. First, a novel fluorescence method for direct comparison of a model and living system is demonstrated by utilizing the membrane partitioning and environmental sensitivity of propidium iodide. Second, measurements are presented comparing the temporal dynamics and local equilibrium concentrations of the different antimicrobial agents in the membrane and internal matrix of the described systems. Finally, we discuss how the data lead to a deeper understanding of the roles of membrane penetration and permeabilization in the action of these AMPs.

A multifunctional thin film Au electrode surface formed by consecutive electrochemical reduction of aryl diazonium salts.

A multifunctional thin film surface capable of immobilizing two diverse molecules on a single gold electrode was prepared by consecutive electrodeposition of nitrophenyl and phenylboronic acid pinacol ester (PBA-PE) diazonium salts. Activation of the stacked film toward binding platinum nanoparticles (PtNPs) and yeast cells occurred via chemical deprotection of the pinacol ester followed by electroreduction of nitro to amino groups. FTIR spectral analysis was used to study and verify film composition at each stage of preparation. The affect of electrodeposition protocol over the thickness of the nitrophenyl and PBA-PE layers was explored and had a profound impact on the film properties. Thicker nitrophenyl films led to diminished PBA-PE diazonium reduction currents during assembly and decreased phenylboronic acid (PBA) layer thickness while allowing for higher PtNP loading and catalytic currents from PtNP-mediated peroxide reduction. Multilayer PBA films could be formed over the nitrophenyl film; however, only submonlayer PBA films permitted access to the underlying layer. The sequence of functional group activation toward binding was also shown to be significant, as perchlorate used to remove pinacol ester also converted aminophenyl groups accessible to the solution to nitrophenyl groups, preventing electrostatic PtNP binding. Finally, SEM images show PtNPs immobilized in close proximity (nanometers) to captured yeast cells on the PBA-aminophenyl-Au film. Such multibinding functionality films that maintain conductivity for subsequent electrochemical measurements hold promise for the development of electrochemical and/or optical platforms for fundamental cell studies, genomic and proteomic analysis, and biosensing.

Luminescent investigations of terbium(III) biosorption as a surrogate for heavy metals and radionuclides.

We describe a metal transport system for investigating the interfacial interactions between the anionic surface charge of a gram-negative bacterium (Escherichia coli) and a trivalent cationic metal, Tb3+. We believe this is the first description of the uptake kinetics, sub- and intracellular distribution, and temporal fate of Tb3+ ion in E. coli. We used the luminescence of the terbium-dipicolinic acid chelate to study metal ion transport. The bacteria had a high tolerance for the metal (IC(50) = 4 mM Tb3+). Metal ion transport was passive and metabolism independent. The uptake kinetics rapidly reached a maximum within 15 min, followed by a stasis for 60 min, and declining thereafter between 120 and 240 min, resulting in a biphasic curve. During this period, greater than one-third of the metal ion was sequestered within the cell. Our choice of a safe Biosafety Level I E. coli bacteria and the relatively non-toxic Tb3+ metal represents a model system for luminescent investigations of biosorption, for studying bacterial-water interfacial chemistry and for the bioremediation of heavy metals and radionuclides.

Electrically addressable diazonium-functionalized antibodies for multianalyte electrochemical sensor applications.

The direct electrically addressable deposition of diazonium-modified antibodies is examined for electrochemical immunosensing applications. The immobilized antibodies can be detected by the use of electroactive enzyme tags and nanoparticle-gold labeling. Control over antibody functionalization density and minimal spontaneous grafting of diazonium-antibody adducts is shown. The utility of the technique for a sandwich immunoassay as well as the ability to individually and selectively address closely spaced microelectrodes for multi-target protein detection in an array format is demonstrated.

Diazonium-functionalized horseradish peroxidase immobilized via addressable electrodeposition: direct electron transfer and electrochemical detection.

A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 +/- 0.7 s-1 and a DeltaEp of 8 mV (v = 150 mV/s). The large ks and low DeltaEp are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.