Nanosecond Laser-Fabricated Monolayer of Gold Nanoparticles on ITO for Bioelectrocatalysis.

Redox enzymes can be envisioned as biocatalysts in various electrocatalytic-based devices. Among factors that play roles in bioelectrochemistry limitations, the effect of enzyme-enzyme neighboring interaction on electrocatalysis has rarely been investigated, although critical in vivo. We report in this work an in-depth study of gold nanoparticles prepared by laser ablation in the ultimate goal of determining the relationship between activity and enzyme density on electrodes. Nanosecond laser interaction with nanometric gold films deposited on indium tin oxide support was used to generate in situ gold nanoparticles (AuNPs) free from any stabilizers. A comprehensive analysis of AuNP size and coverage, as well as total geometric surface vs. electroactive surface is provided as a function of the thickness of the treated gold layer. Using microscopy and electrochemistry, the long-term stability of AuNP-based electrodes in the atmosphere and in the electrolyte is demonstrated. AuNPs formed by laser treatment are then modified by thiol chemistry and their electrochemical behavior is tested with a redox probe. Finally, enzyme adsorption and bioelectrocatalysis are evaluated in the case of two enzymes, i.e., the Myrothecium verrucaria bilirubin oxidase and the Thermus thermophilus laccase. Behaving differently on charged surfaces, they allow demonstrating the validity of laser treated AuNPs for bioelectrocatalysis.

Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons.

We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A) channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by RuBi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A) receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A) channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A)-Rs mediated signals in small cell compartments.