Electrochemical Detection of Single Microbeads Manipulated by Optical Tweezers in the Vicinity of Ultramicroelectrodes

Latex micrometric beads are manipulated by optical tweezers in the vicinity of an ultramicroelectrode (UME). They are optically trapped in solution and approached the electrode surface. After the electrochemical measurement, they are optically removed from the surface. The residence time of the particle on the electrode is thus controlled by the optical tweezers. The detection is based on diffusional hindrance by the insulating objects which alters the fluxes of the redox Ru­(NH₃)₆³⁺ species toward the UME and thus its mass-transfer limited current. We have optically deposited successively 1, 2, and 3 beads of 3-μm radius on the UME surface, and we have recorded the variations of the current depending on their landing locations that were optically controlled. Finally we decreased the current by partially blocking the electroactive surface with a six-bead assembly. The variation of the steady-state current and the approach curves allow for the indirect electrochemical localization of the bead in the vicinity of the UME, not only when the bead is in contact but also when it is levitated at distances lower than the UME radius. These experiments show that single particles or more complex structures may be manipulated <i>in situ</i> in a contactless mode near the UME surface. From comparison with simulations, the electrochemical detection affords an indirect localization of the object in the UME environment. The developed approach offers a potential application for interrogating the electrochemical activity of single cells and nanoparticles

Monitoring Metabolic Responses of Single Mitochondria within Poly(dimethylsiloxane) Wells: Study of Their Endogenous Reduced Nicotinamide Adenine Dinucleotide Evolution

It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly­(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion

Spectroscopic and Crystallographic Characterization of “Alternative Resting” and “Resting Oxidized” Enzyme Forms of Bilirubin Oxidase: Implications for Activity and Electrochemical Behavior of Multicopper Oxidases

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in <i>Rhus vernicifera</i> and <i>Trametes versicolor</i> laccase, characterized by “normal” type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu–Cu distance, while the alternative resting form shows unusually small <i>A</i>_∥ and high <i>g</i>_∥ EPR features, lack of 330 nm absorption intensity, and a long T3 Cu–Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis

Spectroscopic and Crystallographic Characterization of “Alternative Resting” and “Resting Oxidized” Enzyme Forms of Bilirubin Oxidase: Implications for Activity and Electrochemical Behavior of Multicopper Oxidases

While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in <i>Rhus vernicifera</i> and <i>Trametes versicolor</i> laccase, characterized by “normal” type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu–Cu distance, while the alternative resting form shows unusually small <i>A</i>_∥ and high <i>g</i>_∥ EPR features, lack of 330 nm absorption intensity, and a long T3 Cu–Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis