Spatial Distribution Of Enzymes Research Articles

Interaction between bilirubin oxidase and Au nanoparticles distributed over dimpled titanium foil towards oxygen reduction reaction

Spatial distribution of enzymes on electrodes is a key point to understand and optimize bioelectrocatalysis, although not largely investigated. In this work, Ti nanodimples serve as a platform for getting a controlled spatial distribution of gold nanoparticles (AuNPs). By varying the thickness of the initial gold layer, AuNPs with average sizes ranging from 13±6, 46±12 to 81±13 nm are obtained. AFM and TEM allow to localize the AuNPs in the dimples. It is in particular highlighted that the smallest NPs are present not only at the bottom but also on the edges of the Ti dimple, with TiO2 layer in their close surrounding. Enzymatic O2 reduction by immobilized Myrothecium verrucaria bilirubin oxidase (Mv BOD) is then investigated on the different materials. After having demonstrated the occurrence of the bioelectrocatalytic reaction on AuNPs excluding the Ti surface, the efficiency of the catalysis is discussed as a function of the AuNP size. The shape and fitting of the cyclic voltammetry curves obtained with the smallest AuNPs suggest a decrease in the electron transfer rate as a consequence of the colocalization of AuNPs and semiconducting TiO2. Interestingly, high stability of the enzymatic current is obtained with the highest AuNPs even in the presence of high concentration of salts.

Chemiluminescent Imaging of Enzyme-Labeled Probes Using an Optical Microscope–Videocamera Luminograph

A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-μm-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens andin situhybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.

Ultrasound accelerates transport of recombinant tissue plasminogen activator into clots

Fibrinolysis is accelerated in vitro in an ultrasound field, and externally applied high frequency ultrasound also accelerates thrombolysis in animal models. Although the mechanism of this effect is not known, ultrasound does not cause mechanical disruption of clots but rather accelerates enzymatic fibrinolysis. To determine if accelerated fibrinolysis could be related to increased transport of enzyme into clot, we have examined the effect of insonification on the distribution of plasminogen activator between clot and surrounding fluid in vitro. Plasma clots were overlayed with plasma containing 125I-radiolabeled, activesite-blocked recombinant tissue plasminogen activator (rt-PA) and incubated in the presence of 1-MHz ultrasound at 4 W/cm 2 or in the absence of ultrasound. The rate of uptake of rt-PA was significantly faster in the presence of ultrasound, reaching 15.5 ± 1.4% at 4 h compared to 8.2 ± 1.0% in the absence of ultrasound ( p < 0.0001). Similarly, ultrasound increased transport of enzyme from the clot into the surrounding fluid. To determine the effect of ultrasound on the spatial distribution of enzyme, plasma clots were overlayed with plasma containing radiolabeled rt-PA and incubated in the presence or absence of ultrasound. The clots were then snap-frozen, and the radioactivity in serial cryotome sections was determined. Exposure to ultrasound altered the rt-PA distribution, resulting in significantly deeper penetration of rt-PA into the clots. We conclude that exposure to ultrasound increases uptake of rt-PA into clots and also results in deeper penetration. These effects of ultrasound on enzyme transport may contribute to the accelerated fibrinolysis observed in an ultrasound field.