Glucose oxidase images

The preparation and application of SAM systems patterned by STM and their use in catalysis was demonstrated by Wittstock and Schuhmann [123]. The patterning (local desorption) of SAMs from alkane thiols on gold was performed by scanning electrochemical microscopy (SECM), followed by the assembly of an amino-deriva-tized disulfide and coupling of glucose oxidase to form a catalytically active pattern of the enzyme. The enzymatic activity could be monitored/imaged by SECM. 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Fig. 19 Rapid lifetime determination imaging of the activity of glucose oxidase. Gray-scale image of the activity of glucose oxidase (left) and resulting calibration curve (right). Experiments were performed in triplicate (rows). The wells in the images contained, from 1 to 12, glucose oxidase activities of 0 (blank), 135, 54.1, 27.1, 13.5, 5.4, 2.7, 1.35, 0.54, 0.27, 0.14, and 0.05 mU ml. 1 respectively, 100 jiL of a 0.2 mmol L-1 EuTc solution, and 15 jiL of a 277.2 mmol I. 1 glucose solution. The total volume was made up to 200 xL with MOPS buffer...

Figure 11. AFM image of a glucose oxidase-modified SWCNT. From reference 89.

Figure 9. AFM images of TPSP-ZnO before (A)and after(B)GOD loading.(C) Cyclic voltammograms ofTPSP-ZnO/Nafion (a), GOD/Nafion (b)GOD/spherical ZnO/ Nafion (c) and GOD/TPSP-ZnO/Nalion (d) modified in 0.1M pH 7.0 PB at 0.1 Vs" ( Reprinted from Biosensors and Bioelectronics, 24, Z. Dai, G. Shao, J. Hong, J. Bao, J. Shen, Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor, 1288,1289, Copyrights (2009) with permission fom Elsevier.

As stated above, SECM can be used to image the reactivity of surface features. A feedback detection scheme was used to observe the localized reaction of glucose oxidase and mitochondria-bound NADH-cytochrome c reductase (7). The spatial resolution of the imaging is high for enzymatic... 

FIG. 18 (A) Constant current image showing several glucose oxidase (MW =... 

A dual-analyte fiber optic biosensor for O2 and glucose was developed by Li and Walt [22] based on O2 quenching of a phosphorescent ruthenium dye. Excitation was at X = 480 nm, with fluorescent emission captured by a CCD camera. A relatively large (350 (U,m diameter) imaging fiber with 6000 elements was modified by attaching two separate drops of ruthenium dye encapsulated in poly(hydroxyethyl methacrylate) polymer (HEMA). The ruthenium dye allowed measurements of O2 in both encapsulated drops, which were approximately 50 ixm in diameter. A two-site Stern-Volmer quenching model (equation (4.32) with n — 2) was used to determine O2 concentration from measurements of fluorescence intensity. One of the drops had the enzyme glucose oxidase (EC... 

Fig. 9 Coupled enzymatic test using glucose oxidase (GOx) and peroxidase (POD) in sheU-in-shell capsules, (a) Reaction schemes, (b) Localization of GOx and POD within sheU-in-shell capsules, (c) CLSM imaging in situ of resotufln formation. Reprinted with permission from Angewandte Chemie International Edition [89]...

Enzyme-mediated feedback can be used to image enzyme patterns. To successfully image enzymatic features, lip fouling from oxide formation or adsorption from solution constituents must be avoided. The enzyme reaction at the substrate must not be inhibited by solution species. It must also be able to sustain a level of regeneration activity of the mediator that can compete with its mass transport from the bulk electrode to the tip. In the case of a glucose oxidase catalyzed reaction, a digital simulation of the positive feedback observed from this enzyme quantitatively expresses this limitation (143). 

Proteins. In addition to DNA, it is also possible to image antibody (e.g., a mouse monoclonal IgG), enzyme (e.g., glucose oxidase, GOD), and haemocyanin (e.g., keyhole limpet haemocyanin, KLH) molecules by the same technique (38). All of these protein molecules... 

Figure 13.16 SEM image of the nanofibers prepared by electrospinning after their derivatiza-tion with glutaraldehyde and subsequent immobilization of glucose oxidase...

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