Micropatterned Multienzyme Devices with Adjustable Amounts of Immobilized Enzymes

Abstract

Multienzyme microstructures of glucose oxidase (GOx) and horseradish peroxidase (HRP) were prepared by layer-by-layer deposition inside microfluidic networks on glass substrates in order to allow both site-specific deposition and control of the amount of immobilized enzymes. The obtained microstructures were characterized by scanning force microscopy for the topography of the deposited layers. The local enzyme activity was characterized by the substrate-generation/tip-collection mode and the enzyme-mediated feedback mode of the scanning electrochemical microscope (SECM). These measurements provided quantitative information about the immobilized enzyme activity as a basis for adjusting enzyme loading for multienzyme structures that realize logical operations based on enzymatic conversions. Information about local HRP activity can also be obtained by optical readout using an Amplex UltraRed fluorgenic substrate and reading with a confocal laser scanning microscope with a much higher repetition rate for image acquisition. Using these principles, a layout with HRP and GOx microstructures was realized that showed the functionality of an OR Boolean logic switch.

Catalysis at the room temperature ionic liquid|water interface: H2O2 generation

“…The generation of H 2 O 2 at the RTIL|water interface was also confirmed by an optical readout using the fluorogenic substrate 33 Amplex UltraRed s . This experiment was performed using a pipette (ca.…”

Enzyme immobilization on silicate glass through simple adsorption of dendronized polymer–enzyme conjugates for localized enzymatic cascade reactions

“…[19][20][21] Covalently or non-covalently immobilized enzymes are applied in various areas, ranging from the large scale synthesis of ne chemicals, food additives and pharmaceuticals 10,[20][21][22][23][24][25] to small scale biosynthesis and bioanalysis. [26][27][28][29][30][31][32][33][34] For any novel enzyme immobilization method, the enzymes' catalytic efficiency, the operational stability of the immobilized enzymes and the ease of preparation are important to consider. 16 In addition to the development of miniaturized devices based on immobilized enzymes, there is growing interest in the development of strategies for the co-immobilization of different types of enzymes for carrying out localized enzymatic cascade reactions involving different types of immobilized enzymes.…”

“…16 In addition to the development of miniaturized devices based on immobilized enzymes, there is growing interest in the development of strategies for the co-immobilization of different types of enzymes for carrying out localized enzymatic cascade reactions involving different types of immobilized enzymes. 4,5,7,8,16,17,23,26,29,33,[35][36][37][38][39][40] The work about which we report here contributes to this eld. We present a simple and efficient method for the non-covalent immobilization of different types of enzymes.…”

Electrogeneration of O2.− and H2O2 Using Polymer‐modified Microelectrodes in the Environment of Living Cells

“…Fluorescence microscopy based on the fluorescent product of a selective reaction between a fluorgenic compound with ROS appears to be a facile real‐time approach for the detection and quantification of ROS (Scheme ) . The generation of H 2 O 2 and O 2 .− has been monitored using Amplex UltraRed and NBD‐Cl, respectively . This common approach is indirect and depends on the selectivity of the reaction between the dye and the compound of interest .…”

“…This common approach is indirect and depends on the selectivity of the reaction between the dye and the compound of interest . For the detection of H 2 O 2 , selectivity is provided by peroxidase required for the oxidation of Amplex UltraRed to a strongly fluorescent resorufin derivative (excitation 563 nm, emission 587 nm) .…”