Enzyme deposition

Enhanced thermal stability enlarges the areas of application of protein films. In particular it might be possible to improve the yield of reactors in biotechnological processes based on enzymatic catalysis, by increasing the temperature of the reaction and using enzymes deposited by the LB technique. Nevertheless, a major technical difficulty is that enzyme films must be deposited on suitable supports, such as small spheres, in order to increase the number of enzyme molecules involved in the process, thus providing a better performance of the reactor. An increased surface-to-volume ratio in the case of spheres will increase the number of enzyme molecules in a fixed reactor volume. Moreover, since the major part of known enzymatic reactions is carried out in liquid phase, protein molecules must be attached chemically to the sphere surface in order to prevent their detachment during operation. 

An enzyme deposited on the LAPS surface allows one to observe the spatial distribution of a specific substrate. In a urea-selective sensor urease was immobilized on a pH-selective LAPS [75],... 

Wehtje, E., Adlercreutz, P. and Mattiasson, B. (1993) Improved activity retention of enzymes deposited on solid supports. Biotechnol. Bioeng., 41, 171-178. 

In this way a sonochemically fabricated microelectrode array was used to form an array of conducting microelectrodes [27] containing a genetically modified AChE which had been modified to maximise pesticide sensitivity. Use of an I125-labelled AChE meant that the amount of enzyme deposited could be measured and in this instance corresponded to 0.15 units activity. Measuring the amperometric... 

For enzyme attachment to the silicon microreactor tested, a layer-by-layer technique was employed to build a multilayer system of polyions and enzyme. Deposition of multilayers was accomplished by alternating positively and negatively charged layers of polydimethyldiallyl ammonium chloride (PDDA) and polystyrene sulfonate (PSS), respectively, to which was attached urease enzyme. After depositing in succession three layers of PDDA, PSS, and PDDA, three layers of urease enzyme were alternately deposited with three layers of PDDA. The resulting architecture is described as follows ... 

Lysosomes 0.4% 1% 200-300 Degradation of foreign macromolecules in the cell by means of hydrolytic enzymes, deposition of copper, ferritin, lipofuscin, bile pigment, etc. 

Alternatively, the above authors (Kimura et al., 1989) used an inkjet nozzle as a tool for precise enzyme deposition on a multigate ISFET. 

SubstrateAssay amide, which, when subjected to peroxidase enzyme, deposits... 

In spite of the extensive contacts between both moieties, the structure of SCl-324-bound a-thrombin is essentially unaltered compared to the over 100 crystal structures of the enzyme deposited with the Protein Data Bank. This observation supports the contention that bacterial co-... 

Lakard et al. have described HRP modified PANI nanoparticles-based biosensor for sensing [159]. This biosensor format exhibits improved enzyme deposition and improved signal-to-noise ratio. There is a strong relationship between nano-dimension and biosensing performance. 

LDH-modified electrodes have been also obtained by a layer-by-layer (LbL) deposition technique, by exploiting the net positive charges on the brucite-like layers [47] after delamination of suitably synthesized LDHs [48,49], the positively charged component of the film is thus obtained. The anionic counterpart consists of an organic polymer, such as poly(sodium 4-styrenesulphonate) [48, 49], of negatively charged electroactive species, such as metal porphyrins [50], phthalocya-nines [51], Au NPs surrounded by an anionic cloud [52], or a specific enzyme deposited from buffered solutions at pH values above the isoelectric point [53]. 

The use of different types of membranes for enzyme entrapment is a well-described technique useful for the processes with allosteric enzymes, where the presence of cofactor(s) is required. The main properties of the membrane-bound biocatalysts is the large surface area of the matrix, allowing high amount of enzymes deposited on the surface and in contact with the substrate, allowing to have high substrate conversion rates. However, the main disadvantage is the exposition of the enzyme to the medium where the shear and hydrolytic activities can compromise the enzyme complex stability and activity. 

Enzyme Deposition. Glucose oxidase (type X-S), bovine albumin (fraction V), and glutaraldehyde were obtained from Sigma Chemical Company and used as supplied. A silicone defoaming agent (B emulsion) was from Dow Corning. A nonionic surfactant (Tergitol NP-10) from Union Carbide Corporation was also incorporated. 

Figure 2. Comparison of current output for three sensors in a 100 mg/dL glucose solution in vitro. The sensors were identical in electrode area and meUiod of fabrication, except for the enzyme deposition. One of the sensors was coated using the Ikariyama adsorption technique (16) and the odier two were coated using the method described here utilizing two different current values. The sensors were not covered with an outer membrane. (Reproduced with permission from ref. 19. Copyright 1991 Elsevier Sequoia.)...

Two principle techniques for electrochemical enzyme deposition have been reported, entrapment in an electrochemically grown polymer and electrochemically aided absorption. A wide range of electrochemically grown polymers have been used. The polymer can function as both an entrapment matrix and as an anti-interference layer (7, 12-20), as a matrix for the immobilisation of the protein with an electron transfer mediator (21-23), and as an electron transfer matrix alone (24, 25), Electrochemically aided adsorption has received comparably less attention (26-30), However, in our experience (31) the latter technique results in larger responses and is more appropriate to microelectrodes. Here we will present results on the electrochemically aided adsorption of GOx and BSA, and also of urease. Furthermore to reduce the interferences at the GOx/ BSA electrode we will describe the deposition of an anti-interference layer of polypyrrole, which is grown on the electrode after the deposition of the proteins. 

Figure P6.10 Schematic description of an anisotropic enzyme electrode. The membrane (exaggerated) has active enzyme deposited as a surface layer at the electrode sensor interface. The product flux is the result of the reaction involving analyte diffusing through the membrane.

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