Enzyme immobilization acid /poly

The oxazolidine-2,5-dione heterocycle, perhaps better known as the N-carboxyanhydride of an amino acid, has been incorporated employing a modification of chloromethylated poly(styrene) (192) (76USP3985715). The reaction sequence involved utilization of a masked amino acid, ethyl acetamidocyanoacetate (205). The amino acid was liberated in a subsequent hydrolysis/decarboxylation step (Scheme 98). The cyclized, IV-carboxyanhydride-functional resins (206) were reported to be useful in solid phase peptide synthesis and as supports for enzyme immobilization. 

Figure 9. Schematic fabrication of LbL films comprising poly(vinylsulfonic acid) (PVS)and PAMAM-Au. The sequential deposition of LbL multilayers was carried out by immersing the substrate alternately into (a) PVS and (b) PAMAM-Au solutions for 5 min per step (c) After deposition of 3 bilayers, an ITO-PVS/PAMAM-Au)3 CoHCF electrode was prepared by potential cycling (d) The enzyme immobilization to produce ITO-PVS/PAMAM-Au)3 CoHCF-GOx was carried out in a solution containing BSA, glutaraldehyde and GOx (Adapted from Ref.[124])...

Many other similar applications have been reported such as the electrochemical determination of electroinactive cationic medicines,313 determination of urea,314 uric acid,315 and application to glucose biosensors to decrease interference of ascorbate, urate, and acetaminophen.316 Enzyme immobilized membranes are also sensing membranes, e.g. urea responsive membranes, poly(carboxylic acid) membranes in which urease is immobilized,317 fructose responsive membranes, and polyion complex membranes in which fructose dehydrogenase is immobilized.318 Such applications will expand further in the future and contribute to human life. 

The LbL technique is undoubtedly one of the best methods to incorporate biological components into man-made devices. Therefore, sensor applications must be one of the most promising subjects for LbL assemblies of biomaterials. For example, Leblanc and coworkers used several bilayers of chitosan and poly(thiophene-3-acetic acid) as cushion layers for stable enzyme films [187]. The first five bilayers of the cushion layer allowed for better adsorption of organophosphorus hydrolase than the corresponding adsorption on a quartz slide. The immobilized enzyme becomes more stable and can be used under harsher conditions. The assembled LbL films can be used for spectroscopic detection of paraoxon, an organophosphorus compound. This cushion layer strategy provides a well-defined substrate-independent interface for enzyme immobilization, in which the bioactivity of the enzyme is not compromised. This leads to fast detection of paraoxon and quick recovery times. 

A facile enzymatic polymerization protocol for preparing poly(thio-phene-3-boronic acid biocomposites was established (13BB41). The biocomposites were monitored for mono-/bi-enzyme immobilization and amperometric biosensing. 

Chen, H. and Y.-L. Hsieh (2004). Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts. Biotechnology and Bioengineering 90(4) 405-413. 

A report by Liu and coworkers described layer-by-layer (LBL) coatings that have been assembled on the inner surfaces of the microchip [90]. Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. Trypsin was adsorbed in the multilayer membrane composed of CS/HA assembled multilayers. The resulting peptide analysis has been carried out by MALDI-TOF-MS. The maximum proteolytic velocity of the adsorbed trypsin was 600 mM/min/[ig, thousands of times faster than that in solution. BSA, MYO, and Cyt-c were used as model substrates for the tryptic digestion. The standard proteins were identifled at a low femtomole per analysis at a concentration of 0.5 ng/pl with the digestion time <5 s. This simple technique may offer a potential solution for low-level protein analysis. 

Transition-metal phosphorus trichalcogenides such as MnPS3 are able to intercalate amino acids and peptides by ion exchange. In this way, increases in the basal spacing of 0.7 and 3-4 nm are observed for the intercalation of poly-L-lysine and lysozyme, respectively [224]. Interestingly, the enzymatic activity of the immobilized protein has been detected, suggesting that the enzyme is protected against denaturation. 

Negatively charged species such as carboxylic acid group in acid-treated CNTs can attract positively charged enzymes from solution as long as the pH value of the enzyme solution is controlled to be lower than the iso-electric point of the enzyme thus, multilayer films of the enzyme can be formed by the layer-by-layer technique. For example, five layers of GOx can be immobilized on the electrode surface by alternatively dipping a poly(diallyldimethylammonium chloride (PDDA))-functionalized GC into a CNT solution and a GOx solution (pH 3.8). Figure 15.15 illustrates the preparation process for the formation of a multilayer film of GOx on the electrode. 

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