Enzyme-linked modified with

Figure 23.4 Avidin may be modified with 2-iminothiolane to produce sulfhydryl groups. Subsequent reaction with a maleimide-activated enzyme produces a thioether-linked conjugate.

PAMAM dendrimers or modified PAMAM dendrimers (with oxiamine or sulfhydryl surface functionalities) exclusive of one another. These antisera recognize den-drimers in ELISA (enzyme-linked immunosorbent assays), dot blots and Western blots. The immunogenicity of dendrimer-protein conjugates has implications for therapeutic use of dendrimers as vaccines and we anticipate that antidendrimer antibodies will have applications in patterning and assembling nanostructures containing dendrimers. 

A further approach to electrically wire redox enzymes by means of supramolecular structures that include CNTs as conductive elements involved the wrapping of CNTs with water-soluble polymers, for example, polyethylene imine or polyacrylic acid.54 The polymer coating enhanced the solubility of the CNTs in aqueous media, and facilitated the covalent linkage of the enzymes to the functionalized CNTs (Fig. 12.9c). The polyethylene imine-coated CNTs were covalently modified with electroactive ferrocene units, and the enzyme glucose oxidase (GOx) was covalently linked to the polymer coating. The ferrocene relay units were electrically contacted with the electrode by means of the CNTs, and the oxidized relay mediated the electron transfer from the enzyme-active center to the electrode, a process that activated the bioelectrocatalytic functions of GOx. Similar results were observed upon tethering the ferrocene units to polyacrylic acid-coated CNTs, and the covalent attachment of GOx to the modifying polymer. 

A difficult problem in utilizing enzymes as catalysts for reactions in a non-cellular environment is their instability. Most enzymes readily denature and become inactive on heating, exposure to air, or in organic solvents. An expensive catalyst that can be used only for one batch is not likely to be economical in an industrial process. Ideally, a catalyst, be it an enzyme or other, should be easily separable from the reaction mixtures and indefinitely reusable. A promising approach to the separation problem is to use the technique of enzyme immobilization. This means that the enzyme is modified by making it insoluble in the reaction medium. If the enzyme is insoluble and still able to manifest its catalytic activity, it can be separated from the reaction medium with minimum loss and reused. Immobilization can be achieved by linking the enzyme covalently to a polymer matrix in the same general manner as is used in solid-phase peptide synthesis (Section 25-7D). 

Figure 301 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether cross-links.

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

The first reported preparation of cross-linked enzyme crystals was by Quiocho and Richards in 1964 [1], They prepared crystals of carboxypeptidase-A and cross-linked them with glutaraldehyde. The material they prepared retained only about 5% of the activity of the soluble enzyme and showed a measurable increase in mechanical stability. The authors quite correctly predicted that cross-linked enzyme crystals, particularly ones of small size where the diffusion problem is not serious, may be useful as reagents which can be removed by sedimentation and filtration. Two years later the same authors reported a more detailed study of the enzymic behavior of CLCs of carboxypeptidase-A [2], In this study they reported that only the lysine residues in the protein were modified by the glutaraldehyde cross-linking. The CLCs were packed in a column for a flow-through assay and maintained activity after many uses over a period of 3 months. 

Perhaps the original hope for these polymers was that they would act simultaneously as immobilisation matrix and mediator, facilitating electron transfer between the enzyme and electrode and eliminating the need for either O2 or an additional redox mediator. This did not appear to be the case for polypyrrole, and in fact while a copolymer of pyrrole and a ferrocene modified pyrrole did achieve the mediation (43), the response suggested that far from enhancing the charge transport, the polypyrrole acted as an inert diffusion barrier. Since these early reports, other mediator doped polypyrroles have been reported (44t45) and curiosity about the actual role of polypyrrole or any other electrochemically deposited polymer, has lead to many studies more concerned with the kinetics of the enzyme linked reactions and the film transport properties, than with the achievement of a real biosensor. 

One recent trend has been away from using a photochromic dye itself merely as an individual component of a solution, polymer film or bulk polymer matrix. Instead, the photochromic is chemically linked to a polymer, which may be a natural polymer such as a cellulose derivative, an enzyme, a protein, or synthetic polymers from acrylates, urethanes, and vinyl compounds. The properties of the polymer can then be modified by external irradiation, and conversely, the properties of the photochromic are modified by the polymer. A recent biochemical example is the photocontrolled binding of monosaccharides to concanavalin A (Con A) modified with spiropyran units.208... 

Figure 43. (A) Electron-transfer pathway for the cyclic photoinduced hydrogenation of acetylene by lactate using eosin-modified myoglobin reconstituted with Co(II)-protoporphyrin IX working in cooperation with LDH. (B) Rates of formation of products upon illumination of a photosystem composed of myoglobin reconstituted with Co(II)-protoporphyrin IX and modified with eosin (0.11 mg mL ), LDH (0.6 units mL ), 5 (8 x 10 M) and lactic acid (1 x 10 M) under an atmospheric pressure of acetylene (a) rate of ethylene formation and (b) rate of pyruvic acid formation. (C) Hypothetical improved system for the same photobiocatalytic system, where the two enzymes and electron mediator are covalently linked.

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