Immobilization Schemes

Several approaches have been described for the immobilization of NPs at electrode surfaces. The simplest is direct adsorption of the NP from solution onto an electrode surface. This method takes advantage of favorable electrostatic interactions between the surface and the NPs. This has been used, for example, to immobilize Ti02 NPs 

For cases in which the NPs are not soluble in the supporting electrolyte in which they will be examined, it is possible to solvent-cast a thin film of the NPs on the electrode surface followed by evaporation [44] or to directly apply an insoluble gel containing the NPs [45]. In a related approach, films of anionic Prussian Blue NPs that had been synthesized in a solution containing chitosan (a cationic glucosamine polymer) were drop-cast onto glassy carbon surfaces, giving very stable 

Free Choice in Number of Layers Layering Sequence 

The nature of the LbL film structure puts the NPs into close proximity to one another. Thus, for cases in which the NPs are electroactive, charge propagation through the films seems most likely to occur by electron exchange, first between the 

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The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

The initial hurdle to overcome in the biosensor application of a nucleic acid is that involving its stable attachment on a transducing element which commonly includes a metallic electrode. In the first part of this chapter, we wish to introduce our approach for DNA immobilization (Scheme 1). A detailed characterization of the immobilization chemistry is also presented. In the second part, we follow the development of work from our laboratory on chemical sensor applications of the DNA-modified electrode involving a biosensor for DNA-binding molecules and an electrochemical gene sensor. 

Because enzymes present such an attractive possibility for achieving chemical selectivity, enzyme electrodes were the first enzymatic chemical sensors (or first biosensors) made. The early designs used any available method of immobilization of the enzyme at the surface of the electrode. Thus, physical entrapment using dialysis membranes, meshes, and various covalent immobilization schemes have been... 

It includes a detailed consideration of the impact of different types of surface-immobilization schemes and different denaturation conditions on the results obtained via SM FRET measurements. 

Hyperbranched polymers can also be used for supramolecular immobilization (Scheme 15). Yet another approach for the noncovalent immobilization has been presented by Tzschucke and coworkers who used interactions between fluorous phase silica (FPS) and perfluoro-tagged palladium... 

Calibration profiles of the sensor based on the final nylon-enzyme net (III) were disappointing oonpared with the analogous sensor based on nylon net type II. The lower detection limit was only 0.1 mM glucose and currents produced were about 80% smaller. However, this alternative immobilization scheme serves to illustrate the synthetic versatility of nylon-6,6 in the biosensor field. 

Several useful schemes for attaching nucleic acid probes onto electrode surfaces have thus been developed [2-8]. The exact immobilization protocol often depends on the electrode material used for signal transduction. Common probe immobilization schemes include attachment of biotin-functionalized probes to avidin-coated surfaces [15], self-assembly of organized monolayers of thiol-functionalized probes onto gold transducers [16], carbodiimide covalent binding to an activated surface [17], as well as adsorptive accumulation onto carbon-paste or thick-film carbon electrodes [15-30]. 

Fig. 4 Slide preparation and probe immobilization scheme. A clean slide is treated with mercaptosilane (MPTS), and an intermediate mercaptosilane layer is formed on the surface of the slide. 5 disulfide-modified probes are immobilized onto the slide through thiol/disulfide exchange reactions...

Another work reported the immobilization of nueleie aeids on CNTs previously opened by oxidative treatment and proteeted by a spin-on-glass film [69]. Once the carboxyl residues were obtained they were activated with EDC and NHS and the nucleic acids were immobilized (Scheme 3). The hybridization event in this case was evaluated by using a peptide nucleic acid sequence as probe and a target containing a fluorescent residue. 

Novel materials are thus needed to improve the mechanical and chemical stability of the sensor for practical applications in various conditions and, on the other hand, to improve the immobilization scheme in order to ensure sensor stability and the spatial control of biomolectdes. The most important materials for chemical and biochemical sensors include organic polymers, sol-gel systems, semiconductors and other various conducting composites. This chapter reviews the state-of-the-art biosensing materials and addresses the limitations of existing ones. 

BOX 9-2 Immobilization Schemes for Preparation of Solid Phase Antibodies for Use in Immunoassay... 

Enzymes integrated in a biosensor system catalyze the conversion of metabolite molecules to consume or produce detectable species. The change in concentration of the species resulting from enzyme reaction is detected by a corresponding signal transducer. Thus, the analytical performance of these biosensors should critically depend on the activity and stability of the immobilized enzymes. In many cases, the enzyme immobilization is the most important step that determines whether or not it is successful to develop reliable biosensors. In this respect, it is no wonder that a number of new immobilization schemes and materials have been proposed to improve the analytical capabilities of biosensors. 

Immobilization Theory. Chemical immobilization of enzymes results in the enhanced stability of the enzyme in the presence of harsh conditions, such as pH extremes, high temperature, solvents, or a variety of other potential environmental conditions that might otherwise denature or inhibit the catalytic activity of the enzyme. Additionally, immobilization enables development of materials that can be inserted and removed from reactors, reducing the need for separation to remove enzymes from solutions during purification. Several types of chemistries exist for immobilization of enzymes. Typically, the two main types of immobilization strategies are physical entrapment or entanglement, and covalent immobilization. These immobilization schemes are illustrated in Figure 1. 

The functionalization of the tip and substrate surface is of paramount importance for a successful experiment. In the literamre, various immobilization schemes have been reported. Although immobilization by mere physisorption may provide, in some cases, access to suitably modified surfaces, robust chemical functionalization protocols are generally preferred. This is mainly due to the robust and specific nature of the attachment, for example, by formation of covalent bonds. The established surface chemistry relies on the formation of self-assembled monolayers (SAMs) of organothiols on gold (the tips must be coated with gold) or silane chemistry. ... 

Four different strategies for immobilizing zeoUtes on the surface of an electrode can be identified (106). The desired zeoUte can be (1) dispersed within a solid matrix (2) compressed onto a conductive substrate (3) embedded in a polymerie film or (4) covalently anchored. Figure 8.14 outlines these broad approaches and the many different possible electrode structures. Further elaboration on these immobilization schemes can be found in Tables I and II of references (102) and (106), respectively. These references also contain specific examples of preparation procedures for zeolite modified electrodes. 

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