Trapped Biomolecules

Biologically active molecules are sometimes trapped in PDMS when end-functionalized PDMS chains are linked into a network structure. This method has been done, for example, with a lipase enzyme. The PDMS plays a beneficial role as an activator or protective agent. Similar results were found for the enzyme a-chymotripsin, with some short-chain poly(ethylene oxide) used to enhance enzymatic activity. It is also possible to generate microtopographic patterns that affect Escherichia coU biofilm formation on PDMS surfaces. 

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Another important consideration is the relative size of enzymes versus that of micropores (less than 2nm) in environmental particles which are especially abundant in noncrystalline mineral phases. Simple biomolecules (e.g., glucose, MW = 180 Da) can readily enter into micropores and can become stabilized by reacting with the mineral surface, whereas large macromolecules such as enzymes [e.g., laccase, MW = 60,000Da and diameter = 5nm (Andersen et al., 1996)] cannot enter and react with the trapped biomolecules. 

Electrokinetic phenomena of the second kind have received mostly academic interest, but there may be important and unexpected applications. Already, second-kind electroosmosis has been implicated as a mechanism for superUmiting current in electrodialysis. Space-charge formation at nanochannel/microchannel junctions has also been exploited to trap biomolecules, although in that case the flow is undesirable since it interferes with trapping. The speed of second-kind electrophoresis may be useful in separation or mixing, but the need to apply a large DC voltage may limit its applicability. 

Active enzymes were encapsulated into a sol-gel matrix for the first time in 1990 719 About 60 different types of hybrid bioceramic materials with inotganic matrices made from silicon, titanium, and zirconium oxides Ti02-cellulose composites etc. were described. Recentiy, bioceramic sensors, solid electrolytes, electrochemical biosensors, etc. have been surveyed in a review. The moderate temperatures and mild hydrolytic and polymerization conditions in sol-gel reactions of alkoxides make it possible to trap proteins during matrix formation. This prevent proteins denaturation. The high stability of the trapped biomolecules, the inertness, the large specific surface, the porosity, and the optical transparency of the matrix facilitate use of sol-gel immobilization. The principal approaches ate considered below. 

The paper is organized in three parts. First, the effects of nanoconfinement on the structure of the sol-gel trapped biomolecule are discussed. Second, from the results on apparent reactivity of these biomaterials, the effects of the matrix on the reactivity of trapped enzyme are elucidated. Finally, the interaction of the confined biomolecules with exogenous ligands/substrates and the applications of these materials in the area of molecular biorecognition are discussed. The reaction chemistries of biologically active molecules in die nanostructured materials have been crucial in establishing the role of the matrix upon the structure and reactivity of the confined proteins. 

The effects of sol-gel confinement upon the structure and reactivity of trapped biomolecules are listed in Table n. 

FIGURE 9.3 Typical SCX-RAM column separation profile peaks (a) represent physical exclusion by pore size. Trapped retained biomolecules are separated by a gradient in the second step (b). Conditions column—LiChrospher 60 XDS (S03/Diol), 25 x 4 mm I.D., flow rate— 0.5 mL/min, gradient from 0 to 1M NaCl in 20 mM KH2P04 pH 2.5, containing 5% ACN in 30 min. Sample 100 pL Human Hemofiltrate (3.7mg/mL), UV detection at 214 nm. 

The above historical outline refers mainly to the EPR of transition ions. Key events in the development of radical bioEPR were the synthesis and binding to biomolecules of stable spin labels in 1965 in Stanford (e.g., Griffith and McConnell 1966) and the discovery of spin traps in the second half of the 1960s by the groups of M. Iwamura and N. Inamoto in Tokyo A. Mackor et al. in Amsterdam and E. G. Janzen and B. J. Blackburn in Athens, Georgia (e.g., Janzen 1971), and their subsequent application in biological systems by J. R. Harbour and J. R. Bolton in London, Ontario (Harbour and Bolton 1975). 

Similarly to the above-mentioned entrapment of proteins by biomimetic routes, the sol-gel procedure is a useful method for the encapsulation of enzymes and other biological material due to the mild conditions required for the preparation of the silica networks [54,55]. The confinement of the enzyme in the pores of the silica matrix preserves its catalytic activity, since it prevents irreversible structural deformations in the biomolecule. The silica matrix may exert a protective effect against enzyme denaturation even under harsh conditions, as recently reported by Frenkel-Mullerad and Avnir [56] for physically trapped phosphatase enzymes within silica matrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have been employed for this purpose, as extensively reviewed by Gill and Ballesteros [57], and the resulting materials have been applied in the construction of optical and electrochemical biosensor devices. Optimization of the sol-gel process is required to prevent denaturation of encapsulated enzymes. Alcohol released during the... 

With regard to biosensor applications, a wide variety of electrochemically active species (ferrocene, ruthenium complexes, or carbon and metal (Pt, Pd, Au...) [185,186] were also introduced into the sol-gel matrices or adsorbed to improve the electron transfer from the biomolecules to the conductive support [187,188]. For instance, glucose oxidase has been trapped in organically modified sol-gel chitosan composite with adsorbed ferrocene to construct a low-cost biosensor exhibiting high sensitivity and good stability [189]. 

This chapter will cover all DCC systems that use proteins to influence the population distribution of DCLs, with the exception of Ramstrdm s coupled DCC systems that are discussed separately in Chapter 6. The DCL systems under discussion are loosely grouped according to the chemical reactions used to set up the DCLs. The chemistry used to make protein-directed DCLs is in many ways fundamental, as it defines the conditions in which the protein can or cannot operate as a molecular trap for the DCL. Compatibility of the various synthetic reactions with protein targets will establish whether a genuine adaptive DCL can be constructed, or whether alternative approaches are needed to separate the fragile biomolecule from the DCL chemistry. 

Although many polyphenolic compounds can quench DPPH215 218 and scavenge radicals in competition with spin traps,225 232,278 consideration must also be given to the fate of the resultant phenoxyl radicals, which can include their direct reaction with important biomolecules, further oxidation to cytotoxic quinones and, in the case of semiquinones, their reduction of oxygen to superoxide. Thus,... 

SA McLuckey, GJ Van Berkel, GL Glish, EC Huang, JD Henion. Ion spray liquid chromatography/ion trap mass spectrometry determination of biomolecules. Anal Chem 63 375-383, 1991. 

Fig. 4 Model of cellular uptake of Lac complexes. Complexes adhere to cells due to electrostatics (a) and enter through endocytosis (b, c). Low aM complexes remain trapped in the endosome (d). High aM complexes escape the endosome (e) where released DNA may form aggregates with cationic biomolecules (f) or the complexes are less able to dissociate and less DNA is available (g). Reproduced with permission from [21], Copyright 2005 John Wiley Sons Limited...

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