Biomedical Engineering Reference
In-Depth Information
protein rotational motion was not arrested within a xerogel. Brennan and coworkers
[50] studied the real-time behavior of monellin sequestered within thin TEOS-derived
xerogels as the protein was challenged by the quencher acrylamide and the chemical
denaturant guanidine hydrochloride. The rotational mobility of glucose odixase (GOD)
and its active site fl avin adenine dinucleotide (FAD) have been investigated by Hartnett
et al.
[51]. The rotational mobility of GOD caged in sol-gel is reduced twice compared
to that in solution, while the active site pocket is similar to that in solution. Hence
while optimizing the infl uencing parameter for encapsulated biomolecules, dynamics
of the proteins inside the “cage” should also be taken into consideration for their better
performance.
16.2.5 Interactions and stability of biomolecules in sol-gel
Even though pore size is ideal for diffusion in reaching the enzyme active site, the
interaction between sol-gel matrices and analytes/substrate also plays a key role in
accessibility. This interaction might be electrostatic, hydrogen bonding and/or hydro-
phobic. Badjic and Kostic [52, 53] studied the interaction between polar silica and
organic compounds. Silica monoliths immersed in solution containing styrene were
evenly dispersed, as styrene could not form hydrogen bonding with silica. After soak-
ing of the silica matrix in electrolyte solutions at a pH value at which pore walls were
negatively charged, anions such as [Fe(CN)
6
]
3
were only partially taken up, whereas
cations such as [Ru(NH
3
)
6
]
3
were excessively taken up by the sol-gel matrix from the
surrounding solution. In either case the internal and external concentrations of the ions
were unequal even after the equilibrium was reached [54].
Several bioactive proteins retained their activity and conformation in sol-gel matri-
ces. The sol-gel entrapped heme proteins such as cytochrome
c
and Mb showed good
stability against pH and thermal perturbations compared to protein in solution [29, 55].
The sol-gel caged cytochrome
c
(cyt
c
) showed high thermal stability due to the exact
fi tting of the protein inside the cage, which was controlled by the protein size [56]. Sol-
gel encapsulated acid phosphatase [57] and bovine carbonic anhydrase II (BCA II)
[58] showed improved thermal stability. This enhanced stability was due to the protective
nature of the cage and the rigidity of the SiO
2
matrix, which reduced the freedom of pep-
tide-chain refolding molecular motions [7]. Trypsin and acid phosphatase entrapped
in silicate sol-gel along with PEG had a half-life 100-fold higher than that of enzyme
in solution at 70ºC [58]. More interestingly the creatine kinase encapsulated in TMOS
sol-gel exhibited fourfold-improved activity upon short exposure to the elevated tem-
peratures [59]. Through resonance Raman spectra, Das
et al.
[45] proved that Mb could
be preserved in native form even at low pH by encapsulating them in sol-gel glasses.
Different types of additives have been employed as stabilizers to the entrapped pro-
teins, including ligand-based stabilizers (Cod III parvalbumin [47], oncomodulin [60]),
methyltrimethoxysilane-based materials (to stabilize atrazine chlorohydrolase) [61],
the incorporation of organosilanes and polymers into lipase-doped silica [62], poly-
(ethylene glycol) (to stabilize acetylcholinesterase and butyrylcholinesterase) [63], and
graft copolymers of polyvinylimidazole and polyvinylpyridine (to stabilize entrapped
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