Biomedical Engineering Reference
In-Depth Information
Rich variety of layered structures
(the number of layers and
adsorption sequences)
Most charged proteins
Easy and mild procedure
(avoidance of protein denaturation)
FIGURE 12.19
Advantageous features of protein immobilization by LbL technique.
be seen in the slow diffusion of substrates through the fi lm when rather thick fi lms were used. This
disadvantage may originate from dense packing of the lipid molecules in LB fi lms.
Unlike LB fi lms, LbL fi lms are less dense component molecules [118]. Most proteins, especially
water-soluble proteins, have charged sites on their surface, and thus the electrostatic LbL adsorption
is quite useful for the construction of various protein organizations. It was actually demonstrated that
a large number of water-soluble proteins were assembled in combination with oppositely charged
polyelectrolytes. As summarized in Figure 12.19, several advantages exist in the LbL assembly
of proteins and a wide variety of layered structures can be prepared. For example, the number of
layers and the layering sequence are easily modifi ed. Most charged proteins are applicable in this
assembling technique. Protein denaturation would be minimized because the adsorption process is
conducted under mild conditions.
In order to demonstrate the advantage of the LbL method concerning wide freedom in struc-
tural designs, a multienzyme reactor was prepared (see Figure 12.20) [119]. The adsorption behav-
iors of the two kinds of enzyme, GOD and glucoamylase (GA), were quantitatively evaluated using
a QCM technique, because the resonant frequency of the QCM sensitively changes due to the mass
adsorption on its electrodes. Systematic QCM analyses revealed that adsorption of both the proteins
can be done with appropriately selected counterionized polyelectrolytes, and that layered sequences
can be freely modifi ed between these enzymes without causing any interference in the amount of
adsorption. Multienzyme fi lms with GOD and GA were prepared on porous fi lter paper that was
immobilized at the bottom of solution container [119]. Substrate (starch) solution was added on the
multienzyme fi lm and the solution containing the products was collected as fi ltrate. The reaction
scheme of the multienzyme reactor is shown in Figure 12.20B. Hydrolysis of the glycoside bond in
starch by GA produces glucose. Glucose is converted to gluconolactone by GOD, with H
2
O
2
as a
coproduct. An aqueous solution of water-soluble starch in 0.1 M PIPES buffer (pH 7.0) was placed
on the enzyme-immobilized ultrafi lters in the upper cup. Filtration was started by applying pressure
to the upper cups with a syringe. The fi ltrate was added to a mixed solution of peroxidase (POD) and
indicator dye DA67, and the concentration of the resulting H
2
O
2
was evaluated from the change in
absorbance at 665 nm. The concentration of unreacted starch was assayed by the iodostarch reac-
tion. However, unreacted starch was not detected in the fi ltrate at all. Starch cannot pass through the
fi lter, thus it allows separation of substrate and products without additional procedures. Systematic
research on reactor performance using LbL fi lms in various layer designs was carried out, revealing
the importance of layered order of the two enzymes and appropriate separation of the enzyme layers
for effi cient material conversion. The advantages of the multienzyme reactor preparation by the LbL
technique can be described as follows. The most pronounced advantage is freedom in fi lm construc-
tion, and various fi lm structures are easily obtainable by quite simple procedures, that is, a change
in the dipping sequences provides fi lms with desirable layering structures. The simplicity of the
