Biomedical Engineering Reference
In-Depth Information
9.5
Nanostructured Bacteriorodhopsin Matrices for Liquid Sensing
Starting from the effects of volatile anesthetics on the structure of bacteriorhodopsin (bR)
in the purple membrane (PM) in solution this work tries to investigate the interaction of
ether and hydrocarbon-type anesthetic vapors with self-assembled bR thin films. A dedi-
cated constant flux chamber has been built to maintain the sample in a rather constant
atmosphere of anesthetics, during the absorption measurements. The kinetics of absorp-
tion and desorption have been determined (Figure 9.7). The results obtained have been
compared with those of bR in solution (36).
The suspensions of PM have been obtained by dilution of the stock suspension (5 mg
ml
1
) with distilled water. The concentration was checked spectrophotometrically; in
particular, the peak of proteins (at 280 nm) should not show the characteristic scattering
signal. The concentration chosen for the experiments was 0.3 mg ml
1
.
In the case of bR in a solution, the anesthetic solution was just added to the bR
solution and stirred for 10 min. This was done to equilibrate the system. In the case of
chloroform, not mixable with water, the cuvette was shaken to promote the contact of
PM with the solvent. The self-assembled samples (drop-coating multilayers) were
formed by spreading 1 ml of the original stock suspension of PM over the flat quartz
surface (75).
This operation had to be carried out carefully to obtain a homogeneous distribution of
material over the surface. Moreover, the time of drying, by exposure to the ambient atmos-
phere, was important to have a good sample. After 12 h the self-assembled thick sample
containing 5 mg of PM patches, 80% of which is bR (MW 26,000) assembled over a surface
of 1.5 cm
2
, was ready. In our case, the bR film was exposed to the anesthetic vapors,
coming to the reaction chamber through tubes from the separate vessel containing the
anesthetic in liquid form (saturated steam). The amount of the incoming anesthetic vapors
was qualitatively monitored, injecting by a syringe different amounts of vapor proceeding
from the heated vessel.
In Figure 9.8(a), a self-assembled film of bR in PM was treated with diethylether vapors:
(a) the absorption spectra of the film, pure and treated with diethylether for different peri-
ods of time, is shown; (b) the decrease in the absorbance at 570 nm was recorded during
the injection of successive amounts of vapors of anesthetic. In Figure 9.8(a), the absorbance
maximum of the PM-thick film appears at 563 nm and the intensity of absorbance
decreases with the treatment with anesthetic. At the same time, a broad band occurs at
about 400 nm during the treatment and disappears after relaxation. It is difficult to give an
interpretation of such a band, which cannot be precisely attributed to the anesthetic-
induced bR480 and bR380 forms; further experiments will probably clarify the role of this
band. Nevertheless, this figure reveals either the functionality of the protein or the
reversibility of the process qualitatively comparable with that obtained in solution. Zones
A, B, C, D are evident in Figure 9.8(b), each of them corresponds to a progressive increase
of the anesthetic pressure zone A is more wide than the others, this could be due to a
higher pressure of the vapor injected in the cell or an effect of saturation of the sample that
makes the successive binding in the other zones more difficult. After the decrease of the
absorbance of 0.1 units, compressed air was injected in three steps (regions 1, 2, 3), obtain-
ing almost the total recovery of the structure after 15 min.
The work pointed out the possibility of using the self-assembled bR films as sensitive
layers for optical biosensors for anesthetics. The important point is that the optical
properties of the layer are reversible for not only ether-type anesthetics but also
hydrocarbon-type such as chloroform. It allows one to consider such films as good candi-
dates for biosensors of continuous monitoring of anesthetics.
