Biomedical Engineering Reference
In-Depth Information
Application of the biosensor based on the principles of the SPR [37,
38] will enable professionals engaged in the diabetology, implement a
radically new strategy for diagnosing and studying the pathogenic mecha-
nisms of desease, which is as “the number one problem” in endocrinology.
h e principle of biosensor work was described in detail early [32- 37].
Transducer was as glass plate with the gold (20 nm) deposited on the
previously formed layer of chromium (3 nm). It was connected with the
prism of the measuring device through polyphenyl ether with the refrac-
tive index in 1.6. IgG and insulin dissolved in 1 mM PBS (pH 8,2) at the
concentration of 1 μg/ml were used as specii c Ag. In each experiment it
was registered resonant angle at the successively introduction in the mea-
suring cell: distilled water, specii c Ag, solution of BSA in PBS at the con-
centration of 1 mg/mL and antiserum with the dilution from 1:10000 to
1:200. h e time of the exposition was 20 min and at er each measuring the
cell was washed by distilled water.
It was shown [36] that modii cations metal surface by the adsorption
of the Ag was stable over time and was not destroyed by washing mea-
suring cell with PBS. Immobilization Ag was accompanied by changes in
the resonant angle within 3200-3500 arc sec. In the case of polyelectro-
lytes amount of Ag adsorbed on the surface was slightly larger as well as
biosensor was more stable and reproducible than one with bulk surface.
Application dodecanthiol also helps to stabilize the immobilized layer of
protein and increases their density on the surface. Number of physically
adsorbed biological molecules is limited by surface area of the transducer,
which is amenable to optical registration. h is limitation can be prevented
by the location of biological material in three-dimensional space. Of
course, the amount of immobilized material can be increased, and thus it
is possible to further increase the sensitivity of the biosensor. To achieve of
such situation it was therefore proposed a dif erent scheme using polyca-
tions and polyanions [30, 31]. Solution of insulin at the concentration of
0.5 μg/mL in 1 mM TB (pH 8.2) was introduced into a measuring cell for
20 min at the room temperature. h en the cell was washed by PBS and
i lled successively polyethylenamine chloride (PAA) - polystyrene sul-
phate hydrochloride (PSS) - PAA at a concentration of 1 μg /mL in water.
Cell was washed by water and treated by insulin during 20 min and washed
again. At last it was i lled by 0.5% solution of GA for 15 min and polyelec-
trolytes were removed by 50 mM buf er (pH 4.0 and 8.0) to form two-layer
sensitive surface.
It was established that the formation of a double layer of insulin on the
transducer surface increased the sensitivity of the immune biosensor to
specii c Ab. h is is particularly important in the study of the early stages
