Biomedical Engineering Reference
In-Depth Information
Fig. 6.19
Concept of OS-FET
per molecule (pI=5.2), which is one of routinely used fusion tags for recombinant
protein production in E
scherichiacoli
, partly because it acts as an intramolecular
chaperone and increases the expression yields of the fused proteins. Due to the large
negative charge of MBP, the reassociation of the two chains is expected to induce
the charge density changes at the gate surface. This charge density change can be
detected as a shift of the V
T
of the FET. Thus, the measurement of V
T
allows a
direct, simple, and nonlabeled OS-IA.
The FET chip is immersed in a measurement solution together with an Ag/AgCl
reference electrode with saturated KCl solution. The potential of a measurement
solution is controlled and fixed by the gate voltage (V
G
) through the reference
electrode. We have paid special attention to the buffer concentration used for
measuring charge density change at the gate surface. The potential change induced
by adsorption of proteins at the gate surface was reported to be dependent on the
electrolyte concentration. It is, therefore, important to optimize the Debye length
at the gate insulator/solution interface. In the present study, a 0.025 M phosphate
buffer solution was used for measuring charge density change at the gate surface,
while the conventional reaction mixture was used during OS-IA reaction.
The shift of the gate voltage (V
G
/-drain current (I
D
) characteristic, the V
T
shift,
was measured after reacting BPA with MBP-linked V
L
(MBP-V
L
)andV
H
chains
(Fig.
6.20
). The initial concentration of BPA added to the OS-FET chip was 1M.
The resultant V
T
shifted in the positive direction by the amount of 13.1 mV due to
the negative charge of MBP (Fig.
6.20
a). On the other hand, the effect of nonspecific
adsorption of MBP-VL on the electrical signal was investigated by use of the
control FET (Fig.
6.20
b). When the control FET chip with the immobilized V
H
chains was reacted with the MBP-VL solution without BPA, the V
T
shift was
4.5 mV to the positive direction. The electrical signal of the control FET implies
not only the antigen-independent basal binding of MBP-V
L
but also the presence of
unexpected BPA, which might be included in the conventional disposal tube used in
the experiments. Thus, for the highly sensitive detection of small uncharged antigens
using the OS-FET, it is important to perform the differential measurement by the use
of the control FET and the OS-FET.
