Progress of MB replacement by cholesterol is manifested in a
monotonic increase of the electrochemical signal of MB in DPV
with increase of target cholesterol concentration. It can be seen
that initially, when the cholesterol concentration was zero, there
was no MB peak in the 0 to þ0.4 V regions even when the MB is
present in the Grp-β-CD (Fig. 3). This indicates that the MB is
bound in the cavity of the β-CD therefore, not able to give any
redox signal into the electrode. Though there is some probability
of MB, which is hydrophilic in nature, to get adsorbed on the
graphene sheets, these will not make any difference to the CV and
DPV results, as MB remains intact on the surface of graphene sheets in the beginning as well as at the end of the experiment. A
control experiment carried out to exclude the possibility of
desorption of MB from the graphene surface during cholesterol
detection proved that only the MB molecule coming out from the
CD cavity is responsible for the DPV signal (Fig. S4). After addition
of the cholesterol, it readily moved inside the cavity of the β-CD,
replacing the MB into the solution and hence, a characteristic peak
was obtained at þ0.05 V. Cholesterol can only go inside a hydrophobic
pocket (that is of β-CD) and would replace MB molecule, as
cholesterol has higher affinity towards CD as compared to MB. The
measured binding constant between cholesterol and β-CD-G was
1.6 M1 which is similar to the earlier observed value with free
β-CD (Breslow and Zhang, 1996). The sensing signal of Grp-β-CD
system increased periodically with increasing concentration of
cholesterol, up to 100 mM, as presented in the calibration curve
(Fig. 3 inset). Above 100 mM, the signal reached its saturation.
Detection limit was less than 1 mM which was quite low and
satisfactory with respect to other recently reported articles. Table 1
illustrates few of the recent literatures on cholesterol biosensors,
through both enzymatic and non-enzymatic sensing routes, establishing
the edge of the present work. The detection limit and
sensitivity of the present sensing matrix is comparatively better
than the reported ones, with a decent sensing time. The peak
potential of MB showed a little shift towards the positive potential.
Being a pH dependent redox indicator, the peak potential of MB
shifted from 0.05 to 0.1 V with decreasing pH, as the cholesterol
solution used in DPV was soluble in acidic buffer. Therefore, in the
higher concentration range, the MB peak in DPV shifted slightly
towards right. The cyclic stability of the sensing matrix was also
tested, before and after the addition of 10 mM cholesterol solution,
by performing 100 cycles in cyclic voltammetry at a scan rate of
100 mV/s, in the potential window of 0.6 to þ0.6 V. It is evident
from obtained voltammograms that the sensing matrix was quite
stable towards its cyclability (Figs. S1 and S2).