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We studied the controlled-potential
MT adsorption at the TMF-covered crystal surface.
Figure 3a depicts the time-resolved QCM responses to
the MT adsorption at two different potentials
(curves 1 and 3), together with that recorded under
the open-circuit condition (curve 2). All the
injected samples contained 20 ?M MT dissolved in
Tris?HCl solutions. A control experiment involving
the injection of the buffer solution at –0.9 V was
also carried out (curve 4). The average mass changes
per unit area at –0.9 V, open circuit, and –0.3
V, are 366, 237, and 150 ng/cm2, respectively.
Excellent precisions of these mass changes monitored
under different potentials were also observed (%RSD
ranges 1-3%), suggesting that the
potential-dependent MT adsorption is a highly
reproducible process. As can be seen, the mass
change in curve 1 (ca. 336 ng/cm2 or 4.9 x 10-11
mol/cm2) is greater than that in curve 2 (ca. 243 ng/cm2
or 3.6 x 10-11 mol/cm2). When no external potential
is applied, the MT adsorption is governed
predominantly by chemisorption through the formation
of the Hg-thiolates between the TMF and the cysteine
residues on the MT molecules[9, 10, 14, 20, 27, 32,
33]. Since –0.9 V is more negative than the
PZC of mercury surface (ca. –0.6 V[27, 33]), it
appears that the electrostatic attraction between
the electrode and the MTs in the solution can lead
to further MT attachment. On the other hand, when
the electrode surface was positively charged, not
only did the adsorption of MT through electrostatic
interaction occur to a lesser extent, but also the
amount of chemisorbed MTs was decreased. This is
evidenced by the smaller mass increase in curve 3
(about 150 ng/cm2 or 2.2 x 10-11 mol/cm2) than that
in curve 2. We think that the electrostatic
repulsion between the positively charged MTs and the
electrode polarized at –0.3 V probably altered the
mass transfer of the MTs in the solution toward the
electrode surface. As a result of the electrostatic
repulsion, the number of MT molecules that were
brought into contact with the TMF electrode in the
flow system is less, resulting in the affixation of
MT adsorbates of a smaller extent.
We performed a subsequent voltammetric
experiment in an attempt to verify the extent of MT
adsorption. Shown in Figure 3b are a series of
superimposed differential pulse voltammograms (DPVs,
curves 1, 2, and 3) that were acquired immediately
after recording the corresponding FI-EQCM curves in
Figure 3a. The subsequent DPV scan of the MTs
adsorbed at –0.9 V is also shown (curve 1a). In
these DPVs, an oxidation peak was observed at a
potential around –0.74 V, which was absent when
only the buffer solution was injected (curve 4).
The oxidation peak potential in curve 2 or curve 3
is close to the peak at –0.7 V observed at
dropping mercury electrodes [11, 12, 14-16, 18, 19,
34]. Previously we formed a MT adsorbate at a TMF
electrode through an extensive adsorptive contact
(e.g., 3 h) of the TMF surface with a MT solution of
a relatively high concentration (e.g., 20 ?M) and
observed a reversible voltammetric wave at ca.
–0.63 V. We attributed this wave to the oxidation
of the Hg to form mercury-cysteine thiolates [20].
The oxidation peak of the MT adsorbate film formed
at –0.9 V, as will be discussed below, may
actually constitute the following two faradaic
processes:
Cd (Hg) – 2e- = Cd2+
(1)
(Cys-H)n(Cys)mHg (ads) –
ne- = (Cys)m + nHg (ads) + nH+ (2)
Reaction (1) describes the anodic
stripping of Cd that had been accumulated into the
TMF at a potential more negative than the Cd2+
reduction potential (e.g., –0.9 V). Cd is probably
accumulated from Cd2+ in the solution that
originates from the reduction of MT complex
(possibly via the breakage of the Cd-Cysteine bond)
at a negative potential. Reaction (2) symbolically
shows the oxidation of the mercury to form the
cysteine-mercury thiolates (Cys)mHg with the MT
molecules adsorbed onto the Hg surface via
electrostatic attraction.[20] Here, the formation of
the cysteine-mercury thiolates accompanies the loss
of protons on the cysteines (Cys-H) that were not in
contact with the Hg surface. Upon oxidation, the MT
molecules remain attached to the surface (see below
in connection with the discussion on the consecutive
CV or DPV scans). The strong retention of the MT
molecules by the TMF is also consistent with the
fact that cyclic voltammetric (CV) signals of MT
adsorbates formed under the open-circuit condition
did not degrade upon many cycles (e.g., 50-60
cycles) [20].
Another possible explanation is that the peak
in Figure 3b is due to the MT complex oxidation (or
more specifically, the “Cd-MT” complex oxidation
[11, 12, 14-16, 18, 19, 34]) which causes the
cysteine residues to form the Cys-Cys bonds (or the
cystine analog). This would also result in the
release of the cadmium from the MT molecules into
the solution:
(Cys)nCdx(Cys)mHg – ne- =
(Cys-Cys)n/2(Cys)mHg + xCd2+ (3)
where x represents the number of
Cd that can be released and (Cys)n represents the
cysteines that were complexed with the labile Cd in
MT. In this case, the charges under the MT oxidation
peak at –0.74 V are associated with the breakage
of the (Cys)nCdx complexes. (Cys)mHg in Reaction
(3), like that in Reaction (2), are the cysteines
that have formed the cysteine-mercury thiolates. We
should stress that the values for n and m in
Reactions (2) and (3) are not necessarily
equivalent. We just intend to use the symbol n to
represent the number of cysteine or sulfhydryl
groups involved in the MT oxidation at –0.74V in
either of the two possible mechanisms.
Figure 3a
Figure 3b 
Figure 3a & 3b. (a)
Time-resolved QCM responses to the injection of a 20
?M MT solution into the low-volume QCM cell housing
a carbon-based thin mercury film at different
potentials. Curves 1 and 3 were acquired at –0.9 V
and –0.3 V, respectively, whereas curve 2 was
recorded after the injection of the MT solution
under the open-circuit condition. Curve 4
shows the mass-time relationship when 100 ?L of a
Tris?HCl buffer was introduced into the cell at
–0.9 V. Arrows indicate the times when injections
were made and when the samples were completely
replaced by the carrier solution. (b)
Superimposed differential pulse voltammograms
acquired immediately after the individual FIA-QCM
measurements (curves 1-4 in panel (a)). The
background voltammogram in a MT-free Tris?HCl buffer
solution is shown as curve 4. Experimental
conditions for the differential pulse voltammetry
were: pulse width = 50 ms, amplitude = 50 mV, and
pulse period = 0.2 s.
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