Brazilian Journal of Analytical Chemistry - BRJAC - Brazilian Journal ...

confirmed by the catalytic effect of the prosthetic group
and molecular recognition of 5-HT by the selective cavities
imprinted in MIP.
I / µA
-0.3
-0.2
-0.1
0.0
0.1
Br J Anal Chem
Molecularly Imprinted Polymer with hemin - MIP
Non-Imprinted Polymer with hemin - NIP
Molecularly Imprinted Polymer without hemin
Without Column
0 250 500 750 1000
Time / s
fi g u re 7. am p e r o g r a m s f o r T h e deTeCTion o f T h e p ro d u C T o f T h e o x i d a T i o n
o f 5-hT. measuremenTs w e r e Carried o u T in 500 µm o l l -1 5-hT, [h 2 o 2 ]
= 310 µm o l l -1 , s a m p l e v o l u m e = 200 µm o l, buffer s o l u T i o n f l o w-ra T e
= 2.0 ml m i n -1 , Tris buffer s o l u T i o n = 0.1 m m o l l -1 , ph = 8.0, a n d a
m i n iC o l u m n p a C ke d w iT h 35 m g o f T h e mip w a s used.
3.4. an a l y T i C a l f e a T u re s a n d kineTiC a n a l y s i s o f T h e imprinTed
p o l y m e r
Under optimized conditions, a calibration curve was
obtained for 5-HT concentrations ranging from 1.0 to
1000 µmol L −1 . The sensitivity was 0.4 nA/µmol L −1 ,
and a satisfactory correlation coefficient (r > 0.999)
was observed. The detection and quantification limits
expressed according to IUPAC recommendations were
0.30 and 0.98 µmol L −1 [15].
The kinetic parameters of the imprinted polymer
were evaluated based on the Michaelis–Menten kinetics.
Figure 8 represents the Lineweaver–Burk plot for
the MIP in the presence of different concentrations of
5-HT in 0.1 mmol L -1 Tris-HCl buffer at pH 8.0. It is seen
that a non linear dependence of the cathodic current
on 5-HT concentration in the whole range from 50 to
1500 µmol L -1 is obtained (insert Figure 8), as would be
anticipated from a Michaelis-Menten type process.
∆j -1 / µA -1 cm 2
2.4
2.0
1.6
1.2
0.8
0.4
0
0 250500 750100012501500 [5-HT] / µmol dm-3 -0.005 0.000 0.005 0.010 0.015 0.020 0.025
∆j / µA cm -2
[5-HT] -1 / µmol -1 dm 3
fi g u re 8. lineweaver–bu r k p l o T f o r T h e mip in T h e presenCe o f differe
n T C o n C e n T r a T i o n s o f 5-hT in 0.1 m m o l l -1 Tris-hCl buffer a T ph 8.0.
inserT: TypiCal d y n a m iC p r o f i l e o f T h e C a T a l y T i C re a C T i o n o f 5-hT C a T alyzed
by T h e peroxidase-l i ke mip.
7
6
5
4
3
2
1
synThesis, C h a r a C T e r i z a T i o n an d kineTiC sTudies of
mip - based biomimeTiC Ca T a l y s T fo r seleCTive se r o T o n i n ox i d a T i o n
The apparent K m value represents the affinity of the
polymer for the substrate and a low value indicates
high affinity. In the present case, K m for the MIP represents
the affinity of the active site for the substrate
5-HT. Thus, in the Michaelis–Menten model initial rates
of reaction are plotted against the substrate concentration
(5-HT) based on a hyperbolic relation as follow:
In order to have a linear relation, the Lineweaver–
Burk equation can be written as [26,27]:
where j is the current density in the steady-state
s
after the addition of substrate, [5-HT] is the substrate
concentration in the bulk solution, V is the
max
maximum rate measured under saturated substrate
conditions (i.e. maximum current density measured
(1)
(2)
under saturated substrate conditions), and
K is
app
m
the apparent Michaelis-Menten. A low K m value indicates
a strong substrate affinity. A value of 818
µmol dm -3 and 9.05 µA cm -2 for K m and V max were
obtained, respectively. The obtained K m value is
smaller than those observed for peroxidase K m (1.5
mmol L -1 for oxidation of phenolic compounds) [28],
it indicates a potential for application of MIP-5-HT
catalyst for analytical purposes, for example, serotonin
determination in biological samples. However,
in native enzymes many difficulties in practical use
exist due to their sensitive properties such as instability
against high temperature, organic solvents, and
several different pH conditions, etc. Furthermore, the
K m value for imprinted polymer is lower than those
reported values in the literature for systems containing
naturally immobilized peroxidases [29-36], giving
evidence for the higher sensitivity and stability of the
imprinted polymer. These results show that imprinted
polymer could be used as a peroxidase mimicking
catalyst, indicating that it could be an alternative for
the peroxidase enzyme.
3.5. re p e a T a b i l i T y an d sTabiliTy of Th e mip
The stability of the MIP was investigated. After successive
measurements for a long period, no significant
change was observed after 100 determinations. The
precision of the responses expressed in terms of the
relative standard deviation (RSD), were 1.3 and 1.7%
for n = 6 analyses of standard solutions containing
50 and 750 μmol L −1 5-HT, respectively. This shows
that MIP has good stability and reproducibility.
89