We devised and prepared a conducting molecularly imprinted polymer (MIP) for human serum albumin (HSA) determination using semi-covalent imprinting. The bis(2,2'-bithien-5-yl)methane units constituted the MIP backbone. This MIP was deposited as a thin film on an Au electrode by oxidative potentiodynamic electropolymerization to fabricate an electrochemical chemosensor. The HSA template imprinting, and then its releasing from the MIP was confirmed by the differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), XPS, and PM-IRRAS measurements as well as by AFM imaging. Semi-covalent imprinting provided a very well defined locations of recognition sites in the MIP molecular cavities. These sites populated the imprinted cavities or the MIP surface only. The DPV and EIS response of the MIP film coated electrode to the HSA analyte was linear in the range of 0.8 to 20 and 4 to 80 µg/mL HSA, respectively, with the limit of detection of 16.6 and 800 ng/mL, respectively. The impressively high imprinting factor reached, exceeding 20, strongly confirmed that semi-covalent imprinting resulted in formation of a large number of very well defined molecular cavities with high affinity to the HSA molecules. The MIP selectivity against low-(molecular weight) interferences, common for physiological fluids, such as blood and urea, was very high. There was no response to the presence of these interferences at concentrations encountered in the samples analyzed. Moreover, the chemosensor selectivity to the myoglobin and cytochrome c interferences was excellent while that to lysozyme was slightly lower but still high. The chemosensor was useful for determination of abnormal HSA concentration in a control blood serum.
We
present an improved approach for the preparation of highly selective
and homogeneous molecular cavities in molecularly imprinted polymers
(MIPs) via the combination of surface imprinting and semi-covalent
imprinting. Toward that, first, a colloidal crystal mold was prepared
via the Langmuir–Blodgett (LB) technique. Then, human
chorionic gonadotropin (hCG) template protein was immobilized on the
colloidal crystal mold. Later, hCG derivatization with electroactive
functional monomers via amide chemistry was performed. In a final
step, optimized potentiostatic polymerization of 2,3′-bithiophene
enabled depositing an MIP film as the macroporous structure. This
synergistic strategy resulted in the formation of molecularly imprinted
cavities exclusively on the internal surface of the macropores, which
were accessible after dissolution of silica molds. The recognition
of hCG by the macroporous MIP film was transduced with the help of
electric transducers, namely, extended-gate field-effect transistors
(EG-FET) and capacitive impedimetry (CI). These readout strategies
offered the ability to create chemosensors for the label-free determination
of the hCG hormone. Other than the simple confirmation of pregnancy,
hCG assay is a common tool for the diagnosis and follow-up of ectopic
pregnancy or trophoblast tumors. Concentration measurements with these
EG-FET and CI-based devices allowed real-time measurements of hCG
in the range of 0.8–50 and 0.17–2.0 fM, respectively,
in 10 mM carbonate buffer (pH = 10). Moreover, the selectivity of
chemosensors with respect to protein interferences was very high.
In
this Review, we have summarized recent trends in protein template
imprinting. We emphasized a new trend in surface imprinting, namely,
oriented protein immobilization. Site-directed proteins were assembled
through specially selected functionalities. These efforts resulted
in a preferably oriented homogeneous protein construct with decreased
protein conformation changes during imprinting. Moreover, the maximum
functionality for protein recognition was utilized. Various strategies
were exploited for oriented protein immobilization, including covalent
immobilization through a boronic acid group, metal coordinating center,
and aptamer-based immobilization. Moreover, we have discussed the
involvement of semicovalent as well as covalent imprinting. Interestingly,
these approaches provided additional recognition sites in the molecular
cavities imprinted. Therefore, these molecular cavities were highly
selective, and the binding kinetics was improved.
Homogenous nanostructuration of molecularly imprinted
polymer (MIP)
films for follicle-stimulating hormone (FSH)-sensing was achieved
by using optimized colloidal crystals as a hard mold. Introduction
of a heating step after assembling colloidal crystals of silica beads
promoted their adhesion. Thus, precise assembling of beads was not
disturbed during further multisteps of surface imprinting, and crack-free
hexagonal packing was maintained. Scanning electron microscopy imaging
confirmed hexagonal packing of silica colloidal crystals as well as
homogenous nanostructuration in MIP films. FSH immobilization over
silica beads and later its derivatization with electroactive functional
monomers was confirmed by X-ray photoelectron spectroscopy analysis.
The nanostructured molecular recognition films prepared in this way
were combined with an electrochemical transducer in order to design
a capacitive impedimetry-based chemosensing system. It was tested
for the determination of FSH in the range from 0.1 fM to 100 pM in
10 mM 2-(N-morpholino) ethane sulfonic acid buffer (pH = 4.2). The
detection limit of the chemosensor was 0.1 fM, showing a high selectivity
with respect to common protein interferences as well as other protein
hormones of the gonadotropin family.
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