Impedimetric and electrode kinetic dynamics of DNA aptamer nanobiosensors for estrogeneous endocrine disruptors
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Date
2011
Authors
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Publisher
University of the Western Cape
Abstract
In this work, DNA aptamer biosensor systems were developed for the detection of l7p-estradiol - an estrogeneous endocrine disrupting chemical (EDC). Endocrine disrupting chemicals are group of compounds that impact negatively on the endocrine system of humans and wildlife. High concentrations of l7p-estradiol in water or food chain disrupts the physiology of the endocrine system of various animal species, leading to feminisation in fish and stimulates the proliferation of cancer cells in humans. Aptasensor systems for the determination of l7pestradiol were prepared with three immobilization platforms: (i) poly(3,4- ethylenedioxythiophene) {PEDOT} doped with gold nanoparticles (AuNPs) to form PEDOTIAuNPs polymeric nanocomposite, (ii) generation 1 poly(propylene thiophenoimine)-copoly(
3 ,4-ethy lenedioxythiophene) dendritic star copolymer (G 1PPT -co-PEDOT), and (iii) generation 2 poly (propylene thiophenoimine)-co-poly(3,4-ethylenedioxythiophene) dendritic star copolymer (G2PPT-co-PEDOT). The morphological properties of the sensor platforms were
interrogated by scanning emission microscopy (SEM) and atomic force microscopy (AFM), while their spectroscopic characteristics were studied by Fourier transform infra red spectroscopy (FTIR) and fluorescence spectroscopy. The electrochemical behaviour of the
platforms and the aptasensors were studied by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV). The DNA aptamer developed for detecting 17~-estradiol and which was used in the fabrication of all aptamer biosensors in this study is a 76-mer biotinylated aptamer (5'-BiotinGCTTCCAGCTTATTGAATTACACGCAGAGG TAGCGGCTCTGCGCATTCAATGCTGCGCGCTGAAGCGCGGAAGC-3'). AulPEDOTIAuNPslAptamer (platform 1) was obtained by covalently attaching streptavidin to the polymeric nanocomposite platform using carbodiimide chemistry and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically using cyclic voltammetry and square wave voltammetry in the presence of [Fe(CN)6J 3-/4- as a redox probe. The signal current observed was inversely proportional to the concentration of 17Bestradiol. The aptasensor demonstrated specificity toward 17~-estradiol. The detectable concentration range of the 17B estradiol was 0.01 nM-O .09 nM with a detection limit of 3.2 pM. The 76-mer biotinylated aptamer for 17~-estradiol was incorporated into a generation 1 poly(propylenethiophenoimine )-co-poly(3 ,4-ethylenedioxythiophene) dendritic star copolymer modified Au electrode via biotin-avidin interaction (platform 2). The Bode plot shows that the charge transfer dynamics of the nanoelectrode can be frequency modulated while the AulG 1PPTco- PEDOT nanoelectrode exhibited greater semi-conductor behavior (higher phase angle value) than AulG 1PPT due to the incorporation of charged functionalized dendrimer at low frequencies (100 mHz). The biosensor response to 17~-estradiol was based on the decrease in the SWV current as the EDC binds to the ssDNA aptamer on the biosensor. The dynamic linear range of the sensor was 0.01-0.07 nM with a detection limit of7.27 pM. Synthesis of electro synthetic generation G2PPT-co-PEDOT (platform 3) was performed by copolymerization of PEDOT with G2PPT dendrimer modified electrode immersed in a solution
of 0.1 M LiCI04 containing 0.1 M EDOT monomer and 0.1 M sodium dodecyl sulphate (SDS) for ten (10) cycles. The electrochemical behaviour of the dendritic star copolymer was investigated with CV and EIS in LiCI04 and phosphate buffer solutions. The results show that the electrochemical deposition of G2PPT-co-PEDOT on gold electrode decreased the electrochemical charge transfer resistance when compared to AuiPEDOTILiCI04 and AuiLiCI04 interfaces. Bode impedimetric analysis indicates that G2PPT-co-PEDOT is a semiconductor. The
fabrication of two novel aptasensors (based on platforms 2 and 3) simultaneously on a screen printed micro array electrode of 96-well multichannel electrochemical robotic sensor testing system for the detection of endocrine disrupting l7~-estradiol, was also carried out. The
aptasensors responses to l7~-estradiol, based on the decrease in the SWV current, were evaluated.
Description
Philosophiae Doctor - PhD
Keywords
17b estradiol, DNA aptamers, Electrochemical impedance spectroscopy, Electrode kinetics, Endocrine disrupting chemicals, Estrogens, Gold nanoparticles, Impedimetric nanobiosensor, Self assembled monolayer, SELEX