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- A molecularly imprinted sensor for sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative stress biomarkerPublication . Martins, Gabriela V.; Fortunato, Elvira; Fernandes, Helena R.; Sales, M. Goreti F.Early diagnosis of Oxidative Stress (OS) biomarkers can be used as a crucial tool in cancer prevention, treatment and survival. In this context, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a repair product of oxidized guanine lesions and has been acknowledged as a suitable biomarker of OS1. Under this scope, a simple and sensitive molecularly imprinted (MIP)-based sensor for detection of urinary 8-OHdG has been designed via electrochemical polymerization. The biomimetic film was assembled in-situ on the gold-modified electrode through electropolymerization of phenol monomer combined with the target molecule 8-OHdG. The electropolymerization of phenol was performed by Cyclic Voltammetry (CV) over the potential range 0.1 to 0.9 V in PBS buffer at pH 7.4, enabling the formation of a non-conductive layer. Several experimental parameters, such as, the initial concentration of the monomer and the ratio template-monomer, have been carefully optimized and the electrochemical performance of the designed MIP sensor was investigated by CV and Electrochemical Impedance Spectroscopy (EIS). In parallel, RAMAN and FTIR spectroscopies comproved the formation of polyphenol films on the electrode surface by electrochemical oxidation of phenol. Our results demonstrated that 8-OHdG molecule was successfully entrapped into the polymeric matrix, enabling a three-dimensional structure with numerous imprinted cavities sites. The developed electrochemical biosensor showed high sensitivity and selectivity towards 8-OHdG over the concentration range [0.1 - 100] pg/ml. Moreover, it was employed to detect 8-OHdG in urine samples as a non-invasive approach to assess the extent of DNA oxidative damage. Overall, this label-free biosensor constitutes a promising low-cost tool to be implemented as an easy-to-use protocol for sensitive detection of 8-OHdG in biological samples.
- A biomimetic sensor for monitoring oxidative stress biomarker in point-of-carePublication . Martins, Gabriela V.; Fortunato, Elvira; Fernandes, Helena R.; Sales, M. Goreti F.Free radicals and other reactive species are constantly generated in vivo and can cause oxidative damage to biomolecules, a process that seems to play an important role at the origin of cancer. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a major product of DNA hydroxylation and is considered a biomarker of damage caused by oxidative stress (OS). Thus, early diagnosis of OS biomarkers may be used as a fundamental tool in cancer prevention and in more efficient therapeutic strategies. For this purpose, a biomimetic sensor for 8-OHdG detection and quantification by Electrochemical Impedance Spectroscopy (EIS) is proposed herein. The biomimetic sensor was obtained by modifying a clean gold (Au) electrode with a OH-terminal thiol compound, followed by direct electropolymerization of phenol in the presence of 8-OHdG. The biomimetic/Au acted as working electrode, while glassy carbon and Ag/AgCl were used as counter and reference electrodes, respectively. Electropolymerization of phenol was performed by Cyclic Voltammetry (CV) over the potential range 0.2 to 0.9 V in pH 7.0 PBS buffer, enabling the formation of a non-conductive layer. Non-imprinted materials (NIM) were also performed by removing the template from the procedure and, then, the ability of the polymer to interact non-specifically with the template was measured. Preliminary results showed the development of a direct and label-free biomimetic sensor with good performance, stability and sensibility. In particular, only MIP material was able to rebind to the target molecule and produce a linear response against EIS on the range 0.010 to 10ng/ml. Overall, the biosensor described herein is simple, precise and may allow routine use for biological samples on-site.
- Paper-based (bio)sensor for label-free detection of 3-nitrotyrosine in human urine samples using molecular imprinted polymerPublication . Martins, Gabriela V.; Marques, Ana C.; Fortunato, Elvira; Sales, Maria Goreti FerreiraOver the last years, paper technology has been widely spread as a more affordable, sustainable and reliable support material to be incorporated in the design of point-of-care (POC) diagnostic devices. However, the single work employing a paper-based device for 3-nitrotyrosine (3-NT), a relevant biomarker for oxidative stress (OS) that is a major origin for many diseases, is incapable of reading successfully complex samples because every species that oxidizes before ~0.75 V will also contribute to the final response. Thus, the introduction of a selective element was made into this set-up by including a molecularly-imprinted polymer (MIP) tailored in-situ. Herein, a novel MIP for 3-NT was assembled directly on a paper platform, made conductive with carbon ink and suitable for an electrochemical transduction. The biomimetic material was produced by electropolymerization of phenol after optimizing several experimental parameters, such a scan-rate, number of cycles, range of potential applied, monomer and template concentrations. Under optimal conditions, the label-free sensor was able to respond to 3-NT from 500 nM to 1 mM, yielding a limit of detection of 22.3 nM. Finally, the applicability of the (bio)sensor was tested by performing calibration assays in human urine samples and a good performance was obtained in terms of sensitivity, selectivity and reproducibility. Overall, the attributes of the herein described sensing approach can be compared to a very limited number of other electrochemical devices, that are still using a conventional three electrode system, making this paper-sustained device the first electrochemical (bio)sensor with potential to become a portable and low-cost diagnostic tool for 3-NT. In general, the incorporation of molecular imprinting technology coupled to electrochemical transduction enabled the fabrication of suitable smart sensors for wide screening approaches.