Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

Abstract

a b s t r a c tThis work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Nonoriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen.The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted.Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

8-hydroxy-2′-deoxyguanosine (8-OHdG) biomarker detection down to picoMolar level on a plastic antibody film

“…In general, the detection capability of these devices varies from 1.1 to 97 nM, but some methods have shown severe interference from urine elements (mostly uric acid), solved therein by the addition of specific enzyme prior to the analysis stage. The use of antibodies as biorecognition element attached to gold supports (N. S. Ferreira & Sales, 2014), graphitic surfaces or silicon nanowires (Mohd Azmi et al, 2014), has solved such lack of selectivity problem. The inherent devices also offered simplicity of construction and good detection features, varying from 0.35 to 7.1 nM.…”

Electrochemical reverse engineering: A systems-level tool to probe the redox-based molecular communication of biology

“…Over the last decade redox has emerged as a critical component of homeostasis, stress (i.e., oxidative stress) and disease, and this has stimulated the development of a variety of new experimental tools (e.g., fluorescent probes [212], immunosensors [213], and electron paramagnetic resonance [214]). We contend that electrochemistry offers a unique set of capabilities that are well suited for in vitro studies in redox biology.…”

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol

“…Billah et al used a 4-aminothiophenol self-assembled monolayer (SAM) to covalently link the hinge region of reduced half-antibody fragments [19]. Ferreira et al adopted cysteamine SAM as a link to bind N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (NHS)-activated antibody to control paratope orientation [20]. However, the covalent processes may induce a loss of immunorecognition activity or reduce the biological functions of antibodies, due to the hazardous chemical reactions.…”