Label-Free DNA Biosensor Based on Reduced Graphene Oxide and Gold Nanoparticles.

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free, and cost-effective DNA detection platform developed at screen-printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl4 to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto a AuNPs-RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, a characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor and was particularly monitored by the change in the charge transfer resistance of a [Fe(CN)6]4-/3- redox couple added in the test solution.

Critical Design Factors for Electrochemical Aptasensors Based on Target-Induced Conformational Changes: The Case of Small-Molecule Targets.

Nucleic-acid aptamers consisting in single-stranded DNA oligonucleotides emerged as very promising biorecognition elements for electrochemical biosensors applied in various fields such as medicine, environmental, and food safety. Despite their outstanding features, such as high-binding affinity for a broad range of targets, high stability, low cost and ease of modification, numerous challenges had to be overcome from the aptamer selection process on the design of functioning biosensing devices. Moreover, in the case of small molecules such as metabolites, toxins, drugs, etc., obtaining efficient binding aptamer sequences proved a challenging task given their small molecular surface and limited interactions between their functional groups and aptamer sequences. Thus, establishing consistent evaluation standards for aptamer affinity is crucial for the success of these aptamers in biosensing applications. In this context, this article will give an overview on the thermodynamic and structural aspects of the aptamer-target interaction, its specificity and selectivity, and will also highlight the current methods employed for determining the aptamer-binding affinity and the structural characterization of the aptamer-target complex. The critical aspects regarding the generation of aptamer-modified electrodes suitable for electrochemical sensing, such as appropriate bioreceptor immobilization strategy and experimental conditions which facilitate a convenient anchoring and stability of the aptamer, are also discussed. The review also summarizes some effective small molecule aptasensing platforms from the recent literature.

Electrochemical Detection Platform Based on RGO Functionalized with Diazonium Salt for DNA Hybridization.

In this paper, we propose an improved electrochemical platform based on graphene for the detection of DNA hybridization. Commercial screen-printed carbon electrodes (SPCEs) were used for this purpose due to their ease of functionalization and miniaturization opportunities. SPCEs were modified with reduced graphene oxide (RGO), offering a suitable surface for further functionalization. Therefore, aryl-carboxyl groups were integrated onto RGO-modified electrodes by electrochemical reduction of the corresponding diazonium salt to provide enough reaction sites for the covalent immobilization of amino-modified DNA probes. Our final goal was to determine the optimum conditions needed to fabricate a simple, label-free RGO-based electrochemical platform to detect the hybridization between two complementary single-stranded DNA molecules. Each modification step in the fabrication process was monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using [Fe(CN)6]3-/4- as a redox reporter. Although, the diazonium electrografted layer displayed the expected blocking effect of the charge transfer, the next steps in the modification procedure resulted in enhanced electron transfer properties of the electrode interface. We suggest that the improvement in the charge transfer after the DNA hybridization process could be exploited as a prospective sensing feature. The morphological and structural characterization of the modified electrodes performed by scanning electron microscopy (SEM) and Raman spectroscopy, respectively, were used to validate different modification steps in the platform fabrication process.

The Role of Aryldiazonium Chemistry in Designing Electrochemical Aptasensors for the Detection of Food Contaminants.

Food safety monitoring assays based on synthetic recognition structures such as aptamers are receiving considerable attention due to their remarkable advantages in terms of their ability to bind to a wide range of target analytes, strong binding affinity, facile manufacturing, and cost-effectiveness. Although aptasensors for food monitoring are still in the development stage, the use of an electrochemical detection route, combined with the wide range of materials available as transducers and the proper immobilization strategy of the aptamer at the transducer surface, can lead to powerful analytical tools. In such a context, employing aryldiazonium salts for the surface derivatization of transducer electrodes serves as a simple, versatile and robust strategy to fine-tune the interface properties and to facilitate the convenient anchoring and stability of the aptamer. By summarizing the most important results disclosed in the last years, this article provides a comprehensive review that emphasizes the contribution of aryldiazonium chemistry in developing electrochemical aptasensors for food safety monitoring.

Tailoring the performance of electrochemical biosensors based on carbon nanomaterials via aryldiazonium electrografting.

Carbon nanomaterials (CNs) offer some of the most valuable properties for electrochemical biosensing applications, such as good electrical conductivity, wide electrochemical stability, high specific surface area, and biocompatibility. Regardless the envisioned sensing application, endowing CNs with specific functions through controlled chemical functionalization is fundamental for promoting the specific binding of the analyte. As a versatile and straightforward method of surface functionalization, aryldiazonium chemistry have been successfully used to accommodate in a stable and reproducible way different functionalities, while the electrochemical route has become the favourite choice since the deposition conditions can be readily controlled and adapted to the substrate. In particular, the modification of CNs by electrochemical reduction of aryl diazonium salts is established as a powerful tool which allows tailoring the chemical and electronic properties of the sensing platform. By outlining the stimulating results disclosed in the last years, this article provides not only a comprehensively review, but also a rational assessment on contribution of aryldiazonium electrografting in developing CNs-based electrochemical biosensors. Furthermore, some of the emerging challenges to be surpassed to effectively implement this methodology for in vivo and point of care analysis are also highlighted.

Influence of Graphene Oxide Concentration when Fabricating an Electrochemical Biosensor for DNA Detection.

We have investigated the influence exerted by the concentration of graphene oxide (GO) dispersion as a modifier for screen printed carbon electrodes (SPCEs) on the fabrication of an electrochemical biosensor to detect DNA hybridization. A new pretreatment protocol for SPCEs, involving two successive steps in order to achieve a reproducible deposition of GO, is also proposed. Aqueous GO dispersions of different concentrations (0.05, 0.1, 0.15, and 0.2 mg/mL) were first drop-cast on the SPCE substrates and then electrochemically reduced. The electrochemical properties of the modified electrodes were investigated after each modification step by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), while physicochemical characterization was performed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Finally, the sensing platform was obtained by the simple adsorption of the single-stranded DNA probe onto the electrochemically reduced GO (RGO)-modified SPCEs under optimized conditions. The hybridization was achieved by incubating the functionalized SPCEs with complementary DNA target and detected by measuring the change in the electrochemical response of [Fe(CN)6]3-/4- redox reporter in CV and EIS measurements induced by the release of the newly formed double-stranded DNA from the electrode surface. Our results showed that a higher GO concentration generated a more sensitive response towards DNA detection.

Versatile graphene biosensors for enhancing human cell therapy.

Technological advances in engineering and cell biology stimulate novel approaches for medical treatment, in particular cell-based therapy. The first cell-based gene therapy against cancer was recently approved by the US Food and Drug Administration. Progress in cancer diagnosis includes a blood test detecting five cancer types. Numerous stem cell phase I/II clinical trials showing safety and efficacy will soon pursue qualifying criteria for advanced therapy medicinal products (ATMP), aspiring to join the first stem-cell therapy approved by the European Medicines Agency. Cell based therapy requires extensive preclinical characterisation of biomarkers indicating mechanisms of action crucial to the desired therapeutic effect. Quantitative analyses monitoring critical functions for the manufacture of optimal cell and tissue-based clinical products include successful potency assays for implementation. The challenge to achieve high quality measurement is increasingly met by progress in biosensor design. We adopt a cell therapy perspective to highlight recent examples of graphene-enhanced biointerfaces for measurement of biomarkers relevant to cancer treatment, diagnosis and tissue regeneration. Graphene based biosensor design problems can thwart their use for health care transformative point of care testing and real-time applications. We discuss concerns to be addressed and emerging solutions for establishing clinical grade biosensors to accelerate human cell therapy.

Functionalized single-walled carbon nanotubes/polypyrrole composites for amperometric glucose biosensors.

This article reports an amperometric glucose biosensor based on a new type of nanocomposite of polypyrrole (PPY) with p-phenyl sulfonate-functionalized single-walled carbon nanotubes (SWCNTs-PhSO3-). An environmentally friendly functionalization procedure of the SWCNTs in the presence of substituted aniline and an oxidative species was adopted. The nanocomposite-modified electrode exhibited excellent electrocatalytic activities towards the reduction or oxidation of H2O2. This feature allowed us to use it as bioplatform on which glucose oxidase (GOx) was immobilized by entrapment in an electropolymerized PPY/SWCNTs-PhSO3- film for the construction of the glucose biosensor. The amperometric detection of glucose was assayed by applying a constant electrode potential value necessary to oxidize or reduce the enzymatically produced H2O2 with minimal interference from the possible coexisting electroactive compounds. With the introduction of a thin film of Prussian blue (PB) at the substrate electrode surface, the PPY/GOx/SWCNTs-PhSO3-/PB system shows synergy between the PB and functionalized SWCNTs which amplifies greatly the electrode sensitivity when operated at low potentials. The biosensor showed good analytical performances in terms of low detection (0.01 mM), high sensitivity (approximately 6 µA mM-1 cm-2), and wide linear range (0.02 to 6 mM). In addition, the effects of applied potential, the electroactive interference, and the stability of the biosensor were discussed. The facile procedure of immobilizing GOx used in the present work can promote the development of other oxidase-based biosensors which could have a practical application in clinical, food, and environmental analysis.