Herein, we present an efficient approach for developing electrochemical aptasensing interfaces, by “click” postfunctionalization of phenylethynyl-grafted glassy carbon substrates with mixed monolayers containing biorecognition elements and phosphorylcholine zwitterionic groups. Typically, controlling the composition of multicomponent surface layers by grafting from a mixture of aryldiazonium salts is challenging due to differences in their chemical reactivity. Our approach circumvents this issue by employing the electrochemical reduction of a single aryldiazonium salt containing a silyl-protected alkyne group followed by deprotection, to create phenylethynyl monolayers which can subsequently accommodate the concurrent immobilization of bioreceptors and zwitterionic groups through “click” postfunctionalization. We show that the surface ratio of the components in the bifunctional monolayers, estimated through XPS and electrochemical methods, can be accurately controlled by adjusting the mole ratio of the corresponding azide reagents in the “click” coupling solution. Moreover, electrochemical impedance spectroscopy and fluorescence microscopy investigations on bifunctional monolayers containing ssDNA and phosphorylcholine groups reveal that they effectively prevent nonspecific protein adsorption, while maintaining sufficiently low impedance to facilitate electrochemical detection. Finally, we demonstrate that proof of concept aptasensing interfaces based on binary layers containing a ferrocene-tagged cocaine/quinine aptamer and phosphorylcholine groups exhibit a trade-off between an improved analytical response and antifouling efficiency.

Developing a comprehensive procedure for tuning the surface and interfacial properties of DNA biorecognition interfaces is crucial for a wide range of analytical applications, but proves to be a difficult task. This study aims to address the challenge by exploiting the significant advantages of surface grafting with protected aryldiazonium salts and “click” azide-alkyne functionalization reactions. In order to control the surface density of reactive alkyne moieties over a wide range, suitable for the immobilization of large biomolecules, we explored novel protecting groups such as triphenylsilyl (TPS) and tris(biphen-4-yl)silyl (TBPS), which are bulkier than the triisopropylsilyl (TIPS) group employed in previous studies. We prepare phenylethynyl-grafted glassy carbon substrates using a two-step protocol consisting in electrografting with the corresponding silyl-protected diazonium salt, followed by nucleophilic cleavage of the protecting group. Using appropriate derivatization reagents and a combination of electrochemical and surface analytical techniques, we find a strong correlation between the protecting group size and the surface coverage of phenylethynyl groups. Next, using the phenylethynyl-grafted substrates for the immobilization of an azide-modified cocaine/quinine aptamer as model bioreceptor, we demonstrate that the surface density of ssDNA decreases with increasing size of the protecting group employed in the grafting procedure. Finally, we prove that our strategy can be effectively employed to fine-tune the analytical performance of an aptasensing platform for quinine detection.

This work introduces a novel comparative analysis on the morphology, surface chemistry and electrochemical behavior of electrochemically reduced graphene oxide (ERGO) modified electrodes obtained by two commonly used methods: direct electrodeposition, and electrochemical reduction of GO drop-casted films. We report that electrodes modified through drop -casting are uniformly coated with ERGO layers, unlike electrodes obtained through electrodeposition, which only show isolated ERGO agglomerates on their surface. However, cyclic voltammetry investigations in the presence of soluble redox probes indicate that drop -casting is less reproducible for producing ERGO -modified electrodes, evidenced by 17 -fold increase in the relative standard deviation of the estimated surface area.

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.

It is well known that both the aptamer surface density and surface chemistry can have a strong influence on the analytical performance of aptasensors based on target-induced conformational changes1. In this context, we report here an improved protocol for obtaining aptasensing platforms which allows fine-tuning the DNA aptamer surface coverage. First, glassy carbon substrates were functionalized with ethynylphenyl groups through the electrochemical reduction of silyl-protected ethynylphenyl diazonium tetrafluoroborates followed by deprotection with tetrabutylammonium fluoride2. The successful removal of the protecting groups (triisopropylsilyl-, triphenylsilyl- and tris(biphen-4-yl)silyl-) was confirmed by X-ray photoelectron spectroscopy and cyclic voltammetry in the presence of Fe(CN)6 3-/4- as soluble redox probe. Following “click” post-modification with suitable derivatization reagents, the surface coverage with ethynylphenyl groups was assessed using complementary techniques such as XPS, cyclic voltammetry and chronocoulometry. For example, the F/C surface ratio of substrates derivatized with 1-(2,2,2-trifluoroethoxy)-6-azidohexane provided an indication of the functionalization degree, and the integration of Fc/Fc+ voltammetric peaks for substrates modified with N-(6-azidohexyl)ferrocenecarboxamide allowed a quantitative determination of the surface coverage. Both techniques showed a good correlation between the protective group size and the surface coverage with alkyne groups. Likewise, we observed a similar trend for substrates derivatized with azide-modified oligonucleotides, where the surface packing density was determined based on the chronocoulometric response of Ru(NH3)6 3+, a cationic redox probe which binds with the negatively charged phosphate groups from the oligonucleotide backbone. As proof of concept, we further developed an electrochemical molecular beacon aptasensor employing a ferrocene-labeled quinine aptamer. We demonstrate that the aptamer surface density, and ultimately the analytical performance of molecular beacon aptasensors, can be effectively fine-tuned by employing silyl protecting groups of different sizes. Acknowledgements This work was supported by a grant from the Romanian Ministry of Education and Research, CNCS-UEFISCDI, project number PN-III-P4-ID-PCE-2020-2474, within PNCDI III. References Onaş, A. M., Dascălu, C., Raicopol, M. D. & Pilan, L. Critical Design Factors for Electrochemical Aptasensors Based on Target-Induced Conformational Changes: The Case of Small-Molecule Targets. Biosensors 12, (2022). Leroux, Y. R., Fei, H., Noël, J.-M. M., Roux, C. & Hapiot, P. Efficient covalent modification of a carbon surface: Use of a silyl protecting group to form an active monolayer. J. Am. Chem. Soc. 132, 14039–14041 (2010).

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.

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.

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.

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.

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.

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.

Cobaltocenium diffusion coefficients have been evaluated by electrochemistry in two organic solvents, acetonitrile or dimethyl sulfoxide, at different temperatures in the presence of tetrabutylammonium perchlorate as electrolyte. The data have been obtained by cyclic voltammetry, potential-step chronoamperometry, rotating disk electrode and differential pulse voltammetry for different cobaltocenium concentrations. Randles-Sevick and Cottrell equations have been used to calculate the diffusion coefficients. Validity of Stokes-Einstein equation has been checked in both solvents.

A novel electrochemical approach aimed at developing biosensing platforms based on polypyrrole/sulfonated graphene nanocomposites is reported. Specifically, nanocomposite layers are deposited onto platinum electrodes through the electrochemical polymerization of pyrrole monomer in the presence of reduced graphene oxide bearing phenylsulfonyl groups. Thus, the functionalized graphene nanofiller acts as dopant and balances the positive charges on the polymer chains, leading to an enhancement of the polymer's electrical conductivity and concomitantly increasing the electrode surface area. The polypyrrole/graphene nanocomposite films are further modified with carboxyphenyl groups via electrochemical reduction of 4-carboxyphenyl diazonium tetrafluoroborate. Grafting carboxyphenyl functionalities serves a dual purpose: it permits the covalent immobilization of glucose oxidase via carbodiimide chemistry and also forms an electrode blocking layer which hinders the oxidation of interfering substances. The feasibility of this strategy is demonstrated by the preparation of a glucose biosensor which exhibited an improved performance: wide linear range (0.02-12 mM), good sensitivity (0.56 mu A mM(-1) cm(-2)) and adequate selectivity towards common interferents including ascorbic acid, paracetamol, uric acid, and cysteine.

Although oxidases are selective and specific biocatalysts frequently used in enzyme biosensors, amperometric detection methods can hardly distinguish the current response of enzymatic reaction products from the currents generated by other electroactive species commonly encountered in complex matrices. Therefore, various methods have been developed in order to improve the overall selectivity of enzyme-based biosensors. Here, we employ substituted polyaryl films grafted on platinum electrodes via the electrochemical reduction of a variety of diazonium tetrafluoroborates as blocking layers. Electron transfer kinetics of three redox probes at the bare and modified Pt electrodes were investigated in order to evaluate the blocking effect of the grafted layers. Further, the effect of surface modification on the voltammetric responses of commonly encountered interferents for glucose determination were examined. As proof of concept, interference-free amperometric biosensors employing glucose oxidase were constructed using aryl modified Pt electrodes.

In the present work we report a new strategy to create glucose amperometric biosensors with improved analytical characteristics using graphene as support for glucose oxidase. First, a reduced graphene oxide film was obtained on the glassy carbon electrode surface by direct electrochemical reduction of graphene oxide from a suspension in water. For facilitating the oxidation of the enzymatically generated H2O2, in a second step, Pt nanoparticles were electrodeposited on the graphene modified electrodes. The obtained biosensors showed good analytical performances in terms of high sensitivity and wide linear range.

In the present work we report a new strategy to create glucose amperometric biosensors with improved analytical characteristics using graphene as support for glucose oxidase. First, a reduced graphene oxide film was obtained on the glassy carbon electrode surface by direct electrochemical reduction of graphene oxide from a suspension in water. For facilitating the oxidation of the enzymatically generated H2O2, in a second step, Pt nanoparticles were electrodeposited on the graphene modified electrodes. The obtained biosensors showed good analytical performances in terms of high sensitivity and wide linear range.

In this paper we report a new strategy for obtaining conducting azopolymers having pendant azobenzene moieties via functionalization of polypyrrole by electrochemical reduction of 4-[(4'-nitrophenyl)azo] benzenediazonium tetrafluoroborate (NABDBF(4)(-)). The functionalization of polypyrrole films with 4'-nitroazobenzene (NAB) groups has been evaluated by cyclic voltammetry and by spectroscopic techniques such as XPS, Raman and UV-vis spectroscopy. Moreover, the third-order nonlinear optical response of NAB-functionalized polypyrrole has been investigated using the optical third-harmonic generation technique. All these investigations confirmed the presence at the polymeric films surface of the functional groups introduced by the electrochemical reduction of diazonium salt. The versatility of this methodology allows not only the functionalization of polypyrrole-type materials with chromophore groups, but can also be successfully applied to other conjugated polymer systems in order to create various photoresponsive materials. (C) 2015 Elsevier B.V. All rights reserved.

In this paper we report the functionalization of conductive polypyrrole (PPY) films via electrochemical reduction of the aryl diazonium salts in a manner that is similar to the one employed for other conductive surfaces. To understand the general trends of the grafting behavior of diazonium salts and to establish the optimal conditions for the covalent functionalization of the PPY films, we have compared the grafting behavior of four p-substituted phenyldiazonium salts: p-nitrophenyl diazonium tetrafluoroborate (PNBDBF4-), p-tolyl diazonium tetrafluoroborate (TDBF4-), p-fluorophenyl diazonium tetrafluoroborate (FPDBF4-) and 4-diazo-N,N-dimethylaniline tetrafluoroborate (DDMABF(4)(-)). The selection of the molecules to be grafted was done both for their electroactivity after grafting and the contrasted electronegativity of the substituents at the benzene ring. For all investigated diazonium salts, a linear relationship between their reduction potential at the PPY electrodes and Hammett substituent constants was obtained, suggesting a similar electrochemical reaction mechanism. The functionalization of the polypyrrole films has been evaluated using electrochemical methods like EQCM, CV and EIS. The presence at the polymeric films surface of the functional groups introduced by the electrochemical reduction of diazonium salts was evidenced also by XPS. This approach enables new functionalities on PPY that could otherwise not withstand the polymerization conditions. (C) 2014 The Electrochemical Society. All rights reserved.