Electrochemical and electronic detection of biomarkers in serum: a systematic comparison using aptamer-functionalized surfaces.

Sensitive and selective detection of biomarkers in serum in a short time has a significant impact on health. The enormous clinical importance of developing reliable methods and devices for testing serum levels of cardiac troponin I (cTnI), which are directly correlated to acute myocardial infarction (AMI), has spurred an unmatched race among researchers for the development of highly sensitive and cost-effective sensing formats to be able to differentiate patients with early onset of cardiac injury from healthy individuals with a mean cTnI level of 26 pg mL-1. Electronic- and electrochemical-based detection schemes allow for fast and quantitative detection not otherwise possible at the point of care. Such approaches rely largely on voltammetric and field-effect-based readouts. Here, we systematically investigate electric and electrochemical point-of-care sensors for the detection of cTnI in serum samples by using the same surface receptors, cTnI aptamer-functionalized CVD graphene-coated interdigated gold electrodes. The analytical performances of both sensors are comparable with a limit of detection (LoD) of 5.7 ± 0.6 pg mL-1(electrochemical) and 3.3 ± 1.2 pg mL-1 (electric). However, both sensors exhibit different equilibrium dissociation constant (KD) values between the aptamer-linked surface receptor and the cTnI analyte, being 160 pg mL-1 for the electrochemical and about three times lower for the electrical approach with KD = 51.4 pg mL-1. This difference is believed to be related to the use of a redox mediator in the electrochemical sensor for readout. The ability of the redox mediator to diffuse from the solution to the surface via the cTnI/aptamer interface is hindered, correlating to higher KD values. In contrast, the electric readout has the advantage of being label-free with a sensing limitation due to ionic strength effects, which can be limited using poly(ethylene) glycol surface ligands.

SECM Investigation of Carbon Composite Thermoplastic Electrodes.

Thermoplastic electrodes (TPEs) are carbon composite electrodes consisting of graphite and thermoplastic polymer binder. TPE production is a solvent-based method, which makes it easy to fabricate and pattern into complex geometries, contrary to classical carbon composite electrodes. Depending on the composition (carbon type, binder, and composition ratio), TPEs can give excellent electrochemical performance and high conductivity. However, these TPEs are relatively new electrode materials, and thorough electrochemical characterization is still missing to understand and predict why large differences between TPEs exist. We used scanning electrochemical microscopy (SECM) as a screening tool to characterize TPEs. SECM data treatment based on scanning probe microscopy imaging allows a fast and easy comparison of the numerous images, as well as the optimization of the preparation. Experiments suggest that TPEs behave as a network of interacting microelectrodes made by electrochemically active islands isolated between less active areas. Higher carbon content in TPEs is not always indicative of more uniform electrodes with better electrochemical performances. Using various SECM redox probes, it is possible to select a specific graphite or polymer type for the analyte of interest. For example, TPEs made with COC:3569 are the best compromise for general detection, whereas PMMA:11 µm is better suited for catechol-like polyphenol analysis.

Investigation of Protective Properties of Organic Layers toward Reactive Oxygen Species.

The antioxidant protective properties of polyaromatic organic layers were evaluated toward reactive oxygen species (ROS) using scanning electrochemical microscopy in a foot-printing strategy. The layers were prepared by electrografting of aryldiazonium salts. Where p-(methyl)phenyl films show only weak protective properties toward ROS, p-(ethynyl)phenyl films evidence efficient protection of the covered surfaces. Applied potentials and electrolytes used during oxygen reduction reaction are critical parameters to control, prevent, or reduce the influence of ROS production and hence enhance the device lifetime.

Molecular Sieving and Current Rectification Properties of Thin Organic Films.

For the purpose of preparing well-organized functional surfaces, carbon and gold substrates were modified using electroreduction of a tetrahedral-shape preorganized tetra-aryldiazonium salt, leading to the deposition of ultrathin organic films. Characterization of the modified surfaces has been performed using cyclic voltammetry, X-ray photoelectron spectroscopy, infrared absorption spectroscopy, ellipsometry, atomic force microscopy, and contact angle measurements. The specific design of the tetra-aryldiazonium salts leads to an intrinsic structuring of the resulting organic films, allowing molecular sieving and current rectification properties toward redox probes in solution.

Photo-electrochemical properties of quantum rods studied by scanning electrochemical microscopy.

Scanning electrochemical microscopy (SECM) is used for studying the intrinsic photo-electrochemical properties of CdSe/CdS quantum rods. They are deposited on a transparent and non-conductive glass plate and investigated by SECM in feedback and generator-collector modes using a series of redox mediators. The method allows the interrogation of the quantum rods under illumination without the interference of the substrate, notably that due to the electron photo-ejection from the substrate, a process that is inherent to any polarized electrode material. Beside the methodological demonstration that could easily be extended to the investigations of the photo-redox properties of nanoparticles, studies highlight the strong reductive properties of quantum rods under illumination.

Immobilization of Monolayers Incorporating Cu Funnel Complexes onto Gold Electrodes. Application to the Selective Electrochemical Recognition of Primary Alkylamines in Water.

The immobilization of a copper calix[6]azacryptand funnel complex on gold-modified electrodes is reported. Two different methodologies are described. One is based on alkyne-terminated thiol self-assembled monolayers. The other relies on the electrografting of a calix[4]arene platform bearing diazonium functionalities at its large rim and carboxylic functions at its small rim, which is post-functionalized with alkyne moieties. In both cases, the CuAAC electroclick methodology proved to be the method of choice for grafting the calix[6]azacryptand onto the monolayers. The surface-immobilized complex was fully characterized by surface spectroscopies and electrochemistry in organic and aqueous solvents. The Cu complex displays a well-defined quasi-reversible system in cyclic voltammetry associated with the Cu(II)/Cu(I) redox process. Remarkably, this redox process triggers a powerful selective detection of primary alkylamines in water at a micromolar level, based on a cavitary recognition process.

Amine-terminated monolayers on carbon: preparation, characterization, and coupling reactions.

Aminophenyl and aminomethylphenyl monolayers have been electrografted to glassy carbon and pyrolyzed photoresist film from the corresponding diazonium ions using a protection-deprotection strategy based on Boc (tert-butyloxycarbonyl) and Fmoc (fluorenylmethyloxycarbonyl) groups. After grafting and then deprotecting films of Boc-NH-Ar, Fmoc-NH-Ar, and Fmoc-NH-CH2-Ar, depth profiling by atomic force microscopy confirmed that the resulting amine-terminated films were monolayers. In contrast, after deprotection, Boc-NH-CH2-Ar gave a multilayer film. Electroactive carboxylic acid derivatives were coupled to the monolayers through amide linkages. Electrochemical measurements revealed that the deprotected Fmoc-NH-CH2-Ar monolayer gave the highest surface concentration of coupled nitrophenyl and ferrocenyl groups and DFT calculations established that this monolayer has the highest theoretical surface concentration of those examined.

Tunneling dendrimers. Enhancing charge transport through insulating layer using redox molecular objects.

Charge transport through an insulating layer was probed using ferrocenyl-terminated dendrimers and scanning electrochemical microscopy. Experiments show that the passage through the layer is considerably enhanced when the transferred charges are brought globally to the surface by the ferrocenyl dendrimer instead of a single ferrocene molecule. This result shows that charge tunneling through an insulator could be promoted by a purely molecular nano-object.

High-yield formation of substituted tetracyanobutadienes from reaction of ynamides with tetracyanoethylene.

A high-yielding sequence of [2+2] cycloaddition-retroelectrocyclization of ynamides with tetracyanoethylene (TCNE) is described. The reaction provided tetracyanobutadiene (TCBD) species, which were characterized by various techniques. DFT and TD-DFT calculations were also performed to complement experimental findings.

Covalently anchored carboxyphenyl monolayer via aryldiazonium ion grafting: a well-defined reactive tether layer for on-surface chemistry.

Electrografting of aryl films to electrode surfaces from diazonium ion solutions is a widely used method for preparation of modified electrodes. In the absence of deliberate measures to limit film growth, the usual film structure is a loosely packed multilayer. For some applications, monolayer films are advantageous; our interest is in preparing well-defined monolayers of reactive tethers for further on-surface chemistry. Here, we describe the synthesis of an aryl diazonium salt with a protected carboxylic acid substituent. After electrografting to glassy carbon electrodes and subsequent deprotection, the layer is reacted with amine derivatives. Electrochemistry and atomic force microscopy are used to monitor the grafting, deprotection, and subsequent coupling steps. Attempts to follow the same procedures on gold surfaces suggest that the grafted layer is not stable in these reaction conditions.

Locally induced and self-induced "electroclick" onto a self-assembled monolayer: writing and reading with SECM under unbiased conditions.

Localized "electroclick" was achieved on azido-terminated self-assembled monolayers using Scanning Electrochemical Microscopy (SECM) in feedback mode, in which the substrate is not electrically connected (unbiased conditions). The method allows both the local immobilization of diverse functional moieties and the monitoring of each modification step at a micrometer scale. Conditions of the "click" coupling reaction were optimized especially to avoid the deposit of metallic copper by the choice of a specific ligand to stabilize the Cu(I) species. The catalytic efficiency in localized "electroclick" reaction of Cu(II)TMPA (TMPA: tris(2-pyridylmethyl)amine) as the "click" catalyst was compared with a derivative containing an alkyne group Cu(II)6eTMPA, the same molecule playing the role of the catalyst and the substrate. Evidences for surface self-catalysis propagation are demonstrated through SECM imaging showing a random 2D progression of the catalytic modification.

Synthesis and Optoelectronic Properties of Threaded BODIPYs.

We report on the synthesis of two new threaded BODIPYs 5 and 6 in good yields using boron as a gathering atom and a macrocycle with a 2,2'-biphenol unit. In addition to usual techniques, they were characterized by X-ray crystallography. Their electrochemical and optical properties were investigated. In particular, both compounds are highly emissive with photoluminescence quantum yields of 54 and 81 % respectively. In addition, they both show a high photostability.

Chemically irreversible redox mediator for SECM kinetics investigations: determination of the absolute tip-sample distance.

The use of a chemically irreversible redox probe in scanning electrochemical microscopy (SECM) was evaluated for the determination of the absolute tip-substrate distance. This data is required for a quantitative use of the method in the analysis of functional surfaces with an unknown redox response. Associated with the relevant model curves, the electrochemical response allows an easy positioning of the tip versus the substrate that is independent of the nature of the materials under investigation. The irreversible oxidation of polyaromatic compounds was found to be well adapted for such investigations in organic media. Anthracene oxidation in acetonitrile was chosen as a demonstrative example for evaluating the errors and limits of the procedure. Interest in the procedure was exemplified for the local investigations of surfaces modified by redox entities. This permits discrimination between the different processes occurring at the sample surface as the permeability of the probe through the layer or the charge transfer pathways. It was possible to observe small differences with simple kinetic models (irreversible charge transfer) that are related to permeation: charge transport steps through a permeable redox layer.

Mixed monolayer organic films via sequential electrografting from aryldiazonium ion and arylhydrazine solutions.

Sequential electrografting at glassy carbon from aryldiazonium salt solutions, or an aryldiazonium salt followed by an arylhydrazine, leads to the formation of covalently attached monolayer films incorporating two modifiers. In the first step, a 4-((triisopropylsilyl)ethynyl)phenyl film is electrografted to the surface, followed by removal of the triisopropylsilyl group to give a submonolayer of phenylethynylene groups. Two general strategies can then be applied to "fill in" the sparse monolayer with a second modifier. In the first route, nitrophenyl groups are grafted to the phenylethynylene-modified surface by the oxidation of 4-nitrophenylhydrazine. Ferrocene can be coupled to the terminal alkyne groups on the surface via a click reaction with azidomethylferrocene; an electrochemical measurement of the amount of immobilized ferrocene demonstrates that the phenylethynylene layer retains close to full reactivity after the second grafting step. In the alternative strategy, ferrocene is coupled to the phenylethynylene layer prior to grafting nitrophenyl groups by the reduction of the 4-nitrobenzenediazonium ion or by the oxidation of 4-nitrophenylhydrazine. For all approaches, the optimization of the grafting conditions gives surface concentrations of ferrocene and nitrophenyl groups that are consistent with those of a mixed monolayer. The stepwise generation of mixed monolayers is also monitored by film thickness measurements by depth profiling using the atomic force microscope. Thickness values are consistent with the proposed film structure in each preparation step.

Surface Modification of Thermoplastic Electrodes for Biosensing Applications via Copper-Catalyzed Click Chemistry.

Cu(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC), also known as click chemistry, has been demonstrated to be highly robust while providing versatile surface chemistry. One specific application is biosensor fabrication. Recently, we developed thermoplastic electrodes (TPEs) as an alternative to traditional carbon composite electrodes in terms of cost, performance, and robustness. However, their applications in biosensing are currently limited due to a lack of facile methods for electrode modification. Here, we demonstrate the feasibility of using CuAAC following the diazonium grafting of TPEs to take advantage of two powerful technologies for developing a customizable and versatile biosensing platform. After a stepwise characterization of the electrode modification procedures was performed, electrodes were modified with model affinity reagents. Streptavidin and streptavidin-conjugated IgG antibodies were successfully immobilized on the TPE surface, as confirmed by electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy.

Chemical Surface Grafting of Pt Nanocatalysts for Reconciling Methanol Tolerance with Methanol Oxidation Activity.

A conceptual challenge toward more versatile direct methanol fuel cells (DMFCs) is the design of a single material electrocatalyst with high activity and durability for both oxygen reduction reaction (ORR) and methanol oxidation reaction (MOR). This requires to conciliate methanol tolerance not to hinder ORR at the cathode with a good MOR activity at the anode. This is especially incompatible with Pt materials. We tackled this challenge by deriving a supramolecular concept where surface-grafted molecular ligands regulate the Pt-catalyst reactivity. ORR and MOR activities of newly reported Pt-calix[4]arenes nanocatalysts (Pt CF 3 ${{_{{\rm CF}{_{3}}}}}$ NPs/C) are compared to commercial benchmark PtNPs/C. Pt CF 3 ${{_{{\rm CF}{_{3}}}}}$ NPs/C exhibit a remarkable methanol tolerance without losing the MOR reactivity along with outstanding durability and chemical stability. Beyond designing single-catalyst material, operable in DMFC cathodic and anodic compartments, the results highlight a promising strategy for tuning interfacial properties.

Use of catechol as selective redox mediator in scanning electrochemical microscopy investigations.

The use of catechols, and more specifically of dopamine, as a specific redox mediator for scanning electrochemical microscopy (SECM) investigations was evaluated in the challenging situation of an ultrathin layer deposited on a conductive substrate (carbon materials). Experiments show that dopamine is a well-adapted redox system for SECM in feedback mode and in unbiased conditions. Used as a redox mediator, catechol permits the investigations of modified surfaces without an electrical connection of the sample thanks to fast charge transfer kinetics but with a surface selectivity that does not exist in classical outer-sphere redox mediators. The interest of catechol in SECM as a sensitive redox mediator is exemplified by monitoring several modification steps of an ultrathin (<1 nm) hierarchically porous organic monolayer deposited on carbon substrates. For quantitative analysis, the SECM approach curves using dopamine could simply be characterized with an irreversible electron transfer kinetics model in a large range of pH.

Highly stable silver nanohybrid electrocatalysts for the oxygen reduction reaction.

Silver nanoparticles (AgNPs) were deliberately functionalized via aryl diazonium chemistry with a monolayer of calix[4]arenes. The resulting nanohybrids show high efficiency and high selectivity toward the ORR in alkaline media along with an exceptional durability and a high methanol tolerance.

Design of robust binary film onto carbon surface using diazonium electrochemistry.

The electroreduction of functionalized aryldiazonium salts combined with a protection-deprotection method was evaluated for the fabrication of organized mixed layers covalently bound onto carbon substrates. The first modification consists of the grafting of a protected 4-((triisopropylsilyl)ethynyl)benzene layer onto the carbon surface on which the introduction of a second functional group is possible without altering the first grafted functional group. After deprotection, we obtained an ultrathin robust layer presenting high densities of both active ethynylbenzene groups (available for "click" chemistry) and the second functional group. The strategy was successfully demonstrated using azidomethylferrocene to react with ethynyl moieties in the binary film by "click" chemistry, and NO(2)-phenyl as the second functional group. Two possible modification pathways with different orderings of the various steps were considered to show the influence and importance of the protection-deprotection process on the final surface obtained. Using mild conditions for the grafting of the second layer maintains a concentration of active ethynyl groups similar to that obtained for a one-component monolayer while achieving a high surface concentration of the second modifier. Considering the wide range of functional aryldiazonium salts that could be electrodeposited onto carbon surfaces and the versatility and specificity of the "click" chemistry, this approach appears very promising for the preparation of mixed layers in well-controlled conditions without altering the reactivity of either functional group.

Controlled covalent functionalization of a graphene-channel of a field effect transistor as an ideal platform for (bio)sensing applications.

The controlled covalent functionalization of the graphene channel of a field effect transistor, based on interdigitated gold electrodes (source and drain), via electrochemical grafting, using specifically designed aryl diazonium species is demonstrated to allow the simple fabrication of a general platform for (bio)sensing applications. The electrochemical grafting of a protected ethynylphenyl diazonium salt leads to the deposition of only a monolayer on the graphene channel. This controlled covalent functionalization of the graphene channel results in a charge mobility of the GFET of 1739 ± 376 cm2 V-1 s-1 and 1698 ± 536 cm2 V-1 s-1 for the holes and electrons, respectively, allowing their utilization as (bio)sensors. After deprotection, a dense and compact ethynylphenyl monolayer is obtained and allows the immobilization of a wide range of (bio)molecules by a "click" chemistry coupling reaction (Huisgen 1,3-dipolar cycloaddition). This finding opens promising options for graphene-based (bio)sensing applications.