Screen-Printed Carbon Electrodes with Cationic Cyclodextrin Carbon Nanotubes and Ferrocenyl-Carnosine for Electrochemical Sensing of Hg(II).

The study reports the use of nanoassembly based on cationic cyclodextrin carbon nanotubes (CNT-CDs) and ferrocenylcarnosine (FcCAR) for electrochemical sensing of Hg(II) in aqueous solution. ß-cyclodextrins (CDs) were grafted onto CNTs by a click chemistry reaction between heptakis-(6-azido-6-deoxy)-ß-cyclodextrin and alkyne-terminated CNTs. The cationic amine groups on the CD units were produced by the subsequent reduction of the residual nitrogen groups. The chemical composition and morphology of CNT-CDs were analyzed by X-ray photoelectron spectroscopy, scanning electron microscopy, and thermogravimetric analysis. A N,N-dimethylformamide dispersion of CNT-CDs was cast on the surface of screen-printed carbon electrodes (SPCEs), and the electrochemical response was evaluated by cyclic voltammetry (CV) using [Fe(CN)6]3- as the redox probe. The ability of SPCE/CNT-CD to significantly enhance the electroactive properties of the redox probe was combined with a suitable recognition element (FcCAR) for Hg(II). The electrochemical response of the CNT-CD/FcCAR nanoassembly was evaluated by CV and electrochemical impedance spectroscopy. The analytical performance of the Hg(II) sensor was evaluated by differential pulsed voltammetry and chronoamperometry. The oxidative peak current showed a linear concentration dependence in the range of 1-100 nM, with a sensitivity of 0.12 µA/nM, a limit of detection of 0.50 nM, and a limit of quantification of 1 nM.

Thermodynamic and voltammetric study on carnosine and ferrocenyl-carnosine.

A potentiometric study on the interactions of L-carnosine (CAR) (2-[(3-aminopropanoyl)amino]-3-(1H-imidazol-5-yl)propanoic acid) with two toxic metal cations, Hg2+ and Cd2+, is reported here. The elucidation of the metal (M2+)-CAR interactions in aqueous solution highlighted the speciation model for each system, the dependence of the formation constants of the complex species on ionic strength (0.15 ≤ I/mol L-1 ≤ 1) and temperature (288.15 ≤ T/K ≤ 310.15) and changes in enthalpy and entropy. The sequestering ability of CAR towards the two metal ions was quantified and compared with that with Pb2+, previously determined. Considering the complexing ability of CAR and its unclear electrochemical properties, a more electroactive derivative, the ferrocenyl-carnosine (FcCAR), was synthesized and its complexing ability was evaluated by UV-vis spectroscopy. FcCAR electrochemical properties were investigated by Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) on Screen-Printed Electrodes (SPEs), to evaluate its sensing properties. Electrochemical responses in the presence of Hg2+ and Pb2+ have been shown to be promising for the electrochemical detection of these metal cations in aqueous environment.

Cyclodextrin polymer clean-up method for the detection of ciguatoxins in fish with cell-based assays.

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa, which are transferred through the food webs, reaching humans and causing a poisoning known as ciguatera. The cell-based assay (CBA) is commonly used for their detection because of its high sensitivity and the provided toxicological information. However, matrix effects may interfere in the CBA. In this work, γ-cyclodextrin-hexamethylene diisocyanate (γ-CD-HDI), γ-cyclodextrin-epichlorohydrin (γ-CD-EPI) and γ-CD-EPI conjugated to magnetic beads (γ-CD-EPI-MB) have been evaluated as clean-up materials for fish flesh extracts containing CTXs. The best results were achieved with γ-CD-HDI in column format, which showed a CTX1B recovery of 42% and 32% for Variola louti and Seriola dumerili, respectively, and allowed exposing cells to at least 400 mg/mL of fish flesh. This clean-up strategy provides at least 4.6 and 3.0-fold higher sensitivities to the assay for V.louti and S.dumerili, respectively, improving the reliability of CTX quantification.

Supramolecular Complexes of Plant Neurotoxin Veratridine with Cyclodextrins and Their Antidote-like Effect on Neuro-2a Cell Viability.

Veratridine (VTD) is a plant neurotoxin that acts by blocking the voltage-gated sodium channels (VGSC) of cell membranes. Symptoms of VTD intoxication include intense nausea, hypotension, arrhythmia, and loss of consciousness. The treatment for the intoxication is mainly focused on treating the symptoms, meaning there is no specific antidote against VTD. In this pursuit, we were interested in studying the molecular interactions of VTD with cyclodextrins (CDs). CDs are supramolecular macrocycles with the ability to form host-guest inclusion complexes (ICs) inside their hydrophobic cavity. Since VTD is a lipid-soluble alkaloid, we hypothesized that it could form stable inclusion complexes with different types of CDs, resulting in changes to its physicochemical properties. In this investigation, we studied the interaction of VTD with ß-CD, γ-CD and sulfobutyl ether ß-CD (SBCD) by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Docking and molecular dynamics studies confirmed the most stable configuration for the inclusion complexes. Finally, with an interest in understanding the effects of the VTD/CD molecular interactions, we performed cell-based assays (CBAs) on Neuro-2a cells. Our findings reveal that the use of different amounts of CDs has an antidote-like concentration-dependent effect on the cells, significantly increasing cell viability and thus opening opportunities for novel research on applications of CDs and VTD.

Cyclodextrin polymers as passive sampling materials for lipophilic marine toxins in Prorocentrum lima cultures and a Dinophysis sacculus bloom in the NW Mediterranean Sea.

Cyclodextrins, cyclic oligomers that form a conical structure with an internal cavity, are proposed as new and sustainable materials for passive sampling of lipophilic marine toxins. Two applicability scenarios have been tested. First, disks containing ß-cyclodextrin-hexamethylene diisocyanate (ß-CD-HDI) and ß-cyclodextrin-epichlorohydrin (ß-CD-EPI) polymers were immersed in Prorocentrum lima cultures for different days (2, 12 and 40). LC-MS/MS analysis showed capture of free okadaic acid (OA) and dinophysistoxin-1 (DTX1) by cyclodextrins at contents that increased with immersion time. Cyclodextrins resulted more efficient in capturing DTX1 than OA. In a second experiment, disks containing ß-CD-HDI, ß-CD-EPI, γ-CD-HDI and γ-CD-EPI were deployed in harbor waters of El Masnou (NW Mediterranean Sea) during a Dinophysis sacculus bloom in February 2020. Free OA and pectenotoxin-2 (PTX2) were captured by cyclodextrins. Toxin contents were higher at sampling points and sampling weeks with higher D. sacculus cell abundance. In this case, PTX2 capture with cyclodextrins was more efficient than OA capture. Therefore, cyclodextrins have provided information regarding the toxin profile of a P. lima strain and the spatial and temporal dynamics of a D. sacculus bloom, proven efficient as passive sampling materials for environmental monitoring.

Amperometric biosensor for glyphosate based on the inhibition of tyrosinase conjugated to carbon nano-onions in a chitosan matrix on a screen-printed electrode.

Glyphosate [N-(phosphonomethyl)glycine] is the most frequently used herbicide to date. Due to its indiscriminate use, it has become a globally occurring pollutant of surface waters. A biosensor for glyphosate is described here that consists of a carbon nano-onion/tyrosinase conjugate immobilized in a chitosan matrix on a screen-printed electrode. The analytical principle is based on the inhibition of the enzyme tyrosinase by glyphosate. L-DOPA is used as the enzyme substrate. The presence of the carbon nano-onions has a beneficial effect on the sensitivity of the assay. Glyphosate can be amperometrically quantified in the 0.015 to 10 µM concentration range and with a 6.5 nM (1.1 µg L-1) detection limit. The biosensor is stable more than 2 months at 4 °C. It was applied to the detection of glyphosate in water and soil samples taken from irrigation of a rice field after aerial application. Results were in good agreement with data obtained by a commercial ELISA. Graphical abstract A highly sensitive amperometric biosensor for glyphosate is reported, based on the covalent immobilization of a carbon nano-onion/tyrosinase conjugate on a chitosan matrix.

Development of highly sensitive IgA immunosensors based on co-electropolymerized L-DOPA/dopamine carbon nano-onion modified electrodes.

The development of versatile platforms to construct novel and sensitive immunosensors is nowadays an intense research field. Nanomaterials and polymers are often combined to fabricate new platforms to immobilize capture antibodies. Here we evaluate for the first time the co-electropolymerization of dopamine (DA) and L-3,4-dihydroxyphenylalanine (L-DOPA) on carbon nano-onion (CNO) modified electrodes as versatile platform to develop electrochemical immunosensors. Mixtures of DA and L-DOPA at different molar rations were co-electropolymerized on CNO-modified glassy carbon electrodes to form a poly(L-DOPA/DA) film. Immobilization of aminoferrocene was used to estimate the number of accessible carboxylic acid groups on the surface (11.3 nmol/cm2), a value comparable to three-dimensional matrices. This platform was applied to the electrochemical detection of IgA antibodies using both a HRP-based sandwich type assay and label-free detection based on [Fe(CN)6]3-/4- signal blocking. The sandwich and the label-free assays showed a wide linear response with LOD of 19 and 48 ng/mL, respectively, allowing the detection of serum IgA deficiency. Most remarkably, the incorporation of CNO layer led to a significant improvement (three-orders of magnitude) of the analytical performance of these immunosensors due to a combination of high surface area and increased electron transfer rates provided by the CNO layer.

Kinetic, spectroscopic and computational docking study of the inhibitory effect of the pesticides 2,4,5-T, 2,4-D and glyphosate on the diphenolase activity of mushroom tyrosinase.

The inhibitory effect of 2,4,5-T, 2,4-D, glyphosate and paraquat on the diphenolase activity of mushroom tyrosinase for oxidation of L-DOPA has been investigated by kinetic measurements, fluorescence spectroscopy and computational docking analysis. 2,4,5-T and 2,4-D inhibit the diphenolase activity of the enzyme following a competitive mechanism, while glyphosate is a mixed inhibitor according to Lineweaver-Burk kinetic analysis. The inhibitory activity follows the order glyphosate >2,4,5-T > 2,4-D with IC50 values of 65, 90 and 106 µM, respectively. Intrinsic tyrosinase fluorescence quenching and computational docking analysis suggest that 2,4,5-T and 2,4-D interact with the active site of the enzyme through hydrophobic interactions, while glyphosate also interacts with external residues of the active site of the enzyme by hydrogen bonding and hydrophilic interactions inducing conformational changes in the protein structure.

Preparation and characterization of alkaline phosphatase, horseradish peroxidase, and glucose oxidase conjugates with carboxylated carbon nano-onions.

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO-enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ∼0.5 mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.

Supramolecular biosensors based on electropolymerised pyrrole-cyclodextrin modified surfaces for antibody detection.

The self-assembly of an adamantane-appended polymer bearing an antigen fragment on a polypyrrole-cyclodextrin modified surface provides a highly sensitive immunosensor with low limits of detection for celiac disease related targets. The pyrrole-carboxylic acid films were formed on the surface of gold electrodes by electropolymerisation and followed by covalent attachment of cyclodextrin units. Surface plasmon resonance measurements confirmed the role of the host/guest interactions between adamantane moieties and ß-cyclodextrin hosts in the formation of the supramolecular sensor interface. Furthermore, this novel electrochemical supramolecular platform was effective in the amperometric detection of anti-gliadin antibodies in spiked serum samples with very good signal recovery.

Reactive Carbon Nano-Onion Modified Glassy Carbon Surfaces as DNA Sensors for Human Papillomavirus Oncogene Detection with Enhanced Sensitivity.

Glassy carbon electrodes were modified with small carbon nano-onions (CNOs) and activated by electrografting of diazonium salts bearing terminal carboxylic acid and maleimide groups. The CNO-modified surfaces were characterized by ESEM and AFM microscopy as well as by electrochemical techniques. The modified electrodes were used for the amperometric detection of a model DNA target sequence associated with the human papillomavirus by immobilizing short recognition sequences by amidation or thiol-maleimide reactions. The analytical parameters of the developed biosensors were compared with glassy carbon electrodes without CNOs. In both cases, the incorporation of CNOs resulted in an enhancement in sensitivity and a decrease in detection limits ascribed to a combination of large surface areas and enhanced electron transfer properties of the CNO-modified electrodes. These results offer promise for the construction of other CNO-based biomolecule detection platforms with enhanced sensitivities.

Bleed-to-read disposable microsystems for the genetic and serological analysis of celiac disease markers with amperometric detection.

Celiac disease is an auto-immune disorder induced by ingestion of gluten in genetically predisposed individuals. Its diagnostics is more accurate using a combination of immunologic and genetic tests to detect of high levels of certain auto-antibodies and the presence human leukocyte antigen HLA-DQ2 or HLA-DQ8 genetic markers. In this work, we report the design and testing of automated microsystems combining sample treatment, storage, fluidic transport, and detection in a single platform able to carry out genetic or serologic analysis for detection of celiac disease markers. These microsystems share a common footprint and many fluidic features and are thus able to perform a complete assay. The microsystem for the genetic assay extracts and amplifies the DNA prior to detection, while the serology microsystem contains a filter and chamber for the generation and subsequent dilution of plasma. The performance of both platforms is demonstrated and compared with reference methods with an excellent correlation, which makes the developed platform amenable for clinical studies.

Supramolecular solubilization of cyclodextrin-modified carbon nano-onions by host-guest interactions.

Small carbon nano-onions (CNOs, 6-12 shells) were prepared in high yields and functionalized with carboxylic groups by chemical oxidation and reacted with ßCD-NH2 to yield CNOs decorated with ßCDs. A biocompatibile dextran polymer with graphted ferrocene groups was employed for the supramolecular self-assembly on the ßCD-CNO surfaces. The ßCDs act as hosts and the polymer ferrocene groups as guests (Fc-Dex) by the formation of inclusion complexes. After their assembly these nanostructures were soluble in aqueous solutions. The resulting product was characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and FT-IR and Raman spectroscopies. Moreover, the deposition of successive layers on the surface of the particles was monitored using DLS measurements and zeta potentials. Through-space interactions between the Fc moieties and the CNO cores and the influence of an additional dextran-ßCD outer layer were measured electrochemically.

Peroxidase-encapsulated cyclodextrin nanosponge immunoconjugates as a signal enhancement tool in optical and electrochemical assays.

Cyclodextrin nanosponges bearing carboxylate groups have been prepared by crosslinking ß-cyclodextrin with pyromellitic dianhydride to form a carboxylic acid terminated nanoporous material. The surface of the particles was covalently modified with an anti-IgG antibody and then loaded with horseradish peroxidase. The structures of unmodified and protein modified nanosponge particles were investigated by Raman spectroscopy and imaging methods. Confocal microscopy indicates that the antibody is located in the outside of the particle while HRP is encapsulated in the inner part. The possibility to use these modified nanosponges as a signal enhancement tool in enzyme-linked colorimetric and electrochemical assays was evaluated using a sandwich format comprising immobilised gliadin as an antigen, a target anti-gliadin antibody and an anti-IgG antibody conjugated to the enzyme-loaded nanosponge immunoconjugates.

A compact hybrid-multiplexed potentiostat for real-time electrochemical biosensing applications.

The architecture and design of a compact, multichannel, hybrid-multiplexed potentiostat for performing electrochemical measurements on continuously-biased electrode arrays is presented. The proposed architecture utilises a combination of sequential and parallel measurements, to enable high performance whilst keeping the system low-cost and compact. The accuracy of the signal readout is maintained by following a special multiplexing approach, which ensures the continuous biasing of all the working electrodes of an array. After sampling the results, a digital calibration technique factors out errors from component inaccuracies. A prototype printed circuit board (PCB) was designed and built using off-the-shelf components for the real-time measurement of the amperometric signal of 48 electrodes. The operation and performance of the PCB was evaluated and characterised through a wide range of testing conditions, where it exhibited high linearity (R(2)>0.999) and a resolution of 400pA. The effectiveness of the proposed multiplexing scheme is demonstrated through electrochemical tests using KCl and [Fe(CN)6](3-) in KCl solutions. The applicability of the prototype multichannel potentiostat is also demonstrated using real biosensors, which were applied to the detection of IgA antibodies.

Spectroscopic and atomic force microscopy characterization of the electrografting of 3,5-bis(4-diazophenoxy)benzoic acid on gold surfaces.

The synthesis of a bipodal diazonium salt, 3,5-bis(4-diazophenoxy)benzoic acid, and the study of its electrochemical deposition on gold surfaces is presented. The presence of the organic layer on the gold surface was characterized using atomic force microscopy and X-ray photoelectron spectroscopy, demonstrating the presence of phenyl groups, indicative of the grafted layer as well as the formation of multilayers, dependent on the electrografting conditions.

Evaluation of techniques for generation of single-stranded DNA for quantitative detection.

A simple and efficient method for the generation of clean single-stranded DNA (ssDNA) with a high recovery and purity from a double-stranded polymerase chain reaction (PCR) product is required for nucleic acid sensing and microarray applications. Currently, the most widely used technique is thermal denaturation due to its simplicity and low cost, but this technique has drawbacks in terms of recovery and reproducibility. The work presented here compares this technique with alternative approaches for ssDNA generation exploiting affinity magnetic separation and exonuclease digestion. The ssDNA recovered was evaluated using gel electrophoresis and enzyme-linked oligonucleotide assay. Recoveries of between 50% and 70% of the theoretical maximum of generatable ssDNA were obtained with good reproducibility, demonstrating a marked improvement in performance as compared with thermal denaturation.

Highly sensitive colorimetric enzyme-linked oligonucleotide assay based on cyclodextrin-modified polymeric surfaces.

In this paper, we describe the development of an enzyme-linked oligonucleotide assay for the detection of a human leukocyte antigen allele associated with celiac disease based on cyclodextrin-modified polymeric surfaces. The surface of maleimide-pre-coated plates was modified with a layer of thiolated cyclodextrin polymer and used for the supramolecular capture of adamantane or ferrocene-modified carboxymethylcellulose polymers bearing DNA probes. The assay was optimised in terms of incubation time, temperature, and surface chemistry and applied to the highly sensitive and selective detection of HLA sequences with a limit of detection of 0.7 nM. A real sample analysed using this platform showed an excellent correlation with maleimide-activated plates using thiolated DNA probes.