l-Cysteine-Tuned the Hierarchical Structure Based on Benzimidazole: Synthesis, Characterization, and Application in Ratiometric Electrochemiluminescence Immunoassay.

Efficient and robust electrochemiluminescence (ECL) emitters are crucial for enhancing the ECL immunosensor sensitivity. This study introduces a novel ECL emitter, CoBIM/Cys, featuring a hierarchical core-shell structure. The core of the structure is created through the swift coordination between the sulfhydryl and carboxyl groups of l-cysteine (l-Cys) and cobalt ions (Co2+), while the shell is constructed by sequentially coordinating benzimidazole (BIM) with Co2+. This design yields a greater specific surface area and a more intricate porous structure compared to CoBIM, markedly enhancing mass transfer and luminophore accessibility. Moreover, the l-Cys and Co2+ core introduces Co-S and Co-O catalytic sites, which improve the catalytic decomposition of H2O2, leading to an increased production of hydroperoxyl radicals (OOH•). This mechanism substantially amplifies the ECL performance. Leveraging the competitive interaction between isoluminol and BIM for OOH• during ECL emission, we developed a ratiometric immunosensor for cardiac troponin I (cTnI) detection. This immunosensor demonstrates a remarkably broad detection range (1 pg mL-1 to 10 ng mL-1), a low detection limit (0.4 pg mL-1), and exceptional reproducibility and specificity.

Fe-MOGs-based enzyme mimetic and its mediated electrochemiluminescence for in situ detection of H2O2 released from Hela cells.

Enzyme mimetics have attracted wide interest due to their inherent enzyme-like activity and unique physicochemical properties, as well as promising applications in disease diagnosis, treatment and monitoring. Inspired by the attributes of nonheme iron enzymes, synthetic models were designed to mimic their capability and investigate the catalytic mechanisms. Herein, metal-organic gels (Fe-MOGs) with horseradish peroxidase (HRP) like Fe-NX structure were successfully synthesized though the coordination between iron and 1,10-phenanthroline-2,9-dicarboxylic acid (PDA) and exhibited excellent peroxidase-like activity. Its structure-activity relationship and the in-situ electrochemiluminescence (ECL) detection of H2O2 secreted by Hela cells were further investigated. The highly dispersed Fe-NX active sites inside Fe-MOGs were able to catalyze the decomposition of H2O2 into large amounts of reactive oxygen species (ROS) via a Fenton-like reaction under a low overpotential. Due to the accumulation of ROS free radicals, the luminol ECL emission was significantly amplified. A proof-of-concept biosensor was constructed with a detection limit as low as 2.2 nM and a wide linear range from 0.01 to 40 µM. As a novel metal organic gels based enzyme mimetic, Fe-MOGs show great promises in early cancer detection and pathological process monitoring.

Microcapsule-based biosensor containing catechol for the reagent-free inhibitive detection of benzoic acid by tyrosinase.

A biosensor based on the release of the enzyme substrate from its structure was developed for the inhibitive detection of benzoic acid. A polyurethane support comprising two perforated microcapsules (800 µm in diameter) filled with methylene blue as a model compound and covered with a conductive deposit of multiwalled carbon nanotubes, continuously released this stored dye for 24 h. An increase in methylene blue concentration of 0.5-0.75 µmol L-1 h-1 and 1.5-2 µmol L-1 h-1, in the presence and absence of the multiwalled carbon nanotube coating, respectively, was demonstrated by UV-vis spectroscopy in a 2 mL UV cuvette. The same configuration with microcapsules filled with catechol was modified by a laponite clay coating containing tyrosinase enzyme. The resulting biosensor exhibits a constant cathodic current at -0.155 V vs AgCl/Ag, due to the reduction of the ortho-quinone produced enzymatically from the released catechol. The detection of benzoic acid was recorded from the decrease in cathodic current due to its inhibiting action on the tyrosinase activity. Reagentless biosensors based on different deposited quantity of tyrosinase (100, 200, 400 and 600 µg) were investigated for the detection of catechol and applied to the detection of benzoic acid as inhibitor. The best performance was obtained with the 400 µg-based configuration, namely a detection limit of 0.4 µmol L-1 and a sensitivity of 228 mA L mol-1. After the inhibition process, the biosensors recover 97-100% of their activity towards catechol, confirming a reversible inhibition by benzoic acid.

ATMP-induced three-dimensional conductive polymer hydrogel scaffold for a novel enhanced solid-state electrochemiluminescence biosensor.

Reliable and sensitive detection of xanthine has important medical and biological significance. In this work, a novel three-dimensional (3D) conductive polymer hydrogel of polyaniline (PAni) was feasibly prepared using aniline (Ani), amino trimethylene phosphonic acid (ATMP) and ammonium persulfate ((NH4)2S2O8) as monomer, gelatinizing agent and oxidizing agent, respectively. Protonation of aniline can be achieved by ATMP, inducing good conductivity of the obtained hydrogel. ATMP remained the chelating abilities in the conductive hydrogel, enabling further immobilization with silver nanoparticles (AgNPs) functionalized by a luminol derivative, N-(aminobutyl)-N-(ethylisoluminol) (ABEI). ABEI-Ag@PAni-ATMP exhibited an enhanced performance of solid-state electrochemiluminescence (ECL). Integrated with xanthine oxidase (XOD), the proposed biosensor can be applied in the detection of xanthine via in-situ generated hydrogen peroxide (H2O2), and present a low detection limit of 9.6 nM, a wide linear range (from 0.01 to 200 µM) and excellent stability.

DNA-Mediated Nanoscale Metal-Organic Frameworks for Ultrasensitive Photoelectrochemical Enzyme-Free Immunoassay.

A novel enzyme-free photoelectrochemical (PEC) immunoassay was developed for the ultrasensitive detection of prostate specific antigen (PSA) based on the DNA-mediated nanoscale zirconium-porphyrin MOFs (NMOFs). By virtue of the intrinsic coordination between unsaturated zirconium sites of the NMOFs frameworks and phosphonate groups, the 5'-phosphorylared ss-DNA-tagged antibody (Ab-DNA) conjugate with a consecutive stretch of guanines as a spacer could be loaded on the NMOFs easily, obtaining a novel type of Ab-DNA-functionalized NMOFs complex. Additionally, as a photocathode PEC active nanomaterial, NMOFs exhibited a significant enhanced photocurrent response with the presence of dopamine under oxygen-containing aqueous media at -0.3 V (vs Ag/AgCl). Furthermore, with the aid of the electrochemical grafting of polyamidoamine (PAMAM) dendrimers functionalized interface, the novel type of Ab-DNA-NMOFs further served as a PEC signal nanoprobe for the ultrasensitive PSA immunoassay. Under optimal conditions, the corresponding immunosensor possessed a wide calibration range of 1 pg mL-1 to 10 ng mL-1 and a limit of detection (LOD) of 0.2 pg mL-1. This present work demonstrated the promising application of DNA-mediated NMOFs in developing highly sensitive, environmentally friendly, and cost-effective PEC biosensors.

Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody.

An original impedimetric immunosensor was developed based on carbon nanotube (CNT) deposits with controlled thicknesses for enhanced electroactive surface areas leading to improved sensor performances. Cholera monitoring was chosen as the model immune system for this setup. These CNT deposits were characterized using confocal laser microscopy and electrochemical methods. To form the sensor device, the CNT deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly(pyrrole-NTA)) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B Subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. Each step of the formation of the immunosensor and the subsequent binding of the analyte antibody anti-cholera toxin were investigated with cyclic voltammetry and Electrochemical Impedance Spectroscopy. After optimization, the resulting impedimetric cholera sensor shows excellent reproducibility, increased sensitivities, a very satisfying detection limit of 10-13gmL-1 and an exceptional linear range for anti-cholera detection of 8 orders of magnitude (10-13-10-5gmL-1) and a sensitivity of 24.7 ± 0.4Ω per order of magnitude.

Carbon-Nanotube-Supported Bio-Inspired Nickel Catalyst and Its Integration in Hybrid Hydrogen/Air Fuel Cells.

A biomimetic nickel bis-diphosphine complex incorporating the amino acid arginine in the outer coordination sphere was immobilized on modified carbon nanotubes (CNTs) through electrostatic interactions. The functionalized redox nanomaterial exhibits reversible electrocatalytic activity for the H2 /2 H+ interconversion from pH 0 to 9, with catalytic preference for H2 oxidation at all pH values. The high activity of the complex over a wide pH range allows us to integrate this bio-inspired nanomaterial either in an enzymatic fuel cell together with a multicopper oxidase at the cathode, or in a proton exchange membrane fuel cell (PEMFC) using Pt/C at the cathode. The Ni-based PEMFC reaches 14 mW cm-2 , only six-times-less as compared to full-Pt conventional PEMFC. The Pt-free enzyme-based fuel cell delivers ≈2 mW cm-2 , a new efficiency record for a hydrogen biofuel cell with base metal catalysts.

Ferrocyanide-Ferricyanide Redox Couple Induced Electrochemiluminescence Amplification of Carbon Dots for Ultrasensitive Sensing of Glutathione.

Here we report a novel solid-state ECL sensor for ultrasensitive sensing of glutathione (GSH) based on ferrocyanide-ferricyanide redox couple (Fe(CN)6(3-/4-)) induced electrochemiluminescence (ECL) amplification of carbon dots (C-dots). The electropolymerization of C-dots and (11-pyrrolyl-1-yl-undecyl) triethylammonium tetrafluoroborate (A2) enabled immobilization of the hydrophilic C-dots on the surface of glassy carbon electrode (GCE) perfectly, while the excellent conductivity of polypyrrole was exploited to accelerate electron transfer between them. The Fe(CN)6(3-/4-) can expeditiously convert the C-dots and S2O8(2-) to C-dot(•-) and SO4(•-), respectively. High yields of the excited state C-dots (C-dots*) were obtained, and a ∼10-fold ECL amplification was realized. The C-dots* obtained through the recombination of electron-injected and hole-injected processes may be impeded due to the interference of GSH to K2S2O8. Therefore, the constructed sensor for GSH showed a detection limit down to 54.3 nM (S/N = 3) and a wide linear range from 0.1-1.0 µM with a correlation coefficient of 0.997.

Noncovalently functionalized monolayer graphene for sensitivity enhancement of surface plasmon resonance immunosensors.

A highly efficient surface plasmon resonance (SPR) immunosensor is described using a functionalized single graphene layer on a thin gold film. The aim of this approach was two-fold: first, to amplify the SPR signal by growing graphene through chemical vapor deposition and, second, to control the immobilization of biotinylated cholera toxin antigen on copper coordinated nitrilotriacetic acid (NTA) using graphene as an ultrathin layer. The NTA groups were attached to graphene via pyrene derivatives implying π-π interactions. With this setup, an immunosensor for the specific antibody anticholera toxin with a detection limit of 4 pg mL(-1) was obtained. In parallel, NTA polypyrrole films of different thicknesses were electrogenerated on the gold sensing platform where the optimal electropolymerization conditions were determined. For this optimized polypyrrole-NTA setup, the simple presence of a graphene layer between the gold and polymer film led to a significant increase of the SPR signal.

Biomimetic versus enzymatic high-potential electrocatalytic reduction of hydrogen peroxide on a functionalized carbon nanotube electrode.

We report the non-covalent functionalization of a multi-walled carbon nanotube (MWCNT) electrode with a biomimetic model of the horseradish peroxidase (HRP) active site. By modifying the MWCNT electrode surface with imidazole-modified polypyrrole, a new biomimetic complex of HRP was synthesized on the MWCNT sidewalls via the coordination of imidazole (Im) to the metal centre of iron protoporphyrin IX, affording (Im)(PP)FeIII . Compared to the pi-stacking of non-coordinated (PP)FeIII on a MWCNT electrode, the (Im)(PP)FeIII -modified MWCNT electrode exhibits higher electrocatalytic activity with an Imax = 0.52 mA cm-2 for the reduction of H2O2, accompanied by a high onset potential of 0.43 V vs. Ag/AgCl. The performances of these novel surface-confined HRP mimics were compared to those of a MWCNT electrode modified by HRP. Although the enzyme electrode displays a higher electrocatalytic activity towards H2O2 reduction, the (Im)(PP)FeIII -modified MWCNT electrode exhibits a markedly higher operational stability, retaining 63% of its initial activity after one month.

Label-free photoelectrochemical detection of double-stranded HIV DNA by means of a metallointercalator-functionalized electrogenerated polymer.

The design of photoactive functionalized electrodes for the sensitive transduction of double-stranded DNA hybridization is reported. Multifunctional complex [Ru(bpy-pyrrole)2 (dppn)](2+) (bpy-pyrrole=4-methyl-4'-butylpyrrole-2,2'-bipyridine, dppn=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) exhibiting photosensitive, DNA-intercalating, and electropolymerizable properties was synthesized and characterized. The pyrrole groups undergo oxidative electropolymerization on planar electrodes forming a metallopolymer layer on the electrode. Thanks to the photoelectrochemical and intercalating properties of the immobilized Ru(II) complex, the binding of a double-stranded HIV DNA target was photoelectrochemically detected on planar electrodes. Photocurrent generation through visible irradiation was correlated to the interaction between double-stranded DNA and the metallointercalator polymer. These interactions were well fitted by using a Langmuir isotherm, which allowed a dissociation constant of 2×10(6)  L mol(-1) to be estimated. The low detection limit of 1 fmol L(-1) and sensitivity of 0.01 units per decade demonstrate excellent suitability of these modified electrodes for detection of duplex DNA.

Biofunctionalization of multiwalled carbon nanotubes by electropolymerized poly(pyrrole-concanavalin A) films.

The synthesis and electropolymerization of a pyrrolic concanavalin A derivative (pyrrole-Con A) onto a multiwalled carbon nanotube (MWCNT) deposit is reported. Glucose oxidase was then immobilized onto the MWCNT-poly(pyrrole-Con A) coating by affinity carbohydrate interactions with the polymerized Con A protein. The resulting enzyme electrode was applied to the amperometric detection of glucose exhibiting a high sensitivity of 36 mA cm(-2) mol(-1) L and a maximum current density of 350 µA cm(-2) .

Polypyrrolic bipyridine bis(phenantrolinequinone) Ru(II) complex/carbon nanotube composites for NAD-dependent enzyme immobilization and wiring.

We report the synthesis and electrochemical characterization of a novel electropolymerizable Ru(II) complex containing two phenanthrolinequinone ligands, Ru(II)(PhQ)2(bpy-pyrrole)(PF6)2. This complex was electropolymerized on glassy carbon (GC) and multiwalled carbon nanotube (MWCNT) electrodes. Higher apparent surface concentrations (80 nmol cm(-2)) were obtained on MWCNTs than on GC electrodes and correspond to ∼1000 equivalent compact monolayers of Ru complex. Moreover, the nanostructured metallopolymer exhibits efficient electrocatalytic properties toward oxidation of NADH. This metallopolymer can be electrogenerated in water from the adsorbed Ru(II) monomer. This property was applied to the immobilization of enzymes by coadsorption of Ru complex and enzyme and then electropolymerization of coatings. This two-step procedure leads to the entrapment of 70%-90% of the deposited amount of enzyme in poly-Ru(II)(PhQ)2(bpy-pyrrole) films. Glucose dehydrogenase (GDH) was thus efficiently immobilized in the electrogenerated polymer matrix. In presence of NAD(+) (10 mM), the resulting enzyme electrode exhibits high current densities for glucose oxidation of 1.04 mA cm(-2) at low overpotentials (-0.1 V) with a detection limit of 1 µM of glucose.

Permeability improvements of electropolymerized polypyrrole films using dissolvable nano-CaCO3 particle templates.

The electropolymerisation of N-substituted pyrroles on a dissolvable calcium carbonate nanoparticle template was investigated in order to improve the film permeabilities in aqueous solution. After deposition of CaCO3 nanoparticles on the electrode surface, poly(pyrrole-ammonium) or poly(pyrrole-NTA) (NTA: nitrilotriacetic acid) were electrogenerated around the template structures of the electrodes using potentiostatic methods. The dissolution of nanoparticles in acidic medium leads to the formation of nano-porous structures increasing, therefore, the polypyrrole permeability in aqueous solutions. Histidine-tagged glucose oxidase, chosen as an enzyme model, was immobilised on the modified polypyrrole-NTA via the NTA-Cu(2+)-histidine interactions to validate the proposed method. The described setup led to a twofold increase in the maximum current density from 5 to 10 µA cm(-2) after template dissolution.

Micro- to nanostructured poly(pyrrole-nitrilotriacetic acid) films via nanosphere templates: applications to 3D enzyme attachment by affinity interactions.

We report the combination of latex nanosphere lithography with electropolymerization of N-substituted pyrrole monomer bearing a nitrilotriacetic acid (NTA) moiety for the template-assisted nanostructuration of poly(pyrrole-NTA) films and their application for biomolecule immobilization. The electrodes were modified by casting latex beads (100 or 900 nm in diameter) on their surface followed by electropolymerization of the pyrrole-NTA monomer and the subsequent chelation of Cu(2+) ions. The dissolution of the nanobeads leads then to a nanostructured polymer film with increased surface. Thanks to the versatile affinity interactions between the (NTA)Cu(2+) complex and histidine- or biotin-tagged proteins, both tyrosinase and glucose oxidase were immobilized on the modified electrode. Nanostructuration of the polypyrrole via nanosphere lithography (NSL) using 900- and 100-nm latex beads allows an increase in surface concentration of enzymes anchored on the functionalized polypyrrole electrode. The nanostructured enzyme electrodes were characterized by fluorescence microscopy, 3D laser scanning confocal microscopy, and scanning electron microscopy. Electrochemical studies demonstrate the increase in the amount of immobilized biomolecules and associated biosensor performances when achieving NSL compared to conventional polymer formation without bead template. In addition, the decrease in nanobead diameter from 900 to 100 nm provides an enhancement in biosensor performance. Between biosensors based on films polymerized without nanobeads and with 100-nm nanobeads, maximum current density values increase from 4 to 56 µA cm(-2) and from 7 to 45 µA cm(-2) for biosensors based on tyrosinase and glucose oxidase, respectively.

Efficient direct oxygen reduction by laccases attached and oriented on pyrene-functionalized polypyrrole/carbon nanotube electrodes.

We report the functionalization of multi-walled carbon nanotube (MWCNT) electrodes by oxidative electropolymerization of pyrrole monomers bearing pyrene and N-hydroxysuccinimide groups. Both polymers were applied to the immobilization and electrical wiring of Trametes versicolor laccase via chemical grafting or non-covalent binding. A "pseudo" host-guest interaction of polymerized pyrene with a hydrophobic cavity of laccase was used for the oriented enzyme immobilization on MWCNT electrodes. The latter leads to higher catalytic current for oxygen reduction (1.85 mA cm(-2)) and higher electroenzymatic stability (50% after one month).

Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film.

A label-free and highly sensitive impedimetric aptasensor was developed based on electropolymerized film for the determination of thrombin. The first step is the electrogeneration of a poly(pyrrole-nitrilotriacetic acid) (poly(pyrrole-NTA)) film onto the surface of electrodes followed by complexation of Cu(2+) ions. Then, the histidine labeled thrombin aptamer was immobilized onto the electrode through coordination of the histidine groups on the NTA-Cu(2+) complex. The aptamer sensor was applied for the detection and quantification of thrombin via impedimetric detection without a labeling step. A linear quantification of thrombin was obtained in the range 4.7×10(-12)-5.0×10(-10) mol L(-1) with a sensitivity of 2838 Ω/log unit (R(2)=0.9984). The impedance modulus at 0.3 Hz as a function of thrombin concentration was used to elaborate a similar linear relationship from 4.7×10(-12) to 5×10(-10) mol L(-1). In addition, aptamer-poly(pyrrole-NTA) electrodes incubated for 40 min in aqueous solutions of bovine serum albumin (BSA), lysozyme and IgG (5×10(-7) mol L(-1)) did not exhibit non-specific adsorption of proteins. Moreover, it has been demonstrated that the selective sensor can be regenerated several times with a good reproducibility.

Electrogenerated poly(pyrrole-lactosyl) and poly(pyrrole-3'-sialyllactosyl) interfaces: toward the impedimetric detection of lectins.

This paper reports on the impedimetric transduction of binding reaction between polymerized saccharides and target lectins. The controlled potential electro-oxidation of pyrrole-lactosyl and pyrrole-3'-sialyllactosyl at 0.95 V vs. Ag/AgCl, provides thin and reproducible poly(pyrrole-saccharide) films. The affinity binding of two lectins: Arachis hypogaea, (PNA) and Maackia amurensis (MAA) onto poly(pyrrole-lactosyl) and poly(pyrrole-3'-sialyllactosyl) electrodes, was demonstrated by cyclic voltammetry in presence of ruthenium hexamine and hydroquinone. In addition, rotating disk experiments were carried out to determine the permeability of both polypyrrole films and its evolution after incubating with lectin target. Finally, the possibility of using the poly(pyrrole-lactosyl) or poly(pyrrole-3'-siallyllactosyl) films for the impedimetric transduction of the lectin binding reaction, was investigated with hydroquinone (2 × 10(-3) mol L(-1)) as a redox probe in phosphate buffer. The resulting impedance spectra were interpreted and modeled as an equivalent circuit indicating that charge transfer resistance (R ct) and relaxation frequency (f°) parameters are sensitive to the lectin binding. R ct increases from 77 to 97 Ω cm(2) for PNA binding and from 93 to 131 Ω cm(2) for MAA binding. In parallel, f° decreases from 276 to 222 Hz for PNA binding and from 223 to 131 Hz for MAA binding. This evolution of both parameters reflects the steric hindrances generated by the immobilized lectins towards the permeation of the redox probe.

Electrogenerated trisbipyridyl Ru(II)-/nitrilotriacetic-polypyrene copolymer for the easy fabrication of label-free photoelectrochemical immunosensor and aptasensor: application to the determination of thrombin and anti-cholera toxin antibody.

A bifunctional copolymer was electrogenerated, which allows efficient bioreceptor immobilization and transduction of the biorecognition event. This copolymer was formed using pyrenebutyric acid Nα',Nα-bis(carboxymethyl)-L-lysine amide (NTA-pyrene) and [tris-(2,2'-bipyridine) (4,4'-bis(4-pyrenyl-1-ylbutyloxy)-2,2'-bipyridine] ruthenium(II) hexafluorophosphate (Ru(II)-pyrene) complex. The pyrene groups, present in both compounds, undergo oxidative electropolymerization on platinum electrodes. The resulting copolymer contains NTA moieties, which were used as a versatile immobilization system for biotin- and histidine-tagged biomolecules, while Ru(II)-pyrene was employed as a photoelectrochemical transducing molecule. The efficiency of this copolymer for biomolecule anchoring was investigated with biotin- and histidine- tagged glucose oxidases, biotin-tagged cholera toxin and a histidine-tagged thrombin aptamer. The constructed enzyme electrodes exhibited an amperometric response toward glucose at 0.6 V vs SCE, demonstrating the anchoring of this enzyme via two coordination systems. An immunosensor configuration based on the immobilization of biotin-tagged cholera toxin was applied to the detection of anti-cholera antibody while the aptasensor based on the immobilization of histidine-tagged thrombin aptamer was tested for thrombin determination. The biorecognition events were monitored via the evolution of the photocurrent intensity generated by the polymerized Ru(II)-pyrene in the presence of visible light and a sacrificial donor (ascorbate). The binding of the targets hinders the diffusion of the sacrificial donor, inducing thus a photocurrent decrease. The constructed immunosensor presents a specific label-free photoelectrochemical response to anti-cholera antibody without labeling step, the detection limit being 0.2 µg mL⁻¹. The label-free photoelectrochemical response of the aptasensor varies linearly with thrombin concentrations up to 10 pmol L⁻¹, the detection limit being 1×10⁻¹³ mol L⁻¹.

Biotin-ß-cyclodextrin: a new host-guest system for the immobilization of biomolecules.

The formation of stable supramolecular interactions between biotin and ß-cyclodextrin was studied. An association constant of 3 × 10(2) M(-1) could be determined by NMR measurements by mapping the high field shift differences of the ß-cyclodextrin protons (H-3) at different biotin concentrations. With the aim to demonstrate a new alternative for the immobilization of bioreceptors, biotin and ß-cyclodextrin tagged biomolecules were immobilized on transducer surfaces, which were functionalized with the correspondent host-guest partner. The reliability of this new affinity system was investigated using two enzymes (glucose oxidase and polyphenol oxidase) as biomolecule models. This supramolecular inclusion complex shows clear advantages to the classic biotin-(strept)avidin-biotin system due to a detrimental effect of the additional avidin layer reducing the transduction efficiency. A 7-fold increase in the maximum current density and an almost 20 times higher sensitivity were exhibited by the immobilized biological layer obtained using this new host-guest system.