Electrochemiluminescence (ECL) Biosensors based on Tris(2,2'-bipyridyl)ruthenium(II) with Glucose and Lactate Dehydrogenases encapsulated within Alginate Hydrogels

Two dehydrogenase enzymes (glucose, GDH, and lactate, LDH, dehydrogenases) encapsulated within alginate hydrogels were deposited on glassy carbon electrodes. The as-prepared enzyme modified alginate hydrogels were utilized as electrochemiluminescence (ECL)-based biosensors for the indirect detection of glucose and lactic acid upon reaction between NADH and tris(2,2′-bipyridyl) ruthenium (II) [Ru(bpy)3]2+. The ECL response was obtained from the redox reaction between the substrate, the cofactor NAD+ and the encapsulated enzyme. The production of NADH resulting from the enzymatic reaction led to the ECL emission upon reaction with [Ru(bpy)3]2+. The biosensors showed good stability and repeatability, with linear range between 0.56-4.2 µM and limit of detection of 0.84 µM for glucose, and linear range between 5-30 µM with a limit of detection of 2.52 µM for lactic acid. These ECL-based biosensors showed good sensitivity when tested in the presence of common interfering species. These biosensors were utilized in artificial sweat and were characterized by good reproducibility and repeatability. The results herein presented suggest that the dehydrogenases encapsulated within alginate hydrogels have potential for the development of biocompatible sensors for detection of glucose and lactic acid in physiological fluids

Voltammetry at Hexamethyl-P-Terphenyl Poly(Benzimidazolium) (HMT-PMBI)-Coated Glassy Carbon Electrodes: Charge Transport Properties and Detection of Uric and Ascorbic Acid

We describe the voltammetric behavior of an anion-exchange membrane, hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI). The anion-exchange properties of HMT-PMBI chemically modified electrodes were investigated using K4Fe(CN)6 and K2IrCl6 as redox probes. The permselectivity properties of HMT-PMBI chemically modified electrodes were ascertained using tris(2-2’)bipyridyl-ruthenium(II) chloride Ru(bpy)32+. Cyclic voltammetry and chronoamperometry were utilized to extract parameters such as the concentration of the redox mediators inside the films and the apparent diffusion coefficients. We found the concentration of K4Fe(CN)6 and K2IrCl6 redox species within HMT-PMBI-coated films to be on the order of 0.04–0.1 mol·dm−3, and values of Dapp ca. 10−10–10−9 cm2·s−1. To evaluate the possibility of using such a polymer coating in electroanalysis, HMT-PMBI-modified electrodes were utilized for the voltammetric detection of uric acid in artificial urine, Surine® and ascorbic acid in Vitamin C samples. The results showed that HMT-PMBI-coated electrodes can detect uric acid in Surine® with a limit of detection (LoD) of 7.7 µM, sensitivity of 0.14 µA·µM−1·cm−2, and linear range between 5 μM and 200 μM, whereas for Vitamin C tablets, the LoD is 41.4 µM, the sensitivity is 0.08 µA·µM−1·cm−2, and the linear range is between 25 μM and 450 μM

Enzymes Encapsulated within Alginate Hydrogels: Bioelectrocatalysis and Electrochemiluminescence Applications

A simple procedure to incorporate enzymes (horseradish peroxidase, HRP, and lactate oxidase, LOx) within alginate hydrogels is reported with electrochemiluminescence (ECL) used to detect the enzymatic reactions with the corresponding substrates. First, HRP and LOx were successfully immobilized into CaCO3 microspheres, followed by the electrostatic layer-by-layer deposition of a nanoshell onto the microspheres, and finally by their dispersion into alginate solution. The as-prepared dispersion was drop cast onto the glassy carbon electrodes and cross-linked by the external and internal gelation methods using Ca2+ cations. The enzymes encapsulated within the alginate hydrogels were characterized using cyclic voltammetry and kinetic studies performed using ECL. The results showed that the enzymatic activity was significantly maintained as a result of the immobilization, with values of the apparent Michaelis–Menten constants estimated as 7.71 ± 0.62 and 8.41 ± 0.43 μM, for HRP and LOx, respectively. The proposed biosensors showed good stability and repeatability with an estimated limit of detection of 5.38 ± 0.05 and 0.50 ± 0.03 μM for hydrogen peroxide and lactic acid, respectively. The as-prepared enzymes encapsulated within the alginate hydrogels showed good stability up to 28 days from their preparation. The sensitivity and selectivity of the enzymes encapsulated within the alginate hydrogels were tested in real matrices (HRP, hydrogen peroxide, in contact lens solution; LOx, lactic acid in artificial sweat) showing the sensitivity of the ECL detection methods for the detection of hydrogen peroxide and lactic acid in real samples

Preparation and voltammetric characterization of electrodes coated with Langmuir-Schaefer ultrathin films of Nafion®

Ultrathin films of Nafion® perfluorinated polymer were deposited on indium-tin oxide electrodes (ITO) by using Langmuir-Schaefer (LS) technique, after optimization of the subphase composition conditions. Morphological characteristics of these coatings were obtained by Atomic Force Microscopy (AFM). Nafion® LS films showed a good uniformity and complete coverage of the electrode surface, however a different organization degree of the polymer layer was evidenced with respect to thin films deposited by spin-coating. ITO electrodes modified with Nafion® LS coatings preconcentrate by ion-exchange electroactive cations, such as Ru[(NH3)6]3+, dissolved in diluted solutions. The electroactive species is retained by the Nafion® LS coated ITO also after transfer of the modified electrode into pure supporting electrolyte. This allowed the use of the ruthenium complex as voltammetric probe to test diffusion phenomena within the Nafion® LS films. Apparent diffusion coefficients (Dapp) of Ru[(NH3)6]3+ incorporated in Nafion® LS films were obtained by voltammetric measurements. Dapp values decrease slightly by increasing the amount of ruthenium complex incorporated in the ultrathin film. They are significantly lower than values typical for recasted Nafion® films, in agreement with the highly condensed nature of the Nafion® LS fims. Filmes ultrafinos do polímero perfluorado Nafion® foram depositados em eletrodos de óxido índio-titânio (ITO) usando a técnica de Langmuir-Scaefer (LS), depois da otimização das condições de composição da subfase. As características morfológicas das camadas foram obtidas por Microscopia de Força Atômica (AFM). Filmes de Nafion® LS mostraram boa uniformidade e cobertura completa da superfície do eletrodo. No entanto, diferentes graus de organização das camadas poliméricas ficaram evidentes com respeito a filmes finos depositados por revestimento rotacional. Eletrodos ITO modificados com camadas de Nafion® LS, pré-concentrados por cátions eletroativos de troca iônica, como o Ru[(NH3)6]3+, dissolveram-se em soluções diluídas. A espécie eletroativa foi retida pelo ITO com camada de Nafion® LS, mesmo depois da mudança do eletrodo modificado para eletrodo de suporte purificado. Isto possibilitou o uso do complexo de rutênio como padrão voltamétrico para o teste do fenômeno de difusão nos filmes de Nafion® LS. Os coeficientes de difusão aparentes (Dapp) do Ru[(NH3)6]3+ incorporado no filme de Nafion® LS foram obtidos por medidas voltamétricas. Os valores de Dapp diminuíram levemente com o aumento da quantidade de complexo de rutênio incorporado no filme ultrafino e foram significativamente mais baixos do que os valores para filmes de Nafion® recapados, em acordo com a natureza altamente condensada dos filmes de Nafion® LS

Tris(2,4,6-trimethoxyphenyl)polysulfone-methylene quaternary phosphonium chloride (TPQPCl) ionomer chemically modified electrodes: An electroanalytical study towards sensing applications

We report for the first time the redox properties of an anion-exchange ionomer membrane, tris(2,4,6-trimethoxyphenyl)polysulfone-methylene quaternary phosphonium chloride (TPQPCl). TPQPCl was solubilized in ethanol/water solutions and films of TPQPCl at different concentrations were drop casted on glassy carbon electrodes. The thickness of TPQPCl films in dry conditions was evaluated using profilometer. The anion-exchange properties and charge transport properties of TPQPCl coated electrodes were investigated using K4Fe(CN)6 as anionic redox probe. The permselectivity properties of TPQPCl coated electrodes were assessed using hexaammine ruthenium(II) chloride. Cyclic voltammetry performed at low scan rate was utilized to determine the concentration of the redox mediator inside the films, with the apparent diffusion coefficient values of different TPQPCl coating estimated using both the Randles-Sevcik equation and the Anson's method. We found the concentration of the ferro/ferricyanide couple redox within the TPQPCl films in the order of 10−2-10−1 mol dm−3, with values of Dapp in the order of 10−9 cm2 s−1. TPQPCl drop casted films evidenced good preconcentration capabilities towards incorporation of anions. To ascertain the suitability of the ionomer coated films towards the electroanalytical detection of anions, TPQPCl coated electrodes were utilized for the voltammetric detection of nitrites in tap and river waters. The results showed that TPQPCl coated electrodes are suitable to quantify nitrites in fresh waters with a limit of detection, LoD, of 1.07 μM, sensitivity as 0.267 μA μM−1 cm−2 and linear range between 1 μM and 500 μM

Poly(ethylene glycol)(PEG)-cryogels: A novel platform towards enzymatic electrochemiluminescence (ECL)-based sensor applications

Enzymes-based electrochemical biosensors require the immobilisation of the enzymes on the electrode surfaces as well as their storage in aqueous environments to maintain the enzymatic activity. Herein, we described an enzyme-based electrochemiluminescence biosensor fabricated by incorporating oxidase enzymes (horseradish peroxidase, HRP; glucose, GOx, lactate, LOx, and cholesterol oxidases, ChOx) within poly(ethylene glycol)diacrylate (PEGDA) cryogels, which retain their activity when stored in dry conditions. The redox reactions between the oxidase enzymes and their corresponding substrates produce hydrogen peroxide that can be detected in the presence of a layer of polyluminol deposited on the electrode surface. These oxidases PEG-based cryogels were characterized using cyclic voltammetry and electrochemiluminescence (ECL) to assess the redox reactions between the enzymes and the corresponding substrates. The proposed biosensors were characterised by good stability and repeatability with a calculated limit of detections (LODs) in the micromolar concentration range. The performances of PEG cryogels over the time evidenced the stability of the as-prepared materials up to 30 days in dry conditions, confirming good retention of the encapsulated enzymes. Furthermore, the biosensors were tested in the presence of interferent species showing good selectivity. Finally, these oxidases-PEG cryogels were tested in real samples (commercial contact lenses, artificial sweat and commercial milk) confirming the suitability of such material for the detection of hydrogen peroxide with calculated LoDs as 10.37 ± 0.4 µM for HRP/contact lenses liquid; 3.87 ± 0.3 µM for GOx/artificial sweat; 1.09 ± 0.6 µM for LOx/artificial sweat; and 6.59 ± 0.5 for ChOx/milk

Electrochemiluminescence (ECL) sensing properties of water soluble core-shell CdSe/ZnS quantum dots/Nafion composite films

Water soluble positively charged 2-(dimethylamino) ethanethiol (DAET)-protected core-shell CdSe/ZnS quantum dots (QDs) were synthesized and incorporated within negatively charged Nafion polymer films. The water soluble QDs were characterized using UV-visible and fluorescence spectroscopies. Nafion/QDs composite films were deposited on glassy carbon electrodes and characterized using cyclic voltammetry. The electrochemiluminescence (ECL) using hydrogen peroxide as co-reactant was enhanced for Nafion/QDs composite films compared to films of the bare QDs. Significantly, no ECL was observed for Nafion/QDs composite films when peroxydisulfate was used as the co-reactant, suggesting that the permselective properties of the Nafion effectively exclude the co-reactant. The ECL quenching by glutathione depends linearly on its concentration when hydrogen peroxide is used as the co-reactant, opening up the possibility to use Nafion/QDs composite films for various electroanalytical applications

Efficient Microwave-Assisted Extraction of Nitrites from Cured Meat and Their Voltammetric Detection at Chemically Modified Electrodes Based on Hexamethyl-p-Terphenyl Poly(methylatedbenzimidazolium) Incorporating Nitrogen-Doped Graphite Nanoplatelets

We describe a fast and reliable procedure for the efficient extraction of nitrites in cured meat using microwave-assisted heat and report their in situ determination via voltammetry using an anion-exchanger ionene, hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI), and nitrogen-doped graphite nanoplatelets (NGNPs). Cyclic voltammetry and chronoamperometry were utilized to evaluate the concentration of the redox mediator within the film and apparent diffusion coefficient. To investigate the suitability of the composite material for sensing applications, HMT-PMBI/NGNPs were tested for their detection of nitrite in bacon samples without the need of any pretreatments or dilutions. HMT-PMBI/NGNP coated electrodes showed enhanced sensitivity in the detection of nitrite ions in bacon with a limit of detection (LoD) of 0.64 µM, sensitivity 0.52 µA µM−1 cm−2, and operating in a linear range between 1–300 μM. The results highlight that the determination of nitrites in cured meat using microwave extraction is in good agreement with standard procedures such as the ISO 2918 and the AOAC International 973.31 methods

Quaternary phosphonium-based (TPQPCl)-ionomer/graphite nanoplatelets composite chemically modified electrodes: a novel platform for sensing applications

Ionomers have attracted considerable interest in electroanalysis due to the possibility of fabricating electrode coatings capable of preconcentrating sub-micromolar concentrations of cations or anions of analytical relevance. In this work, we describe the electroanalytical performances of an ionomer (TPQPCl)/graphite nanoplatelets composite material towards the development of an amperometric sensor for detection of ascorbic acid. Graphite nanoplatelets at different concentrations were dispersed in ethanolic solutions containing TPQPCl. The as-prepared TPQPCl/graphite nanoplatelet-coated electrodes were characterised using Fe(CN)64−/3− as an anionic redox probe. The results evidence the good preconcentration capability of the positively charged TPQPCl towards the incorporation of negatively charged species. By tuning the ionomer/graphite nanoplatelets ratio, it is possible to detect simultaneously ascorbic acid even in the presence of dopamine as an interference species. The TPQPCl/graphite nanoplatelet-coated electrodes were able to detect ascorbic acid in the linear range of 5–10 000 μM with a limit of detection calculated as 4.8 μM using linear sweep voltammetry. Finally, the TPQPCl/graphite nanoplatelet-coated electrodes were tested towards detection of ascorbic acid in vitamin C tablets and orange juice without any sample pretreatment