A photoelectrochemical enzyme biosensor based on functionalized hematite microcubes for rutin determination by square-wave voltammetry.

A photoelectrochemical biosensing strategy for the highly sensitive detection of the flavonoid rutin was developed by synergizing the photoelectrocatalytic properties of hematite (α-Fe2O3) decorated with palladium nanoparticles (PdNPs) and the biocatalysis towards laccase-based reactions. The integration of α-Fe2O3.PdNPs with a polyphenol oxidase as a biorecognition element yields a novel biosensing platform. Under visible light irradiation, the photoactive biocomposite can generate a stable photocurrent, which was found to be directly dependent upon the concentration of rutin. Under the optimal experimental conditions, the cathodic photocurrent, measured at 0.33 V vs. Ag/AgCl, from the square-wave voltammograms presented a linear dependence on the rutin concentration within the range of 0.008-30.0 × 10-8 mol L-1 (sensitivity: 1.7 µA·(× 10-8 M-1)·cm-2), with an experimental detection limit (S/N = 3) of 8.4 × 10-11 mol L-1. The proposed biosensor device presented good selectivity towards rutin in the presence of various organic compounds and inorganic ions, demonstrating the potential application of this biosensing platform in complex matrices. This bioanalytical device also exhibited excellent operational and analytical properties, such as intra-day (standard deviation, SD = 0.21%) and inter-day (SD = 1.30%) repeatability, and long storage stability (SD = 2.80% over 30 days).Graphical abstract.

Electrochemical Characterization of the Laccase-Catalyzed Oxidation of 2,6-Dimethoxyphenol: an Insight into the Direct Electron Transfer by Enzyme and Enzyme-Mediator System.

The oxidation process of 2,6-dimethoxyphenol (2,6-DMP) by laccase from Botryosphaeria rhodina MAMB-05 and the corresponding enzyme-mediator systems was studied using cyclic voltammetry (CV). The enzyme was classified as a high oxidation potential laccase (> 0.70) V vs. NHE) based on its Redox potential at different pHs. The cyclic voltammograms for 2,6-DMP (- 58.7 mV pH-1) showed that its oxidation potential decreased more significantly compared to the enzyme (- 50.2 mV pH-1) by varying the pH. The 2,2'-azino-bis[3-ethyl-benzothiazoline-6-sulfonic acid] diammonium salt (ABTS) and 2,2,6,6-tetramethylpiperidine 1-oxyl radical (TEMPO) mediators were effectively oxidized by laccase from B. rhodina MAMB-05. The influence of laccase on the comproportionation of ABTS and the ionic step of the oxidation of TEMPO was also studied using CV. A higher potential difference was observed between laccase and the substrate, and correlated with higher enzyme activity. For the laccase-mediator systems, there was no clear correlation of potential difference between laccase and mediators with enzyme activity towards 2,6-DMP. This observation suggests that there are other limiting parameters for enzyme activity despite Redox potential difference, especially during ionic steps of the mechanism.

Covalent attachment of laccase to carboxymethyl-botryosphaeran in aqueous solution for the construction of a voltammetric biosensor to quantify quercetin.

Laccase from Botryosphaeria rhodina MAMB-05 was covalently immobilized on carboxymethyl-botryosphaeran by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) in aqueous solution. This approach was employed to fabricate a novel laccase-based biosensor to electrochemically quantify quercetin (QCT), using a simple carbon black paste electrode as a transducer. The proposed biosensor was characterized by electrochemical impedance spectroscopy and Nyquist plots were used to evaluate the immobilization of the enzyme. For determining QCT, variables were optimized, that included experimental conditions for laccase immobilization, pH of the supporting electrolyte, and instrumental parameters of the electroanalytical technique. From square-wave-voltammograms, a linear dependence between the cathodic current peak and QCT concentration was observed within the range 4.98-50.0 × 10-8 mol L-1, with a theoretical detection limit of 2.6 × 10-8 mol L-1. The proposed method was successfully applied to determine QCT in beverages, pharmaceuticals, and biological samples. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in chemically complex solutions. Operational and analytical stability of the laccase-biosensor were evaluated, and good intra-day (SD = 1.23%) and inter-day (SD = 2.32%) repeatability, and long storage stability (SD = 3.47%) are presented.

Chemometric-assisted construction of a biosensing device to measure chlorogenic acid content in brewed coffee beverages to discriminate quality.

In this work we propose the use of statistical mixture design in the construction of a biosensor device based on graphite oxide, platinum nanoparticles and biomaterials obtained from Botryosphaeria rhodina MAMB-05. The biosensor was characterized by electrochemical impedance spectroscopy. Under optimized experimental parameters by factorial design, the biosensor was applied to the voltammetric determination of chlorogenic acid (CGA) measured as 5-O-caffeoylquinic acid (5-CQA). The biosensor response was linear (R2 = 0.998) for 5-CQA in the concentration range 0.56-7.3 µmol L-1, with limit of detection and quantification of 0.18 and 0.59 µmol L-1, respectively. The new biosensing device was applied to quality control analysis based upon the determination of CGA content in specialty and traditional coffee beverages. The results indicated that specialty coffee had a significantly higher content of CGA. Principal component analysis of the voltammetric fingerprint of brewed coffees revealed that the laccase-based biosensor can be used for their discrimination.

Carboxymethyl-botryosphaeran stabilized carbon nanotubes aqueous dispersion: A new platform design for electrochemical sensing of desloratadine.

The polysaccharide carboxymethyl-botryosphaeran (CMB) was used to improve the dispersion of multi-walled carbon nanotubes (MWCNTs) in water. This feature was applied in modifying a glassy carbon electrode (GCE) to construct a sensitive voltammetric sensor for the determination of desloratadine (DESL), a tricyclic antihistamine. The morphology and spectroscopic behavior of the sensor were evaluated. The modified sensor was characterized as homogeneous, and presented a higher electroactive area and a lower charge transfer resistance compared to the unmodified GCE. Using linear sweep voltammetry at 25 mV s-1, the developed sensor presented a sensitivity of 1.018 µA L µmol-1 in the linear working range of 1.99-32.9 µmol L-1, with a detection limit of 0.88 µmol L-1 of DESL in 0.10 mol L-1 potassium hydrogen-phosphate solution (pH 8.0). In addition, the sensor showed excellent repeatability with a relative standard deviation of only 1.02% for a sequence of 10 measurements. The sensor was successfully applied in the analysis of pharmaceutical preparations containing DESL, with equivalent results compared to a validated spectrophotometric method at the 95% confidence level. The sensor was also employed in the analysis of a spiked sample of DESL in rat serum.

Exploring the exocellular fungal biopolymer botryosphaeran for laccase-biosensor architecture and application to determine dopamine and spironolactone.

Laccase was immobilized on a glassy carbon electrode layered with multi-walled carbon nanotubes using a film of botryosphaeran, a fungal (1 → 3)(1 → 6)-ß-D-glucan. This novel biosensing platform was characterized by electrochemical impedance spectroscopy and scanning electron microscopy, and applied for the determination of dopamine. Experimental variables such as enzyme concentration, pH value and operational parameters of the electroanalytical technique were optimized. Using square-wave voltammetry, there was a linear dependence of peak current and dopamine concentration within the range of 2.99-38.5 µmol L-1 with a limit of detection of 0.127 µmol L-1. The biosensor was successfully applied in the determination of dopamine in pharmaceutical injection and synthetic biological samples, and presented good selectivity even in the presence of uric acid and ascorbic acid, as well as other phenolic compounds. The different aspects regarding the operational stability of the laccase biosensor were evaluated, demonstrating good intra-day and inter-day repeatability, and long-storage stability. Furthermore, this biosensor was evaluated in the indirect determination of spironolactone by using the analytical signal of dopamine, presenting a limit of detection of 0.94 µmol L-1. The results obtained in the analysis of spironolactone in commercial pharmaceutical samples were satisfactory.

Laccase stabilized on ß-D-glucan films on the surface of carbon black/gold nanoparticles: A new platform for electrochemical biosensing.

In this study, (1→3)(1→6)-ß-D-glucan (botryosphaeran) from Botryosphaeria rhodina MAMB-05 was used, for the first time, to immobilize laccase on a carbon black paste electrode modified with gold nanoparticles. The physicochemical characterization of the proposed laccase-biosensor was performed using transmission electron microscopy and electrochemical impedance spectroscopy. The performance of this novel bio-device was evaluated by choosing hydroquinone as a typical model of a phenolic compound. For hydroquinone determination, experimental variables such as enzyme concentration, pH and operational parameters of the electroanalytical technique were optimized. From square-wave voltammograms, a linear dependence between the cathodic current peak and the hydroquinone concentration was observed within the range 2.00-56.5µmolL-1, with a theoretical detection limit of 0.474µmolL-1. The proposed method was successfully applied to determine hydroquinone in dermatological cream, and samples from biological and environmental niches. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in complex matrices. Operational and analytical stability of the laccase biosensor were evaluated, and demonstrated good intra-day (SD=0.3%) and inter-day (SD=3.4%) repeatability and long storage stability (SD=4.9%).