Biosensors for phenol derivatives using biochemical signal amplification

Two different approaches, both exploiting two enzymes cooperative functioning, to enhance the sensitivity of tyrosinase (PPO) based biosensor for amperometric detection of phenols have been compared. For this purpose, one monoenzyme electrode (PPO) and two bienzyme electrodes (PPO and d-glucose dehydrogenase, GDH; PPO and horseradish peroxidase, HRP) were constructed using agar-agar gel as enzyme immobilization matrix. The biosensors responses for l-tyrosine detection were recorded at −50 mV versus saturated calomel electrode (SCE). The highest sensitivity (74 mA M⁻¹) was observed for the PPO-GDH couple, while that recorded for PPO-HRP couple system was only 32 times higher than that measured for monoenzyme electrode (0.01 mA M⁻¹). The ability of the PPO-, PPO-GDH-, PPO-HRP-based biosensors to assay phenols was demonstrated by quantitative determination of phenol, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 2-amino-3 (4-hydroxyphenyl) propanoic acid, 2-hydroxytoluene, 3-hydroxytoluene, 4-hydroxytoluene, 4-clorophenol, 3-clorophenol, 2-clorophenol, 4-hydroxybenzoic acid.     

Disposable biosensors for determination of biogenic amines

This work reports monoamine oxidase (MAO)/horseradish peroxidase (HRP) and diamine oxidase (DAO)/horseradish peroxidase (HRP) based biosensors using screen-printed carbon electrodes for the determination of biogenic amines (BA). The enzymes have been covalently immobilized onto the carbon working electrode, previously modified by an aryl diazonium salt, using hydroxysuccinimide and carbodiimide. The detection has been performed by measuring the cathodic current due to the reduction of the mediator hydroxymethylferrocene at a low potential, 250 mV vs screen-printed Ag/AgCl reference electrode. The experimental conditions for the enzymes immobilization, as well as for the main variables that can influence the chronoamperometric current have been optimized by the experimental design methodology. Under these optimum conditions, the disposable biosensors have been characterized. A linear response range from 0.2 up to 1.6 μM and from 0.4 to 2.4 μM of histamine was obtained for DAO/HRP and MAO/HRP based biosensors, respectively. The biosensor construction was highly reproducible, yielding relative standard deviations of 10% and 11% in terms of sensitivity for DAO/HRP and MAO/HRP based biosensors, respectively. The capability of detection, 0.18 ± 0.01 μM in the case of DAO/HRP and 0.40 ± 0.04 μM (α = 0.05 and β = 0.005) for MAO/HRP based biosensors, and the biosensor sensitivity towards different BA has also been analyzed. Finally, the developed biosensors have been applied to the determination of the total amine content in fish samples.     

Monitoring of malolactic fermentation in wine using an electrochemical bienzymatic biosensor for l-lactate with long term stability

l-lactic acid is monitored during malolactic fermentation process of wine and its evolution is strongly related with the quality of the final product. The analysis of l-lactic acid is carried out off-line in a laboratory. Therefore, there is a clear demand for analytical tools that enabled real-time monitoring of this process in field and biosensors have positioned as a feasible alternative in this regard. The development of an amperometric biosensor for l-lactate determination showing long-term stability is reported in this work. The biosensor architecture includes a thin-film gold electrochemical transducer selectively modified with an enzymatic membrane, based on a three-dimensional matrix of polypyrrole (PPy) entrapping lactate oxidase (LOX) and horseradish peroxidase (HRP) enzymes. The experimental conditions of the biosensor fabrication regarding the pyrrole polymerization and the enzymes entrapment are optimized. The biosensor response to l-lactate is linear in a concentration range of 1 × 10⁻⁶–1 × 10⁻⁴ M, with a detection limit of 5.2 × 10⁻⁷ M and a sensitivity of – (13500 ± 600) μA M⁻¹ cm⁻². The biosensor shows an excellent working stability, retaining more than 90% of its original sensitivity after 40 days. This is the determining factor that allowed for the application of this biosensor to monitor the malolactic fermentation of three red wines, showing a good agreement with the standard colorimetric method.     

Determination of L- and D-enantiomers of methotrexate using amperometric biosensors

In order to determine the enantiopurity of methotrexate (Mtx), seven biosensors were proposed for the assay of l-Mtx and three biosensors for the assay of d-Mtx. The biosensors were designed using physical and chemical immobilization of glutamate oxidase and/or l-amino acid oxidase (l-AAOD) and/or horseradish peroxidase (HRP) for the assay of l-methotherexate, and d-amino acid oxidase (d-AAOD) and HRP for the assay of d-Mtx. Electrode characteristics were obtained and compared for the different carbon paste based biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol l⁻¹ to pmol l⁻¹, magnitude order with limits of detection in the fmol l⁻¹ to nmol l⁻¹ concentration range. All biosensors were successful for the determination of the enantiopurity of Mtx as raw material, and in its pharmaceutical formulations (tablets and injections).     

Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10⁻⁶ to 2.0 × 10⁻⁵ or 1.0 × 10⁻⁶ to 1.0 × 10⁻⁵ M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M⁻¹ were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10⁻⁷ and 3.1 × 10⁻⁷ M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.     

Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of l-malic and l-lactic acids were developed by coimmobilization of the enzymes l-malate dehydrogenase (MDH) and diaphorase (DP), or l-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at −50 mV were used for the monitoring of the enzyme reactions involved in l-malic and l-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for l-malic and l-lactic were obtained with linear ranges of 5.2 × 10⁻⁷ to 2.0 × 10⁻⁵ and 4.2 × 10⁻⁷ to 2.0 × 10⁻⁵ M, respectively. The calculated detection limits were 5.2 × 10⁻⁷ and 4.2 × 10⁻⁷ M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748^T into a synthetic wine. Samples collected during MLF were assayed for l-malic and l-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.     

Characterization of graphite electrodes modified with laccase from Trametes versicolor and their use for bioelectrochemical monitoring of phenolic compounds in flow injection analysis

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 μM, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min⁻¹ and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP).     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414 nA μM⁻¹) compared to 378 nA μM⁻¹ for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74 μM) than the HRP biosensor (83 μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.     

Potentiometric determination of the total acidity and concentration of citric acid in wines

The method of potentiometric titration with a copper electrode is used for the determination of the total acidity and concentration of citric acid (CA) in identifying the adulteration of wines. The procedure is suitable for the determination of citric acid in wines in the range from 0.1 to 3.5 g/L in the presence of 30-fold amounts of tartaric, acetic, malic, succinic acids and a 10-fold amount of oxalic acid after the separation of organic carboxylic acids on an AV-17-8 anion exchanger. The procedure was developed and certified for the potentiometric determination of the mass fraction of citric acid in table wines and wine materials with an error not exceeding 20%. The criteria (mass fraction of citric acid, the percentage of citric acid in the total acidity, and the shape of the curves of potentiometric titration) were proposed for revealing the adulteration of the acid composition of wines.     

Phenylboronic acid self-assembled layer on glassy carbon electrode for recognition of glycoprotein peroxidase

We have successfully fabricated a phenylboronic acid self-assembled layer on glassy carbon electrodes (GCE), where 3-aminophenylboronic acid (APBA) is covalently bound to the electrochemical pretreated GCE surface with glutaraldehyde linkage. The specific binding of glycoprotein peroxidase with the self-assembled layer has been studied using horseradish peroxidase (HRP) as a model glycoprotein. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays show that the affinity interaction of HRP with the APBA modified GCE surface includes specific and nonspecific bonding. The specific binding is attributed to the boronic acid–diols interaction where the boronic acid specifically binds the glycosylation sites of the HRP. This specific binding is reversible and can be split by sorbitol and glucose or released in an acidic buffer. The catalytic current of the HRP-loaded electrode, due to the catalytic oxidation of thionine in the presence of hydrogen peroxide, is proportional to HRP concentrations of the incubation solution. This work offers a new way to build novel sensors by specific binding of glycoproteins to a boronic acid self-assembled layer for determination of glycated proteins.     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

Disposable Horseradish Peroxidase Biosensors for the Selective Determination of Tyramine

This work reports the development of horseradish peroxidase based biosensors using screen‐printed carbon electrodes for the determination of tyramine (tyr). A novel procedure based on the insertion of the enzyme in the screen‐printing process (SPC_HRPEs) has been compared with the cross‐linked immobilization into the carbon working electrode (HRP/SPCEs). Both biosensors were characterized obtaining good capability of detection (2.1±0.2 and 0.2±0.01 µM for SPC_HRPEs and HRP/SPCEs, respectively). The reproducibility was 3.4 % and 6.8 % for SPC_HRPEs and HRP/SPCEs, respectively. The repeatability was 2.2 % and 7.1 % for SPC_HRPEs and HRP/SPCEs, respectively. The specificity towards different biogenic amines was analyzed. The developed biosensors were applied to the determination of tyr content in cheese samples.     

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au nanocomposite for horseradish peroxidase immobilization and biosensing

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au (PEDOT-PSS/Au) nanocomposite was prepared via one-step chemical synthesis and the matrix was characterized by UV-vis spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and transmission electron microscope (TEM). Due to the excellent aqueous compatibility and biocompatibility, the PEDOT/PSS-Au nanocomposite can be used as biomaterial for enzymes immobilization. In this system, redox enzyme, horseradish peroxidase (HRP) was integrated with PEDOT/PSS-Au nanocomposite and the direct electron transfer of HRP was observed. Moreover, we find that the HRP/PEDOT-PSS/Au modified electrode shows excellent electrocatalytic ability for H₂O₂ and the formal Michaelis-Menten constant was 0.78 mmol/L. The response currents have good linear relation with the concentrations of H₂O₂ with a linear range from 2.0 × 10⁻⁷ to 3.8 × 10⁻⁴ mol/L.     

Sequential analysis of malic acid and both enantiomers of lactic acid in wine using a high-performance liquid chromatographic column-switching procedure.

A liquid chromatographic column-switching method for the sequential determination of malic acid and both enantiomers of lactic acid in wine is described. The procedure involves the heart cutting of lactic acid enantiomers from a reversed-phase high-performance liquid chromatography chromatogram, retaining them, and back-flushing them through a chiral ligand-exchange column in which they are separated. The method is used to determine the concentration of lactic acid enantiomers in commercial wines. The results are in satisfactory agreement with those of other methods. The malic acid contents of various wines are also determined. The total analysis time for one experiment is approximately 10 min.     

Oxidase/peroxidase bilayer-modified electrodes as sensors for lactate,pyruvate, cholesterol and uric acid

Enzyme heterobilayer-modified electrodes were fabricated by successively covalently binding to the surface of a tin(IV) oxide plate horseradish peroxidase (HRP), then an oxidase (lactate, pyruvate or cholesterol oxidase or uricase), which liberates hydrogen peroxide by reaction with the respective substrate. The cooperative action of oxidase-HRP leads to an efficient amperometric sensor system with the minimum amount of enzyme immobilized on an electrode.     

Electrografted Poly(N‐mercaptoethyl acrylamide) and Au Nanoparticles‐Based Organic/Inorganic Film: A Platform for the High‐Performance Electrochemical Biosensors

In this study, we describe the use of the combination of eletrografting poly(N‐mercaptoethyl acrylamide) and Au nanoparticles in the construction of high‐performance biosensors. The poly(N‐mercaptoethyl acrylamide) was electrografted onto the glassy carbon electrode surface, which provided a strongly adhering primer film for the stable attachment of Au nanoparticles and horseradish peroxidase (HRP) enzymes. The performances of the biosensors based on the HRP immobilized in the Au/poly(N‐mercaptoethyl acrylamide) composite film were investigated. A couple of redox peaks were obtained, indicating that the Au nanoparticles could facilitate the direct‐electron transfer between HRP and the underlying electrode. The biosensor showed an excellent electrocatalytic activity toward the reduction of hydrogen oxide and long‐term stability, owing to the stable electrografted film and biocompatible Au nanoparticles. Our results demonstrate that the combination of electrografting and Au nanoparticles provides a promising platform for the immobilization of biomolecules and analysis of redox enzymes for their sensing applications.     

Immobilization of Glycosylated Enzymes on Carbon Electrodes,and its Application in Biosensors

A novel approach was used to immobilize glycosylated enzymes on a glassy carbon electrode (GCE) based on the interaction of boronic acid and carbohydrate moiety within the glycoproteins. 4-Aminomethylphenylboronic acid (4-AMBA) was covalently grafted on a glassy carbon electrode (GCE) by amine cation radical formation in the electrooxidation process of the amino-containing compound. The boronic acid group immobilized in this way could recognize glycoproteins such as glucose oxidase, horseradish peroxidase, dehydrogenase and others. X-ray photoelectron spectroscopy measurement proved the presence of a 4-AMBA monolayer on the GCE. The adsorptions of three kinds of enzymes were investigated by cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The activity of the immobilized horseradish peroxidase was also studied.     

Hydrogen peroxide and peracetic acid determination in waste water using a reversible reagentless biosensor

During the reversible reaction between peroxidase (HRP) and peroxides, several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for their determination. On this basis, a reversible reagentless optical biosensor based on HRP for hydrogen peroxide and peracetic acid determinations has been developed. The biosensor (which can be used for at least 3 months and/or more than 200 measurements) is prepared by HRP entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the analyte concentration is also presented. Both peroxides show similar responses in the sensor film. Under the recommended working conditions, the biosensor shows linear response ranges from 6 × 10⁻⁷ to 1.0 × 10⁻⁴ M using FIA mode, and from 2 × 10⁻⁷ to 1.5 × 10⁻⁵ M using continuous mode for both peroxides; the precision, expressed as R.S.D., is about 4%. This biosensor has been applied for peroxide determination in waste water samples previously treated with peroxides.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hyaluronic acid and single walled carbon nanotubes composite film

Direct electrochemistry and electrocatalysis of horseradish peroxidase (HRP) immobilized on a hyaluronic acid (HA)-single walled carbon nanotubes (SCNs) composite film coated glassy carbon electrode (GCE) was studied for the first time. HRP entrapped in the SCNs-HA composite film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks in a 0.1 M phosphate buffer solution (pH 7.0). Formal potential vs. standard calomel electrode (E°′) was −0.232 V, and E°′ was linearly dependent on the solution pH indicating that the electron transfer was proton-coupled. The current is linearly dependent on the scan rate, indicating that the direct electrochemistry of HRP in that case is a surface-controlled electrode process. UV-VIS spectrum suggested HRP retained its original conformation in the SCNs-HA film. Immobilized HRP showed excellent electrocatalysis in the reduction of hydrogen peroxide (H₂O₂).