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.     

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.     

Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor

This paper presents an analysis method for organophosphorus insecticides based on AChE biosensors coupled with a preconcentration and oxidation on a solid phase column. Three organic solvents, acetonitrile (ACN), ethanol and methanol were tested for their influence on AChE activity, insecticide inhibition and their ability to elute the adsorbed insecticides. Our results showed that ACN in a concentration of 5% (v/v) had the less negative effect on biosensor analysis and was the most appropriate organic solvent for the column elution. The presence of the organic solvent in the incubation media of the biosensor was found to induce a reduction of the inhibition percentages. The inhibition of the biosensors was performed in phosphate buffer with 5% (v/v) ACN, while the initial and remaining response of the biosensors were measured in PBS. In these conditions, the LODs of paraoxon and dichlorvos were measured with or without a preconcentration step. The LODs of the AChE biosensor without sample preconcentration were 8 × 10⁻⁸ M for paraoxon and 1 × 10⁻⁷ M dichlorvos and the LOD obtained after the preconcentration step were 2.5 × 10⁻⁸ M for paraoxon and 2.5 × 10⁻⁸ M for dichlorvos. Moreover, the use of the column allowed the heterogeneous oxidation of organophosphorus insecticides for improved LOD.     

One-step construction of reagentless biosensor based on chitosan-carbon nanotubes-nile blue-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of novel reagentless biosensors by in situ formation of chitosan-carbon nanotubes-nile blue-horseradish peroxidase (CS-CNTs-NB-HRP) biocomposite film on electrode surface. The mediator effect of NB, conducting performance of CNTs and the biocompatible microenvironment of CS were combined by such one-step non-manual process. NB could interact with CNTs and resulted in good dispersion of CNTs-NB nanocomposites in aqueous solution. Cyclic voltammetry measurements demonstrated that electrons were efficiently shuttled between HRP and the electrode mediated by NB. The developed reagentless biosensor exhibited a fast amperometric response for the determination of H₂O₂ and 95% of the steady-state current was obtained within 2 s. The linear response of the reagentless biosensor for the determination of H₂O₂ ranged from 1.0 × 10⁻⁶ to 2.4 × 10⁻⁴ mol l⁻¹ with a detection limit of 1.2 × 10⁻⁷ mol l⁻¹. The biosensor exhibited high reproducibility and long-time storage stability. The as-prepared biosensor also showed effective anti-interference capability. The ease of the one-step non-manual technique and the promising feature of the biocomposite could serve as a versatile platform for fabricating electrochemical biosensors.     

Electrochemical biosensors for monitoring malolactic fermentation in red wine using two strains of Oenococcus oeni

Amperometric biosensors for the determination of l-malic and l-lactic acids were optimised and used to monitor micro-malolactic fermentations (micro-MLFs) in red wine. Platinum-based probes, coupled with appropriate enzymes, were assembled in electrochemical flow-injection analysis systems. A classical lactate oxidase based sensor was used for l-lactic acid, while l-malic acid was detected via a new biosensor based on the malic enzyme immobilised in a reactor using phenazine methosulphate as mediator. After a preliminary optimisation phase, a recovery study to evaluate the effect of the matrix (red wine) on biosensor performance was carried out by the addition of different standard solutions of the two analytes to the samples. Recoveries from 93 to 100% and from 94 to 102% were observed for l-malic acid and l-lactic acid, respectively. These optimised biosensors were finally employed to monitor micro-MLFs induced by inoculation of two different strains of Oenococcus oeni into red wine. During the micro-MLFs, samples of wine were collected and assayed for l-malic, l-lactic, and citric acids by use of both biosensors and spectrophotometric techniques. In parallel the viable bacterial cell count was also evaluated. The kinetics of bacterial growth, degradation of l-malic and citric acids, and production of l-lactic acid was found to be a function of the strains inoculated.     

Quartz crystal microbalance biosensor for recombinant human interferon-beta detection based on antisense peptide approach

Quartz crystal microbalance (QCM) biosensors for recombinant human interferon-β (rhIFN-β) were constructed by utilizing antisense peptides adhering to the QCM gold surfaces. Two antisense peptides, both corresponding to the N-terminal fragment 1-14 of rhIFN-β, were used in this study. Antisense peptide AS-1 was the original antisense peptide and AS-2 was the modified antisense peptide based on the antisense peptide degeneracy. Both antisense peptides were immobilized on the gold electrodes of piezoelectric crystals, respectively, via a self-assembling monolayer of 1,2-ethanedithiol. The binding affinity between rhIFN-β and each immobilized antisense peptide in solution was evaluated using a quartz crystal microbalance-flow injection analysis (QCM-FIA) system. The dissociation constant of rhIFN-β on the antisense peptide AS-1 and AS-2 biosensor was (1.89 ± 0.101) × 10⁻⁴ and (1.22 ± 0.0479) ×10⁻⁵ mol L⁻¹, respectively. The results suggested that AS-2 had a higher binding affinity to rhIFN-β than AS-1. The detection for rhIFN-β using each biosensor was precise and reproducible. The linear response ranges of rhIFN-β binding to both biosensors were same with a concentration range of 0.12-0.96 mg mL⁻¹. The results demonstrated the successful construction of highly selective QCM biosensors using antisense peptide approach, and also confirmed the feasibility of increasing antisense peptide binding affinity by appropriate sequence modification.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

An aptasensor for sensitive detection of human breast cancer cells by using porous GO/Au composites and porous PtFe alloy as effective sensing platform and signal amplification labels

A novel aptamer biosensor for cancer cell assay has been reported on the basis of ultrasensitive electrochemical detection. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for MCF-7 cells detection. Functionalized nanoporous materials, porous graphene oxide/Au composites (GO/Au composites) and porous PtFe alloy have been introduced into the biosensor. Owing to the large surface area and versatile porous structure, the use of nanoporous materials can significantly improve the analysis performance of the biosensors by loading of large amounts of molecules and accelerating diffusion rate. Under the optimized experimental conditions, the proposed aptamer biosensor exhibited excellent analytical performance for MCF-7 cells determination, ranging from 100 to 5.0 × 10⁷ cells mL⁻¹ with the detection limit of 38 cells mL⁻¹. The biosensor showed good selectivity, acceptable stability and reproducibility, and developed a highly sensitive and selective method for cancer cells detection.     

Examination of performance of glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection

A composite electrode was prepared by modifying glassy carbon microparticles with gold nanoparticles (Au-nps) and xanthine oxidase enzyme (XOD) for xanthine (X) and hypoxanthine (Hx) detection. After the optimization of the system for X, the biosensor was characterized for X and Hx. A linearity was obtained in the concentration range between 5.00 × 10⁻⁷ and 1.00 × 10⁻⁵ M for X with equation of y = 0.24x + 0.712 and 5.00 × 10⁻⁶ to 1.50 × 10⁻⁴ M for Hx, with equation of y = 0.014x + 0.575, respectively. Obtained results were compared to X and/or Hx biosensors including/not including Au-np in the structure. The developed system was also applied for detection of Hx in canned tuna fish sample and very promising results were obtained.     

A new amperometric bienzymatic biosensor based on biocomposites for the determination of gluconic acid in wines

A new amperometric bienzymatic biosensor for gluconic acid based on the coimmobilization of gluconokinase (EC 2.7.1.12) and phosphogluconate dehydrogenase (EC 1.1.1.44) by polysulfone membrane entrapment onto the surface of a graphite-epoxy composite is reported. This biosensor represents an alternative to gluconate dehydrogenase (EC 1.1.99.3) based methods, which is no longer commercially available. Measurements were done at an applied potential of +0.800 V, room temperature and phosphate buffer pH 7.50; obtaining a linear response range for gluconic acid extended from 7.0 × 10⁻⁶ to 2.5 × 10⁻⁴ M. Constructed biosensors showed good reproducibility for calibrations using different electrodes (RSD of 1.74%). Finally, biosensor was applied to real wine samples, and the results obtained were validated by comparison with those provided by a reference laboratory. Good correlation was found when the biosensor results were plotted vs. the reference values (slope = 1.03 ± 0.04, intercept = 0.01 ± 0.02, r² = 0.995).     

Electrochemiluminescent disposable cholesterol biosensor based on avidin–biotin assembling with the electroformed luminescent conducting polymer poly(luminol-biotinylated pyrrole)

An electrochemiluminescent cholesterol disposable biosensor has been prepared by the formation of assembled layers on gold screen-printed cells. The detection layer is based on the electro-formation of new luminol copolymers with different synthesized biotinylated pyrroles prepared by click-chemistry, offering a new transduction layer with new electroluminescent properties on biosensors. The electrochemiluminescence (ECL) luminol copolymers are electroformed by cyclic voltammetry (five cycles) at pH 7.0 uses a10⁻³ M biotinylated pyrrole–luminol ratio of 1:10 in PBS buffer. With respect to the recognition layer, cholesterol oxidase was biotinylated by incubation with biotin vinyl sulfone, and immobilized on the copolymer by avidin–biotin interaction. The analytical signal of the biosensor is the ECL enzymatic initial rate working in chronoamperometric mode at 0.5 V excitation potential with 10 s between pulses at pH 9.5. The disposable device offers a cholesterol linear range from 1.5 × 10⁻⁵ M to 8.0 × 10⁻⁴ M with a limit of detection of 1.47 × 10⁻⁵ M and accuracy of 7.9% for 9.0 × 10⁻⁵ M and 14.1% for 2.0 × 10⁻⁴ M, (n = 5). Satisfactory results were obtained for cholesterol determination in serum samples compared to a reference procedure.     

Microwave plasma treated carbon nanotubes and their electrochemical biosensing application

A convenient microwave plasma treatment method with ammonia precursor was proposed to enhance the solubility of carbon nanotubes (CNTs). The SEM, XRD and FTIR spectra clearly demonstrated that the carbon skeleton structure of the resultant ammonia plasma-treated CNTs (ammonia PT-CNTs) was not destroyed and amine groups of different forms were successfully coupled to CNTs in the MWP treatment process. The ammonia PT-CNTs have excellent solubility in water and are insoluble in nonpolar tetrahydrofuran, and the cyclic voltammograms suggest that the enhanced wetting properties clearly favor faster electron transfer kinetics on the ammonia PT-CNT electrodes. By choosing glucose oxidase as a model enzyme, the application of the ammonia PT-CNTs in construction of biosensors was further investigated. Due to the biocompatibility and electron transfer capability of the ammonia PT-CNTs, the resultant GOD biosensor displayed a good sensing performance. The biosensor has a fast response of less than 10 s, and the response current linearly increases with the glucose concentration in the range of 1.2 × 10⁻⁴ to 7.5 × 10⁻³ M with a detection limit of 1.0 × 10⁻⁵ M.     

Alumina sol-gel/sonogel-carbon electrode based on acetylcholinesterase for detection of organophosphorus pesticides

Two new amperometric biosensors based on immobilization of acetylcholinesterase on a sonogel-carbon electrode for detection of organophosphorous compounds are proposed. The electrodes were prepared applying high-energy ultrasounds directly to the precursors. The first biosensor was obtained by simple entrapping acetylcholinesterase in Al₂O₃ sol-gel matrix on the sonogel-carbon. The second biosensor was produced in a sandwich configuration. Its preparation involved adsorption of the enzyme and modification via a polymeric membrane such as polyethylene glycol and the ion-exchanger Nafion. The optimal enzyme loading was found to be 0.7 mIU. Both biosensors showed optimal activity in 0.2 M phosphate buffer, pH 7.0, at an operating potential of 210 mV. The detection limit achieved for chlorpyriphos-ethyl-oxon was 2.5 × 10⁻¹⁰ M at a 10-min incubation time.     

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.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Oxidized multiwalled carbon nanotubes for quantitative determination of cationic surfactants in water samples using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A new biosensor for detection of phenols, based on tyrosinase immobilization with alumina sol-gel on Sonogel-Carbon transducer, has been developed. The electrode was prepared using high energy ultrasounds directly applied to the precursors. The alumina sol-gel provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenols are oxidized by tyrosinase biosensor to form a detectable product, which was determined at −300 mV vs. Ag/AgCl reference electrode. For phenol, the sensor exhibited a fast response which resulted from the porous structure and high enzyme loading of the sol-gel matrix. The linear range was from 5 × 10⁻⁷ M to 3 × 10⁻⁵ M, with a detection limit of 3 × 10⁻⁷ M. The stability of the biosensor was also evaluated.     

Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

Determination of thiodicarb using a biosensor based on alfalfa sprout peroxidase immobilized in self-assembled monolayers

A biosensor based on alfalfa sprout (Medicago sativa) homogenate as a source of peroxidase is proposed for the determination of thiodicarb by square-wave voltammetry. This enzyme was immobilized in self-assembled monolayers of l-cysteine on a gold electrode. Several parameters were investigated to evaluate the optimum conditions for operation of the biosensor. The analytical curve was linear for thiodicarb concentrations of 2.27 × 10⁻⁶ to 4.40 × 10⁻⁵ mol L⁻¹ with a detection limit of 5.75 × 10⁻⁷ mol L⁻¹. The lifetime of the Au-alfalfa sprout-SAMs was 20 days (at least 220 determinations). The average recovery of thiodicarb from samples of vegetable extracts ranged from 99.02 to 101.04%. The results obtained for thiodicarb in vegetable extracts using the proposed method are in close agreement with those using a high performance liquid chromatography procedure at the 95% confidence level.     

Seed-mediated synthesis of copper nanoparticles on carbon nanotubes and their application in nonenzymatic glucose biosensors

In this paper, for the first time, Cu nanoparticles (CuNPs) were prepared by seed-mediated growth method with Au nanoparticles (AuNPs) playing the role of seeds. Carbon nanotubes (CNTs) and AuNPs were first dropped on the surface of glassy carbon (GC) electrode, and then the electrode was immersed into growth solution that contained CuSO₄ and hydrazine. CuNPs were successfully grown on the surface of the CNTs. The modified electrode showed a very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium, which was utilized as the basis of the fabrication of a nonenzymatic biosensor for electrochemical detection of glucose. The biosensor can be applied to the quantification of glucose with a linear range covering from 1.0 × 10⁻⁷ to 5 × 10⁻³ M and a low detection limit of 3 × 10⁻⁸ M. Furthermore, the experiment results also showed that the biosensor exhibited good reproducibility and long-term stability, as well as high selectivity with no interference from other oxidable species.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.