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.     

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.     

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.     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

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.     

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).     

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.     

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.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

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.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10^–6 M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

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.     

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.     

A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum

Different techniques have been used (physical adsorption, physically entrapped sandwich and microencapsulation) for the immobilization of urease enzyme in tetramethylorthosilicate (TMOS) derived sol-gel matrix on the sensing surface of glass-pH-electrode. No significant leaching of enzyme occurs from the microencapsulated and physically entrapped enzyme sandwich films. Potentiometric techniques have been used for the estimation of urea concentration in each instance. Various parameters of biosensor performance have been studied which indicates that microencapsulation technique is a better method of enzyme immobilization in sol-gel films derived from TMOS. The advantage of microencapsulated biosensor over others include higher sensitivity (dpH/dp(C) = 2.4), lower detection limit of 52 μg mL⁻¹, larger linear range (0.01-30 mM) of urea determination and reasonably long-term stability of about 25 days with 80% response signal.     

Immobilization of laccase on polymer grafted polytetrafluoroethylene membranes for biosensor construction

In this study, Trametes versicolor laccase was immobilized on polytetrafluoroethylene (PTFE) membranes using two different techniques, entrapment to gelatin and covalent immobilization to the surface. For surface immobilization, functional groups were formed on PTFE surface by radiofrequency (RF) plasma treatment followed by polymer grafting. Two different polymers, polyacrylamide (pAAm) and polyacrylic acid (pAAc) were tried. For polyacrylamide grafted PTFE, a two-step polymerization process was used. The membranes were first treated with hydrogen plasma and pAAm grafted PTFE (pAAm-g-PTFE) was then formed by argon plasma treatment. To produce pAAc grafted PTFE (pAAc-g-PTFE), the surface was first treated with argon plasma and AAc was then attached to the surface by heat treatment (70 °C, 6 h). For both cases, an optimized carbodiimide coupling reaction was used for laccase immobilization. Enzyme activity was measured by an oxygen electrode using guaiacol as substrate. All three biosensing membranes were characterized and compared in terms of optimum working conditions, storage stability and reusability. Our study concluded that although a higher activity was obtained by gelatin entrapped laccase, its mechanical instability and poor storage life makes the gelatin biosensor unattractive for multiple usages and for field measurements. pAAc-g-PTFE biosensor was found to be more stable and highly reusable (ca. 50 times) when compared with the other two biosensors. In addition, its sensitivity was suitable for field applications. Therefore, the pAAc-g-PTFE biosensor could be proposed as an alternative on-site detection tool for phenolic compound monitoring.     

A novel amperometric biosensor based on single walled carbon nanotubes with acetylcholine esterase for the detection of carbaryl pesticide in water

Amperometric biosensor is fabricated for the detection of carbaryl based on single walled carbon nanotubes (SWCNTs) and acetylcholine esterase (AchE). The dispersion of SWCNTs in positively charged polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA), possibly takes place due to weak supramolecular interaction between them, which then binds electrostatically to the negatively charged AchE at pH 7.4 using layer-by-layer (LbL) self-assembly technique. The optical intensity of UV/vis spectra increased with the number of layers, indicating the build up of a multilayer coating on the electrode. The activity of acetylcholine esterase on modified electrode of 3 mm in diameter was found to be 0.2 U. The biosensor showed good sensitivity and stability towards the monitoring of carbaryl pesticides in water with the detection limit of 10⁻¹² g L⁻¹ and recovery of 99.8 ± 2.7% to 10⁻¹⁰ g L⁻¹. This protocol can be used for the immobilization of other enzymes to fabricate a range of biosensors.     

Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor

A novel disposable third-generation hydrogen peroxide (H₂O₂) biosensor based on horseradish peroxidase (HRP) immobilized on the gold nanoparticles (AuNPs) electrodeposited indium tin oxide (ITO) electrode is investigated. The AuNPs deposited on ITO electrode were characterized by UV-vis, SEM, and electrochemical methods. The AuNPs attached on the ITO electrode surface with quasi-spherical shape and the average size of diameters was about 25 nm with a quite symmetric distribution. The direct electron chemistry of HRP was realized, and the biosensor exhibited excellent performances for the reduction of H₂O₂. The amperometric response to H₂O₂ shows a linear relation in the range from 8.0 μmol L⁻¹ to 3.0 mmol L⁻¹ and a detection limit of 2 μmol L⁻¹ (S/N = 3). The value of HRP immobilized on the electrode surface was found to be 0.4 mmol L⁻¹. The biosensor indicates excellent reproducibility, high selectivity and long-term stability.     

Platinum nanoparticles functionalized nitrogen doped graphene platform for sensitive electrochemical glucose biosensing

In this work, we reported an efficient platinum nanoparticles functionalized nitrogen doped graphene (PtNPs@NG) nanocomposite for devising novel electrochemical glucose biosensor for the first time. The fabricated PtNPs@NG and biosensor were characterized using transmission electron microscopy, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, static water contact angle, UV–vis spectroscopy, electrochemical impedance spectra and cyclic voltammetry, respectively. PtNPs@NG showed large surface area and excellent biocompatibility, and enhanced the direct electron transfer between enzyme molecules and electrode surface. The glucose oxidase (GOx) immobilized on PtNPs@NG nanocomposite retained its bioactivity, and exhibited a surface controlled, quasi-reversible and fast electron transfer process. The constructed glucose biosensor showed wide linear range from 0.005 to 1.1 mM with high sensitivity of 20.31 mA M⁻¹ cm⁻². The detection limit was calculated to be 0.002 mM at signal-to-noise of 3, which showed 20-fold decrease in comparison with single NG-based electrochemical biosensor for glucose. The proposed glucose biosensor also demonstrated excellent selectivity, good reproducibility, acceptable stability, and could be successfully applied in the detection of glucose in serum samples at the applied potential of −0.33 V. This research provided a promising biosensing platform for the development of excellent electrochemical biosensors.     

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.