Cholinesterase immobilisation on the surface of screen-printed electrodes based on concanavalin A affinity

This paper reports a new method for the immobilisation of acetylcholine esterase (AChE) on the surface of screen-printed electrodes (SPEs) based on the affinity between the glycoprotein enzyme and concavalin A (Con A). The surface of the working electrode has been modified with a Nafion layer that contains graphite, the mediator 7,7,8,8-tetracyanoquinodimethane (TCNQ) and heptylamine. The enzyme-free SPEs were characterised by cyclic voltammetry in buffer solutions and amperometry using cysteamine as analyte. The AChE immobilisation process leads to the sandwich structure: electrode-carbohydrate-Con A-enzyme. The first step of the immobilisation is the covalent activation of an amino group bound in a Nafion layer. The following steps are based on the affinity. The non-specific adsorption has been totally eliminated using BSA solutions at two different pHs. Various amounts of enzyme, from 0.1 to more than 2 mIU AChE, have been loaded on the electrode surface. The method offers the advantage of a free diffusion, which allows obtaining a response time of less than 2 min. An operational stability of more than 10 measurements was registered, while the active surface of the electrode was successfully reloaded for three consecutive times without any important change of the analytical performances.     

Manufacture and evaluation of carbon nanotube modified screen-printed electrodes as electrochemical tools

Carboxylated multiwalled carbon nanotubes (MWCNT-COOH) dissolved in a mixture of DMF:water were used to modify the surfaces of commercially available screen-printed electrodes (SPEs). The morphology of the MWCNT-COOH and the modified SPEs was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. SEM analysis showed a porous structure formed by a film of disordered nanotubes on the surface of the working electrode.The modification procedure with MWCNT-COOH was optimised and it was applied to unify the electrochemical behaviour of different gold and carbon SPEs by using p-aminophenol as the benchmark redox system. The analytical advantages of the MWCNT-COOH-modified SPEs as voltammetric and amperometric detectors as well as their catalytic properties were discussed through the analysis, for instance, of dopamine and hydrogen peroxide. Experimental results show that the electrochemical active area of the nanotube-modified electrode increased around 50%. The repeatability of the modification methodology is around 6% (R.S.D.) and the stability of MWCNT-COOH-modified SPEs is ensured for, at least, 2 months.     

Facile and Low-Cost SPE Modification Towards Ultra-Sensitive Organophosphorus and Carbamate Pesticide Detection in Olive Oil

Despite the fact that a considerable amount of effort has been invested in the development of biosensors for the detection of pesticides, there is still a lack of a simple and low-cost platform that can reliably and sensitively detect their presence in real samples. Herein, an enzyme-based biosensor for the determination of both carbamate and organophosphorus pesticides is presented that is based on acetylcholinesterase (AChE) immobilized on commercially available screen-printed carbon electrodes (SPEs) modified with carbon black (CB), as a means to enhance their conductivity. Most interestingly, two different methodologies to deposit the enzyme onto the sensor surfaces were followed; strikingly different results were obtained depending on the family of pesticides under investigation. Furthermore, and towards the uniform application of the functionalization layer onto the SPEs’ surfaces, the laser induced forward transfer (LIFT) technique was employed in conjunction with CB functionalization, which allowed a considerable improvement of the sensor’s performance. Under the optimized conditions, the fabricated sensors can effectively detect carbofuran in a linear range from 1.1 × 10^?9 to 2.3 × 10^?8 mol/L, with a limit of detection equal to 0.6 × 10^?9 mol/L and chlorpyrifos in a linear range from 0.7 × 10^?9 up to 1.4 × 10^?8 mol/L and a limit of detection 0.4 × 10^?9 mol/L in buffer. The developed biosensor was also interrogated with olive oil samples, and was able to detect both pesticides at concentrations below 10 ppb, which is the maximum residue limit permitted by the European Food Safety Authority.     

Ruthenized screen-printed choline oxidase-based biosensors for measurement of anticholinesterase activity

Plastic disposable choline biosensors based on ruthenized-carbon screen-printed electrodes were prepared and their use for monitoring organophosphorus pesticides and carbamates is described. The presence of 0.5% ruthenium on activated carbon mixed to form a simple graphite-based ink for the working electrode surface increased the sensitivity towards hydrogen peroxide. The choline biosensor is based on such an electrode coupled with choline oxidase immobilized by adsorption and was used to detect the inhibition effect of carbamates and organophosphorus pesticides on acetylcholinesterase. With the optimized procedure described (pH, buffer composition, incubation time, substrate concentration), concentrations of pesticides (Carbofuran) as low as 1 nM could be detected.     

Disposable screen-printed electrode coupled with recombinant Drosophila melanogaster acetylcholinesterase and multiwalled carbon nanotubes for rapid detection of pesticides

Recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE), multiwalled carbon nanotubes (MWCNTs), and Prussian blue have been combined for development of a three-electrode biosensor with more rapid responses and higher stability than in our previous study. A new disposable screen-printed electrode (SPE) was developed for rapid detection of organophosphate and carbamate pesticides. After optimization, 10 microg MWCNT and 5 microL enzyme immobilization solution consisting of 0.2% glutaraldehyde, 0.1% Nafion, 0.2% bovine serum albumin, 0.1 g/L MWCNT, and 1.5 mU R-DmAChE were fixed on each of the R-DmAChE/MWCNT SPEs. The LOD of this biosensor was 0.5 microg/L for pesticide standards of dichlorvos (DDV) and carbofuran. The performance of this biosensor was tested for vegetable and water samples at various spiked levels, and good stability and sensitivity were found. The obtained recoveries were from 82.6 to 110.5% for DDV at levels of 0.5-5 microg/L and 73.4 to 118.4% for carbofuran at 1-10 microg/L in lake and sea water samples, demonstrating that the proposed approach is an alternative means for rapid detection of pesticide residues and contaminants in food safety and environmental monitoring.     

Determination of Anticholinesterase Activity for Pesticides Monitoring Using a Thiocholine Sensor

Abstract

An acetylthiocholine sensor based on a disposable screen-printed carbon electrode has been assembled for measuring organophosphorus and carbamate pesticides in river water samples through the degree of inhibition of the enzyme acetylcholinesterase (AChE). The carbon working electrode surface was modified by deposition of a mediator, tetracyanoquinodimethane (TCNQ), and Nafion. Acetylcholinesterase catalyses the cleavage of acetylthiocholine to thiocholine, which is measured by differential pulse voltammetry and directly related to the enzyme activity. The scan speed, the pulse amplitude of the differential pulse voltammetry and several parameters in the procedure were optimised. An inhibition calibration curve was obtained using carbofuran. The method was also applied to water samples, showing its suitability as a rapid screening assay (15 min per test) for anticholinesterase activity detection.     

Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes

A simple and automated methodology for a sensitive electrochemical detection of enzyme immunoassays that employ alkaline phosphatase (AP) as label has been developed. A flow injection system with programmable pump, valve and cell functions, amperometric detection of indigo and screen-printed electrodes (SPEs) are responsible for the advantages of this methodology. Amperometric detection at a low potential of indigo, the product of the enzymatic hydrolysis of the substrate 3-indoxyl phosphate (IP), is combined with a flow injection system. This incorporates in the flow cell a disposable screen-printed board provided with a graphite working electrode. No electrode pretreatment is necessary to obtain reproducible signals. The system was applied to the determination by an enzyme-linked immunosorbent assays (ELISA) of pneumolysin (PLY), a toxin related to respiratory infections. Linear calibration curves for low and high concentration ranges were obtained. These were also performed in a proteic matrix and linearity was also obtained.     

Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents

A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91x10(-8) M paraoxon and 1.24x10(-9) M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile.     

A rational design of the multiwalled carbon nanotube–7,7,8,8-tetracyanoquinodimethan sensor for sensitive detection of acetylcholinesterase inhibitors

A new, simple and effective amperometric acetylcholinesterase biosensor was developed using screen-printed carbon electrodes modified with carbon nanotubes (MWCNTs)–7,7,8,8-tetracyanoquinodimethane (TCNQ). The design of the biosensor was based on the supramolecular arrangement resulted from the interaction of MWCNTs and TCNQ. This arrangement was confirmed by spectroscopic and electrochemical techniques. Two different supramolecular arrangements were proposed based on different MWCNTs:TCNQ ratios. The synergistic effect of MWCNTs and TCNQ was, for the first time, exploited for detection of thiocholine at low potential with high sensitivity. The biosensor developed by immobilization of acetylcholinesterase (AChE) in sol–gel allowed the detection of two reference AChE inhibitors, paraoxon-methyl and chlorpyrifos with detection limits of 30 pM (7 ppt) and 0.4 nM (0.1 ppb), respectively. Efficient enzyme reactivation was obtained by using obidoxime.     

Recent developments in the field of screen-printed electrodes and their related applications

The development of analytical methods that respond to the growing need to perform rapid ‘in situ’ analyses shows disposable screen-printed electrodes (SPEs) as an alternative to the traditional electrodes. This review presents recent developments in the electrochemical application of disposable screen-printed sensors, according to the types of materials used to modify the working electrode. Therefore, unmodified SPE, film-modified SPE, enzyme-modified SPE and antigen/antibody-modified SPE are described. Applications are included where available.     

Comparative immobilization of antibodies on modified screen-printed graphite electrodes

Immobilization of polyclonal antibodies was studied on native screen-printed graphite electrodes (SPEs) and variously modified electrodes. SPEs coated with didodecylammonium bromide (DDAB, a synthetic membranelike substance) films with gold nanoparticles gave the maximum electrochemical response. DDAB and gold nanoparticle films strongly changed the surface morphology, and the electrochemical signal became more intense and stable. This immobilization method increased the concentration of immobilized antibodies while their activity was retained. The detection limit of the enzymatic label (horseradish peroxidase) was 0.02 ng/L of sample.     

Graphene-based Disposable Electrochemical Sensor for Chlorhexidine Determination

In this work, simple, reliable, eco-friendly, and quantitative electrochemical sensors were developed to detect chlorhexidine Digluconate (CHX) in a variety of dosage forms, including mouthwashes and intimate douches, as well as chlorhexidine in spiked human saliva. Without any sample pre-treatment or extraction processes, CHX was measured in colored aqueous formulations. Based on carbon screen-printed electrodes, two potentiometric sensors (sensors I and II), utilizing graphene nanocomposites (Gr-NC), were designed (SPEs). An ionophore, 2-hydroxypropyl-β-cyclodextrin, was doped into the Poly Vinyl Chloride (PVC) polymeric membrane to improve sensor selectivity.     

Chemically modified screen-printed electrodes as efficient potentiometric sensors for cyclobenzaprine hydrochloride determination in pure and pharmaceutical preparations

Chemically modified screen-printed electrodes (SPEs) with 3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt [Alizarin Red (ALZ.R)] and tricresylphosphate (TCP) were fabricated and examined for potentiometric determination of cyclobenzaprine hydrochloride (CZP.HCl) in pure and pharmaceutical samples. The content of ALZ.R in the prepared SPEs varied, which significantly affected their potentiometric response during CZP.HCl estimation. The measured potential value was dependent on the pH of the studied solution. A stable modified SPE sensor was prepared with reproducible readings within 22 days. A wide linear concentration range could be measured with Nernestian slope value of 60.12 ± 0.18 mV decade⁻¹. Based on the recorded analytical parameters of these modified SPEs, a sensitive and reliable detection of CZP.HCl in pharmaceutical samples could be attained.     

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase.In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection.Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.     

Towards the development of a portable sensor based on a molecularly imprinted membrane for the rapid determination of salbutamol in pig urine

A new type of conductometric probe based on a molecularly imprinted membrane (MIM) for the detection of salbutamol has been designed and fabricated. The probe consists of two parallel screen-printed electrodes (SPE). One of the SPEs was coated with a molecularly imprinted membrane using salbutamol as the template, and the other was modified with a non-molecularly imprinted membrane (N-MIM). Measurements of salbutamol were conducted after the conductometric probe had been connected to a commercial portable conductometer. Multi-sample or successive detections could be easily accomplished by replacing the one-off SPE coated with the salbutamol molecularly imprinted membrane with a new one. The conductometric response of the sensor to the concentration of salbutamol displayed a linear correlation over a range from 50 to 280 nM, with a detection limit of 13.5 nM. The recoveries reached 92.1-98.3% based on pig urine samples. In addition, the sensor based on this new type of probe demonstrated high sensitivity and selectivity for salbutamol.     

Sensitive and rapid amperometric magnetoimmunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at −0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H₂O₂ was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.     

An electrochemical cell coupled with disposable screen-printed electrodes for use in flow injection analysis.

An electrochemical cell coupled with disposable screen-printed electrodes (SPEs) that is specifically designed for use in flow injection analysis (FIA) is described in this study. The cell is made of foldable polyoxymethylene (acetal) thick platelets with the bottom portion consisting of a cavity track to drag the SPEs in position and the top portion having predrilled T-like holes to arrange the Ag/AgCl reference electrode and stainless steel inlet & outlet. An "O ring" is suitably fixed on the top of the working electrode to form a thin-layer space where the electrochemical reaction can take place. Hydrodynamic characterization was validated by using a benchmark hexacyanoferrate redox couple. The results of practical analysis of glucose in human plasma clearly demonstrate the characteristics and applicability of the proposed wall-jet electrochemical cell in FIA.     

Optimization of acetylcholinesterase immobilization on microelectrodes based on nitrophenyl diazonium for sensitive organophosphate insecticides detection

The immobilization of acetylcholinesterase on platinum microelectrodes modified with p-nitrobenzenediazonium is optimized. In the first step, a layer of p-nitrophenyl groups was deposited on the surface and then reduced to p-aminophenyl groups. Finally, the enzyme was linked to the amino groups on the surface using glutaraldehyde. Each step of the electrode modification was characterized by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) at acidic and neutral pH to modify the electric charges of different bound moieties. The deposition of diazonium groups was attempted by potentiometry, amperometry or CV, but only potentiometry proceeded without passivation of the surface. The use of microelectrodes improved the limit of detection of ethylparaoxon measurements to 20 nM (compared to 100 nM in case of screen-printed electrodes based on the same method of immobilization). The method allowed the production of stable and reproducible amperometric microbiosensors and may be adapted to other enzymes and electrode materials.     

Potentiometric determination of cetylpyridinium chloride using a new type of screen-printed ion selective electrodes

A new type of screen-printed ion-selective electrode for the determination of cetylpyridinium chloride (CPC) is presented. These new electrodes involve in situ, modified and unmodified screen-printed ion-selective electrodes for the determination of CPC. The screen-printed electrodes (SPEs) show a stable, near-Nernstian response for 1 × 10⁻² to 1 × 10⁻⁶ M CPC at 25 °C over the pH range 2-8 with cationic slope 60.66 ± 1.10. The lower detection limit is found to be 8 × 10⁻⁷ M and response time of about 3 s and exhibit adequate shelf-life (6 months). The fabricated electrodes can be also successfully used in the potentiometric titration of CPC with sodium tetraphenylborate (NaTPB). The analytical performances of the SPEs are compared with those for carbon paste electrode (CPE) and polyvinyl chloride (PVC) electrodes. The method is applied for pharmaceutical preparations with a percentage recovery of 99.60% and R.S.D. = 0.53. The frequently used CPC of analytical and technical grade as well as different water samples has been successfully titrated and the results obtained agreed with those obtained with commercial electrode and standard two-phase titration method. The sensitivity of the proposed method is comparable with the official method and ability of field measurements.     

Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples

The development of rapid, low-cost and reliable diagnostic methods is crucial for the identification and treatment of many diseases. Screen-printed gold electrodes (Au/SPEs), coated with a ternary monolayer interface, involving hexanedithiol (HDT), a specific thiolated capture probe (SHCP), and 6-mercapto-1 hexanol (MCH) (SHCP/HDT/MCH) are shown here to offer direct and sensitive detection of nucleic acid hybridization events in untreated raw biological samples (serum, urine and crude bacterial lysate solutions). The composition of the ternary monolayer was modified and tailored to the surface of the Au/SPE. The resulting SHCP/HDT/MCH monolayer has demonstrated to be extremely useful for enhancing the performance of disposable nucleic acid sensors based on screen-printed electrodes. Compared to common SHCP/MCH binary interfaces, the new ternary self-assembled monolayer (SAM) resulted in a 10-fold improvement in the signal (S)-to-noise (N) ratio (S/N) for 1 nM target DNA. The SHCP/HDT/MCH-modified Au/SPEs allowed the direct quantification of the target DNA down to 25 pM (0.25 fmol) and 100 pM (1 fmol) in undiluted/untreated serum and urine samples, respectively, and of 16S rRNA Escherichia coli (E. coli) corresponding to 3000 CFU μL⁻¹ in raw cell lysate samples. The new SAM-coated screen-printed electrodes also displayed favorable non-fouling properties after a 24 h exposure to raw human serum and urine samples, offering great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests.