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.     

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.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

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.     

Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection

A novel and ultrasensitive sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Core/shell Fe₃O₄/Au magnetic nanoparticles (AuMNPs) loading aptamer1 (Apt1) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5–100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in protein detection and disease diagnosis.     

Sulfite oxidase biosensors based on tetrathiafulvalene modified screen-printed carbon electrodes for sulfite determination in wine

Screen-printed carbon electrodes have been modified with tetrathiafulvalene and sulfite oxidase enzyme for the sensitive and selective detection of sulfite. Amperometric experimental conditions were optimized taking into account the importance of quantifying sulfite in wine samples and the inherent complexity of these samples, particularly red wine. The biosensor responds to sulfite giving a cathodic current (at +200 mV vs screen-printed Ag/AgCl electrode and pH 6) in a wide concentration range, with a capability of detection of 6 μM (α = β = 0.05) at 60 °C. The method has been applied to the determination of sulfite in white and red samples, with averages recoveries of 101.5% to 101.8%, respectively.     

Determination of asulam by fast stopped-flow chemiluminescence inhibition of luminol/peroxidase

An efficient, sensitive and fast stopped-flow method has been developed to determine asulam in water, based on its inhibition effect on the horseradish peroxidase-luminol-hydrogen peroxide chemiluminescence reaction, (HRP-luminol-H₂O₂). Ultra fast data acquisition (0.20 s) facilitates excellent selectivity because no interferences from concomitants in the matrix act in such short time scale. The precision as repeatability (expressed as relative standard deviation, n = 10) was 0.4% at a 40 pM level. The detection limit was 1.5 pM (0.35 ng/L) and 7.15 pM in pure and raw water, respectively. The calibration data over the range 5-60 pM present a correlation coefficient of r = 0.9993. The proposed method has been applied to determine asulam in water samples by using solid-phase extraction (SPE). Mean recovery value was 98.1 ± 2% at 50 pM level.     

Determination of phenolic acids using Trametes versicolor laccase

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0 μM caffeic acid, 0.05-0.2 μM ferulic acid, 2.0-14.0 μM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0 μM caffeic acid, 2.0-10.0 μM ferulic acid, 4.0-30.0 μM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.     

Sensitivity enhancement of SPR biosensor with silver mirror reaction on the Ag/Au film

Based on well-known silver mirror reaction the Ag film was formed on Au film modified by self-assembled monolayer (SAM) of 1,6-hexanedithiol (HDT). The sensitivity of the biosensor based on this Ag/Au film is enhanced compared to that based on Au film. When the surface plasmon resonance (SPR) biosensor based on this Ag/Au film was used to determine human IgG, the range of concentrations of human IgG that could be determined is 0.30-40.00 μg mL⁻¹. The lowest concentration (0.30 μg mL⁻¹) that could be detected was about 8 times lower than that obtained by the biosensor without modification by Ag film (2.50 μg mL⁻¹), which demonstrated that the biosensor based on Ag/Au film could make the resonant wavelength move to longer wavelength following with the sensitivity enhancement of the SPR biosensor.     

Screen-printed biosensors for the control of wine quality based on lactate and acetaldehyde determination

Biosensors for d-lactate and acetaldehyde were developed, based on screen-printed electrodes and NAD⁺-dependent dehydrogenases. Modification of screen-printed electrodes with the mediator Meldola Blue or with Meldola Blue-Reinecke salt resulted in sensitive, low cost and reliable NADH detectors. The biosensors were realised in two configurations, as disposable and reusable devices. Single-use sensors were obtained by simple deposition of enzyme and cofactor on the surface of mediator-modified electrodes. Chronoamperometry was used for the detection of substrates in small volumes of samples (25 μl). Immobilisation of dehydrogenases by entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) allowed sensors to be obtained with sufficient operational stability. Amperometry in stirred solutions was the detection technique with biosensors for multiple use. The 3σ detection limits for acetaldehyde were 1 μM by amperometry and 6 μM by chronoamperometry and for d-lactate-0.03 μM and 0.05 μM for reusable and disposable biosensors respectively. The biosensors were applied in the analysis of some French and Romanian wines.     

Amperometric immunosensor for detection of antibodies of Salmonella typhi in patient serum

An amperometric sensor for detection of antibodies to Salmonella typhi in the serum of patients was developed. This involved usage of screen-printed electrodes and recombinant flagellin fusion protein. An indirect enzyme-linked immunosorbant assay was used for detection of antibodies to S. typhi in the patient serum. The screen-printed electrodes were made using polystyrene and graphite. These electrodes were tested for their ability to detect 1-naphthol, which is the product formed due to the hydrolysis of the substrate 1-naphthyl phosphate by the enzyme alkaline phosphatase. These electrodes were coated with recombinant flagellin fusion protein made by recombinant DNA technology and blocked with bovine serum albumin (BSA). Further they were incubated with patient serum and goat anti-human alkaline phosphatase conjugate. The immunosensing was performed by using amperometric method. Pooled human serum samples from apparently healthy individuals were used as control. Both the pooled healthy human serum samples and patient sera were subjected to Widal agglutination test and amperometric method. A 100% correlation was found between the Widal test and amperometric method. The time taken for the detection by electrochemical method is 1 h and 15 min, while the time taken by Widal test is 18 h.     

Characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes and their application to the development of impedimetric immunosensors

This paper describes the characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes (SPEs) using electrochemical impedance spectroscopy (EIS) and the application of this approach to the development of impedimetric immunosensors. After applying −0.1 V for 45 s, the amount of electrodeposited silver quantitatively changes the magnitude of two elements of the electrical equivalent circuit: the interface capacitance, Ci, and the charge-transfer resistance, R_CT. Better correlations have been found when considering the R_CT since this parameter is almost exclusively dependent on the amount of deposited silver under these experimental conditions. This approach has been successfully applied to the development of an impedimetric immunosensor for aflatoxin M₁. The R_CT magnitude shows good correlation with the amount of gold immobilized on the electrode surface after a competitive assay and thus, with the toxin concentration. This approach has been found sensitive in a wide range of concentrations, from 15 to 1000 free-AFM₁ ppt with a limit of detection of 12 ppt.     

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.     

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.     

Amperometric simultaneous sensing system for d-glucose and l-lactate based on enzyme-modified bilayer electrodes

An amperometric biosensor system which uses screen-printed electrodes to simultaneously detect d-glucose and l-lactate has been developed and applied for simple and rapid determination of d-glucose and l-lactate levels in lactic fermenting beverages. The system was constructed from three-dimensionally layered electrodes. Taking into consideration the effects of easily oxidized substances contained in the samples, ferricyanide ions, which are electrochemically oxidized at a lower voltage, were chosen as a mediator. A linear relationship between steady-state current and concentration was found over a range of 1-100 mM (d-glucose) and 1-50 mM (l-lactate); the variation coefficients were 1.43% (n = 10) and 3.50% (n = 10) for the d-glucose and l-lactate sensors, respectively. When applied to lactic fermenting beverages, there was good agreement between the results obtained by the proposed sensing system and those obtained by the HPLC method. Using the proposed method, assays were completed within 5 min.     

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H₂O₂ (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.     

Construction of novel simple phosphate screen-printed and carbon paste ion-selective electrodes

The construction and performance characteristics of different phosphate ion-selective electrodes are described. Three types of electrodes are demonstrated, namely screen-printed, carbon paste and the conventional PVC membrane electrodes. The cited electrodes are based on bisthiourea ionophores and show a considerable selectivity towards hydrogenphosphate with Nernstian slopes depending on the type of the electrode and the ionophore used. Matrix compositions of each electrode are optimised on the basis of effects of type and concentration of the ionophore as well as influence of the selected plasticizers. The screen-printed electrodes work satisfactorily in the concentration range 10⁻⁵ to 10⁻² mol L⁻¹ with anionic Nernstian compliance (32.8 mV/decade activity) and detection limit 4.0 × 10⁻⁶ mol L⁻¹. The screen-printed electrodes show fast response time of about 2.2 s and exhibit adequate shelf-life (4 months). The fabricated electrodes can be also successfully used in the potentiometric titration of HPO₄²⁻ with Ba²⁺.     

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.     

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.     

Speciation of chromium using chronoamperometric biosensors based on screen-printed electrodes

Chronoamperometric assays based on tyrosinase and glucose oxidase (GOx) inactivation have been developed for the monitoring of Cr(III) and Cr(VI). Tyrosinase was immobilized by crosslinking on screen-printed carbon electrodes (SPCEs) containing tetrathiafulvalene (TTF) as electron transfer mediator. The tyrosinase/SPC_TTFE response to pyrocatechol is inhibited by Cr(III). This process, that is not affected by Cr(VI), allows the determination of Cr(III) with a capability of detection of 2.0 ± 0.2 μM and a reproducibility of 5.5%. GOx modified screen-printed carbon platinised electrodes (SPC_PtEs) were developed for the selective determination of Cr(VI) using ferricyanide as redox mediator. The biosensor was able to discriminate two different oxidation states of chromium being able to reject Cr(III) and to detect the toxic species Cr(VI). Chronoamperometric response of the biosensor towards glucose decreases with the presence of Cr(VI), with a capability of detection of 90.5 ± 7.6 nM and a reproducibility of 6.2%. A bipotentiostatic chronoamperometric biosensor was finally developed using a tyrosinase/SPC_TTFE and a GOx/SPC_PtE connected in array mode for the simultaneous determination of Cr(III) and Cr(VI) in spiked tap water and in waste water from a tannery factory samples.