Acetylcholinesterase- and Butyrylcholinesterase-Based Biosensors for the Detection of Quaternary Ammonium Biocides in Food Industry.

A sensitive and robust electrochemical cholinesterase-based sensor was developed to detect the quaternary ammonium (QAs) biocides most frequently found in agri-food industry wash waters: benzalkonium chloride (BAC) and didecyldimethylammonium chloride (DDAC). To reach the maximum residue limit of 28 nM imposed by the European Union (EU), two types of cholinesterases were tested, acetylcholinesterase (AChE, from Drosophila melanogaster) and butyrylcholinesterase (BChE, from horse serum). The sensors were designed by entrapping AChE or BChE on cobalt phthalocyanine-modified screen-printed carbon electrodes. The limits of detection (LOD) of the resulting biosensors were 38 nM for DDAC and 320 nM for BAC, using, respectively, AChE and BChE. A simple solid-phase extraction step was used to concentrate the samples before biosensor analysis, allowing for the accurate determination of DDAC and BAC in tap water with limits of quantification (LOQ) as low as 2.7 nM and 5.3 nM, respectively. Additional assays demonstrated that the use of a phosphotriesterase enzyme allows for the total removal of interferences due to the possible presence of organophosphate insecticides in the sample. The developed biosensors were shown to be stable during 3 months storage at 4 °C.

Development of a new dual electrochemical immunosensor for a rapid and sensitive detection of enrofloxacin in meat samples.

A novel dual electrochemical immunosensor was fabricated for the rapid and sensitive detection of enrofloxacin (EF) antibiotic in meat. Anti-quinolone antibody was immobilized onto screen-printed dual carbon electrodes via carbodiimide coupling. A new electrochemical probe was synthesized by conjugating difloxacin and aminoferrocene, whose oxidation was measured at + 0.2 V vs. Ag/AgCl by differential pulse voltammetry. The detection principle was based on the competitive binding of this conjugate and free EF on immobilized antibodies. The proposed immunosensor allowed detection of EF at concentrations ranging from 0.005 µg.mL-1 to 0.01 µg.mL-1 with a detection limit of 0.003 µg.mL-1. The immunosensor was stable for at least 1 month at 4 °C and displayed a good specificity for other fluoroquinolones. The new dual electrode design offered an improved accuracy as one electrode was used as negative control. The efficiency of the sensor and the adequacy of the extraction process were finally validated by detecting EF in different meat samples.

A signal-on electrochemical aptasensor based on silanized cellulose nanofibers for rapid point-of-use detection of ochratoxin A.

An innovative ultrasensitive electrochemical aptamer-based sensor was developed for ochratoxin A (OTA) detection in cold brew coffee through revolutionary combination of nanofibers, electrochemical method, and aptamer technologies. The assembly of the aptasensor was based on the activation of silanized cellulose nanofibrous membranes as a supporting matrix for methylene blue (MB) redox probe-labeled aptamer tethering. Cellulose nanofibrous membranes were regenerated by deacetylating electrospun cellulose acetate nanofibrous membranes with deacetylation efficacy of 97%, followed by silanization of the nanofiber surfaces by using (3-aminopropyl)triethoxysilane (APTES). A replacement of conventionally casted membranes by the nanofibrous membranes increased the active surface area on the working electrode of a screen-printed three-electrode sensor by more than two times, consequently enhancing the fabricated aptasensor performance. The developed aptasensor demonstrated high sensitivity and specificity toward OTA in a range 0.002-2 ng mL-1, with a detection limit of 0.81 pg mL-1. Moreover, the assembled aptamer-based sensor successfully detected OTA in cold brew coffee samples without any pretreatment. The aptasensor exhibited good reusability and stability over long storage time. Graphical abstract.

Ultrasensitive label-free electrochemical immunosensor based on PVA-co-PE nanofibrous membrane for the detection of chloramphenicol residues in milk.

An ultrasensitive label-free amperometric immunosensor for the detection of chloramphenicol (CAP) residues in milk has been developed by using a screen-printed carbon electrode laminated with a layer of poly (vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane that is covalently immobilized with a CAP antibody (anti-CAP). The performance of the PVA-co-PE nanofiber membrane (PVA-co-PE NFM) on the electrode was compared with a PVA-co-PE casted membrane (PVA-co-PE CM), necessary fabrication steps and performance of the sensors were investigated by electrochemical impedance spectroscopy (EIS). The application of the PVA-co-PE NFM decreased the electron-transfer-resistance by about 4 times compared with a conventional PVA-co-PE casted membrane. Under the optimal conditions, the established immunosensor exhibited high sensitivity for determination of CAP in a range 0.01-10 ng mL-1, with a limit of detection of 0.0047 ng mL-1. In addition to the good selectivity, reusability and stability over time, the prepared immunosensor was successfully used in the detection of CAP in milk samples without any pretreatment.

Detection of Bisphenol A in aqueous medium by screen printed carbon electrodes incorporating electrochemical molecularly imprinted polymers.

Electrochemical molecularly imprinted polymers (e-MIPs) were for the first time introduced in screen-printed carbon electrodes (SPCE) as the sensing element for the detection of an organic pollutant. To play this sensing role, a redox tracer was incorporated inside the binding cavities of a cross-linked MIP, as a functional monomer during the synthesis step. Ferrocenylmethyl methacrylate was used for this purpose. It was associated with 4-vinylpyridine as a co-functional monomer and ethylene glycol dimethacrylate as cross-linker for the recognition of the endocrine disruptor, Bisphenol A (BPA), as a target. Microbeads of e-MIP and e-NIP (corresponding non-imprinted polymer) were obtained via precipitation polymerization in acetonitrile. The presence of ferrocene inside the polymers was assessed via FTIR and elemental analysis and the polymers microstructure was characterized by SEM and nitrogen adsorption/desorption experiments. Binding isotherms and batch selectivity experiments evidenced the presence of binding cavities inside the e-MIP and its high affinity for BPA compared to carbamazepine and ketoprofen. e-MIP (and e-NIP) microbeads were then incorporated in a graphite-hydroxyethylcellulose composite paste to prepare SPCE. Electrochemical properties of e-MIP-SPCE revealed a high sensitivity in the presence of BPA in aqueous medium compared to e-NIP-SPCE with a limit of detection (LOD) of 0.06 nM. Selectivity towards carbamazepine and ketoprofen was also observed with the e-MIP-SPCE.

Chemiluminescence immunoassays for estradiol and ethinylestradiol based on new biotinylated estrogen derivatives.

New chemiluminescence-based immunoassays for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies. Under optimized conditions, the chemiluminescence immunoassays showed a highly sensitive response to E2 and EE2, with respective detection limits of 0.5 and 1.2 ng L-1. The LOD achieved using biotinylated E2 was in the same order of magnitude as those obtained using commercially available E2-bovine serum albumin conjugate (E2-BSA). The developed devices were successfully applied to analysis wastewater treatment plants effluents (WWTP) with negligible matrix effect.

Biosensor-assisted selection of optimal parameters for designing molecularly imprinted polymers selective to phosmet insecticide.

Molecularly imprinted polymers (MIPs) for phosmet insecticide were synthesized by batch polymerization. The affinity of functional monomers to phosmet was tested using an original method involving an electrochemical biosensor based on acetylcholinesterase inhibition. It was demonstrated that association of phosmet with appropriate functional monomers resulted in a decrease of enzyme inhibition. Using this method, it was shown that N,N-methylenebis(acrylamide) displayed the highest interactions with phosmet using DMSO as solvent. These results were in good accordance with those obtained by conventional computational modeling. Molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs) were synthesized and adsorption isotherms were studied to describe their interaction with phosmet. Freundlich isotherm was able to fit phosmet adsorption on MIPs with good agreement (R2 = 0.9). The pre-exponential factor KF determined for MIPs was 1.439mg(1-N)g-1LN, more that 10 times higher than for NIPs (0.125mg(1-N)g-1. LN), indicating an increase of binding sites number and average affinity.

Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor.

This paper describes for the first time the selection of aptamers selective to penicillin. Aptamers were selected using a specific process called Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique is based on the selection of DNA aptamers using penicillin G in solution while the ssDNA library is fixed on a support. One aptamer showing a good affinity to penicillin was finally selected and tested in electrochemical sensor configuration, using electrochemical impedance spectroscopy as detection technique. The developed aptasensor allowed the detection of penicillin in a wide concentration range, comprised between 0.4 and 1000µgL-1 Such performance was compatible with milk analysis, as the maximum residue limit tolerated in this matrix is 4µgL-1. The selectivity of the developed sensor was also studied, showing that the sensor was also able to bind other beta-lactam antibiotics, although with a weaker affinity. Finally the sensor was used for detection of penicillin G in milk. It was shown that a simple sample treatment with isopropanol followed by filtration was sufficient to eliminate matrix effects, allowing the determination of penicillin in milk at concentrations compatible with legislation requirements.

Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample.

The discovery of in-vitro systematic evolution of ligands by exponential enrichment (SELEX) process has considerably broaden the utility of aptamer as bio-recognition element, providing the high binding affinity and specificity against the target analytes. Recent research has focused on the development of structure switching signaling aptamer assay, transducing the aptamer- target recognition event into an easily detectable signal. In this paper, we demonstrate the development of structure switching aptamer assay for determination of aflatoxin M1 (AFM1) employing the quenching-dequenching mechanism. Hybridization of fluorescein labelled anti-AFM1 aptamer (F-aptamer) with TAMRA labelled complementary sequences (Q-aptamer) brings the fluorophore and the quencher into close proximity, which results in maximum fluorescence quenching. On addition of AFM1, the target induced conformational formation of antiparallel G-quadruplex aptamer-AFM1 complex results in fluorescence recovery. Under optimized experimental conditions, the developed method showed the good linearity with limit of detection (LOD) at 5.0ngkg(-1) for AFM1. The specificity of the sensing platform was carefully investigated against aflatoxin B1 (AFB1) and ochratoxin A (OTA). The developed assay platform showed the high specificity towards AFM1. The practical application of the developed aptamer assay was verified for detection of AFM1 in spiked milk samples. Good recoveries were obtained in the range from 94.40% to 95.28% (n=3) from AFM1 spiked milk sample.

Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor.

In this work, we propose for the first time a sensitive Ochratoxin A (OTA) detection in cocoa beans using competitive aptasensor by differential pulse voltammetry (DPV). In the proposed method, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE), and percentage binding was calculated. The detection was performed after adding avidin-ALP to perform avidin-biotin reaction; the signal was generated through a suitable substrate 1-naphthyl phosphate (1-NP), for alkaline phosphatase (ALP). The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns specifically designed for OTA. The developed aptasensor showed a good linearity in the range 0.15-5 ng/mL with the limit of detection (LOD) 0.07 ng/mL and 3.7% relative standard deviation (RSD). The aptasensor displayed good recovery values in the range 82.1-85% with 3.87% RSD, thus, demonstrated the efficiency of proposed aptasensor for such matrices.

Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk.

An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2-150 ng/L (LOD=1.15 ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2 µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000 ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50 ng/kg) and infants (25 ng/kg).

Biosensor-based real-time monitoring of paracetamol photocatalytic degradation.

This paper presents for the first time the integration of a biosensor for the on-line, real-time monitoring of a photocatalytic degradation process. Paracetamol was used as a model molecule due to its wide use and occurrence in environmental waters. The biosensor was developed based on tyrosinase immobilization in a polyvinylalcohol photocrosslinkable polymer. It was inserted in a computer-controlled flow system installed besides a photocatalytic reactor including titanium dioxide (TiO2) as photocatalyst. It was shown that the biosensor was able to accurately monitor the paracetamol degradation with time. Compared with conventional HPLC analysis, the described device provides a real-time information on the reaction advancement, allowing a better control of the photodegradation process.

Molecularly imprinted polymer cartridges coupled to high performance liquid chromatography (HPLC-UV) for simple and rapid analysis of fenthion in olive oil.

A combination of molecular modelling and a screening of the library of non-imprinted polymers (NIPs) was used to identify acrylamide as a functional monomer with high affinity towards fenthion, organophosphate insecticide, which is frequently used in the treatment of olives. A good correlation was found between the screening tests and modelling of monomer-template interactions performed using a computational approach. Acrylamide-based molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) were thermally synthesised in dimethyl formamide (porogen) using ethylene glycol dimethacrylate as a cross-linker and 1,1-azo-bis (isobutyronitrile) as an initiator. The chemical and physical properties of the prepared polymers were characterised. The binding of fenthion by the polymers was studied using solvents with different polarities. The developed MIP showed a high selectivity towards fenthion, compared to other organophosphates (dimethoate, methidathion malalthion), and allowed extraction of fenthion from olive oil samples with a recovery rate of about 96%. The extraction of fenthion using MIPs was much more effective than traditional C18 reverse-phase solid phase extraction and allowed to achieve a low detection limit (LOD) (5 µg L(-1)).

Detection of glycoalkaloids using disposable biosensors based on genetically modified enzymes.

In this work we present a rapid, selective, and highly sensitive detection of α-solanine and α-chaconine using cholinesterase-based sensors. The high sensitivity of the devices is brought by the use of a genetically modified acetylcholinesterase (AChE), combined with a one-step detection method based on the measurement of inhibition slope. The selectivity was obtained by using butyrylcholinesterase (BChE), an enzyme able to detect these two toxins with differential inhibition kinetics. The enzymes were immobilized via entrapment in PVA-AWP polymer directly on the working electrode surface. The analysis of the resulting inhibition slope was performed employing linear regression function included in Matlab. The high toxicity of α-chaconine compared to α-solanine due to a better affinity to the active site was proved. The inhibition of glycoalkaloids (GAs) mixture was performed over AChE enzyme wild-type AChE and BChE biosensors resulting in the detection of synergism effect. The developed method allows the detection of (GAs) at 50 ppb in potato matrix.

Computational and experimental investigation of molecular imprinted polymers for selective extraction of dimethoate and its metabolite omethoate from olive oil.

This work presents the development of molecularly imprinted polymers (MIPs) for the selective extraction of dimethoate from olive oil. Computational simulations allowed selecting itaconic acid as the monomer showing the highest affinity towards dimethoate. Experimental validation confirmed modelling predictions and showed that the polymer based on IA as functional monomer and omethoate as template molecule displays the highest selectivity for the structurally similar pesticides dimethoate, omethoate and monocrotophos. Molecularly imprinted solid phase extraction (MISPE) method was developed and applied to the clean-up of olive oil extracts. It was found that the most suitable solvents for loading, washing and elution step were respectively hexane, hexane-dichloromethane (85:15%) and methanol. The developed MIPSE was successfully applied to extraction of dimethoate from olive oil, with recovery rates up to 94%. The limits of detection and quantification of the described method were respectively 0.012 and 0.05 µg g(-1).

Acetylcholinesterase immobilized on magnetic beads for pesticides detection: application to olive oil analysis.

This work presents the development of bioassays and biosensors for the detection of insecticides widely used in the treatment of olive trees. The systems are based on the covalent immobilisation of acetylcholinesterase on magnetic microbeads using either colorimetry or amperometry as detection technique. The magnetic beads were immobilised on screen-printed electrodes or microtitration plates and tested using standard solutions and real samples. The developed devices showed good analytical performances with limits of detection much lower than the maximum residue limit tolerated by international regulations, as well as a good reproducibility and stability.

Molecular imprinting solid phase extraction for selective detection of methidathion in olive oil.

A specific adsorbent for extraction of methidathion from olive oil was developed. The design of the molecularly imprinted polymer (MIP) was based on the results of the computational screening of the library of polymerisable functional monomers. MIP was prepared by thermal polymerisation using N,N'-methylene bisacrylamide (MBAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. The polymers based on the itaconic acid (IA), methacrylic acid (MAA) and 2-(trifluoromethyl)acryl acid (TFMAA) functional monomers and one control polymer which was made without functional monomers with cross-linker EGDMA were also synthesised and tested. The performance of each polymer was compared using corresponding imprinting factor. As it was predicted by molecular modelling the best results were obtained for the MIP prepared with MBAA. The obtained MIP was optimised in solid-phase extraction coupled with high performance liquid chromatography (MISPE-HPLC-UV) and tested for the rapid screening of methidathion in olive oil. The proposed method allowed the efficient extraction of methidathion for concentrations ranging from 0.1 to 9 mg L(-1) (r(2)=0.996). The limits of detection (LOD) and quantification (LOQ) in olive oil were 0.02 mg L(-1) and 0.1 mg L(-1), respectively. MIPs extraction was much more effective than traditional C18 reverse-phase solid phase extraction.

Chronoamperometric determination of lead ions using PEDOT:PSS modified carbon electrodes.

A new simple chronoamperometry methodology was developed for the ultrasensitive determination of lead ions using a PEDOT:PSS coated graphite carbon electrode. The polymer was directly coated on a graphite carbon electrode and characterized using simple cycle voltammetric measurements. The presence of lead ions induced a cathodic peak starting at -550 ± 10 mV vs. Ag/AgCl, and an anodic peak starting at -360 ± 10 mV vs. Ag/AgCl. Electroaccumulation of lead ions onto the PEDOT:PSS modified electrode was performed at -650 mV vs. Ag/AgCl for 30s in a pH 2.2 hydrochloric acid solution. Chronoamperometry measurements were carried out at -350 mV vs. Ag/AgCl allowing the oxidation of accumulated lead. Using this method, lead ions were detected for concentrations ranging between 2.0 nmol L(-1) and 0.1 µmol L(-1) (R(2)=0.999). The detection limit was calculated to be 0.19 nmol L(-1) and the quantification limit of 0.63 nmol L(-1). The method was shown to be highly precise and sensitive, negligible interference was detected from other metal ions. The proposed method was successfully applied for the detection of lead ions in vegetables.

Screen-printed poly(3,4-ethylenedioxythiophene) (PEDOT): A new electrochemical mediator for acetylcholinesterase-based biosensors.

This work describes the use of a PEDOT:PSS-based conductive polymer for designing AChE-based biosensors. The transducers were obtained directly by screen-printing a PEDOT:PSS suspension on the surface of thick film carbon electrodes. The obtained working electrodes showed a high conductivity when compared with electrodes modified with conventional mediators like cobalt phthalocyanine or tetracyanoquinodimethane. The PEDOT:PSS polymer was shown to be suitable for thiocholine oxidation, allowing the measurement of AChE activity at 100 mV vs Ag/AgCl. The high conductivity of PEDOT:PSS allowed the accurate detection of the organophosphate insecticide chlorpyrifos-oxon at concentrations as low as 4x10(-9)M, corresponding to an inhibition ratio of 5%.

Biosensors as analytical tools in food fermentation industry.

The food industries need rapid and affordable methods to assure the quality ofproducts and process control. Biosensors, combining a biological recognition element and a sensitive transducer, are versatile analytical tools that offer advantages as classical analytical methods due to their inherent specificity, selectivity and simplicity. This paper reviews the recent trends in the development and applications of biosensors used in food fermentation industry, focusing on amperometric enzymatic and microbial sensors.