Selection of spore-specific aptamers for Geobacillus stearothermophilus, a food spoilage bacterium.

Due to their ability to form extremely heat resistant spores, anaerobic bacteria are responsible for frequent food spoilage. The development of rapid and specific methods for the detection and quantification of spore contamination is therefore of major interest. In this paper, we describe for the first time the selection of aptamers specific to spores of Geobacillus stearothermophilus (Gbs), which induce flat sour spoilage in vegetable cans. Eighteen Spore-SELEX cycles were performed including 4 counter-selections with 12 bacteria commonly found in cannery. To optimise candidate amplification, PCR in emulsion was performed, and high-throughput sequencing analysis was applied to follow candidate evolution. Sequencing of aptamers from cycle 18 revealed 43 overrepresented sequences whose copy number exceeds 0.15% of the total obtained sequences. Within this group, the A01 aptamer presented a much higher enrichment with a relative abundance of 17.71%. Affinity and specificity for Gbs spores of the 10 most abundant candidates at cycle 18 were confirmed by PCR assay based on aptamer-spore complex formation and filtration step. Obtaining these aptamers is the starting point for the future development of biosensors dedicated to the detection of Gbs spores.

Synthesis and Characterization of Bis-1,2,3-Triazole Ligand and its Corresponding Copper Complex for the Development of Electrochemical Affinity Biosensors.

The bis-triazole ligand and its corresponding copper complexes were synthesized and characterized for the first time and proposed as new labels for the development of electrochemical aptasensors. The bis-triazole ligand was prepared from methyl 1,6-heptadiyne-4-carboxylate and 2-(azidomethyl)phenol using classical CuAAC in presence of different copper salts. The X-ray structure of bis-triazole showed a symmetry center (C1). UV-Vis and X-band EPR spectra showed that the coordination capacity of the bis-triazole ligand was improved in the presence of triethylamine due to deprotonation of the triazole and phenolate moieties. After complexation with copper, the obtained complex was successfully attached to an anti-estradiol aptamer through thiol-maleimide coupling, and the resulting labelled aptamer was immobilized on a carbon screen-printed electrode by carbodiimide coupling. The electrochemical response of the resulting sensor was shown to decrease in the presence of estradiol, demonstrating that the developed complexes can be applied for the development of aptasensors.

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.

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.

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of ß-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 µM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.

Immunosensors for estradiol and ethinylestradiol based on new synthetic estrogen derivatives: application to wastewater analysis.

Novel electrochemical immunosensors for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of magnetic beads (MBs) as solid support and screen-printed electrodes as sensing platforms. Four synthetic estrogen derivatives containing either a carboxylic group or an amine group at the C-3 position were synthesized and covalently bound to MBs functionalized with amine or carboxyl groups, respectively. The assay was based on competition between the free and immobilized estrogen for the binding sites of the primary antibody, with subsequent revelation using alkaline phosphatase-labeled secondary antibody. Preliminary colorimetric tests were performed in order to validate the applicability of the synthetic estrogens to immuno-recognition and to optimize different experimental parameters. In a second step, electrochemical detection was carried out by square wave voltammetry (SWV). Under the optimized working conditions, the electrochemical immunosensors showed a highly sensitive response to E2 and EE2, with respective detection limits of 1 and 10 ng/L. Cross-reactivity evaluated against other hormones demonstrated an excellent selectivity. The developed devices were successfully applied to analysis of spiked and natural water samples. These new immunosensors offer the advantages of being highly sensitive, easy, and rapid to prepare, with a short assay time.

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.

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.

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.

Spectroelectrochemistry as a new tool for the quantification of UV filters in sun creams.

This study focuses on the spectroelectrochemical quantification of four UV filters, butylmethoxy dibenzoylmethane (BM), benzophenone-3 (BP3), ethylhexyl methoxycinnamate (EM) and octocrylene (OC) used as sunscreens in cosmetics. Three of them exhibited electrochemical activity resulting in the modification of their absorption spectrum under the application of an oxidizing potential of +1.8 V vs. Ag. When working with a mixture containing both electroactive and nonelectroactive UV filters, UV-vis absorption spectra recorded before and after the application of the potential differed. The combination of spectral deconvolution of the spectra pair allowed a more accurate identification and quantification of UV filters than spectral deconvolution of the initial absorbance spectrum alone. This method was effective for the assessment of UV filters in model mixtures and commercial sun creams.

Monoclonal Antibody-Based Immunosensor for the Electrochemical Detection of Chlortoluron Herbicide in Groundwaters.

Chlortoluron (3-(3-chloro-p-tolyl)-1,1-dimethyl urea) is an herbicide widely used in substitution to isoproturon to control grass weed in wheat and barley crops. Chlortoluron has been detected in groundwaters for more than 20 years; and dramatic increases in concentrations are observed after intense rain outbreaks. In this context; we developed an immunosensor for the determination of chlortoluron based on competitive binding of specific monoclonal antibodies on chlortoluron and immobilized biotinylated chlortoluron; followed by electrochemical detection on screen-printed carbon electrodes. The optimized immunosensor exhibited a logarithmic response in the range 0.01-10 µg·L-1; with a calculated detection limit (LOD) of 22.4 ng·L-1; which is below the maximum levels allowed by the legislation (0.1 µg·L-1). The immunosensor was used for the determination of chlortoluron in natural groundwaters, showing the absence of matrix effects.

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.

Detection of Salmonella in Food Matrices, from Conventional Methods to Recent Aptamer-Sensing Technologies.

Rapid detection of the foodborne pathogen Salmonella in food processing is of crucial importance to prevent food outbreaks and to ensure consumer safety. Detection and quantification of Salmonella species in food samples is routinely performed using conventional culture-based techniques, which are labor intensive, involve well-trained personnel, and are unsuitable for on-site and high-throughput analysis. To overcome these drawbacks, many research teams have developed alternative methods like biosensors, and more particularly aptasensors, were a nucleic acid is used as biorecognition element. The increasing interest in these devices is related to their high specificity, convenience, and relative rapid response. This review aims to present the advances made in these last years in the development of biosensors for the detection and the quantification of Salmonella, highlighting applications on meat from the chicken food chain.

Salen/salan metallic complexes as redox labels for electrochemical aptasensors.

This work presents the synthesis and characterization of salen/salan metal complexes for their future application as electrochemical labels in affinity sensors. Due to its stability and electrochemical properties, an oxovanadium salan complex was selected and coupled to an estradiol-specific aptamer. The response of the resulting aptasensor was shown to decrease with increasing estradiol concentration.

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples.

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.

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.

Pharmaceuticals released from senior residences: occurrence and risk evaluation.

One of the main pursuits, yet most difficult, in monitoring studies is to identify the sources of environmental pollution. In this study, we have identified health-care facilities from south European countries as an important source of pharmaceuticals in the environment. We have estimated that compounds consumed in by the elderly and released from effluents of senior residences can reach river waters at a concentration higher than 0.01 µg/L, which is the European Medicines Agency (EMA) threshold for risk evaluation of pharmaceuticals in surface waters. This study has been based on five health institutions in Portugal, Spain, and France, with 52 to 130 beds. We have compiled the pharmaceuticals dispensed on a daily base and calculated the consumption rates. From 54.9 to 1801 g of pharmaceuticals are consumed daily, with laxatives, analgesics, antiepileptics, antibiotics, and antidiabetic agents being the main drug families administered. According to excretion rates, dilution in the sewerage system, and elimination in wastewater treatment plants, macrogol, metformin, paracetamol, acetylcysteine, amoxicillin, and gabapentin, among others, are expected to reach river waters. Finally, we discuss the risk management actions related to the discharge of pharmaceuticals from senior residences to surface waters.

Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase.

This work presents a biosensor for organophosphorus pesticides based on immobilisation of a highly sensitive genetically engineered acetylcholinesterase (B394) by affinity interactions on metal chelate-functionalised magnetic microbeads. The developed sensor has been compared with those based on the widely used Electric eel cholinesterase and a classical entrapment procedure in a polyvinylalcohol-based matrix. The use of the B394 enzyme allowed lowering both IC50 and LOD by a factor of 100 when compared with Electric eel enzyme sensor. The oriented and site-specific immobilisation combined with the high specificity of the B349 mutant allows a more sensitive detection of insecticides, concentrations as low as 1.31(-11)M (IC10) being detected for both pesticides chlorpyriphos-oxon and chlorfenvinphos.

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.