Οπτικοί ανοσοαισθητήρες για την ταυτόχρονη ανίχνευση αλλεργιογόνων ουσιών σε τρόφιμα

Η τροφική αλλεργία επηρεάζει μεγάλο ποσοστό των ανθρώπων παγκοσμίως. Για το λόγο αυτό, διεθνείς οργανισμοί έχουν θεσπίσει κανόνες τόσο για τον έλεγχο παρουσίας αλλεργιογόνων όσο και για την υποχρεωτική αναγραφή τους στις ετικέτες των επεξεργασμένων προϊόντων εκ μέρους των βιομηχανιών τροφίμων. Για την αποφυγή ανεπιθύμητης επιμόλυνσης των τροφίμων με αλλεργιογόνα λόγω της χρήσης κοινών γραμμών παραγωγής για διαφορετικά προϊόντα είναι απαραίτητος ο έλεγχος παρουσίας αλλεργιογόνων τόσο στο νερό καθαρισμού/ έκπλυσης των σωληνώσεων και μηχανών όσο και στα ίδια τα τελικά προϊόντα και τις πρώτες ύλες που χρησιμοποιούνται. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η ανάπτυξη μεθόδου για τον ταυτόχρονο προσδιορισμό της κ-καζεΐνης, της πρωτεΐνης φιστικιού, της πρωτεΐνης σόγιας και της γλιαδίνης σε νερό έκπλυσης σωληνώσεων από βιομηχανίες τροφίμων σε πραγματικό χρόνο, μέσω οπτικού ανοσοαισθητήρα χωρίς τη χρήση ιχνηθετών. Ο οπτικός ανοσοαισθητήρας που χρησιμοποιήθηκε ήταν πλήρως ολοκληρωμένος σε ψηφίδες πυριτίου και περιλάμβανε συστοιχία δέκα συμβολομέτρων Mach-Zehnder (MZI) καθώς και τις αντίστοιχες οπτικές πηγές παρέχοντας τη δυνατότητα ταυτόχρονης ανίχνευσης πολλών αναλυτών στο ίδιο δείγμα. Για την επίτευξη του στόχου, αναπτύχθηκαν και βελτιστοποιήθηκαν ανοσοενζυμικοί προσδιορισμοί (ELISA) για όλα τα στοχευόμενα αλλεργιογόνα καθώς και μέθοδοι ανίχνευσης μονού και πολλαπλών αναλυτών με τον αισθητήρα. Οι αναπτυχθείσες μέθοδοι ταυτόχρονου προσδιορισμού των αλλεργιογόνων ήταν ταχείες (6,5 min) και ακριβείς με όρια ανίχνευσης εφάμιλλα ή χαμηλότερα των εμπορικά διαθέσιμων μεθόδων που χρησιμοποιούνται από τις βιομηχανίες. Η δυνατότητα εφαρμογής του αισθητήρα σε πραγματικές συνθήκες αξιολογήθηκε μέσω ανάλυσης δειγμάτων νερού έκπλυσης σωληνώσεων γαλακτοβιομηχανίας και διαπιστώθηκε ότι οι τιμές που προσδιορίστηκαν ήταν σε καλή συμφωνία με αυτές που ελήφθησαν τόσο με τις μεθόδους ELISA που αναπτύχθηκαν όσο και από εξωτερικά διεθνή εργαστήρια. Επιπλέον, ο αισθητήρας αξιοποιήθηκε για την ανίχνευση νοθείας κατσικίσιου γάλακτος με αγελαδινό, μέσω προσδιορισμού βοείου κ-καζεΐνης, και παρείχε αξιόπιστες και ταχείες (5 min) μετρήσεις με ευαισθησία ανίχνευσης <0,1% ( ο/ο).Food allergy affects a large percentage of people worldwide. For this reason, international organizations have addressed directives which make mandatory the control and labeling of processed products so as to indicate the presence of allergenic compounds by the food industries. In order to avoid undesirable contamination of food with allergens due to the use of common production lines for different products, it is necessary to control the presence of allergens in both the rinsing water of pipelines and machines, the finished products and the raw materials used as well. The purpose of this PhD dissertation was to develop an optical immunosensor for the simultaneous, real-time, label-free determination of κ-casein, peanut protein, soy protein and gliadin in rinsing water from food industry. The immunosensor was based on an optoelectronic platform fully integrated onto silicon chips and comprised an array of ten Mach-Zehnder (MZI) interferometers along with the corresponding optical sources, enabling the simultaneous detection of multiple analytes in the same sample. To achieve this goal, enzyme-immunoassays (ELISAs) were developed and optimized for all target allergens as well as methods for single and multiple analytes detection with the sensor. The methods developed for the simultaneous determination of allergens were fast (6.5 min) and accurate with detection limits equal to or lower than those provided by the commercially available kits used by food industry. The potential of the sensor to perform in real conditions was evaluated by analyzing rinsing water samples from a dairy indystry and it was found that the results were in good agreement with those obtained by the ELISA methods developed or provided by external international analytical laboratories. In addition, due to its excellent performance for allergen detection in rinsing water, the immunosensor was also evaluated for the detection of goat milk adulteration with cow milk (through bovine k-casein determination). The immunosensor developed found to be reliable and rapid (5 min) with high detection sensitivity (<0.1%, v / v)

Optical immunosensors for the simultaneous detection of allergens in food

Food allergy affects a large percentage of people worldwide. For this reason, international organizations have addressed directives which make mandatory the control and labeling of processed products so as to indicate the presence of allergenic compounds by the food industries. In order to avoid undesirable contamination of food with allergens due to the use of common production lines for different products, it is necessary to control the presence of allergens in both the rinsing water of pipelines and machines, the finished products and the raw materials used as well. The purpose of this PhD dissertation was to develop an optical immunosensor for the simultaneous, real-time, label-free determination of κ-casein, peanut protein, soy protein and gliadin in rinsing water from food industry. The immunosensor was based on an optoelectronic platform fully integrated onto silicon chips and comprised an array of ten Mach-Zehnder (MZI) interferometers along with the corresponding optical sources, enabling the simultaneous detection of multiple analytes in the same sample. To achieve this goal, enzyme-immunoassays (ELISAs) were developed and optimized for all target allergens as well as methods for single and multiple analytes detection with the sensor. The methods developed for the simultaneous determination of allergens were fast (6.5 min) and accurate with detection limits equal to or lower than those provided by the commercially available kits used by food industry. The potential of the sensor to perform in real conditions was evaluated by analyzing rinsing water samples from a dairy indystry and it was found that the results were in good agreement with those obtained by the ELISA methods developed or provided by external international analytical laboratories. In addition, due to its excellent performance for allergen detection in rinsing water, the immunosensor was also evaluated for the detection of goat milk adulteration with cow milk (through bovine k-casein determination). The immunosensor developed found to be reliable and rapid (5 min) with high detection sensitivity (<0.1%, v / v).Η τροφική αλλεργία επηρεάζει μεγάλο ποσοστό των ανθρώπων παγκοσμίως. Για το λόγο αυτό, διεθνείς οργανισμοί έχουν θεσπίσει κανόνες τόσο για τον έλεγχο παρουσίας αλλεργιογόνων όσο και για την υποχρεωτική αναγραφή τους στις ετικέτες των επεξεργασμένων προϊόντων εκ μέρους των βιομηχανιών τροφίμων. Για την αποφυγή ανεπιθύμητης επιμόλυνσης των τροφίμων με αλλεργιογόνα λόγω της χρήσης κοινών γραμμών παραγωγής για διαφορετικά προϊόντα είναι απαραίτητος ο έλεγχος παρουσίας αλλεργιογόνων τόσο στο νερό καθαρισμού/ έκπλυσης των σωληνώσεων και μηχανών όσο και στα ίδια τα τελικά προϊόντα και τις πρώτες ύλες που χρησιμοποιούνται. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η ανάπτυξη μεθόδου για τον ταυτόχρονο προσδιορισμό της κ-καζεΐνης, της πρωτεΐνης φιστικιού, της πρωτεΐνης σόγιας και της γλιαδίνης σε νερό έκπλυσης σωληνώσεων από βιομηχανίες τροφίμων σε πραγματικό χρόνο, μέσω οπτικού ανοσοαισθητήρα χωρίς τη χρήση ιχνηθετών. Ο οπτικός ανοσοαισθητήρας που χρησιμοποιήθηκε ήταν πλήρως ολοκληρωμένος σε ψηφίδες πυριτίου και περιλάμβανε συστοιχία δέκα συμβολομέτρων Mach-Zehnder (MZI) καθώς και τις αντίστοιχες οπτικές πηγές παρέχοντας τη δυνατότητα ταυτόχρονης ανίχνευσης πολλών αναλυτών στο ίδιο δείγμα. Για την επίτευξη του στόχου, αναπτύχθηκαν και βελτιστοποιήθηκαν ανοσοενζυμικοί προσδιορισμοί (ELISA) για όλα τα στοχευόμενα αλλεργιογόνα καθώς και μέθοδοι ανίχνευσης μονού και πολλαπλών αναλυτών με τον αισθητήρα. Οι αναπτυχθείσες μέθοδοι ταυτόχρονου προσδιορισμού των αλλεργιογόνων ήταν ταχείες (6,5 min) και ακριβείς με όρια ανίχνευσης εφάμιλλα ή χαμηλότερα των εμπορικά διαθέσιμων μεθόδων που χρησιμοποιούνται από τις βιομηχανίες. Η δυνατότητα εφαρμογής του αισθητήρα σε πραγματικές συνθήκες αξιολογήθηκε μέσω ανάλυσης δειγμάτων νερού έκπλυσης σωληνώσεων γαλακτοβιομηχανίας και διαπιστώθηκε ότι οι τιμές που προσδιορίστηκαν ήταν σε καλή συμφωνία με αυτές που ελήφθησαν τόσο με τις μεθόδους ELISA που αναπτύχθηκαν όσο και από εξωτερικά διεθνή εργαστήρια. Επιπλέον, ο αισθητήρας αξιοποιήθηκε για την ανίχνευση νοθείας κατσικίσιου γάλακτος με αγελαδινό, μέσω προσδιορισμού βοείου κ-καζεΐνης, και παρείχε αξιόπιστες και ταχείες (5 min) μετρήσεις με ευαισθησία ανίχνευσης <0,1% ( ο/ο)

Label-Free Biosensors Based onto Monolithically Integrated onto Silicon Optical Transducers

The article reviews the current status of label-free integrated optical biosensors focusing on the evolution over the years of their analytical performance. At first, a short introduction to the evanescent wave optics is provided followed by detailed description of the main categories of label-free optical biosensors, including sensors based on surface plasmon resonance (SPR), grating couplers, photonic crystals, ring resonators, and interferometric transducers. For each type of biosensor, the detection principle is first provided followed by description of the different transducer configurations so far developed and their performance as biosensors. Finally, a short discussion about the current limitations and future perspectives of integrated label-free optical biosensors is provided

Optical Immunosensors for Bacteria Detection in Food Matrices

Optical immunosensors are one of the most popular categories of immunosensors with applications in many fields including diagnostics and environmental and food analysis. The latter field is of particular interest not only for scientists but also for regulatory authorities and the public since food is essential for life but can also be the source of many health problems. In this context, the current review aims to provide an overview of the different types of optical immunosensors focusing on their application for the determination of pathogenic bacteria in food samples. The optical immunosensors discussed include sensors based on evanescent wave transduction principles including surface plasmon resonance (SPR), fiber-optic-, interferometric-, grating-coupler-, and ring-resonator-based sensors, as well as reflectometric, photoluminescence, and immunosensors based on surface-enhanced Raman scattering (SERS). Thus, after a short description of each transduction technique, its implementation for the immunochemical determination of bacteria is discussed. Finally, a short commentary about the future trends in optical immunosensors for food safety applications is provided

Simultaneous Detection of SARS-CoV-2 Nucleoprotein and Receptor Binding Domain by a Multi-Area Reflectance Spectroscopy Sensor

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications

Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach&ndash;Zehnder Interferometers Monolithically Integrated on Silicon Chips

The consumption of water and milk contaminated with bacteria can lead to foodborne disease outbreaks. For this reason, the development of rapid and sensitive analytical methods for bacteria detection is of primary importance for public health protection. Here, a miniaturized immunosensor based on broadband Mach&ndash;Zehnder Interferometry for the simultaneous determination of S. typhimurium and E. coli O157:H7 in drinking water and milk is presented. For the assay, mixtures of bacteria solutions with anti-bacteria-specific antibodies were run over the chip, followed by solutions of biotinylated anti-species-specific antibody and streptavidin. The assay was fast (10 min for water, 15 min for milk), accurate, sensitive (LOD: 40 cfu/mL for S. typhimurium; 110 cfu/mL for E. coli) and reproducible. The analytical characteristics achieved combined with the small chip size make the proposed biosensor suitable for on-site bacteria determination in drinking water and milk samples

A 3D-Printed Electrochemical Immunosensor Employing Cd/Se ZnS QDs as Labels for the Rapid and Ultrasensitive Detection of <i>Salmonella typhimurium</i> in Poultry Samples

Salmonella is one of the leading causes of foodborne illnesses worldwide, with poultry products being a major source of contamination. Thus, the detection of salmonella in commercial poultry products is crucial to minimize the effects on public health. Electrochemical sensors are promising tools for bacteria detection due to their sensitivity, simplicity, and potential for on-site analysis. In this work, a three-dimensional (3D) printed electrochemical immunosensor for the determination of Salmonella typhimurium in fresh chicken through a sandwich immunoassay employing biotinylated anti-S. typhimurium antibody followed by streptavidin labeled with Cd/Se ZnS quantum dots (QDs) is presented. The device features three carbon-black polylactic acid electrodes and a holder, and the quantification of S. typhimurium is performed by anodic stripping voltametric (ASV) determination of the Cd(II) released after acidic dissolution of the QDs. To enhance sensitivity, an electroplated bismuth film was deposited on the working electrode, achieving a detection limit of 5 cfu/mL in a total assay time of 25 min, whereas 5 h of sample pre-enrichment was required for the detection of 1 cfu/25 mL of chicken rinse and chicken broth. The method is accurate, with %recovery values ranging from 93.3 to 113% in fresh chicken samples, and repeatable with intra- and inter- assay coefficient of variations S. typhimurium at the point-of-need

Lab-on-a-membrane foldable devices for duplex drop-volume electrochemical biosensing using quantum dot tags

<p>This work describes a new type of integrated lab-on-a-membrane foldable device suitable for on-site duplex electrochemical biosensing using drop-size sample volumes. The devices are fabricated entirely by screen-printing on a nylon membrane and feature two assay zones which are located symmetrically on either side of a three-electrode voltammetric cell with a bismuth citrate-loaded graphite working electrode. After the completion of two spatially separated drop-volume competitive immunoassays on the assay zones using biotinylated antibodies labeled with streptavidin-conjugated Pb- and Cd-based quantum dots (QDs), respectively, the QD labels are dissolved releasing Pb(II) and Cd(II) in the assay zones. Then, the two assay zones are folded over, and they are brought in contact with the voltammetric cell for simultaneous anodic stripping voltammetric (ASV) determination of Pb(II) and Cd(II) at the bismuth nanostructured layer formed on the working electrode by reduction of the bismuth citrate during the preconcentration step. The fabrication of the devices is discussed in detail, and their operational characteristics are exhaustively studied. In order to demonstrate their applicability to the analysis in complex matrices, duplex ASV-QDs-based determination of bovine casein and bovine immunoglobulin G is carried out in milk samples yielding limits of detection of 0.04 μg mL^-1 and 0.02 μg mL^-1, respectively. The potential of the devices to detect milk adulteration is further demonstrated. These new membrane devices enable duplex biosensing with distinct advantages over existing approaches in terms of cost, fabrication, and operational simplicity and rapidity, portability, sample size, disposability, sensitivity, and suitability for field analysis.</p