Molecular Identification through Membrane Engineering as a Revolutionary Concept for the Construction of Cell Sensors with Customized Target Recognition Properties: the Example of Superoxide Detection

AbstractMembrane-engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor molecules on the cell surface. We have previously reported the construction of an ultra-sensitive superoxide anion (O2•−) sensor based on immobilized cells, which have been membrane-engineered with superoxide dismutase (SOD). In the present study, we provide evidence that superoxide dismutation triggered changes to the membrane potential of membrane-engineered fibroblast cells, as confirmed by electrophysiological and fluorescence assays. In addition, by conducting selective inhibition assays, we show that electroinserted SOD molecules retained their characteristic catalytic properties. We also investigated the effect of the concentration of electroinserted SOD molecules. Finally, we increased the sensitivity of the sensor by hundredfold to a detection limit of 1 pM O2•− by changing the intensity of the electrical field during electroinsertion and the concentration of immobilized cells on the performance of the biosensor

Pesticide residue screening using a novel artificial neural network combined with a bioelectric cellular biosensor

We developed a novel artificial neural network (ANN) system able to detect and classify pesticide residues. The novel ANN is coupled, in a customized way, to a cellular biosensor operation based on the bioelectric recognition assay (BERA) and able to simultaneously assay eight samples in three minutes. The novel system was developed using the data (time series) of the electrophysiological responses of three different cultured cell lines against three different pesticide groups (carbamates, pyrethroids, and organophosphates). Using the novel system, we were able to classify correctly the presence of the investigated pesticide groups with an overall success rate of 83.6%. Considering that only 70,000-80,000 samples are annually tested in Europe with current conventional technologies (an extremely minor fraction of the actual screening needs), the system reported in the present study could contribute to a screening system milestone for the future landscape in food safety control

Noninvasive Superoxide Monitoring of In Vitro Neuronal Differentiation Using a Cell-Based Biosensor

Membrane-engineered cells bearing superoxide dismutase (SOD) molecules on their surface offer the capability of ultrarapid and ultrasensitive detection of the superoxide anion (O2-) through the measurement of changes of their cell membrane potential. We herewith report the application of this technology for the noninvasive determination of superoxide levels during the in vitro differentiation of PC12 cells. We were able to detect changes in O2- accumulation in the culture medium, which were closely associated with the progress of neuronal differentiation

Development of the membrane-engineering technology on the cell-based biosensor for the detection of superoxide and its application on the study of cellular division and cell differentiation mechanisms

Membrane-engineering is a generic novel methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor molecules on the cell surface. The starting point of the present dissertation is the previously reported construction of an ultra-sensitive superoxide anion (O2−) sensor based on immobilized cells, which have been membrane-engineered with superoxide dismutase (SOD). In the present study, evidence is provided that superoxide dismutation triggered changes to the membrane potential of membrane-engineered fibroblast cells, as confirmed by electrophysiological and fluorescence assays. These changes were associated with changes in [Ca2+]cyt, as revealed by the selective inhibition of intracellular calcium ion traffic. In addition, by conducting selective inhibition assays, it is demonstrated that electroinserted SOD molecules retained their characteristic catalytic properties. The effect of the concentration of electroinserted SOD molecules on the performance of the superoxide assay was also investigated. Finally, the sensitivity of the sensor was increased by hundredfold to a detection limit of 1 pM O2− by changing the concentration of immobilized cells on the performance of the biosensor.The next step was the investigation of a possible relationship between the levels of reactive oxygen species (ROS) and the stimulation of frond division of the aquatic plant Spirodela polyrrhiza (duckweed) during a 7-day experimental culture period. In particular, superoxide concentration was monitored using a state-of-the-art cell biosensor. A considerable reduction in ROS and superoxide concentration was observed during the first two days of culture, whereas duckweed cultures achieved near exponential growth rates after the second day. In addition, apoptotic markers such as the cytoplasmic concentration of cytochrome C, mitochondrial membrane depolarization and the activity of caspase-3 declined during the culture period and at least before daughter frond maturation. It is suggested that S. polyrrhiza frond division may have been stimulated by the observed reduction of free radicals and the associated avoidance of cell apoptotic pathways in cultured plants.Also ROS have been shown to have an important role in cell differentiation. In the present study, N2a murine neuroblastoma cells were differentiated by treatments with forskolin or dibutyryl cyclic AMP. The differentiation was expressed as the development of neurites, observed using coomassie brilliant blue after 48h treatment. This was accompanied by an increase in the concentration of ROS and specifically mitochondrial superoxide, both detected by flow cytometry. At the same time the superoxide concentration was monitored using the novel biosensor, whereas a high correlation between the two different methods of superoxide detection (flow cytometry-biosensor) was observed.In an additional approach, the biosensor was applied to yet another complex biological system, i.e. in clinical medicine as a diagnostic method to patients in order to detect their post-operative status. It has been frequently suggested that oxidative stress is involved in post-operative stress. The results, though preliminary, due the small patient sample size, showed that the levels of O2− in patients with worst recovery rate were highest. In conclusion, the results of the individual partial experimental sections of the present study (1) elucidated the deepest aspects of the working principle of the molecular identification through membrane engineering and (2) confirmed the efficiency and considerable application perspectives of the superoxide biosensor based on this technology.Η μεμβρανική μηχανική είναι μια γενική, καινοτόμος μεθοδολογία για την αύξηση της ευαισθησίας των κυτταρικών βιοαισθητήρων έναντι των μορίων-στόχων, ηλεκτροεισάγοντας τον ειδικό υποδοχέα στην κυτταρική επιφάνεια. Σημείο εκκίνησης της παρούσας διδακτορικής διατριβής είναι η ανάπτυξη, σε προηγούμενη μελέτη, ενός ευαίσθητου αισθητήρα για την ανίχνευση σουπεροξειδίου (O2−) βασισμένου σε ακινητοποιημένα κύτταρα, τα οποία τροποποιήθηκαν μεμβρανικά με το ένζυμο δισμουτάση του σουπεροξειδίου (SOD). Στην παρούσα μελέτη, τεκμηριώνεται ότι η αντίδραση του O2− με την SOD προκαλεί αλλαγές στο μεμβρανικό δυναμικό των μεμβρανικώς τροποποιημένων κυττάρων Vero, όπως επιβεβαιώνεται από τις ηλεκτροφυσιολογικές και φθορισμομετρικές μετρήσεις. Αυτές οι αλλαγές συσχετίστηκαν με αλλαγές της συγκέντρωσης του ενδοκυτταρικού ασβεστίου, όπως φαίνεται μετά την εκλεκτική παρεμπόδιση του ενδοκυτταρικού μονοπατιού ασβεστίου. Επιπλέον, εφαρμόζοντας εκλεκτικούς παρεμποδιστές, αποδεικνύεται ότι τα ηλεκτροεισαχθέντα μόρια της SOD διατηρούν τις καταλυτικές τους ιδιότητες. Ερευνήθηκε η επίδραση της συγκέντρωσης της ηλεκτροεισαχθείσας SOD. Τέλος, αυξήθηκε η ευαισθησία του αισθητήρα κατά 100 φορές, με όριο ανίχνευσης 1 pM O2−, σαν συνέπεια της μεταβολής της συγκέντρωσης των ακινητοποιημένων κυττάρων στον αισθητήρα.Μελετήθηκε η πιθανή σχέση ανάμεσα στα επίπεδα των ελευθέρων ριζών (ROS) και της κυτταρικής διαίρεσης κατά την in vitro επταήμερη καλλιέργεια του υδρόβιου φυτού Spirodela polyrrhiza. Πιο συγκεκριμένα, καταγράφτηκε η συγκέντρωση του σουπεροξειδίου χρησιμοποιώντας τον εξελιγμένο βιοαισθητήρα. Μια σημαντική μείωση των ROS αλλά και της συγκέντρωσης του O2−, παρατηρήθηκε κατά τις δύο πρώτες ημέρες της καλλιέργειας, ενώ η S. polyrrhiza βρίσκονταν κοντά στην εκθετική φάση ανάπτυξης. Επιπλέον, αποπτωτικοί δείκτες όπως είναι η συγκέντρωση του κυτοπλασματικού κυτοχρώματος C, η αποπόλωση της μιτοχονδριακής μεμβράνης και η δραστικότητα της κασπάσης-3 φθίνουν κατά τη περίοδο καλλιέργειας τουλάχιστον πριν την ωρίμανση των φυλλιδίων. Θεωρείται ότι η διαίρεση των φυλλιδίων του S. polyrrhiza μπορεί να δραστηριοποιήθηκε από παρατηρούμενη μείωση των ελευθέρων ριζικών και τη σχετική αποφυγή των κυτταρικών αποπτωτικών μονοπατιών των καλλιεργούμενων φυτών.Επίσης οι ROS έχει φανεί να παίζουν σημαντικό ρόλο στην κυτταρική διαφοροποίηση. Στην παρούσα μελέτη, τα κύτταρα νευροβλαστώματος διαφοροποιήθηκαν με φορσκολίνη και κυκλική μονοφωσφορική διβουτυρυλαδενοσίνη (db-cAMP) . Η διαφοροποίηση εκφράστηκε σαν ανάπτυξη των νευριτών χρησιμοποιώντας τη χρωστική coomassie brilliant blue μετά από 48 ώρες επώασης των ουσιών διαφοροποίησης. Η διαφοροποίηση συνοδεύτηκε με αύξηση των ROS και του μιτοχονδριακού O2−, και τα δύο ανιχνεύθηκαν με κυτταρομετρία ροής. Παράλληλα έγινε καταγραφή της συγκέντρωσης O2− με τον βιοαισθητήρα για την αξιολόγηση του συγκρίνοντάς τον με μια κλασική μέθοδο όπως είναι η κυτταρομετία ροής. Όπως παρατηρήθηκε, υπήρξε μεγάλη συσχέτιση των αποτελεσμάτων.Επιπροσθέτως, ο βιοαισθητήρας δοκιμάστηκε στην κλινική ιατρική, με σκοπό την καταγραφή των επιπέδων O2− σε ασθενείς σε μεταγχειριτική κατάσταση. Έχει συχνά προταθεί ότι το οξειδωτικό στρες είναι πιθανός εμπλεκόμενος παράγοντας του μετεγχειριτικού στρες. Τα αποτελέσματα, αν και πρώϊμα, λόγω του μικρού ακόμα μεγέθους δείγματος, κατέδειξαν ότι τα επίπεδα του Ο2- ήταν πιο αυξημένα στους ασθενείς με δυσμενή μετεγχειρητική εξέλιξη.Συμπερασματικά, τα αποτελέσματα των ανεξαρτήτων επιμέρους πειραματικών ενοτήτων της παρούσας διατριβής (1) αποσαφήνισαν τις βαθύτερες πτυχές του μηχανισμού λειτουργίας της μοριακής αναγνώρισης μέσω μεμβρανικής μηχανικής και (2) επιβεβαίωσαν την αποτελεσματικότητα και τις σημαντικές προοπτικές εφαρμογής του βιοαισθητήρα σουπεροξειδίου που αναπτύχθηκε με βάση τη συγκεκριμένη τεχνολογία

Comparative Study of a Cell-Based and Electrochemical Biosensor for the Rapid Detection of 2,4,6-Trichloroanisole in Barrel Water Extracts

(1) Background: Fungal metabolites such as haloanisoles (especially 2,4,6-tribromoanisole/ 2,4,6-TCA) are contaminants of cork and wood barrels, materials that are widely used in the wine industry. Thus, the accurate and timely detection of these substances is very important for this sector of beverage industry. (2) Methods: Potentiometry was used for the Bioelectric Recognition Assay (BERA)-based experimental approach, where changes in the electric properties of the Vero cells modified with anti-TCA antibodies were recorded in response to the presence of 2,4,6-TCA in different concentrations. Furthermore, a second electrochemical biosensor system based on the cyclic voltammetric (CV) measurement of a reaction taking place on a screen printed electrode was developed in parallel to the customized application and configuration of the cell-based system. (3) Results: The BERA cell-based biosensor was able to quantitatively differentiate among the lower 2,4,6-TCA concentrations (control, 0.25 and 1.25 ng/L) from spiked oak barrel water extracts in an entirely distinct and reproducible manner. In contrast, the CV method was not sensitive enough to differentiate between the samples. (4) Conclusions: The present study indicates that the BERA-based biosensor after further development and optimization could be used for the routine, high throughput detection of 2,4,6-TCA in oak barrel water extracts

Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen

One of the key challenges of the recent COVID-19 pandemic is the ability to accurately estimate the number of infected individuals, particularly asymptomatic and/or early-stage patients. We herewith report the proof-of-concept development of a biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus. The biosensor is based on membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody. We demonstrate that the attachment of the protein to the membrane-bound antibodies resulted in a selective and considerable change in the cellular bioelectric properties measured by means of a Bioelectric Recognition Assay. The novel biosensor provided results in an ultra-rapid manner (3 min), with a detection limit of 1 fg/mL and a semi-linear range of response between 10 fg and 1 μg/mL. In addition, no cross-reactivity was observed against the SARS-CoV-2 nucleocapsid protein. Furthermore, the biosensor was configured as a ready-to-use platform, including a portable read-out device operated via smartphone/tablet. In this way, we demonstrate that the novel biosensor can be potentially applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing, therefore offering a possible solution for the timely monitoring and eventual control of the global coronavirus pandemic

Assessment of Organophosphate and Carbamate Pesticide Residues in Cigarette Tobacco with a Novel Cell Biosensor

The conventional analysis of pesticide residues in analytical commodities, such as tobacco and tobacco products is a labor intensive procedure, since it is necessary to cover a wide range of different chemicals, using a single procedure. Standard analysis methods include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by GC and HPLC to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. As a consequence, current methods of analysis provide a limited sample capacity. In the present study, we report on the development of a novel cell biosensor for detecting organophosphate and carbamate pesticide residues in tobacco. The sensor is based on neuroblastoma N2a cells and the measurement of changes of the cell membrane potential, according to the working principle of the Bioelectric Recognition Assay (BERA). The presence of pesticide residues is detected by the degree of inhibition of acetylcholine esterase (AChE). The sensor instantly responded to both the organophoshate pesticide chlorpyriphos and the carbamate carbaryl in a concentration-dependent pattern, being able to detect one part per billion (1 ppb). Additionally, tobacco leaf samples (in blended dry form) were analyzed with both the novel biosensor and conventional methods, according to a double-blind protocol. Pesticide residues in tobacco samples caused a considerable cell membrane hyperpolarization to neuroblastoma cells immobilized in the sensor, as indicated by the increase of the negative sensor potential, which was clearly distinguishable from the sensor’s response against pesticide-free control samples. The observed response was quite reproducible, with an average variation of +5,6%. Fluorescence microscopy observations showed that treatment of the cells with either chlorpyrifos or carbaryl was associated with increased [Ca2+]cyt . The novel biosensor offers fresh perspectives for ultra-rapid, sensitive and low-cost monitoring of pesticide residues in tobacco as well as other food and agricultural commodities

Assessment of organophosphate and carbamate pesticide residues in cigarette tobacco with a novel cell biosensor

Abstract: The conventional analysis of pesticide residues in analytical commodities, such as tobacco and tobacco products is a labor intensive procedure, since it is necessary to cover a wide range of different chemicals, using a single procedure. Standard analysis methods include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by GC and HPLC to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. As a consequence, current methods of analysis provide a limited sample capacity. In the present study, we report on the development of a novel cell biosensor for detecting organophosphate and carbamate pesticide residues in tobacco. The sensor is based on neuroblastoma N2a cells and the measurement of changes of the cell membrane potential, according to the working principle of the Bioelectric Recognition Assay (BERA). The presence of pesticide residues is detected by the degree of inhibition of acetylcholine esterase (AChE). The sensor instantly responded to both the organophoshate pesticide chlorpyriphos and the carbamate carbaryl in a concentration-dependent pattern, being able to detect one part per billion (1 ppb). Additionally, tobacco leaf samples (in blended dry form) were analyzed with both th

An Ultra-Rapid Biosensory Point-of-Care (POC) Assay for Prostate-Specific Antigen (PSA) Detection in Human Serum

Prostate-specific antigen (PSA) is the established routine screening tool for the detection of early-stage prostate cancer. Given the laboratory-centric nature of the process, the development of a portable, ultra rapid high-throughput system for PSA screening is highly desirable. In this study, an advancedpoint-of-care system for PSA detection in human serum was developed based on a cellular biosensor where the cell membrane was modified by electroinserting a specific antibody against PSA. Thirty nine human serum samples were used for validation of this biosensory system for PSA detection. Samples were analyzed in parallel with a standard immunoradiometric assay (IRMA) and an established electrochemical immunoassay was used for comparison purposes. They were classified in three different PSA concentration ranges (0, <4 and ≥4 ng/mL). Cells membrane-engineered with 0.25 μg/mL anti-PSA antibody demonstrated a statistically lower response against the upper (≥4 ng/mL) PSA concentration range. In addition, the cell-based biosensor performed better than the immunosensor in terms of sensitivity and resolution against positive samples containing <4 ng/mL PSA. In spite of its preliminary, proof-of-concept stage of development, the cell-based biosensor could be used as aninitiative for the development of a fast, low-cost, and high-throughput POC screening system for PSA