Determination of carbamate residues in crop samples by cholinesterase-based biosensors and chromatographic techniques

An amperometric biosensor based on cholinesterase (ChE) has been used for the determination of selected carbamate insecticides in vegetable samples. The linear range of the biosensor for the N-methylcarbamates (aldicarb, carbaryl, carbofuran, methomyl and propoxur) varied from 5 x 10(-5) to 50 mg kg(-1). Limits of detection were calculated on the basis that the ChE enzymes were 10% inhibited and varied, depending of the combination ChE (as acetyl- or butyrylcholinesterase) vs. inhibitor (pesticide), from 1 x 10(-4) to 3.5 mg kg(-1). The biosensor-based carbamate determination was compared to liquid chromatography/UV methods. Three vegetable samples were spiked with carbofuran and propoxur at 125 mu g kg(-1) followed by conventional procedures. Good correlations were observed for carbofuran in the vegetable extracts (79, 96 and 91% recoveries for potato, carrot and sweet pepper, respectively), whereas for propoxur unsatisfactory results were obtained. Potato and carrot samples were spiked with 10, 50 and 125 mu g kg(-1) carbofuran, followed by direct determination by the amperometric biosensor. The fortified sampler; resulted in very high inhibition values, and recoveries were: 28, 34 and 99% for potato, and 140, 90 and 101% for carrot, respectively, at these three fortification levels. (C) 1998 Elsevier B.V. B.V.

Polishable and Renewable DNA Hybridization Biosensors

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.

Melanin as an active layer in biosensors

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC (R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Amperometric urea biosensors based on the entrapment of urease in polypyrrole films

Urease (Urs) was immobilized in electrochemically prepared polypyrrole (PPy) and the resulting films were characterized by cyclic voltammetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and ultraviolet visible spectroscopy (UV-VIS). The enzymatic activity of Urs entrapped in the PPy matrix was confirmed by the catalytic conversion of urea into carbon dioxide and ammonia, when urea was detected amperometrically at different concentrations in standard samples and commercial fertilizers. The PPy/Urs biosensors exhibited selectivity, a relatively high efficiency at urea concentrations below 3.0 mmol L-1, and a sensitivity to urea of 2.41 mu A cm(-2) mmol(-1) L (C) 2011 Elsevier Ltd. All rights reserved.

Electrochemically prepared polypyrrole-2-carboxylic acid films: synthesis protocols and studies on biosensors

The process for obtaining polypyrrole-2-carboxylic acid (PPY-2-COOH) films in acetonitrile was investigated using cyclic voltammetry, electrochemical quartz crystal microgravimetry (EQCM), and infrared spectroscopy (FTIR). Different potential ranges were applied during cyclic voltammetry experiments with the aim of obtaining films without and with the presence of controlled amounts of water added in acetonitrile. The FTIR spectra of the films have evidenced that cations and anions from the electrolyte solution were incorporated into the PPY-2-COOH structure, with a preferential adsorption of cations. After chemically immobilizing polyphenoloxidase (tyrosinase, PPO), PPY-2-COOH/PPO films were build for amperometric detection of catechol, establishing a linear limit of concentrations ranging from 5.0 x 10-4 to 2.5 x 10-2 mol L-1.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

Real time monitoring of DNA hybridization and replication using optical and acoustic biosensors

Während in den letzten Jahren zahlreiche Biosensoren zum spezifischen Nachweis von DNA entwickelt wurden, ist die Anwendung oberflächen-sensitiver Methoden auf enzymatische Reaktionen ein vergleichsweise neues Forschungsgebiet. Trotz der hohen Empfindlichkeit und der Möglichkeit zur Echtzeit-Beobachtung molekularer Prozesse, ist die Anwendung dieser Methoden nicht etabliert, da die Enzymaktivität durch die Nähe zur Oberfläche beeinträchtigt sein kann. Im Rahmen dieser Arbeit wurde die enzymatische Verlängerung immobilisierter DNA durch eine DNA Polymerase mit Hilfe von Oberflächenplasmonen-Fluoreszenzspektroskopie (SPFS) und einer Quarzkristall-Mikrowaage (QCM) untersucht. Die Synthese von DNA wurde im Fall der QCM als Massenzuwachs detektiert, der sich im Abfall der Resonanzfrequenz des Schwingquarzes und einem Anstieg seiner Dissipationsenergie ausdrückte. Die viskoelastischen Eigenschaften der DNA-Schichten wurden bestimmt, indem die erhaltenen Daten mit einem auf Voigt basierenden Modell ausgewertet wurden. SPFS nutzt das evaneszente elektromagnetische Feld, das mit Oberflächenplasmonen einhergeht, zur oberflächen-sensitiven Anregung von Chromophoren. Auf diese Weise wurde der Einbau von Farbstoff-markierten Nukleotiden in die entstehende DNA-Sequenz als Indikator für das Voranschreiten der Reaktion ausgenutzt. Beide Meßtechniken konnten erfolgreich zum Nachweis der DNA-Synthese herangezogen werden, wobei die katalytische Aktivität des Enzyms vergleichbar zu der in Lösung gemessenen war.

Development of bionanotechnological strategies for signal enhancement in nucleic acids biosensors

Nucleic acid biosensors represent a powerful tool for clinical and environmental pathogens detection. For applications such as point-of-care biosensing, it is fundamental to develop sensors that should be automatic, inexpensive, portable and require a professional skill of the user that should be as low as possible. With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an amplification step must be implemented. Often this type of determinations should be performed with simple, automatic and inexpensive hardware: the use of a chemical (or nanotechnological) isothermal solution would be desirable. My Ph.D. project focused on the study and on the testing of four isothermal reactions which can be used to amplify the nucleic acid analyte before the binding event on the surface sensor or to amplify the signal after that the hybridization event with the probe. Recombinase polymerase amplification (RPA) and ligation-mediated rolling circle amplification (L-RCA) were investigated as methods for DNA and RNA amplification. Hybridization chain reaction (HCR) and Terminal deoxynucleotidil transferase-mediated amplification were investigated as strategies to achieve the enhancement of the signal after the surface hybridization event between target and probe. In conclusion, it can be said that only a small subset of the biochemical strategies that are proved to work in solution towards the amplification of nucleic acids does truly work in the context of amplifying the signal of a detection system for pathogens. Amongst those tested during my Ph.D. activity, recombinase polymerase amplification seems the best candidate for a useful implementation in diagnostic or environmental applications.

Detektion neuroaktiver Substanzen durch Modifikation des Aktivitätsmusters neokortikaler Netzwerke auf Multielektrodenarrays – Charakterisierung und Optimierung eines neuronalen Biosensors

In dieser Arbeit wurden kortikale neuronale Netzwerke auf Multielektrodenarrays auf ihre Tauglichkeit als zellbasiertes Biosensorsystem untersucht. Der Schwerpunkt der pharmakologischen Untersuchungen an den ausgereiften kortikalen Netzwerken lag auf dem Einsatz von Substanzen, welche auf den GABAA-Rezeptor einwirken. Die Modifikation des spontan generierten Aktivitätsmusters ließ dabei Rückschlüsse auf die Wirksamkeit und den Wirkungsmechanismus der Testsubstanzen zu. Ferner war in den meisten Fällen eine Diskriminierung der auf den gleichen Rezeptor einwirkenden Substanzen möglich. Die Analyse der Spikerate und verschiedener auf Bursts beruhender Messparameter machte deutlich, dass die Burstrate bei den extrazellulären Ableitungen auf Netzwerkebene den sensitivsten und verlässlichsten Parameter zum Nachweis der Substanzeffekte darstellte. Durch die Verwendung kortikaler Netzwerke unter optimierten Kulturbedingungen und einer auf das System abgestimmten Analysesoftware konnte die Reproduzierbarkeit und Sensitivität im Vergleich zu anderen Studien deutlich verbessert werden. Um die extrazelluläre Signalableitung von einer möglichst geringen Zellanzahl und damit einem überschaubaren zellulären Netzwerk auf Multielektrodenarrays zu ermöglichen, wurden die Oberflächeneigenschaften der Substrate so modifiziert, dass die Lokalisation der Zellsomata und das Auswachsen der Neurite einer geometrischen Kontrolle unterlag. Die kontrollierte Substratbeschichtung des Adhäsionspromotors Poly-D-Lysin in einem triangulären Muster konnte dabei durch die Methode des Mikrokontaktstempelns realisiert werden. Durch das kontrollierte Zellwachstum konnte die extrazelluläre Ableitung von Netzwerken einer geringen Zelldichte über einen Zeitraum von mehreren Wochen ermöglicht werden. Die Untersuchung struktureller und morphogenetischer Eigenschaften, sowie elektrophysiologische Untersuchungen der strukturierten Netzwerke bewiesen, dass die kontrollierte Substratbeschichtung sich nicht negativ auf das Wachstum, die Synaptogenese und die Funktionalität auswirkte.

Aktivitätsabhängige Kontrolle der Apoptose in neonatalen kortikalen Neuronen und Etablierung eines Biosensors zur Echtzeitanalyse der Caspase-3-Aktivierung

Die Apoptose spielt eine entscheidende Rolle während der normalen Entwicklung des zentralen Nervensystems. Elektrische Aktivität und die Versorgung mit trophischen Faktoren sind ausschlaggebend für das Überleben von Neuronen. Um zu untersuchen, welche zellulären Prozesse die aktivitätsabhängige Apoptose in organotypischen Schnittkulturen des neugeborenen Neokortex beeinflussen, wurde in der vorliegenden Arbeit immunzytochemisch das Auftreten aktivierter Caspase-3, nach pharmakologischer Beeinflussung von Ionenkanälen und membranständigen Rezeptoren analysiert. Die Unterdrückung neuronaler Aktivität durch den Natriumionenkanalblocker TTX führte zu einem signifikanten Verlust kortikaler Neuronen. Ein ähnlicher Anstieg der Zahl apoptotischer Neurone konnte durch Applikation von Antagonisten ionotroper Glutamatrezeptoren, GABAA-Rezeptoren oder neuronaler Gap Junctions induziert werden. Jedoch konnte bei einigen Antagonisten die apoptosefördernde Wirkung erst nach längerer Einwirkung beobachtet werden. Im Weiteren wurde eine Methode etabliert, mit deren Hilfe eine Echtzeitanalyse der Apoptose kortikaler Neurone unter dem Entzug trophischer Faktoren in Gegenwart unterschiedlicher extrazellulärer Kaliumkonzentrationen ermöglicht wurde. Dazu wurden dissoziierte kortikale Kulturen mit dem pCaspase3-sensor Vektor transfiziert. Das durch dieses Plasmid codierte fluoreszente Protein wird Caspase-3 abhängig gespalten. In der vorliegenden Arbeit konnte gezeigt werden, dass der Caspase3-sensor spezifisch für die Aktivierung der Caspase-3 ist, und dass die Überlebensfähigkeit der transfizierten Neurone durch das Transfektionsprotokoll nicht beeinflusst wird.

Dynamics of epithelial monolayers assessed by acoustic and impedimetric whole cell biosensors

Mit der Zielsetzung der vorliegenden Arbeit wurde die detailierten Analyse von Migrationsdynamiken epithelilaler Monolayer anhand zweier neuartiger in vitro Biosensoren verfolgt, der elektrischen Zell-Substrat Impedanz Spektroskopie (electrical cell-substrate impedance sensing, ECIS) sowie der Quarz Kristall Mikrowaage (quartz crystal microbalance, QCM). Beide Methoden erwiesen sich als sensitiv gegenüber der Zellmotilität und der Nanozytotoxizität.rnInnerhalb des ersten Projektes wurde ein Fingerprinting von Krebszellen anhand ihrer Motilitätsdynamiken und der daraus generierten elektrischen oder akkustischen Fluktuationen auf ECIS oder QCM Basis vorgenommen; diese Echtzeitsensoren wurdene mit Hilfe klassicher in vitro Boyden-Kammer Migrations- und Invasions-assays validiert. Fluktuationssignaturen, also Langzeitkorrelationen oder fraktale Selbstähnlichkeit aufgrund der kollektiven Zellbewegung, wurden über Varianz-, Fourier- sowie trendbereinigende Fluktuationsanalyse quantifiziert. Stochastische Langzeitgedächtnisphänomene erwiesen sich als maßgebliche Beiträge zur Antwort adhärenter Zellen auf den QCM und ECIS-Sensoren. Des weiteren wurde der Einfluss niedermolekularer Toxine auf die Zytoslelettdynamiken verfolgt: die Auswirkungen von Cytochalasin D, Phalloidin und Blebbistatin sowie Taxol, Nocodazol und Colchicin wurden dabei über die QCM und ECIS Fluktuationsanalyse erfasst.rnIn einem zweiten Projektschwerpunkt wurden Adhäsionsprozesse sowie Zell-Zell und Zell-Substrat Degradationsprozesse bei Nanopartikelgabe charackterisiert, um ein Maß für Nanozytotoxizität in Abhangigkeit der Form, Funktionalisierung Stabilität oder Ladung der Partikel zu erhalten.rnAls Schlussfolgerung ist zu nennen, dass die neuartigen Echtzeit-Biosensoren QCM und ECIS eine hohe Zellspezifität besitzen, auf Zytoskelettdynamiken reagieren sowie als sensitive Detektoren für die Zellvitalität fungieren können.

Advanced schemes for surface plasmon resonance and plasmon-enhanced fluorescence biosensors

Advanced optical biosensor platforms exploiting long range surface plasmons (LRSPs) and responsive N-isopropylacrylamide (NIPAAm) hydrogel binding matrix for the detection of protein and bacterial pathogen analytes were carried out. LRSPs are optical waves that originate from coupling of surface plasmons on the opposite sites of a thin metallic film embedded between two dielectrics with similar refractive indices. LRSPs exhibit orders of magnitude lower damping and more extended profile of field compared to regular surface plasmons (SPs). Their excitation is accompanied with narrow resonance and provides stronger enhancement of electromagnetic field intensity that can advance the sensitivity of surface plasmon resonance (SPR) and surface plasmon-enhanced fluorescence spectroscopy (SPFS) biosensors. Firstly, we investigated thin gold layers deposited on fluoropolymer surface for the excitation of LRSPs. The study indicates that the morphological, optical and electrical properties of gold film can be changed by the surface energy of fluoropolymer and affect the performance of a SPFS biosensor. A photo-crosslinkable NIPAAm hydrogel was grafted to the sensor surface in order to serve as a binding matrix. It was modified with bio-recognition elements (BREs) via amine coupling chemistry and offered the advantage of large binding capacity, stimuli responsive properties and good biocompatibility. Through experimental observations supported by numerical simulations describing diffusion mass transfer and affinity binding of target molecules in the hydrogel, the hydrogel binding matrix thickness, concentration of BREs and the profile of the probing evanescent field was optimized. Hydrogel with a up to micrometer thickness was shown to support additional hydrogel optical waveguide (HOW) mode which was employed for probing affinity binding events in the gel by means of refractometric and fluorescence measurements. These schemes allow to reach limits of detection (LODs) at picomolar and femtomolar levels, respectively. Besides hydrogel based experiments for detection of molecular analytes, long range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) was employed for detection of bacterial pathogens. The influence of capture efficiency of bacteria on surfaces and the profile of the probing field on sensor response were investigated. The potential of LRSP-FS with extended evanescent field is demonstrated for detection of pathogenic E. coli O157:H7 on sandwich immunoassays . LOD as low as 6 cfu mL-1 with a detection time of 40 minutes was achieved.rn