Elektrokemijski biosenzori za procjenu onečišćivača radi poboljšanja kakvoće hrane

This paper describes the application of electrochemical disposable biosensors in food analysis, which have recently been developed in our laboratory. The disposable biosensors, based on acetylcholinesterase inhibition potential, were exploited for testing the presence of organophosphorus and carbamatepesticides in water, fruit, and vegetable samples. The paper further describes preliminary tests for the detection of genetically modified organisms and hybridisation by coupling the DNA biosensors with the polymerase chain reaction.U radu su opisane neke primjene jednokratnih elektrokemijskih biosenzora za analizu hrane koji su nedavno razvijeni u našem laboratoriju. Biosenzori za jednokratnu upotrebu, bazirani na inhibiciji aktivnosti kolinesteraze primijenjeni su za ispitivanje prisutnosti organofosfornih i karbamatnih pesticida u uzorcima voda, voća i povrća. Razvijena je skupina DNK-biosenzora, a u ovom radu prikazani su rezultati preliminarnih ispitivanja detekcije genetički modificiranih organizama i hibridizacije vezanjem PCR i DNK-biosenzora

Surface plasmon resonance imaging for affinity-based biosensors

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity

DNA-Metallodrugs Interactions Signaled by Electrochemical Biosensors: An Overview

The interaction of drugs with DNA is an important aspect in pharmacology. In recent years, many important technological advances have been made to develop new techniques to monitor biorecognition and biointeraction on solid devices. The interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases. The propensity of a given compound to interact with DNA is measured as a function of the decrease of guanine oxidation signal on a DNA electrochemical biosensor. Covalent binding at N7 of guanine, electrostatic interactions, and intercalation are the events that this kind of biosensor can detect. In this context, the interaction between a panel of antitumoral Pt-, Ru-, and Ti-based metallodrugs with DNA immobilized on screen-printed electrodes has been studied. The DNA biosensors are used for semiquantitative evaluation of the analogous interaction occurring in the biological environment

Aptamers for biosensors

Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance as transducers. The work was focused on two case studies, i.e. the Thrombin-binding aptamer and the aptamer against C-Reactive Protein. From an analytical point of view, the work was devoted to the optimisation of the analytical performance of a piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection, respectively. Efforts towards the application of these aptasensors in complex matrices, such as human plasma and serum, were also undertaken, in order to demonstrate the wide applicability of aptamers, as an alternative to antibodies. In this work, the possibility of introducing a computationally-assisted method to study aptamer-protein interaction and aptamer selection was also evaluated. For this purpose, the Thrombin-binding aptamer was chosen as a model and a retrospective docking study was performed by comparing the affinity of mutated sequences for thrombin with that of the Thrombin-binding aptamer, on the basis of a computationally-derived binding score. Finally, the reliability of computational results was tested by experimental measurements. For this purpose, the Thrombin-binding aptamer and other mutated sequences, selected on the basis of their binding score, were employed for the development of optical biosensors and the resulting analytical performances were compared. Even if further studies should be carried out in order to validate the proposed computational approach to aptamer selection, this work can have a significant impact on future aptamers selection for sensors and diagnostics.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A novel optical chip for affinity biosensors based on fluorescence anisotropy

The subject of this thesis relates to the realisation of a novel biochip for sensors based on optical principles. In particular, affinity biosensors for antigens (IgG/anti-IgG), for a transcription factor, and for mRNA were studied and developed. The interrogation of the biosensors implied the development of a novel and innovative optical platform based on fluorescence which was designed, implemented and thoroughly characterised. The biosensors developed were integrated within an optical biochip connected to an innovative platform. The optical biochip was fabricated from polymethylmethacrylate (PMMA) formed by two pieces of PMMA complementary shaped in order to obtain four micro-channels. The lower part included the micro-channels and the inlet and outlet for the fluidic, while the sensing biolayer was immobilised on the upper part. The optical signal comprised the fluorescence emitted by the layer, which was anisotropically coupled to the cover and suitably guided by the chip. Several chemical treatments of the surface were investigated to obtain the most effective distribution of carboxylic groups for the covalent immobilisation of antibodies. Deposition of the polymers, in particular by Langmuir-Blodgett method, can enhance the performances of the chip going through lower detection limits. The potential of the optical chip as a biosensor was investigated in depth by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. Following this, bioassays for the determination of the NF-kB transcription factor and for the mRNA that codifies for MGMT protein were implemented on the optical platform. The development and characterisation of the biosensors, the identification of the protocol for the bioassays and the design and characterisation of the optical platform were performed at the Institute of Physics Applied Carrara of National Research Council (IFAC-CNR), Firenze, Italy. The research devoted to the development of biosensing surfaces for the realisation of affinity biosensors able to detect and quantify antigens, transcription factors and RNA sequences were carried out at the laboratories of the Institute of Clinical Physiology of National Research Council (IFC-CNR), in Pisa, Italy. Langmuir-Blodgett deposition of polymers, Atomic Force Microscopy imaging and further fluorescence measurements of proteins covalently bound on the surfaces were performed at Cranfield University in Silsoe, England. The overall project starts from a first characterisation of the optical system, and comprehend the enhanced optimisation of the biochip performances until the realisation of the biosensors previously mentioned.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

BIOSENSORS FOR CONTINUOUS EX-VIVO DETERMINATION IN CONJUNCTION WITH AN ARTIFICIAL PANCREAS

Glucose, lactate, and pyruvate can be continuously measured in whole blood by using three extracorporeal electrochemical biosensors. We fixed two newly developed sensors, for lactate and pyruvate, in series with an endocrine artificial pancreas ("Betalike") without affecting its performance. The new sensors for whole blood should be able to clarify the fate of the intermediary metabolites of glucose in diabetic patients when drugs or insulin is infused