Timely coronary artery reperfusion is essential to prevent myocyte death following myocardial infarction. The act of restoring blood flow however, paradoxically reduces the beneficial effects of reperfusion. This phenomenon, termed myocardial reperfusion injury, refers to the injury of cardiac myocytes that were viable immediately before reperfusion. Recent studies have shown that the timing and hemodynamic sequence of events which govern reperfusion can help to minimize the severity of reperfusion injury. The term postconditioning describes a modified form of reperfusion that involves a series of flow interruptions which confer significant cardioprotection to the heart. This thesis investigates ischemic postconditioning and endothelial nitric oxide synthase (eNOS) phosphorylation as cardioprotective therapies against reperfusion injury. In the first half of this thesis, we test the hypothesis that phosphorylation of eNOS serves as a cardioprotection nodal point for ischemic postconditioning. We show that phosphorylation of eNOS increases enzyme activity and that its product, nitric oxide, plays a critical role in cardioprotection. A number of cardiac dysfunctions arise after reperfusion and we address the effects of postconditioning on infarct size and myocardial blood flow. The second half of this thesis introduces the use of magnetic relaxometry sensors to detect cardiac biomarkers. The ability to non-invasively measure infarct size in small animals would be helpful in studying models of myocardial ischemia-reperfusion injury. We investigate the use of implantable biosensors in vivo and show that the cumulative detection of cardiac biomarkers correlates with infarct severity. / Engineering and Applied Sciences

Σκοπός της παρούσης διπλωματικής εργασίας είναι η εξαγωγή πληροφορίας για τη βιοχημική αντίδραση που συμβαίνει στην επιφάνεια ενός βιοαισθητήρα μέσω της ανάλυσης του φάσματος της ηλεκτρικής ποσότητας εξόδου. Αυτό γίνεται με τη διάσπαση του τελευταίου στα βασικά του συστατικά, ήτοι τη ΦΠΙ (Φασματική Πυκνότητα Ισχύος) της στοχαστικής διαδικασίας που λαμβάνει χώρα στην επιφάνεια –και την οποία θα αντιμετωπίσουμε ως διαταραχή- και τον ενδογενή θόρυβο της συσκευής. Αρχικά γίνεται μια παρουσίαση της βασικής αρχής λειτουργίας των βιοαισθητήρων και διαμορφώνεται ένα μοντέλο που συνδέει την αντίδραση με το ρεύμα εξόδου του αισθητήρα. Στη συνέχεια παρουσιάζονται δύο βασικά είδη διαταραχών (RTS και 1/f θόρυβος), η σύνδεσή τους με την περίπτωσή μας καθώς και προκύπτοντα πρακτικά ζητήματα. Έπειτα παρουσιάζεται ένα μακροσκοπικό μοντέλο της αντίδρασης από τη βιοχημεία και διαμορφώνεται ένα μικροσκοπικό μοντέλο από τη σκοπιά της στοχαστικής διαδικασίας μέσω του οποίου εξάγεται και η ΦΠΙ των βιοχημικών διαταραχών. Η τελευταία συγκρίνεται με την παρατηρούμενη στη βιβλιογραφία και με τη χρήση εργαλείων που ερμηνεύουν διαταραχές γίνεται μια προσπάθεια ερμηνείας . Τέλος, υλοποιούμε, προσομοιώνουμε και αξιολογούμε έναν εκτιμητή που ανιχνεύει είδη και ποσότητες αναλυτών αναλύοντας την απόκριση του αισθητήρα και εκμεταλλευόμενος το μοντέλο που έχει παρουσιαστεί. / The goal of this diploma thesis is to infer data about the biochemical reaction taking place on the surface of a biosensor through analyzing the spectrum of the electrical output quantity. This is achieved through decomposing the aforementioned spectrum to its constituents, namely the PSD (Power Spectral Density) of the stochastic process that takes place on the sensor surface (treated as a fluctuation) and the inherent device noise. We initially describe the basic principles by which biosensors operate and we formulate a model that relates reaction quantities to the sensor output current. Two basic kinds of fluctuations (namely RTS and 1/f noise) are then presented and related to our case. Some practical aspects are addressed as well. A macroscopic model of the reaction which is commonly used in biochemistry is then described. Moreover, we describe a microscopic model of the reaction treating it as a stochastic process and we subsequently obtain the PSD of the biochemical fluctuations. The aforementioned PSD is then compared to PSDs extracted from experimental data and an attempt is made to interpret the latter using fluctuation analysis concepts. Finally, we develop, simulate and evaluate an estimator which detects analyte species as well as analyte concentrations through processing of the biosensor response and making use of our model.

This dissertation develops biosensors for rapid detection of pathogens for environmental monitoring and food safety applications and utilizes the multidisciplinary and multi-application characteristics of biosensors to develop a lesson plan that can be implemented in secondary education classrooms. The detection methods evolve from particle immunoagglutination assay, PDMS optofluidic lab-on-a-chip, and spectrum analysis to smartphone and image analysis without any reagent; the potential application in secondary education also underlines the extended value of biosensors. In the first paper presented here, an optofluidic lab-on-a-chip system and subsequent sampling procedure were developed for detecting bacteria from soil samples utilizing Mie scattering detection of particle immunoagglutination assay. This system and protocol detected the presence of Escherichia coli K12 from soil particles in near real-time (10 min) with a detection limit down to 1 CFU mL⁻¹ and has the potential to be implemented in the field. We also compared the interaction between E. coli and soil particles to the two-step protein-surface interaction. In the second paper, a smartphone-utilized biosensor consisting of a near-infrared (NIR) LED (wavelength of 880 nm) and a digital camera of a smartphone was developed for detecting microbial spoilage on ground beef, without using any reagents. The method was further improved by programming a smartphone application that allows the user to position the smartphone at an optimum distance and a range of angles utilizing its internal gyro sensor to measure a series of scatter intensities against the detection angle. This handheld device can be used as a preliminary screening tool to monitor microbial contamination on meat products. In the third paper, we designed a lesson plan for secondary education classrooms using biosensors as a core and branching out to different applications and fields of study with the goal of heightening students' interest and motivation toward attaining degrees and careers in STEM fields. Results revealed that the lesson was more effective in affecting younger students than older students, and more effective in teaching about the applications of biosensors than about the techniques of biosensor development.

In the United States and other developed countries, despite great efforts in time and funding for the prevention of foodborne and airborne diseases, there is still an unacceptable level of common pathogens spread via food, water, and air. To this end, lab-on-a-chip (LOC) technologies were developed for field-deployable assays and point of care diagnostics. These devices have potential applications in hospitals, agricultural farms, processing plants, and even on fields of battle. Two successful types of assays in the recent years towards point of care diagnostics are immunoassays and nucleic acid detection assays. In the Appendix A, we demonstrated a complete, field-deployable particle immunoassay encased within a microfluidic chip that detects small quantities of Salmonella Typhimurium in poultry fluid samples. Because the necessary reagents are pre-loaded and the test and negative control channels are fed by a single sample inlet, single pipetting of sample is possible. This assay demonstrated a 10 CFU/mL limit of detection, which is considerably lower than PCR and enzyme-linked immunosorbent assay (ELISA). Total assay time, including sample reading in an integrated handheld device, was 10 minutes, which was much lower than conventional methods. Because of the simplified protocol and assay time, this biosensor has potential in clinical and field diagnostic applications. In Appendix B, we fit the particle immunoassay to test for Influenza A H1N1/2009 virus and included aerosol sampling from a scaled-down mock classroom. To make the assay field deployable, we used an iPhone for signal detection. The detection limit of the assay was 1 pg/mL (10 pg/mL using the iPhone), which is well below the limit of detection for RT-PCR. This protocol demonstrated that immunoassays can be effective in the presence of interfering dust particles and that viruses can be collected from aerosol with minimal sample preparation. In Appendix C, we demonstrated that paper microfluidics, a newer vision of microfluidics, is a cheap and easy method to extract nucleic acid from S. Typhimurium in a variety of samples, including poultry packaging liquid, whole blood, and feces. Fluorescent detection with an iPhone allows for field and clinical testing. This protocol interfaces with rapid PCR and is a true diagnostic tool.

This dissertation develops biosensors for rapid detection of pathogens for environmental monitoring and food safety applications and utilizes the multidisciplinary and multi-application characteristics of biosensors to develop a lesson plan that can be implemented in secondary education classrooms. The detection methods evolve from particle immunoagglutination assay, PDMS optofluidic lab-on-a-chip, and spectrum analysis to smartphone and image analysis without any reagent; the potential application in secondary education also underlines the extended value of biosensors. In the first paper presented here, an optofluidic lab-on-a-chip system and subsequent sampling procedure were developed for detecting bacteria from soil samples utilizing Mie scattering detection of particle immunoagglutination assay. This system and protocol detected the presence of Escherichia coli K12 from soil particles in near real-time (10 min) with a detection limit down to 1 CFU mL⁻¹ and has the potential to be implemented in the field. We also compared the interaction between E. coli and soil particles to the two-step protein-surface interaction. In the second paper, a smartphone-utilized biosensor consisting of a near-infrared (NIR) LED (wavelength of 880 nm) and a digital camera of a smartphone was developed for detecting microbial spoilage on ground beef, without using any reagents. The method was further improved by programming a smartphone application that allows the user to position the smartphone at an optimum distance and a range of angles utilizing its internal gyro sensor to measure a series of scatter intensities against the detection angle. This handheld device can be used as a preliminary screening tool to monitor microbial contamination on meat products. In the third paper, we designed a lesson plan for secondary education classrooms using biosensors as a core and branching out to different applications and fields of study with the goal of heightening students' interest and motivation toward attaining degrees and careers in STEM fields. Results revealed that the lesson was more effective in affecting younger students than older students, and more effective in teaching about the applications of biosensors than about the techniques of biosensor development.

The contamination of drinking water by microbial pathogens is recognized as one of the most pressing water supply problems of our day. To minimize the impact of pathogens and parasites on the environment and public health, accurate methods are needed to evaluate their presence and concentration. Although various techniques exist to detect certain pathogens in water (e.g., immunofluorescence or PCR techniques), these are time- and labor-intensive. A direct, real-time method for detection and quantification of target organisms would thus be very useful for rapid diagnosis of water safety. A quartz crystal microbalance with dissipation monitoring (QCM-D) based biosensor for detection of waterborne pathogens (i.e., Escherichia coli O157:H7) was developed. The detection platform is based on the immobilization of affinity purified antibodies onto gold coated QCM-D quartz crystals via a cysteamine self-assembled monolayer. The results show that the optimal sensor response is the initial slope of the dissipation shift. A highly log-log linear response is obtained for detection of E. coli O157:H7 over a broad range of cell concentration from 3 x 105 to 1 x 109 cells/mL. The prepared biosensor also exhibits a log-log linear working range from 107 to 109 cells/mL for E. coli K12 D21, a non-pathogenic model organism. The biosensor also shows satisfactory selectivity using Bacillus subtilis . To our knowledge, this is the first study demonstrating the use of the slope of the dissipation shift as a sensor response when using QCM-D technology. / Keywords: Biosensor, QCM-D, E. coli O157:H7, polyc1onal antibodies, dissipation slope, cysteamine, self-assembled monolayer

A bioactive paper is a paper that can detect, capture and deactivate water and airborne pathogens. In this project, we presented a model "bioactive paper" made by attaching T4 bacteriophages to a cellulose substrate. T4 bacteriophages can be genetically engineered to possess copies of cellulose-binding modules (CBM) on their capsids. This allows them to bind specifically onto cellulose surfaces. Our model surface is a thin film of regenerated cellulose made by spin coating a glass or quartz substrate with a cellulose triacetate and subsequently hydrolyzing the surface back to cellulose. We successfully demonstrated the attachment of the CBM-T4 bacteriophages onto cellulose substrates by the phage viability test. The deposition kinetics were measured using an impinging jet apparatus combined with an evanescent wave light scattering (EWLS) system. We first tested the apparatus by using amidine latex particles deposited on the cellulose at different flow rates and found them to be in a good agreement with the constant potential double-layer model. The adhesion experiments were also performed in an impinging jet apparatus in which the CBM-T4 bacteriophages and the unassembled protein complexes from a suspension of 4.08 x 10 8 PFU/mL were allowed to diffuse to the cellulose surface, The competitive diffusion kinetics were again studied by the EWLS technique. For CBM-T4, the blocking time was found to be around 58 minutes and the maximum surface number density of phages was 5.9 x 1010 per m 2. / Key phrases: bioactive paper, cellulose film, cellulose binding module, bacteriophage T4, evanescent wave light scattering, unassembled protein complex, diffusion kinetics

Biosensors are analytical devices made up of a combination of a biological entity, usually an enzyme, that recognizes a specific analyte (substrate) and the transducer that translates the biorecognition event into a signal. In order to create new types of biosensors the corresponding experimental studies are necessary. Computational experiments could very well replace very expensive physical ones. However, the multi-step character of a chemical reaction scheme must be considered and modeled accordingly. In this thesis such reaction schemes were studied in great details. Original mathematical models were developed for optical peroxidase-based and amperometric laccase-based biosensors. The deterministic nature of model construction allows the automated models to be built. Based on this assumption flexible model for computational modeling of different practical multistep biosensors was developed. In order to optimize the numerical solution of the reaction-diffusion type equations common finite difference schemes were compared. The comparison shows that the fastest schemes to achieve the required relative error are implicit and Hopscotch schemes. For the problems where accuracy is not a significant factor but the speed is, the simplest explicit scheme should be used. Applying the new flexible model a computational modeling of the multi-step biosensors were produced. The modeling of laccase biosensor explained and confirmed the synergistic effect. The computational modeling of the... [to full text] / Biojutikliai yra analitiniai įtaisai sudaryti iš biologiškai aktyvios bei selektyviai atpažįstančios substratą medžiagos, dažniausiai fermento, ir keitiklio formuojančio makroskopinį fizinį signalą. Naujų įtaisų kūrimui būtini lygiagretūs eksperimentiniai tyrimai. Skaitiniai eksperimentai gali patikimai pakeisti fizinius. Modeliuojant tokius biojutiklius, būtina atsižvelgti į juose vykstančių procesų daugiapakopį pobūdį. Šiame darbe nuodugniai ištirtos tokių reakcijų schemų savybės. Sudaryti originalūs matematiniai modeliai optiniam peroksidaziniam bei amperometriniam lakaziniam daugiapakopiams biojutikliams. Deterministinė modelių sudarymo proceso prigimtis leidžia jį automatizuoti. Remiantis šiuo principu sukurtas bendras įrankis kompiuteriniam daugiapakopių biojutiklių modeliavimui. Siekiant optimizuoti skaitinį sprendimą palygintos dažniausiai naudojamos baigtinių skirtumų skaitinio sprendimo schemos sprendžiant reakcijos - difuzijos lygtis. Pastarasis palyginimas parodė, kad greičiausiai reikiamas sprendinio tikslumas pasiekiamas taikant neišreikštinę bei Hopscotch schemas. Uždaviniams, kuriems sparta svarbesnė už tikslumą, turėtų būti taikoma išreikštinė schema. Taikant naują įrankį atliktas kompiuterinis daugiapakopių biojutiklių modeliavimas. Kompiuterinis lakazinio biojutiklio modeliavimas teoriškai paaiškino eksperimentiškai stebėtą sinergetinę mediatoriaus įtaką biojutiklio atsakui. Peroksidazinio biojutiklio kompiuterinio modeliavimo rezultatai parodė, kad plataus... [toliau žr. visą tekstą]

Biojutikliai yra analitiniai įtaisai sudaryti iš biologiškai aktyvios bei selektyviai atpažįstančios substratą medžiagos, dažniausiai fermento, ir keitiklio formuojančio makroskopinį fizinį signalą. Naujų įtaisų kūrimui būtini lygiagretūs eksperimentiniai tyrimai. Skaitiniai eksperimentai gali patikimai pakeisti fizinius. Modeliuojant tokius biojutiklius, būtina atsižvelgti į juose vykstančių procesų daugiapakopį pobūdį. Šiame darbe nuodugniai ištirtos tokių reakcijų schemų savybės. Sudaryti originalūs matematiniai modeliai optiniam peroksidaziniam bei amperometriniam lakaziniam daugiapakopiams biojutikliams. Deterministinė modelių sudarymo proceso prigimtis leidžia jį automatizuoti. Remiantis šiuo principu sukurtas bendras įrankis kompiuteriniam daugiapakopių biojutiklių modeliavimui. Siekiant optimizuoti skaitinį sprendimą palygintos dažniausiai naudojamos baigtinių skirtumų skaitinio sprendimo schemos sprendžiant reakcijos - difuzijos lygtis. Pastarasis palyginimas parodė, kad greičiausiai reikiamas sprendinio tikslumas pasiekiamas taikant neišreikštinę bei Hopscotch schemas. Uždaviniams, kuriems sparta svarbesnė už tikslumą, turėtų būti taikoma išreikštinė schema. Taikant naują įrankį atliktas kompiuterinis daugiapakopių biojutiklių modeliavimas. Kompiuterinis lakazinio biojutiklio modeliavimas teoriškai paaiškino eksperimentiškai stebėtą sinergetinę mediatoriaus įtaką biojutiklio atsakui. Peroksidazinio biojutiklio kompiuterinio modeliavimo rezultatai parodė, kad plataus... [toliau žr. visą tekstą] / Biosensors are analytical devices made up of a combination of a biological entity, usually an enzyme, that recognizes a specific analyte (substrate) and the transducer that translates the biorecognition event into a signal. In order to create new types of biosensors the corresponding experimental studies are necessary. Computational experiments could very well replace very expensive physical ones. However, the multi-step character of a chemical reaction scheme must be considered and modeled accordingly. In this thesis such reaction schemes were studied in great details. Original mathematical models were developed for optical peroxidase-based and amperometric laccase-based biosensors. The deterministic nature of model construction allows the automated models to be built. Based on this assumption flexible model for computational modeling of different practical multistep biosensors was developed. In order to optimize the numerical solution of the reaction-diffusion type equations common finite difference schemes were compared. The comparison shows that the fastest schemes to achieve the required relative error are implicit and Hopscotch schemes. For the problems where accuracy is not a significant factor but the speed is, the simplest explicit scheme should be used. Applying the new flexible model a computational modeling of the multi-step biosensors were produced. The modeling of laccase biosensor explained and confirmed the synergistic effect. The computational modeling of the... [to full text]

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