Protozoa and bacteria can easily cause disease in humans, specifically E. coli, Plasmodium falciparum, and Cryptosporidium parvum. These three pathogens are associated with large public health concerns that span the globe. The variety of locations in which these can be found is extremely high. Cryptosporidium spp. are extremely resilient when in oocyst form, P. falciparum is in the Anopheles spp. mosquito, while E. coli can be found on anything from food and water, to the skin and gut. The diverse range of locations these can be found in means that a portable sensor for their detection is necessary. In detecting Cryptosporidium, microscopy is the preferred method of identification currently. This requires a trained lab technician as well as calibrated and expensive optical equipment. Technician error can lead to false negative or positive diagnoses as well as sample destruction. A method to remove this technician interaction is thus necessary. This method must allow for objective results that are not open to interpretation. Particle immunoagglutination assays with Mie scatter allow for such an approach using inexpensive components. Particle immunoagglutination relies on the principles of antibody-antigen interaction and antibody conjugated latex particles. Using highly carboxylated latex particles, it is possible to attach IgG antibodies that are specific to a target antigen. Mie scatter is governed by particle size rather than wavelength as other forms of scatter. These two combined allow for an increase in light scatter based on particle size. This is correlated in a linear manner as long as the number of antibody-conjugated particles is higher than the number of antigens. Microfluidics is an ever growing field in the field of lab-on-a-chip that works very well with particle immunoagglutination. In this paper, a method to rapidly identify the presence of Cryptosporidium using microfluidics and particle immunoagglutination is discussed and analyzed. This method allows for a low detection limit of 1-10 oocysts/sample and an assay time of approximately 10 minutes. Results are displayed on a computer screen as the value of light scatter intensity and, when compared to a standard curve, is an objective way to identify the concentration and presence of oocysts in a diverse range of samples. These samples include PBS, pool water, and sump water. This system also works with P. falciparum, which causes malaria in rural and urban poor regions of the world. With the low income and remote nature of these locations, a portable microfluidic device is necessary. Smartphones allow for a portable microfluidic device that can detect P. falciparum antigens in 10% whole blood. This system is capable of detecting as little as 1 pg/mL antigen. The microfluidic chip is inexpensive and disposable, allowing for a portable and inexpensive system. Using a single smartphone, a lab technique requiring a spectrometer, light source, and laptop can be made portable and less expensive, while maintaining sensitivity and specificity. In order to identify biological agents, there are commonly 3 methods for doing so: PCR, culturing, and ELISA. Culturing can take more than 24 hours, but results in a high signal to initial target ratio, while ELISA has poor sensitivity due to a 1:1 signal to target ratio, though is much quicker than culturing at usually 3 hours or less. PCR manages to solve both these problems by exponentially increasing the number of copies of target genetic material in a relatively short time frame of 1-3 hours. PCR relies on 4 basic components: target genetic material, primers to set a start and end location for duplication, polymerase to add base pairs to the strand beginning at the primers, and heat. PCR has worked very well during the past 31 years. It has worked so well that it is often the gold standard. However, there are flaws built into today's systems. These largely come in the form of inefficient heat transfer via conduction and large sample volumes due to unnecessary additions of nuclease free water (NFW). Both of these can be easily overcome by droplet PCR. Droplet PCR relies on small sample volumes of between 8 and 12 μL and convection in oil rather than conduction through plastic. In this study, it was found that droplet PCR could be performed on genomic E. coli DNA in as little as 15 minutes for 30 cycles. Sensitivity was also analyzed and found to be 2.62 pg DNA/μL or about 5 x 10² cfu/sample. PCR has a theoretical lower limit of 1 copy of genetic material and this is only 2 orders of magnitude above that. The system was also tested for portability and resistance to shock and vibration. It was found that the surface heated, thermocouple guided system is more shock and vibration resistant than standard wire guided, hanging droplet PCR systems. It was also found that the use of coconut oil allows for the system to be transported without fear of the contents spilling out and contaminating other samples. This is because of coconut oil's high melting temperature.

En studie i hur känsligheten avtar från ytan hos biosensorer med höga frekvenser presenteras. Med ny teknologi som avancerade elektroakustiska tunnfilms komponenter, så kallade FBARs, blir tidigare outforskade områden som decay längden möjliga att studera. För att undersöka hur frekvenssvaret och känsligheten påverkas av interaktioner långt ut från en sensoryta används proteinkemi. Ett protokoll har optimerats innehållande aktivering med EDC/NHS och fibrinogen för att säkerställa en jämn tjocklek och fördelning av ett adsorberat proteinlager över en yta. Dessa ytor kontrollerades först med hjälp av ellipsometri och sedan i ett QCM instrument. Alla experiment med de högfrekventa FBAR sensorerna utfördes vid Ångströmslaboratoriet i Uppsala där pågående forskning inom området finns. Resultaten bekräftar teorin om en avtagande känslighet i och med ett ökat avstånd från ytan. En experimentell genomförd och beräknad tjocklek för decay längden uppskattades som inte helt stämde överens med den teoretiskt beräknade. En ny term föreslås då frekvenssvaret hos en biosensor planar ut. Detta är en effekt som sker vid dubbla tjockleken av den teoretisk beräknade tjockleken av decay längden och har fått namnet; detection length. Efter denna längd eller gräns observeras en inverterad signal som det än så länge inte finns någon förklaring till. / A study of the sensitivity decrease of biosensors working at high frequencies is presented. With new technology such as film bulk acoustic resonators (FBAR), issues like the decay length is no longer irrelevant theory but may cause limitation in the system as well as it offers new detection possibilities. To investigate the frequency response and sensitivity, layer-on-layer construction chemistry was used. A protocol involving activation with EDC/NHS and coupling chemistry with fibrinogen was optimized to ensure accurate thickness and uniformly distribution of each layer over the surface. Surfaces were characterized using null ellipsometry and the protocol was tested in a traditional quartz crystal microbalance (QCM). Experiments with the FBAR were preformed at the Ångström laboratory in Uppsala were there is ongoing research and development in FBAR technology. The results confirmed the theory of decreasing frequency and sensitivity further out from the surface. An experimental and estimated thickness was calculated which to some extent correlates to the theoretically calculated decay length. A new terminology is suggested when the frequency levels off. It occurs approximately at twice the distance and thickness of the theoretically calculated decay length and is given the name; detection length. Beyond the detection length an inverted signal is observed which cannot yet be explained for.

L’auto-assemblatge de molècules en nano- i micro-estructures és una àrea de gran interès, sent els lípids particularment atractius en la formació de diverses estructures incloent els liposomes. Hi ha un gran número de mètodes reportats en la literatura per a la preparació de liposomes, però els inconvenients que limiten l’ús generalitzat dels liposomes són; els passos de preparació que requereixen de molt de temps donant lloc a poblacions heterogènies de liposomes de mida incontrolable, l’ús de solvents orgànics i la necessitat de passos per a reduir la mida dels liposomes. Per tant; l’objectiu d’aquest doctorat és la optimització d’un mètode ultra-ràpid per a la preparació de liposomes en un sol pas i lliure de dissolvents orgànics. Anomenat “Curvature tuned preparation method” ha estat implementat en diverses formulacions lipídiques per a la formació de liposomes i d’altres superestructures de lípids. Aquestes estructures s’han emprat en diverses aplicacions, com ara en nanoreactors i plantilles per a la síntesis a mida de nanopartícules d’or, liposomes per encapsular enzims com a potenciadors de senyal en el desenvolupament de immunosensors i finalment, com a vehicles per l’alliberament controlat de fàrmacs. / The self-assembly of molecules into nano- or microstructures is an area of intense interest, with lipids being particularly attractive in the formation several structures including liposomes. There are numerous methods reported for the preparation of liposomes, however, time-consuming preparative steps resulting in heterogeneous liposome populations of incontrollable size, the use of organic solvents and the need of further size-reducing steps are the drawbacks limiting wide-spread use of liposomes. Therefore; the main concern of this PhD thesis is optimization of a one-step, organic solvent-free, ultra rapid method for the preparation of liposomes. So called “Curvature tuned preparation method” was later implemented in several lipid formulations which resulted in liposomes and other lipid superstructures. Those structures were further used in several applications, such as nanoreactors and templates for tailored synthesis of gold nanoparticles, enzyme encapsulating liposomes as signal enhancers in immunosensor development, and finally as carriers for controlled release of drugs

The main purpose of this work is study of electrochemical hydrogen peroxide and ascorbate reactions on electrodes modified by Prussian blue (PB), with the aim to apply these electrodes in creation of sensors and biosensors. For this purpose, a detailed study of electrochemical reduction of hydrogen peroxide, as well as of oxidation of ascorbate at Prussian blue modified rotating disk electrode. In view of the results obtained, a mechanism for hydrogen peroxide reduction at PB modified electrode has been proposed. In accordance with this mechanism, electron transfer appears to be rate-limiting step. The kinetics of decomposition of PB modified electrode in the course of a cathodic reduction of hydrogen peroxide has been studied, and the influence of different factors to this process has been determined. Prototypes of sensors and biosensors, for different analytes have been elaborated and tested. / Darbo tikslas yra elektrocheminių vandenilio peroksido ir askorbato reakcijų tyrimas ant Berlyno mėlynuoju (BM) modifikuotų elektrodų, siekiant pritaikyti šiuos elektrodus jutiklių ir biojutiklių kūrimui. Ištirta vandenilio peroksido redukciją ir askorbato oksidaciją naudojant sukamojo disko elektrodą. Gauti rezultatai galimai įrodo stadijinį vandenilio peroksido katodinės redukcijos mechanizmą vykstantį ant BM modifikuoto elektrodo. Detaliai ištirta BM sluoksnio irimo kinetika vandenilio peroksido elektroredukcijos metu, ir nustatyti faktoriai, įtakojantys irimo proceso greitį. Sukurti jutiklių ir biojutiklių prototipai, kurie galėtų būti panaudoti biologiškai aktyvių medžiagų (vandenilio peroksido, askorbato, gliukozės) nustatymui.

Darbo tikslas yra elektrocheminių vandenilio peroksido ir askorbato reakcijų tyrimas ant Berlyno mėlynuoju (BM) modifikuotų elektrodų, siekiant pritaikyti šiuos elektrodus jutiklių ir biojutiklių kūrimui. Ištirta vandenilio peroksido redukciją ir askorbato oksidaciją naudojant sukamojo disko elektrodą. Gauti rezultatai galimai įrodo stadijinį vandenilio peroksido katodinės redukcijos mechanizmą vykstantį ant BM modifikuoto elektrodo. Detaliai ištirta BM sluoksnio irimo kinetika vandenilio peroksido elektroredukcijos metu, ir nustatyti faktoriai, įtakojantys irimo proceso greitį. Sukurti jutiklių ir biojutiklių prototipai, kurie galėtų būti panaudoti biologiškai aktyvių medžiagų (vandenilio peroksido, askorbato, gliukozės) nustatymui. / The main purpose of this work is study of electrochemical hydrogen peroxide and ascorbate reactions on electrodes modified by Prussian blue (PB), with the aim to apply these electrodes in creation of sensors and biosensors. For this purpose, a detailed study of electrochemical reduction of hydrogen peroxide, as well as of oxidation of ascorbate at Prussian blue modified rotating disk electrode. In view of the results obtained, a mechanism for hydrogen peroxide reduction at PB modified electrode has been proposed. In accordance with this mechanism, electron transfer appears to be rate-limiting step. The kinetics of decomposition of PB modified electrode in the course of a cathodic reduction of hydrogen peroxide has been studied, and the influence of different factors to this process has been determined. Prototypes of sensors and biosensors, for different analytes have been elaborated and tested.

Electrospun, non-woven, fibers have high surface area compared to conventional cast films. The thesis reports on the modification of electrospun fibers to concentrate and/or detect biohazards. In one study, electrospun fibers with affinity for the lectins ricin/concanavalin A were fabricated using molecular imprinting or through binding to immobilized antibodies, aptamers or lectin specific sugars. Attempts to fabricate imprinted electrospun fibers through inclusion of the template during the spinning process proved unsuccessful. However, electrospun fibers with affinity towards biohazards were successfully produced by post-modification with antibodies, aptamers or lectin specific saccharides. With regards to the latter, dextran, mannose and chitosan were immobilized onto nylon electrospun fibers that were partially hydrolyzed or treated with cyanuric chloride. The sugar-modified fibers bound significantly higher amount of lectins. Electrospun fibers were also fabricated, post modified with antibodies to capture and detect Salmonella. The study has illustrated the utility of electrospun fibers for biohazard diagnostics. / The National Center for Food Protection and Defense. USA

Biojutikliai yra įrenginiai, skirti medžiagoms aptikti bei jų koncentracijoms matuoti. Siekiant sumažinti biojutiklių gamybos kaštus yra pasitelkiamas matematinis biojutikliuose vykstančių procesų modeliavimas. Disertacijoje nagrinėjami matematiniai ir kompiuteriniai biojutiklių modeliai, aprašantys biojutiklių, sudarytų iš kelių, skirtingas savybes turinčių dalių, veikimą. Nagrinėjami modeliai yra formuluojami vienmatėje bei dvimatėje erdvėse, aprašomi diferencialinėmis lygtimis dalinėmis išvestinėmis su netiesiniais nariais ir yra sprendžiami skaitiškai, naudojant baigtinių skirtumų metodą. Skaitiniai modeliai yra įgyvendinami kompiuterine programa. Disertacijoje pateikiamas originalus matematinis modelis biojutikliui su anglies nanovamzdelių elektrodu, nustatyti kriterijai, apibrėžiantys, kada biojutiklį su perforuota membrana galima modeliuoti vienmačiu modeliu. Darbe susisteminti elementai, naudojami biojutiklių modelių formulavimui, pagrindinį dėmesį skiriant biojutiklio struktūrinėms savybėms modeliuoti. Apibrėžta biojutiklių modelių aprašo kalba ir sukurta programinė įranga, leidžianti modeliuoti biojutiklių veikimą vienmačiais modeliais arba modeliais, formuluojamais stačiakampėje dvimatės erdvės srityje. Taikant sukurtą biojutiklių modeliavimo programinę įrangą, ištirtas biojutiklio su anglies nanovamzdelių elektrodu modelio adekvatumas ir struktūrinių bei geometrinių savybių įtaka biojutiklio elgsenai. / Biosensors are analytical devices mainly used to detect analytes and measure their concentrations. Mathematical modeling is widely used for optimizing and analyzing an operation of biosensors for reducing price of development of new biosensors. The object of this research is mathematical and computer models, describing an operation of biosensors, made of several parts with different properties. The dissertation covers models, formulated in one and two-dimensional spaces by partial differential equations with non-linear members, and solved numerically, using the method of finite differences. The numerical models are implemented by a computer program. An original mathematical model for a biosensor with a carbon nanotube electrode is presented in the dissertation. The conditions at which the one-dimensional mathematical model can be used instead of two-dimensional one for accurate prediction of the biosensor response are investigated. Elements, used to build models of biosensors with a complex structure, were systemized. The biosensor description language is proposed and the computer software, simulating an operation of biosensors in the one-dimensional space and a rectangular domain of the two-dimensional space, is developed. An adequateness of the model for the biosensor with the carbon nanotube electrode and the impact of structural and geometrical properties on a response of the biosensor were investigated, performing computer experiments using the developed software.

Biosensors are analytical devices mainly used to detect analytes and measure their concentrations. Mathematical modeling is widely used for optimizing and analyzing an operation of biosensors for reducing price of development of new biosensors. The object of this research is mathematical and computer models, describing an operation of biosensors, made of several parts with different properties. The dissertation covers models, formulated in one and two-dimensional spaces by partial differential equations with non-linear members, and solved numerically, using the method of finite differences. The numerical models are implemented by a computer program. An original mathematical model for a biosensor with a carbon nanotube electrode is presented in the dissertation. The conditions at which the one-dimensional mathematical model can be used instead of two-dimensional one for accurate prediction of the biosensor response are investigated. Elements, used to build models of biosensors with a complex structure, were systemized. The biosensor description language is proposed and the computer software, simulating an operation of biosensors in the one-dimensional space and a rectangular domain of the two-dimensional space, is developed. An adequateness of the model for the biosensor with the carbon nanotube electrode and the impact of structural and geometrical properties on a response of the biosensor were investigated, performing computer experiments using the developed software. / Biojutikliai yra įrenginiai, skirti medžiagoms aptikti bei jų koncentracijoms matuoti. Siekiant sumažinti biojutiklių gamybos kaštus yra pasitelkiamas matematinis biojutikliuose vykstančių procesų modeliavimas. Disertacijoje nagrinėjami matematiniai ir kompiuteriniai biojutiklių modeliai, aprašantys biojutiklių, sudarytų iš kelių, skirtingas savybes turinčių dalių, veikimą. Nagrinėjami modeliai yra formuluojami vienmatėje bei dvimatėje erdvėse, aprašomi diferencialinėmis lygtimis dalinėmis išvestinėmis su netiesiniais nariais ir yra sprendžiami skaitiškai, naudojant baigtinių skirtumų metodą. Skaitiniai modeliai yra įgyvendinami kompiuterine programa. Disertacijoje pateikiamas originalus matematinis modelis biojutikliui su anglies nanovamzdelių elektrodu, nustatyti kriterijai, apibrėžiantys, kada biojutiklį su perforuota membrana galima modeliuoti vienmačiu modeliu. Darbe susisteminti elementai, naudojami biojutiklių modelių formulavimui, pagrindinį dėmesį skiriant biojutiklio struktūrinėms savybėms modeliuoti. Apibrėžta biojutiklių modelių aprašo kalba ir sukurta programinė įranga, leidžianti modeliuoti biojutiklių veikimą vienmačiais modeliais arba modeliais, formuluojamais stačiakampėje dvimatės erdvės srityje. Taikant sukurtą biojutiklių modeliavimo programinę įrangą, ištirtas biojutiklio su anglies nanovamzdelių elektrodu modelio adekvatumas ir struktūrinių bei geometrinių savybių įtaka biojutiklio elgsenai.

Established techniques for the detection of pathogens, such as bacteria and viruses, require long timeframes for culturing. State of the art biosensors rely on the diffusion of the target analyte to the sensor surface. AC electric fields can be exploited to enhance the sampling of pathogens and concentrate them at specific locations on the sensor surface, thus overcoming these bottlenecks. AC electrokinetic effects like the dielectrophoretic force and electrothermal flows apply forces on the particle and the bulk fluid, respectively. While dielectrophoresis forces pathogens towards a target location, electrothermal flows circulates the fluid, thus replenishing the local concentration. Numerical simulations and experimental proof of principle demonstrate how AC electrokinetics can be used to collect model bioparticles on an antibody functionalized selective surface from a heterogeneous solution having physiologically relevant conductivity. The presence of parallel channels in a quadrupolar microelectrode design is identified as detrimental during the negative dielectrophoretic collection of bioparticles at the centre of the design while simultaneously providing secondary concentration points. These microelectrodes were incorporated onto the surface of a novel cantilever design for the rapid positive dielectrophoretic collection of Escherichia coli bacteria and enabled the subsequent detection of the bacteria by measuring the shift in the resonance frequency of the cantilever. Finally, a proof of principle setup for a Raman coupled, AC electrokinetically enhanced sampling and detection of viruses is shown where the presence of M13 phages are identified on a selective antibody functionalized surface using Raman spectroscopy. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-01-30 19:23:48.958

No description available.