Homogeneous Assays for Simplified Screening of HLA-conferred Genetic Disease Risk

The increasing incidence of type 1 diabetes has led researchers on a quest to find the reason behind this phenomenon. The rate of increase is too great to be caused simply by changes in the genetic component, and many environmental factors are under investigation for their possible contribution. These studies require, however, the participation of those individuals most likely to develop the disease, and the approach chosen by many is to screen vast populations to find persons with increased genetic risk factors. The participating individuals are then followed for signs of disease development, and their exposure to suspected environmental factors is studied. The main purpose of this study was to find a suitable tool for easy and inexpensive screening of certain genetic risk markers for type 1 diabetes. The method should be applicable to using whole blood dried on sample collection cards as sample material, since the shipping and storage of samples in this format is preferred. However, the screening of vast sample libraries of extracted genomic DNA should also be possible, if such a need should arise, for example, when studying the effect of newly discovered genetic risk markers. The method developed in this study is based on homogeneous assay chemistry and an asymmetrical polymerase chain reaction (PCR). The generated singlestranded PCR product is probed by lanthanide-labelled, LNA (locked nucleic acid)-spiked, short oligonucleotides with exact complementary sequences. In the case of a perfect match, the probe is hybridised to the product. However, if even a single nucleotide difference occurs, the probe is bound instead of the PCR product to a complementary quencher-oligonucleotide labelled with a dabcyl-moiety, causing the signal of the lanthanide label to be quenched. The method was applied to the screening of the well-known type 1 diabetes risk alleles of the HLA-DQB1 gene. The method was shown to be suitable as an initial screening step including thousands of samples in the scheme used in the TEDDY (The Environmental Determinants of Diabetes in the Young) study to identify those individuals at increased genetic risk. The method was further developed into dry-reagent form to allow an even simpler approach to screening. The reagents needed in the assay were in dry format in the reaction vessel, and performing the assay required only the addition of the sample and, if necessary, water to rehydrate the reagents. This allows the assay to be successfully executed even by a person with minimal laboratory experience.

Upconverting Phosphor Technology: Exceptional Photoluminescent Properties Light Up Homogeneous Bioanalytical Assays

The aim of the present study was to demonstrate the wide applicability of the novel photoluminescent labels called upconverting phosphors (UCPs) in proximity-based bioanalytical assays. The exceptional features of the lanthanide-doped inorganic UCP compounds stem from their capability for photon upconversion resulting in anti-Stokes photoluminescence at visible wavelengths under near-infrared (NIR) excitation. Major limitations related to conventional photoluminescent labels are avoided, rendering the UCPs a competitive next-generation label technology. First, the background luminescence is minimized due to total elimination of autofluorescence. Consequently, improvements in detectability are expected. Second, at the long wavelengths (>600 nm) used for exciting and detecting the UCPs, the transmittance of sample matrixes is significantly greater in comparison with shorter wavelengths. Colored samples are no longer an obstacle to the luminescence measurement, and more flexibility is allowed even in homogeneous assay concepts, where the sample matrix remains present during the entire analysis procedure, including label detection. To transform a UCP particle into a biocompatible label suitable for bioanalytical assays, it must be colloidal in an aqueous environment and covered with biomolecules capable of recognizing the analyte molecule. At the beginning of this study, only UCP bulk material was available, and it was necessary to process the material to submicrometer-sized particles prior to use. Later, the ground UCPs, with irregular shape, wide size-distribution and heterogeneous luminescence properties, were substituted by a smaller-sized spherical UCP material. The surface functionalization of the UCPs was realized by producing a thin hydrophilic coating. Polymer adsorption on the UCP surface is a simple way to introduce functional groups for bioconjugation purposes, but possible stability issues encouraged us to optimize an optional silica-encapsulation method which produces a coating that is not detached in storage or assay conditions. An extremely thin monolayer around the UCPs was pursued due to their intended use as short-distance energy donors, and much attention was paid to controlling the thickness of the coating. The performance of the UCP technology was evaluated in three different homogeneous resonance energy transfer-based bioanalytical assays: a competitive ligand binding assay, a hybridization assay for nucleic acid detection and an enzyme activity assay. To complete the list, a competitive immunoassay has been published previously. Our systematic investigation showed that a nonradiative energy transfer mechanism is indeed involved, when a UCP and an acceptor fluorophore are brought into close proximity in aqueous suspension. This process is the basis for the above-mentioned homogeneous assays, in which the distance between the fluorescent species depends on a specific biomolecular binding event. According to the studies, the submicrometer-sized UCP labels allow versatile proximity-based bioanalysis with low detection limits (a low-nanomolar concentration for biotin, 0.01 U for benzonase enzyme, 0.35 nM for target DNA sequence).

Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Optimal distance of the light-curing tip for light transmission through the resin composite

Hammaslääketieteessä käytetettävien komposiittien valonläpäisevyys vaihtelee. Samoin LED-valokovettimet eroavat toisistaan valotehonsa ja muotoilunsa perusteella. On yleisesti tiedossa, että valokovettimesta tulevan valon intensiteetti pinta-alayksikköä kohden heikkenee, kun kovettimen etäisyys kasvaa. Toisaalta ei ole tiedossa, miten valokovetettavan kohteen ja valokovettimen kärjen väliin sijoitettu materiaali tarkalleenottaen vaikuttaa valon intensiteettiin eri etäisyyksiä käytettäessä. Tämän tutkimuksen tarkoituksena on selvittää, miten valokovetettavan kohteen ja valokovettimen kärjen väliin asetettava etukäteen polymerisoitu materiaali vaikuttaa valon intensiteettiin eri etäisyyksillä. Tutkimus suoritettiin käyttämällä kahta eri valokovetinta. Jotta etäisyyden vaikutusta valotustehoon voitiin demonstroida, vaihdettiin kovettimen etäisyyttä sensorista 0,2,4,6,8,10mm välillä. Valotehot rekisteröitiin MARC resin calibrator -laitteella. Sensorin ja valokovettimen kärjen väliin asetettavat erilaiset komposiittilevyt olivat valmiiksi kovetettuja,1mm paksuisia, filleripitoisuuksiltaan neljää erilaista muovia. Valotehot rekisteröitiin jokaiselta etäisyydeltä komposiitin ollessa sensorin päällä. Rinnakkaisesti verrattiin myös etäisyyden vaikutusta valotehoon ilman esikovetettua materiaalia kovettimen kärjen ja valoa mittaavan sensorin välissä. Vertailun suorittamiseksi laskettiin intensiteettisuhdeluku muovillisen ja muovittoman arvon välillä aina tietyllä etäisyydellä Valokovettimen kärjen etäisyyden kasvattaminen sensorista (eli valokovetettavasta kohteesta) odotusten mukaisesti pienensi valotehoa. Laittamalla sensorin ja kovettimen väliin komposiittilevy, valoteho pieneni odotetusti vielä enemmän. Tutkittaessa intensiteettisuhdetta (valoteho muovin kanssa : valoteho ilman muovia) kuitenkin huomattiin, että 4-6mm:n kohdalla suhdeluku oli suurempi kuin 0,2,8 ja 10mm kohdalla. Johtopäätöksenä oli, että suurin mahdollinen valokovetusteho saavutetan laittamalla kovetuskärki mahdollisimman lähelle kohdetta. Jos valokovetettavan kohteen ja valokovettimen kärjen välissä oli kiinteä komposiittipalanen, suurin mahdollinen valokovetusteho kohteeseen saavutetaan edelleen laittamalla kovetuskärki kiinni muoviin. Jos etäisyyttä muovin pinnasta sen sijaan kasvatettiin, valokovetusteho ei laskenutkaan niin nopeasti kuin oli odotettu. Tämä voi liittyä siihen, että tehokkaan valokeilan halkaisijan koko on suurempi verrattuna komposiitin sekä sensorin halkaisian kokoon. Toiseksi on arvioitu, että resiinikomposiitin täyteaineet voisivat fokusoida läpi kulkevaa valoa sensoriin. Se, pitääkö tämä ilmiö paikkansa, vaatii kuitenkin enemmän tutkimusta

Methods for Region Based Paper Surface Roughness Measurement

Paperin pinnan karheus on yksi paperin laatukriteereistä. Sitä mitataan fyysisestipaperin pintaa mittaavien laitteiden ja optisten laitteiden avulla. Mittaukset vaativat laboratorioolosuhteita, mutta nopeammille, suoraan linjalla tapahtuville mittauksilla olisi tarvetta paperiteollisuudessa. Paperin pinnan karheus voidaan ilmaista yhtenä näytteelle kohdistuvana karheusarvona. Tässä työssä näyte on jaettu merkitseviin alueisiin, ja jokaiselle alueelle on laskettu erillinen karheusarvo. Karheuden mittaukseen on käytetty useita menetelmiä. Yleisesti hyväksyttyä tilastollista menetelmää on käytetty tässä työssä etäisyysmuunnoksen lisäksi. Paperin pinnan karheudenmittauksessa on ollut tarvetta jakaa analysoitava näyte karheuden perusteella alueisiin. Aluejaon avulla voidaan rajata näytteestä selvästi karheampana esiintyvät alueet. Etäisyysmuunnos tuottaa alueita, joita on analysoitu. Näistä alueista on muodostettu yhtenäisiä alueita erilaisilla segmentointimenetelmillä. PNN -menetelmään (Pairwise Nearest Neighbor) ja naapurialueiden yhdistämiseen perustuvia algoritmeja on käytetty.Alueiden jakamiseen ja yhdistämiseen perustuvaa lähestymistapaa on myös tarkasteltu. Segmentoitujen kuvien validointi on yleensä tapahtunut ihmisen tarkastelemana. Tämän työn lähestymistapa on verrata yleisesti hyväksyttyä tilastollista menetelmää segmentoinnin tuloksiin. Korkea korrelaatio näiden tulosten välillä osoittaa onnistunutta segmentointia. Eri kokeiden tuloksia on verrattu keskenään hypoteesin testauksella. Työssä on analysoitu kahta näytesarjaa, joidenmittaukset on suoritettu OptiTopolla ja profilometrillä. Etäisyysmuunnoksen aloitusparametrit, joita muutettiin kokeiden aikana, olivat aloituspisteiden määrä ja sijainti. Samat parametrimuutokset tehtiin kaikille algoritmeille, joita käytettiin alueiden yhdistämiseen. Etäisyysmuunnoksen jälkeen korrelaatio oli voimakkaampaa profilometrillä mitatuille näytteille kuin OptiTopolla mitatuille näytteille. Segmentoiduilla OptiTopo -näytteillä korrelaatio parantui voimakkaammin kuin profilometrinäytteillä. PNN -menetelmän tuottamilla tuloksilla korrelaatio oli paras.

Mass transfer and particleinteractions in granular systems and suspension flows

The purpose of this study was to investigate some important features of granular flows and suspension flows by computational simulation methods. Granular materials have been considered as an independent state ofmatter because of their complex behaviors. They sometimes behave like a solid, sometimes like a fluid, and sometimes can contain both phases in equilibrium. The computer simulation of dense shear granular flows of monodisperse, spherical particles shows that the collisional model of contacts yields the coexistence of solid and fluid phases while the frictional model represents a uniform flow of fluid phase. However, a comparison between the stress signals from the simulations and experiments revealed that the collisional model would result a proper match with the experimental evidences. Although the effect of gravity is found to beimportant in sedimentation of solid part, the stick-slip behavior associated with the collisional model looks more similar to that of experiments. The mathematical formulations based on the kinetic theory have been derived for the moderatesolid volume fractions with the assumption of the homogeneity of flow. In orderto make some simulations which can provide such an ideal flow, the simulation of unbounded granular shear flows was performed. Therefore, the homogeneous flow properties could be achieved in the moderate solid volume fractions. A new algorithm, namely the nonequilibrium approach was introduced to show the features of self-diffusion in the granular flows. Using this algorithm a one way flow can beextracted from the entire flow, which not only provides a straightforward calculation of self-diffusion coefficient but also can qualitatively determine the deviation of self-diffusion from the linear law at some regions nearby the wall inbounded flows. Anyhow, the average lateral self-diffusion coefficient, which was calculated by the aforementioned method, showed a desirable agreement with thepredictions of kinetic theory formulation. In the continuation of computer simulation of shear granular flows, some numerical and theoretical investigations were carried out on mass transfer and particle interactions in particulate flows. In this context, the boundary element method and its combination with the spectral method using the special capabilities of wavelets have been introduced as theefficient numerical methods to solve the governing equations of mass transfer in particulate flows. A theoretical formulation of fluid dispersivity in suspension flows revealed that the fluid dispersivity depends upon the fluid properties and particle parameters as well as the fluid-particle and particle-particle interactions.