Photon Upconverting Nanophosphors: Unique Reporters for Optical Biosensing

Upconversion photoluminescence is a unique property of mostly certain inorganic materials, which are capable of converting low-energy infrared radiation into a higher-energy emission at visible wavelengths. This anti-Stokes shift enables luminescence detection without autofluorescence, which makes the upconverting materials a highly suitable reporter technology for optical biosensing applications. Furthermore, they exhibit long luminescence lifetime with narrow bandwidths also at the optical window of biomaterials enabling luminescence measurements in challenging sample matrices, such as whole blood. The aim of this thesis was to study the unique properties and the applicability of nano-sized upconverting phosphors (UCNPs) as reporters in biosensing applications. To render the inorganic nanophosphors water-dispersible and biocompatible, they were subjected to a series of surface modifications starting with silica-encapsulation and ending with a bioconjugation step with an analyte-recognizing biomolecule. The paramagnetism of the lanthanide dopants in the nanophosphors was exploited to develop a highly selective separation method for the UCNP-bioconjugates based on the magnetic selectivity of the high gradient magnetic separation (HGMS) system. The applicability of the nano-sized UCNPs as reporters in challenging sample matrices was demonstrated in two homogeneous sensing applications based on upconversion resonance energy transfer (UC-RET). A chemosensor for intracellular pH was developed exploiting UC-RET between the UCNP and a fluorogenic pH-sensitive dye with strongly increasing fluorescence intensity in decreasing pH. The pH-independent emission of the UCNPs at 550 nm was used for referencing. The applicability of the pH-nanosensor for intracellular pH measurement was tested in HeLa cells, and the acidic pH of endosomes could be detected with a confocal fluorescence microscope. Furthermore, a competitive UC-RET-based assay for red blood cell folic acid was developed for the measurement of folate directly from a whole blood sample. The optically transparent window of biomaterials was used in both the excitation and the measurement of the UC-RET sensitized emission of a near-infrared acceptor dye to minimize sample absorption, and the anti-Stokes detection completely eliminated the Stokes-shifted autofluorescence. The upconversion photoluminescence efficiency is known to be dependent on crystallite size, because the increasing surface-to-volume ratio of nano-sized UCNPs renders them more susceptible to quenching effects of the environment than their bulk counterpart. Water is known to efficiently quench the luminescence of lanthanide dopants. In this thesis, the quenching mechanism of water was studied using luminescence decay measurements. Water was found to quench the luminescence of UCNPs by increasing the non-radiative relaxation of the excited state of Yb3+ sensitizer ion, which had a very strong quenching effect on upconversion luminescence intensity.

Relationship of breast fillet deboning time to shear force, pH, cooking loss and color in broilers stunned by high electrical current

Selostus: Korkealla virranvoimakkuudella tainnutettujen broilereiden rintafileen irroitushetken vaikutus lihaksen leikkausvoiman vastukseen, pH:hon, keittohävikkiin ja väriin

Reactivity of silicate liming materials from Northern Europe assessed by Soil Incubation and two pH Stat methods

Selostus: Pohjois-Euroopan silikaattisten kalkitusaineiden reaktiivisuus astiakoemenetelmällä ja kahdella pH-staattisella menetelmällä arvioituna

Fabrication and characterization of silicon position sensitive particle detectors

Position sensitive particle detectors are needed in high energy physics research. This thesis describes the development of fabrication processes and characterization techniques of silicon microstrip detectors used in the work for searching elementary particles in the European center for nuclear research, CERN. The detectors give an electrical signal along the particles trajectory after a collision in the particle accelerator. The trajectories give information about the nature of the particle in the struggle to reveal the structure of the matter and the universe. Detectors made of semiconductors have a better position resolution than conventional wire chamber detectors. Silicon semiconductor is overwhelmingly used as a detector material because of its cheapness and standard usage in integrated circuit industry. After a short spread sheet analysis of the basic building block of radiation detectors, the pn junction, the operation of a silicon radiation detector is discussed in general. The microstrip detector is then introduced and the detailed structure of a double-sided ac-coupled strip detector revealed. The fabrication aspects of strip detectors are discussedstarting from the process development and general principles ending up to the description of the double-sided ac-coupled strip detector process. Recombination and generation lifetime measurements in radiation detectors are discussed shortly. The results of electrical tests, ie. measuring the leakage currents and bias resistors, are displayed. The beam test setups and the results, the signal to noise ratio and the position accuracy, are then described. It was found out in earlier research that a heavy irradiation changes the properties of radiation detectors dramatically. A scanning electron microscope method was developed to measure the electric potential and field inside irradiated detectorsto see how a high radiation fluence changes them. The method and the most important results are discussed shortly.

Ethnic sensitive substance abuse work : services for non-Finnish speaking clients in substance abuse treatment units in Finland today

The purpose of this study was to examine the current situation in substance abuse treatment units in Finland in taking non-Finnish speaking clients into consideration. The initiative for this research came from the Development of Alcohol and Drugs Intervention group at Stakes (National Research and Development Centre for Welfare and Health). Their aim was to gather information about the functioning and relevance of the quality assessment forms based on the quality recommendations for substance abuse work, filled in by substance abuse treatment units. The ethnic issue was chosen as the main approach in the study. The aim of this research was to answer the following questions: what is the readiness and competence in substance abuse treatment units in Finland to receive and encounter non-Finnish speaking clients, how is the quality of these services assessed and/or developed in the units, and what has been the role and functioning of the quality recommendations and quality assessment forms in working with non-Finnish speaking clients. The research methods used in the study were both quantitative and qualitative. The information concerning language services provided in the units was gathered from the quality assessment forms and basic information forms found in the database maintained by Stakes. The total amount of units found in the database was 267. In addition to that, semi-structured theme-interviews were carried out in four substance abuse treatment units in order to get a more deep understanding of how the services function in practice. The few number of non-Finnish speaking clients in the units may explain to a certain degree the results of the research. The results however showed that there is still space for improving the services. In the light of quality recommendations, the degree of language options provided in substance abuse treatment units in Finland today is low. Also the quantity of interpreter services provided in the units is scarce. There could also be unified guidelines specially tailored for substance abuse treatment units on how to work with ethnic minorities, as the knowledge is currently adopted from several different instances. The quality recommendations as well as quality assessment forms were valued and applied in the units appropriately and were also perceived to have an effect on the functioning, and quality, in the units.

pH:n stabiilisuus ditioniittivalkaistulla neutraalilla LWC-prosessilla

Tässä työssä Oy Keskuslaboratoriossa sijaitsevalla kiertovesisimulaattorilla on tutkittiu ditioniittivalkaisun ja hylyn määrän vaikutusta pH:n stabiilisuuteen ja häiriöaineiden määrään neutraalissa LWC-prosessissa. Lisäksi on tutkittu ditioniitin määrän seisokkien ja valkaisuviiveiden vaikutusta. Kirjallisuusosassa tutustutaan ditioniittivalkaisuun ja valkaisun kemiaan sekä kalsiumkarbonaatin liukenemiseen. Lyhyesti selvitetään myös paperikoneen vesikiertoja sekä prosessissa olevia häiriöaineita. Karbonaatin liukenemisen osalta tarkastellaan myös mahdollisuutta mallintaa prosessia. Diplomityön aikana ajettiin yhdeksän simulaattoriajoa, jotka ajettiin kahden tai kolmen ajon sarjoissa. Ajoja ajettiin kolmella eri hylkymäärällä. Ditioniittivalkaisun lisäksi suoritettiin referenssiajoja ilman valkaisua. Lyhyen kierron pH pyrittiin pääosin säätämään seitsemään. Valkaisuviiveiden ja yli-yön kestävien seisokkien vaikutusta tutkittiin myös. Kokeissa todettiin ditioniitin alentavan pH tasoa. Tämä pH:n aleneminen johtaa kalsiumkarbonaatin liukenemiseen kalsiumkarbonaatin pyrkiessä puskuroimaan pH:n laskua. Valkaisureaktiossa syntyy negatiivisesti varauruneita yhdisteitä, jotka lisäävät prosessin anionisuutta. Ditioniitin valkaisuvaikutus näkyi lopputuotteen vaaleuden kasvuna sekä kieroveden värillisten yhdisteiden vähenemisenä. Ditioniittivalkaisu näytti poistavan myös noin 30% ligniinistä. Lipofiilisiin uuteaineisiin ditioniitilla ei ollut vaikutusta. Riittävä ditioniittiannos kokeiden perusteella on korkeintaan 1% ditioniittiä käytetyn mekaanisen massan määrästä. Korkeammalla annostuksella ei ollut vaikutusta vaaleuteen. Valkaisuaikana 30 minuuttia on riittävä. Pidempien valkaisuviiveiden ja seisokkien aikana valkaisussa syntyvän sulfiitin todettiin muuttuvan sulfaatiksi. Hylyn määrän lisääminen nosti pH:ta, varausta, johtokykyä ja liuenneen kalsiumin määrään. Hylyn määrän lisäminen paransi myös optisia ominaisuuksia, mikä johtui osin hylyn sisältämästä kalsiumkarbonaatista ja osin hylyn sisältämästä mekaanisesta massasta, joka oli valkaistua.

Puumassapesurin suotautumisolojen kartoitus ja simulointi

Työn tavoitteena oli tutkia sellumassan suotautumiseen liittyviä tekijöitä Ahlstrom Machineryn Drum Displacer™ -puumassapesurissa (DD-pesuri). Teoriaosassa tarkasteltiin aluksi suotautumisen teoriaa kuitu-vesi-suspensiossa, minkä jälkeen esiteltiin suotautumisnopeuteen vaikuttavia fysikaalisia ja kemiallisia vaikutusmekanismeja. Seuraavaksi kuvattiin massan pesun yleisiä perusteita sekä teoriaa puumassapesureissa. Lopuksi tarkasteltiin pesurien kytkeytymistä muuhun kuitulinjaan sekä prosessista johtuvia pesun toiminnan ulkoisia häiriötekijöitä. Kokeellisen osan aluksi tarkasteltiin paine- ja lämpötilamittauksien avulla massapesurissa vallitsevia prosessioloja. Mittaustulosten perusteella pumppausolot pesurin suodoslinjoissa ovat vaikeahkot ja häiriötilanteita voi esiintyä, mutta käytäntö on osoittanut tästä olevan vain harvoin haittaa prosessin toiminnalle. Pesureissa toteutuneet syrjäytysnopeudet laskettiin ja niitä verrattiin syrjäytystestien antamiin tuloksiin. Kuitulinjasta riippuen testin vastaavuus tehdasprosessiin vaihteli suuresti. Syrjäytystesteillä kokeiltiin myös tehdasprosesseissa usein esiintyvien muuttujien vaikutusta sellukakun syrjäytettävyyteen. Kakun paksuus ja syrjäytyslämpötila vaikuttivat syrjäytysnopeuteen Darcyn lain mukaisesti. Alipaineen massakakun alapuolella havaittiin huonontavan syrjäytysnopeutta verrattuna tilanteeseen, jossa kakun alla vallitsi ilmanpaine. Tämä havainto on selvästi ristiriidassa suotautumisen teorian kanssa. Massakakun muodostumis-pH osoittautui ratkaisevaksi lopulliselle syrjäytysnopeudelle, sillä alkalisissa oloissa muodostetun kuitukakun syrjäytysnopeus ei enää parantunut happamalla syrjäytysnesteellä. Happamissa oloissa muodostetun kakun syrjäytysnopeus oli alkalisista parempi, mutta se alkoi hitaasti alentua, kun syrjäytysneste vaihtui alkaliseen. Massan laimentaminen ennen syrjäytystä alkalisella tehdassuodoksella puhtaan veden sijasta alensi ligniinipitoisella massalla lopullista syrjäytysnopeutta. Shirato-Tillerin mallilla ja Jönssonin staattisella mallilla simuloitiin numeerisesti syrjäytystestiä kahdessa eri pH:ssa, ja simulointituloksia verrattiin vastaavissa oloissa tehtyihin syrjäytystesteihin. Shirato-Tillerin mallin antamien syrjäytysnopeuksien havaittiin olevan lähellä syrjäytystestien nopeuksia, kun Jönssonin mallin antamat tulokset jäivät huomattavasti testituloksia alemmiksi. Herkkyystarkastelussa havaittiin mallien olevan varsin herkkiä parametrien virheille. Hajonta vaadittavien kuituparametrien määrityksissä ja menetelmien työläys rajoittavat numeerisen simuloinnin käytettävyyttä, sillä kuituparametrien määrityksen vaatima työmäärä on ainakin toistaiseksi syrjäytystestiä suurempi. Lopuksi todettiin, että oikeiden syrjäytysolosuhteiden käyttö on ensiarvoisen tärkeää oikeiden tulosten saamiseksi sekä kokeellisessa että numeerisessa simuloinnissa. Nykyinen syrjäytystestilaitteisto on pienin muutoksin käyttökelpoinen, kun massan testaus prosessioloissa tulee rutiininomaiseksi.

Sensitive Troponin Assays: Diagnostic and Prognostic Use in Cardiology

Cardiac troponins (cTns) are the recommended biochemical markers in the diagnosis of myocardial infarction (MI). They are very sensitive and tissue-specific but are limited by their delayed appearance in the circulation. Biochemical markers with more rapid release kinetics, e.g. myoglobin and especially heart-type fatty acid-binding protein (H-FABP), have been used to enhance the early identification of MI. The implementation of cTns into clinical practice has shown that cardiomyocyte injury occurs in many other clinical conditions than MI. The aim of this study was to evaluate the impact of modern and highly sensitive cTnI assays on the early diagnosis of MI. In a patient cohort with suspected MI, such a sensitive cTnI assay enhanced the early diagnostic accuracy when compared to a less sensitive cTnI assay and to myoglobin. When compared to H-FABP during the early hours after symptom onset, the sensitive cTnI assay showed at least similar and, after 6 hours, superior diagnostic accuracy. A positive cTnI test result had superior prognostic value when compared to H-FABP, even among early presenters. The prognostic value of cTn in acute heart failure (AHF) was evaluated in 364 patients who participated in the FINN-AKVA study. The patients presented with AHF but no acute coronary syndrome (ACS). Up to half of the patients had elevated cTn levels which were associated with higher 6-month mortality. The magnitude of cTn elevation was directly proportional to mortality. Finally, the clinical spectrum of cTnI elevations was evaluated in 991 cTnI positive emergency department (ED) patients. 83% of the patients had MI and 17% had cTnI elevation due to other clinical conditions. The latter patient group was characterized by lower absolute cTnI levels and – importantly – higher in-hospital mortality when compared to the MI patients. In conclusion, the use of a highly sensitive cTnI assay enhances the early diagnostic accuracy and risk stratification in suspected MI patients. Cardiac troponin elevations are highly prevalent also in other acute clinical conditions and indicate an adverse outcome of these patients.

Simplified sample handing in mass spectrometry based protein research - focus on protein phosphorylation

The human genome comprises roughly 20 000 protein coding genes. Proteins are the building material for cells and tissues, and proteins are functional compounds having an important role in many cellular responses, such as cell signalling. In multicellular organisms such as humans, cells need to communicate with each other in order to maintain a normal function of the tissues within the body. This complex signalling between and within cells is transferred by proteins and their post-translational modifications, one of the most important being phosphorylation. The work presented here concerns the development and use of tools for phosphorylation analysis. Mass spectrometers have become essential tools to study proteins and proteomes. In mass spectrometry oriented proteomics, proteins can be identified and their post-translational modifications can be studied. In this Ph.D. thesis the objectives were to improve the robustness of sample handling methods prior to mass spectrometry analysis for peptides and their phosphorylation status. The focus was to develop strategies that enable acquisition of more MS measurements per sample, higher quality MS spectra and simplified and rapid enrichment procedures for phosphopeptides. Furthermore, an objective was to apply these methods to characterize phosphorylation sites of phosphopeptides. In these studies a new MALDI matrix was developed which allowed more homogenous, intense and durable signals to be acquired when compared to traditional CHCA matrix. This new matrix along with other matrices was subsequently used to develop a new method that combines multiple spectra from different matrises from identical peptides. With this approach it was possible to identify more phosphopeptides than with conventional LC/ESI-MS/MS methods, and to use 5 times less sample. Also, phosphopeptide affinity MALDI target was prepared to capture and immobilise phosphopeptides from a standard peptide mixture while maintaining their spatial orientation. In addition a new protocol utilizing commercially available conductive glass slides was developed that enabled fast and sensitive phosphopeptide purification. This protocol was applied to characterize the in vivo phosphorylation of a signalling protein, NFATc1. Evidence for 12 phosphorylation sites were found, and many of those were found in multiply phosphorylated peptides

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.

Astringent Food Compounds and Their Interactions with Taste Properties

Astringency is traditionally thought to be induced by plant tannins in foods. Because of this current research concerning the mechanism of astringency is focused on tannin‐protein interactions and thus on precipitation, which may be perceived by mechanoreceptors. However, astringency is elicited by a wide range of different phenolic compounds, as well as, some non‐phenolic compounds in various foods. Many ellagitannins or smaller compounds that contribute to astringent properties do not interact with salivary proteins and may be directly perceived through some receptors. Generally, the higher degree of polymerization of proanthocyanidins can be associated with more intense astringency. However, the astringent properties of smaller phenolic compounds may not be directly predicted from the structure of a compound, although glycosylation has a significant role. The astringency of organic acids may be directly linked to the perception of sourness, and this increases along with decreasing pH. Astringency can be divided into different sub‐qualities, including even other qualities than traditional mouth‐drying, puckering or roughing sensations. Astringency is often accompanied by bitter or sour or both taste properties. The different sub‐qualities can be influenced by different astringent compounds. In general, the glycolysation of the phenolic compound results in more velvety and smooth mouthdrying astringency. Flavonol glycosides and other flavonoid compounds and ellagitannins contribute to this velvety mouthdrying astringency. Additionally, they often lack the bitter properties. Proanthocyanidins and phenolic acids elicit more puckering and roughing astringency with some additional bitter properties. Quercetin 3‐O‐rutinoside, along with other quercetin glycosides, is among the key astringent compounds in black tea and red currants. In foods, there are always various other additional attributes that are perceived at the same with astringency. Astringent compounds themselves may have other sensory characteristics, such as bitter or sour properties, or they may enhance or suppress other sensory properties. Components contributing to these other properties, such as sugars, may also have similar effects on astringent sensations. Food components eliciting sweetness or fattiness or some polymeric polysaccharides can be used to mask astringent subqualities. Astringency can generally be referred to as a negative contributor to the liking of various foods. On the other hand, perceptions of astringent properties can vary among individuals. Many genetic factors that influence perceptions of taste properties, such as variations in perceiving a bitter taste or variations in saliva, may also effect the perception of astringency. Individuals who are more sensitive to different sensations may notice the differences between astringent properties more clearly. This may not have effects on the overall perception of astringency. However, in many cases, the liking of astringent foods may need to be learned by repetitive exposure. Astringency is often among the key sensory properties forming the unique overall flavour of certain foods, and therefore it also influences whether or not a food is liked. In many cases, astringency may be an important sub‐property suppressed by other more abundant sensory properties, but it may still have a significant contribution to the overall flavour and thus consumer preferences. The results of the practical work of this thesis show that the astringent phenolic compounds are mostly located in the skin fractions of black currants, crowberries and bilberries (publications I–III). The skin fractions themselves are rather tasteless. However, the astringent phenolic compounds can be efficiently removed from these skin fractions by consecutive ethanol extractions. Berries contain a wide range of different flavonol glycosides, hydroxycinnamic acid derivatives and anthocyanins and some of them strongly contribute to the different astringent and bitterness properties. Sweetness and sourness are located in the juice fractions along with the majority of sugars and fruit acids. The sweet and sour properties of the juice may be used to mask the astringent and bitterness properties of the extracts. Enzymatic treatments increase the astringent properties and fermented flavour of the black currant juice and decrease sweetness and freshness due to the effects on chemical compositions (IV). Sourness and sweetness are positive contributors to the liking of crowberry and bilberry fractions, whereas bitterness is more negative (V). Some astringent properties in berries are clearly negative factors, whereas some may be more positive. The liking of berries is strongly influenced by various consumer background factors, such as motives and health concerns. The liking of berries and berry fractions may also be affected by genetic factors, such as variations in the gene hTAS2R38, which codes bitter taste receptors (V).

Climate driven changes in temperature, pH and food quality : effects on copepod reproduction

Increased emissions of greenhouse gases into the atmosphere are causing an anthropogenic climate change. The resulting global warming challenges the ability of organisms to adapt to the new temperature conditions. However, warming is not the only major threat. In marine environments, dissolution of carbon dioxide from the atmosphere causes a decrease in surface water pH, the so called ocean acidification. The temperature and acidification effects can interact, and create even larger problems for the marine flora and fauna than either of the effects would cause alone. I have used Baltic calanoid copepods (crustacean zooplankton) as my research object and studied their growth and stress responses using climate predictions projected for the next century. I have studied both direct temperature and pH effects on copepods, and indirect effects via their food: the changing phytoplankton spring bloom composition and toxic cyanobacterium. The main aims of my thesis were: 1) to find out how warming and acidification combined with a toxic cyanobacterium affect copepod reproductive success (egg production, egg viability, egg hatching success, offspring development) and oxidative balance (antioxidant capacity, oxidative damage), and 2) to reveal the possible food quality effects of spring phytoplankton bloom composition dominated by diatoms or dinoflagellates on reproducing copepods (egg production, egg hatching, RNA:DNA ratio). The two copepod genera used, Acartia sp. and Eurytemora affinis are the dominating mesozooplankton taxa (0.2 – 2 mm) in my study area the Gulf of Finland. The 20°C temperature seems to be within the tolerance limits of Acartia spp., because copepods can adapt to the temperature phenotypically by adjusting their body size. Copepods are also able to tolerate a pH decrease of 0.4 from present values, but the combination of warm water and decreased pH causes problems for them. In my studies, the copepod oxidative balance was negatively influenced by the interaction of these two environmental factors, and egg and nauplii production were lower at 20°C and lower pH, than at 20°C and ambient pH. However, presence of toxic cyanobacterium Nodularia spumigena improved the copepod oxidative balance and helped to resist the environmental stress, in question. In addition, adaptive maternal effects seem to be an important adaptation mechanism in a changing environment, but it depends on the condition of the female copepod and her diet how much she can invest in her offspring. I did not find systematic food quality difference between diatoms and dinoflagellates. There are both good and bad diatom and dinoflagellate species. Instead, the dominating species in the phytoplankton bloom composition has a central role in determining the food quality, although copepods aim at obtaining as a balanced diet as possible by foraging on several species. If the dominating species is of poor quality it can cause stress when ingested, or lead to non-optimal foraging if rejected. My thesis demonstrates that climate change induced water temperature and pH changes can cause problems to Baltic Sea copepod communities. However, their resilience depends substantially on their diet, and therefore the response of phytoplankton to the environmental changes. As copepods are an important link in pelagic food webs, their future success can have far reaching consequences, for example on fish stocks.