Upconverting diagnostics: Multiplex assays utilizing upconversion luminescence technology

Conventional diagnostics tests and technologies typically allow only a single analysis and result per test. The aim of this study was to propose robust and multiplex array-inwell test platforms based on oligonucleotide and protein arrays combining the advantages of simple instrumentation and upconverting phosphor (UCP) reporter technology. The UCPs are luminescent lanthanide-doped crystals that have a unique capability to convert infrared radiation into visible light. No autofluorescence is produced from the sample under infrared excitation enabling the development of highly sensitive assays. In this study, an oligonucleotide array-in-well hybridization assay was developed for the detection and genotyping of human adenoviruses. The study provided a verification of the advantages and potential of the UCP-based reporter technology in multiplex assays as well as anti-Stokes photoluminescence detection with a new anti- Stokes photoluminescence imager. The developed assay was technically improved and used to detect and genotype adenovirus types from clinical specimens. Based on the results of the epidemiological study, an outbreak of adenovirus type B03 was observed in the autumn of 2010. A quantitative array-in-well immunoassay was developed for three target analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone). In this study, quantitative results were obtained for each analyte and the analytical sensitivities in buffer were in clinically relevant range. Another protein-based array-inwell assay was developed for multiplex serodiagnostics. The developed assay was able to detect parvovirus B19 IgG and adenovirus IgG antibodies simultaneously from serum samples according to reference assays. The study demonstrated that the UCPtechnology is a robust detection method for diverse multiplex imaging-based array-inwell assays.

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.

NIR-Vis Up-Conversion Luminescence in the Yb3+,Er3+ Doped Y2O2S, ZrO2, and NaYF4 Nanomaterials

Since the discovery of the up-conversion phenomenon, there has been an ever increasing interest in up-converting phosphors in which the absorption of two or more low energy photons is followed by emission of a higher energy photon. Most up-conversion luminescence materials operate by using a combination of a trivalent rare earth (lanthanide) sensitizer (e.g. Yb or Er) and an activator (e.g. Er, Ho, Tm or Pr) ion in a crystal lattice. Up-converting phosphors have a variety of potential applications as lasers and displays as well as inks for security printing (e.g. bank notes and bonds). One of the most sophisticated applications of lanthanide up-conversion luminescence is probably in medical diagnostics. However, there are some major problems in the use of photoluminescence based on the direct UV excitation in immunoassays. Human blood absorbs strongly UV radiation as well as the emission of the phosphor in the visible. A promising way to overcome the problems arising from the blood absorption is to use a long wavelength excitation and benefit from the up-conversion luminescence. Since there is practically no absorption by the whole-blood in the near IR region, it has no capability for up-conversion in the excitation wavelength region of the conventional up-converting phosphor based on the Yb3+ (sensitizer) and Er3+ (activator) combination. The aim of this work was to prepare nanocrystalline materials with high red (and green) up-conversion luminescence efficiency for use in quantitative whole-blood immunoassays. For coupling to biological compounds, nanometer-sized (crystallite size below 50 nm) up-converting phosphor particles are required. The nanocrystalline ZrO2:Yb3+,Er3+, Y2O2S:Yb3+,Er3+, NaYF4:Yb3+,Er3+ and NaRF4-NaR’F4 (R: Y, Yb, Er) materials, prepared with the combustion, sol-gel, flux, co-precipitation and solvothermal synthesis, were studied using the thermal analysis, FT-IR spectroscopy, transmission electron microscopy, EDX spectroscopy, XANES/EXAFS measurements, absorption spectroscopy, X-ray powder diffraction, as well as up-conversion and thermoluminescence spectroscopies. The effect of the impurities of the phosphors, crystallite size, as well as the crystal structure on the up-conversion luminescence intensity was analyzed. Finally, a new phenomenon, persistent up-conversion luminescence was introduced and discussed. For efficient use in bioassays, more work is needed to yield nanomaterials with smaller and more uniform crystallite sizes. Surface modifications need to be studied to improve the dispersion in water. On the other hand, further work must be carried out to optimize the persistent up-conversion luminescence of the nanomaterials to allow for their use as efficient immunoassay nanomaterials combining the advantages of both up-conversion and persistent luminescence.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Spectroscopie de luminescence à température et pression variables pour des complexes des lanthanides et de l'or

Ce travail est axé vers la compréhension détaillée des propriétés de luminescence de composés de certains métaux lourds. La première partie de ce mémoire décrit la caractérisation spectroscopique d'un radical de type nitronyle nitroxyde, 2-(2-pyridinyl)-4,4,5,5-tétraméthyl-4,5-dihydro-1H-imidazolyl-1-oxyl-3-oxyde, abrégé (NIT2-Py), et de ses complexes avec les cations Tb(III), [Tb(hfac)3NIT2-Py], et Y(III), [Y(hfac)3NIT2-Py]. La variation de la température affecte les spectres de luminescence qui montrent de la structure vibronique résolue. Les maxima de ces transitions vibroniques se rapprochent au fur et à mesure que la température augmente. Ces variations des maxima en fonction de la température ne correspondent pas à des variations de fréquences vibrationnelles et sont de l'ordre de 200 cm-1 entre 80 K et 240 K. La variation de la température n'a pas d'influence significative sur la structure moléculaire, comme atteste la variation mineure des maxima des spectres Raman entre 80 K et 300 K. La comparaison des spectres expérimentaux à des spectres calculés montre que ces variations peuvent être reproduites par l'utilisation d'une combinaison de fréquences vibrationnelles. Le paramètre dont la variation est très significative est la résolution du spectre de luminescence, représentée par la largeur à mi-hauteur des transitions vibroniques qui forment le spectre de luminescence. La deuxième partie de ce mémoire décrit les propriétés de luminescence d'une série de complexes d’or(I). Elles sont comparées aux changements structuraux à pression et température variable. Les interactions aurophiles ont une grande influence sur la luminescence. La variation de la température et de la pression est une approche efficace pour varier la luminescence. Les effets observés dans les spectres d'émission de ces complexes dépendent des changements de structure induits par variation de la température et de la pression. Ces petites variations structurales mènent à des changements importants, à titre d'exemple à un déplacement du maximum de la bande de luminescence de 60 cm-1/ kbar vers les faibles énergies pour un des complexes de l'or(I) étudiés au cours de ce projet.

INVESTIGATIONS ON THE SOLVENT EXTRACTION AND LUMINESCENCE OF LANTHANOIDS WITH MIXTURES OF HETEROCYCLIC β- DIKETONE S AND VARIOUS NEUTRAL OXO-DONORS

The thesis entitled “ Investigations on the solvent extraction and luminescence of lanthanoids with mixtures of heterocyclic β-diketone S and various neutral oxo-donors” embodies the results of investigations carried out on the solvent extraction of trivalent lanthanoids with various heterocyclic β-diketones in the presence and absence of neutral oxo-donors and also on the luminescent studies of Eu3+-heterocyclic β-diketonate complexes with Lewis bases. The primary objective of the present work is to generate the knowledge base, especially to understand the interactions of lanthanoid-heterocyclic β-diketonates with various macrocyclic ligands such as crown ethers and neutral organophosphorus extractants , with a view to achieve better selectivity. The secondary objective of this thesis is to develop novel lanthanoid luminescent materials based on 3-phenyl-4-aroyl-5-isoxazolones and organophosphorus ligands, for use in electroluminescent devices. In the beginning it describes the need for the development of new mixed-ligand systems for the separation of lanthanoids and the development and importance of novel luminescent lanthanoid- β-diketonate complexes for display devices. The syntheses of various para substituted derivatives of 4-aroyl-5-isoxazolones and their characterization by various spectroscopic techniques are described. It also investigate the solvent extraction behaviour of trivalent lanthanoids with 4-aroyl-5-isoxazolones in the presence and absence of various crown ethers such as 18C6, DC18C6, DB18C6 and B18C6. Elemental analysis, IR and H NMR spectral studies are used to understand the interactions of crown ethers with 4-aroyl-5-isoxazolonate complexes of lanthanoids. The synergistic extraction of trivalent lanthanoids with sterically hindered 1-phenyl-3-methyl-4-pivaloyl-5-pyrazolone in the presence of various structurally related crown ethers are studied. The syntheses, characterization and photyphysical properties of Eu3+-4-aroyl-5-isoxazolonate complexes in the presence of Lewis bases like trictylphosphine oxide or triphenylphosphine oxide were studied.

Luminescence from Surfactant-Free ZnO Quantum Dots Prepared by Laser Ablation in Liquid

Highly transparent, luminescent and biocompatible ZnO quantum dots were prepared in water, methanol, and ethanol using liquid-phase pulsed laser ablation technique without using any surfactant. Transmission electron microscopy analysis confirmed the formation of good crystalline ZnO quantum dots with a uniform size distribution of 7 nm. The emission wavelength could be varied by varying the native defect chemistry of ZnO quantum dots and the laser fluence. Highly luminescent nontoxic ZnO quantum dots have exciting application potential as florescent probes in biomedical applications.

Luminescence tuning and enhanced nonlinear optical properties of nanocomposites of ZnO–TiO2

In this article we present the spectral and nonlinear optical properties of ZnO–TiO2 nanocomposites prepared by colloidal chemical synthesis. Emission peaks of ZnO–TiO2 nanocomposites change from 340 nm to 385 nm almost in proportion to changes in Eg. The nanocomposites show self-defocusing nonlinearity and good nonlinear absorption behaviour. The nonlinear refractive index and the nonlinear absorption increase with increasing TiO2 volume fraction at 532 nm and can be attributed to the enhancement of exciton oscillator strength. ZnO–TiO2 is a potential nanocomposite material for the tunable light emission and for the development of nonlinear optical devices with a relatively small limiting threshold

Visible luminescence mechanism in nano ZnO under weak confinement regime

We describe the structure of luminescence spectrum in the visible region in nano-ZnO in colloidal and thin film forms under weak confinement regime by modeling the transition from excited state energy levels of excitons to their ground state. Measurements on nanocrystallites indicate the presence of luminescence due to excitonic emissions when excited with 255 nm. The relevant energy levels showing the transitions corresponding to the observed peaks in the emission spectrum of ZnO of particle size 18 nm are identified.

Luminescence studies of strontium sulphide based phosphors for display applications

This thesis has focused on the synthesis and analysis of some important phosphors (nano, bulk and thin film) for display applications. ACTFEL device with SrS:Cu as active layer was also fabricated.Three bulk phosphors: SrS:Cu,CI; SrS:Dy,Cl; and SrS:Dy,Cu,Cl were synthesized and their structural, optical and electrical properties were investigated. Special emphasis was given to, the analysis of the role of defects and charge compensating centers, on the structural changes of the host and hence the luminance. A new model describing the sensitizing behaviour of Cu in SrS:Dy,Cu,Cl two component phosphor was introduced. It was also found that addition of NH4CI as flux in SrS:Cu caused tremendous improvement in the structural and luminescence properties.A novel technique for ACTFEL phosphor deposition at low temperature was introduced. Polycrystalline films of SrS:Cu,F were synthesized at low temperature by concomitant evaporation of host and dopant by electron beam evaporation and thermal evaporatin methods.Copper doped strontium sulphide nanophosphor was synthesized for the first time. Improvement in the luminescence properties was observed in the nanophosphor with respect to it' s bulk counterpart.

Defect analysis of semiconductor thin films for photovoltaic applications using photo-luminescence and photo-conductivity

The present thesis can be divided into three areas:1) the fabrication of a low temperature photo-luminescence and photoconductivity measuring unit 2) photo-luminescence in the chalcopyrite CulnSez and CulnS2 system for defect and composition analysis and 3) photo-luminescence and photo-conductivity of In:JS3. This thesis shows that photo-luminescence is one of most essential semiconductor characterization tool for a scientific group working on photovoltaics. Tools which can be robust, non-destructive, requiring minimal sample preparation for analysis and most informative of the device applications are sought after by industries and this thesis is towards establishing photo-luminescence as "THE" tool for semiconductor characterization. The possible application of photo-luminescence as a tool for compositional and quality analysis of semiconductor thin films has been worked upon by this thesis. Photo-conductivity complement photo-luminescence and together they provide all the information required for the fabrication of an opto-electronic device.

Enhanced luminescence and nonlinear optical properties of nanocomposites of ZnO–Cu

In this article, we present the spectral and nonlinear optical properties of ZnOCu nanocomposites prepared by colloidal chemical synthesis. The emission consisted of two peaks. The 385-nm ultraviolet (UV) peak is attributed to ZnO and the 550-nm visible peak is attributed to Cu nanocolloids. Obvious enhancement of UV and visible emission of the samples is observed and the strongest UV emission of a typical ZnOCu nanocomposite is over three times stronger than that of pure ZnO. Cu acts as a sensitizer and the enhancement of UV emission are caused by excitons formed at the interface between Cu and ZnO. As the volume fraction of Cu increases beyond a particular value, the intensity of the UV peak decreases while the intensity of the visible peak increases, and the strongest visible emission of a typical ZnOCu nanocomposite is over ten times stronger than that of pure Cu. The emission mechanism is discussed. Nonlinear optical response of these samples is studied using nanosecond laser pulses from a tunable laser in the wavelength range of 450650 nm, which includes the surface plasmon absorption (SPA) band. The nonlinear response is wavelength dependent and switching from reverse saturable absorption (RSA) to saturable absorption (SA) has been observed for Cu nanocolloids as the excitation wavelength changes from the low absorption window region to higher absorption regime near the SPA band. However, ZnO colloids and ZnOCu nanocomposites exhibit induced absorption at this wavelength. Such a changeover in the sign of the nonlinearity of ZnOCu nanocomposites, with respect to Cu nanocolloids, is related to the interplay of plasmon band bleach and optical limiting mechanisms. The SA again changes back to RSA when we move over to the infrared region. The ZnOCu nanocomposites show self-defocusing nonlinearity and good nonlinear absorption behavior. The nonlinear refractive index and the nonlinear absorption increases with increasing Cu volume fraction at 532 nm. The observed nonlinear absorption is explained through two-photon absorption followed by weak free-carrier absorption and interband absorption mechanisms. This study is important in identifying the spectral range and composition over which the nonlinear material acts as a RSA-based optical limiter. ZnOCu is a potential nanocomposite material for the light emission and for the development of nonlinear optical devices with a relatively small limiting threshold.

On the luminescence characteristics of cerium and copper doped barium sulphide phosphor

Photoluminescence, thermoluminescence and phosphorescence studies of cerium and copper doped BaS phosphors are attempted. Cu+ centres in BaS lattice activate red emission while Ce3+ sensitize the blue emission. Results are explained on the basis of superposition theory involving monomolecular kinetics. In Randall and Wilkins model, the decay and TL studies are found to corelate each other.

Role of structural saturation and geometry in the luminescence of silicon-based nanostructured materials

The structural saturation and stability, the energy gap, and the density of states of a series of small, silicon-based clusters have been studied by means of the PM3 and some ab initio (HF/6-31G* and 6-311++G**, CIS/6-31G* and MP2/6-31G*) calculations. It is shown that in order to maintain a stable nanometric and tetrahedral silicon crystallite and remove the gap states, the saturation atom or species such as H, F, Cl, OH, O, or N is necessary, and that both the cluster size and the surface species affect the energetic distribution of the density of states. This research suggests that the visible luminescence in the silicon-based nanostructured material essentially arises from the nanometric and crystalline silicon domains but is affected and protected by the surface species, and we have thus linked most of the proposed mechanisms of luminescence for the porous silicon, e.g., the quantum confinement effect due to the cluster size and the effect of Si-based surface complexes.