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.

Epäorgaanisten nanopartikkeleiden muokkaaminen orgaanisten yhdisteiden avulla

Epäorgaanisten nanopartikkeleiden dispersioita käytetään laajasti mm. paperi-, maali- ja muoviteollisuudessa esim. optisten, termisten ja mekaanisten ominaisuuksien parantamiseen sekä kustannustehokkuuden nostamiseen. Viime vuosina nanokokoisten partikkeleiden käyttö on kasvanut myös biologisissa sovelluksissa. Dispersioiden parhaan toimivuuden kannalta ensisijaisen tärkeitä ovat niiden pysyvyys ja dispergoitumistaso. Primääristen partikkeleiden yhteenliittyminen eli agglomeraatio johtaa usein lopputuotteen huomattavaan ominaisuuksien huononemiseen. Partikkeleiden yhteenliittyminen voidaan estää neljällä eri tavalla: elektrostaattisesti, steerisesti, elektrosteerisesti ja tyhjennysstabilisaation avulla. Kolmessa viimeisessä tavassa stabilisaatio perustuu partikkeleiden pinnalle adsorboituvien tai liuoksessa vapaana olevien orgaanisten yhdisteiden keskinäisiin repulsiovuorovaikutuksiin. Eniten tutkitut stabilisaatiotavat ovat steerinen ja elektrosteerinen stabilisaatio. Niissä partikkelin pinnalle adsorboitunut orgaanisista molekyyleista koostuva kerros mahdollistaa partikkeleiden välisen repulsion. Adsorboituneen kerroksen ominaisuuksien luotettavalla määrityksellä on siis merkittävä osa dispersioiden stabiiliuden tutkimuksessa. Määrityksiin käytettävät tekniikat perustuvat mm. titrimetriaan, UV-vis-spektroskopiaan, FTIR-spektrofotometriaan tai termogravimetriaan. Työn teoriaosiossa esitetään tärkeimmät dispersioissa esiintyvät primääriset vuorovaikutukset (Lifshitz-van der Waals-voimat, happo–emäs-vuorovaikutukset, elektrostaattiset vuorovaikutukset ja Brownin liikkeen aikaansaamat vuorovaikutukset), minkä jälkeen käydään tarkemmin läpi niiden aikaansaamat stabilointimekanismit. Kolmannessa osiossa keskitytään adsorboituneiden pien- ja makromolekyylien pitoisuuksien, konformaatioiden ja kerrospaksuuksien määrittämiseen soveltuviin menetelmiin. Työn kokeellisessa osiossa etsitään luotettavaa menetelmää nano-CaCO3-partikkeleiden pinnalle adsorboituneen kaupallisen stabilointipolymeerin pitoisuuden määrittämiseksi. Tähän tarkoitukseen sovelletaan FTIR-spektrofotometriaan, UV-vis-spektroskopiaan ja titrimetriaan perustuvia menetelmiä. Tulokset osoittavat, että adsorptio on todennettavissa UV-vis – ja titrimetriamenetelmillä, mutta adsorboituneen makromolekyylin määrän määrittäminen onnistuu vain titrimetrisesti.

Development of 3D super-resolution tomographic STED microscopy and its application to studies on bone resorption

In this doctoral thesis, a tomographic STED microscopy technique for 3D super-resolution imaging was developed and utilized to observebone remodeling processes. To improve upon existing methods, wehave used a tomographic approach using a commercially available stimulated emission depletion (STED) microscope. A certain region of interest (ROI) was observed at two oblique angles: one at a standard inverted configuration from below (bottom view) and another from the side (side view) via a micro-mirror positioned close to the ROI. The two viewing angles were reconstructed into a final tomogram. The technique, named as tomographic STED microscopy, was able to achieve an axial resolution of approximately 70 nm on microtubule structures in a fixed biological specimen. High resolution imaging of osteoclasts (OCs) that are actively resorbing bone was achieved by creating an optically transparent coating on a microscope coverglass that imitates a fractured bone surface. 2D super-resolution STED microscopy on the bone layer showed approximately 60 nm of lateral resolution on a resorption associated organelle allowing these structures to be imaged with super-resolution microscopy for the first time. The developed tomographic STED microscopy technique was further applied to study resorption mechanisms of OCs cultured on the bone coating. The technique revealed actin cytoskeleton with specific structures, comet-tails, some of which were facing upwards and some others were facing downwards. This, in our opinion, indicated that during bone resorption, an involvement of the actin cytoskeleton in vesicular exocytosis and endocytosis is present. The application of tomographic STED microscopy in bone biology demonstrated that 3D super-resolution techniques can provide new insights into biological 3D nano-structures that are beyond the diffraction-limit when the optical constraints of super-resolution imaging are carefully taken into account.

Recognition of nucleic acids by metal ion complexes

Metal-ion-mediated base-pairing of nucleic acids has attracted considerable attention during the past decade, since it offers means to expand the genetic code by artificial base-pairs, to create predesigned molecular architecture by metal-ion-mediated inter- or intra-strand cross-links, or to convert double stranded DNA to a nano-scale wire. Such applications largely depend on the presence of a modified nucleobase in both strands engaged in the duplex formation. Hybridization of metal-ion-binding oligonucleotide analogs with natural nucleic acid sequences has received much less attention in spite of obvious applications. While the natural oligonucleotides hybridize with high selectivity, their affinity for complementary sequences is inadequate for a number of applications. In the case of DNA, for example, more than 10 consecutive Watson-Crick base pairs are required for a stable duplex at room temperature, making targeting of sequences shorter than this challenging. For example, many types of cancer exhibit distinctive profiles of oncogenic miRNA, the diagnostics of which is, however, difficult owing to the presence of only short single stranded loop structures. Metallo-oligonucleotides, with their superior affinity towards their natural complements, would offer a way to overcome the low stability of short duplexes. In this study a number of metal-ion-binding surrogate nucleosides were prepared and their interaction with nucleoside 5´-monophosphates (NMPs) has been investigated by 1H NMR spectroscopy. To find metal ion complexes that could discriminate between natural nucleobases upon double helix formation, glycol nucleic acid (GNA) sequences carrying a PdII ion with vacant coordination sites at a predetermined position were synthesized and their affinity to complementary as well as mismatched counterparts quantified by UV-melting measurements.

Research progress in wood-plastic composites: A review

The interest towards wood-plastic composites (WPCs) is growing due to growing interest in materials with novel properties, which can replace more traditional materials, such as wood and plastic. The use of recycled materials in manufacture is also a bonus. However, the application ofWPCs has been limited because of their often poor mechanical and barrier properties, which can be improved by incorporation of the reinforcing fillers. Nanosized fillers, having a large surface area, can significantly increase interfacial interactions in the composite on molecular level, leading to materials with new properties. The review summarizes the development trends in the use on nanofillers for WPC design, which were reported in accessible literature during the last decade. The effect of the nanofillers on the mechanical properties, thermal stability, flammability and wettability ofWPC is discussed.