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.Käänteisviritteinen fotoluminesenssi on lähinnä tiettyjen epäorgaanisten nanomateriaalien ainutlaatuinen ominaisuus, jossa matalaenergistä infrapunasäteilyä muutetaan korkeampienergiseksi näkyvän aallonpituuden emissioksi. Tämä Stokesin siirtymän vastainen fotoluminesenssi mahdollistaa mittaamisen ilman autofluoresenssia, minkä takia käänteisviritetyt materiaalit soveltuvat leimoiksi optisiin biosensorisovelluksiin. Lisäksi ne tuottavat pitkäikäistä emissiota kapeilla kaistanleveyksillä myös aallonpituusalueilla, jotka biomateriaaleissa ovat optisesti läpinäkyviä, ja mahdollistavat näin luminesenssimittaukset myös hankalista näytemateriaaleista, kuten kokoverestä. Väitöskirjan tarkoituksena oli tutkia nanokokoisten käänteisviritteisten partikkelien ainutlaatuisia ominaisuuksia ja niiden käyttöä leimoina biosensorisovelluksissa. Nanopartikkelit pinnoitettiin silikalla ja analyyttejä tunnistavilla biomolekyyleillä. Partikkelien sisältämien lantanidi-ionien paramagneettisuutta käytettiin hyväksi kehitettäessä erittäin selektiivistä erotusmenetelmää biomolekyylipinnoitettujen partikkeleiden puhdistusta varten. Menetelmä perustui korkean gradientin magneettiseen erotteluun. Nanopartikkeleiden käyttöä leimoina hankalissa näytemateriaaleissa tutkittiin kehittämällä kaksi käänteisviritteisen luminesenssin resonanssienergiansiirtoon perustuvaa, erotusvapaata sovellusta. Väitöskirjassa kehitettiin kemiallinen sensori solunsisäisen pH:n tutkimiseen. Sensori perustui resonanssienergiansiirtoon nanopartikkelin ja pH-herkän pienmolekyylivärin välillä. pH-nanosensorin soveltuvuutta solunsisäisen pH:n mittaamiseen tutkittiin HeLa-soluilla, joiden endosomien hapan pH pystyttiin havaitsemaan fluoresenssimikroskoopin avulla. Väitöskirjassa kehitettiin myös kilpaileva pesuvaiheeton määritys, jolla punasolujen foolihappopitoisuus voidaan mitata suoraan kokoverinäytteestä. Biologisten materiaalien optisesti läpinäkyviä valon aallonpituuksia käytettiin sekä käänteisviritteisten nanopartikkelien virittämiseen että resonanssienergiansiirron vastaanottajapienvärin emission mittaamiseen, jotta pystyttiin minimoimaan kokoverinäytteen valon absorptio. Lisäksi Stokesin siirtymän vastainen fotoluminesenssi mahdollisti mittaamisen ilman autofluoresenssia. Käänteisviritteisen fotoluminesenssin tehokkuuden tiedetään olevan kidekokoriippuvainen, koska nanokokoisten partikkeleiden suuri pinnan ja tilavuuden välinen suhde altistaa partikkelit ympäristön luminesenssia sammuttaville vaikutuksille. Vesi sammuttaa tehokkaasti lantanidien luminesenssia, ja väitöskirjassa tutkittiin sen mekanismia tarkemmin luminesenssin elinikämittausten avulla. Veden havaittiin sammuttavan voimakkaasti käänteisviritteisten nanopartikkeleiden luminesenssia lisäämällä Yb3+ herkistimen virittyneen tilan säteilemätöntä purkautumistaSiirretty Doriast

Investigating dye performance and crosstalk in fluorescence enabled bioimaging using a model system

Detailed imaging of biological structures, often smaller than the diffraction limit, is possible in fluorescence microscopy due to the molecular size and photophysical properties of fluorescent probes. Advances in hardware and multiple providers of high-end bioimaging makes comparing images between studies and between research groups very difficult. Therefore, we suggest a model system to benchmark instrumentation, methods and staining procedures. The system we introduce is based on doped zeolites in stained polyvinyl alcohol (PVA) films: a highly accessible model system which has the properties needed to act as a benchmark in bioimaging experiments. Rather than comparing molecular probes and imaging methods in complicated biological systems, we demonstrate that the model system can emulate this complexity and can be used to probe the effect of concentration, brightness, and cross-talk of fluorophores on the detected fluorescence signal. The described model system comprises of lanthanide (III) ion doped Linde Type A zeolites dispersed in a PVA film stained with fluorophores. We tested: F18, MitoTracker Red and ATTO647N. This model system allowed comparing performance of the fluorophores in experimental conditions. Importantly, we here report considerable cross-talk of the dyes when exchanging excitation and emission settings. Additionally, bleaching was quantified. The proposed model makes it possible to test and benchmark staining procedures before these dyes are applied to more complex biological systems

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging

NIR induced modulation of the red emission from erbium ions for selective lanthanide imaging

Upon direct excitation with green light (522 nm), Er3+ ion doped nanoparticles feature a number of radiative and non-radiative decay pathways, leading to distinct and sharp emission lines in the visible and near-infrared (NIR) range. Here we apply, in addition to continuous 522 nm irradiation, a modulated NIR irradiation (1143 nm) to actively control and modulate the red emission intensity (around 650 nm). The modulation of red Er3+ ion emission at a chosen frequency allows us to reconstruct fluorescence images from the Fourier transform amplitude at this particular frequency. Since only the emission from the Er3+ ion is modulated, it allows to selectively recover the lanthanide specific signal, removing any non-modulated auto-fluorescence or background emission resulting from the continuous 522 nm excitation. The modulated emission of specific lanthanides can open up new detection opportunities for selective signal recovery

Electronic Energy Levels of Dysprosium(III) ions in Solution. Assigning the Emitting State and the Intraconfigurational 4f-4f Transitions in the Vis-NIR Region and Photophysical Characterization of Dy(III) in Water, Methanol, and Dimethyl Sulfoxide

Dysprosium­(III) ions are the third most luminescent lanthanide­(III) ions. Dy­(III) is used as dopant in optical fibers and as shift reagent in NMR imaging and is the element at the forefront of research in single-molecule magnets. Nonetheless, the excited state manifold of the dysprosium­(III) ion is not fully mapped and the nature of the emitting state has not been unequivocally assigned. In the work reported here, the photophysical properties of dysprosium­(III) triflate dissolved in H2O, MeOH, and DMSO have been studied in great detail. The solvates are symmetric, all oxygen donor atom complexes where the coordination number is 8 or 9. By comparing protonated and deuterated solvents, performing variable temperature spectroscopy, and determining the excited state lifetimes and luminescence quantum yields, the solution structure can be inferred. For the three complexes, the observed electronic energy levels were determined using absorption and emission spectroscopy. The Dy­(III) excited state manifolds of the three solvates differ from that reported by Carnall, in particular for the low lying 6F-states. It is shown that dysprosium­(III) complexes primarily luminesce from the 4F9/2 state, although thermal population of, and subsequent luminescence from the 4I15/2 state is observed. The intrinsic luminescence quantum yield is moderate (∼10%) in DMSO-d6 and is significantly reduced in protonated solvent as both C–H and O–H oscillators act as efficient quenchers of the 4F9/2 state. We are able to conclude that the emitting state in dysprosium­(III) is 4F9/2, that the mJ levels must be considered when determining electronic energy levels of dysprosium­(III), and that scrutiny of the transition probabilities may reveal the structure of dysprosium­(III) ions in solution

Versatile and Validated Optical Authentication System Based on Physical Unclonable Functions

Counterfeit consumer products, electronic components, and medicines generate heavy economic losses, pose a massive security risk, and endanger human lives on a daily basis. Combatting counterfeits requires incorporation of uncopiable or unclonable features in each and every product. By exploiting the inherent randomness of stochastic processes, an optical authentication system based on physical unclonable functions (PUFs) was developed. The system relies on placing unique tagsPUF-tagson the individual products. The tags can be created using commercial printing and coating technologies using several combinations of carrier materials and taggant materials. The authentication system was found to be independent of how contrast was generated, and examples of PUF-tags based on scattering, absorption, and luminescence were made. A version of the authentication using the combination of scattering-based PUF-tags and a smartphone-based reader was validated on a sample size of 9720 unique codes. With zero false positives in 29 154 matches, an encoding capacity of 2.5 × 10120, and a low cost of manufacture, the scattering-based authentication system was found to have the potential to solve the problem of counterfeit products