Ewa M. Goldys

Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence

Upconversion nanocrystals convert infrared radiation to visible luminescence, and are promising for applications in biodetection, bioimaging, solar cells and three-dimensional display technologies. Although the design of suitable nanocrystals has improved the performance of upconversion nanocrystals, their emission brightness is limited by the low doping concentration of activator ions needed to avoid the luminescence quenching that occurs at high concentrations. Here, we demonstrate that high excitation irradiance can alleviate concentration quenching in upconversion luminescence when combined with higher activator concentration, which can be increased from 0.5 mol% to 8 mol% Tm(3+) in NaYF₄. This leads to significantly enhanced luminescence signals, by up to a factor of 70. By using such bright nanocrystals, we demonstrate remote tracking of a single nanocrystal with a microstructured optical-fibre dip sensor. This represents a sensitivity improvement of three orders of magnitude over benchmark nanocrystals such as quantum dots.

Non-specific cellular uptake of surface-functionalized quantum dots

We report a systematic empirical study of nanoparticle internalization into cells via non-specific pathways. The nanoparticles were comprised of commercial quantum dots (QDs) that were highly visible under a fluorescence confocal microscope. Surface-modified QDs with basic biologically significant moieties, e.g. carboxyl, amino, and streptavidin, were used, in combination with surface derivatization with polyethylene glycol (PEG) for a range of immortalized cell lines. Internalization rates were derived from image analysis and a detailed discussion about the effect of nanoparticle size, charge and surface groups is presented. We find that PEG derivatization dramatically suppresses the non-specific uptake while PEG-free carboxyl and amine functional groups promote QD internalization. These uptake variations displayed a remarkable consistency across different cell types. The reported results are important for experiments concerned with cellular uptake of surface-functionalized nanomaterials, both when non-specific internalization is undesirable and when it is intended for material to be internalized as efficiently as possible.

Plasmonic Approach to Enhanced Fluorescence for Applications in Biotechnology and the Life Sciences

One of the most rapidly growing areas of physics and nanotechnology is concerned with plasmonic effects on the nanometer scale; these have applications in sensing and imaging technologies. Nanoplasmonic colloids such as Ag and Au have been attracting active interest, and there has been a recent explosion in the use of these metallic nanostructures to modify the spectral properties of fluorophores favorably and to enhance the fluorescence emission intensity. In this feature article, we summarize our work over a range of nanoplasmonics-assisted biological applications such as flow cytometry, immunoassays, cell imaging and bioassays where we use custom-designed plasmonic nanostructures (Ag and Au) to enhance fluorescence signatures. This fluorophore-metal effect offers unique advantages in providing improved photostability and enhanced fluorescence signals. We discuss the plasmonic enhancement of lanthanide fluorophores whose long and microsecond lifetimes offer the advantage of background-free fluorescence detection, but low photon cycling rates lead to poor brightness. We also show that plasmonic colloids are capable of enhancing the emission of fluorescent nanoparticles, including upconverting nanocrystals and lanthanide nanocomposites.

Metal-enhanced fluorescence in the life sciences: here, now and beyond.

We discuss the phenomenon of enhanced fluorescence in the proximity of metal nanostructures addressing the question of how much fluorescence signal can be obtained from fluorophores in such altered environments. We review its applicability for the methodologies used in the life science, such as immunoassays, flow cytometry and bioimaging. Experimental and theoretical scenarios employing various metal nanostructures - such as homogeneous enhancing substrates, fluorescence-enhancing microbeads, and metal core-dielectric shell nanocomposites - are described.

Fluorescence upconversion in Sm-doped Gd2O3

We report the observation of efficient fluorescence upconversion in Sm-doped Gd2O3 nanopowders prepared by the spray pyrolysis method. The blue upconversion emission was observed with low-power continuous-wave excitation at 514, 561, 594, and 633nm and with a pulsed femtosecond at 710nm, in a laser scanning confocal microscope. This result indicates that Sm-doped Gd2O3 has the potential as a fluorescent label that may be excited in red, yellow, and green with blue emission.

On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays

Significant multiplexing capacity of optical time-domain coding has been recently demonstrated by tuning luminescence lifetimes of the upconversion nanoparticles called ‘τ-Dots’. It provides a large dynamic range of lifetimes from microseconds to milliseconds, which allows creating large libraries of nanotags/microcarriers. However, a robust approach is required to rapidly and accurately measure the luminescence lifetimes from the relatively slow-decaying signals. Here we show a fast algorithm suitable for the microsecond region with precision closely approaching the theoretical limit and compatible with the rapid scanning cytometry technique. We exploit this approach to further extend optical time-domain multiplexing to the downconversion luminescence, using luminescence microspheres wherein lifetimes are tuned through luminescence resonance energy transfer. We demonstrate real-time discrimination of these microspheres in the rapid scanning cytometry, and apply them to the multiplexed probing of pathogen DNA strands. Our results indicate that tunable luminescence lifetimes have considerable potential in high-throughput analytical sciences.

Upconversion in NaYF4:Yb, Er nanoparticles amplified by metal nanostructures

Upconversion (UC) fluorescence in NaYF(4):Yb, Er nanoparticles amplified by metal nanostructures was compared in two nanostructure geometries: gold nanoshells surrounding nanoparticles and silver nanostructures adjacent to the nanoparticles, both placed on a dielectric silica surface. Enhanced UC luminescence signals and modified lifetimes induced by these two metals were observed in our study. The UC luminescence intensities of green and red emissions were enhanced by Ag nanostructures by a factor of approximately 4.4 and 3.5, respectively. The corresponding UC lifetimes were reduced ∼ 1.7-fold and ∼ 2.4-fold. In NaYF(4):Yb, Er nanoparticles encapsulated in gold nanoshells, higher luminescence enhancement factors were obtained (∼9.1-fold for the green emission and ∼ 6.7-fold for the red emission). However, the Au shell coating extended the red emission by a factor of 1.5 and did not obviously change the lifetime of green emission. The responsible mechanisms such as plasmonic enhancement and surface effects are discussed.

Use of fluorescence spectroscopy to differentiate yeast and bacterial cells

This study focuses on the characterization of bacterial and yeast species through their autofluorescence spectra. Lactic acid bacteria (Lactobacillus sp.), and yeast (Saccharomyces sp.) were cultured under controlled conditions and studied for variations in their autofluorescence, particularly in the area representative of tryptophan residues of proteins. The emission and excitation spectra clearly reveal that bacterial and yeast species can be differentiated by their intrinsic fluorescence with UV excitation. The possibility of differentiation between different strains of Saccharomyces yeast was also studied, with clear differences observed for selected strains. The study shows that fluorescence can be successfully used to differentiate between yeast and bacteria and between different yeast species, through the identification of spectroscopic fingerprints, without the need for fluorescent staining.

Simultaneous Concentration and Separation of Proteins in a Nanochannel

Molecular separation technologies such as gel electrophoresis and liquid chromatography coupled with mass spectrometry detection have been the foundations of biomarker discovery. This is because medically significant biomarkers, for example in blood, can be as much as 10 fold less common than the most abundant protein, albumin and detecting these low abundance molecules requires high sensitivity and selective depletion of the dominant species. Conventional approaches including antibody depletion remove selected molecules by less than 3 orders of magnitude only. This prevents the isolation, characterization and discovery of millions of new proteins where key disease markers could be identified. Overcoming this barrier requires new approaches to analytical detection that minimize sample pre-processing steps while achieving high throughput with very high levels of sensitivity. Here we describe a new device that demonstrates simultaneous concentration and separation of proteins by conductivity gradient focusing without membranes, external pumps, temperature gradients or ampholytes. Concentration and separation take place in an electric field driven, 120-nm deep nanochannel, supporting a stable salt and conductivity gradient. Conductivity gradient focusing is one of many techniques that use opposing convective flow and electrophoretic forces to focus molecules to an equilibrium position. These methods include a step change in chromatographic packings, electrochromatorgraphy, varying the molecular charge (as in isoelectric focusing), temperature gradient focusing, varying the cross section through which the electric current flows, and varying the buffer conductivity. In contrast to all of these approaches, the device presented here does not require ampholytes, matrices or gels, membranes, temperature gradients, or an external pump. Electrokinetic phenomena at the nanoscale have recently been shown to produce rapid and high preconcentration of proteins and peptides in physiological buffers. In these reports nanochannels in microfluidic devices create gradients in the electric field by their charge selective transport characteristics. By combining this with a transport mechanism, often electro-osmosis, charged molecules can be trapped and accumulated owing to a balance in the viscous drag force and the electrophoretic force. The interaction of surface charges, mobile charges, and water molecules with each other and the electric field is complex but our understanding has been advanced by a number of excellent fundamental studies. Concentration polarization, as it is sometimes known, at the entrance to a nanochannel, gives rise to a gradient in the concentration of salt ions, which, in turn, perturbs the electric field creating a trap. Typically these traps cause sample stacking on the microchannel side of the microto nanochannel junction. In such cases the electric field gradient is very abrupt, causing all molecules to accumulate in a tightly confined region with limited scope for separation.

Ultrabright Eu–Doped Plasmonic Ag@SiO2 Nanostructures: Time-gated Bioprobes with Single Particle Sensitivity and Negligible Background

Fluorescence has become one of the key detection methods in genomics, proteomics and cell biology and its applications extend as far as biomedical diagnostics. [ 1–4 ] Advances in this area have been accelerated by the development of new nanotechnology-inspired bioprobes such as quantum dots and silica or polymer encapsulated nanoparticles as well as smart detection strategies. [ 2 , 3–7 ] In parallel, photonic techniques have been pursued to take advantage of special optical properties, such as long fl uorescence decay times or infrared multi-photon excitation wavelength (to name just a few) to suppress background noise in fl uorescence detection. [ 2 , 8 ] However, these developments have often occurred in isolation from one another, and, as a result, an ideal fl uorescence detection strategy is yet to be developed. It should combine ultrabright bioprobes that have high absorption coeffi cients and high quantum yields and are clearly distinguishable and stable (free from bleaching and/or blinking). It should also provide high signal to noise ratio achievable by using photonics approaches such as timegating. Lanthanide fl uorophores with their long lifetimes are desirable as bioprobes for ultrasensitive detection, since they can be used in a time-gating mode that offers exceptionally high background rejection. [ 5 , 9–12 ] However the fl uorescence intensity of all lanthanide-based fl uorophores is very low compared with traditional fl uorescent dyes, due to long (ms) fl uorescence lifetimes. Recent efforts have been made to encapsulate lanthanide-based molecular complexes into nanoparticles, which produce amplifi ed signals due to increased number of lanthanide ions per nanoparticle, [ 7 ] but the limitation of slow radiative rates remains. The application of metallic nanostructures, particularly silver, provides a way to favorably modify the luminescence properties of close lanthanide fl uorophores and to alleviate their classical constraints. [ 13 ] The metal-enhanced fl uorescence (MEF) effect