Long-Lifetime Luminescent Europium(III) Complex as an Acceptor in an Upconversion Resonance Energy Transfer Based Homogeneous Assay

Long-lifetime luminescent Eu^III complexes are widely used as donors in Förster resonance energy transfer to enable time-gated detection of sensitized emission from an intrinsically short-lived acceptor. Here we report a unique energy-transfer system, where the sensitized acceptor emission has prolonged luminescence lifetime compared to the donor and the long lifetime is not cut short upon high energy-transfer efficiency. The infrared-excited, ultraviolet-emitting, Tm^III-doped upconverting nanoparticles were used as donors, and a luminescent Eu^III-chelate was used as an acceptor. Upon excitation the sensitized acceptor emission, which is already spectrally resolved from the donor, can be measured even after the donor luminescence has decayed. Because of anti-Stokes characteristics, the time-gated detection is not needed to avoid the autofluorescence. Thus, the long luminescence lifetime can be further modulated and utilized, e.g., in background-free molecular sensing, rendering the system extremely attractive

Homogeneous Detection of Avidin Based on Switchable Lanthanide Luminescence

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moietiesa light-absorbing antenna ligand and a lanthanide ion carrier chelatewere conjugated to separate biotins, which is known as avidin’s natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle

Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb³⁺-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb³⁺-LBP–GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases

Background-Free Referenced Luminescence Sensing and Imaging of pH Using Upconverting Phosphors and Color Camera Read-out

Fluorescence background and problems with proper signal referencing severely disrupt the read-out of luminescence sensors and images. We present a pH sensor film in combination with a simple read-out technique that overcomes issues of background signals and autofluorescence. It consists of micrometer-sized upconversion phosphors (UCPs) and a pH indicator (Neutral Red) that absorbs their green emission. Both are embedded in a proton permeable hydrogel matrix. The UCPs generate green and red luminescence upon excitation with IR light of 980 nm wavelength. Solely the green light of the UCPs is affected by the pH indicator, while the red emission acts as inert reference signal for ratiometric measurements. The emission peaks of the UCPs match the red and green color channels of standard digital cameras. Thereby, the devised sensor film can be used for referenced ratiometric sensing and 2D imaging of pH using a color camera read-out. The sensor setup using common and hand-held devices is cheap and straightforward and allows for point-of-care measurements. Finally, pH measurements in human serum samples show the potential of this sensor for imaging free of interfering background signals

Distance and Temperature Dependency in Nonoverlapping and Conventional Förster Resonance Energy-Transfer

Förster resonance energy-transfer (FRET) is a powerful and widely applied bioanalytical tool. According to the definition of FRET by Förster, for energy-transfer to take place, a substantial spectral overlap between the donor emission and acceptor excitation spectra is required. Recently also a phenomenon termed nonoverlapping FRET (nFRET) has been reported. The nFRET phenomenon is based on energy-transfer between a lanthanide chelate donor and a spectrally nonoverlapping acceptor and thus obviously differs from the conventional FRET, but the mechanism of nFRET and resulting implications to assay design have not been thoroughly examined. In this work, a homogeneous DNA-hybridization assay was constructed to study the distance and temperature dependency of both nFRET and conventional FRET. Capture oligonucleotides were labeled at the 5′-end with a Eu(III)-chelate, and these conjugates hybridized to complementary tracer oligonucleotides labeled with an organic fluorophore at various distances from the 3′-end. The distance dependency was studied with a fluorometer utilizing time-resolution, and the temperature dependency was studied using a frequency-domain (FD) luminometer. Results demonstrated a difference in both the distance and temperature dependency between conventional FRET and nFRET. On the basis of our measurements, we propose that in nFRET thermal excitation occurs from the lowest radiative state of the ion to a higher excited state that is either ionic or associated with a ligand-to-metal charge-transfer state

Environmental Impact on the Excitation Path of the Red Upconversion Emission of Nanocrystalline NaYF₄:Yb³⁺,Er³⁺

The mechanism for red upconversion luminescence of Yb–Er codoped materials is not generally agreed on in the literature. Both two-photon and three-photon processes have been suggested as the main path for red upconversion emission. We have studied β-NaYF₄:Yb³⁺,Er³⁺ nanoparticles in H₂O and D₂O, and we propose that the nanoparticle environment is a major factor in the selection of the preferred red upconversion excitation pathway. In H₂O, efficient multiphonon relaxation (MPR) promotes the two-photon mechanism through green emitting states, while, in D₂O, MPR is less effective and the three-photon path involving back energy transfer to Yb³⁺ is the dominant mechanism. For the green upconversion emission, our results suggest the common two-photon path through the ⁴F_9/2 energy state in both H₂O and D₂O

Effective Shielding of NaYF₄:Yb³⁺,Er³⁺ Upconverting Nanoparticles in Aqueous Environments Using Layer-by-Layer Assembly

Aqueous solutions are the basis for most biomedical assays, but they quench the upconversion luminescence significantly. Surface modifications of upconverting nanoparticles are vital for shielding the obtained luminescence. Modifications also provide new possibilities for further use by introducing attaching sites for biomolecule conjugation. We demonstrate the use of a layer-by-layer surface modification method combining varying lengths of negatively charged polyelectrolytes with positive neodymium ions in coating the upconverting NaYF₄:Yb³⁺,Er³⁺ nanoparticles. We confirmed the formation of the bilayers and investigated the surface properties with Fourier transform infrared and reflectance spectroscopy, thermal analysis, and ζ-potential measurements. The effect of the coating on the upconversion luminescence properties was characterized, and the bilayers with the highest improvement in emission intensity were identified. In addition, studies for the nanoparticle and surface stability were carried out in aqueous environments. It was observed that the bilayers were able to shield the materials’ luminescence from quenching also in the presence of phosphate buffer that is currently considered the most disruptive environment for the nanoparticles

Photon Upconversion in a Molecular Lanthanide Complex in Anhydrous Solution at Room Temperature

Molecular photon upconversion luminescence was observed from an ion-associated complex of an erbium chelate of 2-thenoyltrifluoroacetone and a near-infrared-emitting cyanine dye in anhydrous solution at room temperature. In the complex erbium was sensitized by the organic antenna dye excited at 808 nm. The result was characteristic erbium emission at 510–565 nm with second-order dependence on the excitation power, suggesting a dye-sensitized energy transfer upconversion mechanism. Compared to inorganic upconverting nanoparticles, the organic molecular dye-sensitized complexes are expected to offer higher molar absorptivity, smaller well-defined size, and simpler addition of functional groups

Disintegration of Hexagonal NaYF₄:Yb³⁺,Er³⁺ Upconverting Nanoparticles in Aqueous Media: The Role of Fluoride in Solubility Equilibrium

The disintegration of hexagonal NaYF₄:Yb³⁺,Er³⁺ upconverting nanoparticles (UCNP) was studied by incubating various nanoparticle concentrations in aqueous suspensions over time while monitoring the upconversion emission intensity and measuring the dissolved particle-constituting ion concentrations. The results revealed that the ions dissolved into water resulting apparently in anisotropic structural disintegration of the UCNPs as observed with transmission electron microscopy. The UCNP disintegration caused partial loss of active ions Yb³⁺ and Er³⁺ from the host matrix and therefore decrease in the upconversion luminescence intensity. The decrease, however, was strongly dependent on the UCNP concentration, and dramatic drop in the intensity was observed especially at diluted nanoparticle suspensions, where the nanoparticles disintegrated almost completely until the solubility equilibrium was achieved. At the concentrated suspensions the equilibrium was achieved already with minimal disintegration, and the change in the luminescence intensity was negligible. Further, due to the high impact of fluoride ions on the solubility equilibrium the disintegration of the UCNPs could be prevented by adding fluoride to the suspension. The reported disintegration of NaYF₄:Yb³⁺,Er³⁺ nanoparticles in diluted aqueous suspensions should be taken into consideration when the UCNPs are used at low concentrations in analytical applications and in guiding the design of improved shell-stabilized UCNPs

Quantitative Multianalyte Microarray Immunoassay Utilizing Upconverting Phosphor Technology

A quantitative multianalyte immunoassay utilizing luminescent upconverting single-crystal nanoparticles as reporters on an antibody array-in-well platform was demonstrated. Upconverting nanoparticles are inorganic rare earth doped materials that have the unique feature of converting low energy infrared radiation into higher energy visible light. Autofluorescence, commonly limiting the sensitivity of fluorescence-based assays, can be completely eliminated with photon upconversion technology because the phenomenon does not occur in biological materials. Biotinylated antibodies for three analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone) were printed in an array format onto the bottom of streptavidin-coated microtiter wells. Analyte dilutions were added to the wells, and the analytes were detected with antibody-coated upconverting nanoparticles. Binding of the upconverting nanoparticles was imaged with an anti-Stokes photoluminescence microwell imager, and the standard curves for each analyte were quantified from the selected spot areas of the images. Single analyte and reference assays were also carried out to compare with the results of the multianalyte assay. Multiplexing did not have an effect on the assay performance. This study demonstrates the feasibility of upconverting single-crystal nanoparticles for imaging-based detection of quantitative multianalyte assays