Terhi Rantanen

Photochemical Characterization of Up-Converting Inorganic Lanthanide Phosphors as Potential Labels

We have characterized commercially available up-converting inorganic lanthanide phosphors for their rare earth composition and photoluminescence properties under infrared laser diode excitation. These up-converting phosphors, in contrast to proprietary materials reported earlier, are readily available to be utilized as particulate reporters in various ligand binding assays after grinding to submicron particle size. The laser power density required at 980 nm to generate anti-Stokes photoluminescence from these particulate reporters is significantly lower than required for two-photon excitation. The narrow photoluminescence emission bands at 520–550 nm and at 650–670 nm are at shorter wavelengths and thus totally discriminated from autofluorescence and scattered excitation light even without temporal resolution. Transparent solution of colloidal bead-milled up-converting phosphor nanoparticles provides intense green emission visible to the human eye under illumination by an infrared laser pointer. In this article, we show that the unique photoluminescence properties of the up-converting phosphors and the inexpensive measurement configuration, which is adequate for their sensitive detection, render the up-conversion an attractive alternative to the ultraviolet-excited time-resolved fluorescence of down-converting lanthanide compounds widely employed in biomedical research and diagnostics.

Fluorescence-Quenching-Based Enzyme-Activity Assay by Using Photon Upconversion†

Enzyme-activity assays are used, for example, for screening enzyme inhibitors and activators to discover novel drug candidates. A homogeneous assay principle for hydrolyzing enzymes based on a double-labeled fluorogenic substrate is commonly employed and is suitable for high-throughput screening. This separation-free assay concept relies on the strong distance dependency of fluorescence resonance energy transfer (FRET), which takes place only at distances below 10 nm. A synthetic internally quenched substrate for the enzyme is labeled with a fluorophore at one end and a quencher at the other end of the molecule. When the enzyme digests the substrate, the two labels are separated and fluorescence is recovered. The performance of fluorescence-quenching-based homogeneous assays is still limited due to the autofluorescence originating from biological materials. This problem can be solved by a novel label technology based on upconverting phosphors (UCPs), which have the unique property of photoluminescence emission at visible wavelengths under near-infrared (NIR) excitation. No autofluorescence is detected at shorter wavelengths, because the upconversion phenomenon requires sequential multiphoton absorption not observed in nature. Due to the NIR excitation, UCP technology is also applicable to strongly colored samples (for example, whole blood), which absorb at ultraviolet and visible wavelengths, a process that interferes with other fluorescence technologies. The aim of this study was to combine the advantageous features of UCP donors and fluorescence-quenching assays to construct a sensitive enzyme-activity assay. Wang et al. have quenched around 70% of the emission of nanosized UCPs by using gold particles. More efficient quenching, however, is required for a practical assay as described above since the best theoretical signal-to-background ratio with these components would be as poor as 3:1. It is not possible to entirely quench the anti-Stokes photoluminescence originating from multiple dopant ions within submicrometer-sized UCPs because only those emitter ions located near the surface of UCP can be quenched. We have now solved this problem with a sequential energy-transfer-based assay concept (Figure 1a).

Upconverting phosphors in a dual-parameter LRET-based hybridization assay

Upconverting phosphors (UCPs) are lanthanide-doped sub-micrometer-sized particles, which produce multiple narrow and well-separated anti-Stokes emission bands at visible wavelengths under infrared excitation (980 nm). The advantageous features of UCPs were utilized to construct a dual-parameter, homogeneous sandwich hybridization assay based on a UCP donor and lanthanide resonance energy transfer (LRET). UCPs with two emission bands (540 nm and 653 nm) were exploited together with two appropriate fluorophores as acceptors. The energy transfer excited emissions of the acceptors were measured at 600 nm and 740 nm without any significant interference from each other. The autofluorescence limitation associated with conventional fluorescence was totally avoided as the measurements were carried out at shorter wavelength relative to the excitation. In the sandwich hybridization assay two different single-stranded target-oligonucleotide sequences were detected simultaneously and quantitatively with a dynamic range from 0.03 to 0.4 pmol (corresponding 0.35-5.4 nM). The UCPs enable multiplexed homogeneous LRET-based assay requiring only a single excitation wavelength, which simplifies the detection and extends the applicability of upconversion in bioanalytical measurements.

Photon upconversion in homogeneous fluorescence-based bioanalytical assays.

Upconverting phosphors (UCPs) are very attractive reporters for fluorescence resonance energy transfer (FRET)‐based bioanalytical assays. The large anti‐Stokes shift and capability to convert near‐infrared to visible light via sequential absorption of multiple photons enable complete elimination of autofluorescence, which commonly impairs the performance of fluorescence‐based assays. UCPs are ideal donors for FRET, because their very narrow‐banded emission allows measurement of the sensitized acceptor emission, in principle, without any crosstalk from the donor emission at a wavelength just tens of nanometers from the emission peak of the donor. In addition, acceptor dyes emitting at visible wavelengths are essentially not excited by near‐infrared, which further emphasizes the unique potential of upconversion FRET (UC‐FRET). These characteristics result in favorable assay performance using detection instrumentation based on epifluorometer configuration and laser diode excitation. Although UC‐FRET is a recently emerged technology, it has already been applied in both immunoassays and nucleic acid hybridization assays. The technology is also compatible with optically difficult biological samples, such as whole blood. Significant advances in assay performance are expected using upconverting lanthanide‐doped nanocrystals, which are currently under extensive research. UC‐FRET, similarly to other fluorescence techniques based on resonance energy transfer, is strongly distance dependent and may have limited applicability, for example in sandwich‐type assays for large biomolecules, such as viruses. In this article, we summarize the essentials of UC‐FRET, describe its current applications, and outline the expectations for its future potential.