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.

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay.

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L(-1) and 1.3 ng L(-1) using two different up-converting phosphors and 0.16 ng L(-1) using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformly sized UCP-particles to increase the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays.

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.

Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol.

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.

Homogeneous assay technology based on upconverting phosphors.

Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.

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.

Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay.

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.

Lactobacilli carry cryptic genes encoding peptidase-related proteins: characterization of a prolidase gene (pepQ) and a related cryptic gene (orfZ) from Lactobacillus delbrueckii subsp. bulgaricus.

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp. lactis prolidase gene (pepQ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate specificity characteristic of prolidase enzymes, no enzymic activity for the orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used. It is noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.