Article pubs.acs.org/ac
Ligand Enabling Visible Wavelength Excitation of Europium(III) for Fluoroimmunoassays in Aqueous Micellar Solutions Timo Valta,*,†,§ Eeva-Maija Puputti,† Iko Hyppan̈ en,† Jouko Kankare,‡ Harri Takalo,†,∥ and Tero Soukka† †
Department of Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland Department of Chemistry, University of Turku, FI-20014 Turku, Finland
‡
ABSTRACT: Fluorescent reporters based on lanthanide ions, such as europium chelates, enable highly sensitive detection in immunoassays and other ligand binding assays. Unfortunately they normally require UV-excitation produced by a xenon flash or nitrogen laser light source. In order to use modern solid state excitation sources such as light emitting diodes (LEDs), these reporters need to be excited at wavelengths longer than 365 nm, where high-powered ultraviolet LEDs are available. A novel ligand, 9-ethyl-3,6-bis(5′,5′,5′,4′,4′-pentafluoro-1′,3′dioxopentyl)carbazole (bdc), was synthesized to efficiently excite europium(III) at wavelengths up to 450 nm in micellar solutions, and its performance was compared to a commercially available DELFIA enhancement solution. The detection limit of Eu(III) with the bdc-ligand using 365 nm excitation was determined to be 63 fM, which is 3 times lower than with the DELFIA solution. The bdc-ligand enabled sensitive detection of europium(III) ions in solution using 365 nm excitation and displayed similar sensitivity and functionality as commercially available DELFIA enhancement solution. Therefore, this novel enhancement solution might be a feasible alternative in producing time-resolved fluorescence under LED-excitation.
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TOPO. Together with the detergent (Triton X-100), these chelates form highly fluorescent micellar complexes.4 The drawback of this methodology is the UV excitation ( 3) signal from all the wells containing Eu(III)-SA, either due to the binding of Eu-SA through bio-BSA or unspecific binding. Using either DES with a 340 nm excitation or bdc-based enhancement solution with a 365 nm excitation resulted in the highest S/Nratios that were almost identical (Figure 4). This indicates that
Figure 3. Excitation spectra of the enhancement solutions with a 5−40 μM bdc-ligand concentration and DES solution, both with 1 μM Eu(III). Different bdc-ligand concentrations are 5.00 μM (solid black line), 7.50 μM (dashed line), 10.0 μM (dashed dotted), 20.0 μM (dashed dotted dotted), 40.0 μM (dotted line), and DES (solid gray line). a.u., arbitrary unit.
affected by the concentration of the ligand. Excitation efficiency in this area increased in comparison to the 335 nm peak with the increased ligand concentration. There is no experimental data to explain what causes this, but we speculate that a high ligand concentration leads to a situation where a single ligand molecule participates in the chelation of only one Eu(III) ion whereas with lower ligand concentrations single ligand may be attached to two different ions. Fluorescence Intensities and Sensitivities. The intensities and sensitivities were measured with a plate reader and from 96-well microtiter plates. The 7.5 μM bdc-ligand concentration was chosen for further comparison because it gave the highest signal when measured from a microtiter well (data not shown). Apparently the higher absorbance in the higher ligand concentrations interferes with the epifluorescence measurement and reduces the detected signal. When the different enhancement solutions were excited with a 340 nm wavelength, which was optimal for DES, the bdc-based enhancement solution with 7.50 μM ligand concentration gave on average a 2.5-fold higher specific signal than those determined with DES. When the excitation wavelength was changed to 365 nm, this ratio was increased to 3.0. The sensitivities of both enhancement solutions were determined with titration of Eu(III). The detection limits (3σ) are listed in Table 2. The results were identical with incubation times of 5 and 15 min.
Figure 4. Signal-to-noise ratios obtained with the heterogeneous binding assay for biotinylated BSA using Eu(III)-labeled streptavidin as a tracer. The enhancement of Eu(III) was done by using either commercial DES or bdc-based enhancement solution. The different symbols represent DES with 340 nm excitation (■), DES with 365 nm excitation (□), bdc with 340 nm excitation (●), and bdc with 365 nm excitation (○).
the bdc-enhancement solution excited at 365 nm works equally well as DES excited at its optimal wavelength in the ligand binding assay. Therefore the bdc enhancement solution might be a feasible alternative to be used with LED excitation.
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CONCLUSIONS We have demonstrated the synthesis and usage of a new ligand that can be used to efficiently excite Eu(III) ions in visible wavelengths in order to produce intense long lifetime fluorescence. The sensitivities achieved with the new ligand in the detection of Eu(III) ions are competitive with a commercially available DELFIA enhancement solution that requires excitation in the higher energy UV-range. The ability to excite europium chelates in visible wavelengths opens up the possibility to use light emitting diodes as an excitation source. With the addition of a semiconductor detector, such as a photodiode, it would be possible to utilize lanthanide chelates in miniaturized assay formats or even in home testing using hand-held fluorescence readers. The evaluation of this feature will be the focal point of future research.
Table 2. Detection Limits for Eu(III) and Relative Intensities with 1.0 μM Eu(III) in DES and bdc-Based Enhancement Solution When Excited at a 340 or 365 nm Wavelengtha relative intensity
a
enhancement solution
excitation maximum (nm)
DES 7.5 μM bdc
340 335, 380
LOD (fM)
340 nm 365 nm 340 nm 365 nm 1.0 2.5
0.4 3.0
32 64
190 63
LOD, limit of detection. 7711
dx.doi.org/10.1021/ac3008913 | Anal. Chem. 2012, 84, 7708−7712
Analytical Chemistry
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail: timo.valta@utu.fi. Phone: +49-621-759-3447. Present Addresses
§ Roche Diagnostics GmbH, Sandhofer Straße 116, D-68305 Mannheim, Germany. ∥ DHR Finland Oy, Innotrac Diagnostics Oy, Biolinja 12, 20750 Turku, Finland.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by the Finnish Funding Agency for Technology and Innovation (TEKES). REFERENCES
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dx.doi.org/10.1021/ac3008913 | Anal. Chem. 2012, 84, 7708−7712
