EXAMPLE 1
Fluorexon was commercially obtained and used without further purification. The lanthanide ions were added from stock solutions of YbCl3.6H2O, NdCl3.6H2O, and ErCl3.6H2O in D2O or H2O. Fluorexon, a well-known fluorescence indicator for Ca2+ ions, was used to sensitize the near-IR (NIR) emission of trivalent ytterbium, neodymium, and erbium ions. Its absorption spectrum was similar to that of fluorescein, with an absorption maximum at 490 nm.
Solutions were prepared consisting of 5×10−6 M of the fluorexon and an equimolar amount of lanthanide ions (Yb3+, Nd3+, or Er3+) in D2O at pD 7. The pD was carefully controlled using an ISFET-based pH meter and concentrated solutions of DCl and NaOD.
The NlR luminescence excitation spectra of the respective Nd3+ and Er3+ complexes are identical to the spectrum of the fluorexon/Yb3+ complex, and all match the corresponding absorption spectra, with an excitation maximum at 490 nm. These results indicate that energy transfer from the fluorexon to the lanthanide ion is the dominant route to the observed rare-earth ion NIR luminescence and that this process is several orders of magnitude more efficient than direct excitation of the lanthanide. In the used concentration range no luminescence was observed when an absorption band of the rare-earth ion was excited.
EXAMPLE 2
Methylthymol blue was commercially obtained and used without further purification. The lanthanide ions were added from stock solutions of YbCl3.6H2O, NdCl3.6H2O and ErCl3.6H2O in D2O and H2O. Methylthymol blue (MTB) was demonstrated to be a luminescence sensitizing agent for ytterbium(III), which emits light in a band around 1000 nm.
Solutions of Yb3+ or Er3+ 1×10−5 M and 1×10−5 M MTB in D2O (pD 5) and H2O (pH 5) were prepared. When ytterbium or erbium ions were added to the solution of MTB, the color changed from yellow to blue, which indicates the formation of a complex between MTB and the ion. The spectra in H2O are similar, but the luminescence is less intense due to more efficient quenching of the Yb3+ excited state by H2O.
MTB does not sensitize Nd(III). This was demonstrated in an experiment where both fluorexon (see Example 1) and MTB were brought into contact with Nd3+ ions. From the absorption spectrum it was clear that both complexed MTB and fluorexon were present, but in the luminescence excitation spectrum (emission at 1060 nm, the principal Nd3+ emission line) only fluorexon sensitization was observed.
EXAMPLE 7
DTPAA was commercially obtained and used without further purification. 5-aminoeosin was commercially purchased. The lanthanide ions were added from stock solutions of YbCl3.6H2O, NdCl3.6H2O and ErCl3.6H2O in D2O and H2O.
The procedure for the preparation of AMFLU-DTPA was repeated using 5-aminoeosin instead of 5-aminofluorescein and yielded AMEO-DTPA, which has its absorption maximum at 515 nm.
The 1:1 complexation was confirmed in an experiment similar to that used in Example 1. The luminescence excitation and emission spectra of AMEO-DTPA/Ln3+ complexes were recorded under the same conditions as the corresponding AMFLU-DTPA complexes and showed similar photophysical characteristics. For the Nd3+ and Yb3+ complexes, luminescence was also observed in H2O (Tris/HCl, pH 8).
General: Characterization Methods
Spectroscopic Measurements
Steady state luminescence measurements were performed on a PTI Alphascan spectrofluorimeter, using a 75-W quartz-tungsten-halogen lamp followed by a SPEX 1680 double monochromator for excitation and a PTI 0.25-m single monochromator for separation of the emitted light, detected under an angle of 90°. The emitted light was converted into an electric signal with a Northcoast 817L liquid nitrogen cooled Germanium detector. For detection a lock-in amplifier (SRS530) was applied; the excitation light was modulated at 70 Hz with an optical chopper. For time-resolved luminescence measurements an Edinburgh Analytical Instruments LP900 system was used, which consisted of a pulsed Xe-lamp followed by a 0.25 m monochromator for excitation and another 0.25 m monochromator positioned at an angle of 90° with respect to the first for separation of the emitted light. The photons were converted into electric signals by means of a Northcoast 817P liquid nitrogen cooled germanium detector (lifetimes >250 ns) or, alternatively, via a Hamamatsu R2658 thermoelectrically cooled photomultiplier tube (lifetimes<250 ns), and fed to a Tektronix fast digital oscilloscope.
