1676
Anal. Chem. lQ85, 57, 1676-1681
358-nm excitation wavelength in the 2000-cm-' scan, 2methylanthracene can be identified from the spectral peak at the 380 nm, 411 nm excitation, emission wavelength pair. It is of interest to note the increase in the spectral complexity of the 2000-cm-' scan in Figure 4B over the 1400-cm-' scan in Figure 4A. The overall spectral complexity of these mixtures increased as the constant energy difference varied from 1400 cm-' to 2000 cm-'. For the PAH mixtures analyzed, the 1400-crnL1scan was the best choice for obtaining a spectrally reduced LTCESFS spectrum and spectrally separated all but two compounds. The selection of other constant energy differences for scanning aided in the separation of PAHs and was used for this purpose. LTCESFS offers a method for analysis of mixtures of PAH isomers and alkyl homologues. Registry No. Anthracene, 120-12-7; 2-methylanthracene, 613-12-7;9-methylanthracene,779-02-2;9,10-dimethylanthracene, 781-43-1; benz[a]anthracene, 56-55-3;pyrene, 129-00-0;benzo[alpyrene, 50-32-8; benzo[b]fluoranthene, 205-99-2; benzo[k]fluoranthene, 207-08-9;benzo[ghi]perylene, 191-24-2.
LITERATURE CITED (1) Personov, R. I. In "Excitation Dynamics of Condensed Molecular Systems"; Agranovich, V. M., Maradudin, A. A., Eds.; North-Holland Publishing: New York, 1983; Vol. 4. (2) Wehry, E. L.; Mamantov, G. In "Modern Fluorescence Spectroscopy"; Wehry, E. L., Ed.; Plenum Press: New York, 1981; Vol. 4.
(3) Kirkbright, G. F.; dellma, C. G. Analyst (London) 1974, 99,338. (4) Wehry, E. L.; Mamantov, G. Anal. Chem. 1979, 51, 643A. (5) Causey, B. S.; Kirkbright, G. F.; deLima, C. G. Analyst (London) 1978, 101. . . . , 367. ... . Farooq, R.; Klrkbright, G. F . Ana/yst(London) 1978, 101, 586. Drake, J. A. G.; Jones, D. W.; Causey, B. S.; Kirkbrlght, G. F. fuel 1978, 57, 863. Shabad, L. M.; Smlrnov, G. A. Atmos. Environ. 1972, 6, 153.
COlmsJo,A.; Stenberg, U. Anal. Chem. 1979, 57, 145. Renkes, 0.D.; Walters, S. N.; Woo, C. S.; Iles, M. K.; D'Silva, A. P.; Fassel, V. A. Anal. Chem. 1983, 55, 2229. D'Silva, A. P.; Fassel, V. A. Anal. Chem. 1984, 56, 985A. Conrad, V. B., Carter, W. J.: Wehry, E. L.; Mamantov, G. Anal. Chem. 1883, 55, 1340. Perry, M. B.; Wehry, E. L.; Marnantov, G. Anal. Chem. 1983, 55, is93
Diciinson, R. B.; Wehry. E. L. Anal. Chem. 1979, 51, 778. Brown, J. C.; Edelson, M.; Small, G. J. Anal. Chem. 1978, 50, 1394. Heislg, V.; Jeffrey, A. M.; McGalde, M. J.; Small, G. J. Science 1984, 223, 289. Warren, J. A.; Hayes, J. M.; Small, G. J. Anal. Chem. 1982, 5 4 , 138. Amirav, A.; Even, U.;Jortner, J. Anal. Chem. 1982, 5 4 , 1668. Hayes, J. M.; Small, G. J. Anal. Chem. 1983, 55, 565A. Hayes. J. M.; Small, G. J. Anal. Chem. 1982, 5 4 , 1202. Inman, E. L.; Wlnefordner, J. D. Anal. Chem. 1982, 5 4 , 2018. Inman. E. L.; Winefordner, J. D. Anal. Chlm. Acta 1982, 141. 241. Kerkhoff, M. J.; Inman, E. L.; Voigtman, E.; Hart, L. P.; Wlnefordner, J. D. Appl. Spectrosc. 1984, 38, 239.
RECEIVED for review January 28,1985. Accepted March 29, 1985. This research was supported by NIH-GM11373-21.
Time-Resolved Fluorometric Determination of Terbium in Aqueous Solution Ilkka Hemmila Wallac Biochemical Laboratory, P.O. Box 10, SF-20201 Turku, Finland
The fluorescent properties of water-soluble binary and ternary complexes of terbium( I I I ) were studied and their applications In tlme-resolved fluorometric analysls were tested. Solutions composed of different Bdiketones, trl-noctyiphosphine oxide as the synergistic agent, and Triton X-100 as the detergent were optimized to maximize fluorescence emission In Tb measurement. The results were then compared wlth seven published methods which included the use of the following respective solutions, ethylenediamine-# ,#'-bis[ ( 0 -hydroxyphenyi)acetic acid], dipicolinic acld, iminodiacetic acid with Tlron, EDTA wlth Iron, EDTA wlth 2,3dihydroxynaphthalene, EDTA with sulfosallcyilc acid, and EDTA with salicylate. Fluorinated allphatlc Bdiketones showed the most promlsing propertles in acidic solution. They were especially suitable for use In tlme-resolved fluorometric analyses where Tb was used as the label after belng conjugated to the anaiyte via bifunctional complexones. An acidlc pH Is required for Tb release before conversion into a fluorescent chelate. The applicability of the developed measurement soiutlons to the measurement of Eu was also tested.
Recently, the development of bifunctional chelating agents for protein labeling with metal ions ( l ) dissociative , fluorescence enhancement solutions (2),and a time-resolved fluorometer optimized for Eu measurement (3) has opened up new application areas for fluorescent lanthanides in the field of
biochemistry (4)and clinical immunology (5-7). These methods employ the unique fluorescent properties of Eu-its wide Stokes shift (270 nm) and exceptionally long fluorescence lifetime (500 ws)-for highly sensitive time-resolved fluorometric detection. In addition to Eu, some other lanthanides (Sm, Tb, Dy) also form fluorescent chelates. T b is especially interesting because it forms many highly fluorescent chelates. As a fluorescent ion it has been used in protein and DNA research (8) and efforts have been made to use its fluorescent chelates in time-resolved fluoroimmunoassays (9-1 I). There are several published methods for the fluorometric detection of T b in aqueous solution based on water-soluble binary chelates, ternary chelates with aminopolycarboxylates as solubilizing ligands, and micellar chelate solutions of pdiketonates and tri-n-octylphosphine oxide (TOPO) (12-19). The applicability of several 0-diketones in time-resolved fluorometric detection of terbium was studied, and the solutions developed for these measurements were compared to published methods. A prerequisite for the use of lanthanides as fluorescent probes is the strong binding of the metal to analytes, e.g., proteins, which is carried out via bifunctional complexones which are themselves normally only weakly fluorescent when conjugated to the anal@. Rapid conversion of lanthanide ions from this weakly fluorescent conjugate into potentially highly fluorescent chelates requires either binding of an additional ligand to the complexed lanthanide (mixed ligand formation) or, preferably, dissociation of the ion before development of the fluorescent chelate (2).
0003-2700/85/0357-1676$01.50/00 1985 American Chemical Society
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Table I. Published Conditions for the Spectrofluorometric Determination of Tb reagent 1
concn, M
EDA-b-HPA Tiron Tiron Tiron SSA DHN DPA salicylate
1.5 x 10-5 20 x 10-6 20 x 10-6 20 x 10-5 50 x 10-5 5 x 10-5 io x 10-5 5 x 10-5
reagent 2
concn, M
PH
EDTA IDA EDTA EDTA
20 x 10-5 20 x 10" 50 X lo-' 5 x 10"
7.7 12.5 12.0 12.7 11.8 11.0
EDTA
10 x 10-5
12.5
5.5
wavelengths, nm excitation emission 295 320 320 320 320 330 270 320
545 546 546 546 545 545 544 545
ref 12 13 14 15 16 17 18
EXPERIMENTAL SECTION Table 11. Structure and Absorptivity of Some P-Diketones The P-diketones, acetylacetone (AcA), l,l,l-trifluoroacetylabsorbance, (HFAcA), acetone (TFAcA),1,1,1,5,5,5-hexafluoroacetylacetone RI0 RZa max, nm L trifluoroacetyhhxmphor (TFA-C),and 2,2,6,6-tetramethyl-3,5heptanedione (TMH) were obtained from Fluka; the 25 000 AcA 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-wtanedione (HFDMO), 15 000 TFAcA benzoylacetone (BA), benzoyltrifluoroacetone (BTA), and the4 700 HFAcA noyltrifluoroacetone (TTA) were obtained from Ega Chemie; and 17 000 PFH the 2-naphthoyltrifluoroacetone (P-NTA)as a ready solution with 9 600 TFMH TOPO and Triton X-100(DELFIA Enhancement Solution) was 21 000 DMH obtained from LKB-Wallac. Other tested P-diketones, 2,2-di22 000 TMH methyl-3,5-hexanedione (DMH), l,l,l-trifuoro-6-methyl-2,410 500 TFDMH heptanedione (TFMH), l,l,l-trifluoro-5.5-dimethyl-2,4-hexane- PFDMH 19 000 dione (TFDMH), 1,1,1,2,2-pentafluoro-6,6-dimethyl-3,5-hepta- HFDMO 23 000 nedione (PFDMH), 1,1,1,2,2-pentafluoro-3,5-hexanedione (PFH), 13 500 TF-TD (TFTD) were prepared by and l,l,l-trifluoro-2,4-tridecanedione General formula of p-diketones,RI-CO-CHz-CO-R2. Claisen condensation from the corresponding ketones and ethyl esters of trifluoroacetic acid or pentafluoropropionic acid using NaH as condensing agent (20). The cyclic p-diketone, TFA-C, and aromatic P-diketones Other chelating agents, iminodiacetic acid (IDA), 1,2-di(BA, BTA, TTA, NTA) which have absorption maxima within hydroxybenzene-3,5-disulfonicacid (Tiron), sulfosalicylic acid the 320-350 nm range (2) did not produce detectable (SSA), and sodium salicylate were obtained from Merck, 2,3fluorescenceunder the conditions tested. The low fluorescence dihydroxynaphthalene (DHN), tri-n-octylphosphine oxide (TOdetected from aromatic 0-diketone chelates of T b is consistent PO), and ethylenediamine-N,"-bis[ (0-hydroxypheny1)aceticacid] with the measured triplet levels of those P-diketones (22) (EDTA-b-HPA) were obtained from Fluka, and dipicolinic acid which lie only slightly above or even below the emission level (DPA) was obtained from Sigma. Standard solutions of TbC13 and EuC13 were prepared by of T b (5D4). dissolving Tb407or EuzO3 (Sigma) in hydrochloric acid. After The optimal conditions of pH and concentration of P-dievaporation of the acid, the residue was dissolved in water and ketones, TOPO, and Triton X-100 revealed that a correlation the metal concentrationswere determined by EDTA titration (21). might exist among them to a certain extent. However, because Labeling of IgG (anti-mouse-IgG)with Tb and a model sandof their fairly wide usable concentration ranges, they were wich immunoassay was performed as described for Eu-labeled optimized in turn by varying the concentration of the relevant antibodies (2) by using Tb instead of Eu and TFTD as a 0-disolution in the presence of fixed concentrations of all the ketone instead of P-NTA for fluorescence measurement. others. On average, the optimal concentrations of P-diketones The buffers used in Tb measurements were 0.1 M acetate varied between 10 and 100 pM (Figure lA), whereas the opbuffer, pH 3-6, 0.05M Tris-HC1, pH 7-9, and 0.05 M glycineNaOH, pH 9-12. The Tb measurement solutions made from timal concentration of TOPO varied between 25 and 200 p M non-@-diketone solutions were prepared according to the references (Figure lB), and Triton X-100 is best used a t a concentration given in Table I. greater than 0.2 % v/v (Figure IC). Although there were The absorption spectra of P-diketones in alkaline ethanol soconsiderable differences in the enhancing effects of TOPOlution were determined with a Beckman Model 35 spectrophodepending on the pH and P-diketone used (enhancement ratio tometer. Fluorescence intensities and fluorescence spectra were ranged from 2X to about 1000X)-optimal fluorescence yields measured with a Perkin-Elmer Model 3000 fluorescence specwere obtained within the 25-200 NM concentration range. trometer and delayed fluorescence and decay times were measured The optimal pH for chelate formation and fluorescence with a time-resolved fluorometer (3) equipped with a xenon flash development varied greatly depending on the P-diketone ~ dulamp of 1000 Hz frequency and of approximately 1 - w flash ration. The fluorometer also incorporated adjustable delay and structure. Figure 2A shows the pH curves of four different counting times, wide excitation filter (250-400nm) with quartz P-diketones in a detergent solution without TOPO being inlenses, and an emission filter for Tb measurement in the 545-nm cluded. TMH required a very high pH (over 10) for maximal range. Delay times of 100 ps and counting times of 200 ps were fluorescence. TFTDH had quite a restricted pH optimum used for Tb chelates having decay times below 100 ps. The near pH 6, whereas TFTD and PFDMH were less affected respective values for Tb chelates having decay times above 100 by the pH within the range 5-10. Both the size of the hyp s were 200 and 400 ps. drocarbon side chain (Rz)and especially the amount of RESULTS AND DISCUSSION fluorine in R1(Table 111)greatly affected the optimum pH. Properties of &Diketones and Their Tb Chelates. The As an electronegative substituent, fluorine increases the acidity absorption spectra of P-diketones were measured after their and enolization of P-diketones making them more strongly conversion to enolic forms by adding potassium hydroxide to chelating especially a t lower pH values (20). their ethanolic solutions. P-Diketones which formed fluorThe change in Rzfrom a short -C(CH3)9group in TFDMH escent T b chelates have a single absorption maximum within to a long chain -(CHZ)&H3 in TFTD resembles the effect of the range 290-300 nm (Table 11). TOPO found in all the tested P-diketones (Figure 2B), that
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Table 111. Fluorescence Properties of Tb /3-Diketone Chelates"
AcA
TFAcA HFAcA PFH TFMH DMH
TMH TFDMH PFDMH HFDMO TFTD TFTD
excitation
emission
decay time,
re1
PH
(max), nm
(max), nm
PS
fluorescence
9.0 7.5 4.0 4.0 5.5 9.0 11.0 5.5 4.0 4.0 4.0 11.0
305 300 312 295 295 255, 293 294 290 296 297 294 294
543.0 543.0 543.0 543.0 544.0 543.0 547.0 544.0 543.5 543.5 543.5 543.5
390 73 30 36 62 550 380
0.5 13 16 65 467 0.6 369 650 380 290 540 340
100
50 35 100 100
"Fluorescences of 500 nM Tb were measured in 50 pM P-diketone,50 r M TOPO, and 0.2% Triton X-100 solution at the stated pH value usinn a Perkin-Elmer sDectrofluorometer.
6ot ./ X
10
20
50
100
200
500
Diketone. pM
25
Y
50
100
200
f I
500
TOPO - p M
8otf--*
'I
PH
pH optimum of Tb-@-diketonatesolutions without (A) and with (B) 50 pM TOPO. The 50 pM @diketonestested in a 0.2% Triton X-100 solution were TFDMH (O), TFTD (0), PFDMH (W), and TMH (0). Flgure 2.
I
Triton, a/o
Flgure 1. Tb
fluorescenceyield vs. concentrations of @diketones(A),
were 50 p M 5.5.
@diketone,50 p M TOPO, and 0.2% Triton X-100 at pH
TOPO (e), and detergent Triton X-100 (C) with TFDMH (O), PFDMH (O), and HFDMO (0). The constant concentrations used in the experiments
of greatly enhancing fluorescence at all pH values, especially at acidic pHs. As a synergistic agent, TOPO is likely to contribute to the transference of the newly formed chelates from the acidic water solution into nonpolar detergent micelles. Before its penetration into micelles, Eu should, however, be in a chelated form and a similar pH dependence is also found with TOPO solutions although generally at much lower pH values. The optimal pH or the lowest usable pH values for different @-diketonesare presented in Table 111. The effect of buffer ions on the fluorescence of two p-diketone chelates at pHs 9 and 5.5 were measured with and
without including 50 pM TOPO (Table IV). Many ions had a remarkable quenching effect on the fluorescence produced in T b by the less strongly chelating @-diketones(TMH), while with TFTD, only phosphate, carbonate, and phthalate had any effect. Addition of TOPO further decreased the quenching effect. Nonchelating buffers such as Tris, acetate, glycine, and MOPS can be used without negative effects on the fluorescence of fluorinated /3-diketone-Tb chelates. Optimal fluorometric measurement solutions for T b were prepared from 11 @-diketonesin a 0.2% aqueous buffered Triton X-100 solution containing 50 pM TOPO using either the optimal pH or lowest usable pH still capable of producing near maximal fluorescence. The fluorescent characteristics were measured with 250 nM T b and the results are presented in Table 111. The highest signals were obtained in acidic solutions of trifluoro-substituted @-diketoneshaving decay times of about 100 ps. More fluorine lowered the usable pH of the measurement solution but at the same time the fluorescenceyield also decreased. In the tested solutions the amount of fluorine in the @-diketonestructure affects with some unknown mechanism the decay time which decreased
ANALYTICAL CHEMISTRY, VOL. 57,
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B
A 2
1
3 1:
Y
0
z Y 0 v)
Y
a 0 3 A U
W A V E L E N G T H , NM
Figure 3. Excitation (A) and emission (8)spectra of three dlfferent Tb chelates, Tb-DPA (I), Tb-TFDMH (2), and Tb-EDTA-DHN (3). The concentrations of Tb were 10, 1, and 10 pM, respectlveiy. DPA and DHN-EDTA solutions were as described in Table I. The concentration of TFDMH was 50 pM in a 50 pM TOPO, 0.2% Triton X-100 solution at pH 5.5.
Table IV. Effects of Buffer Ions on the Fluorescence of Tb-TMH and Tb-TFTD Chelates with and without Including TOPO' TMH, pH 9.0
TFTD, pH 5.5
TFTD, pH 9.0
buffer ions (50 mM)
with TOPO
without TOPO
with TOPO
without TOPO
with TOPO
without TOPO
Tris phosphate acetate carbonate phthalate glycine MOPS borate
151.5 1.9 96.3 6.3 4.2 257.2 123.0 113.0
29.1 0.03 2.7 0.03 0.02 11.3 4.8 1.8
191.0 73.8 184.5
96.6 54.9 97.4
133.8 175.1 169.2 124.8
70.0 73.4 88.4 82.2
170.1 30.0 177.2 170.1 128.1 164.0 150.2 163.4
79.5 27.0 70.8 58.2 62.0 82.0 69.2 85.2
"Fluorescence measured using 5 nM Tb with a time-resolved fluorometer, value counts a-diketones 50 p M TOPO 0 and 50 pM; Triton X-100 0.2%.
Table V. Fluorescence Properties of Tb Chelate8 Used for Tb Detection
reagent EDA-b-HPA Tiron Tiron-EDTA Tiron-IDA SSA-EDTA DHN-EDTA DPA salicylateEDTA
decay excitation emission time, re1 pH (max), nm (max), nm ps fluorescence
7.7 12.5 12.0 12.7 11.8 11.0 5.5 12.5
287 320 320 320 325 252,332 270 320
544 546 547 547 544 546 543 545
685 175 710 300 974 90 1320 614
34.8 62.0 45.0 38.5
