Anal. Chem. IBS7, 59, 423-427
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Homogeneous Immunochemical Technique for Determination of Human Lactoferrin Using Excitation Transfer and Phase-Resolved Fluorometry Kasem Nithipatikom and Linda B. McGown*
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
A homogeneous immunofluorometrlc technlque for the determlnatlon of human lactoferrin is described, In whlch fluoresceln-labeled antibody and tetramethylrhodamlne-labeled antlbody bind to the muitlvalent lactoferrin antlgen. Excitatlon transfer causes quenching of the fluorescein fluorescence, whlch is also quenched by antlgen-antibody binding. The decreases In fluorescence Intensity and llfetime of the fluord n label are reflected In measurements of phase-resoived fluorescence Intensity. Optimization of determination condltions and factors affecting the dynamic range and sensitivity of callbratlon curves are discussed. Callbratlon curves were reproduclble to withln less than 2 % standard devlatlon. Determinations of three synthetic unknowns gave an average relative error of -2.5% and an average relatlve standard deviation of 2.4%. Other competitive and noncompetitive approaches are also dlscussed, Including the use of labeled antigens and Texas Red label.
Fluoroimmunoassay and immunofluorometric techniques have become increasingly important as an alternative to the use of radiometric immunochemical techniques, and a wide variety of heterogeneous and homogeneous approaches have been explored (1,2,3). Homogeneous fluorometric techniques, which are possible because of the sensitivity of fluorescence to the chemical microenvironment of the fluorophore, are especially interesting since the time-consuming and errorpropagating separation of antibody-bound from free analyte is eliminated. Fluorometric techniques developed for homogeneous analyses have been based on polarization, intensity enhancement or quenching (direct or indirect), release of a hydrolyzable fluorescent group from the antigen species by hydrolytic action of the antibodies or an enzyme (in which case the antibody protects the antigen from the hydrolytic enzyme), quenching enhancement by excitation transfer, and fluorescence lifetime changes. The use of excitation transfer has been described for competitive and noncompetitive fluorometric immunochemical determinations of both haptens and antigenic analytes (4). In this paper we describe a noncompetitive, homogeneous immunofluorometric technique for the determination of human lactoferrin which combines the effects of direct quenching and excitation transfer. Changes in the fluorescence intensity and fluorescence lifetime of fluorescein-labeled anti-lactoferrin antibody (Ab*F) result from excitation transfer to tetramethylrhodamine-labeled anti-lactoferrin antibody (Ab*R) as well as from direct effects of binding to lactoferrin (Ag). Other immunochemical approaches to the lactoferrin determination were also studied, including both competitive and noncompetitive schemes involving excitation transfer or, in some cases, direct quenching alone. Fluorescein, tetramethylrhodamine, and Texas Red (a sulforhodamine derivative (5))were used as labels for the anti-lactoferrin antibody, and fluorescein and Texas Red as labels for the lactoferrin
antigen. The noncompetitive excitation transfer approach with Ab*F and Ab*R gave the best selectivity for a homogeneous analysis and was chosen for development into the technique described in this paper. The combined fluorescence intensity and lifetime changes are incorporated into a single phase-resolved intensity measurement by phase-resolved fluorometry, which has been described in detail elsewhere (6, 7). Phase-resolved fluoroimmunoassay techniques in which fluorescent-labeled antigen (or hapten) competes with the nonlabeled analyte for nonlabeled antibody have been described for phenobarbital (8) and human serum albumin (9). Unlike the immunofluorometric technique described here, the phase-resolved fluoroimmunoassay techniques have been based solely on the small changes in fluorescence intensity and lifetime that occur upon binding of the labeled antigen to the antibody. Human lactoferrin (red protein or lactotransferrin) is a single chain, iron-binding glycoprotein. It has a molecular weight of approximately 75000-85000 (IO, l l ) ,with two ferric ion binding sites per protein. The concentration ranges of human lactoferrin are 3-4 mg/mL in early colostrum and 1-2 mg/mL in mature milk (12,13). Lactoferrin helps to prevent the growth of iron-requiring bacteria in milk and may also aid newborns in absorbing iron more efficiently from human milk than from other types of milk and formulas, which have lower concentrations of lactoferrin (14).
THEORY Phase-resolved fluorometry is based on the phase-modulation approach to the determination of fluorescence lifetimes. Amplitude-modulated excitation is used with phase-sensitive detection to measure the ac emission signal which is phasedelayed and demodulated relative to the excitation to an extent determined by the fluorescence lifetime(s) of the emitter(s) and the modulation frequency. The ac emission signal is integrated over a variable phase half-cycle, alternated with a nonintegration half-cycle. The resulting phase-resolved fluorescence intensity (PRFI) has the form where +D is the position of the integration half-cycle (variable from OD to 360°),A is the dc (steady-state) intensity, which is a function of the concentration(s) and spectral characteristics of the emitter(s) and of steady-state instrumental parameters, mexis the modulation depth (ac to dc intensity ratio) of the exciting light, m is the demodulation of the emission (the ratio of the modulation depth of the emission signal to that of the excitation beam), and + is the phase-delay of the emission signal. Therefore, F ( $ J ~depends ) upon the spectral intensity and lifetime characteristics and concentrations of the emitter(s) and on the modulation frequency and wavelengths used.
EXPERIMENTAL SECTION Human lactoferrin (ChromPure) and anti-human lactoferrin (AffiniPure F(ab’), fragment from rabbit) reagents were all
0003-2700/87/0359-0423$01.50/00 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
purchased from Jackson Immunochemical Laboratories, Inc. (Avondale, PA), as solids and reconstituted with water according to the manufacturer’s instructions. Distilled demineralized water was used for all solutions in these studies. The antibody solutions were further diluted with water to make stock solutions (0.109 mg/mL) that included anti-lactoferrin antibody unconjugated (Ab) and conjugated with dichlorotriazinylamino fluorescein (Ab*F),tetramethylrhodamine isothiocyanate (Ab*R),and Texas Red (Ab*T). Lactoferrin solutions, including unconjugated (lactoferrin(Ag), 17.6 mg/mL), fluorescein-conjugated (Ag*F, 3.9 mg/mL), and Texas Red-conjugated (Ag*T, 6.0 mg/mL) were also prepared in water. Standard and test solutions for the calibration curves and related studies were prepared by the addition 30 pL of Ab*F, 90 p L of Ab*R, 50 pL of unconjugated lactoferrin (Ag) of appropriate concentration (or buffer, for zero Ag), and 2830 pL of buffer (0.10 M phosphate, pH 7.4) to the cuvette and incubated for 30 min. Solutions for the studies involving other reagents were similarly prepared in buffer. Disposable polyethylene cuvettes (Precision Cells) were used for all fluorescence measurements. Fluorescence measurements were made with an SLM 4800s spectrofluorometer (SLM Instruments, Inc.) with a 450-W xenon arc lamp source and photomultiplier tube (Hamamatsu R928) detection. An Apple IIe microcomputer was used for on-line data acquisition and for data analysis. A frequency of 30 MHz was used for excitation modulation. The sample compartment was maintained at 25 f 0.1 OC with a Haake A81 temperature control unit. Both the “10-average”and the “100-average”data averaging modes were used, in which the measurement is the average of 10 or 100 samplings, averaged internally by the instrument’s electronics over a period of approximately 3 or 30 s, respectively. Steady-state excitation and emission spectra were obtained in the 10-average mode with slit settings of 16 nm for the excitation monochromator entrance and 2 nm for all other slits. Phaseresolved and steady-state intensities were measured in triplicate in the 100-average mode. Fluorescence lifetimes are reported as the average of five determinations from phase-delay and demodulation measurements made in the 100-average mode. A kaolin scatteringsolution was used as the reference for the lifetime determinations. For PRFS measurements, slits were set at 16 nm and 0.5 nm for the excitation monochromator entrance and exit, respectively, and at 0.5 nm for the modulation tank chamber exit. The emission monochromator, when wed for phase-resolved measurements, had entrance and exit slits set at 8 nm (or at 16 nm for lifetime determinations). Alternatively,band-pass filters (10 nm half-width, Oriel) were used in the emission beam, including 520 nm for Ab*F, 570 nm for Ab*R, and 610 nm for Ab*T. Excitation for measurements with the emission filters was set at 10 nm below the excitation maximum to minimize any scattered light.
RESULTS AND DISCUSSION Systems Studied. The systems studied (summarized in Table I) can be categorized as either competitive or noncompetitive. In the competitive techniques (fluoroimmunomays), labeled antigen (Ag*F or Ag*T) competes with unlabeled lactoferrin (Ag) analyte for antibody. Within this category, the direct quenching technique is based solely on quenching of the Ag* fluorescence upon binding to unlabeled Ab, and the excitation transfer technique employs quenching due to energy transfer between the Ag* and labeled antibody (Ab*R) in addition to direct quenching effects. Unfortunately, rhodamine-labeled antigen was not available, so the only systems investigated in the latter category were energy transfer from Ag*F to Ab*R and Ab*R to Ag*T, measuring donor fluorescence in both cases. The noncompetitive (immunofluorometric) techniques involve binding of Ag analyte to labeled antibodies. In the direct quenching noncompetitive technique, the fluorescence characteristics of Ab*F, Ab*R, or Ab*T are changed upon binding to Ag. In the excitation transfer noncompetitive technique, a pair of labeled antibodies (Ab*F and Ab*R, or Ab*R and Ab*T) can undergo energy transfer upon binding of both the donor and acceptor to the multivalent Ag. The fluorescence of the donor Ab* is
Table I. Fluorescedce Lifetimes for the Different Systems in the Absence and Presence of Lactoferrin Analyte Tb
reagents
label” measd
reagents reagents + Ag
Excitation Transfer noncompetitive Ab*F, Ab*R Ab*R, Ab*T competitive Ab*R, Ag*F Ab*R, Ag*T noncompetitive Ab*F Ab*R
Ab*T competitive Ag*F, Ab
F R
3.43
F R
1.11
F R T
3.47 2.32 4.06
F
1.25
2.37
1.93 Direct Quenching
2.92 2.12
-0.51
1.31 2.14
0.20 0.21
3.20 3.68
-0.27 -0.18 -0.38
1.31
0.06
2.14
-0.25
Each label measured at its excitation/emission maxima (in nm): fluorescein (F)at 490/515; tetramethylrhodamine (R) at 550/575; Texas Red (T) at 590/610. *Fluorescence lifetime and standard deviation ( n = 5), in ns, determined by phase-delay with a scattering solution as a reference. Each value is the mean of five determinations, with standard deviations ranging from 20 to 80 ps. The maximum fluorescence lifetime difference obtained for the measured label in a solution of the reagents in the absence and presence of An. quenched as a result of both energy transfer to the acceptor Ab* and binding to Ag. Fluorescence Characteristics and Comparison of the Techniques. The fluorescence lifetimes ( T ~ of ) the relevant labeled reagents in the absence and presence of unlabeled lactoferrin (Ag) are shown in Table I for each of the systems studied. Each noncompetitive technique showed a decrease in the fluorescence lifetime of the measured Ab* in the presence of Ag,reflecting the quenching processes that occur upon binding of Ag to the Ab*. In the competitive techniques, the lifetime of the measured Ag* or Ab* increases in the presence of Ag,which replaces Ag* on the antibody. The Ag* is dequenched in the free form relative to the Ab-bound form, or in the Ab*R-Ag*T excitation transfer case, the Ag*T is replaced by Ag on the Ab*R, resulting in dequenching of the latter. The noncompetitive techniques show greater lifetime differences than the competitive techniques, probably due primarily to the higher degree of labeling and the position of the labels on the antigens compared to the antibodies. For example, there are 20.4 pg of fluorescein per milligram of protein (3:l molar ratio) for the Ag*F in contrast to 12.0 pg of fluorescein per milligram of protein (21molar ratio) for the Ab*F according to the manufacturer. The close proximity of the multiple labels on Ag* promotes self-quenching, as evidenced by the low fluorescence lifetimes of the labeled antigens relative to antibodies conjugated to the same label. Intensities of the Ag* species were also considerably lower than those of the similarly labeled Ab*. The fluorescence intensity of a solution of Ab*F gave an intensity 6 times higher than that of a solution containing Ag*F adjusted to the same analytical concentration of the fluorescein label. The noncompetitive excitation transfer approach with Ab*F and Ab*R showed the greatest difference in fluorescence lifetime in the absence and presence of Ag, as well as the largest intensity difference (not shown). This system was therefore chosen for further study and is the subject of the remainder of this paper. The emission spectrum of Ab*F overlaps extensively with the excitation spectrum of Ab*R (Figure I), permitting energy transfer between the two labels
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
425
DETECTOR PHASE ANGLE (DEG)
Figure 1. Emission spectrum (A, = 490 nm) of Ab'F (-) excitation spectrum (A, = 575 nm) of Ab'R
e-).
3*10
I l
a
0
1
and
Figure 3. PRFI as a function of detector phase angle ($D) for Ab"F in the presence of Ab'R (Ab'FIAb'R = 0.33, total Ab* = 6.54 mg/L): (0)no Ag present; (0)1.43 mg/L Ag.
W
L
2.80 2
3
4
5
6
7
MOLES Ab*B / MOLES Ab*P
Figure 2. Fluorescence lifetime of Ab'F as a function of the molar ratio of Ab'R to Ab'F In the presence of (0)1.15 mg/L Ag and (0) 2.00 mg/L Ag.
0.60
I
40
0
80
I
120
160
DETECTOR PHASE ANGLE .(DEG)
which causes the large fluorescence lietime decrease observed upon the addition of unlabeled lactoferrin to solutions containing the two Ab* species. Optimization of Ab*F to Ab*R Ratio. The 7F of Ab*F in the presence of unlabeled lactoferrin (Ag) as a function of the ratio of Ab*F:Ab*R is shown in Figure 2 for two different concentrations of Ag. Two regions of decreasing slope occur. The first is probably due to increased energy transfer as an Ab*R is added to each Ab*F-Ag complex. The second decrease is caused by the large excess of free Ab*R present in the solution and may result from energy transfer or other interactions between the free Ab*R and the Ab*F moiety of the Ab*F-Ag complex. A ratio of 3 Ab*R to 1 Ab*F (Ab*F/Ab*R = 0.33) was chosen as optimal since it gives a maximal lifetime difference between the two Ag concentrations and occurs in the region of zero slope, thereby minimizing the effect of experimental imprecision in the Ab*F:Ab*R ratio from solution to solution. In the absence of Ag, no significant difference in TF was observed between Ab*F alone and Ab*F in the presence of Ab*R (Ab*F/Ab*R = 0.50). Choice of Detector Phase Angle. A phase difference of 3.29O was observed between free Ab*F and Ab*F in the presence of unlabeled lactoferrin (Ag)for solutions containing an Ab*F:Ab*R ratio of 0.33. Plots of PRFI as a function of detector phase angle for the two solutions are shown in Figure 3. The phase maxima for the two species occur a t detector phase angle settings of approximately 70' in Figure 3 (the detector phase angle settings have an arbitrary phase and therefore have only a relative, rather than an absolute, significance). The detector phase angle dependence is a function of both the fluorescence lifetime of Ab*F and the modulation frequency f used (Equation 1 and related discussion). Although the instrument used in this study does not permit further optimization off beyond the use of 30 MHz (the best of the three frequencies available, including 6, 18, and 30 MHz), a continuously variable instrument could be used to improve sensitivity by the selection off that maximizes the detector phase angle difference between the free and Ag-bound Ab*F.
Figure 4. Ratio of the PRFI of Ab'F in the presence of Ag to the PRFI in the absence of Ag as a function of detector phase angle. The solid horizontal line indicates the steady-state intensity ratio. Solution conditions were the same as those for Figure 3.
b 0.60 -5.00
-4.00
-3.00
Figure 5. Effect of detector phase angle on calibration curves. PRFI ratio (as in Figure 4) as a function of log CAgat detector phase angles (0)O', (0) 45', (A)90°, ( 0 )135'. Solution conditions were the same as those for Figure 3.
Figure 4 shows the ratio of the PRFI of Ab*F in the presence of Ag to that of free Ab*F in solutions with an Ab*FAb*R ratio of 0.33 as a function of detector phase angle (corresponding to the detector phase angles in Figure 3). The ratio of steady-state intensities is also shown (horizontal line). Detector phase angles from 0" to 4 5 O improve sensitivity relative to steady state by increasing the intensity difference between the two Ab*F forms. Detector phase angles in the range from 45' to 135' have ratios higher than the steady-state value so sensitivity will be reduced relative to steady state. Sensitivity as a function of detector phase angle is illustrated in Figure 5, in which PRFI ratios are plotted as a function of the log Ag concentration for the four detector phase angles shown in Figure 4. Maximum sensitivity is obtained at de-
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Table 11. Determination of Synthetic Unknown Solutions sample
true value4
foundb
errorc
RSDd
1 2 3
71.8 363 818
70.6 336 830
-1.7 -7.4 1.5
1.3 1.2 4.8
-2.6
2.4
av
a Concentration added to cuvette, #g!L. Experimentally determine value, averaged for four identically prepared solutions, gg/L in cuvette. 'Relative determination error (a) for average concentration found. Relative standard deviation ( W)of the results for the four solutions.
0.60
-5.00
-4.00
LOG CAg
-3.00
(CAP in g / l )
3.50r
Flgure 6. Effect of Ab'F/Ab'R and total Ab* on calibration curves. PRFI ratio (as in Figure 4)as a function of log C ,, for the following: (0)Ab*F/Ab*R = 0.33,total Ab' = 2.18 mg/L; (0)Ab*F/Ab*R = 0.33,total Ab* = 6.54 mg/L: (A)Ab'F/Ab'R = 0.50, total Ab* = 6.54mg/L. Measurements were made at dD= 45".
O
0.80.
5
0.70.
! !!
3.001 0.00
0.60,
0.50
1.00 g Ag
2.00
g Ab*F
Figure 8. Fluorescence lifetime of Ab'F as a function of the weight ratio of Ag to Ab'F.
0.50
0.00
1.50
1.00
2.00
3.00
Flgure 7. Phase-resolved (e)and steady-state (M) calibration curves, obtained by using both C,, (solid lines) and log CAg(dashed lines) scales. Measurements made at q5D = O", Ab'F/Ab*R = 0.33,and total CAb= 4.36 mg/L. Error bars indicate f l standard deviation. tector phase angle settings near Oo; i.e., dD = +Ab*F - 70". Calibration Curves. Calibration curves were constructed by plotting the ratio of the PRFI of each standard solution to that of the zero unlabeled lactoferrin Ag standard as a function of either Ag concentration or log Ag concentration. The PRFI ratio was used to normalize the curves and minimize day-to-day variability and variations between different sets of standard solutions and reagents. The effects of total C A b (the sum of the concentrations of Ab*F and Ab*R) and of Ab*FAb*R on the shapes and ranges of the calibration curves are shown in Figure 6. Increasing the total C A b %fold increases the linear range of the curve and shifts it to higher C, (higher determination limits). Increasing the Ab*F:Ab*R from 0.33 to 0.50 shifts the linear range to even higher , C and decreases sensitivity. Therefore, the total CAband Ab*F:Ab:R can be adjusted to give either a wider dynamic range or a lower determination limit, depending upon the needs of the particular analysis. Steady-state and phase-resolved calibration curves are shown in Figure 7 . The curves are shown with both Ag concentration and log Ag concentration scales. Each curve shown is the average of four curves, each generated from triplicate measurements in the 100-average mode per point. A pooled standard deviation of 8.39 X intensity ratio units
was calculated for the points in the four phase-resolved curves, which range from 0.50 to 1.00 intensity ratio units. This represents an 0.8-1.7% relative standard deviation over the range of the calibration curve. The greatly increased sensitivity and linear range of the phase-resolved curve relative to the steady-state curve are evident from the figure. The total C A b was chosen midway between the two concentrations used in Figure 6, compromising between lower determination limits and a wider dynamic range as discussed above. Intensity ratios in the calibration curves begin to increase a t high Ag concentrations to form the characteristic "Ushaped" calibration curves typical of noncompetitive antibody-labeled determinations (4), as shown in the log [Ag] scale curves (Figure 7 ) . In order to circumvent the ambiguous results from such a curve, samples can be run twice, a t two different dilutions. Alternatively, two calibration curves can be used with their combined dynamic ranges appropriately adjusted by choice of total C A b and Ab*F:Ab*R. The sample is run at a single dilution under the two sets of conditions (one for each curve). Determination of Synthetic Unknowns. Results for the determination of three different synthetic unknown concentrations of lactoferrin (Ag) are shown in Table 11. Four solutions were prepared at each concentration, and the results shown are the mean and standard deviation for each concentration. Effect of Ag on the Fluorescence of Ab*F. There are several possible reasons for the observed changes in fluorescence lifetime and intensity of Ab*F in the presence of Ag and Ab*R. Energy transfer to Ab*R is certainly an explanation
Anal. Chem. 1987, 59, 427-432
for some of the observed quenching since the lifetime change is much larger in the presence of Ag if Ab*R is also present (Table I). However, as shown in Figure 8, the lifetime of Ab*F is also significantly reduced by Ag alone, without Ab*R. The first decrease in T~ occurs in the presence of small amounts of Ag and may be due to self-quenching by fluorescein labels on different Ab molecules brought together on a single multivalent Ag molecule (4), as well as to quenching of Ab*F upon binding to Ag. As Ag concentration increases and the binding ratio approaches 1 Ab*F per Ag, TF increases. A second region of decreasing lifetime is then observed at higher Ag concentrations, perhaps due to the formation of clusters of Ab*F on the antigen surfaces (4), again bringing fluorescein labels on different antibodies in closer proximity to facilitate quenching.
CONCLUSIONS The selectivity of the noncompetitive immunofluorometric technique described for homogeneous determinations of lactoferrin is greatly enhanced first by the use of phase-resolution to exploit fluorescence lifetime differences between the free and antigen-bound labeled antibody and, second, by the use of energy transfer from the monitored donor label to a receptor label on a separate antibody also bound to the multivalent antigen. Of the various competitive and noncompetitive techniques studied, the excitation transfer noncompetitive approach gave the best selectivity and sensitivity. Excellent precision was observed for calibration curves, with a relatively large dynamic range. Good accuracy and reproducibility were obtained for the determination of synthetic unknowns. Lactoferrin was chosen as the model system for these studies because of the availability of both lactoferrin and antilactoferrin antibody (in a highly purified form) labeled with several different fluorophores capable of excitation transfer. Because rhodamine-labeled lactoferrin was not
427
available, we could not evaluate the competitive technique with excitation transfer from Ab*F to Ag*R. This system would be an interesting subject for future studies. It would also be worthwhile to study the competitive schemes with antigens that have a similar degree of labeling. However, because labeled antibodies are often easier to obtain or prepare than labeled antigens, the noncompetitive excitation transfer technique with two labeled antibody species may prove more convenient and widely applicable than the competitive approach.
LITERATURE CITED (1) Nakamura, R. M. Ciin. Lab. Assays: [Pap. Annu. Clin. Lab. Assays COnf.1 4th 1983, 33-60. (2) Smith, D. S.; Hassan, M.; Nargessl, R. D. I n Modern Fiuorescence Spectroscopy; Wehry, E. L., Ed.; Plenum: New York, 1981; Chapter 4. (3) Hemmiia, I. Ciin. Chem. (Winston-Salem, N.C.)1985, 31, 359-370. (4) Ullman, E. F.; Schwartzberg, M.; Rubenstein, K. E. J. Biol. Chem. 1976, 251, 4172-4178. ( 5 ) Tltus, J. A.; Haugland, R.; Sharrow, S. 0.; Segal, D. M. J . Immunol. Methods 1982, 50. 193-204. (6) Mattheis, J. R.; Mitchell, G. W.; Spencer, R. D. I n New Directions in Molecular Luminescence; ASTM STP 822; Eastwood, D., Ed.; American Society for Testing and Materials: Philadelphia, PA, 1963; pp
50-64. (7) Lakowicz, J. R. Principles of Fiuorescence Spectroscopy; Plenum: New York, 1983; Chapter 4. (8) Bright, F. V.; McGown, L. B. Taianta 1985, 32, 15-18. (9) Tahboub, Y.; McGown, L. B. Anal. Chim. Acta 1988. 182, 185-191. (10) Blackberg. L.; Hernell. 0. FEBS Left. 1980, 109, 180-184. (11) Querinjean, P.;Masson, P. L.; Heremans, J. F. Euf. J . Biochem. 1071. . .20. ~ . 420-425. . -. _. (12) Nagasawa, T.; Kiyosawa, I.; Kuwahara. K. J . Dairy Sci. 1972, 55, 1651-1659. (13) Lonnerdal. B.; Forsum, E.: Hambraeus, L. Am. J. Clln. Nutr. 1976, 2 9 , 1127-1133. (14) Saarinen, U. M.; Slimes, M. A. Pediatr. Res. 1979, 13, 143-147.
RECEIVED for review August 25,1986. Accepted October 16, 1986. This work was supported by the National Science Foundation (Grant CHE-8403759).
Determination of Heavy Siderophile Elements in Geological Samples via Selective Excitation of Probe Ion Luminescence R. J. Haskell' and J. C. Wright* Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
Previously developed techniques for selective excitation of probe ion luminescence (SEPIL) for Re and Os ultratrace measurements are appiled to a variety of geobgicai samples. The ions arqobserved via their incorporatlon into anttfluorlte A,SnX, (A = K, Rb, Cs; X = CI, Br) host lattices. The effect of the host on the optical characteristlcs of the dopant is discussed in the framework of possible trade-offs that need to be made in real sample analysis. Sample preparation procedures that have been optimized for the determination of these elements in varlous sampks are described and Justified. Good agreement wRh several NBS, USBM, and NIM standards is reported.
Heavy siderophilic elements, such as rhenium and osmium, are of interest to geochemists to identify differentiation 'Present address: Control Division, The Upjohn Co., Kalamazoo, MI 49001. 0003-2700/87/0359-0427$01.50/0
processes in primordal earth (1). Low abundances of these elements on planetary crusts allow them to become indicators for the extraterrestrial origin of spherules in some deep-sea sediments (2), stratospheric dust (2), and the lunar surface (3). For this reason, the high noble-metal concentration in the cretateous-tertiary boundary layer, deposited 65 million years ago, was taken as evidence for a meteor that resulted in mass extinctions (4). In addition, the intrinsic value of these elements makes their recovery economically feasible from materials even where they are present at parts per billion levels (5).
While all of the traditional spectrometric techniques have been applied to these elements (6, 7),results are generally disappointing. Instrumental detection limits are poor, and interferences from base (8)and precious (9) metals are a severe problem, particularly for flame methods. Electrothermal techniques (IO) are limited by the refractory nature of the elements (Re bp 5600 "C).Fire assay, a frequently employed decomposition/preconcentration technique, is highly de@ 1987 American Chemlcal Society
