2766
Anal. Chem. 1989, 61, 2766-2769
contrast enhancement through the application of a sigmoidshaped look-up table, in which high-intensity regions are further intensified and low-intensity regions are deemphasized. The contrast enhancement makes it possible to observe a correlation between microscopic cracks and ECL intensity. For example, the arrows in Figure 5 point to cracks on the externally illuminated image and corresponding regions of high intensity on the ECL image.
CONCLUSION Results presented here give direct evidence that the electron-transfer kinetics of luminol oxidation are enhanced at regions rich in defects over undisturbed basal plane HOPG. Quantitatively, the electron-transfer rate constant differs between the two regions by over 2 orders of magnitude. Electrochemical pretreatment produces a surface that is visibly rich in defects and that behaves electrochemically in a manner similar to a surface possessing obvious mechanical defects. The correlation between defects and electron transfer activity on electrochemically pretreated surfaces reinforces the conclusion that activation during oxidation/reduction pretreatment is due to fracturing of the graphite lattice and formation of edge plane defects, rather than being due to oxide formation (18). At least for the current case of luminol, it is unnecessary to invoke surface oxides to explain enhanced activity. Registry No. KNO,, 7757-79-1;graphite, 7782-42-5;luminol, 521-31-3.
LITERATURE CITED (1) Evans, J. F.; Kuwana, T. Anal. Chem. 1977,49, 1632. (2) Elliot, C. M.; Murray, R. W. Anal. Chem. 1988. 250, 173.
Stulik, K.; Brabcova. D. J. Electroanal. Chem. 1988. 250, 173. Poon, M.; McCreery, R. L. Anal. Chem. 1988, 58, 2745. Hershenhart, E.; McCreery, R. L.; Knight, R. D. Anal. Chem. 1979, 51, 358. Hu, I.-F.; Karweik, D. H.; Kuwana, T. J. Nectfoanal. Chem. 1985, 188, 59. Stuns, K. J.; Kovach. M.; Kuhr, W. G.; Wightman. R. M. Anal. Chem. 1883, 55, 1632. Fagan, 0. T.; Hu, I.-F.; Kuwana, T. Anal. Chem. 1985, 57, 2759. Hu, I.-F.; Kuwana, T. Anal. Chem. 1088,58, 3235. Engstrom, R. C. Anal. Chem. 1982,5 4 , 2310. Engstrom, R. C.; Strasser, V. A. Anal. Chem. 1084, 5 6 , 136. Wang, J.; Hutchins, L. 0.Anal. Chim. Acta 1985, 167, 325. Evans, J. F.; Kuwana, T. Anal. Chem. 197% 5 1 , 358. Park, S.; Quate, C. F. Appl. Phys. Len. 1988,48,112. Koenig, J. L.; Tuinstra, F. J. Chem. Phys. 1970, 53, 1126. Bowling, R. J.; Packard, R. T.; McCreery, R. L. J. Nectrochem. SOC. 1988, 6 0 , 1459. Bowling, R. J.; Packard, R. T.; McCreery, R. L. J. Am. Chem. SOC. 198& 1 1 1 , 1217. Bowling. R. J.: Packard, R. T.; McCreery, R. L. Langmuk 1989,5 , 683. McQulllan, A. J.; Hester. R. F. J. Raman Spechosc. 1984, 15, 1, 15. Engstrom, R. C.; Johnson, K. W.; OesJarlais, S. Anal. Chem. lS87, 59, 670. Petersen, S. L.; Weisshaar, D. E.;Tallman, D. E.; Schulze, R. K.; Evans, J. F.; DesJarlais. S.; Engstrom, R. C. Anal. Chem. l98S9 6 0 , 2358. Engstrom, R. C.; Pharr, C. M.; Koppang, M. D. J. flectroanal. Chem. 1987,221, 251. Engstrom, R. C.; Pharr, C. M.; Tople, R. A.; Unzelman, P. L., in press. Wick, R. A. Appl. Opt. 1987,2 6 , 3210. Haapakka, K. E.;Kankare, J. J. Anal. Chim. Acta 1982, 138. 263. Kepiey. L. J.; Bard, A. J. Anal. Chem. 1988, 6 0 , 1459.
RECEIVED for review June 16, 1989. Accepted September 25, 1989. This work was supported by the Air Force Office of Scientific Research (R.L.M.) and the National Science Foundation, Grant No. CHE-8703018 (R.C.E.). C.M.P. was the recipient of a Sigma Xi Grant-In-Aid of Research.
Fumed Silica Substrates for Enhanced Fluorescence Spot Test Analysis of Benzo[ a Ipyrene-DNA Adduct Products Randy W. Johnson and Tuan Vo-Dinh* Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 -6101
Amorphous fumed sl#ca has been found to enhance both the lntenslty and resolutlon of the emlslon, excitation, and synchronous fluorescence spectra of benzo[a]pyrene-r-7,t8,9,1O-tetrahydrotetroi (BPT) on a fllter paper substrate. Experimental parameters including the type and concentration of fumed slllca were investigated as well as the method of application. The use of fumed slllca as an enhancing agent in lumlnescence detection of polynuclear aromatic hydrocarbons Is attractive since the procedure is dmple, rqM, and cost-effective, and therefore suitable for routine analysis.
INTRODUCTION Polynuclear aromatic (PNA) compounds are generally recognized as potential carcinogens and mutagens. The process is believed to be initiated by conversion of these compounds via chemical or metabolic activation into highly reactive electrophiles which undergo attack by nucleophilic
* Author
t o whom correspondence should be addressed.
centers in DNA ( I , 2). The formation of complexes between electrophiles and specific sites in DNA may lead to induction of mutation and consequently other manifestations of genetic alterations. Benzo[a]pyrene and (BP) has often been investigated as the model compound for the carcinogenic PNAs. I t is metabolized to a number of oxiranes, phenols, and quinones (3-5). The ultimate carcinogenic product of B P is believed to be BP-'I,€i-dihydrodiol9,10-epoxide (BPDE) which binds to DNA (6). Methods for the measurement of BPDEDNA complexes are therefore important for biomonitoring of human exposure to genotoxic PNA chemicals. The highly fluorescent aromatic nucleus of the PNA molecule has allowed the detection of BPDE-DNA adducts by luminescence. Extremely sensitive assays are required due to the low levels of environmental exposure, which lead to low levels of adduct formation. Radioactive methods such as the 32Ppostlabeling technique can detect DNA adducts of PNA at extremely low levels (7). Nonradioactive techniques such as surface-enhanced Raman scattering (SERS) spectrometry (8),fluorescence line-narrowing (FLN) spectroscopy (9), room temperature phosphorimetry (RTP) ( l o ) ,and fluorometric detection for liquid chromatography (11 ) have been utilized
0003-2700/89/0361-2766$01.50/0t Z 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989
to detect minute amounts of BPDE-DNA adducts. The synchronous luminescence (SL) technique has been used for the selective characterization of mixtures of different PNAs (12-14). The SL technique involves scanning excitation and emission synchronously with a fixed wavelength difference (Ax). A signal is seen only when AA matches the interval between one absorption and one emission band; in many cases, a single peak is observed. Since DNA quenches the fluorescence of the covalently bound BPDE, acid hydrolysis induced relhoval of the pyrenyl moieties from the DNA as tetrols is generally required with each of these luminescence assays with the exception of the FLN, SERS, and R T P techniques. In this paper we report a simple technique for increasing the sensitivity of fluorescence detection for the benzo[a]pyrene tetrol (BPT) derivative. In previous studies, sensitized luminescence by energy transfer has been extensively investigated (15-19) for improving the sensitivity of luminescence analysis. In this work we now report the development of a simple method for fluorescence enhancement of a PNA compound (BPT) on filter paper by a substance with no intrinsic fluorescencefumed silica. There have been many diverse applications of silica and its derivatives in analytical spectroscopy. The use of silica and its derivatives as absorbants of analyte compounds has been widely documented (20-25). A Cls-derivative chromatographic silica wag used to preconcentrate pyrene from methanol-water solutions (26). The hydrophobic interactions between the PNA and the silica surface largely determine the amount of preconcentration. Utilizing a sensitive laser fluorometric system, Kirsch and co-workers (27) were able to achieve a detection limit of approximately 8000 molecules of Rhodamine 6G adsorbed onto the surface of small silica spheres. They point out two major advantages: (1) there is no solvent fluorescence or Raman scatter and (2) the particles are viewed from a stationary position. Cellulose substrates generally have highly reflective surfaces and can produce scattered light. Front surface illumination/detection geometry used with a solid surface can produce higher levels of scattered light than do solution measurements where a right-angle geometry is used. In this paper we report the effectiveness of fumed silica as a fluorescence enhancer on a paper support utilizing the biologically important compound B P T as a model. Experimental factors such as the type and concentration of fumed silica and method of application are discussed in detail. The results indicate that fumed silica induces an efficient enhancement of the fluorescence emission signals from BPT on a filter-paper background and is therefore potentially useful for the luminescence detection of PNA-DNA adducts. EXPERIMENTAL S E C T I O N Instrumentation. All fluorescence spectra were obtained with a Perkin-Elmer 650-40 luminescence spectrometer equipped with a 150-W xenon lamp excitation source and an R928 Hamamatsu photomultiplier tube. An IBM-XT personal computer was linked to the spectrophotometer through a RS-232 interface and appropriate software was written to control the spectrophotometer, collect and process the data, and display the results in graphic forms. Special laboratory-constructed sample holders were used. These interchangeable, finger-type sample holders allow frontal excitation and right-angle detection (28). Procedure. Two different procedures were investigated to obtain enhanced fluorescence. In the first method, equal volumes of 1 X lO+ M benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol(BPT) in ethanol and 10% (w/w) aqueous suspensions of fumed silica were combined and thoroughly mixed. With a Gilson micropipet, 2.5 mL of this solution was applied to filter paper (within the sample holder) and allowed to dry for approximately 2 min prior to measurement. In another procedure, fumed silica or analyte solution was applied to the filter paper first and upon drying the BPT solution was spotted onto the pretreated paper substrate.
300
310
320
330
340
2767
350
360
WAVELENGTH (nm)
Excitation scan of 1 X loJ M ethanolic BPT mixed with an equal volume of aqueous 10% (w/w) grade LM 190 fumed silica (-) compared to the excitation scan of 1 X M BPT mixed with an equal volume of water (---) on filter-paper substrate. Figure 1.
-'"
10
5
5
E
4
1
1
6p \ CI
370
I
\
390
410
430
450
470
490
WAVELENGTH (nm)
Flgure 2. Emission scan of 1 X M ethanolic BPT mixed with an equal volume of aqueous 10% (w/w) grade LM-130 fumed silm (-) compared to the emission scan of 1 X lod M BPT mixed with an equal volume of water (- - -) on filter-paper substrate.
In either case, the fluorescence of BPT without sensitizer was measured for comparison. Appropriate background spectra were obtained and subtracted for all measurements. For excitation measurements, the emission wavelength was set at 380 nm; for emission measurements, the excitation wavelength was set at 349 nm. For synchronous luminescence analyses, AA was set at 17 nm. Spectral resolutions were set at 1.5 nm for excitation, emission, and synchronous scans. Materials. Amorphous fumed silica (grades EH-5, LM-130, L-90, and M-5) was graciously provided by the CABOT Corp. BPT was acquired from the National Institues of Health Repository. Spectral-grade ethanol was purchased from Warner Graham Co., and distilled water was purchased from American ScientificProducts. Grade 2043A filter paper from Scheicher and Schuell, Inc., was used for all measurements. RESULTS AND DISCUSSION Enhanced Fluorescence Spectra. The experimental parameters investigated in luminescence enhancement by fumed silica included both the type and concentration of the fumed silica as well as the method of application. The effect of these parameters on the fluorescence of BPT was determined. Figures 1, 2, and 3 show the excitation, emission, and synchronous spectra of ethanolic 1 X M B P T premixed with an equal volume of aqueous 10% grade LM-130 fumed silica (final BPT concentration 4 x lo4 g) prior to application onto fiiter paper. For comparison, spectra obtained with BPT on paper substrate without fumed silica are also shown (dashed curve). It was observed that premixing the sample with fumed silica significantly enhanced the fluorescence of B P T by almost 1order of magnitude. In all cases, the signal has been increased -5 times. The limit of detection for BPT
2768
ANALYTICAL CHEMISTRY, VOL. 61, NO. 24, DECEMBER 15, 1989
I
5 t
10 I
13 t
c Gi a
a
4
k m a
>
Q
m
>
-
4> t fn
z fn
E
5
0 Y
z
m : : w
0 y1
5
Ma 2
a
s 2
i: 320
340
360
380
400
420
440
460
480
500
370
390
WAVELENGTH
410
430 450 WAVELENGTH (nm)
470
490
M ethanoic BPT mixed with Figure 3. Synchronous scan of 1 X an equal volume of aqueous 10% (w/w) grade LM-130 fumed silica (-) compared to the emission scan of 1 X M BPT mixed with an equal volume of water (- - -) on filter-paper substrate. Ah as set to 17 nm.
Figure 4. Comparison of the emission spectral intensitles of 1 X M BPT premixed with equal volumes of 10% solutions of different grades of fumed silica: curve A, grade M 5 ; curve B, grade EK5; curve C, grade LM-130; curve D, grade L-90. Excitation wavelength was set to 349 nm.
Table I. Fluorescence Intensity as a Function of Fumed Silica Size"
Table TI. Fluorescence Intensity as a Function of Fumed Silica Concentrationa
fluorescence
fumed silica
typeb L-90 LM-130 M-5
surface area,b mZjg 100 f 160 f 200 f 380 f
15 15 25 30
nominal particle diameter,*pm
intensity,c
0.027 0.017 0.014 0.007
5.95 f 0.86 9.24 f 0.45 9.59 f 0.72 8.60 f 1.84
arbitrary
units
EH-5 a Fumed silica materials were suspended in aqueous solution to give 10% solutions and were premixed with equal volumes of 1 X M ethanolic BPT solution prior to application onto filter paper. bData supplied by vendor. 'Measured at 380 nm with A,, = 349 nm.
is 2 pg. The dynamic range is approximately 2-3 orders of magnitude. The relative standard deviation of the measurement technique is approximately 10%. Figure 3 also illustrates the utility of the SL technique. While the emission and excitation spectral profiles of B P T are relatively broad, the synchronous spectrum exhibits narrower bandwidths, thereby simplifying detection of B P T in a mixture of PNA or other compounds with significant emission overlap. The synchronous spectra also show the presence of a small impurity, as indicated by the peak at 395 nm. It is noteworthy from Figures 1and 2 that besides the intensity enhancement, the spectral characteristics of B P T in the presence of fumed silica are very similar to those in the absence of fumed silica. The lack of significant structure alteration in the fluorescence excitation (Figure 1)and emission (Figure 2 ) spectra indicates that spectral changes due to absorption onto fumed silica are minimal for the excited singlet and ground state. The different commercially available grades of fumed silica vary according to surface areas and nominal particle diameters. For the grades used in this study, the surface areas ranged from 100 to 380 m2/g and nominal particle diameters from 0.007 to 0.027 mm (Table I). Figure 4 depicts the fluorescence intensity of 1 X M B P T measured a t 380 nm (hex= 349 nm) when premixed with equal volumes of 10% (w/w) of each of the four grades of fumed silica utilized in this study. Grade L-90 with the largest particle diameter (0.027 wm) and smallest surface area (100 m2/g) gives the weakest fluorescence signal, while the other grades give statistically equivalent signals. It is interesting to note that in previous investigations of silver-coated fumed silica as a substrate for surface-enhanced Raman scattering (SERS),Grade L-90 with the largest particle diameter provided the maximum SERS intensity while the smaller silica sizes gave weaker signals (29).
fluorescence intensity,b arbitrary units ?k fumed
1 2 3 4 5 6 7 8 9 10
silica
LM-130'
EH-5d
2.21 2.03 1.88 1.87 2.44 1.84 2.62 2.10 2.37 2.10
2.53 2.17 1.80 1.70 1.72 1.90 1.42 1.73 1.92 1.69
a Fumed silica materials were suspended in aqueous solution to give 5% solutions and applied to filter paper. Upon drying, 2.5 pL of 5 X lo4 M ethanolic BPT was applied to the same spot. bMeasured at 380 nm with A, = 349 nm. cRelative standard deviation = f 9.8%. dRelativestandard deviation = f 11.8%.
The fluorescence intensity of 5 x lo4 M B P T (applied on top of dried silica spot) with different concentrations of grades LM-130 and EH-5 fumed silica was measured at 380 nm (Aex = 349 nm). The concentrations ranged from 1% to 10% (w/w). Use of higher concentrations was prohibited due to aggregate formation of fumed silica particles. While Alak and Vo-Dinh (29) found an optimum concentration of 3% for SERS enhancement, the fluorescence signals from 1% to 10% on paper varied by only 10-12% and no clear pattern was observed (Table 11). Effect of Sample Preparation and Delivery. While the nominal particle size of the fumed silica (as indicated by grade) and the concentration (from 1%to 10%) appeared to play minor roles in fluorescence enhancement of BPT, the method of sample preparation and delivery seemed to be a key factor. Figure 5 shows the emission spectra of 1 X M BPT premixed with equal volumes of grade LM-130 fumed silica prior to application (curve A), application of fumed silica to the filter paper substrate prior to B P T (curve B), and application of of BPT to the paper substrate prior to fumed silica (curve C). For comparison, the emission spectra obtained with RPT on paper substrate without fumed silica is shown (curve D). Premixing equal volumes of enhancing agent and analyte solution gave the best enhancement of the B P T fluorescence signal, followed by application of analyte onto previously applied enhancing agent. Application of fumed silica onto analyte gave a comparatively small enhancement of fluorescence signal. The two most effective methods, premixing of
ANALYTICAL CHEMISTRY, VOL. 61,NO. 24, DECEMBER 15, 1989 10
I
1
>
a m IU
4 > t
z
z
0 Y
z
5: 0 U
3 370
390
410
430 WAVELENGTH
450
470
490
(nm)
Figure 5. Comparison of the emission spectral intensities produced by different sample application methods: curve A, 1 X M ethanolic BPT premixed with 10% (w/w) aqueous grade LM-130 fumed silica; curve 6, 5% (w/w) aqueous fumed silica applied first to filter paper with 5 X lo-' M BPT spotted on top; curve 5 X lo-' BPT applied to filter paper first with aqueous fumed silica on top; curve D, 5 X lo-' M BPT solution applied without fumed silica. Excitation wavelength was set to 349 nm.
analyte and enhancing agent solution and application of fumed silica preceding analyte application both allow the agent to have extensive contact with the paper. When the fumed silica is applied onto the analyte, incident light is prevented from fully reaching the BPT, thus weaker fluorescence enhancement is observed. Fumed silica has no intrinsic fluorescence of its own; therefore, it is not functioning in the same manner by which anthracene or naphthalene enhances luminescence: that is, a transfer of excitation energy. While further investigation is underway to more fully explain the phenomena, several possibilities exist. As a manifestation of the chromatographic effect as noted in preconcentration studies (26),the fluorescence enhancement might be due to improved adsorption of the BPT onto the surface treated with fumed silica. Fumed silica can also retain more BPT molecules onto the surface. The microstructure of fumed silica particles might also induce surface-enhanced mechanisms in fluorescence emission. This latter possibility is under current investigation. The use of amorphous fumed silica as a luminescence sensitizing enhancing in this type of analysis is attractive for several reasons. One of the problems related to analysis of complex mixtures by the sensitized (energy transfer) technique is that not all compounds will be sensitized. The degree of sensitization is determined by the amount of spectral overlap between analyte and sensitizer. Since fumed silica is inducing an increase in fluorescence signal in another manner, spectral overlap between sensitizers and analyte molecules is not required and the applicability of the technique is more general. The potential exists therefore for the resolution of minute quantities within a multicomponent mixture of PNA compounds. As demonstrated, fumed silica enhances both intensity and resolution of luminescence on filter paper. It is inert and gives no interfering fluorescence emission. Very small amounts of sample are required (4 X g final concentration on paper). The procedure is simple, fast, and
2769
cost-effective and therefore suitable for routine analyses. Fumed silica is an inexpensive, easy-to-handle material. We have demonstrated the application of fumed silica enhancement of fluorescence for BPT and are currently investigating its utility for the detection of a number of PNAs. This technique also has potential for other analytical applications where detection of minute concentrations is required, such as DNA sequencing utilizing fluorophores (30).
ACKNOWLEDGMENT The authors acknowledge the assistance of G . H. Miller for preliminary fluorescence measurements. The computer software was written by Mr. Peter St. Wecker. Registry No. BPT, 61490-68-4; silica, 7631-86-9. LITERATURE CITED (1) Heidelberger, C. Annu. Rev. Biochem. 1975, 44, 79. (2) Jerina, D. M.; Daly, J. W. Science 1974, 785, 573. (3) Nemoto, N.; Gelboin, H. Arch. Biochem. Biophys. 1975, 170, 739. (4) Nemoto, N.; Gelboin, H. V. Biochem. pharmacal. 1978, 25, 1221. (5) Nemoto, N.; Takayama, S.; Gelboin, H. V. Biochem. Pharmacal. 1977, 25, 1825. (6) Levin, W.; Wood, A. W.; Yagi, H.; Jerina, D. M.; Conney, A. H. Roc. Natl. Acad. Sci. U . S . A . 1978, 73, 3867. (7) Gupta, R. C.; Reddy, M. V.; Randerath, K. Carcinogenesis 1982, 3 , 1081. (8) Vo-Dinh, T.; Uziel. M.; Morrison, A. L., Appl. Spectrosc. 1987, 47, 605. (9) Sanders, M. J.; Cooper, R. S.; Small, G. J.; Heisig. V.; Jeffrey, A. M. Anal. Chem. 1985, 57, 1148. (10) Vo-Dinh, T.; Uziel, M. Anal. Chem. 1987, 59, 1093. (11) Rahn, R. 0.; Chang, S. S.; Holland, J. M.; Shugart, L. R. Biochem. Blophys. Res. Commun. 1982, 709, 262. (12) Vo-Dinh, T. Anal. Chem. 1978, 50, 396. (13) Vo-Dinh, T. I n Modern Fluorescence Specfroscopy;Wehry, E. L., Ed.; Plenum Press: New York, 1981. (14) Vo-Dinh, T. Appl. Specfrosc. 1982, 36, 576. (15) Frenkel, J. Phys. 2. Sowjetunion 1938, 9 , 158. (16) Forster, T. Discuss. Faraday Soc. 1959, 27, 7. (17) Powell, R. C.; Soos, 2. G. J . Lumin. 1975, 7 7 , 1. (18) Hornyak, I. J . Lumin. 1972, 5 , 132. (19) Hornyak, I. J . Lumin. 1975/1976. 7 7 , 241. (20) Watanabe, H.; Goto, K.; Taguchi, S.; McLaren, J. W.; Berman, S. S.; Russell, D. S. Anal. Chem. 1981, 53, 738. (21) Taguchi, S . ; Yai, T.; Shimada, Y.; Goto, K. Talanta 1983, 30, 169. (22) Uden, P. C.; Parees, D. M.;Walters, F. H. Anal. Chem. 1975, 8 , 795. (23) Ryan, D. K.; Weber, J. H. Talanta 1985, 32, 859. (24) Jezorek, J. R.; Faltynski, K. H.; Blackburn, L. G.; Henderson, P. J.; Medina, H. D. Talanta 1985, 32, 783. (25) Leydon, D. E.; Luttrell, G. H. Anal. Chem. 1975, 47, 1812. (26) Carr, J. W.; Harris, J. M. Anal. Chem. 1988, 6 0 , 698. (27) Kirsh, B.; Voigtman, E.; Winefordner, J. D. Anal. Chem. 1985, 57, 2008. (28) Vo-Dinh, T.; Walden, G.; Winefordner, J. D. Anal. Chem. 1977, 49, 1126. (29) Alak, A. M.; Vo-Dinh, T. Anal. Chem. 1989, 6 7 , 656-680. (30) Smith, L. M.; Sanders, J. 2.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Conne4 C. R.; Heiner, C.; Kent S . B. H.; Hood, L. E.; Nature 1988, 321, 674.
RECEIVED for review May 16, 1989. Accepted September 5, 1989. This work was sponsored by the Laboratory Director's R&D Fund and Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05840R321400 with Martin Marietta Energy Systems, Inc. This research was also supported in part by an appointment of R.W.J. to the postgraduate Research Training Program under Contract No. DE-AC05-760R00033 between the U.S. Department of Energy and the Oak Ridge Associated Universities.
