Determination of Technetium and Its Speciation by Surface-Enhanced

Anal. Chem. 2007, 79, 2341-2345

Determination of Technetium and Its Speciation by Surface-Enhanced Raman Spectroscopy Baohua Gu*,† and Chuanmin Ruan‡

Environmental Sciences Division, Oak Ridge National Laboratory, and Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee 37831

Technetium-99 (Tc) is an important radionuclide of concern, and there is a great need for its detection and speciation analysis in the environment. For the first time, we report that surface-enhanced Raman spectroscopy (SERS) is capable of detecting an inorganic radioactive anion, pertechnetate (TcO4-), at ∼10-7 M concentration levels. The technique also allows the detection of various species of Tc such as oxidized Tc(VII) and reduced and possibly complexed Tc(IV) species by use of gold nanoparticles as a SERS substrate. The primary Raman scattering band of Tc(VII) occurs at about 904 cm-1, whereas reduced Tc(IV) and its humic and ethylenediaminetetraacetic acid (EDTA) complexes show scattering bands at about 866 and 870 cm-1, respectively. Results also indicate that Tc(IV)-humic complexes are unstable and reoxidize to TcO4- upon exposure to oxygen. This study demonstrates that SERS could potentially offer a new tool and opportunity in studying Tc and its speciation and interactions in the environment at low concentrations. Technetium-99 (Tc) is a fission product of uranium-238 and a β emitter with a maximum energy of 292 keV. It is a radionuclide of concern because of its long half-life (2.1 × 105 yr) and high mobility under oxic conditions in the subsurface environment.1-3 Its contamination mainly results from enrichment activities during the cold war and, in some cases, from uses in radiobiology and nuclear medicine.1-5 In oxygenated and suboxygenated environments, Tc occurs as Tc(VII) in the form of the pertechnetate anion (TcO4-), which is highly soluble, poorly retained by sediments, and thus readily transported with groundwater. Under reducing conditions, however, Tc(VII) is readily reduced, either chemically or biologically, primarily to Tc(IV) species, which have low solubility and thus are retained by sediments.1,2,6-9 However, reduced Tc(IV) is known to form complexes with a number of organic and inorganic ligands such as carbonate, citrate, and * To whom correspondence should be addressed. Phone: 865-574-7286. Fax: 865-576-8543. E-mail: [email protected]. † Environmental Sciences Division, Oak Ridge National Laboratory. ‡ Oak Ridge Institute for Science and Education. (1) Wildung, R. E.; Garland, T. R.; McFadden, K. M.; Cowan, C. E. Technetium sorption in surface soils; Elsevier Applied Science. Publishers: London, 1986. (2) Bondietti, E. A.; Francis, C. W. Science 1979, 203, 1337-1340. (3) Gu, B.; Dowlen, K. E.; Liang, L.; Clausen, J. L. Sep. Technol. 1996, 6, 123132. (4) Mikelsons, M. V.; Pinkerton, T. C. Appl. Radiat. Isot. 1987, 38, 569-570. (5) Wilson, G. M.; Pinkerton, T. C. Anal. Chem. 1985, 57, 246-253. 10.1021/ac062052y CCC: $37.00 Published on Web 02/09/2007

© 2007 American Chemical Society

natural humic substances,8-10 rendering reduced Tc(IV) also soluble and potentially mobile in the subsurface. Sekine et al.10 have suggested that, under reducing environments, Tc(IV)-humic complexes could potentially be the dominant chemical species occurring in deep groundwater containing humic substances and might thus control the mobility and fate of Tc. Evidence of Tc(IV)-humic complexation mainly comes from indirect measurements by gel-permeation or ion-exchange chromatography and precipitation techniques.9-12 Until recently, analyses by extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge spectroscopy (XANES) provided direct proof of the complexation reactions between Tc(IV) and humic substances.11,13 However, these techniques required the use of a relatively high concentration of Tc and had only limited uses to date, perhaps because of the accessibility and high radioactivity of Tc. Conventional Raman spectroscopy also has been used to study the complexation reactions between Tc(IV) and synthetic organic ligands such as hydroxyethylidene diphosphonate (HEDP) and ethylenediaminetetraacetic acid (EDTA) in the field of radiopharmaceuticals and nuclear medicine.4,14 But the technique is insensitive unless relatively high concentrations of Tc (millimoles) and high power lasers are used for the measurements. To date, no studies have reported the use of surface-enhanced Raman spectroscopy (SERS) to detect and analyze Tc and its species, although SERS has been widely used as an important tool in areas such as trace chemical and biological detection and characterization.15-20 SERS refers to the observation that the (6) Francis, A. J.; Dodge, C. J.; Meinken, G. E. Radiochim. Acta 2002, 90, 791797. (7) Lloyd, J. R.; Sole, V. A.; Van, Praagh, C. V. G.; Lovley, D. R. Appl. Environ. Microbiol. 2000, 66, 3743-3749. (8) Wildung, R. E.; Gorby, Y. A.; Krupka, K. M.; Hess, N. J.; Li, S. W.; Plymale, A. E.; McKinley, J. P.; Fredrickson, J. K. Appl. Environ. Microbiol. 2000, 66, 2451-2460. (9) Abdelouas, A.; Grambow, B.; Fattahi, M.; Andres, Y.; Leclerc-Cessac, E. Sci. Total Environ. 2005, 336, 255-268. (10) Sekine, T.; Asai, N.; Mine, T.; Yoshihara, K. Radiochemistry 1997, 39, 309311. (11) Maes, A.; Geraedts, K.; Bruggeman, C.; Vancluysen, J.; Rossberg, A.; Hennig, C. Environ. Sci. Technol. 2004, 38, 2044-2051. (12) Sekine, T.; Watanabe, A.; Yoshihara, K.; Kim, J. I. Radiochim. Acta 1993, 63, 87-90. (13) Geraedts, K.; Bruggemann, C.; Maes, A.; Van, Loon, L. R.; Rossberg, A.; Reich, T. Radiochim. Acta 2002, 90, 879-884. (14) Davison, A.; Perlstein, R. M.; Mabrouk, P. A.; Jones, A. G.; Morelock, M. M. J. Nucl. Med. Allied Sci. 1985, 29, 194-194. (15) Dai, S.; Lee, Y. H.; Young, J. P. Appl. Spectrosc. 1996, 50, 536-537. (16) Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106.

Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2341

apparent Raman cross section for target molecules adsorbed on roughened metal (e.g., silver or gold) surfaces is greatly enhanced over their normal surface values.16,17,21 In other words, the Raman spectral signal could be enhanced by many orders of magnitude, allowing detection up to single molecular concentration levels. SERS has overcome several major limitations of conventional Raman spectroscopy such as low signal intensity and interference from environmental matrixes (e.g., fluorescence by dissolved humic materials). The technique is nondestructive, and water does not interfere with spectral collection, thereby allowing direct analysis of environmental aqueous samples. We have recently developed a SERS technique to detect an important environmental pollutant, perchlorate (ClO4-), at ultralow concentrations with silver or modified gold nanoparticles as a SERS substrate.18,19 Here we report the detection and analysis of Tc using similar techniques. In particular, we found that this new technique is capable of not only detecting Tc unambiguously at low concentrations but also providing chemical species of reduced or oxidized forms of Tc in aqueous solution. MATERIALS AND METHODS Preparation of Gold SERS Substrates. SERS substrates used for technetium and its speciation analysis were prepared following similar techniques reported previously for the detection of perchlorate anions.19 In brief, colloidal Au nanoparticles with an average size of about 54 nm in diameter were prepared following the methods of Olson et al.22 with minor modifications. A seed colloidal Au suspension was first prepared by mixing 1 mL of 1% aqueous HAuCl4‚3H2O in 100 mL of purified water with vigorous stirring, followed by the sequential addition of 1 mL of 1% trisodium citrate and 1 mL of 0.075% NaBH4 in 1% trisodium citrate. This seed Au suspension was stirred continuously for an additional 5 min and stored at 4 °C. Colloidal Au nanoparticles (∼54 nm) were then prepared by heating 4 mL of 1% HAuCl4‚3H2O in 900 mL of deionized water to boiling, followed by the addition of 1 mL of the “seed” colloid and 3.6 mL of a 1% trisodium citrate solution. The solution mixture was refluxed for an additional 10 min before it was cooled under agitation. The particle size of resulting Au nanoparticles was determined to be ∼54 nm by means of dynamic light scattering on a ZetaPlus particle size analyzer (Brookhaven Instruments Co., New York). To enhance the sorption and therefore the detection of TcO4and organo-Tc(IV) complexes, surfaces of Au nanoparticles were further modified with positively charged dimethylamine functional groups.19 The Au colloidal suspension was then centrifuged at 14 000 rpm, and the concentrated Au nanoparticles were collected, resuspended in 0.01 M HCl solution, and used as SERS substrates. This concentrated Au nanoparticle suspension was found to be stable for at least 8 months (since it was prepared), although ultrasonification is necessary to ensure well-dispersed Au colloids before use. All chemicals used in this study were reagent-grade. (17) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 87, 2957-2975. (18) Gu, B.; Tio, J.; Wang, W.; Ku, Y.; Dai, S. Appl. Spectrosc. 2004, 58, 741744. (19) Ruan, C. M.; Wang, W.; Gu, B. Anal. Chim. Acta 2006, 567, 114-120. (20) Vo-Dinh, T.; Allain, L. R.; Stokes, D. L. J. Raman Spectrosc. 2002, 33, 511514. (21) Zhao, K.; Xu, H.; Gu, B.; Zhang, Z. J. Chem. Phys. 2006, 125, 081102.

2342

Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

Preparation of TcO4- and Reduced Tc(IV)-Ligand Complexes for SERS Detection. Aqueous TcO4- solutions in the concentration range of 3 × 10-4 to 3 × 10-7 M were prepared by a series of 10× dilution of the stock ammonium pertechnetate (NH4TcO4) (3 mM) in water. After mixing of each TcO4- solution (0.4 mL) with 0.1 mL of the concentrated Au colloids, approximately 5 µL of the suspension was transferred onto a clean glass slide, dried to a wet consistency until any visible liquid water was evaporated, and then immediately analyzed by micro-Raman spectroscopy. Reduced Tc(IV) and its organic ligand complexes were prepared by the reduction of NH4TcO4 (3 × 10-4 M) with NaBH4 in the presence of excess amounts of various organic ligands,4 including EDTA (2.5 mM) and a humic acid (FRC-HA, 2.5 mM as carbon) isolated from the U.S. Department of Energy (DOE) Oak Ridge Field Research Center (FRC) in Oak Ridge, TN.23 The reduction was carried out at room temperature in small vials under open atmosphere, and an excess amount of NaBH4 (0.2 M) was used to ensure complete reduction of Tc(VII) initially. However, no attempt was made to avoid the reoxidation of reduced Tc(IV) or Tc(IV)-ligand complexes, so that Tc speciation changes during the reduction and oxidation processes could be evaluated. These samples were analyzed similarly to TcO4- samples after mixing with Au nanoparticles. Raman Spectroscopic Analysis. Raman spectra of samples (∼5 µL) on glass slides were collected on a Renishaw microRaman spectrometer system equipped with a 300 mW nearinfrared diode laser at a wavelength of 785 nm for excitation (Renishaw Inc., New Mills, U.K.). The laser beam was set in position with a Leica imaging microscope and a long-workingdistance (LWD) objective (50×) at a lateral spatial resolution of ∼2 µm. The use of a LWD objective is necessary in order to view and analyze samples in liquid by focusing onto clusters of Au nanoparticles. It is advantageous to collect spectra while the sample is wet but without free-standing water; it appears to give a low background signal of modified Au nanoparticles. A chargecoupled device (CCD) array detector was used to achieve signal detection from a 1200 grooves/mm grating light path controlled by Renishaw WiRE software and analyzed by Galactic GRAMS software. Laser power at the exit of the microscope objective was ∼1 mW by using a set of neutral density filters. The acquisition time is 10 s, and the spectral resolution is 2-4 cm-1. RESULTS AND DISCUSSION Analysis of Aqueous TcO4- Concentrations. Detection of TcO4- in aqueous samples was performed initially to determine if SERS could be used as a quantitative technique for the analysis of TcO4- at varying concentrations. Results indicate that we were able to detect TcO4- at concentrations as low as 3 × 10-7 M (Figure 1). The primary Raman scattering peak (due to symmetric stretching vibration) of TcO4- occurred at about 904 cm-1, which is consistent with literature data from conventional Raman spectroscopy.4 No Raman signal could be detected directly from the aqueous solution at such low concentrations or even by using (22) Olson, L. G.; Lo, Y. S.; Beebe, T. P.; Harris, J. M. Anal. Chem. 2001, 73, 4268-4276. (23) Gu, B.; Yan, H.; Zhou, P.; Watson, D.; Park, M.; Istok, J. D. Environ. Sci. Technol. 2005, 39, 5268-5275.

Figure 1. Detection of pertechnetate (TcO4-) on modified gold nanoparticles by surface-enhanced Raman spectroscopy (SERS). SERS spectra were acquired by use of laser excitation at 785 nm (∼1 mW at the exit of a 50× microscope objective) without background corrections. Concentrations of TcO4- were (a) 0, (b) 3 × 10-7, (c) 3 × 10-6, (d) 3 × 10-5, and (e) 3 × 10-4 M.

the stock solution at 3 mM without the addition of Au nanoparticles (data not shown). By using a more powerful argon ion laser at 3000 mW as an excitation source, Mikelsons and Pinkerton4 reported detection of TcO4- at ∼5 mM. These observations suggest that SERS yielded an enhancement factor of at least 104 for TcO4- detection by use of surface-functionalized Au nanoparticles as a SERS substrate. Results also indicate that the primary Raman scattering band at 904 cm-1 for TcO4- increased consistently with an increase of aqueous TcO4- concentrations (Figure 1), although a plot between the peak height and the concentration did not yield a linear relationship (Figure 2). These observations are common for vibrational spectroscopic techniques and may be partially attributed to a wide concentration range (4 orders of magnitude) used in the study. A semilogarithmic plot, however, yielded a nearly straight line (Figure 2, inset), suggesting that the technique could potentially be used for quantitative or semiquantitative analysis of TcO4- in aqueous solution, particularly within a small concentration range. Similarly to what was observed for the detection of perchlorate (ClO4-) ions,19 the reproducibility of these measurements is fairly good, usually within (15% for different specimens prepared under the same experimental conditions. For every drop of sample analyzed, we also examined three or more spots within the same specimen and found that the intensity of resulting Raman spectra is highly reproducible. The surface modification of Au nanoparticles with dimethylamine functional groups is essential in achieving maximum SERS enhancement and good reproducibility. These observations can be attributed to the fact that these amine functional groups carry positive charges and thus act as anion-exchange or molecular recognition sites, rendering TcO4- ions adsorbed or distributed evenly on modified Au nanoparticles. Additionally, the amine functional groups may have facilitated the aggregation of Au nanoparticles, which is necessary to obtain strong plasmon enhancement on gold metal sur-

Figure 2. Plot of the pertechnetate (TcO4-) concentration against peak height of SERS spectra shown in Figure 1. The inset was plotted on a semilog scale on average.

faces.16,17,21 Without the surface modification, the analyte is unlikely to be evenly distributed when it dries or crystallizes on metal nanoparticle surfaces. In fact, our initial studies with bare gold or silver nanoparticles gave no SERS signals at all, even at millimolar concentrations. As reported previously, the likelihood of detecting an analyte by SERS depends on several characteristics of the metal colloid aggregates, such as position, neighboring electromagnetic interactions, size, and surface morphology.16,17 Classic calculations of isolated colloids in SERS assume an average contribution per metal colloid, but Nie and Emory16 hypothesized that the total SERS enhancement factor could be correlated to the existence of “hot spots”. These authors have shown that between 1 in 100 and 1 in 1000 silver colloids were SERS-active or “hot”. The probability of finding these “hot spots” becomes even lower as the analyte concentration decreases. This has been a major limiting factor for the use of SERS as a routine analytical tool. Therefore, our technique using surface-modified Au nanoparticles provides better and more reproducible detection of TcO4- and other ions.19 Speciation Analysis of Tc and Its Complexes. One great advantage of using SERS is that not only can the presence of TcO4- be unambiguously identified but also different species of Tc may be determined in aqueous solution. As illustrated earlier, the Raman spectrum of TcO4- ions (i.e., the oxidized form of Tc) contains a primary scattering peak at about 904 cm-1. However, the reduced Tc(IV)O2 and its complexes with EDTA and FRCHA were found to have Raman bands at about 870 and 866 cm-1, respectively (Figure 3). The spectrum in the presence of FRCHA also showed a doublet at 904 and 866 cm-1, suggesting the presence of both oxidized TcO4- ions and reduced Tc(IV) species. These observations are consistent with previously published data4 that reduced Tc(IV)O2 has a strong Raman scattering peak at about 877 cm-1. Reduced Tc(IV) is known to form complexes with various synthetic organic ligands and, in fact, Tc(IV)-ligand complexes have been widely used in diagnostic imaging applicaAnalytical Chemistry, Vol. 79, No. 6, March 15, 2007

2343

Figure 3. SERS speciation analysis of Tc (10-4 M) and its complexes with EDTA (2.5 mM) and FRC-HA (2.5 mM as C) following the reduction by NaBH4. SERS spectra were acquired by use of laser excitation at 785 nm (∼1 mW at the exit of a 50× microscope objective) without background corrections.

tions in nuclear medicine.4,14,24-26 An even greater Raman shift at 748 cm-1 has been reported when reduced Tc(IV) forms a dimeric -TcO2Tc- ring structure due to its complexation with organic ligands such as TCTA (1,4,7-triazayclononane-N,N′,N′′-triacetate), H2-EDTA, and oxalate.27,28 The slight shift in frequencies observed in this study may be attributed to the complexation reactions between organic ligands and monomeric or colloidal Tc(IV) because the formation of dimeric ligand-TcO2Tc-ligand complexes would have caused a greater shift in frequencies.4,27 Using EXAFS and XANES techniques, Geraedts et al.13 and Maes et al.11 also reported that interactions between reduced Tc(IV) and humic substances involve the association of humics with hydrated TcO2‚ xH2O polymers (or colloids), especially at relatively high Tc(IV) concentrations. They called this reaction mechanism hydrophobic sorption or colloidal interaction with humic substances. The fact that the spectrum of Tc(IV)-humic complexes showed doublet peaks (at 866 and 904 cm-1, Figure 3) indicates partial reoxidation of reduced Tc(IV) or Tc(IV)-humic complexes. As stated earlier, these samples were prepared and analyzed under open atmosphere about 1 h following the reduction by NaBH4. Reoxidation did not occur in the presence of EDTA, suggesting that reduced Tc(IV) is perhaps less strongly bound to humics than EDTA. Similarly in a study of the interaction of Tc(IV) colloids with humic substances, Maes et al.11 reported the presence of both TcO4- and Tc(IV) species in one of the samples that was taken in the course of the reduction process. To further verify the oxidation of Tc(IV)-humic complexes, the same SERS specimens were re-examined after 24 h. Results (Figure 4) clearly indicate an increased band intensity at 904 cm-1 for TcO4- but a (24) Ben Said, K.; Fattahi, M.; Musikas, C.; Revel, R.; Abbe, J. C. Radiochim. Acta 2000, 88, 567-571. (25) Davison, A.; Jones, A. G.; Abrams, M. J. Inorg. Chem. 1981, 20, 43004302. (26) Selig, H.; Fried, S. Inorg. Nucl. Chem. Let. 1971, 7, 315-320. (27) Linder, K. E.; Dewan, J. C.; Davison, A. Inorg. Chem. 1989, 28, 38203825. (28) Alberto, R.; Anderegg, G.; Albinati, A. Inorg. Chim. Acta 1990, 178, 125130.

2344 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

Figure 4. SERS analysis of Tc(IV) complexes with EDTA and FRCHA after 1-day drying on glass slides in open atmosphere (the same sample used for SERS analysis in Figure 3). SERS spectra were acquired by use of laser excitation at 785 nm (∼1 mW at the exit of a 50× microscope objective) without background corrections.

diminished band intensity for reduced Tc(IV) species. Only a shoulder at about 866 cm-1 could be seen in the spectrum of Tc(IV)-humic complexes. On the other hand, the complexes between Tc(IV) and EDTA remained stable after 24 h. These observations are consistent with the notion that stability of Tc(IV)-ligand complexes depends on, among other factors, the chemical nature and structural properties of organic ligands themselves. Humics are naturally occurring, complex, and heterogeneous mixtures of organic compounds (with different functional groups) that can form complexes with varying stabilities. They also contain electron-rich (or electron-donating) and electrondeficient (electron-accepting) moieties, making them redox-active for reactions with metals and radionuclides.23,29 Therefore, the initial partial oxidation of Tc(IV)-humics (Figure 3) may be attributed to the oxidation of Tc(IV) or TcO2‚xH2O colloids that were bound to humic moieties with relatively low stabilities, and all Tc(IV) was then oxidized with an increase of exposure time. This work clearly demonstrates that the SERS technique could be used for the determination of Tc species in micromolar concentration ranges. Although techniques such as liquid scintillation counting and inductively coupled plasma mass spectrometry (ICP-MS) can give a low detection limit at nanomolar concentration ranges,3,30,31 they provide no information on the chemical speciation of Tc. The SERS technique could potentially offer a new tool and opportunity in future studies to study the reaction kinetics and mechanisms of Tc with various ligands and their speciation in the environment. However, it is noted that reactions between Tc(IV) and organic ligands also could cause some degree of peak broadening (Figure 3), which could make it difficult to distinguish different organic complexes, especially in a mixed ligand system. Future studies are obviously warranted in order to optimize the technique so that SERS could be utilized for (29) Cory, R. M.; McKnight, D. M. Environ. Sci. Technol. 2005, 39, 8142-8149. (30) Bartosova, A.; Rajec, P.; Klimekova, A. Chem. Pap. 2006, 60, 125-131. (31) Richter, R. C.; Koirtyohann, S. R.; Jurisson, S. S. J. Anal. At. Spectrom. 1997, 12, 557-562.

chemical and structural analyses because of its simplicity and ability to provide molecular fingerprint information.

Directed Research and Development SEED Program of Oak Ridge National Laboratory, which is managed by UT-Battelle LLC for the U.S. Department of Energy under DE-AC05-00OR22725.

ACKNOWLEDGMENT This research was supported in part by the DOE Environmental Remediation Sciences Program (ERSP) of the Office of Science Biological and Environmental Research and by the Laboratory

Received for review November 2, 2006. Accepted January 16, 2007. AC062052Y

Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

2345