A Portable Raman Acoustic Levitation Spectroscopic System for the

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Anal. Chem. 2005, 77, 4955-4961

A Portable Raman Acoustic Levitation Spectroscopic System for the Identification and Environmental Monitoring of Algal Cells Bayden R. Wood,*,† Philip Heraud,†,‡ Slobodanka Stojkovic,‡ Danielle Morrison,† John Beardall,†,‡ and Don McNaughton†

Centre for Biospectroscopy and School of Chemistry, Monash University, 3800 Victoria, Australia, and School of Biological Sciences, Monash University, 3800 Victoria, Australia

We report the coupling of a portable Raman spectrometer to an acoustic levitation device to enable environmental monitoring and the potential taxonomic identification of microalgae. Spectra of living cells were recorded at 785 nm using a fiber-optic probe coupled to a portable Raman spectrometer. The spectra exhibit an excellent signal-tonoise ratio and clearly show bands from chlorophyll a and β-carotene. Spectra of levitated photobleached microalgae clearly show a reduction in chlorophyll a concentration relative to β-carotene after 10 min of exposure to a quartz halogen lamp. Spectra recorded from levitated nitrogenlimited cells also show a significant reduction in bands associated with chlorophyll a, as compared to nitrogenreplete cells. To investigate the diagnostic capability of the technique, four species of microalgae were analyzed. Good quality spectra of all four species were obtained showing varying ratios of β-carotene to chlorophyll. The combination of an acoustic levitation device and a portable Raman spectrometer shows potential as a taxonomic and environmental monitoring tool with direct application to field studies in remote environments. The ability to detect chemical analytes and monitor nutrient status of living cells is of critical importance in environmental monitoring and biotechnology applications.1 To date, a number of different sample handling procedures and spectral monitoring techniques have been proposed to deal with commercial microorganisms.2,3 In this context, FT-IR spectroscopy shows enormous potential.3 One major limitation in developing FT-IR for on-line bioprocessing applications is the interference from water deformation modes in the 1700-1550-cm-1 region of the FT-IR spectrum, where the strong and analytically useful amide bands absorb. Raman spectroscopy has the advantage that water is a weak * Corresponding author. Phone: +61-3-9905-5721. Fax: +61-3-9905-4597. E-mail: [email protected]; http://web.chem.monash.edu.au/ BioSpec/biospectroscopy.html. † Centre for Biospectroscopy and School of Chemistry. ‡ School of Biological Sciences. (1) Beardall, J.; Berman, T.; Heraud, P.; Kadiri, M.; Light, B.; Patterson, G.; Roberts, S.; Sahan, E.; Schulzberger, B.; Urlinger, U.; Wood, B. R. Aq. Sci. 2001, 63, 107-121. (2) Baena, J. R.; Lendl, B. Curr. Opin. Chem. Biol. 2004, 8, 534-539. (3) Jarute, G.; Kainz, A.; Schroll, G.; Baena, J. R.; Lendl, B. Anal. Chem. 2004, 76, 6353-6358. 10.1021/ac050281z CCC: $30.25 Published on Web 06/21/2005

© 2005 American Chemical Society

Raman scatterer and, therefore, does not interfere with or obscure bands in the Raman spectrum. Hitherto, most Raman measurements of living cells have required a microscope facility, and in many cases, single cell analysis is either impractical or often results in a very poor signal-to-noise ratio. Moreover, the technique has little practicality in the field, where the ability to diagnose and monitor algal blooms is paramount to the preservation of delicate ecosystems. Acoustic levitation provides a containerless environment for monitoring and is now an established miniaturization technique that has found a myriad of analytical and bioanalytical applications.4 The technique has been applied to investigate the evaporation,5,6 drying,7 temperature,8 and stability of liquid drops.4 It has also been used to monitor the formation of ice particles,9 as well as in titration and crystallization studies of pharmaceuticals10 and proteins.11 Although a number of applications have involved liquid droplets and crystals, few studies have focused on living cells with a notable exception.12 To date, the analysis of cells with an acoustic levitation device has entailed incorporating a fluorescence imaging detector system to monitor single-cell responses from fat cells and other related cells.12 The coupling of an acoustic levitation system with Raman spectroscopy was first demonstrated by Kiefer and Popp and coworkers,13,14 who investigated chemical reactions of microcrystals. Subsequently, Lendl and co-workers15 further demonstrated the scope of the technique in vibrational spectroscopy. Their method (4) Santesson, S.; Nilsson, S. Anal. Bioanal. Chem. 2004, 378, 1704-1709. (5) Trinh, E. H.; Martson, P. L.; Robey, J. L. J. Colloid Interface Sci. 1998, 124, 95-103. (6) Bayazitoglu, Y.; Mitchell, G. F. J. Thermophys. Heat Transfer 1995, 9, 694701. (7) Yarin, A. L.; Brenn, G.; Kastner, O.; Tropea, C. Phys. Fluids 2002, 14, 22892298. (8) Omrane, A.; Santesson, S.; Alden, M.; Nilsson, S. Lab Chip 2004, 4, 287291. (9) Tuckerman, R.; Neidhart, B.; Lierke, E. G.; Bauerecker, S. Chem. Phys. Lett. 2002, 363, 349-354. (10) Santesson, S.; Johansson, J.; Taylor, L. S.; Levander, I.; Fox, S.; Sepaniak, M.; Nilsson, S. Anal. Chem. 2003, 75, 2177-2180. (11) Santesson, S.; Cedergren-Zeppezauer, E. S.; Johansson, T.; Laurell, T.; Nilsson, J.; Nilsson, S. Anal. Chem. 2003, 75, 1733-1740. (12) Santesson, S.; Andersson, M.; Degerman, E.; Johansson, T.; Nilsson, J.; Nilsson, S. Anal. Chem. 2000, 72, 3412-3418. (13) Musick, J.; Popp, J. Phys. Chem. Chem. Phys 1 1999, 24, 5497-5502. (14) Musick, J.; Kiefer, W.; Popp, J. Aplp. Spectrosc. 2000, 54, 1136-1141. (15) Leopold, N.; Haberkorn, M.; Laurell, T.; Nilsson, J.; Baena, J. R.; Frank, J.; Lendl, B. Anal. Chem. 2003, 75, 2166-2171.

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involved surface enhanced Raman scattering (SERS) using silver colloidal sols to detect several organic test molecules. The Raman signal in this case is generated by the chemical and electromagnetic interaction between the organic molecule and the surface of the colloidal nanoparticles. In this paper, we report on a portable system that combines acoustic levitation and Raman spectroscopy with a fiber-optic probe to directly monitor algal cells. In addition to the portability, the system has the advantage of enabling the measurement of cells in their native media in a contactless and fixed position and without the use of chemical probes that may disturb the system. EXPERIMENTAL SECTION Cells and Chemicals. Four species of microalgae, namely, the green alga Dunaliella tertiolecta, two diatoms Chaetoceros muelleri and Phaeodactylum tricornutum, and the red alga Porphyridium purpureum (CSIRO Marine Laboratories, Hobart, strains CS 175, CS 176, CS 29, and CS-25, respectively) were grown in 100-mL batch cultures with artificial seawater, based on the “D” medium of Provasoli et al.16 Cultures were maintained under cool white fluorescent tubes at a photon flux of 100 µmol quanta m-2 s-1 at 18 °C, and the cells were analyzed when cultures were in the midexponential phase. Nitrogen-limited (N-limited) cultures of D. tertiolecta were obtained by transferring cells from a nutrientreplete, midexponentially growing culture into artificial seawater medium without nitrogen, in which they were maintained for 4 days before the cells were subjected to levitation. Suspensions (10-mL) containing ∼107 cells were centrifuged for 2 min at 5000 rpm, and the supernatant was removed. Aliquots (10 µL) of concentrated cells were transferred by pipet into an acoustic node of the levitation device. For D. tertiolecta, ∼104 cells were transferred to the acoustic node. In a similar manner, a similar number of cells were deposited in a quartz microcuvette for spectral acquisition. Chlorophyll a (chla) and β-carotene were purchased from Sigma-Aldrich (Clayton, Victoria, Australia) and used as supplied. Approximately 20 mg of each compound was dissolved in 1 mL of MilliQ water, and 10 µL was transferred by pipet into the acoustic node. Apparatus. A photograph of the experimental apparatus is shown in Figure 1. The standard frequency of the ultrasonic levitator (Dantec/Invent Measurement Technology, Erlangen, Germany) is 56 kHz, the standard wavelength is 5.9 mm, the largest drop diameter is 2.5 mm, and the smallest is 15 µm. The instrument, which is based on a piezoelectric vibrator, generates a standing acoustic wave with equally spaced nodes and antinodes by multiple reflections between a solid reflector and an ultrasonic transducer.8 The samples are levitated below pressure nodes, due to axial radiation pressure and radial Bernoulli stress. There are four to five pressure nodes, but only the inner two to three are used for stable levitation. Destabilization effects caused by the ultrasonic transducer and reflector influence the outer two nodes, and hence, the central or inner nodes are used for sample levitation. The cell suspension placed in the node appears like a flattened doughnut rather than a spherical droplet (Figure 1A). For long experiments (i.e., >150 s) water must be added to avoid evaporation and, ultimately, cell death, which may influence the (16) Provasoli, L.; McLaughlin, J. J. A.; Droop, M. R. Archiv. Microbiol. 1957, 25, 392-428.

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Figure 1. Experimental apparatus: (A) Dantec ultrasonic acoustic levitation device showing cells trapped in a node, (B) Raman fiberoptic probe, (C) control unit for acoustic levitation device, (D) quartz halogen light source, and (E) InPhotonics portable 785-nm Raman spectrometer.

spectra. The Raman spectra were acquired with an InPhotonics portable InPhotote Raman spectrometer with a 785-nm diode laser and an InPhotonics RamanProbe. The fiber-optic sampling probe is a cylindrical device that is 12.7 mm in diameter. Laser light is transmitted to the probe via a 90-µm excitation fiber. The light is collimated into the probe using a collimating lens before it is transmitted through a band-pass filter. Approximately 85% of the light then passes through a dichroic filter, and extraneous light is reflected. Another lens focuses the light onto the sample and also collects the backscattered light from the sample and focuses this light back into the probe. The dichroic filter then reflects the scattered light to the opposite side of the probe to where it is redirected parallel to the original path but in the opposite direction. The light is then transmitted to a long-pass filter assembly consisting of three filters which remove the Rayleigh and antiStokes scattered light. A final lens focuses the light into a 200µm collection fiber, which transmits the signal to the spectrometer. Power at the sample was measured at 50 mW, and unless otherwise stated, each spectrum was collected with 20 s of laser exposure. Spectra were baseline-corrected using OPUS spectroscopic software at minimum turning points (1800, 1409, 1080, 928, 763, and 600 cm-1). Spectra were also recorded of cells in the microcuvette using the same parameters adopted for the levitation experiment. Effect of Laser Exposure and High White Light Flux. In the first experiment, the effect of constant laser exposure was investigated by irradiating the cells with 20 and 120 s of laser light and recording spectra during this time interval. In the second experiment, 10-µL samples of D. tertiolecta were levitated and then

Effect of Nitrogen Limitation. In a third experiment, Raman spectra of nitrogen-replete (N-replete) and nitrogen-limited (N-limited) levitated algal cells were compared. Samples of both N-replete and N-limited cultures of D. tertiolecta were prepared as described above. Aliquots (10 mL) were centrifuged at 5000 rpm for 5 min, and the supernatant was removed. Samples (10 µL) were levitated, and spectra were acquired at full laser power with an acquisition time of 20 s. Ten spectra for both the N-replete and N-limited cells were recorded from 10 different levitated droplets, and the spectra were compared. Preliminary Taxonomic Identification Experiments. In the final experiment, spectra from three species of eukaryotic algae and one species of cyanobacteria were compared. For each species, five spectra were recorded from five levitated droplets. Each spectrum was acquired with 20 s of laser exposure with 50 mW of power.

Figure 2. Representative Raman spectra recorded of a levitated suspension of living D. tertiolecta cells along with spectra of levitated solutions of β-carotene and chla. Each spectrum was acquired in 20 s with ∼50 mW power at the sample.

Figure 3. (A) Spectrum of levitated living D. tertiolecta cells with 20 s of laser exposure and (B) 120 s of laser exposure. The arrows highlight chla bands that diminish in intensity relative to ν2 of β-carotene after 120 s of laser exposure.

exposed to a high flux of white light (2000 µmol quanta m-2 s-1), provided via the fiber optic of a Schott KL 1500 lamp (Schott AG, Mainz, Germany), for 10 min prior to recording a 20-s-exposure Raman spectrum. At 2.5-min intervals, 10 µL of water was added to the suspension in the node to prevent evaporation. Consequently, the size and shape of the droplet changed over time, resulting in a change in cell density and the overall signal intensity but no change in the relative intensities of the bands. Three samples exposed to high white light flux and three controls with no light exposure were measured; representative spectra are presented in Figure 3. After spectral acquisition, the samples were placed in an Eppendorf tube containing 1 mL of 90% acetone and centrifuged for 7 min at 5000 rpm to extract the chla from the cells. UV measurements of the chla in acetone were taken before and after removal of the Mg. Calculations based on the UV measurements and spectrophotometric equations devised by Lorenzen17 provided a relative measure of chla concentration versus pheophytin.

RESULTS AND DISCUSSION The combination of a portable Raman spectrometer and an acoustic levitation device was used to analyze living algal cells. The combination enabled the analysis of living algal cells in a contactless environment resulting in a 15-fold increase in signal-to-noise ratio, as compared to spectra recorded of a similar number of cells in a microcuvette (refer to Supporting Information). Mechanism of Band Enhancement and Spectroscopic Assignments. Figure 2 depicts Raman spectra recorded from D. tertiolecta, chla, and β-carotene from levitated droplets in an acoustic node generated by the 56-kHz levitation device. The nonprocessed spectrum can be viewed on the computer screen in Figure 1, which shows a large sloping baseline due to fluorescence but an excellent signal-to-noise ratio. The spectrum of D. tertiolecta is dominated by enhanced bands from chla and β-carotene with no signals evident from other macromolecular components, including proteins. The strong enhancement of many β-carotene bands at 785 nm, which is well away from the major electronic transitions, has been explained by a π-electron/phonon coupling mechanism advanced by Castilioni et al.18 and experimentally verified by Parker et al.19 using near-IR Raman excitation wavelengths and inelastic neutron scattering measurements. Enhancement of chla is not expected through normal preresonant enhancement with the Q-band of chla centered at 660 nm. It is possible that the observed enhancement in chla results from excitonic interactions in the highly ordered environment of the chloroplasts. Excitons in molecular crystals and aggregates have been important in elucidating the functionality of photopigments in photosynthesis.20 In biological systems such as algae, light energy is absorbed by special chlorophyll pigments assembled in protein matrixes in so-called antenna complexes and is transferred via an exciton mechanism to the reaction center.21 The exciton model is based on the quantum mechanical precept that electronic energy is distributed throughout the chromophoric environment.22 This arises because interactions between induced (17) Lorenzen, C. J. Limnol. Oceanogr. 1967, 12, 343-346. (18) Castiglioni, C.; Del Zoppo, M.; Zerbi, G. J. Raman Spectrosc. 1993, 24, 485494. (19) Parker, S. F.; Tavender, S. M.; Dixon, M.; Herman, H.; Williams, K. P.; Maddams, W. F. Appl. Spectrosc. 1999, 53, 86-91. (20) Renger, T.; Volkhard, M. J. Phys. Chem. B 1997, 101, 7232-7240. (21) Sturgis, J.; Robert, B. J. Phys. Chem. B 1997, 101, 7227-7231.

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Table 1. Band Assignments for D. Tertiolecta wavenumbera (cm-1)

mode assignmentb,c

1554 (m) 1524 (vs) 1495 (vw) 1437 (m) 1388 (w) 1348 (vw) 1325 (m) 1308 (vw) 1289 (m) 1250 (vw) 1233 (m) 1212 (vw) 1191 (w) 1186 (m) 1155 (vs) 1142 (sh) 1006 (m) 986 (m) 915 (m) 757 (s)

chla, νC2-C3, νC4-C5, νC10-C11, νC15-C16 β-car (ν1), [ν(C13dC14), ν(C11dC12)], chla (1535 cm-1), νC1-C20, νC9-C10, νC14-C15, νC7-C8 chla, νC12-C13, νC1-C20, νC5-C6(81 + 82 + 121)CH3 bend chla, (71 + 121)CH3 bend, νC8-C9, νC19-C20, νC13-C14 β-car [δas(9Me), δas(13Me)] chla, (5 + 20)δ(CH), (81 + 82)CH3 bend chla, (20 + 5 + 10)δ(CH), νN21-C1 β-car [δa(9Me), δa(13Me)] chla, (81 + 82 + 71)CH3 bend, (20 + 5 + 10)δ(CH), νN21-C4 chla, [ν(CaN), δ(CmH)] chla, (17 + 18)δ(CH), (171 + 172) CH3 bend chla, (81 + 171 + 172)CH3 bend, (10 + 5)δ(CH), νN23-C14 β-car, 8H (30), 7H (27), 5-Meβ (12) chla, (10 + 132 + 2)δ(CH), νC16-C17, νN23-C14 β-car [ν(C12-C13), δ(C14-H)], chla, (132 + 5 + 20)δ(CH), νC8-C9, νC6-C7 β-car [δ(C10-H), δ(C11-H)] chla, 20 δ(CH), νN21-C4 β-car (ν2), [ν(C14-C15), δ(C15-H)] chla, 20δ(CH), νN22-C9, νN21-C1, νN24-C16, β-car(ν3) [F (13Me), ν(C12-C13)] chla, (71 + 81 + 82) CH3 bend, δ(C8-1C8-C9) chla, δ(N24-C19-C20), δ(C17-1C17-2C173) chla, 31δ(HCO), δ(N24-C19-C20), δ(N21-C4-C3)

a Band intensities are defined as vw ) very weak, w ) weak, m ) medium, s ) strong, vs ) very strong, ν ) in-plane stretch, γ ) out-of-plane stretch, δ ) deformation mode, Me ) methyl. b Assignment of chla from ref 33. c Assignment of β-carotene from ref 34.

transition dipole moments form a superposition of states, resulting in an electronic band of states that enables the movement of electrons throughout the chla molecules within the chloroplast. Akins et al. advanced the theory of aggregated enhanced Raman scattering (AERS) by incorporating molecular excitonic concepts in a quantum theory analytical expression for aggregated molecules.22-25 They proposed that the enhancement of vibrational modes could be explained in terms of an increase-size effect and near-resonance terms in the polarizability.22 Excitonic coupling will essentially split the electronic states into a broad band of states with different energies, geometries, and oscillator strengths. The Raman intensities for a particular wavelength will then reflect the extent of the excitonic coupling in the chromophoric lattice. We have previously reported the possibility of such interactions occurring in heme aggregates, including hemozoin (malaria pigment) at 780 and 830 nm.26,27 The dramatic enhancement of the totally symmetric modes that are normally only enhanced when excitation occurs in the vicinity of the Soret band at ∼400 nm where a Franck-Condon enhancement mechanism dominates was reported.26 In this case, the enhancement was via a very small z-polarized charge-transfer band centered at 867 nm and known as band I.26 We have also observed unusual enhancement of heme moieties in red blood cells using 633-nm excitation that appeared to be independent of resonance or preresonance Raman scattering.28-32 Only vibrational modes of the porphyrin macrocycle were observed to be enhanced at this excitation wavelength, (22) Akins, D. L.; O ¨ zcelik, S.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1997, 101, 3251-3259. (23) Akins, D. L.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1994, 98, 3612-3618. (24) Akins, D. L.; O ¨ zcelik, S.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1996, 100, 14390-14396. (25) Akins, D. L.; Zhu, H.-R.; Guo, C. J. Phys. Chem. 1996, 100, 5420-5425. (26) Wood, B. R.; Langford, S.; Cooke, B. M.; Lim, J.; Glenister, F. K.; Duriska, M.; Unthank, J.; McNaughton, D. J. Am. Chem. Soc. 2004, 126, 92339239. (27) Wood, B. R.; Langford, S.; Cooke, B. M.; Glenister, F. K.; Lim, J.; Duriska, M.; McNaughton, D. FEBS Lett. 2003, 554, 247-252. (28) Wood, B. R.; McNaughton, D. J. Raman Spectrosc. 2002, 33, 517-523.

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and there was no contribution from amide I or III protein modes. We have no direct proof of an excitonic mechanism giving rise to the enhancement of bands observed in the Raman spectra of living algal cells; however, the fact that there is no enhancement of the amide modes would indicate that an excitonic mechanism is plausible. Band assignments for D. tertiolecta are detailed in Table 1 and are based for chla on those proposed by Chen et al.33 for chlorophyll d using a combination of Raman spectroscopy and density functional theory predictions and by Saito and Tasumi34 for β-carotene. The band assignments are complex because of the many overlapping vibrational modes associated with the porphyrin moiety in chlorophyll. In general, C-C stretching vibrations from chla appear between 1600 and 1500 cm-1 along with the band at 1524 cm-1 assigned to ν1 from CdC double bonds in β-carotene. Bands in the 1500-1400 cm-1 spectral window arise mainly from C-C stretching, CH3 bending, and in-plane C-H bending from methine groups in chla along with CH2 and CH3 deformation modes from β-carotene. Bands in the 1400-1300 cm-1 result primarily from CH3 bends along with methine in-plane C-H bending contributions as well as N-C stretching of chla.33 Bands between 1300 and 1200 cm-1 arise from combinations of CH3 deformations, in-plane CH methine vibrations, and N-C stretching vibrations of chla. Peaks observed in the 1200-1000 cm-1 are mainly due to C-O stretching vibrations of the propionate groups of chla. The band at 1155 cm-1 is assigned to ν2 from C-C bonds of β-carotene, whereas ν3 appears at 1006 cm-1.35 Bands below (29) Wood, B. R.; Tait, B.; McNaughton, D. Biochim. Biophys. Acta 2001, 1539, 58-70. (30) Wood, B. R.; Hammer, L.; McNaughton, D. Vibr. Spectrosc. 2004, available on line April 18, 2005. (31) Wood, B. R.; Hammer, L.; Davis, L.; McNaughton, D. J. Biomed. Opt. 2004, 10, 014005. (32) McNaughton, D.; Lim, J.; Langford, S.; Collie, J.; Wood, B. R. Proc. SPIE Smart Mater., Nano-, Micro-Smart Syst. 2005, 5651, 52-60. (33) Chen, M.; Zeng, H.; Larkum, A. W. D.; Zheng-Li, C. Spectrochim. Acta, Part A 2004, 60, 527-534. (34) Saito, S.; Tasumi, M. J. Raman Spectrosc. 1983, 140, 310-321.

Figure 4. Raman spectra of levitated D. tertiolecta cells exposed to 10 min of white light produced by a quartz halogen lamp and the control with no light exposure but using the same levitation time (10 min). Each spectrum was acquired with 20 s of laser exposure.

Figure 6. Raman spectra of nitrogen-limited and nitrogen-replete suspensions of levitated D. tertiolecta cells. The arrows highlight chla bands that diminish in response to nitrogen limitation.

Figure 5. Plot of the ratio of chla to pheophytin based on the spectrophotometric equations devised by Lorenzen.17 Error bars indicate standard errors on the means (n ) 3).

1000 cm-1 result from N-C-C and C-C-C in-plane bending vibrations, whereas bands below 800 cm-1 are associated with out-of-plane C-H deformations and O-C-O vibrations, mainly from chla.33 Effect of High Light Exposure. Figure 3 compares spectra acquired with 20 and 120 s of laser exposure. Spectra recorded at the longer acquisition time show a dramatic intensity decrease in bands at 1324, 1233, 1185, and 742 cm-1 relative to the 1155 cm-1 band from β-carotene. We chose the 1155 cm-1 band as an internal standard because constant laser and white light exposure results in negligible photodamage to β-carotene. The abovementioned bands are associated with the porphyrin skeletal vibrations and indicate photo- or heat damage, or both, to the porphyrin moieties at the longer exposure time; however, the cells still appeared very motile and green after the long exposure. To investigate the effects of photobleaching, spectra were recorded before and after 10 min of exposure to a quartz halogen lamp. Figure 4 shows average spectra from four trials recorded after 10 (35) Wagner, W.-D. J. Raman Spectrosc. 1986, 17, 51-53.

Figure 7. Representative Raman spectra of different species of levitated algal cells. Each spectrum was acquired with 20 s of laser exposure.

min of exposure with the quartz halogen lamp along with a control sample levitated for the same time but not exposed to the lamp. Even after 10 min of acoustic levitation and white light exposure, the cells were still motile, although they were not as visibly green as the control. The spectra of the cells exposed to the halogen light exhibit a profile similar to that of the spectra of cells after prolonged laser exposure in Figure 3. In particular, bands at 1324, 1289, 1233, 1185, and 757 cm-1 are significantly decreased in intensity as compared to the control. To corroborate these results, UV measurements of the chla in acetone were taken before and after the addition of 10 µL of HCl. Treatment of chla with acid degrades it to pheophytin through removal of the Mg ion. Calculations based on the UV measurements and spectrophotometric equations devised by Lorenzen17 enable one to determine the concentration of chla in the cell suspension. Figure Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Table 2. Description of Algae Investigated in Taxonomic Experiment along with Concentration Data for Chlorophyll a and β-Carotene

c

species

algal type

cell size (µm)

cell shape

chla (pg/cell)

β-carotene (pg/cell)

D. tertiolecta P. tricornutum Chaetoceros sp. P. purpureum

green diatom diatom red

6-9 6-10 7-10 5-8

oblate spheroid cylinder cylinder oblate spheroid

1.33a 0.278a 0.98b 0.286c

0.048a 0.0076a unavailable unavailable

a Concentrations quoted from ref 40. b Concentration of chla in C. muelleri is based on the related organism Chaetoceros gracilis from ref 41. Concentration of chla in P. purpureum is based on data reported for Porphyridium cruentum from ref 42.

5 shows a plot of the ratio of chla to pheophytin based on the UV measurements, which shows a 2-fold decrease in concentration of chla as compared to the control, which is consistent with the Raman spectra. Destruction of chla molecules in photosystem II core complexes from chlorophyte microalgae exposed to photoinhibitory fluxes of visible light has been previously described.36 Formation of singlet oxygen under high light results in damage to chla and appears to be a primary mechanism causing the inhibition of photosynthesis under these conditions. Nitrogen Limitation Experiment. In the past, a number of techniques have been employed to determine factors limiting growth and production of algae. These include bioassays; estimation of a range of physiological parameters, such as nutrient uptake rates; analysis of elemental and macromolecular composition; and measurement of specific molecular markers for nutrient limitation. All of these techniques have particular advantages and disadvantages.1,38 Figure 6 depicts that cells under N-limited conditions show a decrease in intensity of chlorophyll bands at 1324, 1233, 1185, 993, and 757 cm-1 relative to the 1155 cm-1 peak of β-carotene. It is important to note that there also may be a concomitant decrease in β-carotene in N-limited cells, as evinced by the decease in ν1 at 1524 cm-1. Unfortunately, it was not possible to use an independent internal standard in the current experiment. Nevertheless, it appears that the concentration of functional chla is significantly less for the N-limited cells, as compared to N-replete cells. This is supported by other work using conventional methods,38 which shows that N-limitation in D. tertiolecta caused a decline in total chla per cell as well as an increase in β-carotene to chla ratio relative to replete cultures. Future studies will involve performing a similar experiment for phosphorus-replete and phosphorus-limited algal suspensions. Taxonomic Identification. The Raman acoustic levitation technique also shows potential as a tool for the taxonomic identification of algae in remote environments. Figure 7 shows spectra recorded of levitated suspensions of C. muelleri, P. tricornutum, D. tertiolecta, and P. purpureum. Table 2 shows the approximate cell size and shape, along with chlorophyll a and β-carotene concentrations per cell based on previous studies.39-41 (36) Bumann, D.; Oesterhelt, D. Proc. Natl Acad. Sci. 1995, 92, 12195-12199. (37) Beardall, J.; Young, E.; Roberts, S. Aq. Sci. 2001, 63, 44-69. (38) Geider, R. J.; MacIntyre, H. L.; Graziano, L. M.; McKay, R. M. L. Eur. J. Phycol. 1998, 33, 315-332. (39) Jeffrey, S. W.; Wright, S. W. In Phytoplankton Pigments in Oceanography; Jeffrey, S. W., Mantoura, R. F. C., Wright, S. W., Eds.; UNESCO: Paris, 1977; pp 343-360. (40) Fujiki, T.; Taguchi, S. J. Plankton Res. 2002, 24, 859-874.

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It is important to note that it is virtually impossible to record a spectrum of living P. purpureum. C. muelleri, or P. tricornutum with the Raman microscope attachment due to their small cytoplasmic volume. The spectrum of P. purpureum has a poor signal-to-noise ratio as compared to D. tertiolecta and C. muelleri because the concentration of both chromophores is much lower in the former. The signal-to-noise ratio of the P. purpureum is lower than that of P. tricornutum because the P. purpureum has a much larger cytoplasmic volume and, hence, fewer cells in the droplet. The P. purpureum has a pronounced peak at 1286 cm-1 that readily distinguishes it from the other microalgae. This peak is possibly due to phycobilins, including phycoerythrin and phycocyanin, both of which have a similar chromophoric structure and are abundant in high concentrations in red algae.42 The extraction and spectroscopic analysis of these proteins is the subject of future studies. The spectra of all four species show different ratios of β-carotene and chlorophyll and also variation in the intensity of ν1 and ν2, possibly because of the different chromophoric environments of the β-carotene in each of the species. The ratio of the intensities for the 1155/1525 cm-1 bands is much greater in the green alga D. tertiolecta than in the diatoms C. muelleri and P. tricornutum. The P. tricornutum lacks structural detail in the 700-600 cm-1 region, which readily distinguishes it from C. muelleri. The results indicate that the technique has potential for the identification of algae; however, a rigorous statistical approach with organisms in a variety of different of growth phases and under different nutrient conditions needs to be established before any conclusions regarding the taxonomic identification potential of the technique can be ascertained. CONCLUSION The combination of a portable Raman spectrometer with fiberoptic probe coupled to an acoustic levitation device provides a new avenue to diagnose and identify microorganisms in their native environments. The high signal-to-noise ratio achieved by suspending the cells in air with no attenuation from surfaces provides a distinct advantage over conventional Raman measurements performed in macrochambers or cuvettes. More work is required to investigate the effects on drop size, evaporation, and cell death, which is the subject of future investigations. Given the potentially deleterious effects of blue-green algal blooms in inland waters and of other harmful algal blooms (e.g., “red tides”) in (41) Stramski, D.; Bricaud, A.; Morel, A. AppL. Opt. 2001, 40, 2929-2945. (42) Tandeau de Marsac, N. Photosynth. Res. 2003, 76, 197-205.

marine systems, the benefit of reliable and quick ways to determine nutrient status and possible changes in microalgal populations in situ could be large. ACKNOWLEDGMENT This work is supported by an Australian Research Council Large Grant, an Australian Research Council Linkage Grant, and an Australian Synchrotron Research Program Fellowship Grant. Mr. Finlay Shanks is acknowledged for instrumental support and maintenance. Dr. Sigurd Bauerecker, GKSS, Germany, kindly lent the acoustic levitation device, and the InPhotote system was kindly provided on loan by Dr. Jim Pearson of the Victorian Forensic Science Centre, Macleod, Melbourne.

SUPPORTING INFORMATION AVAILABLE Figure 1S shows a spectrum of D. tertiolecta recorded using a normal microcuvette along with a spectrum of a similar number cells recorded using the acoustic levitation device. The spectrum recorded using the acoustic levitation device shows an ∼15× increase in the signal-to-noise ratio as compared to the microcuvette using the same instrumental parameters. This material is available free of charge via the Internet http://pubs.acs.org.

Received for review February 16, 2005. Accepted May 19, 2005. AC050281Z

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