36 <strong>Spectroscopy</strong> 26(6) June 2011 www.spectroscopyonline.com Table I: Key parameters of the Pistorius method (7) used in this study Sample X Start X End Slope Intercept Protein 1477 1590 12.72 -0.27 Lipid 2780 2984 78.96 -2.3 Carbo 1133 1180 2.05 0.07 spectra when the amount of sample is limited to less than a milligram. The sampling area in the ATR experiment and transmission is 2–5 mm, while the sampling area with an infrared microscope is routinely 100 µm and can be less than 10 µm. The Array Automation software would permit the analysis of micro-arrays including the SBS standard 1536-well plate format. A final, very promising, sampling technique is automated reflectance analysis. This involves depositing the algae samples onto a highly reflecting surface, such as aluminum foil, in an array format. In this example, the samples were deposited in a 96-well format and analyzed using the reflectance option with the well plate reader. The optical layout of the reflectance module that is positioned over the X,Y stage is shown in Figure 5. The 96-spot foil containing the samples was taped to a metal plate that mounted in the automated X,Y stage. A spectrum from a well with no sample was acquired as a background. Spectra were then automatically acquired from the original samples. Figure 6 shows the screen after the software has acquired the data. The color of each well in the schematic in the upper left corresponds to the magnitude (result) of the chemometric analysis selected. This could be as simple as the intensity of the amide band from the protein, the similarity to a target spectrum, or the amount of lipid in the sample. In this case, the color corresponds to the amount of lipid in the sample based on this implementation of the Pistorius method (7). The wells colored red or orange correspond to samples containing more lipid material. Conclusions Many of the FT-IR techniques that have been reported for classifying and characterizing clinical samples can be applied to analyzing materials developed for use in biofuels. In this feasibility study spectra from algae samples were obtained using a number of different infrared sampling techniques, forming a basis for developing rapid screening methods to determine the lipid content of microbiological species intended for biofuels. These techniques would prove valuable during several steps in the development process including optimizing the algae strains through the actual production of biomass to ensure that the algae species are remaining true and are not contaminated by wild organisms. For a small number of samples, ATR spectroscopy provides an easy nondestructive way to quickly determine the chemical composition of dried samples. For applications with a large number of samples, the use of a “well plate” based on the microporous membrane would provide a low-cost way to rapidly analyze multiple samples. Multivariate statistical techniques could be employed to eliminate interference from the spectral features because of the polymer film, which might otherwise limit its general use. One interesting possibility of using a microporous film as the sample substrate is filtering samples directly onto the substrate using an automated 96-sample filtering system. The robust nature and relatively low cost of silicon make it an excellent choice as a substrate for a 96- well plate. Transmission sampling plates can be produced from a single piece of silicon (120 × 85 mm) with 96 sample positions or a plate that mounts 96 individual disposable windows. The results of the reflectance analysis using the Array Automation system are also very promising, and this appears to be an easy method for analyzing multiple samples. As with any analytical application, the best method depends on the details of the analysis. However, FT-IR spectroscopy has the flexibility and specificity to deliver a robust solution optimized to the specific problem. References (1) J. Sheehan, T. Dunahay, J. Benemann, and P. Roessler, “A Look Back at the U.S. Department of Energy’s Aquatic Species Program-Biodiesel from Algae”, NREL/TP-580–24190 (1998). (2) S.R. Lowry, Cellular and Molecular Biology 44, 169–177 (1998). (3) D. Naumann, D. Helm, and H. Labischinski, Nature 351, 81–82 (1991). (4) K. Stehfest, J. Toepel, and C. Wilhelm, Plant Physiology and Biochemistry 43, 717–726 (2005). (5) A.P. Dean, M.C. Martin, and D.C. Sigee, Phycologia 46, 151–159 (2005). (6) D.L. Wetzel, A.J. Eilert, L.N. Pietrzak, S.S. Miller, and J.A. Sweat, Cellular and Molecular Biology 44, 145–168 (1998). (7) A.M. Pistorius, W.J. DeGrip, and T.A. Egorova-Zachernyuk, Biotechnology and Bioengineering 103, 123–129 (2009). (8) M.M. Mossoba, F.M Khambaty, and F.S. Fry, Applied <strong>Spectroscopy</strong> 56, 732–736 (2002). Steve Lowry is Application Scientist, Molecular <strong>Spectroscopy</strong>, with Thermo Fisher Scientific, Waltham, Massachusetts. ◾ For more information on this topic, please visit our homepage at: www.spectroscopyonline.com
2012 Photonics West 21–26 January 2012 Call for Papers Submit your abstract by 11 July 2011 Applications of optoelectronics, lasers, micro/nanophotonics, and biomedical optics Location The Moscone Center San Francisco, California, USA spie.org/pw Conference dates 21–26 January 2012 Exhibition dates BiOS: 21–22 January Photonics West: 24–26 January Technologies - BiOS–Biomedical Optics - OPTO–Integrated Optoelectronics - LASE–Lasers and Applications - MOEMS-MEMS–Micro & Nanofabrication - Green Photonics
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