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Detection of Spores Using Electric Field-Assisted FTIR-ATR He Li,† Luke D. Doucette,§ Doug Bousfield,‡ and Carl P. Tripp*,†,§ Laboratory for Surface Science and Technology, Department of Chemistry, and Department of Chemical and Biological Engineering, University of Maine, Orono, Maine 04469, and Orono Spectral Solutions, Old Town, Maine 04468 An approach that integrates an electric field with an attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) flow through cell was used to detect spores in aqueous environments. A “proof of concept” in terms of the principle features of the method is described. It is shown that under an electric field, the negatively charged spores migrate and are concentrated on the surface of a ZnSe internal reflection element (IRE). No coating on the IRE is required, and a maximum amount adsorbed was obtained within the time needed to record the first spectrum. The amount adsorbed depends on both the pH and the ionic strength. Lowering the pH decreases the charge density and reduces the lateral-lateral repulsion force, leading to a higher packing density on the IRE. Reversal of the field does not overcome the strong attraction between the spores and the IRE. However, repeated measurements can be performed as the spores are completely and rapidly removed from the IRE by simply adding the next sample. The intensity of the infrared bands is due to mass loading of the spores on the IRE and setting a minimum value of 1 × 10-3 absorbance; this requires a total of ∼4 × 106 spores/ cm2. The theoretical detection limit in terms of spore concentration for our cell cavity height of 1.5 mm is ∼10 ppm or 2.5 × 107 spores/cm3. The use of infrared spectroscopy for direct detection of chemical and biological targets in water at a low parts per million level is not possible because the opacity of water in the infrared region limits the beam path length to ∼25-50 µm.1 Attenuated total reflection (ATR) is a common technique used in infrared spectroscopy for aqueous-based studies because the finite penetration of the evanescent wave defines the amount of water probed by the IR beam which, in turn, circumvents the need for narrow path length cells. However, the use of ATR is limited to detecting and analyzing aqueous-based materials that are above certain threshold concentrations, e.g., at least millimolar. Therefore, to achieve low detection levels in ATR, the target compound * To whom correspondence should be addressed. E-mail:
[email protected]. † Laboratory for Surface Science and Technology and Department of Chemistry, University of Maine. ‡ Department of Chemical and Biological Engineering, University of Maine. § Orono Spectral Solutions. (1) Freda, M.; Piluso, A.; Santucci, A.; Sassi, P. Appl. Spectrosc. 2005, 59, 1155– 1159. 10.1021/ac902984s 2010 American Chemical Society Published on Web 05/18/2010
is usually concentrated on a thin film coating on the internal reflection element (IRE) composed of high-surface area metal oxide powders, polymers, and sol-gels.2-9 Recently, we applied the approach of coated IRE’s for the detection of spores.10 The aim was to develop a nonassay and solventless detection method for use by the military and homeland defense community. A ZnSe IRE was coated with alumina, and the negatively charged spores electrostatically adsorbed on the positively charged surface. While this approach led to the concentration of spores on the surface of the IRE, it did not address other fundamental issues with practical usage of coated IRE’s in a detection platform. One issue is that the time required for maximum adsorption is dictated by mass transport to the coated IRE. When using flow cells, the direction of the flow is tangential to the surface and the time required to achieve maximum adsorption was ∼20 min. This issue of mass transport also applies to chemical adsorption on coated IRE’s.8,11 Furthermore, IRE’s are relatively expensive, and thus, a cleaning procedure is required to reuse the coated IRE. On alumina-coated IRE’s, a method for removing the spores once adsorbed could not be found, and thus, the IRE had to be removed and manually cleaned between experiments. Coatings in which adsorption is reversible have been used in chemical detection,12 but in this case, the amount adsorbed is dictated by the equilibrium partitioning of the adsorbate between the surface and the solution. In previous work,13 we developed an electric field-assisted ATR flow cell to concentrate spores from water onto the surface of the IRE for detection. An ultrathin palladium oxide film was deposited onto the surface of the IRE that was in contact with water and (2) Rivera, D.; Poston, P. E.; Uibel, R. H.; Harris, J. M. Anal. Chem. 2000, 72, 1543–1554. (3) Rivera, D.; Harris, J. M. Anal. Chem. 2001, 73, 411–423. (4) Howley, R.; MacCraith, B. D.; O’Dwyer, K.; Masterson, H.; Kirwan, P.; McLoughlin, P. Appl. Spectrosc. 2003, 57, 400–406. (5) Howley, R.; MacCraith, B. D.; O’Dwyer, K.; Kirwan, P.; McLoughlin, P. Vib. Spectrosc. 2003, 31, 271–278. (6) Han, L.; Niemczyk, T. M.; Haaland, D. M.; Lopez, G. P. Appl. Spectrosc. 1999, 53, 381–389. (7) Haibach, F. G.; Sanchez, A.; Floro, J. A.; Niemczyk, T. M. Appl. Spectrosc. 2002, 56, 398–400. (8) Li, H.; Tripp, C. P. Langmuir 2002, 18, 9441–9446. (9) Chen, C.; Tripp, C. P. Biochim. Biophys. Acta 2008, 1778, 2266–2272. (10) Li, H.; Tripp, C. P. Appl. Spectrosc. 2008, 62, 963–967. (11) Ninness, B. J.; Bousfield, D. W.; Tripp, C. P. Appl. Spectrosc. 2001, 55, 655–662. (12) Collette, T. W.; Williams, T. L.; Urbansky, E. T.; Magnuson, M. L.; Hebert, G. N.; Strauss, S. H. Analyst (Cambridge, U.K.) 2003, 128, 88–97. (13) Doucette, L. D.; Li, H.; Ninness, B. J.; Tripp, C. P. Int. J. High Speed Electron. Syst. 2007, 17, 729–737.
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Figure 1. (A) Diagram of the E-field flow cell. (B) Side view of the E-field flow cell. (C) Inner cavity side of the E-field flow cell.
functioned as the active collector electrode. Although collection and detection occurred within minutes, the electrode thin film degraded with repeated use due to electrochemical changes caused by electric field application. In this paper, we describe an improved approach for utilizing an electric field for ATR detection of spores Specifically, the spores are concentrated on an uncoated IRE using an external electric field, and this circumvents issues associated with electrode and absorptive film degradation and reuse. A maximum in the amount of spores adsorbed occurs rapidly, and the spores are easily removed when water is passed through the cell with a reverse field for repeated use. EXPERIMENTAL SECTION Electric Field-Assisted Flow Cell. A diagram and picture of the E-field ATR cell is shown in Figure 1. A Teflon ATR flow cell from Harrick was modified to enable application of an electric field. As purchased, the standard flow cell consists of two Teflon blocks each containing inner cavities [42 mm (length) × 4 mm (width) × 1.5 mm (height)] that mate to each side of the (50 mm × 20 mm × 2 mm, 45°) ZnSe internal reflection element (IRE) through compressed O-rings. Inlet and outlet channels to these cavities allow fluids to be passed across the IRE surface. The modification consisted of milling an external cavity on each cell block to accommodate two flat plate electrodes (35 mm × 8 mm) that cover the entire cavity area in contact with the IRE. The depth of the milled outer cavity was chosen to minimize the thickness 5054
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of the Teflon wall between the inner cavity and the electrode, and this was ∼1 mm. The total distance between electrodes is approximately 7 mm (see Figure 1A), consisting of a 1 mm thick Teflon wall, a 1.5 mm inner cavity containing the BG suspension, the 2 mm thick ZnSe IRE, a second 1.5 mm inner cavity that contained air, and a second 1 mm thick Teflon wall. Contact between the clamping pin of the flow through cell and the electrode was prevented using a Teflon spacer placed on the back of the electrode. The electrodes were connected to a direct current power supply that could be adjusted between ±0.1 and ±40 V. The BG spores have an isoelectric point near pH 2.5 and thus are negatively charged for experiments reported here. By convention, a positive voltage applied to the electrodes refers to the case in which the electrode on the back of the ZnSe IRE is positively charged. This scenario is depicted in Figure 1A. When a positive voltage is applied, the negatively charged spores move in the direction of the IRE surface. Materials. Bacillus globigii (BG) spores were supplied by American Type Culture collection (ATCC lot 19076-03268, batch 25). A fresh stock suspension was prepared prior to each experiment via addition of 10 mg of BG into 100 g of deionized water followed by ultrasonication for 5-10 min. The desired concentration was achieved by dilution with deionized water. All ATR experiments were begun within 30 min of the preparation of the suspension. Dilute solutions of sodium chloride, sodium hydroxide, and hydrochloride acid were obtained from Aldrich and used to adjust the ionic strength and pH. ATR Studies. In a typical experiment, a bare ZnSe ATR crystal was mounted in the flow cell and a peristaltic pump (Master-Flex L/S computerized drive pump) was used to pass deionized (DI) water adjusted to the desired pH and ionic strength at a specified flow rate (typically 5 mL/min). Spectral changes were often observed during the initial 10-30 min of water being passed through the cell. These changes were monitored by recording a temporary reference spectrum through the cell with water flowing and then absorbance spectra (100% baseline) as a function of time under continuous flowing conditions. We attribute these initial changes in the spectrum to removal of bubbles from the cell. This water was passed through the system until no changes were observed in the 100% baseline spectrum. After this point, a new reference spectrum was recorded and this reference spectrum was used for the remainder of the experiment. After this point, it is noted that no spectral changes were obtained when the water flow was stopped and restarted or when ±40 V was applied to the electrode plates. A stirred BG suspension in a 150 mL beaker was then passed through the cell, and spectra were recorded as a function of time under varying conditions of pH, ionic strength, flow or no flow, and applied voltage. IR spectra were recorded on a Bomem MB series FTIR instrument equipped with a liquid N2-cooled MCT detector. Typically, 100 scans were co-added at a resolution of 4 cm-1. All experiments were performed at least in triplicate. RESULTS AND DISCUSSION Figure 2 shows a typical IR spectrum of BG spores adsorbed on the ZnSe internal reflection element (IRE) for a 100 ppm (∼2.5 × 108 spores/cm3) BG suspension in deionized water at pH 5.5, no flow conditions, and an applied voltage of 20 V. The
Figure 2. FTIR spectra of BG spores. Transmission spectra of (A) the dry power in a KBr pellet, (B) a 100 ppm suspension on the bare ZnSe IRE at 20 V, and (C) the same suspension on the bare ZnSe IRE with no voltage applied for 1 h.
Figure 3. Intensity of the band at 1100 cm-1 as a function of time under varying conditions: (A) flow and 20 V, (B) no flow and 20 V, (C) no flow and 0 V, (D) no flow and -20 V, (E) flow of DI water and -20 V, and (F) DI water, no flow, and 20 V. Points labeled A′-E′ refer to a second cycle of the same procedure.
assignments of the bands are well-established,14 and for the purposes of this study, we use the intensity of an IR band at 1100 cm-1 to monitor the relative amount of BG spores within the penetration depth of the bare ZnSe IRE. At 1100 cm-1, the penetration depth of a ZnSe IRE in contact with water is calculated at approximately 1.34 µm. In the first experiment, a suspension of 100 ppm BG spores in deionized water at pH 5.8 was passed through the cell and spectra were recorded as a function of time. Figure 3 is a plot of the timedependent change in the peak intensity for the band at 1100 cm-1. No band at 1100 cm-1 was observed when the suspension was passed through the cell with 20 V applied to the cell (region labeled A in Figure 3). The flow was then stopped at point B, and this was accompanied by a rapid and immediate increase in the intensity of the 1100 cm-1 band to a plateau value. In separate (14) Mantsch, H. H., Chapman, D., Eds. Infrared Spectroscopy of Biomolecules; Wiley: New York, 1996.
experiments conducted with no flow and no applied voltage, a band was not observed at 1100 cm-1 for long hold times (i.e., >1 h). However, when 20 V was applied, a rapid increase in the intensity of the 1100 cm-1 band occurs that reaches a plateau at the same value obtained in Figure 3. This rapid increase in the magnitude of the signal that occurs at point B in Figure 3 demonstrates two of the three important features of the E-field ATR method. First, concentration of the spores occurs on an uncoated IRE, and second, mass transport of the spores to the surface occurs rapidly within the time needed to record a spectrum (
