Airborne Bacterial Spore Counts by Terbium-enhanced Luminescence

Mar 18, 2008 - dipicolinic acid (DPA) in-line with hot aqueous dodecylamine, .... airborne bacterial spore concentrations in a real environment in a r...
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Environ. Sci. Technol. 2008, 42, 2799–2804

Airborne Bacterial Spore Counts by Terbium-enhanced Luminescence Detection: Pitfalls and Real Values QINGYANG LI,† P U R N E N D U K . D A S G U P T A , * ,‡,§ A N D HENRY K. TEMKIN§ Department of Chemistry and Biochemistry and Nano Tech Center, Texas Tech University, Lubbock, Texas 79409-1061 and Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019-0065

Received October 6, 2007. Revised manuscript received January 13, 2008. Accepted January 29, 2008.

Bacterial spore determination by terbium(III)-dipicolinate luminescence has been reported by several investigators. We collected spore samples with a cyclone and extracted dipicolinic acid (DPA) in-line with hot aqueous dodecylamine, added Tb(III) in a continuous-flow system and detected the Tb(III)DPA with a gated liquid core waveguide fluorescence detector with a flashlamp excitation source. The absolute limit of detection (LOD) for the system was equivalent to 540 B. subtilis spores (for a 1.8 m3 sample volume (t ) 2 h, Q ) 15 L/min), concentration LOD is 0.3 spores/L air). Extant literature suggests that, from office to home settings, viable spore concentrations range from 0.1 to 10 spores/L; however, these data have never been validated. Previously reported semiautomated instrumentation had an LOD of 50 spores/L. The present system was tested at five different location settings in Lubbock, Texas. The apparent bacterial spore concentrations ranged from 9 to 700 spores/L and only occasionally exhibited the same trend as the simultaneously monitored total optical particle counts in the g0.5 µm size fraction. However, because the apparent spore counts sometimes were very large relative to the 0.5+ µm size particle counts, we investigated potential positive interferences. We show that aromatic acids are very likely large interferents. This interference typically constitutes ∼70% of the signal and can be as high as 95%. It can be completely removed by prewashing the particles.

Introduction Because Anthrax is an agent of bioterrorism, rapid detection of airborne bacterial spores is presently of great interest (1). Early warning systems (EWSs) of spore release in areas with high traffic are badly needed for routine monitoring. Microscopy, the most obvious way to determine the nature and identity of airborne spores, may someday address this in rapid automated form with pattern recognition. The classical approach of impaction of the sample on nutrient agar plates and culturing to examine colonies formed is too slow to provide an EWS. Flow cytometry is promising (2); mass spectrometric methods target characteristic species * Corresponding author e-mail: [email protected]. † Department of Chemistry and Biochemistry, Texas Tech University. ‡ The University of Texas at Arlington. § Nano Tech Center, Texas Tech University. 10.1021/es702472t CCC: $40.75

Published on Web 03/18/2008

 2008 American Chemical Society

from bacterial spores elicited by laser ablation (3) or desorption (4) and pyrolysis (5). Gas chromatography/ion mobility spectrometry after pyrolysis has also been studied (6). A real EWS based on any of these methods is still far off. Online polymerase chain reaction (PCR) to confirm a suspected bioagent is a very promising approach; this was proposed for routine monitoring in airports and post offices. However, the capital cost and the cost of expensive unstable bioreagents are very high. The U.S. Postal Service planned to spend ∼$250 million to install ∼1700 such PCR-based biohazard detection systems. The annual operating cost for nationwide implementation was projected to be at least $ 100 million (7). Even if deployed, such approaches ideally are best used as confirmatory systems for EWSs. EWSs based on the fluorescence of nicotinamide adenine dinucleotide (NADH) and tryptophan may be too nonspecific (8); an EWS that exhibits at least some specificity is desired. Conservative estimates of human LD50 for well-aerosolized viable anthrax spores range from 4100 (9) to 8000 (10) spores. The average breathing rate is 15/min; with a breath volume of 0.5 L, this equals a breathing rate of 7.5 L/min. For an 1 min exposure to equal LD50, the air concentration must be 550–1050 spores/ L, with the very conservative (and likely unrealistic) assumption that all spores inhaled are viable and are deposited in the lung. If we consider an EWS capable of detecting 10% of this LD50, 55–105 spores/L within 1 min, an analytical system with a sampling rate of 10 L/min will be able to meet this goal if the absolute LOD of the system was e550 spores. Dipicolinic acid (DPA, 2,6-pyridinedicarboxylic acid) makes up 5–15% of the mass of all bacterial spores (11). DPA can chelate lanthanides, Tb(III) in particular, thereby enhancing the weak fluorescence of Tb(III) by >20 000 times (12). Further, the fluorescence lifetime of Tb(III) is extended upon formation of Tb(DPA)n3–2n (n ) 1–3, τ ) 0.6–2.0 ms) (13, 14). This permits time-gated fluorescence detection, which greatly enhances sensitivity and selectivity. Lester et al. (15) reported an “anthrax smoke” detector based on DPA detection with a gated charge-coupled device detector. The general strategy involves continuous aerosol collection (16). DPA is released by microwave heating (15) or pyrolysis (16). After adding Tb(III), a gated detector measures the fluorescence. Although not specific for anthrax, the method is thought to be specific for bacterial spores and is therefore promising as a simple autonomous EWS with a PCR for confirmation. An ideal EWS will monitor the background bacterial spore concentration and issue warnings upon abnormal excursions. Based on an impaction and culture based technique, one study reports that the background concentration range is from ∼0.1 viable spores/L in offices to 1 viable spore/L in homes (17). Although the concentration of total spores (viable and nonviable) will be higher, presently reported LOD values of 50 spores/L may be inadequate to reasonably document background concentrations. Here, we aimed to develop a system that can monitor background airborne bacterial spore concentrations in a real environment in a reasonable period and that can determine excursions when purposely deployed with a much faster response time. We previously reported a gated fluorescence detector with an LOD of 0.4 nM DPA (sample volume ∼0.25 mL, 100 fmol DPA) (18). The early generation UV LED used therein was a weak light source. Presently, we report a much more sensitive and integrated collection/analysis system with a flash lamp excited liquid core waveguide (LCW) based gated fluorescence detector (19). Sufficient sensitivity to determine VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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background spore counts is attained. The results have led to an understanding of intrinsic problems and a practical solution.

Experimental Section For materials and spectra, see the Supporting Information. Gated Detection System with LCW Based Flow-through Cell. Small volume flow-through capabilities make a LCW fluorescence detector (19–21) ideal. Briefly, new UVtransparent low refractive index polymers have made watercore optical fibers possible. If excitation light is incident from the radial direction, then the unabsorbed light simply passes out through the walls. An axially located detector sees very little stray excitation light. If a fluorophore is present, a significant part of the fluorescence emission will proceed axially to register on an axially located detector, which can thus detect the fluorescence without interference by the excitation light. The detector is shown in Supporting Information (Figure S1), along with relevant spectra in Supporting Information (Figure S2). Signal Timing. The timing diagram is presented in Supporting Information (Figure S3), and a detailed description appears in the Supporting Information. Briefly, the PMT is gated on 250 µs after the flash is triggered (duration 10 L/min, this device had a 50% cutoff well below 1 µm mass median aerodynamic diameter (MMAD) and would efficiently collect spores or other particles larger than 1 µm MMAD. We operated this device at a flow rate of 15 L/min to absolutely ensure that spores are quantitatively collected. Referring to Figure 1, as supplied, the cyclone (CY) has a removable cap at the bottom where the particles are deposited. An acrylic bottom cap (BC) with a slightly conical bottom was machined for the present work to replace this removable cap. The cap bottom was provided with a 10–32 threaded liquid outlet. To collect the deposited particles into 2800

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FIGURE 1. Schematic for the spore collection/detection system. a liquid, a 3 mL wash solution aliquot was injected at preprogrammed intervals after each sampling period through a stainless steel capillary SSC (HTX-28X, www.smallparts.com) inserted through the sample air inlet (SI). An unbalanced load motor (V, cellular phone vibrator, G13566, www.goldmine-elec-products.com) was turned on for 1 min to help dislodge any adhering water droplets on the PTFE-coated cyclone inner walls and drain to the bottom and for particles collected at the bottom to be slurried. All operation and preprogrammed turn on/off operations are controlled by the laptop computer through the digital outputs in the PC card using relays or MOSFET switches (IRLI530N, www.irfi. com) as required. During aerosol sampling, the peristaltic pump (PP) and the flash lamp are turned off. After the desired sample period, the air pump is turned off; PP, the flash lamp, and the 3-way solenoid valve (V1) (075T3MP12–32–5P, www.biochemvalve.com) are turned on. The DDA solution flows for 3 min at 1 mL/min and V1 is switched back so that the DDA solution is merely recirculated to its reservoir. The vibrator (V) is turned on for 1 min to slurry the collected aerosol. The three-way pinch-type valve (V2) (075P3MP12–01S, Biochem Valve, can handle particles) is now turned on to aspirate the collected slurry at 0.4 mL/min through the heated extractor HE (residence time 5 min). The processed extract is drawn through the membrane filter (F) and is delivered to the 0.4 mL loop of the motorized 6-port injection valve (V3) (C22–3186EH, www.vici.com) so that the sample suspension completely fills the loop. At 7.5 min after starting the pump, the loop is completely full with the heart of the sample plug; V3 is switched to inject the extracted DPA into the 50 mM NaOAc buffer stream (0.4 mL/min). PP continues to draw the liquid for another 8 min so the collector bottom and the heated extractor lines are completely cleared and washed, and the last 3 min of this V1 is turned on and off at 30s intervals to wash the system with fresh DDA. During the last 30s, V2 is off, so PP draws filtered air through air filter AF and clears out the injection valve sample line with air. The sampling and analysis systems are essentially disconnected from each other now so air sampling can begin again. In the analyzer, the injected sample merges with the DDA carrier stream that is then mixed with an equal flow of a TbCl3 solution (20 µM, except as stated) at a tee and flows through a mixing coil MC (0.66 mm i.d. × 500 mm, PTFE tube, 22 SW, Zeus). The luminescence of the Tb(DPA)n3–2n chelate is then detected by the gated fluorescence detector (GFD). The injection valve is switched to the load position after 8 min; PP and the flash lamp are also turned off. The above steps constitute one complete sampling-extractionmeasurement cycle (normally 2 h or less for sampling and 16.5 min for processing and measurement) this repeats indefinitely. We have also operated the device continuously where the wash liquid is continuously introduced into and pumped out of BC and no injection valve is used. It is also possible to incorporate additional sampling trains and multiplex to the same extractor/detector system.

Field Airborne Spore Measurements. Initially, laboratory air samples were collected with the sampler intake at a height of 1.5 m above the floor. Initial outdoor samples were also collected from within the laboratory, via a grounded sampling conduit (0.8 cm diameter × 150 cm). We then collected outdoor samples from another 3 sites in Lubbock, Texas (a) Citibus Plaza (33°35′18′′ N, 101°50′44′′ W) in downtown, a bus terminus where buses come and go every ∼30 min with readily discernible changes in ambient particle concentrations; (b) Science Spectrum (33°31′44′′ N, 101°52′31′′ W), a hands-on children’s exploratory museum and theater — upwind of a nearby well traveled highway — and (c) Legacy playground baseball field (33° 32′10′′ N, 101°57′58′′ W) located ∼1 mile west of the major loop highway that surrounds the city of Lubbock and downwind of major road construction activities during the sampling period. For the laboratorybased experiments, we also measured particle concentrations and the aerosol size distribution by a laser-based multichannel optical particle counter (OPC, model A2212–01–115–1, www.metone.com) every 10 min. The data were integrated and averaged to match the sampling period of the endospore analyzer.

Results and Discussions Performance of the Cyclone Sampler. The Collison nebulizer was filled with a suspension of B. subtilis spores in DI water (80 mg/50 mL) for aerosol generation. This is a very high feed concentration (9 × 107 spores/mL) and was so used to determine the breakthrough with greater accuracy. The aerosol was sampled through the present system with a glass fiber filter as the back-up collector. This experiment was repeated thrice with 1 h collection periods. The spore concentration measured by the system and that of the glass fiber filter extract (the latter manually injected into the analytical system) were compared. The collection efficiency of the sampler was consistently >99% at the operational sampling rate of 15 L/min. DPA Extraction Optimization. Newly prepared B. Subtilis spore suspension (4200 spores/mL, 4.5 orders of magnitude lower than the concentration used in the breakthrough experiment above) was used for all of the following experiments. The spore suspension was spiked with DDA to attain various concentrations of DDA in the final solution, injected into the heated extractor, and allowed to remain. After desired extraction periods, the solution was taken out; TbCl3 was added to attain a Tb concentration of 10 µM, and the luminescence was measured by the GFD. Detailed data can be found in Supporting Information (Figures S5 and S6). At 60 °C, the extraction is nearly complete within 5 min with 0.8 mM DDA. These conditions were chosen. Briefly, DPA release rates increase with increasing temperature and increasing [DDA], studied up to 70 °C and 0.8 mM, respectively. However, the gains are marginal above t ) 60 °C and [DDA] ) 0.4 mM. Calibration of the Detection System Using DPA. Figure 2 shows detector calibration data for standard DPA solutions injected through V3 in the analysis system of Figure 1. The peak height of the luminescence signal is linear with the DPA concentration with an r2 value of 0.9994 in the tested range of 0–5 nM with a slope of 187.5 mV/nM. The relative standard deviation at the 5 nM level is 1.3% (n ) 7). On the basis of the observed signal-to-noise (S/N) ratio of a 300 pM DPA sample, the S/N ) 3 LOD is calculated to be 90 pM. This is the best reported LOD for DPA by a significant margin. The upper end of the linear range extends to at least 100 nM. Calibration Using B. Subtilis Spore Suspension. B. subtilis spore suspensions of known concentrations were diluted 1:1 with 1.0 mM DDA, and this was supplied to the inlet of the heated extractor (60 °C) and repeatedly analyzed by the system of Figure 1 (see Supporting Information, Figure

FIGURE 2. Typical performance of the system for determination of aqueous DPA. S7). In the tested range of 0–35 500 spores/mL, the luminescence intensity increased linearly with the spore concentration (slope: 60.8 µV/(spore/mL), r2 ) 0.9980). The relative standard deviation at the 35 500 spores/mL level was 1.7% (n ) 7). The S/N ) 3 LOD was calculated to be 263 spores/mL in the original suspension. The lowest reported LOD thus far using the same chemistry was 10 000 B. subtilis spores/mL with a 10 mm path length standard cuvette on a Perkin-Elmer LS-50 fluorometer (12), a large benchtop instrument also using a flashlamp. Thus, the concentration LOD is ∼40× better. Furthermore, the actual illuminated volume in this work is ∼0.5 µL. The mass LOD corresponding to this illuminated volume actually corresponds to a small fraction (13%) of the DPA contained in a single B. subtilis spore. In the previous section, the calibration slope for DPA was stated to be 187.5 mV/nM, whereas the calibration slope for the B. subtilis spore suspension is 60.8 µV/(spore/mL). If we assume that our procedure is 100% effective in extracting the DPA from the spores, the average DPA content of each B. subtilis spore is 0.324 fmol, this is comparable to the figures (0.365 (12) or 0.43 fmol/spore (23)) reported by others. Determination of Laboratory Generated Airborne B. Subtilis Spores. B. subtilis spore aerosol concentration generated can be estimated from the consumption rate (9.0 mL/h) of the spore suspension used as the nebulizer feed and the total air flow rate (42 LPM), discounting any loss of particles in the aerosol generation/transmission system. With 2 h collection at 15 SLPM, the measured luminescence intensity was linear with the spore concentration in the tested range of 0–63.4 spores/L with an r2 value of 0.9980, a slope of 24.4 mV/(spores/L air), and S/N ) 3 LOD being 0.3 spore/L air. This can be improved with higher air/liquid volumetric ratios (ALVR), which is 5000 for the present system for a 1 min sample; a typical commercial bioaerosol sampler operating at 450 SLPM and collecting into 10 mL of liquid (24) exhibits an ALVR of 45 000 for a 1 min sample. Also note that the LOD computed above is conservative; because of transmission losses in the system, the actual aerosol concentration must be lower than the estimate made from feed spore concentration, feed consumption rate, and the total airflow. The aerosol concentrations can also be calculated by the calibration slope obtained directly with aqueous B. subtilis spore suspension, assuming that the spores are evenly suspended in the 3 mL collection solution. For feed concentrations ranging between 3550 and 71 000 VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Illustrative data. Continuous monitoring of aerosols.

FIGURE 4. Soluble compounds contribute to the signal.

TABLE 1. Tb(III)-Fluorescence Based Spore Counts, Total Particle Extract*

Plaza, Science Spectrum, and Legacy Field, respectively. For the experiments where the optical particle counts were measured, there was no correlation between 0.5+ µm particle counts and the spore counts (r2 ) 0.003). However, in one series of experiments when the instruments sampled air over a 27 h period in one of the hallways of the Chemistry building, particle concentrations increased over a several hour period, and the increase in spore counts was highly correlated with this (Figure 4). Prima facie, the results presented above would indicate that background spore concentrations are 2 orders of magnitude higher than the values in the literature (0.1–1 spores/L). However, the earlier results pertain to culture-based methods (17) and represent colony forming units (cfu)/L. For airborne bacteria, it is generally known that the vast majority of sampled bacteria cannot be grown in culture (25, 26), and the viable population can be as little as 1% of the total population. Whether this is because the organism is nonviable to begin with or becomes nonviable as a result of sampling is not known. More importantly, it is not known whether this applies to bacterial spores as well. The DPA-based assay cannot distinguish between viable and nonviable entities. It is reasonable that this assay will produce higher apparent spore counts than a culture-based method. Nevertheless, sometimes the apparent spore counts appeared to us to constitute an improbably large fraction of the 0.5+ µm OPC counts; therefore, we considered possible interferences. Interference from Compounds with Short-lived Fluorescence. Fluorescence emission from typical compounds present in an aerosol sample typically have a lifetime 150 µs on the present gated detector, no measurable fraction of such fluorescence will remain. However, the detection system is electronically gated and , unlike with a mechanical chopper, light is not physically blocked. When exposed to light, all PMTs suffer from “afterpulse” or photocathode memory effects, which means that an initial light pulse from a short-lived fluorescence event may have a long tail, even after significant time and even though the PMT was electronically “off” during that event. We explored this possibility with aqueous fluorescein, a high quantum efficiency fluorophore with short-lived fluorescence. Fluorescein 5 µM) produced clear false positive signals; 5 µM fluorescein produced a signal equivalent to 1.7 nM DPA. Native fluorescence of the aerosol extract to be equivalent to >5 µM fluorescein is very unlikely. We actually measured the native short-lived fluorescence of the collected aerosol

Date

Spores/L 0.1 µm 0.2 µm 0.3 µm 0.5 µm 1.0 µm 3.0 µm

A. Sampling Site: TTU Chemistry Building (In Laboratory) 11/29/05 51 11881 9913 7801 848 72 39 11/30/05 26 10684 6094 777 149 28 19 12/16/05 19 4747 2618 6420 3778 266 12 12/18/05 33 6688 2880 6052 2752 173 26 12/19/05 35 4736 687 692 482 45 30 12/21/05 76 9052 1380 856 505 43 6 B. Sampling Site: TTU 12/14/05 37 4348 12/15/05 108 10406 12/19/05 8.8 5178 12/20/05 722 10453

Chemistry Building (Outdoor Air) 2698 712 278 26 9 1276 433 163 16 2 670 269 114 25 10 1652 664 260 21 11

* The standard deviation in spore counts is about 3–5%, whereas that for optical particle counts is 10–15%.

spores/mL, the spore concentrations determined based on aqueous calibration was 80.7–68.1% (generally lower with increasing feed concentration), with mean ( standard deviation being 75.9 ( 4.5% (n ) 6) relative to calculated concentrations based on nebulized solution consumption assuming no system losses. No static discharger was used; 20–30% aerosol transmission loss in the entire system is reasonable. Field-measured Airborne Bacterial Spore Data. Sampling was conducted for an extended period to calculate the daily average background spore concentrations. The spore concentration was calculated by converting the luminescence signal data into spore counts using the calibration slope obtained using laboratory generated airborne B. subtilis spores (Figure 3). The particle distribution was also measured using a laser particle counter. Representative data are shown in Table 1. The measurements of laboratory air on 6 different days indicated apparent spore concentrations of 19–76/L (mean ( sd: 40 ( 21 spores/L). These apparent concentrations were very poorly correlated with 0.5+ µm optical particle counts (r2 < 0.25). For all outdoor samples (outside laboratory air and three field locations), with the exception of one sample that measured an order of magnitude greater concentration than all the other samples, the range of the apparent spore concentration (9–108 spores/L, mean ( sd; 55 ( 34 spores/ L) was similar to that of the laboratory air measurements. Daytime samples (8–10 h measurements) at the three outdoor locations averaged 45 ( 15, 76 ( 13, and 58 ( 10 at Citibus 2802

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extract in several instances and found it to be much lower, simply not enough for interference from this source to be significant. Interference from Aromatic Ligands. A second possibility is interference from compounds in ambient aerosol that behave very much like DPA in that they can (a) absorb UV light, (b) coordinate to a metal center, and, therefore, (c) lead to long-lived fluorescence via intersystem crossing. Benzoic acid and its derivatives widely exist in the ambient aerosols with an approximate concentration of ∼100 ng/ m3 (27). Although benzoic acid itself has been previously examined and does not significantly interfere (28), we observed that the excitation/emission spectra of Tb(III)adducts of benzoic acid derivatives where chelation can occur is nearly the same as Tb(III)-DPA; thus, such compounds would cause strong interference (see Supporting Information, Figure S8). With Tb(III)-DPA having a reference fluorescence intensity of unity, the Tb(III) adducts of 1, 2-benzenedicarboxylic acid, 1,2,4,5-benzenetetracarboxylic acid, p-amino-salicylic acid, and pamino-benzoic acid exhibited fluorescence intensities of 0.001, 0.16, 0.49, and 0.57, respectively. Prewashing Technique to Remove Interference. Because interfering compounds such as aromatic carboxylic acids are water-soluble, and although the DPA in the bacterial spores is not readily extracted by water, it should be possible to remove the interferences by filtration and prewashing. Initially, this was manually conducted to determine how much artifact signal was produced. After the aerosol sample was collected using the dry cyclone sampler, two 1.5 mL aliquots of 0.8 mM DDA solution were used to wash the cyclone collector, and the washings were transferred to a clean centrifuge tube. A small amount of ethanol (0.2 mL) was added to the washings to help solubilize organic acids, and the mixture was shaken. The suspension was filtered by syringe through a polyvinyledene fluoride (PVDF) demountable membrane filter (Durapore, 0.10 µm pore size, diameter 2.5 cm, type VVPP, Millipore), and the filtrate was capped into a glass vial and stored capped. The filter was further washed with 10 mL aliquots of 10% ethanol and deionized water, and the washings were discarded. The filter was then removed from the filter holder, put into a glass vial, and 3.0 mL DDA solution (0.8 mM) and 0.2 mL ethanol were added to it. In a third empty vial, the same amount of DDA-ethanol solution was used as control. All three vials were then put into a 60 °C water bath for 15 min. In each case, the extract supernatant was injected via V3 in the analytical system of Figure 1, where it merged with the TbCl3 solution in-line, and the resulting fluorescence was detected by the GFD. The control solution exhibited no detectable signal in any of the experiments. The data obtained using the procedure to continuously monitor air outside our laboratory building is shown in Figure 4. It is readily apparent that the fluorescence elicited by the soluble matter is typically the major part of the signal (mean ( sd: 0.69 ( 0.16, range 0.42–0.94), and sometimes it represents nearly all the signal. It is obvious that direct measurement without prewashing cannot provide a meaningful measure of DPA. An automated system that incorporates washing and backflushing of the filter is shown in Supporting Information (Figure S9); more extensive field data on spore counts will be reported in the future. Initial results suggest that these counts are within the same range as those shown for the insoluble fraction in Figure 4.

Acknowledgments This research was supported by the Defense Advanced Research Projects Agency (DARPA) under a program monitored by Dr. J. Carrano. This work was also supported by US

EPA STAR grant No. RD- 83107401-0 and the J. F Maddox Foundation. We thank Jason V. Dyke for help.

Supporting Information Available Experimental Section: materials; design of flow-through LCWbased gated fluorescence detector; excitation/emission spectra of Tb(DPA) 33- and transmission filter spectrum; signal timing and diagram; spore aerosol generation system schematic; temperature effect on DPA extraction with dodecylamine; dodecylamine concentration effect on DPA extraction; typical system output with B. subtilis spore suspension; excitation/emission spectra for 1,2,4,5-benzenetetracarboxylic acid and phthalic acid when mixed with Tb(III); and arrangement for automated wash and backwash. This material is available free of charge via the Internet at http:// pubs.acs.org.

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