Detection of Bacillus globigii Spores Using a Fourier TransformInfrared–Attenuated Total Reflection Method

HE LI and CARL P. TRIPP*Laboratory for Surface Science & Technology, The Department of Chemistry, University of Maine, Orono, Maine 04469

The use of an alumina-coated ZnSe internal reflection element (IRE) to

detect spores by attenuated total reflection infrared spectroscopy (FTIR-

ATR) was investigated. Two methods for coating the IRE with alumina are

described. It is shown that the adsorption proceeds through an interaction

of the carboxylate groups on Bacillus globigii (BG) and positively charged

sites on the alumina. The amount adsorbed is highly dependent on

solution pH and passes through a maximum value near pH 5, which is

dictated by the charge density on the spores and the charge density on the

alumina surface. Furthermore, it is shown that lateral–lateral repulsion

between the spores limits the maximum adsorbed amount, giving rise to a

detection limit of 107 spores per cm2 of the IRE.

Index Headings: Infrared spectroscopy; IR spectroscopy; Zeta potential;

Attenuated total reflection; ATR; Spores.

INTRODUCTION

The development of sensors for the detection of air-borneand water-based spores is largely driven by homeland securityand military needs. Current sensors are assay based, usingapproaches such as polymerase chain reaction (PCR) toprovide identification and low level (104 spores) detectionlimits. These bioassays or immunoassays require reagents suchas primers and buffers, and analysis times are typicallyperformed in slightly less than one hour.1 However, a desiredfeature of next-generation spore detection systems is that theydo not require reagents. In essence, the sensor must operate byanalyzing intact spores. This is a difficult task given therequirement of extremely low false alarm rates.

Infrared (IR) spectroscopy could form the basis of a sporedetection platform that does not require the use of reagents.Recently, the identification of bacterial spores using Fouriertransform infrared photoacoustic2 and transmission spectros-copy3 has been demonstrated. Analyzing forty specimens,representing five different strains of Bacillus spores, Foster etal.2,3 achieved classification success rates of 87% or better atthree different levels: (1) bacteria/nonbacterial, (2) membershipwithin the spore library, and (3) bacterial strain. Also, lookingat two closely related Bacillus globigii spore samples notcontained in the library, along with eight nonbacterial samples,they reported 100% accuracy at classifying the samples at thethree indicated levels. While low false alarm rates underbattlefield conditions remain to be demonstrated, it is clear thatto achieve detection sensitivity of 104 spores or better, an IR-based approach will require the development of samplingmethods that can concentrate spores and that are amenable forinterrogation by the IR beam.

Collection and concentration of the spores is needed becausecurrent systems are unable to detect the agents at the low doselevels present in the environment. Collection methods such as

impingement into a liquid or centrifugation are commonlyemployed techniques for the sampling/concentrating of bio-aerosols. In these concentration methods, the airborne sporesare transferred from the aerosol phase to a liquid phase. Thus,the development of IR sampling approaches in water could beused as a common method for both air-borne and water-basedspore detection.

The approach to developing a water-based IR samplingmethod is clearly dependent on the properties of the spores.Bacterial spores are dormant cells produced by a variety ofbacteria including Bacillus subtilis and Bacillus anthracis.These spores are encased within a complex multi-layeredprotein structure, whose primary function is to help the sporeendure a wide range of harsh environmental stresses. Onceconditions are favorable for germination, these spores retain theability to return to a vegetative state. Irrespective of theparticular strain (B. anthracis, B. subtilis, B. cereus), the sporedimensions are typically confined to a narrow range ofapproximately 1.2 lm in length by 0.75 lm wide.4 Therefore,bacterial spores, based on their size, can be consideredcolloidal particles. Most colloidal particles when suspendedin aqueous media will acquire a surface charge due toionization of surface amine, carboxylate, and phosphatefunctionalities or due to the adsorption of other ions.5 Thesurface chemistry of the outer protein coat will determine theextent of charge acquired at various solution conditions (pH,ionic strength). The isoelectric point for typical spores is in therange of pH 4–5, indicating that negatively charged sites on thespore surface dominate at pH levels above this range.6

Therefore, a straightforward approach to concentrate andcollect bacterial spores is to take advantage of their inherentsurface charging characteristics and to use an oppositelycharged surface as the collection medium.

In this paper, we investigate a sampling approach for the insitu recording of IR spectra of spores concentrated on apositively charged attenuated total reflection (ATR) ZnSecrystal. Specifically, the surface of a ZnSe crystal was given apositive charge by applying an alumina coating. Since water isa strong absorber of infrared radiation, an ATR approach isused because there is a finite penetration of the IR beam intothe aqueous medium, enabling in situ measurements. Thepenetration depth of the evanescent wave in the ATR approachis 1–5 lm, which is a good match for ‘‘monolayer or bilayer’’coverage of micrometer-sized bacteria. ZnSe was selected asthe internal reflection element (IRE) because it is commonlyused in ATR studies and is transparent in the important 1800–600 cm�1 region of the infrared spectrum.

EXPERIMENTAL

Bacillus globigii (BG) spores were supplied by AmericanType Culture Collection (ATCC). Polyethylene (MW 35 000)powder, sodium chloride, cetyltrimethylammonium bromide

Received 19 December 2007; accepted 13 June 2008.* Author to whom correspondence should be sent. E-mail: ctripp@maine.edu.

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� 2008 Society for Applied Spectroscopy

(CTAB), and toluene were supplied by Aldrich. Aluminapowder (AlonC, 100 m2/g) was obtained from Degussa. Dilutesolutions of sodium hydroxide and hydrochloride acid weresupplied by Aldrich and used to adjust pH.

Infrared spectra were recorded using a standard ATR liquidflow cell arrangement from Harrick equipped with ZnSe IREs(50 3 20 3 2 mm, 45 degrees). Two methods were used to coatthe ZnSe with an alumina layer. In the first approach, theAlonC powder was deposited on the ZnSe IRE. The simplestapproach was to disperse the alumina powder in toluene andthen add the suspension drop-wise to the ZnSe IRE. However,coatings produced in this manner did not adhere to the ZnSeIRE and were easily removed by flowing water. To overcomethis problem, a polyethylene/AlonC suspension was depositedon the ZnSe IRE. The polyethylene (PE) acted as the binderand was selected because it has few IR bands and these bandsdo not interfere with detection of the spores.

The alumina/polymer suspension was prepared by adding 50mg polyethylene to 15 mL toluene at 105 8C for 30 minuteswith stirring. Next, 20 mg of alumina was added to the solutionand the suspension was ultrasonicated for 10 minutes. A 20 lLaliquot of the suspension was deposited evenly on one side ofthe ZnSe crystal using a pipette and the solvent was thenallowed to evaporate. Films produced in this manner wereabout 70 nm to 100 nm in thickness and showed no change inthe intensity of bands due to polyethylene or alumina whenplaced in contact with flowing water solutions for periods ofdays.

In the second approach, a thin alumina film on top of a ZnSeIRE was prepared using a sputter deposition technique.Aluminum oxide (Al2O3) was deposited in a high vacuumchamber, base pressure 5 3 10�8 torr, using reactive magnetronsputter. A pure aluminum target was used in a three inch AJAmagnetron. The target was pre-sputtered for ten minutes at 3mtorr using argon. Conditions for the film deposition wereargon flowing at 10 standard cubic centimeters per minute(sccm) and oxygen flowing at 2 sccm at a pressure of 3 mtorr.The substrate (ZnSe IRE) was at room temperature. The Al2O3

was deposited using pulsed DC with an MDX 1.5K source anda Sparc-le 20 at 300 Watts at a rate of 0.02 nm per second. Therate was monitored using an Inficon quartz crystal monitor.This approach resulted in a contiguous alumina film ofapproximately 10 nm thickness.

The coated ZnSe (either the AlonC/PE or thin Alumina film)was mounted in the flow-through ATR accessory and aperistaltic pump was used to flow deionized (DI) water at a rateof 1 mL/min adjusted to the specified pH for 2 hours. Areference spectrum was then recorded with the water flowingthrough the cell. A suspension was prepared by adding 10 mgBG to 100 g of deionized water followed by ultrasonication for30 minutes. Dilute hydrochloride acid and sodium hydroxidesolutions were used to adjust the pH of the system. Thesuspension containing the BG spores was then flowed throughthe cell and spectra were recorded as a function of contact time.

The intensity of an IR band at 1100 cm�1 due to spores wasused to estimate the number of spores per cm2 area of the IREusing the following procedure. A suspension containing theBG spores was flowed through the ATR cell and spectra wererecorded until a maximum intensity in the bands due to BG wasobtained. DI water was then flowed through the cell for 1minute. The IRE was then removed and dried, and atransmission spectrum was recorded through the coated IRE.

In recording this transmission spectrum, the reference wasrecorded through the alumina/polyethylene coated IRE. Theamount of spores (mg/cm2) was determined for the transmis-sion spectrum using a Beer-Lambert plot derived from knownquantities of spores pressed into KBr pellets. This value is thenconverted to number of spores/cm2 using density (1.45 g/cm3)and volume (approximate 2.73 3 10�13 cm3)7 of a BG spore.Using this calibration procedure, we find that a value of 1 310�3 absorbance for the 1100 cm�1 peak in the ATR spectrumrepresents approximately 4.5 3 107 spores/ cm2.

It is realized that the calibration procedure could lead to anunderestimation of the quantity of spores/cm2 when the sporesstack on top of each other on the alumina. The stacked pilewould be probed by the light beam in the recording of atransmission spectrum but would lie outside the penetrationdepth of the evanescent wave in the corresponding ATRspectrum. However, as we show later, a stacked layer of sporeson the surface is unlikely because of the strong repulsionbetween the negatively charged spores and because atomicforce microscopy (AFM) images reveal a single layer ofpacked spores on the alumina.

All spectra were recorded on a Bomem MB-series Fouriertransform infrared (FT-IR) spectrometer equipped with a liquidN2 cooled mercury cadmium telluride (MCT) detector.Typically 100 scans were co-added at a resolution of 4 cm�1.The zeta potential of the alumina powder and BG spores wasmeasured on a Malvern Zetasizer 3000 system. Atomic forceimages were recorded on an Asylum Research MFP-3DTM

atomic force microscope with commercial tetrahedral Si tips ona cantilever with a length of 240 lm. A resonance frequency ofabout 70 kHz in air and a nominate spring constant of 2 N/mwere used. All measurements were performed in AC mode inair to avoid damage to the surface. The images were visualizedin height and amplitude mode.

RESULTS AND DISCUSSION

The amount of BG spores adsorbed on the coated ZnSe IREvaries with solution pH because the surface charge on thealumina and BG spores are pH dependent. To determine theoptimum pH to conduct the experiments, the zeta potential ofthe alumina and BG spores were measured as a function of pH.The results are shown in Fig. 1. BG spores are negativelycharged above pH 2.5 and reach a maximum value near pH 6.

FIG. 1. Zeta potential of alumina and BG spores measured at various solutionpH values.

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In contrast, the zeta potential of alumina is positive below pH9.6 and reaches a maximum positive value near pH 6. Based onthese curves, we anticipate that a maximum in the amount ofBG spores adsorbed would occur over a narrow range near pH6. In this narrow pH range, the zeta potential of the aluminaand BG are at the maximum positive and negative values,respectively.

In the first ATR experiment, spectra were recorded as afunction of contact time when a suspension of BG spores at pH6 was flowed across an alumina/PE coated ZnSe IRE. Figure 2ashows a typical spectrum obtained for BG spores adsorbed onthe alumina/PE coated IRE. For comparison, the spectrum ofBG spores recorded in transmission in a KBr pellet is providedin Fig. 2b. The major bands associated with BG are clearlyidentifiable, including amide I (1650 cm�1), amide II (1540cm�1), and the C–O stretching mode around 1100 cm�1.

Additional experiments were performed that showed that theBG spores adsorb on the positively charged alumina and notwith the ZnSe or polyethylene. In two control experiments, thesame BG suspension at pH 6 was flowed across a bare ZnSeIRE and a ZnSe IRE coated only with polyethylene. In bothcases, IR bands due to BG were not observed.

There is one clear difference in the spectrum of pure spores(Fig. 2b) and the spectrum recorded for the same sporesadsorbed on the alumina/PE coating. Figure 2a shows a band at1720 cm�1 (C¼O stretching mode of COOH) that is not presentin Fig. 2b. A band at 1720 cm�1 was also observed by Parikhand Chorover8 for adsorption of bacteria on iron oxide. Theappearance of a band at 1720 cm�1 was attributed to thebridging of carboxylate groups to OH groups on the iron oxideand this assignment applies in our case for the adsorption ofBG spores with the OH groups on alumina.

There is the possibility that the band at 1720 cm�1 at low pHis due to acidification of the carboxylate groups on the sporeitself. It is noted that the change in zeta potential with pHshown in Fig. 1 is primarily due to the existence of carboxylategroups on the spore surface. However, control experimentswere performed that show that this is not the case. Specifically,a suspension containing BG spores was incubated at pH 3.4and an IR spectrum of this suspension was measured on a

horizontal ATR equipped with a bare ZnSe IRE. In addition, analiquot of the suspension was dried rapidly by evaporation andan IR spectrum of the dried powder in a KBr pellet wasacquired. In both spectra, a band at 1720 cm�1 was notdetected. Thus, the appearance of a band at 1720 cm�1 in Fig.2a arises from adsorption of the spores on the alumina.

In preparing the alumina powder coated IRE, a mass ratio of2:5 alumina:PE was used. Assuming a uniform dispersion, thismass ratio would result in only about 12% coverage of the IREsurface as alumina. Since the PE functions only as a binder, ahigher amount of BG adsorbed would be anticipated byincreasing the surface coverage of alumina relative topolyethylene. For this reason, the adsorption of BG spores ona sputtered deposited Al2O3 film was performed. A sputteredfilm of alumina on the ZnSe IRE would result in a 100%alumina surface exposed to the spore.

Figure 3 shows the time-dependent change in the intensity ofthe 1100 cm�1 peak as a function of time while a BGsuspension is flowed through the ATR cell containing thealumina/PE film and the thin alumina film. It is found that themaximum amount of BG spores adsorbed is similar (within10%) on both coatings. The intensity of the 1100 cm�1 peakcan be converted to the number of spores/cm2 using thecalibration procedure described in the Experimental section.For example, the absorbance value in the plateau region of thecurve for deposition on the alumina/PE film corresponds to3.25 3 108 spores/cm2. Assuming single-layer coverage ofspores, this value in the number of spores per cm2 area of theIRE would correspond to a tightly packed layer on the alumina.This was confirmed by obtaining AFM images of the alumina/PE coated IRE after deposition of the spores (see Fig. 4), whichshow a uniform and highly packed layer of spores on thesurface.

It is noted that there would be an intensity increase with a 10nm film compared to the thicker (70–100 nm) alumina/PE film.This could account for the 10% higher value obtained for theabsorbance values for BG adsorbed on the alumina film. Usinga three-layer refractive index model,9 we anticipate about 12%higher value in the strength of the electric field at the aluminasurface compared to the alumina/PE surface. Therefore, the lowsurface coverage of alumina in the alumina/PE coating (and by

FIG. 2. IR ATR spectra of (A) Bacillus Globigii adsorbed on alumina/PEcoated ZnSe and (B) Bacillus Globigii as a dry powder in a KBr pellet. Theordinate scale is for spectrum B. Spectrum A has been expanded by a factor of40 for clarity in presentation.

FIG. 3. Integrated intensity of the 1100 cm�1 band due to BG spores adsorbedon sputtered alumina and alumina/PE versus incubation time.

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inference, the charge density on the coated ATR) is not themajor factor in determining the maximum in the amount ofspores adsorbed on the coated ZnSe IRE.

One possible explanation for the similarity in adsorbedamounts of BG on the alumina/PE and the sputtered depositedalumina films is that the maximum amount adsorbed is dictatedby the packing density of the micrometer-sized spores, which,in turn, is affected by the lateral charge repulsion betweenspores. This explanation is supported by additional experi-ments in which the maximum amount of BG spores adsorbedwas measured as a function of pH.

Figure 5a is a plot of the integrated intensity of the 1100cm�1 band due to maximum amount of BG spores adsorbed onthe alumina/PE films as a function of pH. As discussed earlier,a maximum in Fig. 5a was anticipated at or near pH 6. This wasbased on the curves in Fig. 1, which shows a maximumdifference in zeta potential between alumina and BG at pH 6.However, Fig. 5a shows that the amount of BG spores detectedat pH 5 is about two times higher than at pH 6. Figure 5a alsoshows that the amount of BG spores adsorbed at pH 4 is 60%higher than at pH 6. In contrast, Fig. 1 shows that the zetapotential for the alumina is constant between pH 4 and 6,whereas the zeta potential for the BG spores is 50% lower atpH 5, and 2.5 times lower in value at pH 4.

From this pH dependence, it is concluded that the position ofthe maximum for the adsorbed amount results from a balancebetween electrostatic attraction on the charged surface andlateral–lateral repulsion between the spores. In essence,reducing the charge density on the spores lowers the lateral–lateral repulsion between the spores (1.2 lm diameter),

enabling a higher packing density on the surface. It is notedthat by adjusting the pH with HCl and NaOH there is also achange in ionic strength of the solution. However, this has littleeffect on the position of the maximum in Fig. 5a. The pHdependence on the adsorbed amount was also measured for BGsuspensions in 0.1 M NaCl solutions and is shown in Fig. 5b.In this case, the maximum in adsorbed amount of BG alsooccurs at pH 5.

FIG. 4. AFM picture of BG spores on the alumina/PE surface.

FIG. 5. Integrated intensity of the 1100 cm�1 band due to BG spores adsorbedon alumina/PE versus solution pH, (A) using DI water and (B) in 0.1 M NaClsolution.

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To confirm that the amount of BG adsorbed on the surface isaffected by lateral–lateral repulsion between spores, weconducted the following experiment. The BG spores wereflowed across an IRE coated with alumina/PE at pH 4 for 10minutes. As shown in Fig. 6, a 10 minute contact time results inapproximately 50% of the maximum amount of BG spores onthe surface. At the 10 minute mark, a 2.74 3 10�4 M solutionof the cationic surfactant, cetyltrimethylammonium bromide(CTAB) solution, was flowed through the cell. This concen-tration was well below the critical micelle concentration of 9.33 10�4 M for CTAB in pure water.10 Spectra were recorded asa function of time until a maximum in the amount of CTABadsorbed on the spores was obtained. The amount of CTABwas measured from the increase in the methylene stretchingmode of CTAB at 2920 cm�1. During contact with the CTABsolution, there was no decrease in bands due to BG spores,showing that CTAB did not displace BG spores from thecoated IRE. Furthermore, in a separate control experiment,bands due to CTAB were not observed when the CTABsolution was placed in contact with an alumina/PE coated IREat pH 4. This control experiment showed that the CTABadsorbed only with the negatively charged BG spores.

The introduction of the CTAB at the 10 minute mark in the

ATR cell results in a decrease in the charge density of the BGspores adsorbed on the IRE. This is consistent with knownbehavior in solution. For example, when a 100 ppm BGsuspension at pH 4 is mixed with a 5.0 3 10�4 M CTABsolution at 1:1 and 1:2 volume ratios, the measured zetapotential is lowered from �40 mV to �20 and �11 mV,respectively. Once a maximum in amount of CTAB wasachieved the cell was flushed with DI water for 5 minutes. Atthis point, the alumina/PE contains 50% coverage of BG sporesthat have a lower charge density due to the adsorbed CTAB.

The BG suspension is then reintroduced to the cell at pH 4and spectra were recorded as a function of contact time. Theamount of BG spores adsorbed was measured and is plotted inFig. 6. The curves in Fig. 6 show a 40% increase in the amountof spores adsorbed relative to the maximum amount obtainedwith addition of BG spores only. It is the reduction in thecharge density on the spores at 50% coverage by adding CTABthat leads to a higher amount of spores adsorbed with thereintroduction of BG spores at the 10 minute mark.

CONCLUSION

It was shown that deposition of alumina powder with apolyethylene binder on a ZnSe crystal adsorbs Bacillus globigiifor detection by infrared spectroscopy. The maximum adsorbedamount is highly dependent on solution pH and primarilydictated by the charge density on the spores. This factor resultsin single-layer coverage and a detection limit of about 107

spores per cm2 of the IRE.

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FIG. 6. Integrated intensity of the 1100 cm�1 band due to BG spores adsorbedas a function of contact time for (A) spores treated with CTAB at the 10 minutemark and (B) no addition of CTAB.

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