ieee sensors journal, vol. 8, no. 6, june 2008 963 ...academic.uprm.edu/ccsde/pdf/nanotechnology...

IEEE SENSORS JOURNAL, VOL. 8, NO. 6, JUNE 2008 963

Nanotechnology-Based Detection of Explosives andBiological Agents Simulants

Oliva M. Primera-Pedrozo, Jackeline I. Jerez-Rozo, Edwin De La Cruz-Montoya, Tatiana Luna-Pineda,Leonardo C. Pacheco-Londoño, and Samuel P. Hernández-Rivera

Abstract—Nanotechnology based detection of threat agents,such as explosives and biological agents, has been a top researchpriority at the Center for Chemical Sensors Development at the De-partment of Chemistry of the University of Puerto Rico-Mayagüez(UPRM). Nanoparticles are of fundamental interest since theypossess unique size-dependent properties are quite different fromthe bulk state. When a bulk metal is reduced in size, its propertiesbegin to change dramatically because the constituent electronsbegin to suffer the effects of quantum confinement. One of theseimportant properties deals with the extraordinary enhancementof the intensities of Raman scattering events in chemical systemscalled surface enhanced Raman scattering (SERS). Until very re-cently, only aromatic moieties containing strong chromophores orhighly delocalized electrons would experience such an enhance-ment, when in close proximity to a silver or gold nanometallicassembly. In other cases, this SERS condition was not sufficient tosatisfy the enhanced Raman scattering requirements because ofCoulombic repulsions do not allow an intimate contact with thecolloidal suspension of nanoparticles. Recent work in the researchgroup includes optimization of particle size, agglomeration rateand ionic strength of the SERS active aqueous colloidal metallicsuspensions. Results have led to extend existing benchmarkslimits of detection of �� to �� in DNTand to �� in the case of TNT. Other worksinclude preparation and testing of bimetallic nano-interalloys:Au/Ag and metallic-semiconductor SERS active colloidal sub-strates: ��

�. Group members have prepared silver and

gold nanorods and nanolayers in an effort to change the sensingplatform: from aqueous media to solventless detection.

Index Terms—Biological warfare agents, explosives, Ramanspectroscopy, surface enhanced Raman scattering (SERS).

I. INTRODUCTION

ANALYTICAL technologies are in high demand for De-fense applications, while being a very important part of

National Security. Domestic preparedness requires a wide arrayof detection capabilities for a range of potential attack scenarios.An alternative approach conventional analysis (MS, IMS, andothers) of threat agents including explosives, chemical and bi-ological agents, and other threat agents such as toxic indus-trial compounds (TICs) is the application of vibrational spectro-

Manuscript received August 16, 2007; revised January 3, 2008; accepted Feb-ruary 14, 2008. This work was supported in part by the U.S. Department of De-fense, University Research Initiative Multidisciplinary University Research Ini-tiative (URI)-MURI Program under Grant DAAD19-02-1-0257. The associateeditor coordinating the review of this paper and approving it for publication wasDr. Dennis Polla.

The authors with the Department of Chemistry, University of PuertoRico-Mayagüez, Mayaguez, PR 00681 USA (e-mail: [email protected];[email protected]; [email protected]; [email protected]; [email protected]; sp_hernandez_uprm @yahoo.com).

Color versions of one or more of the figures in this paper are available onlineat http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/JSEN.2008.923936

scopic techniques [FTIR, Raman, and surface enhanced Ramanscattering (SERS)] in which the vibrational spectrum displaysa fingerprint of the chemical composition of each chemical orbiological agent together with detection capability. While anIR spectroscopic investigation of bacterial cells requires a fewhundred cells from controlled cultivation conditions for analysisand drying steps, this is not necessary when applying Ramanspectroscopy. In particular, when only a small sample amountis available, SERS spectroscopy is well suited [1].

SERS involves enhancement of Raman signals from targetmolecules adsorbed on roughened metal surfaces. Recent ad-vances have made this a versatile technique in diverse fields ofapplication not only in analytical science but also in biomedi-cine [2], environmental monitoring [3]–[5], and analysis of ex-plosives [6]. The appeal of this technology lies in its ability toidentify nearly unequivocally many different compounds. Theprocess is nondestructive, sensitive, fast, and repeatable [7]. Thesize of the nanoparticles prepared by chemical methods is gen-erally governed by factors such as the ratio of the capping agentto the reducing agent [8], the nature of the reducing and cap-ping agents [6], [9] the temperature of the reaction, and the rateof addition of the reducing agent [10]. Colloidal particles areattracted to each other by van der Waals forces, ultimately re-sulting in the coagulation and precipitation of the sol [11].

Citrate-capped nanoparticles are negatively charged and theyattract positively charged countercations from the solution.This arrangement results in the formation of a diffuse electricaldouble layer and, consequently, a Coulombic repulsion betweenthe particles. As long as the electric potential associated with thedouble layer is high, electrostatic repulsion between the particleswill prevent agglomeration. However, the double layer is verysensitive to changes in temperature and, in particular, to the ionicstrength of the solution. An increase in the ionic strength by theaddition of a salt causes a compression of the double layer andshortens the range of repulsion. Reduction of the charge on thecolloid by the addition of a neutral, strongly binding adsorbate,which displaces the adsorbed citrate anions, would also resultin agglomeration [12]. When a bulk metal is reduced in size, itsproperties begin to change dramatically because the constituentelectrons begin to suffer the effects of quantum confinement.This causes nanoparticles to behave very distinctly from thebulk as well as the atomic state. Some of the properties suchas optical, electronic, chemical, magnetic, and mechanical areaffected by these size dependent effects. The phenomenon ofSERS or “giant Raman effect” first noted in the 1970s by Fleis-chmann and co-workers and is related to an anomalous increasein intensity of Raman signals of organic molecules adsorbedon electrochemically roughened Ag electrode surfaces [13].

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964 IEEE SENSORS JOURNAL, VOL. 8, NO. 6, JUNE 2008

The signal enhancement has been shown to be equivalent to anenhancement of the Raman scattering cross section for a singlemolecule by a factor of [14], [15]. Creighton andco-workers demonstrated that colloidal metallic suspensions ofAg, Au, and Cu that show sharp surface plasmon bands in thevisible region are excellent candidates for SERS studies. Therequirement of large concentrations of sample, a main disadvan-tage of Raman spectroscopy, is overcome by SERS. During thepast few decades, many different methods have been proposedin the preparation of metal nanostructured substrates valid forSERS. Colloidal nanoscale particles have been the most fre-quently used SERS substrates. Metal colloids, in particular Agand Au, were first used as substrates used for SERS [16]. Amongthe possible metals with applications in SERS (specifically Ag,Au, and Cu), silver is the more universal substrate for severalreasons: plasmon resonance in the visible (VIS) region of theelectromagnetic spectrum with excellent match with laserlines located at 514.5 and 488 nm and solid-state diode laserat 532 nm; highest SERS effect in metallic sols (10 to 100 foldhigher than Au); and simplest preparation methods available.There are a few drawbacks for the use of colloidal silver sols: lowstability and poor shelf life when compared with Au counterpartsand low reproducibility (has high as 30% intensity variability).

The behavior of the dielectric constant of silver near theFrohlich frequency gives rise to an intense surface Plasmonabsorption in the visible wavelength region [17], [18]. Ag andAu colloids display different optical properties which have tobe considered from the point of view of the electromagneticmechanism associated with the SERS effect; whereas Ag solsdisplay an absorption maximum ca 400–420 nm, the Au solsshow a maximum at above 500 nm. The aggregated colloids shownew absorption bandswhich appear shifted toward the red region:500–600 nm for Ag colloids and 700–900 nm for Au colloids.These new absorption bands are due to the plasmon excitationin aggregated particles that govern the SERS excitation profilesof such colloids [17]. Due to this difference, it is important tomake the correct choice of excitation source at an appropriatewavelength, depending on the nature of the metal sol employed.Whereas Ag colloids usually have a higher activity in the visibleregion, Au sols are more active in the red and near infrared (NIR)regions. The preparation of Ag/Au mixed colloids allows forthe combination of the SERS activities of both metals in abroader interval of the electromagnetic spectrum [19].

There are several methods to prepare Ag metal colloids em-ployed in SERS. Among these are: chemical reduction (wetchemistry synthesis), photoinduced reduction [20] or laser ab-lation [21]. The preparation of Ag colloids in suspension bychemical reduction is the most widely used method for SERSexperiments. It is usually performed by using silver nitrate asthe starting Ag salt, which is reduced by a chemical reducingagent to produce colloidal suspensions composed of nanoparti-cles with varying size ( 10–120 nm) depending on the methodof preparation. The most frequently used reducing agents aretrisodium citrate [22], [23] and sodium borohydride [16]. Thestability of the sols is due to the adsorption of the counter ionsof the salts employed in the colloidal preparation which donatea high electric charge to the nanoparticles. However, these col-loids display several disadvantages such as the existence of im-

purities resulting from the incomplete oxidation and residualredox species and the counter ions of the salts employed [16],[24]. Recently, other chemical reducing agents have been usedsuch as hydroxylamine [25] with the advantages of easier prepa-ration, since the reduction reaction can be done at room tem-perature, and the elimination of the residual oxidation products,which are gases, in the case of hydroxylamine colloids molec-ular nitrogen and nitrogen oxides.

Despite the higher stability of the chemically prepared Aucolloids, a partial aggregation is necessary for them to becomeactive in SERS [26]. The size of the colloids prepared by chem-ical methods is generally governed by factors such as the ratioof the reducing agent, the nature of the reducing agent, temper-ature, and the rate of addition of the reducing agent. Nucleationand growth are two closely interlinked processes that dependmarkedly on the nature of the reducing agents. Strong reducingagents such as borohydrides and superhydrides induce the for-mation of nucleation centers which then grow into small clus-ters whose final size is determined by the nature of the reducingagent. In the case of weak reducing agents such as citrate, therate of reduction is slow. Hence, growth and not the nucleationprocess dominate, resulting in large-sized particles.

In the case of analysis of microorganisms such as bacteria,spores, and viruses by conventional methods, a large number oftests are often needed for a definitive identification. Classifica-tion and speciation require even more lengthy microbiologicalassays. Among new methods under development, vibrationalRaman Spectroscopy and SERS provide reagentless proceduresin which there is no need to add chemical dyes or labels foridentification. There are many advantages in using SERS inthis application. Amid the other techniques available, it is afast, specific, and noninvasive analytical method that promisesto deliver analysis capability of detection and identificationof a single bacterium in the not so distant future. Thus, theaim of the present research was to combine Raman and SERSspectroscopies and multivariate statistical analysis for reliableand robust discrimination of bacteria. Eleven bacterial specieswere used to demonstrate the capability of vibrational spec-troscopy techniques in the identification and discrimination ofbacterial cells between various species. The study initiated withbacterial discrimination species at stationary phase using themultivariate analysis technique of principal component analysis(PCA). Then, spectroscopic discrimination of bacterial specieswas continued with other statistical analyses: discriminatefactor analysis (DFA) and hierarchical cluster analysis (HCA).

II. EXPERIMENTAL METHODS

A. Reagents

(99.98%) was purchased from Strem Chemicals,Newburyport, MA. Adenine ( , 99%), trisodiumcitrate hydrate, , nitric acid ( ,70%), sulfuric acid ( , 98%), were obtained from FisherScientific, International, Chicago, IL. Hydroxylamine hy-drochloride, sodium chloride (NaCl, USP/FCC granular), and1,2-Bis(4-pyridyl)ethane (BPE, , 99%) were pur-chased from Sigma-Aldrich Chemical Company, Milwaukee,WI. 2,4,6-trinitrotoluene (min 30 wt% water; 98% TNT purity)


PRIMERA-PEDROZO et al.: NANOTECHNOLOGY-BASED DETECTION OF EXPLOSIVES AND BIOLOGICAL AGENTS SIMULANTS 965

was obtained from ChemService, West Chester, PA. Glycerol(99.5%), Lennox broth (LB: tryptone 10 g, yeast extract 5 g,sodium chloride 5 g/liter solution) were purchased from FisherScientific International. Aqueous solutions were prepared usingpurified, 18.2 MOhm water. The following bacterial strains wereobtained from the sources indicated and used in the research: a)Bacillus subtilis (ATCC number 6633); b)Bacillus cereus (ATCCnumber 14579); c)Staphylococcus aureus (ATCC number 6538);d) Escherichia coli (ATCC number 8789); e) Staphylococcusepidermidis (ATCC number 2228); f) Salmonella tennessee(ATCC number 93311); g) Klebsiella pneumoniae (ATCCnumber 3882); h) Pseudomonas aeruginosa (ATCC number9721) were provided by Prof. Carlos Rios-Velazquez (Depart-ment of Biology, UPRM); i) Bacillus thuringiensis (ATCCnumber 35646); j) Enterobacter aerogenes (ATCC number13048); and k) Proteus mirabilis (ATCC number 25933) wereobtained from Fisher Scientifics. Pure cultures were stored at

in micro vials containing 20% glycerol (cryoprotectant)until use. Bacterial cells were grown in 5 mL of LB (Lennox)broth for approximately 15 h, centrifuge for 20 min to 6000 rpmand retirement the broth, washed four times with 5 mL of NaCl0.80% w/v, and obtained a pellet. Standard biosafety level 2 labprocedures were employed for handling the strains studied hereeven though they are no virulent. Spore-forming bacteria weredestroyed by exposure to 95% ethanol for 20 min and UV.Non spore-forming bacteria and contaminated glassware wereautoclaved prior to disposal.

B. Instrumentation

The nanoparticles were characterized using techniques suchas UV-VIS spectrophotometry using a Varian Cary-100, doublebeam spectrophotometer and a Scanning Electron Microscope(JEOL JSM 6460LV). Images were taken with 10 ofsilver colloids and drying at room temperature. Colloids wereprepared for the identification of traces of nitroexplosives. ARenishaw Raman Microspectrometer RM2000 system wasemployed for the vibrational spectroscopy measurements. Thesystem was equipped with a Leica microscope. TNT detec-tion was achieved at a wavelength of 532 nm, laser powerused 10 mW. The excitation source was a diode-pumped532 nm green laser with a variable output power of up to 6 W(Coherent, VERDI); Renishaw Raman MicrospectrometersRM-2000 equipped with spectroscopic CCD camera, Leicamicroscope and 10 magnification objective. The spectra wereobtained in the range of 100–3500 Raman Shift (wavenumbers,

), three scans, and 30 s of integration time.The size and shape of the gold/silver bimetallic nanoparticles

alloys were investigated by transmission electron microscopy(TEM). A Zeiss High Resolution TEM, model 922 was usedoperated at 200 kV for the measurements. The chemical com-position of nanoparticles was investigated by energy-dispersiveX-ray (EDX) microanalysis with a UTW + Sapphire X-ray de-tector and 15 tilt stage. Other chemical studies included theline scan map and mapping in scanning TEM (STEM) by EDX.Samples for TEM analysis were prepared by dipping a carbon-coated copper grid (CF200-Cu, 200 Mesh, Electron MicroscopySciences) in the colloidal solution and allowing drying in air.

C. Synthesis of Metallic Colloidal Suspensions

Methods have been developed for the preparation of silvercolloids by chemical synthesis. Among these metal specimensthere are roughened metal surfaces considered to be essential forobserving SERS. All glassware was vigorously cleaned usingaqua regia (strong acid mixture) of 4:1, which washandled with extreme care as copious amounts of gas were liber-ated, which could cause explosions if the acid is stoppered andbad burns occur if it comes into contact with skin, the glass-ware was then rinsed with water before washing with deter-gent. Finally, the glassware was washed well with triple dis-tilled water. Ag colloid preparation followed a modified Lee andMeisel method [19]. Briefly, 100 mL of a aqueous so-lution of was heated to boiling and then 25 mL of a1% trisodium citrate solution was added. Themixture was kept boiling for 1 h, and then was allowed to cooldown. The resultant colloidal mixture was had a dark grey color.For the Ag-coated Au colloids, different aliquots (between 1and 8 mL) of a 1 mM solution were added dropwiseto 20 mL of a gold sol. After adding an appropriate aliquot oftrisodium citrate solution, the mixture was boiled and stirred for1 h.

The preparation of nanoparticles can be consid-ered as a combination of two processes that occur sequentiallyin a single reaction mixture, the formation of the silver core,and the coating with . The starting reaction mixturewas prepared from two solutions. The first solution was a2.8 mM in sodium citrate. The second solution con-tains equimolar amounts of isopropoxide and isopropanol inwater in a concentration approximated of 5.75 mM for each ofboth components. Freshly prepared 20 mL of the first solutionand 5 mL of the second one were mixed in a round-bottom flaskand stirred while boiling at reflux. After 90 min, the colloidsattained a green-black coloration.

For sample analysis by SERS, 800 of the silver colloids,100 of sample ( to TNT solutions; to

DNT solutions), 100 of NaCl were combined in aplastic vial, and then stirred in a mini vortex stirrer for 30 s. Asample was taken of the resulting clear solution to fill a capillarytube of diameter of 1.65 90 mm for analysis. The interrogationvolume in a capillary tube as sample container and using a 10objective is of the order of 400 pL . For asolution this represents 4 fmol or 2400 millionmolecules. A typical high explosive (HE) has a molar mass of200–250 . The upper limit of the mass contained inthe interrogation volume for a solution of TNT is 1 pg

.For analysis of microorganisms, the pellet obtained from cen-

trifugation was resuspended in 50 of NaCl 0.80% w/v. Then,10 of bacterial cells suspension and 50 of silver colloidwere mixed in a microtube. A 10 aliquot of this mixture wastransferred to microscope glass slides covers, and then placedon the microscope stage for Raman analysis. The SERS spectrawere obtained with 0.5 mW at 532 nm with three scans and20 s of acquisition time. Spectra were acquired in the RamanShift range of 200–4000 wavenumber . Visual inspec-tion of these spectra showed that the most information-rich area



was between 400 and 1800 , and this was used for dataanalysis. Calibration was periodically checked by recording theposition of know Raman lines of silicon (521 ). All datawere obtained from the WIRE 2.0 software.

D. Data Analysis

To assess the ability of Raman and SERS, a data discrim-inate between the different species, the spectra of each tech-nique was analyzed by multivariate statistical techniques. Thisprocess involved three different analyses: principal componentanalysis (PCA) was employed to reduce the dimensionality ofthe data, while preserving most of the variance. DiscriminantFactor Analysis (DFA) was then used to discriminate betweengroups on the basis on the retained principal components (PC)and, finally, the Hierarchical Cluster Analysis (HCA) was ap-plied to the data. The PCA, DFA and HCA were all performedin the Statgraphics version 9.0.

III. RESULTS AND DISCUSSION

A. Plasmon Resonance Absorption for Metallic Sols

The absorption spectra of many metallic colloidal nanopar-ticles are characterized by a strong broad absorption band thatis absent in the bulk metal spectra. Classically, this giant dipoleband, referred to as the plasmon resonance absorption band isascribed to a collective oscillation of the conduction electronsin response to optical excitation [25]. The optical absorption ofAu and Ag colloids in particular has been a subject of study fordecades. These colloids show absorption maxima at about 400and 540 nm for Ag and Au, respectively, because of the differentplasmon excitation resonance of each metal [26].

The absorption maximum of the metallic sols provides in-formation on the average particle size, whereas its full-width athalf-maximum (FWHM) gives an estimation of particle size dis-tribution. In some cases, aggregation occurs during preparationor storage. Strict adherence to preparation protocols and the useof rigorously clean apparatus is therefore imperative. Typical Agcolloids prepared with citrate have absorption maxima between395 and 420 nm. By preparing very small particle size Ag “seeds”or core particles and then reducing more on the seeds, itwas possible to prepare Ag/Ag nanoparticles of larger averagediameter.

UV-VIS spectra were obtained by diluting 1 of the col-loidal suspensions in 3 mL water. Neat silver sols are highlyabsorbing suspensions and will cause overrange of most spec-trophotometers. A Varian Cary 100 was used for the absorbancemeasurements. Scan range was from 200 to 800 nm. Fig. 1shows the UV-VIS absorption spectra of several of the colloidalsuspensions prepared. Ag colloids prepared from four differentconcentrations of citrate show differences in their spectral ab-sorption characteristics, which may be attributed to the morpho-logical differences.

The citrate-reduced Ag/Ag sols had an absorption maximumat 440–460 nm. The red shift is probably due to an increasein particle size. The absorption spectra of Ag-coated Au col-loids showed only an absorption band of maximum absorptionat 515 nm, which can be attributed to plasmon resonance of Auparticles. The fact that gold/silver colloids were blue shifted

Fig. 1. UV-VIS spectra of colloids: (a) silver colloid � � �� ; (b)silver/gold colloid, � � �� ; and (c) gold colloid � � �� .

from normal colloidal gold suspensionssuggests the formation of the Au/Ag bimetallic alloy nanoparti-cles colloidal suspension (Fig. 1). On the other hand, a FWHMof 65 nm for gold and 44 nm for gold/silver alloy is indicativeof colloidal suspensions with modest monodispersity. However,for silver a FWHM of 117 nm indicates poor monodispersionof nanoparticles in these colloidal metallic suspensions.

The appearance of only one absorption band correspondingto Au indicates that homogeneous mixed colloidal particles ofboth metals are formed without significant formation of inde-pendent particles. The narrowing of the band indicates that thereis a change from a more polydispersed system (Ag) to a highermonodispersed system (Au). There is a blue shift in the positionof the surface plasmon, with a corresponding increase in inten-sity. In order to convert this slow reduction into a kineticallyfavorable one, temperatures ranging between 80 and 100are required. At elevated temperatures, the partially oxidized in-termediate, acetone dicarboxylate, is formed, which is in turn agood reducing agent [27].

B. Morphology Silver and Gold Colloids

The shapes and sizes of these particles are better character-ized using Transmission Electron Microscopy (TEM). Fig. 2contains some of the results obtained by TEM measurements:(a) for silver, (b) for gold, and (c) and (d) for bimetallic Au/Agparticles. According to TEM images, the average particle size ofsilver nanoparticles was 60 11 nm. Silver particles in sols pre-pared were highly polydispersed varying both in size and shape,as can be seen in Fig. 2(a). The average size and size distribu-tion nanoparticles in the silver colloids are dependent on tem-perature increase. A fine control in the temperature is requiredto maintain steady growth of nanoparticles. As the size distribu-tion sharpens, it is important to maintain a steady temperaturein order to achieve the size and shape that would produce largeelectromagnetic enhancement at the metallic colloidal surface.Colloids prepared by chemical methods tend to form clustersbecause of their complicated double layer structure. These clus-ters have much higher morphological heterogeneity, since theyare composed by nanoparticles with a wide spectrum of sizes



Fig. 2. TEM images of metallic nanoparticles: (a) Ag and (b) Au (100 nmscale on both images); (c) TEM dark-field image of bimetallic Au/Ag alloys;(d) high-resolution bright field TEM image of Au/Ag (20 nm scale); and (e) en-ergy dispersive X-ray analysis (EDX) of Au/Ag.

and shapes. This correlates with the broader plasmon resonanceabsorption band.

In contrast, Au nanoparticles were very nearly monodisperseand almost spherical in shape. Gold nanoparticles were 54

5 nm, most of them very close in size to the average size.Bimetallic Au/Ag nanoparticles prepared were very similar inshape and size to the Au seed nanoparticles although they weresignificantly smaller that the gold particles (about 35 nm in sizeand nearly spherical in shape).

Energy dispersive X-ray (EDX) analysis of different nanopar-ticles indicated that the Au/Ag nanoparticles were bimetallicalloy nanoparticles. A typical EDX run is shown in Fig. 2(e).Atomic Au and Ag emission lines have been marked. The ele-mental lines intensity ratio Au-L/Ag-K was 10.7 (not calibratedfor instrumental response). Thus, the particles are much richerin the gold component. Moreover, silver coverage of gold seedparticles seems to be incomplete and diffuse. This was demon-strated when an X-ray line map was performed using the AuMand AgL lines. These results are shown in Fig. 2. The gold andsilver X-ray emissions detected and mapped emerge from thenanoparticles shown in the white line drawn. Gold emissionsmap two particles of roughly of 20–30 nm (core substrate).Silver emissions correspond to a much thinner atomic “shell.”

Fig. 3. X-ray fluorescence line scan mapping performed on Au/Ag nanoparti-cles using the Ag-L and Au-M atomic emission lines.

Fig. 4. SERS spectra of TNT: (a) spectrum without adjusting pH (typically,�� –9) and (b) spectrum with pH adjusted to high values.

C. SERS of 2,4,6-Trinitrotoluene (TNT) on Metallic Sols

The objective of this study was to modify the ionic strengthand pH of colloidal suspensions of metallic colloids interactingwith nitroaromatic explosives bond in an effort to improveHE-nanoparticles bond to stimulate improved SERS signaturesin these systems.

The electrical double layer structure on the nanoparticles sur-face is very sensitive to changes in temperature, and in partic-ular the ionic strength of the solution. An increase in the ionicstrength by the addition of a salt causes a compression of thedouble layer and shortens the range of repulsion. Reduction ofthe charge on the colloid by the addition of a neutral, stronglybinding adsorbate, which displaces the adsorbed citrate anions,would also result in agglomeration.

Modification of the ionic strength of the media surroundingthe colloidal suspension interacting with TNT had a unique out-come on the SERS spectra obtained. Illustrated in Fig. 4 is theeffect of changing the pH from the normal value: pH 8.5–9, tohighly alkaline pH ( 12.5), where maximum SERS intensitywas observed.



Fig. 5. TNT SERS spectra: (a) on Au colloids; (b) on Au/Ag colloids; and(c) on Ag colloids.

Two things could be happening. First, the adsorption geom-etry could be different at pH 9, as suggested from the bandsthat are enhanced, compared with the SERS spectrum at pH12.5. Splitting of signal at ca. 1600 ( asymmetricstretch) at pH 12.5 suggests conformation geometry for theTNT-nanoparticles adsorbate-substrate pair with nonequivalent

groups attached to the surface. Another possible expla-nation is the formation of TNT degradation products in thehighly alkaline media. Some of these compounds are coloredand seem to be involved in surface enhanced resonance Ramanscattering (SERRS) [28]. Detection limits achieved under thepostulated SERRS event were (tenths of femtogram)or solutions.

Fig. 5 illustrates the SERS effect on TNT for Ag, Au andAu/Ag sols. The spectra were obtained for a TNT solu-tion at . Fig. 5(a) shows TNT-Au SERS spectrum ofsolution excited at 785 nm. SERS spectrum using silver coatedgold seeds colloidal suspensions is presented in Fig. 5(b). Thisspectrum was obtained at the same conditions as for the Aucolloid. Silver colloids are not SERS active in the NIR, so thetrace shown in Fig. 5(c) was excited at 532 nm. Fig. 4 illus-trates that TNT nitroaromatic explosive shows enhancement ofthe out-of-plane bending in the 820–850 region andthe symmetric stretching mode in the 1300–1370-region for gold colloids. The symmetric nitro stretching modeis observed at 1356 for TNT, the aromatic ring breathingmode near 1006 [10]. The key spectral regions are thenitrate stretching region about 1350 and the out-of-planebending mode c.a. 820 .

D. SERS of TNT and DNT on Sols

SERS spectra of TNT solutions obtained withsols at different pH values are shown in Fig. 6.

These spectra were excited by a 532 nm solid state diodelaser. The maximum increase of the stretching mode at1365 was observed at , when compared withspectra at the other pH measured. Spectral characteristics, suchas narrow peaks, large number of enhanced vibrations andpresence of luminescence background (as observed in trace d inFig. 8) demonstrate surface enhanced Raman scattering in the

system. In addition, the band at 1213 is shifted

Fig. 6. TNT SERS spectra on �� sols exited by 532 nm laser at pH:(a) pH not adjusted (8.5–9); (b) �� ; (c) �� ; and (d) �� .

Fig. 7. TNT SERS spectra on colloids: (a) �� colloid and (b) Agcolloid.

Fig. 8. DNT SERS spectra on �� colloids. Left: spectra with 532 nmlaser at for different pH: (a) 12.3; (b) 10.4; (c) 5.3; (d) 4.2. Right:spectra with 488 nm laser at pH 12.3: (a) ; (b) ; (c) ;(d) .

to higher wavenumbers and symmetric stretch band c.a.1365 shift to lower wavenumbers: 1330–1350 .Fig. 7 shows a comparison of TNT SETS spectra on Ag and

sols. The spectra are nearly identical except for spe-cific band enhancements favored by a particular nanosurface.

Fig. 8(a) shows the spectrum SERS of solution ofDNT at different pH values with colloids excited by



TABLE IMICROORGANISMS AND IDENTIFIERS USED

532 nm laser. An increase of the intensities of the asym-metric stretch mode (1583 ), symmetric stretchingvibration (1348 ) and 744 vibrational mode (as-signed to out-of plane C-H and C-N bend) was detected at pH12.3, relative to unmodified colloids and other pH modifiedcolloids.

SERS spectra excited at 488 nm line (blue) from an laserfor a concentration range from to DNT at

are shown in Fig. 8(b). Vibrational modes correspondingto the nitro group , asymmetric stretching mode at the1583 , symmetric stretching vibration at 1348are observed at pH of 12.3 with a large enhancement, except thevibration 744 corresponding to the out-of plane C-H andC-N bend, which was observed in the dispersion with 532 nm.These results where acquired even for concentrationsof DNT.

E. Raman and SERS Spectra of Microorganisms

Many bacterial cells were analyzed in Raman and SERS spec-troscopies to determine which spectroscopic technique could bebetter used to detect, identify, and discriminate between closelyrelated bacteria populations. In addition, Colony Forming Unitswere calculated for each bacterial species by dilution from orig-inal suspension. Table I contains a description of bacterial cellsused in the study and the identifier given to each microorganism.The relation of bacterial cells analyzed by Raman and SERS wasapproximately 17:1. For each species, three independent culturereplicates were grown and three bacterial cells from each repli-cate culture were analyzed.

Raman cross-section enhancements due to proximity to thenanostructure metal surfaces are typically cited in the rangeof per molecule from ensemble averaged SERSmeasurements. These enhancements depend on the nature of

Fig. 9. Representative Raman and SERS spectra for E. coli: (a) normal or spon-taneous Raman (RS) spectrum (intensity �15) and (b) SERS spectrum.

the SERS active substrate, excitation wavelength and targetelectronic structure. Single molecule enhancements at selected“hot spots,” however, as large as have been reported [29].To provide some quantitative measure of the magnitude ofthe bacterial SERS enhancement on the silver colloids sub-strates used, the relative SERS and Normal Raman scatteringcross-sections, i.e., scattered power per bacterium, normalizedfor data collection time, and incident laser power of gram-posi-tive, B. thuringiensis, bacteria were determined. If the strongestvibrational band in each spectrum is compared with the Ramancross section of B. thuringiensis is found to be enhanced bya factor of on the silver colloidal SERS substrates.For the gram-negative specie E. coli enhancement due to theSERS substrate was . The observed correspondingnormalized SERS and Normal Raman spectra are compared inFig. 9 for E. coli.

Simple visual inspection of the Raman and SERS spectraof all bacteria examined (Fig. 10) shows they are qualitativelyvery similar. Nonetheless, on closer examination, subtle quanti-tative differences were observed and several bands that appear inthe spectra could be tentatively assigned using literature values.Bands were found to match features reported in the literature forsamples of larger amounts of bacterial biomass or their molec-ular components [30].

Most bands assigned correspond to functional groups inthe main constituents of a microbial cell: proteins, carbohy-drates, lipids, and nucleic acids [31], [32]. Some spectroscopicwindows are more specific of smaller molecular compounds,such as the very characteristic sharp band of phenylalanine at

, which is present in all protein-containingsamples. It is apparent that simple visual inspection of thesespectra will not allow determining the relationship between thebacteria based on their Raman and SERS “fingerprints.” Thus,alternative strategies such as multivariate statistical methodshad to be implemented. These included Principal ComponentsAnalysis (PCA), Discriminant Factor Analysis (DFA), andHierarchical Cluster Analysis (HCA). Table II contains thetentative Raman and SERS band assignments, adapted from theliterature [33]–[39].



Fig. 10. Left: Gram+bacteria; Right: Gram-bacteria. Top: Raman spectra.Bottom: SERS spectra.

F. Statistical Data Analysis

To determine whether Raman and SERS could discriminatebetween the bacterial strains, all spectra were analyzed usingmultivariate methods. Initially, spectral data were normalizedand then PCA was performed. Then, significant principal com-ponents (PCs) were retained and DFA and HCA were applied tothe data set using Ward’s method and Eucledian distances. DFAand HCA show the relationships between the whole collectionsof bacterial species analyzed in the research.

For Raman data, the PCs were calculated using a range be-tween 600 and 1800 and seven principal componentswere retained and used to obtain the DFA. Results of Ramandiscrimination studies are illustrated in Fig. 11. This first stepallowed the discrimination between S. aureus, S. tennessee, K.pneumoniae, P. aeruginosa, B. thuringiensis, E. aerogenes, andP. mirabilis, but B. subtilis, B. cereus, S. epidermidis, and E. colispecies could not be fully discriminated.

Thus, a second PCA-DFA of these four species was per-formed in the range of 1000–1800 , with the objective ofreducing the degrees of freedom of the data and concentratingin the most representative and significant spectroscopic regionin these spectra. In this second analysis, the discriminationbetween these four species was very successful, demonstratingthat Raman microscopy is a technique that may used to identifyand discriminate the eleven bacterial species used in this study.

SERS spectral data were analyzed similarly to Raman spec-tral data: same range and used the data of 11 bacterial species(Fig. 12). In this first analysis, low-order discrimination be-tween all species was not achieved, but discrimination amongGram-negative and Gram-positive species was observed. Thetop region of the PC-DFA plot shows the Gram-negative speciesand the bottom part contains the Gram-positive species.

When a second analysis performed, the Gram-positivespecies and Gram-negative species were analyzed separately

TABLE IITENTATIVE BAND ASSIGNMENT FOR RAMAN AND SERS ANALYSIS

Fig. 11. PC-DFA plot of Raman spectra of all bacterial species.

(Fig. 13). Complete discrimination between the species wasobserved. Therefore, SERS spectroscopy can be used effec-tively to discriminate and classify between eleven bacterial



Fig. 12. SERS discrimination between microorganisms: Top: all Gram-positivemicroorganisms; bottom: all Gram-microorganisms.

Fig. 13. Complete SERS discrimination of microorganisms. Top: Gram-posi-tive; Bottom: Gram-negative microorganisms.

used in this research in two consecutive steps. First, the sampledata is analyzed with all set data to find it is Gram-negativeor Gram-positive. The second step allows locating the datacorresponding to the particular microorganism and finding itsspecies.

IV. CONCLUSION

The benefits of forming colloidal binary alloys by dopingexisting metal suspensions of very small size nanoparticles(seeds) has been clearly established since plasmon excitationthese systems can be selected in wavelength range and allowsfor coupling with exciting single line lasers more suitably.When working with neat gold colloid at 532 nm (green line ofsolid-state diode laser) TNT is not Raman enhanced. However,when this sol is doped with silver, several bands of TNT aresurface enhanced at the excitation wavelength (532 nm). Au/Agalloy sols have been successfully used as substrates for SERS

studies, due to the fact that they combine the advantages ofboth Au and Ag colloids and, thus, serve as interesting systemsfor further research.

In the present work, methods to prepare silver colloids re-duced with different chemical agents: sodium citrate and hy-droxylamine hydrochloride were investigated. Changing severalparameters of the synthesis such agitation, rate of addition of thereducing agent and the reducing agents used resulted in silversols of high signal enhancement reproducibility and improvedstability and shelf life. Gold, silver/silver, and gold/silver col-loidal metallic suspensions were also prepared and used suc-cessfully for the task proposed.

It was demonstrated that at in aqueous solution of2,4,6-trinitrotoluene, it was possible to detect the nitroaromaticHE in silver roughened surfaces (metallic sols). Enhanced sig-nals due to the presence of the out-of-plane bending modeswere detected at 820 and 850 and the symmetricstretch mode in the 1350–1395 region. The aromatic ringbreathing mode near 1000 was also enhanced. A detec-tion limit lower than of TNT sample in the laser pathand giving rise to SERS could be easily and reproducibly de-tected. Titanium dioxide coated silver seeds were also used todetect nitroaromatic high explosives DNT and TNT. High signalenhancements were observed for 488 nm excitation. Highly al-kaline media (pH 10.3 for TNT and 12.3 for DNT) produced thehighest SERS enhancements.

The results of this study also illustrate the enormous poten-tial of vibrational spectroscopic methods for rapid microbialidentification. The methodologies used are of practical interest,as they require very simple sample preparation consisting ofwashing the cells with saline solution and placed on the mi-croscope stage. No chemical reagents are necessary to specif-ically tag or mark cell components. The added advantage isthat these techniques are nondestructive and noninvasive for mi-crobial analysis. Therefore, the techniques may be useful toolsfor classifying bacterial cultures, without requiring strict stan-dardization of growth conditions. The spectra contain multidi-mensional information on all major substance classes present inbacterial cells. The work also showed that when the SERS isused, signal enhancements of at least are easily observed,when compared with the Normal or Spontaneous Raman Spec-troscopy. This contribution is important because it demonstratesthat the methods developed are capable of assessing the het-erogeneity of microbial cells, providing spectral information onchemical composition within a bacterial cell population.

ACKNOWLEDGMENT

The authors would like to acknowledge contributions fromS. Grossman and A. LaPointe of the Department of Defense,Night Vision and Electronic Sensors Directorate, Fort Belvoir,VA.

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Oliva M. Primera-Pedrozo was born in Colombia.She received the B.S. degree in chemistry fromthe University of Cartagena, Colombia and theM.S. degree in chemistry from University of PuertoRico, Mayagüez (UPRM). She is currently workingtowards the Ph.D. degree in materials chemistry atUPRM.

Her work includes the synthesis of gold nanorodsand nanocubes for surface enhanced raman spec-troscopy (SERS) applications. During the last threeyears, she has participated in several projects related

to vibrational spectroscopy (IR, Raman, and SERS) for detection of explo-sives, chemical warfare agents biological warfare agents, and pharmaceuticalingredients.



Jackeline I. Jerez-Rozo graduated from the Indus-trial University of Santander, Santander, Colombia.She is currently working towards the M.S. degree atthe Chemistry Department, University of Mayagüez,Mayagüez, Puerto Rico, where she works in theResearch Group of Chemical Sensors Developmentunder the supervision of Dr. S. P. Hernandez. HerM.S. thesis is entitled “Enhanced Raman Scatteringof TNT on Nanoparticles Substrates: Ag and AuColloids and Au-Ag Alloys Prepared by ReductionWith Hydroxylamine Hydrochloride and Sodium

Citrate.”She plans to begin her doctoral studies next year and her goal is to continue

working in the area of nanochemistry.

Edwin De La Cruz-Montoya, photograph and biography not available at thetime of publication.

Tatiana Luna-Pineda, photograph and biography not available at the time ofpublication.

Leonardo C. Pacheco-Londoño, photograph and biography not available atthe time of publication.

Samuel P. Hernández-Rivera received the B.S. andM.S. degrees from the University of Puerto Rico,Mayagüez (UPRM) and the Ph.D. degree from TheJohns Hopkins University, Baltimore, MD.

He is a Professor of Physical Chemistry at theDepartment of Chemistry, University of PuertoRico-Mayagüez (UPRM). He is the Director ofthe DoD-MURI sponsored Center for SensorsDevelopment and the UPRM based ChemicalImaging Center. Research interests are in IR/RamanMicrospectroscopies in applied and fundamental

studies and standoff and point detection of explosives, chemical and biologicalagents using vibrational spectroscopy. Other interests are in SERS and inchemometrics enhancement of spectroscopic data and discriminant and clusteranalysis of populations.

Dr. Hernández-Rivera is a member of the American Chemical Society, theAmerican Physical Society, Sigma Xi, Phi Kappa Phi, the Society for Photo-Op-tical Instrumentation Engineers (SPIE), and the Puerto Rico Chemists Asso-ciation. He is also member of the editorial board of Springer’s Sensing andImaging: An International Journal.


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