Characterization of the Ag Mediated Surface-Enhanced RamanSpectroscopy of Saxitoxin

WILLIAM F. PEARMAN,* S. MICHAEL ANGEL, JOHN L. FERRY, and SHERWOOD HALLDepartment of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208 (W.F.P., S.M.A., J.L.F.); and Food

and Drug Administration, HFS-716, CHCB DBC ORS CFSAN, College Park, Maryland 20740 (S.H.)

The rapid detection and quantification of saxitoxin (STX) is reported

using surface-enhanced Raman spectroscopy (SERS) with a colloidal

hydrosol of silver nanoparticles. Under the conditions of our experiments,

the limit of detection (LD) for STX using SERS is 3 nM, with a limit of

quantification (LQ) of 20 nM. It is shown that the SERS method is rapid,

with spectra being collected in as little as 5 seconds total integration time

for a 40 nM STX sample. In order to improve the signal-to-noise ratio,

SERS spectra were generally collected with a total integration time of 1

minute (6 accumulations of 10 seconds each), with no need for extensive

sample work-up or substrate preparation. Based on these results, the

SERS technique shows great promise for the future detection and

quantification of STX molecules in aqueous solutions.

Index Headings: Surface-enhanced Raman spectroscopy; SERS; Saxitoxin;

STX; Silver colloid; Paralytic shellfish poison; PSP; Harmful algal bloom

toxin.

INTRODUCTION

Saxitoxins are a family of toxins produced by cyanobacteriaand dinoflagellates1 that are found in harmful algal blooms(HAB) in both the world’s oceans2 and in freshwater lakes,1,3

of which saxitoxin is the nominal parent. Saxitoxin (Fig. 1) is anaturally produced algal toxin (marine or fresh water) that isoften associated with shellfish bed closings and surface watercontamination during harmful algal blooms.1,2,4–6 Humanexposure to this deadly toxin, mostly through the consumptionof contaminated shellfish,7 can lead to paralysis,4 respiratoryfailure, and/or death.2 Aside from the obvious health relatedconcerns generated by this toxin, there are also environmental(fish kills, shellfish contamination) and economic (commercialfisheries, recreation and tourism) impacts to consider.8

To date, several analytical techniques have been utilized todetect saxitoxin. These techniques can be separated into twobroad, if not overlapping, categories: bioassay and spectro-scopic techniques. The current standard for the detection andquantification of aqueous saxitoxin is the mouse injectionassay,9 which, though accurate, is impractical for on-siteapplications and is hampered by the need for a large number oflive animals. A current alternative to the mouse injection assayis a membrane potential assay; the reported sensitivity of thisassay is ;500 pg of STX.10 Kulagina et al. describe a neuronalnetwork biosensor (NNB) that utilizes cultured mammalianneurons grown over microelectrode arrays, where a limit ofdetection (LD) for STX in a seawater matrix was reported at0.33 nM.11 For the NNB, measurements typically stabilized in10–20 min after exposure to the toxin depending on the toxinconcentration.11

Current spectroscopic techniques include those described in

works by Kele, Gawley, and co-workers who use coumarinbased photo-induced electron transfer (PET) sensors.12–14 Inthis approach, the binding of these coumarin fluorophores tothe STX leads to enhanced fluorescence signal. The enhancedfluorescence is used to quantify and qualify saxitoxin atconcentrations ranging from 10�5 to 10�7 M.12–14 In their mostrecent work, Kele et al. reported a limit of detection for STX at10�5 M,12 but they had detected 10�7 M STX in previouswork.13,14 Other techniques for saxitoxin detection includemass spectrometry15 and surface plasmon resonance (SPR),16

with the latter reporting a detection limit of 1 nM for STX.16

A portable Enzyme-Linked ImmunoSorbent Assay (ELISA),which tests samples on an immunochromatographic platform,has the capability to provide either a positive or a negativeresponse to the presence of paralytic shellfish poisons (PSP) inless than 20 minutes.17 This assay is sensitive to the currentregulatory limit of 80 lg STX equivalents 100 g�1.5,17

However, the ELISA can only provide qualitative information,and thus a gap still remains in terms of rapid, on-sitequantitative analysis of saxitoxin.

Raman spectroscopy, specifically surface-enhanced Ramanspectroscopy (SERS), is an effective technique for thenoninvasive analysis of biologically relevant molecules.18–21

The normal Raman effect is very weak: only one in ;109

photons will be inelastically or Raman scattered. However,SERS significantly enhances the detectable Raman signal byseveral orders of magnitude through both electromagnetic andchemical enhancement factors.22,23 It is also a rapid techniquerequiring little sample preparation. Thus, a major benefit ofSERS is the ability to qualify and quantify molecules atbiologically relevant concentrations in near real time. Theprimary purpose for this work was to determine the viability ofSERS as a rapid technique for the detection and quantificationof low levels of saxitoxin in an aqueous environment. This newcapability offers the potential to detect and then monitor thegrowth of an STX contamination on site, allowing the initiationof proactive measures that can reduce the severity of a potentialSTX outbreak.

EXPERIMENTAL

Materials. A stock 9.93 mg/L saxitoxin solution (3 mMaqueous HCl) was provided by the United States Food andDrug Administration (USFDA). NaBH4, 30% H2O2, andAgNO3 were purchased from Sigma, and H2SO4 and 2,20-bipyridine were purchased from Fisher Scientific. All reagentswere used without further purification or modification. Allsamples and dilutions were prepared in 18 MX H2O (BarnsteadNanopure).

Instrumentation. A 532 nm continuous wave laser beam(Spectra Physics, Millennium) was delivered to the samplevials via a fiber-optically coupled Raman probe (Kaiser Mark

Received 22 January 2008; accepted 25 April 2008.* Author to whom correspondence should be sent. E-mail: william.pearman@us.army.mil.

Volume 62, Number 7, 2008 APPLIED SPECTROSCOPY 7270003-7028/08/6207-0727$2.00/0

� 2008 Society for Applied Spectroscopy

II) and focused into the sample vial by a 103 microscopeobjective (Olympus MPLAN) delivering ;100 mW of laserpower to the sample. The collection fiber was coupled to aHolospec f/1.8 imaging spectrometer (Kaiser Optical SystemsInc.) that utilized a 532 nm SuperNotch Plus filter for furtherlaser line rejection and a low frequency (LF) holographicgrating covering the spectral range of 50–2400 cm�1. Ramanspectra were recorded using a 16-bit, liquid nitrogen cooledcharge-coupled device (CCD) (Princenton Instruments) withWinSpec/32 software. Daily calibrations of the system wereperformed using cyclohexane.24 Colloidal particle sizing wasperformed via dynamic light scattering (Brookhaven Instru-ments Corp., ZetaPlus Particle Sizing Software Version 3.57).Finally, ultraviolet–visible (UV/Vis) spectra of the Ag colloidwere obtained using a Cary 500 UV/Vis Spectrometer.

Borohydride Stabilized Silver Colloidal Synthesis. Allglassware was cleaned using a 4:1 H2SO4:30% H2O2 solutionprior to colloid synthesis. Borohydride stabilized particles wereprepared using a slightly modified procedure originallydescribed by Cermakova et al.25 Briefly, 150 mL of 1.2 mMNaBH4(aq) was placed in a 250 mL round-bottom flasksubmerged in an ice bath. Then, 20 mL of 2.2 mM AgNO3(aq)were prepared in a separate flask and also chilled in an ice bath.After approximately one half hour, a stir bar was added and theNaBH4 solution was vigorously stirred. The AgNO3 was thenadded drop wise with a disposable pipette to the NaBH4. Thebalance of the AgNO3 solution remained in the flask in the icebath until use. Once added, the drops would turn dark gray anddisperse in the NaBH4, ultimately turning the bulk of thesolution a pale yellow. Once all of the AgNO3 was added, thecolloidal solution was removed from the ice bath and stirringwas continued for an additional 45 minutes. The hydrosol usedherein was synthesized for a previous, unrelated experimentand was stored for a period of three months prior to use in thisstudy. Prior to use with the STX, the SERS response for thecolloidal hydrosol was verified with 3.2 3 10�4 M 2,20-bipyridine. At the time of use, this Ag hydrosol had a kMAX¼404 nm and an average particle size of 39.2 nm 6 0.4 nm.

Sample Preparation. Aqueous dilutions of the stock STXsolution were performed prior to addition to the Ag colloidalhydrosol. Samples were generally prepared by the addition ofan aliquot of the STX solution to 500 lL of the Ag colloid. In

order to obtain some of the lower STX concentrations, the STXsolution was added to 1500 lL of the colloidal hydrosol. AtSTX concentrations .100 lM, an instantaneous color changewas observed upon the addition of the STX to the colloid aftermixing. Colors ranged from a deep yellow to a reddish orange.After 24 hours, these solutions became colorless, yet stillproduced intense Raman spectra.

RESULTS AND DISCUSSION

Determination of Saxitoxin Concentrations Required fora Saxitoxin Monolayer. The STX molecule was modeledusing Gaussian 98Wt

26 (Gaussian Inc.) and ChemDraw 3DUltra 10 (CambridgeSoftt) software in order to obtain anestimation of the molecular ‘‘footprint,’’ the area that a singleSTX molecule occupies on the colloidal surface. After theminimization of both energy and geometry (Hartree Foch/631G), we estimated the ‘‘footprint’’ of a single STX moleculeby halving the total Connolly Molecular Surface Area ascalculated by Gaussian 98W, to be ;104 A2. By determiningboth the total number of STX molecules at a given STXconcentration (1 mM) and the number of Ag nanoparticlesavailable, reasonable predictions are possible for the determi-nation of STX concentrations required to form a monolayer onthe colloidal surface. Since a 1 mM STX/Ag colloid sample (10lL 26 mM STX and 250 lL Ag colloid) contains 1.6 3 1017

STX molecules and the 5.7 3 109 Ag nanoparticles have anaggregate surface area of 2.9 3 1015 A2, STX molecules in thissample require approximately 5.5 3 103 times more surfacearea than is available from the Ag nanoparticles in solution.Therefore, simply dividing the initial concentration (mM) bythe required surface area gives a good estimate of theconcentrations required to form a monolayer of STX moleculeson the colloidal surface. For the collection of SERS spectra onthe silver colloid used in this study, final STX concentrations ofless than ;20 lM were required.

Verification of the Surface-Enhanced Raman ScatteringActivity of Saxitoxin. A 94 lM STX sample was used to testthe SERS activity of STX. Spectra were collected of the 94 lMSTX sample, the Ag colloid blank solution, and three replicateSERS samples (20 lL of 94 lM STX combined with 500 lLAg colloid) with a final STX concentration of 4 lM. Figure 2

FIG. 2. Verification of the SERS response of STX. The lower trace (b) is of anaqueous 94 lM STX sample without the presence of Ag nanoparticles. Thethree upper traces (a) are from three replicates of a 20 lL aliquot of the same 94lM STX sample added to 500 lL of Ag nanoparticles. The final STXconcentration for these replicates is 4 lM. The variation in the replicates isindicative of the sample-to-sample variability experienced with SERS.FIG. 1. Chemical structure of the saxitoxin (STX) molecule.

728 Volume 62, Number 7, 2008

clearly shows the dramatic enhancement observed with the 4lM STX samples in the presence of the Ag nanoparticles (Fig.2a) compared to the 94 lM STX solution with no Agnanoparticles (Fig. 2b). Also shown in Fig. 2a is the inherentsample-to-sample variability of SERS with respect to theintensity of the measured signal from replicates of identicallyprepared samples. The blank colloid spectrum is not shown asit is indistinguishable from that of the 94 lM STX sample.Total integration time for all the spectra was one minute (sixaccumulations of 10 s each) at ;100 mW of 532 nm excitation.

Tentative Assignments of Observed Peaks in Sub-monolayer Spectra. A complete list of the tentative peakassignments from the SERS spectra is found in Table I. Asystematic literature search served as the primary source for the

vibrational assignments of the SERS structure;27–33 however,most peaks did correlate well with the calculated vibrationsusing Gaussian 98Wt for the bulk STX molecule. At first theintense peaks at 1397 and 1604 cm�1 were thought to be theamide I and amide III peaks, commonly seen in biologicallyrelevant molecules, yet this does not appear to be the case withSTX. Although dramatic shifts in frequency can occur due tovarious solvent and electronic effects, the major peak at 1604cm�1 is outside the acceptable frequency shift values of 40–60cm�1 for amide I.28,32

The intense peak 1604 cm�1 is the strongest observed in allof the SERS spectra. It is believed that this peak originatesfrom the interaction of the three vibrations (ring str/NH2

þ def./C¼N str) within the guanidinium groups of the STX molecule.

TABLE I. Proposed peak assignments for SERS spectra of STX. Scaled positions are the ab initio positions corrected for the particular method and basisset used in the calculations. All literature values were taken from Ref. 29 unless otherwise noted.

Experimentalposition (cm�1)

Literatureposition (cm�1) Scaled position (cm�1) Proposed assignment Ratio observed/calculated Reference

553.43 557 558 C–N3 sym in-plane def 0.992 (34)678.76 600–750 694 NH2 deformation (amide) 0.978760.73 750–795 779 Ring out-of-plane bend 0.977 (32)807 ;800 793 NH2

þ rocking 1.018948.87 820–980 942 C–C–N sym str 1.007996.15 990-1050 981 C–N3 str 1.015

1026.5 1000–1090 1050 C–N sym str (5-member ring) 0.978 (34)1100.1 ;1150 1118 NH2 in-plane rocking 0.9841191 1180–1280 1216 C–N–C asym str 0.9791248.8 1180–1280 1222 C–N str (N–C¼ unsat amine) 1.0221292.9 1250–1360 C–N str (N–C¼ unsat amine)1397.1 1350–1450 1414 Ring str (guanamine) 0.988 (32)1532.3 ;1500 1535 N–H def. from NH2

þ 0.998 (30)1603.8 1560–1660 1589 Ring str/NH2

þ def./C¼N str 1.009 (32)/(29)

FIG. 3. Calibration curve constructed using the baseline-corrected peak intensity of the SERs peak at 1604 cm�1. Curves were constructed using both the 1604 and1397 cm�1 peaks as indicated by the asterisks in the inset. Each data point represents an average of three spectra collected from three replicate samples. The error barsdenote 61 standard deviation of the baseline-corrected peak intensities.

APPLIED SPECTROSCOPY 729

Several other vibrations are assigned to the guanidinium groupsas well and appear to originate from the five-membered ringguanidinium group when compared with the ab initiocalculations. This may be due to the fact that the five-memberguanidinium group is more flexible with respect to rotation,34

which may facilitate better binding to the colloidal surface. Ofnote are the peaks at 1397 cm�1 (ring stretch), 1532 cm�1 (N–H deformation from NH2

þ), and 807 cm�1 (NH2þ rocking).

Some contribution from the NH2 group of the carbamate wasalso observed in the SERS spectra at 679 cm�1 (NH2

deformation) and 1100 cm�1 (NH2 in-plane rocking).These results suggest that the STX molecule binds to the Ag

colloidal surface at the guanidinium sites. Although notconclusive, a lack of significant contribution from the diolgroup coupled with the apparent affinity of nitrogen to bind toan Ag colloidal surface35 appears to support this assumption.

Determination of a Limit of Detection. The constructionof a calibration curve was conducted using a maximum STXconcentration of 288 nM. This value was chosen since theintensity of the Raman signal of a single 10 s acquisitionsaturated the CCD down to STX concentrations of 340 nM.Three replicate samples for each concentration (288, 194, 98,49, and 39 nM) were made and three acquisitions of oneminute (six acquisitions of 10 s) were collected for eachsample. All data collected were included in the construction of

the calibration curve shown in Fig. 3, in which error barsdenote 61 standard deviation of the peak intensity at 1604cm�1. Curves were constructed using both the 1604 and 1397cm�1 peaks (indicated by * in the inset in Fig. 3); however,only the curve constructed with the 1604 cm�1 data ispresented herein. Solving the equation of the linear fit producesa y-intercept of 10 nM, but there is still a fair amount of inter-sample variability (see Fig. 2a) that must be considered.Equation 1 is used to calculate the LD, where rb is defined asthe standard deviation of the true colloidal blank and m is theslope of the calibration curve:

LD ¼3rb

mð1Þ

Using Eq. 1, a LD of 3 nM is predicted; however, with therelatively large sample-to-sample variability at a givenconcentration, it is important to determine the limit ofquantitation (LQ). The LQ is based on the maximum acceptablerelative standard deviation, commonly 10%, of a reportedvalue.36,37 LQ is calculated using Eq. 2, where (lb) is theaverage of the blank and (rb) is again the standard deviation ofthe true colloidal blank:

LQ ¼ lb þ 10rb ð2Þ

The result of Eq. 2 is in terms of intensity ‘‘counts’’; therefore,dividing the LQ (counts) by the slope of the calibration curve m(counts/nM) produces a LQ of 20 nM. Figure 4 clearlyillustrates the plausibility and relative conservativeness of boththe LD and LQ where a single 5 s acquisition of the same 40 nMSTX sample used in the construction of the calibration curvewas collected. This spectrum represents 1/12 the totalacquisition time used in the collection of the Raman spectraused in the construction of the calibration curves.

Collection of .20 lM Saxitoxin Spectra. As mentionedpreviously, STX concentrations below 20 lM are required toform a monolayer on the colloidal surface for the particle sizeof Ag colloid used. However, the possibility may exist for STXconcentrations to exceed the lM range in environmentaloutbreaks. Therefore, the spectra from 1 mM STX sampleswere also collected during this study. At these higherconcentrations, the colloidal particles become saturated withSTX molecules. In a saturated or multilayer situation, theresultant Raman spectrum resembles a normal Raman spectrum

FIG. 4. A single 5 second acquisition of a 40 nM STX solution; 40 nMrepresented the lowest concentration measured in the construction of thecalibration curve, while 5 s represents 1/12 the total integration time of thespectra used in construction of the calibration curve.

FIG. 5. Growth of the Raman spectra as a function of time. The first trace inthe foreground was taken immediately after mixing 10 lL of 26 lM STX with250 lL of Ag colloid (thus, at approximately 1 minute). Spectra were takenevery 5 minutes for 90 minutes total. Final STX concentration was 1 mM.

FIG. 6. The 1 mM STX multilayer spectrum taken at (a) 1 minute after mixingand (b) 90 minutes after mixing.

730 Volume 62, Number 7, 2008

of a bulk liquid or solid rather than a monolayer on an analytebound to metal substrate.38

As seen in the time series plot in Fig. 5, the Raman responsefor these 1 mM STX samples was relatively fast, about oneminute, but a period of up to ninety minutes was required forthe spectra to stabilize. The growth of the Raman STX signalupon addition to the colloid was steady and intense for the firsttwenty minutes. After this time period, the Raman signalcontinued to increase, albeit not as dramatically. To furtherillustrate this observation, Fig. 6 clearly shows the drasticchange in spectral intensity from one minute (Fig. 6a) to ninetyminutes (Fig. 6b). Understanding that a multilayer or saturatedcondition exists, it is not surprising to observe either the initialdramatic increase or the long period of time required for themolecules to build upon one another on the colloidal surface.

Tentative Assignments of Observed Peaks in MultilayerSpectra. To aid in the identification of the multilayer peaks,the Raman spectrum was again modeled using Gaussian 98Wt

(Hartree Foch/631G)26 and the actual SERS spectra at lowerconcentrations provided additional insight into the possiblebinding orientation of the STX molecule to the colloidalsurface as previously discussed. The majority of the observedRaman peaks shown in Fig. 6b appear to originate from thering structures of the molecule, which includes the diol andguanidinium groups. There appears to be no contribution fromthe carbamate group in the multilayer spectra. This is not anentirely surprising observation when the multilayer stacking ofthe STX molecules on the colloidal surface is considered. Atabulated list of the tentative peak assignments for themultilayer Raman spectra is found in Table II. The diol group(see Fig. 1) is evident in several assignable peaks. The O–Hout-of-plane deformations at 682 cm�1 and 1380 cm�1 alongwith the O–H deformation and C–H wag at 1422 cm�1 areclearly identifiable. Finally, the C–C–O stretch from the diol at1043 cm�1 shows up as an intense peak in the spectra. Theamine vibrations from the guanidinium group yield low-intensity shoulders at 1254 cm�1 and 1362 cm�1 from the N–C¼ stretching vibrations. Further amine peaks are found at 735cm�1 (N–H wag), 847 cm�1 (NH2

þ rocking), 1167 cm�1 (C–Nstretch), and 1196 cm�1 (C–N–C symmetric stretch). Finally,the methylene groups are identified by the CH2 wag at 1311cm�1 and the characteristic CH2 scissor vibration is identifiedat 1479 cm�1. As can be seen in Table II, all assigned peaks arein fairly good agreement with those predicted with the use ofthe ab initio calculations.

CONCLUSIONS

It is clear that the STX molecule exhibits a very strong SERSresponse and that the SERS technique is a viable alternative forSTX detection and monitoring. The conservative LD and LQ

reported herein for STX using SERS (3 nM and 20 nM,respectively) are clearly within the proposed health alertstandards for drinking water of 10 nM.39 The guanidiniumportion of the molecule appears to preferentially bind to thecolloidal surface at low concentrations and is stable for severaldays to weeks. This fact facilitates the possibility of collectingsamples on site, adding aliquots of the sample to a colloidalhydrosol where binding of the STX to the colloidal surface canoccur, and then interrogating the sample on site or at a laterdate. Samples analyzed on site can be returned to the lab forfurther or corroborative testing. It is presently unclear whatmatrix effects there may be or how the matrix might effect theability of SERS to quantify and qualify STX in complexmatrices such as contaminated shellfish or sea water. Matrixeffects will be the focus of future studies along with tests todetermine the qualitative ability of SERS to discriminatedifferent toxins belonging to the saxitoxin family. Thisadditional data would greatly strengthen SERS as a viableand accurate discriminatory test for STX in the environment.

ACKNOWLEDGMENTS

The authors wish to thank the United States Army and the United StatesMilitary Academy for their support and the National Science Foundation forfunding under CHE 0526821.

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TABLE II. Proposed peak assignments for multilayer STX Raman spectra. Scaled positions are the ab initio positions corrected for the particularmethod and basis set used in the calculations. All literature values were taken from Ref. 29 unless otherwise noted.

Experimentalposition (cm�1)

Literatureposition (cm�1)

Scaledposition (cm�1) Proposed assignment Ratio observed/calculated Reference

681.71 510–710 654 C–O–H out-of-plane deformation 1.042 (28)734.52 700–750 718 N–H wag 1.023847.14 ;800 831 NH2

þ rocking 1.0191043 1000–1090 1050 C–C–O str (alcohol) 0.9931167.1 1130–1145 1168 C–N str (secondary amine) 0.9991196.3 1170–1190 1216 C–N–C asym str 0.9841243.6 ;1260 1222 C–N str (N–C¼ unsat amine) 0.9581310.9 1295–1365 1316 CH2 wag 0.9961379.5 ;1385 1372 O–H out-of-plane deformation 1.0051422.1 ;1420 1414 O–H def þ C–H wag 1.006 (30)1478.9 1465 1498 CH2 deformation bound to N 0.987 (31)1603.8 1560–1660 1589 Ring str/NH2

þ def./C¼N str 1.009 (32)/(29)

APPLIED SPECTROSCOPY 731

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