An electrochemical surface-enhanced Raman spectroscopy approach to anthrax detection

Xiaoyu Zhang, Chanda Ranjit Yonzon, Richard P. Van Duyne

Chemistry Department, Northwestern University, 2145 Sheridan road, Evanston, IL. 60201

ABSTRACT

Metal film over nanosphere (MFON) electrodes are excellent substrates for surface-enhanced Raman scattering (SERS) spectroscopy. These surfaces are produced by vapor deposition of a metal film over nanospheres that are assembled in a hexagonally close packed arrangement. The efficiency and reproducibility of AgFON electrode as SERS substrates are confirmed by the repeatability of the electrochemical surface enhanced Raman scattering spectra of pyridine and the Ru(bpy)3

3+/Ru(bpy)32+ complexes adsorbed on AgFON electrodes. The Raman signal for AgFON

electrodes is observed to be extremely stable even at extremely negative potentials in both aqueous and nonaqueous electrolytes. Recent reports have indicated that SERS enhancement factors of up to 14 orders of magnitude can be achieved, providing the sensitivity requisite for ultra trace level detection of target analytes. For this reason, we are developing a method for bacterial endospore SERS detection based on the endospores marker -- dipicolinic acid (DPA). The SERS spectra of dipicolinic acid in aqueous solutions are reported. The dipicolinate vibrational features could be observed in the SERS spectra at the concentration as low as 8 × 10-5 M in 5 minutes. These limits of detection are entirely controlled by the thermodynamics and kinetics of DPA binding to the AgFON surface.

Keyword: electrochemistry, surface-enhanced Raman, Ag film over nanosphere, dipicolinic acid, anthrax, endospore

1. INTRODUCTION

1.1 Surface-enhanced Raman scattering and metal film over nanosphere surfaces

Surface-enhanced Raman scattering (SERS) spectroscopy is an important technique that can display intrinsic interfacial sensitivity and selectivity. SERS, discovered by Van Duyne and Jeannaire1 in 1977, produces very large enhancements in the effective Raman cross section of species located at (or close to) nanostructured noble metal surfaces. The Raman signals of ensemble-averaged molecules show enhancement up to 8 orders of magnitude,2 while the signals from single molecules can show an increase by 14 or 15 orders of magnitude in single molecule SERS (SMSERS) cases.3,4 This effect enables SERS spectroscopy to be one of the most sensitive ultra trace analytical method.

SERS is the first vibrational spectroscopy applicable to in situ metal-solution interfaces,5 and has widespread applications to electrochemical interfaces.6-8 For example, when the potential of an electrode is modulated between two values, SERS can be used to monitor the surface species at the two modulated potentials. SERS has also been successfully applied to the characterization of molecular species formed during electrochemical reduction processes.9-11

The most challenging characteristic of SERS spectroscopy is that the surface enhancement factor (EF) is extreme sensitive to the sizes, shapes and orientations of nanostructured SERS-active surfaces. Traditional SERS-active surfaces are typically metal oxidation-reduction cycle roughened electrodes (MORC electrodes) or metal colloids. The MORC electrodes are generally produced in electrolyte solutions by applying an oxidizing potential to dissolve the metal and a reducing potential to redeposit the active metal. The reproducibility of these nanostructured metal surfaces, however, is limited by using electrochemical oxidation-reduction cycles. The colloid solutions are principally synthesized by reducing metal salt solutions. In colloid, the metal particles size and the aggregate size are extremely influenced by the initial chemical concentrations, temperature, pH, and rate of mixing.12 The nanostructured morphology of these traditional SERS-active surfaces has a variety of sizes, shapes and orientations.13

Proceedings of SPIE Vol. 5221 Plasmonics: Metallic Nanostructures and Their Optical Properties,edited by Naomi J. Halas, (SPIE, Bellingham, WA, 2003) · 0277-786X/03/$15.00

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Van Duyne and coworkers have developed MFON technique to fabricate SERS-active electrode surfaces of well-defined nanostructured morphology. The surface roughness produced by vapor deposition of a SERS-active metal on top of a nanosphere arrangement is reproducible.14 In particular, these surfaces are useful for SERS studies under electrochemical conditions because they are not restricted to specific electrolyte environments during the SERS-active generation. Therefore, new SERS experiments can be carried out with MFON electrodes that were previously not possible with MORC electrodes. For example, the use of MFON electrodes enables a variation of the concentration or chemical nature of the supporting electrolyte in electrochemical surface enhanced Raman scattering (ECHEM-SERS) spectroscopy experiment.15

1.2 Dipicolinic acid and Bacillus anthracis (anthrax)

Bacillus anthracis, a dangerous pathogen for the disease anthrax, is an endospore-forming bacterial species. Due to the threat posed by the aerosolization of spores during a bioterrorist attack, the identification of Bacillus anthracis would provide vital information to community emergency workers and would facilitate a timely and appropriate response to an attack.

The Bacillus anthracis bacteria can exist in two different forms: rod-shaped organisms and endospores. Rod-shaped organisms can grow and divide in favorable environment. In adverse nutrients and conditions, the organisms can turn to endospores. Endospore is so named because it initially develops inside the rod shaped form. Endospores are composed of a central spore cell that is surrounded by various protective layers. Dipicolinic acid (pyridine-2,6-dicarboxylic acid) exists in these protective layers and can make up 5-14% of the spores dry weight.16 The first discovery of DPA in a biological system was reported by Udo in 1936.17 Subsequently DPA has been found to be produced by both the Bacillus and Clostridium genera bacterial endospores18 and by some fungi.19,20

Dipicolinic acid, a chemical substance characteristics of endospores, is often referred to as “the biomarker” 21 and therefore can be used for the rapid detection of endospores. It is worth noting that the detection of DPA is not specific to Bacillus anthracis because other endospore-forming bacterial species also produce DPA. However, the detection based on DPA is a very useful screening tool for substances that are suspected to be concentrated forms of anthrax endospores in a suspicious place. Current chemical detections of endospores based on dipicolinic acid biomarker include: the study of the theoretical fluorescence of dipicolinic acid and its anion;22 the investigation of terbium Tb(III) dipicolinate photoluminescence;23-25 the research of vibrational spectroscopic methods including UV resonance Raman spectroscopy26 and Fourier transform infrared spectroscopy21; and the exploration of mass spectrometric analyses.21 In developing these previous studies of endospore detection, the sensitivities can be achieved only by terbium dipicolinate photoluminescence technique,25 which relies on the enhanced luminescence of [Tb(DPA)]+ compared to Tb(III) alone. The limit of detection (LOD) of photoluminescence was 5000 colony-forming-units/mL (about 5 × 103 ~ 104 spores/mL). Considering 3.65 × 10-16 mole DPA in an average spore,27 the LOD of photoluminescence corresponds to 2 × 10-9 ~ 4 × 10-9 M DPA. However increases in the luminescence intensity of Tb(III) can be also achieved by aromatic complexes other than dipicolinic acid.28 Alternative techniques with improved spectral selectivities must be developed to overcome the poor spectral specificity of luminescent terbium probe. The vibrational spectroscopic techniques that can yield highly compound-specific information, therefore, have much better spectral specificity. In comparison with other vibrational spectroscopic methods, such as FTIR and normal Raman, SERS spectroscopy enjoys both highly sensitivity for trace level detection and the advantages of application in aqueous media. There are several reviews on the use of SERS spectroscopy for the detection of environmental pollutants, explosives, and chemical warfare agents or simulants.29,30 For these reasons, we are investigating the utility of AgFON electrodes and SERS for rapid identification of dipicolinic acid anion.

2. EXPERIMENTAL SECTION

2.1 Materials

All the chemicals used were of reagent grade or better. Ag (D. F. Goldsmith, Evanston, IL), polystyrene aqueous suspensions (Duke Scientific Co., Palo Alto, Ca), pyridine (Fisher Scientific, Fairlawn, VA), potassium chloride (Mallinckrodt Laboratory Chemicals, Phillipsburg, NJ), tetra-n-butylammonium hexafluorophosphate (TBAH) (Aldrich Chemical Co., Milwaukee, WI), acetonitrile (Fisher Scientific, Fairlawn, VA), dipicolinic acid (Aldrich Chemical Co., Milwaukee, WI), and potassium phosphate buffer solution (KPi) (Sigma, St. Louis, MO) were used as purchased. Ru(bpy)3(PF6)2 was prepared by using literature procedures.31 Water of 18 MΩ resistivity was

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obtained with a series of purifying cartridges (Milli-Q, Millipore, Marlborough, MA). A stock solution of dipicolinic acid was prepared in KPi, pH 7.6. All the solutions were N2 deoxygenated for 20 minutes before they were used.

Figure 1. Schematic and AFM image of AgFON surfaces. (A) Top view of close-packed array of nanosphere, (B) side view along “S” axis after metal deposition, and (C) an AFM image of a AgFON surface with Ag mass thickness of 200 nm and nanosphere diameter of 390 nm.

2.2 AgFON substrate fabrication

The AgFON substrates were fabricated in two steps. First polystyrene spheres (390 nm) self-assembled in a hexagonally close packed arrangement on smooth Ag electrode surfaces. Next, a Ag film (200 nm) was thermally deposited onto the nanosphere coated electrodes. Ag was deposited in a modified Consolidated Vacuum Corporation vapor deposition system with a base pressure of 10-7 Torr. The mass thickness and deposition rate for each film were measured using a Leybold Inficon XTM/2 quartz-crystal microbalance (East Syracuse, NY). The fabrication of AgFON surfaces is schematically depicted in Figure 1. Along line “S”, the metal overlayer contacts the substrate, providing electrical contacts to the substrate material (Figure 1B), which is necessary for electrochemical studies.

2.3 Atomic force microscopy (AFM)

AFM was used to obtain topographic images of the AgFON surface. The images were taken under ambient conditions with Digital Instruments Nanoscope IV microscope with a Nanoscope IIIa controller operating in tapping mode. Etched Si nanoprobe tips (TESP, Digital Instruments, Santa Barbara, CA) were used. These tips had resonance frequencies between 280 and 320 kHz and are conical in shape, with cone angle of 20º and an effective radius of curvature at the tip of 10 nm.

2.4 Ultraviolet-visible absorption spectroscopy

UV-vis absorption measurements were taken using an Ocean Optics (Dunedin, FL) SD2000 fiber optically coupled with spectrometer with a CCD detector. All spectra in this study are from macroscopic measurements obtained from transmission mode using unpolarized white light with probe diameter of 4 mm.

2.5 Electrochemistry

The home-built smooth Ag working electrode was embedded into a glass body using Torr Seal (Varian Vacuum Products, Lexington, MA). Prior to use, surfaces were polished with 0.3, 0.05 µm alumina successively (Buehler Ltd., Lake Bluff, IL) and sonicated in MQ water. The Ag/AgCl reference electrode and the BAS 100B/W electrochemical workstation were purchased from Bioanalytical System Inc. (West Lafayette, IN). The potential of the BAS Ag/AgCl reference electrodes is -35 mV relative to the saturated calomel electrode (SCE). The locally constructed Raman spectroelectrochemical cell has been previously described,32 which consisted of three electrodes with a Pt wire (D. F. Goldsmith, Evanston, IL) as the auxiliary electrode. The cell was also designed to enable a laser beam to enter and exit the cell after reflecting off of the electrode surface while traveling a minimal solution volume.

2.6 Surface-enhanced Raman spectroscopy

A Spectra Physics model 120 HeNe laser was used for excitation wavelength of 632.8 nm. The SERS measurement system consists of interference filter (Edmund Scientific, Barrington, NJ), a 1” holographic notch filter (Ann Arbor, MI), a single-grating monochromator with the entrance slit set at 100 µm (Acton Research Corp. model

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VM-505), a liquid N2 cooled CCD detector (Roper Scientific, Trenton, NJ), and a data acquisition system (Photometrics, Tucson, AZ). The spectral positions of the CCD pixels were calibrated using the emission lines of known wavelengths from a Ne lamp. Spectral analysis and data graphics were performed using a Grapher 3, and CorelDraw 10.

3. RESULTS AND DISCUSSION

ECHEM-SERS of pyridine and Ru(bpy)32+ on AgFON electrodes were studied first to evaluate the

electrochemical performances and SERS activities of AgFON electrodes. Each of these adsorbates represents a different class of adsorption behavior. Pyridine, the universal model system for SERS studies, is a molecule that can adsorb reversibly to Ag surfaces. Ru(bpy)3

2+ shows very strong and irreversible adsorption to Ag electrode surfaces.

3.1 ECHEM-SERS of a reversible adsorbate on AgFON electrodes

50 mM pyridine in 0.10 M KCl aqueous solution and Ag/AgCl reference electrode were used in this experiment. The SERS spectra of pyridine on AgFON were taken under cyclic voltammetric potential voltage conditions. Specifically, a sequence of 10-second exposures was collected continuously during a slow (10 mV/sec) potential sweep from -0.3 to -2.2 then back to -0.3 V (Figure 2). The spectral intensity of pyridine on AgFON reached the maximum value at -0.7 ~ -0.8 V with an excitation wavelength of 632.8 nm. This result is consistent with the studies on other surfaces but under otherwise similar conditions.33-35 Figure 2 also showed the Raman signal remained at -2.2 V even as H2 gas evolved from the electrode surface. When the potential on the AgFON electrode reversed at –2.2 V and then came back to the initial potential at -0.3 V, the SERS enhancement could be observed. Similar studies on AgORC electrodes showed no SERS signal of pyridine at -1.7 V vs. SCE (~ -2.0 V vs. Ag/AgCl) 36 and an irreversible SERS signal loss phenomenon.37,38

Figure 2. ECHEM-SERS spectra of 50 mM pyridine in 0.10 M KCl aqueous solution on AgFON. Laser excitation was 632.8 nm, 1~2 mW. Collection time for each spectrum was 10 sec. The voltage potential scanned from -300 to -2200, then back to -300mV, at 10 mV/s.

Compared to AgORC electrodes, AgFON electrodes exhibited stable Raman signals to extremely negative potential excursions, and therefore these nanostructured electrodes extend the potential voltage window for ECHEM-SERS measurements.

3.2 ECHEM-SERS of irreversible adsorbates on AgFON electrode

The SERS of Ru(bpy)32+ has been examined extensively,39-41 although detailed ECHEM-SERS studies have

not.42 In this study, ECHEM-SERS, coupled with cyclic voltammetry (CV) and differential pulse voltammetry (DPV), was used to characterize the one-electron reduction of Ru(bpy)3

2+ irreversibly adsorbed on AgFON electrode surfaces.

1 mM Ru(bpy)32+ in acetonitrile has three continuous one-electron reduction peaks on smooth Ag electrodes

with 0.10 M TBAH as the supporting electrolyte (Figure 3A). The reduction of Ru(bpy)32+ to Ru(bpy)3

+ occurs at

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about -1.3 V vs. Ag quasi reference electrode. To probe the reduction potential of adsorbed Ru(bpy)32+ on AgFON

electrodes, we incubated the substrates for several hours in the electrolyte solution containing 10-6 M Ru(bpy)3(PF6)2 (these electrodes are referred to as coated AgFON electrode later); we then placed the electrodes into 10-9 M Ru(bpy)3(PF6)2 and 0.10 M TBAH solution. The more dilute solution was used to avoid the deabsorption of Ru(bpy)3(PF6)2 from the electrode surfaces.43 Surface CV and DPV were both measured on coated AgFON electrodes. DPV is a more sensitive

Figure 3. (A) CV of 1 mM Ru(bpy)32+ in CH3CN at a smooth Ag electrode with 0.10 M TBAH as the supporting electrolyte, (B)

surface CV of adsorbed Ru(bpy)32+ on AgFON electrode, and (C) DPV of adsorbed Ru(bpy)3

2+ on AgFON electrode, from -1000 to -1950 mV.

technique than surface CV for examining the electrochemical behavior of electroactive reactants attached to electrode surfaces.43 Differential pulse voltammograph (Figure 3C) indicated that the one-electron reduction of the adsorbed Ru(bpy)3

2+ occurred around -1.2 V. There was about 0.1 V positive shift of the one-electron reduction peak of the adsorbed Ru(bpy)3

2+ compared to that of the bulk Ru(bpy)32+ in solution. This shift is due to different diffusion

conditions in these two cases.

The UV-vis absorption spectrum of Ru(bpy) 32+ showed the major absorption band at 453 nm (Figure 4A).

The emission spectrum of Ru(bpy) 32+ indicates a major band at about 600 nm.44 The excited state of Ru(bpy) 3

2+ absorbs at 310 nm and 360 nm.45 The ECHEM-SERS spectra of adsorbed Ru(bpy)3

2+ and electrochemically generated Ru(bpy)3

+ on coated AgFON electrodes were taken with an excitation wavelength of 632.8 nm. The metallic surfaces leads to the quenching of the strong luminescence of the Ru(bpy) 3

2+, and therefore we did not observe the broaden

Figure 4. (A) The UV-vis spectrum of 1.0 × 10-5 M Ru(bpy)32+ indicates the relationship between the absorption band and the

excitation laser. (B) ECHEM-SERS spectra of adsorbed Ru(bpy)32+ on AgFON with 0.10 M TBAH acetonitrile solution in the

Raman spectroelectrochemical cell. The potential was altered from 0 to -1.3 V vs. Ag. Laser excitation was 632.8 nm, 1~2 mW. The collection time for each spectrum was 15 sec.

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Raman spectra background that was usually caused by the emission. The SERS spectra at 0.0 V and –0.7 V belong to Ru(bpy)3

2+ since no electrochemical reduction occurs at these potentials. The SERS signal intensity does not change significantly at these potentials. When Ru(bpy)3

+ was electrochemically generated at -1.2 V, the SERS spectrum had notable changes in relative intensity pattern (Figure 4B). In particular, the intensity of the ring-breathing mode at 1041 cm-1 decreased sharply, and shifted to 1037 cm-1. At the same time, the intensities of in-plane δ(CCH) at 1173 and ν(C-C) inter-ring at 1274 cm-1 increased, and shifted to 1168 and 1268 cm-1, respectively. A summary of representative vibrational bands is given in Table I. Assignments were based on normal resonance Raman spectra (RRS) of Ru(bpy)3

2+ and its excited state in aqueous solution.46,47

Table I. Positions (cm-1) and assignments of the bands observed in the SERS of Ru(bpy)32+/Ru(bpy)3

+ and RRS of Ru(bpy)32+ and

its excited state *Ru(bpy)32+

SERS RRS46 Ru(bpy)3

2+ Ru(bpy)3+ Ru(bpy)3

2+ *Ru(bpy)32+

632.8 nm 632.8 nm 468.0 nm 350.6 nm Assignments47

1318 s 1316 s 1316 s 1317 s

1274 m 1268 s 1272 m 1276 w ν(C-C) inter-ring

1254 w

1173 w 1168 s 1171 s 1173 m

1110 w 1110 w 1109 vw δ(CCH) in plane

1066 w 1067 w 1067 w

1041 vs 1037 w 1043 w 1041 m

1026 w 1024 w 1025 m 1027 w ring breathing

m: medium, s: strong, v: very, w: weak.

3.3 ECHEM-SERS of dipicolinic acid anion on AgFON electrode

The solution phase normal Raman spectrum of calcium dipicolinate has been studied.48 Detailed normal mode

Table II. Positions (cm-1) and assignments of the bands observed in the SERS and normal Raman of dipicolinic acid ions

SERS Normal Raman48 Proposed Assignments48

1572 m 1453 sh 1430 s 1384 vs 1232 w 1189 m 1155 w 1114 w 1090 vw 1013 vs

1572 s, p 1566 sh, p 1452 sh 1447 s, p 1383 vs, p 1370 sh 1193 sh 1187 m, p 1149 w, dp 1082 m, p 998 vs, p

νas(OCO) , A1 R14, B2 R5, A1 νs(OCO), A1 νs(OCO), B2

δ(CH), B2 δ(CH),A1 δ(CH), B2

R7, A1 R9, A1

Figure 5. A) Raman spectrum on AgFON, with 23 mM DPA solution in the cell; B) SERS on AgFON, with 0.10 M Kpi buffer solution in the cell; C) SERS on AgORC electrode, with 23 mM DPA and 0.10 M KCl solution in the cell. Laser excitation was 632.8 nm, 1.9 mW. The collection time for each spectrum was 60 sec.

p: polarized, dp: depolarized

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assignments (Table II) have been made using the analysis of isotopically labeled species. The Raman spectra shown in Figure 5 were 23 mM dipicolinic acid in KPi buffer solution (pH = 7.6) on a AgFON electrode and a AgORC electrode. Figure 5A is the Raman spectrum on AgFON electrode with 23 mM dipicolinic acid KPi buffer solution in the spectroelectrochemical cell. After rinsing both the cell and the AgFON electrode thoroughly by KPi solution to remove the interference from dipicolinic acid ions in the bulk solution, we took the SERS spectrum on the AgFON electrode with KPi buffer solution in the cell (Figure 5B). The relative intensity pattern in Figure 5B is consistent with that in Figure 5A, which proves the SERS ability of detecting dipicolinic acid ions. The overall SERS intensity in Figure 5B, however, is ~ 2 times weaker than in Figure 5A. This intensity loss may be caused by the loss of adsorbed DPA ions in rinsing process. The deabsorption of DPA anions from AgFON is slow because the Raman signal intensities in Figure 5B almost kept the same after 20 hours (not shown). Figure 5C is the Raman spectrum on AgORC electrode with 23 mM dipicolinic acid and 0.10 M KCl in the cell. Compared to Figure 5A, the overall intensity in Figure 5C is ~10 times weaker. The possible reason is that the absorption of DPA ions to Ag surfaces competes with that of halide ions to Ag surfaces, other than a coabsorption process for pyridine case. The assignments of the primary bands in the SERS spectra, as listed in Table II, were based on previous normal Raman studies of calcium dipicolinate solution.48

Figure 6. ECHEM-SERS spectra of adsorbed DPA ions on AgFON electrode with 0.10 M NaClO4 in the cell. The AgFON electrodes were incubated in 32 mM DPA KPi buffer solution for several hours prior to use. Laser excitation was 632.8 nm, 1~2 mW. The collection time for each spectrum was 60 sec. The voltage potentials were referred to Ag/AgCl electrode.

The ECHEM-SERS spectra of adsorbed DPA ions on AgFON electrodes with 0.10 M NaClO4 in the cell are shown in Figure 6. New band at 935 cm-1 due to ClO4

- appeared. The dipicolinate vibrational intensity and relative intensity pattern do not change significantly when the potential altered from -0.2 to -0.5 V vs. Ag/AgCl. This is one of the characteristics of irreversible absorption to AgFON electrode, as shown in Ru(bpy) 3

2+ case. When the concentration of NaClO4 was increased to 0.50 M, only the 935 cm-1 peak could be observed (Figure 7). The absorption of DPA ions to Ag surfaces, therefore, is also a competing process with the absorption of ClO4

- ions.

Figure 7. Raman spectrum of adsorbed DPA ions on AgFON electrode with 0.50 M NaClO4 in the cell. All the other conditions were the same as in Figure 6.

Figure 8. SERS spectrum of adsorbed DPA ions on AgFON surface. The AgFON surface was incubated in 8 × 10-5 M DPA solution for 5 minutes prior to use. Laser excitation was 632.8 nm, 1~2 mW. Collection time was 60 sec.

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As a first attempt to test the ability of SERS to detect low concentration DPA in a short time, Figure 7 shows the SERS spectrum on the AgFON surface that was incubated in 8 × 10-5 M DPA KPi buffer solution for 5 minutes prior to use. The Raman spectroelectrochemical cell was not used so that a laser beam could reflect off of the surface without traveling through any solution. New vibrational peaks appeared in comparison to Figure 5, which were attributed to background “contaminants”. The dipicolinate vibrational features, at 1572 (m), 1430 (s), 1384 (vs), 1189 (m) and 1013 cm-1 (vs), are observed. This promises SERS spectroscopy as a rapid dipicolinic acid detection method.

4. CONCLUSIONS

We have shown that AgFON electrodes are viable surfaces for the study of ECHEM-SERS because of their suppression of irreversible SERS enhancement loss and their stability in both aqueous and nonaqueous electrolytes. The spectra of dipicolinic acid ions on AgFON electrode surfaces exhibit substantial intensity compared to on AgORC electrode. The ECHEM-SERS studies on dipicolinic acid ions indicate that the absorption of DPA ions to Ag surfaces is irreversible and can be competed by other ions easily. The dipicolinate vibrational features are observed in the SERS spectra at the concentration as low as 8 × 10-5 M in 5 minute, which promises SERS spectroscopy as a rapid DPA identification technique. These limits of detection are entirely controlled by the thermodynamics and kinetics of DPA binding to AgFON surfaces. One approach to a lower the LOD would result from the use of self-assembled monolayers (SAMs) in combination with AgFON surfaces, although this was not done in the present study. The power of this approach in biocompatible ECHEM-SERS spectroscopy has been illustrated by the previous studies of our group.49 Therefore, by choosing an appropriate terminal functional group for the SAM to maximize the interfacial binding between DPA ions and AgFON surfaces, one could decrease the LOD.

5. ACKNOWLEDEGMENTS

We gratefully acknowledge the support from National Science Foundation (DMR-0076097) and the Air Force Office of Scientific Research MURI program (F49620-02-1-0381).

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