Microarray‐Based Detection of Dye‐Labeled DNA by SERRS Using Particles Formed by Enzymatic Silver Deposition

The growing interest in DNA diagnostics is addressed today by microarrays with fluoresence detection. In our approach, we utilize spatially defined arrays of short oligonucleotides on a modified glass surface. Surface enhanced resonance Raman scattering (SERRS) is used to obtain molecularly specific spectra of the Raman‐active dye‐labeled DNA. Nanoparticles produced by enzymatic silver deposition are used as SERS‐active substrate. They grow directly on the modified oligonucleotides and only in the spatially defined areas on the chip. Furthermore, they potentially offer several advantages for SERS detection. The nanoparticles are characterized and their ability for use as SERS‐ and SERRS‐active substrate is estimated. Three different Raman‐active dyes are investigated for their potential for involvement in sequence specific DNA analysis.     

Surface- and resonance-enhanced micro-Raman spectroscopy of xanthene dyes: from the ensemble to single molecules.

Surface-enhanced resonance Raman scattering (SERRS) spectra of various rhodamine dyes, of pyronine G and thiopyronine adsorbed on isolated silver clusters were recorded at the ensemble level and at the single-molecule level with a high-resolution confocal laser microscope equipped with a spectrograph and a CCD-detector. Comparing single-molecule spectra with ensemble spectra, various inhomogeneous spectral features, such as line splitting, spectral wandering, spectral diffusion and abrupt spectral jumps between different metastable spectral states, are revealed positions and the relative intensities of the vibronic bands. Resonance enhancement is investigated with respect to single-molecule surface-enhanced Raman scattering (SERS) spectroscopy and is found to be responsible for approximately three orders of magnitude in sensitivity. A significant influence of the substituents on the single-molecule SERRS sensitivity is found, showing that various chemical effects are responsible for surface enhancement in addition to the electromagnetic enhancement effect.     

Surface enhanced raman and resonance raman spectroscopy in a non-aqueous electrochemical environment: Tris(2,2′-bipyridine)ruthenium(II) adsorbed on silver from acetonitrile

This letter reports the first observation of both surface enhanced Raman scattering (SERS) and surface enhanced resonance Raman scattering (SERRS) from the transition metal complex tris(2,2′-bipyridine)ruthenium (II), Ru(bpy)₃²⁺, adsorbed on a silver electrode from acetonitrile (ACN). The assignment of these spectra as valid examples of SERS and SERRS in a non-aqueous environment is based on the following criteria: (1) in situ demonstration of monolayer surface coverage of Ru(bpy)₃²⁺ using double potential step chronocoulometry (DPSCC); (2) the Raman signals are most intense after surface roughening by anodization; (3) the Raman spectra are potential dependent in the non-faradaic potential region; (4) the measured enhancement factors are greater ilian 10⁶; (5) the surface spectra are frequency shifted relative to their bulk counterpart; and (6) several other molecules also exhibit non-aqueous SERS and SERRS behavior. These results are highly significant in that generality of surface enhanced Raman spectroscopy has been extended into the rich domain of nonaqueous electrochemistry.     

Electrochemical pathway for the quantification of SERS enhancement factor

This communication presents a new pathway for the more precise quantification of surface-enhanced Raman scattering (SERS) enhancement factor via deducing resonance Raman scattering (RRS) effect from surface-enhanced resonance Raman scattering (SERRS). To achieve this, a self-assembled monolayer of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-Co^IITAPc) is formed on plasmon inactive glassy carbon (GC) and plasmon active GC/AuNP surface. The surfaces are subsequently used as common probes for electrochemical and Raman (RRS and SERRS) studies. The most crucial parameters required for the quantification of SERS substrate enhancement factor (SSEF) such as real surface area of GC/AuNPs substarte and the number of 4α-Co^IITAPc molecules contributing to RRS (on GC) and SERRS (on GC/AuNPs) are precisely estimated by cyclic voltammetry experiments. The present approach of SSEF quantification can be applied to varieties of surfaces by choosing an appropriate laser line and probe molecule for each surface.     

Semi-quantitative analysis of indigo by surface enhanced resonance Raman spectroscopy (SERRS) using silver colloids

In this paper we report for the first time semi-quantitative analysis of indigo using surface enhanced Raman spectroscopy (SERS) and surface enhance resonance Raman spectroscopy (SERRS). Indigo, a dye widely used today in the textile industry, has been used, historically, both as a dye and as a pigment; the latter in both paintings and in printed material. The molecule is uncharged and largely insoluble in most solvents. The application of SERS/SERRS to the semi-quantitative analysis of indigo has been examined using aggregated citrate-reduced silver colloids with appropriate modifications to experimental protocols to both obtain and maximise SERRS signal intensities. Good linear correlations are observed for the dependence of the intensities of the SERRS band at 1151 cm(-1) using laser exciting wavelengths of 514.5 nm (R=0.9985) and 632.8 nm (R=0.9963) on the indigo concentration over the range 10(-7)-10(-5) and 10(-8)-10(-5) mol dm(-3), respectively. Band intensities were normalised against an internal standard (silver sol band at 243 cm(-1)). Resonance Raman spectra (RRS) of aqueous solutions of indigo could not be collected because of its low solubility and the presence of strong fluorescence. It was, however, possible to obtain RS and RRS spectra of the solid at each laser excitation wavelength. The limits of detection (L.O.D.) of indigo by SERS and SERRS using 514.5 and 632.8 nm were 9 ppm at both exciting wavelengths. Signal enhancement by SERS and SERRS was highly pH dependent due to the formation of singly protonated and possibly doubly protonated forms of the molecule at acidic pH. The SERS and SERRS data provide evidence to suggest that an excess of monolayer coverage of the dye at the surface of silver colloids is observed at concentrations greater than 7.85x10(-6) mol dm(-3) for each exciting wavelength. The data reported herein also strongly suggest the presence of multiple species of the indigo molecule.     

Characterization and surface-enhanced Raman spectral probing of silver hydrosols prepared by two-wavelength laser ablation and fragmentation

A four step Ag foil laser ablation-Ag nanoparticle fragmentation procedure in ultrapure water was carried out both under argon and in air. Pulses of a high power Nd/YAG laser were used for laser ablation (1064 nm) and for the three step Ag hydrosol treatment in the absence of Ag foil in the sequence 1064-532-1064 nm. Transmission electron microscopy (TEM) and surface plasmon (SP) extinction spectra provide evidence of Ag nanoparticle fragmentation in the second and third step of the procedure carried out under argon. While polydispersity of Ag hydrosol increases in the second step, both the polydispersity and the mean size of the nanoparticles are reduced in the third step. Qualitative and quantitative surface-enhanced Raman scattering (SERS)/surface-enhanced resonance Raman scattering (SERRS) spectral probing of systems with Ag hydrosols and the selected adsorbates at 514.5 nm excitation shows that Ag hydrosols obtained in the second step of the preparation procedure carried out in air are the most suitable substrates for SERS/SERRS experiments performed at this excitation wavelength.     

Surface-enhanced Raman scattering of EDOT and PEDOT on silver and gold nanoparticles

Surface-enhanced Raman scattering (SERS) spectra of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and its monomer 3,4-ethylenedioxythiophene (EDOT) on Ag and Au nanoparticles presenting different morphologies and stabilizing agents have been obtained using the excitation radiation at 633 nm. The SERS spectra of the monomer and polymer are strongly dependent both on the metal and capping agent of the substrate. SERS spectra of EDOT on Au nanospheres indicates that adsorption occurs with the thiophene ring perpendicular to the metal surface. In contrast, polymerization takes place on the silver surface of Ag nanospheres. EDOT adsorption on Ag nanoprisms with polyvinylpyrrolidone (PVP) as capping agent occurs similarly to that observed on gold. Surface-enhanced resonance Raman scattering (SERRS) spectra of PEDOT on gold nanostars that present a thick layer of PVP show no chemical interaction of PEDOT with the metal surface; however, when PEDOT is adsorbed on citrate stabilized gold nanospheres, the SERRS spectra suggest that thiophene rings are perpendicular to the surface. Oxidation of PEDOT also is observed on Ag nanospheres. The investigation of the interface between PEDOT and metal surface is crucial for the development in polymer-based optoelectronic devices since this interface plays a crucial role in their stability and performance.     

Excitation wavelength dependent surface-enhanced Raman spectra of a dipping film of azobenzene-containing long-chain fatty acid on a silver mirror

Surface-enhanced Raman scattering (SERS) of dipping films of azobenzene-containing long-chain fatty acids, nAmH (n=8, 12, m=3, 5), on silver mirrors measured with a wide range of excitation wavelengths in the 457.9-1064 nm region is reported. The obtained Raman spectra show great SERS effect even with the 1064 nm excitation, and the excitation with 457.9, 476.5, and 488.0 nm gives surface-enhanced resonance Raman scattering (SERRS) due to the resonance effect of the symmetry-forbidden n-pi* transition of the azo group. Of particular note in the present study is that the SERS spectra with the excitation in the 532-1064 nm region yield Raman bands whose frequencies are almost identical to those bands in Raman spectra of nAmH in solid state while the SERRS spectra with the excitation in the 457.9-514.5 nm region show not only a set of bands which correspond to those of nAmH in the solid state but also a set of bands whose frequencies show a significant shift from those of the bands of nAmH in the solid state. These observations lead us to conclude that there are two kinds of molecular aggregates in the dipping films of azobenzene-containing long-chain fatty acid in which azobenzene moieties are condensed to form small bundles.     

Practical understanding and use of surface enhanced Raman scattering/surface enhanced resonance Raman scattering in chemical and biological analysis

The unique ability to obtain molecular recognition of an analyte at very low concentrations in situ in aqueous environments using surface enhanced Raman scattering (SERS) and surface enhanced resonance Raman scattering (SERRS) detection makes these spectroscopies of considerable interest. Improved understanding of the effect coupled to improvements in practical techniques make the use of SERS/SERRS much simpler than has been the case in the past. This article is designed as a tutorial review targeted at aiding in the development of practical applications.     

Semi-quantitative trace analysis of nuclear fast red by surface enhanced resonance Raman scattering

The dye nuclear fast red has been detected and determined semi-quantitatively by means of surface enhanced resonance Raman scattering (SERRS) and surface enhanced Raman scattering (SERS), using laser exciting wavelengths of 514.5 and 632.8 nm, respectively, by employing a citrate-reduced silver colloid. A good linear correlation is observed for the dependence of the intensities of the SERRS bands at 989 cm⁻¹ (R=0.9897) and 1278 cm⁻¹ (R=0.9872) on dye concentration over the range 10⁻⁹ to 10⁻⁷ M, when using an exciting wavelength of 514.5 nm. At dye concentrations above 10⁻⁷ M, the concentration dependence of the SERRS signals is non-linear. This is almost certainly due to the coverage of the colloidal silver particles being in excess of a full monolayer of the dye. A linear correlation is also observed for the dependence of the intensities of the SERS bands at 989 cm⁻¹ (R=0.9739) and 1278 cm⁻¹ (R=0.9838) on the dye concentration over the range 10⁻⁸ to 10⁻⁶ M when using an exciting wavelength of 632.8 nm. Strong fluorescence prevented collection of resonance Raman scattering (RRS) spectra from powdered samples or aqueous solutions of the dye using an exciting wavelength of 514.5 nm, but weak bands were observed in the spectra obtained from both powdered and aqueous samples of the dye using an exciting wavelength of 632.8 nm. A study of the pH dependence of SERRS/SERS and UV–VIS absorption spectra revealed the presence of different ionisation states of the dye. The limits of detection for nuclear fast red by SERRS (514.5 nm), SERS (632.8 nm) and visible spectroscopy (535 nm) are 9, 89 and 1000 ng ml⁻¹, respectively.     

Surface-enhanced Raman scattering and surface-enhanced resonance Raman scattering excitation profiles of Ag-2,2'-bipyridine surface complexes and of [Ru(bpy)3]2+ on Ag colloidal surfaces: manifestations of the charge-transfer resonance contributions to the overall surface enhancement of Raman scattering

Excitation profiles of SERS (surface-enhanced Raman scattering) and/or SERRS (surface-enhanced resonance Raman scattering) spectral bands of two forms of a Ag-bpy (bpy = 2,2'-bipyridine) surface complex and of [Ru(bpy)3]2+ on Ag nanoparticle (hydrosol) surfaces were determined from the spectra excited in the 458-600 nm region and are reported together with the FT-SERS spectra of the Ag-bpy surface complex and FT Raman spectra of [Ru(bpy)3] Cl2. Seven of the observed 11 fundamentals as well as their first overtones and combination bands are selectively enhanced in SERS of the Ag-bpy surface complex formed in the Ag colloid/HCl/bpy system. The profiles of these bands show a common maximum at approximately 540 nm. The selectively enhanced bands of the Ag-bpy surface complex have nearly the same wavenumbers as those enhanced in the SERRS and resonance Raman spectra of [Ru(bpy)3]2+ upon excitation close to the 453 nm maximum of its MLCT absorption band. Moreover, the intensity patterns of the bpy vibrations of the two species match both in resonance (541 nm excitation for Ag-bpy, 458 nm for [Ru(bpy)3]2+) and in off-resonance (458 and 1064 nm for Ag-bpy, 1064 nm for [Ru(bpy)3]2+). The distinct band shapes of the excitation profiles of the selectively enhanced vibrational modes of the Ag-bpy surface complex, as well as the observation of overtones and combination bands in the SERS spectra upon excitation into this "band", are interpreted in terms of a charge-transfer resonance contribution to the overall SERS enhancement. In view of the near-coincidence of the vibrational modes coupled to the resonant electronic transition of Ag-bpy with those coupled to the MLCT transition of [Ru(bpy)3]2+, the resonant electronic transition is tentatively assigned to a Ag metal to bpy (pi*) CT transition.     

Reproducible SERRS from structured gold surfaces

Metallic substrates with ordered spherical cavities have been shown to be very effective for surface-enhanced Raman scattering (SERS) and can be fabricated reproducibly using electrodeposition. The sensitivity of detection is increased by several orders of magnitude by using surface-enhanced resonance Raman scattering (SERRS). In this report we demonstrate SERRS for the first time on electrodeposited gold films templated with colloidal spheres and demonstrate the reproducibility of the response. We also obtain a direct comparison between SERRS and SERS by choosing two dyes, Cy5 and Cy3, which are similar in structure but differ in their excitation maxima, such that one is resonant and the other non-resonant with our laser excitation. As expected, the resonant enhancement is found to be of the order of 10(3) over and above that for SERS. The net SERRS enhancements are shown to be of the order of 10(9). We also find that the resonant enhancement profile of the different peaks for the chromophore follows the plasmonic resonance absorption spectrum obtained for the structured surface.     

黄曲霉素B1在银团簇表面吸附的表面增强拉曼光谱

采用密度泛函理论(DFT)的B3LYP方法和6-311g(d, p)(C, H, O)/LanL2DZ(Ag)基组, 优化得到黄曲霉素分子AFB₁与Ag小团簇形成的复合物AFB₁-Ag_n (n=2, 4, 6)的稳定结构, 并计算了三种复合物的表面增强拉曼光谱(SERS)和预共振拉曼光谱(SERRS), 与实验结果相一致. 计算结果显示: 三种复合物表面增强拉曼光谱中C＝O伸缩振动模的增强因子约为10²-10³, 是由于极化率改变引起的静化学增强. 根据含时密度泛函理论(TDDFT)方法计算得到的吸收光谱, 分别选择407.5、446.2和411.2 nm作为入射光, 计算三种复合物的共振拉曼光谱, 发现在SERRS光谱中, Ag―O伸缩振动的增强因子达到10⁴量级, 主要是由电荷转移产生的共振增强引起的.     

Cetylpyridinium Chloride Activated Trinitrotoluene Explosive Lights Up Robust and Ultrahigh Surface‐Enhanced Resonance Raman Scattering in a Silver Sol

Surface‐enhanced resonance Raman scattering (SERRS) is not realized for most molecules of interest. Here, we developed a new SERRS platform for the fast and sensitive detection of 2,4,6‐trinitrotoluene (TNT), a molecule with low Raman cross section. A cationic surfactant, cetylpyridinium chloride (CPC) was modified on the surface of silver sols (CP‐capped Ag). CPC not only acts as the surface‐seeking species to trap sulfite‐sulfonated TNT, but also undergoes complexation with it, resulting in the presence of two charge‐transfer bands at 467 and 530 nm, respectively. This chromophore absorbs the visible light that matches with the incident laser and plasmon resonance of Ag sols by the use of a 532.06 nm laser, and offered large resonance Raman enhancement. This SERRS platform evidenced a fast and accurate detection of TNT with a detection limit of 5×10^?11 M under a low laser power (200 μW) and a short integration time (3 s). The CP‐capped Ag also provides remarkable sensitivity and reliable repeatability. This study provides a facile and reliable method for TNT detection and a viable idea for the SERS detection of various non‐resonant molecules.     

Surface enhanced fluorescence and Raman imaging of Langmuir-Blodgett azopolymer films

The spectroscopic properties and surface-enhanced spectra of Langmuir-Blodgett (LB) films of methacrylic homopolymer (HPDR13) are presented. It is shown that LB film displays strong fluorescence attributed to the spatial restrictions imposed by its structure. The emission is observed in conjunction with photoisomerization, a process clearly demonstrated by the formation of surface-relief gratings in the LB film [C.R. Mendon?a et al., Macromolecules 32 (1999) 1493]. Surface-enhanced Raman scattering (SERS), Surface-enhanced resonance Raman scattering (SERRS) and surface-enhanced fluorescence (SEF) were observed for LB films of HPDR13 deposited onto silver island films. SERS measurements were also carried out on a sample fabricated with one monolayer LB film deposited onto silver islands followed by one overlayer of silver (LB sandwiched between two layers of silver islands). The polymer interacts very weakly with the metal surface (physisorption), and the enhancement effect is determined by the local electric field enhancement. The strong SERS and SERRS signals were suitable for micro-Raman imaging. Line, area mapping and global images of the LB monolayer on silver island are reported. The transfer ratio in the fabrication of the LB suggests a homogeneous coating of the silver islands, thereby the chemical images show the variation of the SERS intensity due to surface enhancement.     

Surface-enhanced raman spectra of dyes and organic acids in silver solutions: chloride ion effect

We have recorded surface-enhanced Raman (SER) spectra of two different classes of compounds, cationic dyes and organic acids, and studied their chloride ion effects on the surface-enhanced Raman scattering (SERS) activities of the silver solution. For the positive charge dyes, rhodamine 6G (R6G) and 1,1'-dimethyl-2,2'-cyanine iodide (DECI), no SERS could be observed without the addition of chloride ions because of lack of the electrostatic interaction between the dye species and the silver particles in the silver solution. The chloride ions served to enlarge silver particles and to contribute the existence of the surface active sites, making the silver solution SERS active to the dye samples. Surface-enhanced resonance Raman scattering (SERRS) intensity of the dye molecules increased with the chloride ion concentration. After reaching a maximum intensity, a Cl- quenching effect on the intensity took place. For the organic acids, benzoic acid and p-aminobenzoic acid (PABA), SERS could be observed without the coexistence of chloride ions. The intensity of the Raman scattering did not vary significantly in the presence of small amount of chloride ion. At high Cl- concentration, quenching SERS intensity began to take effect.     

Silver-particle-based surface-enhanced resonance Raman scattering spectroscopy for biomolecular sensing and recognition

We demonstrate in this work that 2-μm-sized Ag (μAg) powders can be used as a core material for constructing biomolecular sensing/recognition units operating via surface-enhanced resonance Raman scattering (SERRS). This is possible because μAg powders are very efficient substrates for both the diffuse reflectance IR and the surface-enhanced Raman scattering–SERRS spectroscopic characterization of molecular adsorbates prepared in a similar manner on silver surfaces. Besides, the agglomeration of μAg particles in a buffer solution can be prevented by the layer-by-layer deposition of cationic and anionic polyelectrolytes such as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). In this particular study, we used rhodamine B isothiocyanate (RhBITC) as a SERRS marker molecule, and μAg powders adsorbed consecutively with RhBITC and PAH–PAA bilayers were finally derivatized with biotinylated poly(l-lysine). On the basis of the nature of the SERRS peaks of RhBITC, those μAg powders were confirmed to selectively recognize streptavidin molecules down to concentrations of 10⁻¹⁰ g mL⁻¹. Since a number of different molecules can be used as SERS–SERRS marker molecules, the present method proves to be an invaluable tool for multiplex biomolecular sensing/recognition via SERS and SERRS.     

Silica-coated gold nanostars for combined surface-enhanced Raman scattering (SERS) detection and singlet-oxygen generation: a potential nanoplatform for theranostics

This paper reports the synthesis and characterization of surface-enhanced Raman scattering (SERS) label-tagged gold nanostars, coated with a silica shell containing methylene blue photosensitizing drug for singlet-oxygen generation. To our knowledge, this is the first report of nanocomposites possessing a combined capability for SERS detection and singlet-oxygen generation for photodynamic therapy. The gold nanostars were tuned for maximal absorption in the near-infrared (NIR) spectral region and tagged with a NIR dye for surface-enhanced resonance Raman scattering (SERRS). Silica coating was used to encapsulate the photosensitizer methylene blue in a shell around the nanoparticles. Upon 785 nm excitation, SERS from the Raman dye is observed, while excitation at 633 nm shows fluorescence from methylene blue. Methylene-blue-encapsulated nanoparticles show a significant increase in singlet-oxygen generation as compared to nanoparticles synthesized without methylene blue. This increased singlet-oxygen generation shows a cytotoxic effect on BT549 breast cancer cells upon laser irradiation. The combination of SERS detection (diagnostic) and singlet-oxygen generation (therapeutic) into a single platform provides a potential theranostic agent.     

Single molecule analysis by surfaced-enhanced Raman scattering

Our main objective in this tutorial review is to provide insight into some of the questions surrounding single molecule detection (SMD) using surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS). Discovered thirty years ago, SERS is now a powerful analytical tool, strongly tied to plasmonics, a field that encompasses and profits from the optical enhancement found in nanostructures that support localized plasmon excitations. The spectrum of the single molecule carries the quantum fingerprints of the system modulated by the molecule-nanostructure interactions and the electronic resonances that may result under laser excitation. This information is embedded in vibrational band parameters. The dynamics and the molecular environment will affect the bandwidth of the observed Raman bands. In addition, the localized surface plasmon resonances (LSPR) empower the nanostructure with a number of optical properties that will also leave their mark on the observed inelastic scattering process. Therefore, controlling size, shape and the formation of the aggregation state (or fractality) of certain metallic nanostructures becomes a main task for experimental SERS/SERRS. This molecule-nanostructure coupling may, inevitably, lead to spectral fluctuations, increase photobleaching or photochemistry. An attempt is made here to guide the interpretation of this wealth of information when approaching the single molecule regime.     

Doping and metallic‐support effect evidenced on SERS spectra of polyaniline thin films

Surface‐enhanced Raman scattering (SERS) is a process with origins, electromagnetic and chemical. The electromagnetic enhancement consists of the excitation of surface plasmons in the metallic support of the thin film. With only the electromagnetic enhancement mechanism, the surface spectra should not differ from volume Raman spectra. However, between SERS and volume Raman spectra, there are differences resulting from the chemical reactions taking place at the polymer/metal interface, intermediated by solvent molecules, that finally depend on the types of polymers and metallic supports. Polyaniline (PAN) is an excellent material to emphasize the chemical component of SERS. This is due to its particular structure with a repeating unit that contains two entities at different weights—a reduced state and an oxidized state–that, in turn, react differently with a metallic substrate. SERS spectra depend on the oxidizing properties of the metal surface, which involves an intermediate compound of the types Ag₂O and Au₂O₃ when N‐methyl‐2‐pyrrolidinone is used as the solvent. This article presents new results concerning the surface chemical effects that produce variations of the PAN SERS spectra. The SERS spectra of the PAN emeraldine base (PAN‐EB) layered on Au support are characterized by a semiquinoid structure that we believe is induced on the intermediate compound Au₂O₃. In the presence of H₂SO₄, the SERS spectra change gradually as the degree of acid protonation doping increases. The SERS spectra of the fully protonated PAN‐EB are identical to those obtained on PAN emeraldine salt (PAN‐ES) synthesized by cyclic voltammetry in an acid medium and are invariable with the type of metallic support. The SERS spectra show that the emeraldine salt can be partially or totally deprotonated with water or NH₄OH. The deprotonation is complete for the Ag support and partial for the Au support. The SERS spectra of the fully protonated PAN‐EB are characterized by a double band with maxima at about 1330 and 1370 cm⁻¹. Although the generation process of positive charge on the macromolecular chain of PAN‐EB doped in the presence of (C₄H₉)₄NBF₄ is similar to that due to protonic acid doping, involving cation addition (C₄H or H⁺ ions, respectively) in SERS spectra, the complex band situated at about 1330–1370 cm⁻¹ no longer appears. The doping of PAN‐EB with FeCl₃ produces two polymer forms: a salt type characterized by a protonated structure similar to that found for PAN‐ES and a base type similar to the leucoemeraldine form. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 2599–2609, 2000