SERS‐based detection of barcoded gold nanoparticle assemblies from within animal tissue

We have explored the potential of deep Raman spectroscopy, specifically surface‐enhanced spatially offset Raman spectroscopy (SESORS), for non‐invasive detection from within animal tissue, by employing SERS‐barcoded nanoparticle (NP) assemblies as the diagnostic agent. This concept has been experimentally verified in a clinically‐relevant backscattered Raman system with an excitation line of 785 nm under ex vivo conditions. We have shown that our SORS system, with a fixed offset of 2–3 mm, offered sensitive probing of injected 2‐quinolinethiol‐barcoded NP assemblies through animal tissue containing both protein and lipid. In comparison with that of non‐aggregated SERS‐barcoded gold NPs, we have demonstrated that the tailored SERS‐barcoded aggregated NP assemblies have significantly higher detection sensitivity. We report that these NP assemblies can be readily detected at depths of 7–8 mm from within animal proteinaceous tissue with high signal‐to‐noise ratio. In addition, they could also be detected from beneath 1–2 mm of animal tissue with high lipid content, which generally poses a challenge because of high absorption of lipids in the near‐infrared region. We have also shown that the signal intensity and signal‐to‐noise ratio at a particular depth is a function of the SERS tag concentration used and that our SORS system has a 2‐quinolinethiol detection limit of 10⁻⁶ M. Higher detection depths may possibly be obtained with optimization of the NP assemblies, along with improvements in the instrumentation. Such NP assemblies offer prospects for in vivo, non‐invasive detection of tumours along with scope for incorporation of drugs and their targeted and controlled release at tumour sites. These diagnostic agents combined with drug delivery systems could serve as a ‘theranostic agent’, an integration of diagnostics and therapeutics into a single platform. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectral characterization and intracellular detection of Surface‐Enhanced Raman Scattering (SERS)‐encoded plasmonic gold nanostars

Plasmonic gold nanostars offer a new platform for surface‐enhanced Raman scattering (SERS). However, due to the presence of organic surfactant on the nanoparticles, SERS characterization and application of nanostar ensembles in solution have been challenging. Here, we applied our newly developed surfactant‐free nanostars for SERS characterization and application. The SERS enhancement factors (EF) of silver spheres, gold spheres and nanostars of similar sizes and concentration were compared. Under 785 nm excitation, nanostars and silver spheres have similar EF, and both are much stronger than gold spheres. Having plasmon matching the incident energy and multiple ‘hot spots’ on the branches bring forth strong SERS response without the need to aggregate. Intracellular detection of silica‐coated SERS‐encoded nanostars was also demonstrated in breast cancer cells. The non‐aggregated field enhancement makes the gold nanostar ensemble a promising agent for SERS bioapplications. Copyright © 2012 John Wiley & Sons, Ltd.     

Fabrication of periodical Ag–Au compound nanostructure films with controllable Ag nanoparticle aggregate patterns: a study on surface‐enhanced Raman scattering

The Ag–Au compound nanostructure films with controllable patterns of Ag nanoparticle (NP) aggregates were fabricated. A strategy of two‐step synthesis was employed toward the target products. Firstly, the precursor Au NP (17 nm) films were synthesized as templates. Secondly, the Ag NPs (45 nm) were deposited on the precursor films. Three types of Ag NP aggregates were obtained including discrete Ag NPs (discrete type), necklace‐like Ag NP aggregates (necklace type), and huddle‐like Ag NP aggregates (huddle type). The surface‐enhanced Raman scattering (SERS) property was studied on these nanostructures by using the probing molecule of rhodamine 6G under the excitation laser of 514.5 nm. Interestingly, the different types of samples showed different enhancement abilities. A statistical method was employed to assess the enhancement. The relative enhancement factor for each Ag NP was estimated quantitatively under the ratio of 1 : 25 : 18 for the discrete‐type, necklace‐type, and huddle‐type samples at the given concentration of 10⁻⁸ mol/l. This research shows that the enhancement ability of each Ag NP is dependent on the aggregate morphology. Moreover, the different enhancement abilities displayed different limit detection concentrations up to 10⁻⁸, 10⁻¹¹, and 10⁻⁹ mol/l, separately. The understanding of the relationship between the defined nanostructures and the SERS enhancement is very meaningful for the design of new SERS substrates with better performance. Copyright © 2015 John Wiley & Sons, Ltd.     

Peptide‐Targeted Gold Nanoparticles for Photodynamic Therapy of Brain Cancer

Targeted drug delivery using epidermal growth factor peptide‐targeted gold nanoparticles (EGF_pep‐Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGF_pep‐Au NP‐Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGF_pep‐Au NP‐Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs. Targeted drug delivery and treatment success can be imaged via the intrinsic fluorescence of the PDT drug Pc 4. Using Pc 4 fluorescence, it is demonstrated in vivo that EGF_pep‐Au NP‐Pc 4 impacts biodistribution of the NPs by decreasing the initial uptake by the reticuloendothelial system (RES) and by increasing the amount of Au NPs circulating in the blood 4 h after IV injection. Interestingly, in vivo PDT with EGF_pep‐Au NP‐Pc 4 results in interrupted tumor growth when compared with EGF_pep‐Au NP control mice when selectively activated with light. These data demonstrate that EGF_pep‐Au NP‐Pc 4 utilizes cancer‐specific biomarkers to improve drug delivery and therapeutic efficacy over untargeted drug delivery.     

SERS study of Ag nanoparticles electrodeposited on patterned TiO2 nanotube films

In this work, Ag nanoparticles (NPs) were deposited on patterned TiO₂ nanotube films through pulse‐current (PC) electrodeposition, and as a result patterned Ag NPs films were achieved. Scanning electron microscopy (SEM), electron probe microanalysis (EPMA), and X‐ray diffraction (XRD) were used, respectively, to study the morphology, uniformity, and phase structure of the patterned Ag NP films. The size and density of the as‐deposited Ag NPs could be controlled by changing the deposition charge density, and it was found that the patterned Ag NP films produced under a charge density of 2.0 C cm⁻² gave intense UV–vis and Raman peaks. Two‐dimensional surface‐enhanced Raman scattering (SERS) mapping of rhodamine 6G (R6G) on the patterned Ag NP films demonstrated a high‐throughput, localized molecular adsorption and micropatterned SERS effect. Copyright © 2010 John Wiley & Sons, Ltd.     

SERS detection of red organic dyes in Ag‐agar gel

Micro‐Raman spectroscopy has been widely employed in the last few years for the study of artworks, allowing for the characterization of a high class of pictorial materials. However, the detection of organic dyes by conventional Raman spectroscopy is quite difficult, due to the high fluorescence provided by these compounds. Recently, remarkable improvements have been achieved by the introduction of the surface enhanced Raman spectroscopy (SERS) technique for the analysis of organic dyes. In the present work, a new method is presented, based on the use of a SERS probe made of agar‐agar coupled with silver nanoparticles, for a non‐destructive and minimally invasive micro‐extraction of dyes from textiles. Ag‐agar gel has been tested first on textile mock‐ups dyed with alizarin, purpurin and carminic acid. SERS measurements have been performed adopting laser light excitations at 514.5 and 785 nm of a micro‐Raman setup. Highly structured SERS band intensities have been obtained. After having verified the safety of the method by colorimetric, X‐ray fluorescence and attenuated total reflectance Fourier transform infrared techniques, a real case, a pre‐Columbian piece of textile, have been investigated by Ag‐agar gel. This cutting‐edge method offers new possibilities for a sensitive and non‐destructive analysis of fluorescent materials. Copyright © 2012 John Wiley & Sons, Ltd.     

Experimental investigations on the weakening effect of magnetic fields on surface‐enhanced Raman scattering

Currently, because of the wide use of magnetic/metal hybrid materials in the field of surface‐enhanced Raman scattering (SERS), magnetic fields (MFs) as a kind of surrounding environments of SERS substrates have been not ignored. And enormous attentions for the effect of MFs on SERS are very necessary. Our analysis from previous reports shows SERS signals are closely associated with MFs; yet the study of the exact effect of MFs on SERS is still in need of detail and broadening. In this work, we demonstrate that the use of Ni/Au microparticles (MPs) and Au hollow spheres (HSs) to study the effect of MFs on SERS signals. The experiments are preformed from three aspects, including the comparison of SERS signals of Ni/Au MPs with different Ms values, the comparison of SERS signals of Au HSs and Ni/Au MPs under different external MFs, and the comparison of SERS signals of single Au HS particle and single Ni/Au particle in the absence and presence of an external MF. Thereinto, under different external MFs from 0, 0.01, 0.02, 0.04 to 0.08 T, SERS signals of Au HSs and Ni/Au MPs are gradually weaker. According to the experiments, the weakening effect of MFs on SERS is confirmed. It is suggested that the weakening effect is originated from the blue‐shift of surface plasmon resonance and the locking of charge‐transfer from Au to probe molecules. Copyright © 2012 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering (SERS) spectra of chloramphenicol in Ag colloids prepared by microwave heating method

Ag colloids were prepared by the microwave heating method. The UV‐visible spectrum and the transmission electron microscopy (TEM) were employed to characterize the Ag colloids. The surface‐enhanced Raman scattering (SERS) spectra of chloramphenicol (CAP) in Ag colloids were successfully recorded. The Raman spectra of CAP were recorded with good concordance comparing to the theoretical results calculated by the Gaussian'98 program. CAP molecules on the Ag surface are oriented tilted to the particle surface via C20‐O23, N‐O13, and N‐O23, which played an important role in the SERS effect. Copyright © 2009 John Wiley & Sons, Ltd.     

New SERS feature of β‐carotene: consequences for quantitative SERS analysis

While recording SERS spectra of pure β‐carotene at sub‐micromole concentrations for reference purpose, we discovered an unusual spectral response never reported before. In pre‐resonance conditions with the 532‐nm line, SERS of β‐carotene with AgNPs exhibits among the strong υ(CC) mode at 1512 cm⁻¹ unshifted from normal Raman spectrum, additional strong bands at 1649, 1575 and 1387 cm⁻¹ as well as other medium bands not observed in the Raman spectrum of the crystalline powder. Such behavior is explained in terms of selection rules relaxation upon cyclohexene terminal rings of the β‐carotene interaction with the NP surface. AFM images of the SERS system suggested dimers and trimers clustering of the nanoparticles with adsorbed β‐carotene. In light of the new SERS feature the consequences in correct interpretation of the SERS imaging from complex biosystems containing carotenoids are discussed. Relative intensity ratio of the β‐carotene band at 1512 cm⁻¹ and water against concentration allowed a reliable SERS calibration curve for 50 to 500 nmol l⁻¹ concentration range and provided quantitative SERS assessment of the carotenoid content in the sea urchin (Paracentrotus lividus) gonads extracts. Copyright © 2015 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman spectroscopy of DNA from leaves of in vitro grown apple plants

Ultrasensitive Raman measurements of nucleic acids are possible by exploiting the effect of surface‐enhanced Raman scattering (SERS). In this work, the vibrational spectra of eight genomic DNAs from in vitro grown apple leaf tissues (Malus domestica Borkh., Fam Rosaceae, cvs. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) were analyzed using surface‐enhanced Raman spectroscopy, in the wavenumber range 200–1800 cm⁻¹. SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. Strong dependences of the SERS spectra on genomic DNA amount in the measured sample volume and on time were observed. Similarities of the SERS signals of DNAs from Rebra and Romus 3 leaves were detected. To our knowledge, this is the first SERS study on genomic DNA from leaf tissues. The present work provides a basis for future use of surface‐enhanced Raman spectroscopy to analyze specific plant DNA–ligand interactions or DNA structural changes induced by plants' stress conditions associated with their natural environment. Besides, this study will generate information that is valuable in the development of low‐level plant DNA‐based analytical sensors. Copyright © 2010 John Wiley & Sons, Ltd.     

SERS activity and stability of the most frequently used silver colloids

Single‐molecule detection by surface‐enhanced resonance Raman scattering (SERRS) spectroscopy has been demonstrated for a variety of molecules. The detection of single molecules that do not have a resonance contribution, SERS, has been shown in the case of adenine. However, when colloidal particles isolated on planar substrates are used as the enhancing medium, the presence of anomalous signals significantly complicates the analysis of the spectra. Selection of a silver colloid that minimizes these spurious signals should improve the ultra‐sensitive detection of non‐resonant single molecules by SERS. A range of silver colloids, prepared by different methods, were investigated with respect to their activity and stability. Minimal anomalous signals were obtained from hydroxylamine‐reduced silver colloids, which suggests that this colloid will be better for ultra‐sensitive SE(R)RS experiments compared to the more common citrate‐ and borohydride‐reduced silver colloids. Copyright © 2011 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering study of human serum on PVA?Ag nanofilm prepared by using electrostatic self‐assembly

Polyvinyl alcohol (PVA)‐protected silver nanoarchitecture (PVA Ag nanofilm) on the surface of the glass substrate was prepared by using electrostatic self‐assembly at a proper voltage. The two‐dimensional morphology of the PVA Ag nanofilm has been examined by scanning electron microscopy (SEM). The surface‐enhanced Raman scattering (SERS) spectra of human serum (HS) on PVA Ag nanofilms were recorded. The results show that the Raman scattering of HS can be enhanced efficiently based on these PVA Ag nanofilms. However, it also can be seen that the effect of sodium citrate (SC) acting as anticoagulant on the SERS spectrum of HS is unnegligible, which has not been discussed adequately in the previous reports. To discuss the effect of SC on the SERS spectrum of HS, we have studied the normal Raman spectra of solid SC and the SERS spectra of 1.0 × 10⁻³ mol/l aqueous solution of SC adsorbed on the PVA–Ag nanofilms. Meanwhile, Raman wavenumbers of the SC molecule were calculated by using the method of DFT‐B3LYP/6‐31G*, and the dominant assignations of the calculated wavenumbers were performed. It was found that the density functional theory (DFT) calculation of SC Raman spectrum matches well with the experimental results. With the perfect reproducibility and high SERS activity, this method will be useful in the development of HS detection methods. Copyright © 2010 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering sensing on black silicon

Reactive ion etching was used to fabricate black‐Si over the entire surface area of 4‐inch Si wafers. After 20 min of the plasma treatment, surface reflection well below 2% was achieved over the 300–1000 nm spectral range. The spikes of the black‐Si substrates were coated by gold, resulting in an island film for surface‐enhanced Raman scattering (SERS) sensing. A detection limit of 1 × 10^?6 M (at count rate > 10² s^?1 . mW^?1) was achieved for rhodamine 6G in aqueous solution when drop cast onto a ～ 100‐nm‐thick Au coating. The sensitivity increases for thicker coatings. A mixed mobile‐on‐immobile platform for SERS sensing is introduced by using dog‐bone Au nanoparticles on the Au/black‐Si substrate. The SERS intensity shows a non‐linear dependence on the solid angle (numerical aperture of excitation/collection optics) for a thick gold coating that exhibits a 10 times higher enhancement. This shows promise for augmented sensitivity in SERS applications.     

High–Yield Production of Uniform Gold Nanoparticles with Sizes from 31 to 577 nm via One‐Pot Seeded Growth and Size‐Dependent SERS Property

In this work, uniform, quasi‐spherical gold nanoparticles (Au NPs) with sizes of 31–577 nm are prepared via one‐pot seeded growth with the aid of tris‐base (TB). Distinct from the seeded growth methods available in literature, the present method can be simply implemented by subsequently adding the aqueous dispersion of the 17 nm Au‐NP seeds and the aqueous solution of HAuCl₄ into the boiling aqueous TB solution. It is found that at the optimal pH range, the sizes of the final Au NPs and their concentrations are simply controlled by either the particle number of the Au seed dispersion or the concentration of the HAuCl₄ solution, while the latter enables us to produce large Au NPs at very high concentration. Moreover, as‐prepared Au NPs of various sizes are coated on glass substrates to test their surface‐enhanced Raman scattering (SERS) activities by using 4‐aminothiophenol (4‐ATP) molecules as probes, which exhibit “volcano type” dependence on the Au NP sizes at fixed excitation wavelength. Furthermore, the Au NPs with sizes of ≈97 and 408 nm exhibit the largest SERS enhancement at the excitation wavelength of 633 and 785 nm, respectively.     

Comparison of SERRS and RRS excitation profiles of [Fe(tpy)2]2+ (tpy = 2,2':6',2'‐terpyridine) supported by DFT calculations: effect of the electrostatic bonding to chloride‐modified Ag nanoparticles on its vibrational and electronic structure

Nonresonance (or normal) Raman scattering (NRS), resonance Raman scattering (RRS), surface‐enhanced Raman scattering (SERS), and surface‐enhanced RRS (SERRS) spectra of [Fe(tpy)₂]²⁺ complex dication (tpy = 2,2':6',2''‐terpyridine) are reported. The comparison of RRS/NRS and SERRS/SERS excitation profiles of [Fe(tpy)₂]²⁺ spectral bands in the range of 445–780 nm is supported by density functional theory (DFT) calculations, Raman depolarization measurements, comparison of the solid [Fe(tpy)₂](SO₄)₂ and solution RRS spectra, and characterization of the Ag nanoparticle (NP) hydrosol/[Fe(tpy)₂]²⁺ SERS/SERRS active system by surface plasmon extinction spectrum and transmission electron microscopy image of the fractal aggregates (D = 1.82). By DFT calculations, both the Raman active modes and the electronic states of the complex have been assigned to the symmetry species of the D_2d point group. It has been demonstrated that upon the electrostatic bonding of the complex dication to the chloride‐modified Ag NPs, the geometric and ground state electronic structure of the complex and the identity of the three different metal‐to‐ligand charge transfer (¹MLCT) electronic transitions remain preserved. On the other hand, the effect of ion pairing manifests itself by a slight change in localization of one of the electronic transitions (with max. at 552 nm) as well as by promotion of the Herzberg–Teller activation of E modes resulting from coupling of E and B₂ excited electronic states. Finally, the very low, 1 × 10⁻¹¹ M SERRS spectral detection limit of [Fe(tpy)₂]²⁺ at 532‐nm excitation is attributed to a concerted action of the electromagnetic and molecular resonance mechanism, in conjunction to the electrostatic bonding of the complex dication to the chloride‐modified Ag NP surface. Copyright © 2014 John Wiley & Sons, Ltd.     

Nanoporous Ag–GaN thin films prepared by metal‐assisted electroless etching and deposition as three‐dimensional substrates for surface‐enhanced Raman scattering

Three‐dimensional (3D) nanoporous gallium nitride (PGaN) scaffolds are fabricated by Pt‐assisted electroless hydrofluoric acid (HF) etching of crystalline GaN followed by in situ electroless deposition of Ag nanostructures onto the interior surfaces of the nanopores, yielding a large surface area substrate for surface‐enhanced Raman scattering (SERS). The resulting 3D SERS‐active substrates have been optimized by varying reaction parameters and starting material concentration, exhibiting enhanced Raman signals 10–100× more intense than either (1) sputtered Ag‐coated porous GaN or (2) Ag‐coated planar GaN. The increase in SERS signal is attributed to a combination of the large surface area and the inherent transparency of PGaN in the visible spectral region. Overall, Ag‐decorated PGaN is a promising platform for high sensitivity SERS detection and chemical analysis, particularly for reaction and metabolic products that can be trapped inside the highly anisotropic nanoscale pores of PGaN. The potential of this sampling mode is illustrated by the ability to acquire Raman spectra of adenine down to 5 fmol. Additionally, correlated SERS and laser desorption/ionization mass spectrometry spectra can be acquired from same sample spot without further preparation, opening new possibilities for the investigation of surface‐bound molecules with substantially enhanced information content. Copyright © 2012 John Wiley & Sons, Ltd.     

Near‐field optical enhancement by lead‐sulfide quantum dots and metallic nanoparticles for SERS

There is a growing interest in using quantum dots (QDs) and metallic nanoparticles (NPs), both for luminescence enhancement and surface‐enhanced Raman scattering (SERS). Here, we study the electromagnetic‐field enhancement that can be generated by lead‐sulfide (PbS) QDs using three‐dimensional finite‐element simulations. We investigate the field enhancement associated with combinations of PbS QDs with metallic NPs and substrates. The results show that high field enhancement can be achieved by combining PbS QDs with metallic NPs of larger sizes. The ideal size for Ag NPs is 25 nm, providing a SERS enhancement factor of ~5*10⁸ for light polarization parallel to the NP dimer axis and a gap of 0.6 nm. For Au NPs, the bigger the size, the higher is the field for the studied diameters, up to 50 nm. The near‐field values for PbS QDs above metallic substrates were found to be lower compared to the case of PbS QD‐metal NP dimers. This study provides the understanding for the design and application of QDs for the enhancement of near‐field phenomena. Copyright © 2013 John Wiley & Sons, Ltd.     

On‐site detection of phosgene agents by surface‐enhanced Raman spectroscopy coupled with a chemical transformation approach

Phosgene and its analogs are greatly harmful to the public health, environmental safety and homeland security as widely used industrial substances with extremely high toxicity. In order to rapidly evaluate the emergency risk caused by these chemicals, a new highly sensitive method based on surface‐enhanced Raman spectroscopy (SERS) technique for measurement of phosgene agents was developed for the first time. Coupled with a chemical transformation approach, the highly toxic phosgene was conveniently converted to a SERS‐sensitive probe, i.e. iodine (I₂), with low toxicity or non‐toxicity. The characteristic SERS peak in 459 cm⁻¹ was used for quantitation and was presumed as a formation of triiodide anion (I₃⁻), which was induced in an iodide (I⁻)‐aggregation Au NPs system. The total measurement can be completed in ~20 min with the limits of detection of ~60 µg/l (phosgene) and ~30 µg/l (diphosgene), respectively, on a portable Raman spectrometer. This work is the first report of SERS measurement on phosgene and diphosgene in a quantitative level. This method is expected to meet the requirements of on‐site detection of phosgene agents, promote emergency responses and raise more opportunities for the portable SERS applications. Copyright © 2015 John Wiley & Sons, Ltd.     

SHINERS and plasmonic properties of Au Core SiO2 shell nanoparticles with optimal core size and shell thickness

Shell‐isolated nanoparticles (NPs)‐enhanced Raman spectroscopy (SHINERS) can be potentially applied to virtually any substrate type and morphology. How to take a step forward to prepare SHINERS NPs (SHINs) with superior performance is critical for the practical applications of surface‐enhanced Raman scattering (SERS) in the breadth and depth. Here, we present a method to obtain 120 nm diameter gold NPs coated with ultrathin silica shells (1–4 nm). The silica shell can be controlled growth through carefully tuning a series of parameters, such as amount of 3‐aminopropyl triethoxysilane used, pH, reaction time, and reaction temperature. We compare the enhancement factor of the obtained 120 nm Au with a 4 nm silica shell NPs to the 55 nm Au with a 4 nm silica shell NPs, and the activity of a 120 nm SHINs is nearly 24 times that the 55 nm SHIN from a single particle view. We also compare the enhancement factor of 1 nm silica shell Au@SiO₂ NPs with the bare Au NPs. The enhancement factor of 1 nm silica shell Au@SiO₂ NPs was found to be about twice that of the bare particles. For a deeper understanding of the source of the giant enhanced electrical field of the 1 nm silica shell Au@SiO₂ NPs, we study the plasmonic property of single 1 nm silica shell Au@SiO₂ NP on a gold film substrate through correlation of the structure of single NP using SEM with its SPR spectroscopy. We find that the multipolar interaction between the single Au@SiO₂ NP and gold film substrate is important for the SERS. Our studies on the performance of 120 nm SHINs and the plasmonic property of these particles can significantly expand the applications of SHINERS technique and improve the understanding of physical nature of SHINs. Copyright © 2013 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman evidence for Rhodamine 6 G and its derivative with different adsorption geometry to colloidal silver nanoparticle

Some high‐affinity functional groups or resonant molecules were often used as probe molecules adsorbed on silver nanoparticles for Surface‐enhanced Raman scattering (SERS). However, it is still unclear how the attached molecules interact with the silver nanoparticles' surface, and how the anchoring groups affect the optical and electronic properties of molecules. Here, we report that surface‐enhanced Raman studies of two organic compounds; rhodamine 6G (R6G) and its aminated derivative (R‐NH₂) have very different functional groups for surface binding but nearly identical SERS spectroscopic properties at pH = 7 and UV–vis at pH = 3, respectively. A surprise was found that under the same experimental conditions, the SERS signal intensity for R6G is nearly 50‐fold higher than that of R‐NH₂. Furthermore, the pH‐dependent study reveals that the structure of R6G is irreversibly stabilized or ‘locked’ in its form and no longer responsive to pH changes. In contrast, R‐NH₂ is still sensitive to pH, and can be switched between its open‐ring and closed‐ring structures. Copyright © 2013 John Wiley & Sons, Ltd.