Surface-enhanced resonance Raman scattering of cytochrome P450-2D6 on coated silver hydrosols

Surface-enhanced resonance Raman scattering (SERRS) from dilute solutions (down to nanomolar concentrations) of human mono-oxygenase CYP2D6 is observed using aqueous dispersions of Ag nanoparticles (hydrosol) coated with self-assembled monolayers (SAMs) of mercaptoalkanoic acids of two different lengths. From a direct comparison with its resonance Raman spectrum in solution, CYP2D6 appears to fully retain its native structure upon adsorption on coated hydrosol through electrostatic interaction, while a structural change in the active site is observed when uncoated citrate-reduced hydrosol is used. Using SERRS on these biocompatible coated hydrosols, the effects of dextromethorphan on the enzyme's active site can be observed, demonstrating that CYP2D6 ability of binding substrates is preserved. Moreover, by tuning the wavelength of the exciting laser away from the main absorption band of the heme, the vibrational bands of the SAM coating are observed and analyzed to see how the presence of the protein affects the SAM structure.     

Surface enhanced resonance Raman scattering imaging of Langmuir-Blodgett monolayers of bis (benzimidazo) thioperylene.

The synthesis, spectroscopic characterization and surface-enhanced spectroscopy of a new electro active organic material bis (benzimidazo) thioperylene (Monothio BZP) are reported. Langmuir monolayers of Monothio BZP were successfully formed on water subphase and characterized by the pi-A surface-pressure area isotherm. Langmuir-Blodgett (LB) monomolecular layers of Monothio BZP were fabricated onto glass substrates and onto silver island films for surface-enhanced spectroscopic studies. The results of surface-enhanced resonance Raman scattering (SERRS), SERRS imaging and surface-enhanced fluorescence (SEF) studies for Monothio BZP LB monolayers are reported. Raman imaging (global imaging and point-by-point mapping) of the SERRS signal for a single monomolecular layer on silver islands were obtained using the 514.5 nm laser line. The SERRS imaging permits a visualization of the variation of the SERRS intensity across of the rough metal surface. The SEF was recorded for the excimer emission of aggregates in the LB film. The distance dependence and the enhancement factor of SEF were determined using fatty acid spacing layers. A temperature dependence study of the LB monolayer SERRS and SEF spectra was carried out between -190 degrees and + 200 degrees C confirming the thermal stability of the LB monolayer on silver. The specificity and the sensitivity of SERRS signal on metal island films was probed using mixed LB films with 0.01% molecular ratio of Monothio BZP in Arachidic Acid (AA). The micro-Raman SERRS spectra from ca. 10(-3) attomole of the dye were recorded.     

Single molecular detection of a perylene dye dispersed in a Langmuir-Blodgett fatty acid monolayer using surface-enhanced resonance Raman scattering

The Langmuir-Blodgett (LB) monolayer technique was used to fabricate single molecule LB monolayer containing bis(phenethylimido)perylene (PhPTCD), a red dye dispersed in arachidic acid (AA) with an average doping of 1 molecule per microm2. The monolayer was transferred onto Ag island films to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra. The mixed LB monolayers were fabricated with a concentration, on average, of 1, 6, 19 and 118 PhPTCD molecules per microm2 in AA. The AA provides a two-dimensional host matrix whose background signal does not interfere with the detection of the probe molecule's SERRS signal. The properties of the single molecule detection were investigated using micro-Raman with a 514.5-nm laser line. The Ag island surfaces coated with the LB monolayer were mapped with spatial steps of 3 microm and global chemical imaging of the most intense SERRS band in the spectrum was also recorded. The SERRS and surface-enhanced fluorescence (SEF) of the neat and single molecule LB monolayer were recorded in a temperature range from liquid nitrogen to + 200 degrees C. Neat PhPTCD LB monolayer spectra served as reference for the identification of characteristic signatures of the single molecule behavior. The spatial resolution of Raman-microscopy experiments, the multiplicative effect of resonance Raman and SERRS, and the high sensitivity of the new dispersive Raman instruments, allow SERRS to be part of the family of single molecular spectroscopies.     

Voltammetric and surface-enhanced resonance Raman spectroscopic characterization of cytochrome C adsorbed on a 4-mercaptopyridine monolayer on silver electrodes

To combine voltammetric techniques with surface-enhanced resonance Raman scattering (SERRS), cytochrome c (cyt c) was immobilized on a roughened silver electrode chemically modified with a self-assembled monolayer (SAM) of 4-mercaptopyridine (PySH). All measurements were performed on the same electrode in a homemade spectroelectrochemical cell suitable for such applications. Cyt c on a PySH-SAM shows a quasi-reversible, monoelectronic, adsorption-controlled CV response with a formal reduction potential of -0.061 V (vs SCE), which is comparable to the values found for native cyt c adsorbed on different SAMs. SERRS spectra proved that cyt c adsorbed on a PySH monolayer is present in the native conformer (the B1 state). Voltammetric and SERRS experiments at high ionic strength revealed that the interaction between the SAM and the protein is electrostatic in nature. In conclusion, PySH was found to be suitable for adsorption of cyt c at SERRS-active silver surfaces. In comparison with other SAMs, PySH requires less time (10 min vs 12-18 h) to form a long-time durable and reproducible coating on the roughened electrode surface.     

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 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.     

Surface-enhanced Raman spectroscopy of 2,5-dibromopyridine and 2,6-dibromopyridine

The surface-enhanced Raman spectroscopy of 2,6-dibromopyridine and 2,5-dibromopyridine in silver hydrosol has been studied. A satisfactory correlation has been observed between the normal Raman spectra in aqueous solution and their surface-enhanced Raman (SER) spectra in silver hydrosol. The prominent features in the SER spectra of the compounds are the strongly enhanced peaks at 1175 and 1369 cm⁻¹ due to (py)CBr and (CC,CN)(py) stretching vibrational modes respectively in 2,6-dibromopyridine and almost no enhancement of intensities in those vibrations in 2,5-dibromopyridine.     

Identification and characterization of active and inactive species for surface-enhanced resonance Raman scattering

The surface-enhanced resonance Raman scattering (SERRS) activity of a statistically significant number of silver nanoparticles has been studied using a correlated SERRS mapping and transmission electron microscopy (TEM) method. TEM allowed the nature of each entity to be directly identified, and the SERRS activity was obtained from the corresponding SERRS map. Particles in various states of aggregation were analyzed to establish relative activities. It was established that SERRS activity is dependent on the specific batch of colloid tested. By averaging different colloid batches, it was shown that increasing SERRS activity is observed with increasing numbers of particles in the aggregates. By reducing the surface coverage of the particles to the extent that single moieties could be examined optically, the ratio of the relative activities of single particles, dimers, trimers, and larger aggregates was estimated. High-resolution TEM images of a number of active and inactive particles are reported. However, no clear correlation between microstructure and SERRS activity was observed.     

Surface-enhanced resonance Raman scattering and density functional calculations of hemicyanine adsorbed on colloidal silver surface

Resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) of 4'-(N,N'-dimethylaminostyryl)-4-propylpyridinium bromide (hemicyanine, HC dye) in acetonitrile solution and on a colloidal silver surface have been investigated. The structure of the dye in the ground (S0) and excited (S1) electronic states was optimized using density functional calculations along with the B3LYP and the configuration interaction with the singlet excitation (CIS) methods, respectively, using the 6-31G basis set. The vibrational frequencies of the molecule were computed at the optimized geometry and compared with the observed Raman bands. A complete normal-mode analysis has been carried out because it is essential for the accurate assignment of the vibrational spectra. From the observed enhancement along various in-plane and out-of-plane vibrations in the SERRS spectrum and from theoretical calculations, it has been inferred that the interaction with the silver surface occurs via the nitrogen lone pair of the pyridyl or the dimethylamino group of the molecule with a tilted orientation. The observed red-shifts in the SERRS spectrum along various vibrations indicate strong interaction (chemisorption) of the HC dye with the silver surface. This is also supported by the presence of a Ag-N stretching vibration at 241 cm(-1). The effect of the dye concentration on the orientation of the molecule is also discussed.     

Resonance Raman optical activity and surface enhanced resonance Raman optical activity analysis of cytochrome c

High-resolution resonance Raman (RR) and resonance Raman optical activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein backbone. Combining the intrinsic resonance properties of cytochrome c with the surface plasmon enhancement achieved with colloidal silver particles, the surface enhanced resonance Raman scattering (SERRS) and surface enhanced resonance ROA (SERROA) spectra of the protein were successfully obtained at concentrations as low as 1 microM. The assignments of spectral features were based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported, while some disparities were observed between the resonance ROA and SERROA spectra. These differences can be ascribed to perturbations of the physical properties of the protein upon adhesion to the surface of the silver colloids.     

Direct evidence of electron flow via the heme c group for the direct electron transfer reaction of fructose dehydrogenase using a silver nanoparticle-modified electrode

The direct electron transfer reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. on alkanethiol-modified silver nanoparticles (AgNPs) was examined using cyclic voltammetry and surface-enhanced resonance Raman scattering (SERRS). Using cyclic voltammetry, catalytic oxidation currents (based on the direct electron transfer reaction of FDH) were observed from a potential of approximately −100 mV (vs. Ag/AgCl, 3 M NaCl) in the presence of d-fructose, without a mediator. A comparison of the SERRS spectra and the resonance Raman spectra of FDH in solution indicated that the heme c site retained its six-coordinated low-spin heme after immobilization. Moreover, SERRS also demonstrated that the heme c of the adsorbed FDH was the electron transfer site within the enzyme.     

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.     

Direct observation of the gating step in protein electron transfer: electric-field-controlled protein dynamics

Heterogeneous electron transfer of proteins at biomimetic interfaces is characterized by unusual distance dependences of the electron-transfer rates, whose origin has been elusive and controversial. Using a two-color, time-resolved, surface-enhanced resonance Raman spectroelectrochemical approach, we have been able to monitor simultaneously and in real time the structure, electron-transfer kinetics, and configurational fluctuations of cytochrome c electrostatically adsorbed to electrodes coated with self-assembled monolayers. Our results show that the overall electron-transfer kinetics is determined by protein dynamics rather than by tunnelling probabilities and that the protein dynamics in turn is controlled by the interfacial electric field. Implications for interprotein electron transfer at biological membranes are discussed.     

Immunoassay utilizing biochemistry reaction product via surface-enhanced Raman scattering in near field

In recent years, surface-enhanced Raman scattering (SERS) has been a topic of keen interest with regard to the detection of trace biological materials[1―8]. A wide range of SERS studies aimed at investigating structure, topology, and composition of biome…     

A close look at silver oxide colloid for surface-enhanced Raman scattering

A silver oxide hydrosol has been prepared by reacting silver nitrate with sodium hydroxide in an aqueous solution. The colloidal particle composition and particle size have been characterized by X-ray diffractometry and transmittance electron microscopy. We have demonstrated that the sol is surface-enhanced Raman scattering active. One major advantage of this sol is the absence of the spectral interference arising from reaction products of the sol formation process. The mechanism of surface-enhanced Raman scattering activity is essentially contributed by the silver particles generated from the photochemical process during the laser excitation.     

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.     

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.     

pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

SERRS and visible extinction spectroscopy of copper chlorophyllin on silver colloids as a function of pH

A study of sodium copper chlorophyllin adsorbed on silver colloids (CuChl/Ag) is conducted using surface-enhanced resonance Raman scattering (SERRS) and visible extinction spectroscopy to examine how the system changes as a function of pH. Initially at basic pH, SERRS signal is not detected even though CuChl is adsorbed onto the silver surface and deprotonated. Upon decreasing the solution pH slightly, colloidal aggregation is induced, evidenced by the broadening of the visible extinction spectra. The larger aggregates possess a surface plasmon that is in resonance with the laser excitation frequency (633-nm) and SERRS signal is detected. As the acid protonates CuChl, the overall negatively-charged surface approaches neutrality which induces more aggregation. Complete protonation of CuChl by pH 4.6 results in colloidal precipitation. However, when aggregation is halted about pH 5, adding NaOH(aq) to the system maintains the extent of aggregation and an intense SERRS signal is detected at basic pH.     

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.