Raman microprobe spectroscopy: A new tool for biofouling diagnostics

Raman vibrational spectra were obtained from crystalline amino acids and acid molecules adsorbed at the surfaces of silver electrodes in aqueous solutions. Results revealed that aromatic acids such as phenylalanine adsorbed via their aromatic ring while bases such as alanine were coordinated by their amine functional groups. Sulphur containing acids (cysteine and cystine) were found to bond through their sulphur groups. In all cases, adsorption increased towards the point of zero charge of silver, as would be expected for uncharged species. Similar experiments carried out on α‐amylase solutions showed that the enzyme molecule changes its coordination to the silver surface as a function of electrode potential, indicating that C‐S, C‐N, and aromatic ring functional groups are all present on the outer surface of the enzyme structure.     

Raman scattering from nucleic acids adsorbed at a silver electrode

Adsorption of nucleic acids at a silver electrode polarized to -0.6 to -0.1 V (vs. Ag/AgCl) was investigated by means of surface enhanced Raman scattering (SERS) spectroscopy. Single-stranded polyriboadenylic acid and thermally denaturated DNA adsorbed at the silver electrode yield two intense bands at 734 and 1335 cm-1 on the SERS spectra. These bands, assigned to the vibrations of adenine residue rings, were much less intense if the SERS spectra were recorded for double-helical complex polyadenylic X polyuridylic acid and native DNA. Moreover, the courses of alkaline denaturation of DNA and its digestion by deoxyribonuclease I were observed by SERS spectroscopy. The results were interpreted as support for the view that intact double-helical segments of nucleic acids are not denatured or destabilized due to their adsorption at the positively charged and roughened surface.     

Surface interactions of a homologous series of alpha,omega-amino acids on colloidal silver and gold

Surface enhanced Raman spectroscopy (SERS) was used to characterize a homologous series of alpha,omega-amino acids on colloidal gold and silver. Raman and SER spectra of the alpha,omega-amino acids, NH2(CH2)nCOOH (n = 3-7), are presented and analyzed, revealing the probable conformations of the molecules on the metal surfaces. The alpha,omega-amino acids interact with silver and gold through both the amine and carboxylate end groups, and modify the conformation of the molecular backbone in order to maximize these interactions. An odd-even effect is observed for backbone conformations of molecules adsorbed to the silver substrate. The anomolous SER spectrum of 5-aminopentanoic acid on gold suggests the possibility of condensation polymerization at the gold surface.     

聚赖氨酸溴化氢结合物的表面增强拉曼光谱

银溶胶对聚赖氨酸溴化氢结合物(PLys—HBr)表现出极大表面增强拉曼(SER)效应。同PLyS—HBr的普通拉曼光谱相比,表面增强因子提高达6个数量级。实验表明,NH_3~+基是银表面增强效应的强活性基团。但是在相同条件下,聚谷氨酸钠(PGA—Na)在银溶胶中不能获得SER光谱,这可能是由于空间障碍或者COO~-基的活性不如NH_3~+基。     

Surface-enhanced resonance Raman spectroscopy of phycocyanin and allophycocyanin

High quality surface-enhanced resonance Raman (SERR) spectra were recorded from native and denatured phycocyanin and allophycocyanin on ascorbic acid treated silver hydrosols. The visible-excited SERR and resonance Raman (RR) spectra of the phycobiliproteins were very similar, indicating a predominantly electromagnetic surface enhancement mechanism. Investigation of pH-induced denaturation ofx allophycocyanin has shown that even small differences in protein/chromophore conformational are sensitively reflected by the SERR spectra. Concerning the adsorption of the protein to the metal surface, the experiments have shown that: (i) there is limited possibility for changing protein conformation during the adsorption process, (ii) there are no changes after the protein has been adsorbed onto the silver surface and (iii) for each protein an optimal activation of the silver sol has to be found for recording proper SERR spectra. The results obtained on phycobiliproteins are also discussed in connection with the interpretation of phytochrome Raman spectra.     

The chromophore-binding site of bacteriorhodopsin. Resonance Raman and surface-enhanced resonance Raman spectroscopy and quantum chemical study

Surface-enhanced Raman spectra of membrane protein, located in native mem brane, bacteriorhodopsin, adsorbed by silver electrodes and hydrosols have been obtained for the first time. The distance between the retinal Schiff’s base and the external side of purple membrane of Halobacteriim halobiim was shown to be 6–9 A. The possible distribition of the point charges aroind protonated retinal Schiff’s base has been proposed on the basis of the resonance Raman data and quantim chemical CNDO/S-CI calculations. Such a model contains tyrosine residue located near the retinal Schiff’s base and connected with COO^- groipvia hydrogen bond COO^- group acts as a protonated Schiff’s base counterion. The distance between oxygen atoms of COO^- group and retinal Schiff’s base plane is 2.5–3.0A. The hydrogen bond (O-H. . .O^-) length between oxygen atom of OH-group and oxygen atom of COO^- group has been chosen 2.7±0.1Å Tyrosine hydroxyl group is located at 2.8–3.5 A from retinal Schiff’s base plane. It was shown that in contrast to generally accepted Honig and Nakanishi model the spectral properties of Brh570, K610, L550 and M4Ï2 forms of bacteriorhodopsin photocycle as well as observed tyrosine deprotonation and COO^- group protonation during M412 formation can be explained reasonably well by the suggested charge distribution. Furthermore, such a model of bacteriorhodopsin active site microenvironment allows to explain catalyzing of photo-induced protonated retinal Schiff’s base deprotonation observed in our preliminary experiments.     

在银溶胶中甘氨酸和甘氨酸二肽的表面增强拉曼散射

甘氨酸及Gly-Gly和Gly-L-Pro二肽的表面增强拉曼散射谱与溶液内的普通拉曼谱的对比研究表明,银胶对COO、NH_2、C-N和C-C基团的振动有明显的增强.对CH_2基团无增强.Gly-Gly的SERS与pH有关,接近中性pH值时增强效果好,在酸性和碱性溶液内效果差.在 Gly-L-Pro的SERS谱中有强的酰胺Ⅰ带,而 Gly-Gly则几乎观察不到.     

Electrooptical behavior of heme-like molecules adsorbed on nerve membrane

This paper discusses the electrooptical properties of iron tetrasulfonated phthalocyanine (Fe-TsPc) adsorbed on a nerve membrane. The study is based on a comparative analysis of the properties of an aqueous solution of Fe-TsPc and the properties of Fe-TsPc adsorbed on a silver electrode. The resonant Raman spectra were obtained from the adsorbed Fe-TsPc on the interfaces of a silver electrode and the nerve membrane. Considerable optical signal changes have been observed when the interfacial potential is altered. The Raman spectra are sufficiently sensitive to indicate the submolecular mechanisms of electron delocalization. The changes in Raman signals as a function of the interfacial potential provide a means for optically monitoring electrical excitability in nerve.     

Electrooptical behavior of heme-like molecules adsorbed on nerve membrane

This paper discusses the electrooptical properties of iron tetrasulfonated phthalocyanine (Fe-TsPc) adsorbed on a nerve membrane. The study is based on a comparative analysis of the properties of an aqueous solution of Fe-TsPc and the properties of Fe-TsPc adsorbed on a silver electrode. The resonant Raman spectra were obtained from the adsorbed Fe-TsPc on the interfaces of a silver electrode and the nerve membrane. Considerable optical signal changes have been observed when the interfacial potential is altered. The Raman spectra are sufficiently sensitive to indicate the submolecular mechanisms of electron delocalization. The changes in Raman signals as a function of the interfacial potential provide a means for optically monitoring electrical excitability in nerve.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Cytochrome c at charged interfaces. 1. Conformational and redox equilibria at the electrode/electrolyte interface probed by surface-enhanced resonance Raman spectroscopy

The structure and the electron-transfer properties of cytochrome c (cyt c) absorbed on a silver electrode were analyzed by surface-enhanced resonance Raman spectroscopy. It was found that the absorbed cyt c exists in various conformational states depending on the electrode potential. In state I the native structure of the heme protein is fully preserved and the redox potential (+0.02 V vs saturated calomel electrode) is close to the value for cyt c in solution. In state II the heme iron exists in a mixture of five-coordinated high-spin and six-coordinated low-spin configurations. It had been shown that these configurations form a thermal equilibrium [Hildebrandt, P., & Stockburger, M. (1986) J. Phys. Chem. 90,6017]. It is demonstrated that these equilibria strongly depend on the charge distribution within the electrical double layer of the silver electrode/electrolyte interface, indicating that the changes in the coordination shell are induced by electrostatic interactions. The structural alterations in state II are apparently restricted to the heme crevice, which assumes an open conformation compared to the close structure in state I. This leads to a strong decrease of the redox potentials, which were determined to be -0.31 and -0.41 V for the five-coordinated high-spin and six-coordinated low-spin configurations, respectively. On the other hand, gross distortions of the protein structure can be excluded since the reversible proton-induced conformational change of cyt c as found in solution at low pH also takes place in state II of the absorbed cyt c. The linkage of cyt c molecules to the surface is mediated by charged amino acid groups, and it depends on the potential which groups are thermodynamically favored to form such a molecular binding site. The conformational states I and II, which are in potential-dependent equilibrium, therefore refer to two different molecular binding sites. At potentials below zero charge (less than approximately -0.6 V) a rapid denaturation of the absorbed cyt c is noted, which is reflected by drastic and irreversible changes in the surface-enhanced resonance Raman spectrum. Our results are discussed on the background of previous electrochemical studies of cyt c at electrodes.     

Active site structure and dynamics of cytochrome c3 from Desulfovibrio gigas immobilized on electrodes

Cytochrome c3 from Desulfovibrio gigas is electrostatically adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid. The redox equilibria and electron transfer dynamics of the adsorbed four-heme protein are studied by surface enhanced resonance Raman spectroscopy. Immobilization on the coated electrodes does not cause any structural changes in the redox sites. The potential-dependent stationary experiments distinguish the redox potential of heme IV (-0.19 V versus normal hydrogen electrode) from those of the other hemes for which an average value of -0.3 V is determined. Taking into account the interfacial potential drops, these values are in good agreement with the redox potentials of the protein in solution. The heterogenous electron transfer between the electrode and heme IV of the adsorbed cytochrome c3 is analyzed on the basis of time-resolved experiments, leading to a formal electron transfer rate constant of 15 s(-1), which is a factor of 3 smaller than that of the monoheme protein cytochrome c.     

Microperoxidase 8 adsorbed on a roughened silver electrode as a monomeric high-spin penta-coordinated species: characterization by SERR spectroscopy and electrochemistry

Microperoxidase 8 (MP8), a heme octapeptide obtained by hydrolytic digestion of cytochrome c, was adsorbed at the surface of a roughened silver electrode in order to provide a new supported biomimetic system for hemoproteins. A combination of two techniques was used to study its redox and coordination properties: electrochemistry and surface-enhanced resonance Raman (SERR) spectroscopy. This allowed us to show that MP8 could be adsorbed as a monolayer at the surface of the roughened silver electrode, where it could undergo a reversible electron transfer. Under those conditions, a redox potential of –0.4 V vs. SCE (–0.16 V vs. NHE) was measured for MP8, which was almost identical to that reported for N-acetyl-MP8 in aqueous solution. In addition, whereas MP8 appeared to aggregate in solution, and led to a mixture of high-spin penta-coordinated (5cHS) and low-spin hexa-coordinated (6cLS) iron(III) or iron(II) species, it was recovered almost exclusively as a monomeric high-spin penta-coordinated species at the surface of the electrode, both in the reduced and in the oxidized states. This then allowed a free coordination site on the iron, on the distal face of MP8 accessible to ligands. Accordingly, experiments performed in the presence of potassium cyanide demonstrated that MP8 adsorbed on a silver electrode could be ligated by a sixth CN^– ligand. Thus there is the possibility of binding several kinds of ligands such as O₂ or H₂O₂, which will open the way to biocatalysis of oxidation reactions at the surface of an electrode, or ligands such as drugs which will lead to the design of new biosensors for molecules of biological interest.     

Cerbinal,a Pseudoazulene Iridoid,as a Potent Antifungal Compound Isolated from Gardenia jasminoides Ellis

d-Gluconate dehydrogenase isolated from Pseudomonas fluorescens was immobilized on the surfaces of carbon and gold electrodes by irreversible adsorption. The electrodes with the adsorbed enzyme produced anodic currents in solutions containing d-gluconate. The currents were attributable to the electro-enzymic oxidation (direct bioelectrocatalytic oxidation) of d-gluconate; the electrochemical system required no external redox molecules serving as mediators of electron transfer between the electrode and the adsorbed enzyme. A model of the direct bioelectrocatalysis at the enzyme-modified electrodes is presented.     

Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS): a pilot study using evanescent-field optical sensing under voltage control to monitor polycationic polymer adsorption onto indium tin oxide (ITO)-coated waveguide chips

A new technique has been developed that combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. This new technique, termed "electrochemical optical waveguide lightmode spectroscopy" (EC-OWLS), proved efficient in monitoring molecular surface adsorption and layer thickness changes of an adsorbed polymer layer examined in situ as a function of potential applied to a waveguide in a pilot study. For optical sensing, a layer of indium tin oxide (ITO) served as both a high-refractive-index waveguide and a conductive electrode. In addition, an electrochemical flow-through fluid cell was provided, which incorporated working, reference, and counter electrodes, and was compatible with the constraints of optical sensing. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) served as a model, polycation adsorbate. Adsorption of PLL-g-PEG from aqueous buffer solution increased from 125 to 475 ng/cm(2 )along a sigmoidal path as a function of increasing potential between 0 and 1.5 V versus the Ag reference electrode. Upon buffer rinse, adsorption was partially reversible when a potential of >/=0.93 V was maintained on the ITO waveguide. However, reducing the applied potential back to 0 V before rinsing resulted in irreversible polymer adsorption. PLL-g-PEG modified with biotin demonstrated similar adsorption characteristics, but subsequent streptavidin binding was independent of biotin concentration. Applying positive potentials resulted in increased adsorbed mass, presumably due to polymer chain extension and reorganization in the molecular adlayer.     

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.     

Study of interaction between aspartic acid and silver by surface-enhanced Raman scattering on H(2)O and D(2)O sols.

Three different surface-enhanced Raman scattering (SERS) spectra are recorded for aspartic acid on H(2)O silver sols under different concentrations and pH values. The analysis of the results shows that it interacts with the metal surface in its dianionic form in two different ways, depending on the pH and concentration. Moreover, in some cases the fumarate anion is detected, which results from the chemical surface transformation of the aspartate. The N-deuterated aspartic acid adsorbed on the D(2)O silver sols gives rise to only one SERS spectrum as a consequence of the interaction of amino and carboxylate functional groups of the dianion with the metal, independent of the concentration and pD.     

Adsorption of 6-mercaptopurine and 6-mercaptopurine-ribosideon silver colloid: a pH-dependent surface-enhanced Raman spectroscopy and density functional theory study. II. 6-mercaptopurine-riboside

Surface-enhanced Raman spectroscopy (SERS) has been applied to characterize the interaction of 6-mercaptopurine-ribose (6MPR), an active drug used in chemotherapy of acute lymphoblastic leukemia, with a model biological substrate at therapeutic concentrations and as function of the pH value. Therefore, a detailed vibrational analysis of crystalline and solvated (6MPR) based on Density Functional Theory (DFT) calculations of the thion and thiol tautomers has been performed. 6MPR adopts the thion tautomeric form in the polycrystalline state. The SERS spectra of 6MPR and 6-mercaptopurine (6MP) recorded on silver colloid provided evidence that the ribose derivative shows different adsorption behavior compared with the free base. Under acidic conditions, the adsorption of 6MPR on the metal surface via the N7 and possibly S atoms was proposed to have a perpendicular orientation, while 6MP is probably adsorbed through the N9 and N3 atoms. Under basic conditions both molecules are adsorbed through the N1 and possibly S atoms, but 6MP has a more tilted orientation on the silver colloidal surface while 6MPR adopts a perpendicular orientation. The reorientation of the 6MPR molecule on the surface starts at pH 8 while in the case of 6MP the reorientation starts around pH 6. Under basic conditions, the presence of the anionic molecular species for both molecules is suggested. The deprotonation of 6MP is completed at pH 8 while the deprotonation of the riboside is finished at pH 10. For low drug concentrations under neutral conditions and for pH values 8 and 9, 6MPR interacts with the substrate through both N7 and N1 atoms, possibly forming two differently adsorbed species, while for 6MP only one species adsorbed via N1 was evidenced.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Raman spectroscopy of model membrane monolayers of dipalmitoylphosphatidic acid at the air-water interface using surface enhancement from buoyant thin silver films

A novel method for the acquisition of surface enhanced Raman (SER) spectra of model membranes of dipalmitoylphosphatidic acid (DPPA) in Langmuir layers at the air-water interface is reported. The approach is based on the electrochemical formation of a buoyant thin layer of coalesced silver colloids in the vicinity of the phosphatidic acid head groups at the interface. This Ag layer is an excellent platform for SER scattering, which shows the spectral features from all parts of the molecule and water between the Ag surface and the DPPA layer. The observation of the spectral response from the phosphatidic acid head groups is of particular significance, allowing insight into their chemical state and orientation at the air-water interface.