Bombesin-modified 6-14 C-terminal fragments adsorption on silver surfaces: influence of a surface substrate

Surface-enhanced Raman scattering (SERS) spectroscopy has been applied to investigate the interaction with a silver colloidal surface of following seven 6-14 fragments of bombesin (BN) C-terminus: cyclo[D-Phe(6),His(7),Leu(14)]BN(6-14), [D-Phe(6),Leu-NHEt(13),des-Met(14)]BN(6-14), [D-Phe(6),Leu(13)-(R)-p-chloro-Phe(14)]BN(6-14), [D-Phe(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), [D-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), [D-Tyr(6),beta-Phe(11),Phe(13),Nle(14)OH]BN(6-14), and [D-Cys(6),Asn(7),D-Ala(11),Cys(14)]BN(6-14), potent r-GRP-R receptor antagonists used in chemotherapy and potential effective drugs in cancer treatment. The adsorption active sites and molecular orientations on the colloidal silver surface have been determined on the basis of SERS "surface selection rules" subsequent to a detailed SERS analysis. In addition, the similarities and differences of these spectra with the SERS spectra of the peptides immobilized on a roughened silver electrode surface have been examined. From the data, suggestion has been made about structural properties of these peptides on the colloidal surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 941-950, 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.     

Investigation of molecular structure of bombesin and its modified analogues nonadsorbed and adsorbed on electrochemically roughened silver surface

This work describes the molecular structure of bombesin (BN) and its analogs on the basis of the absorption infrared and Raman results described below. In these analogues is replaced one ([D-Phe12]BN, [Tyr4]BN, and [Lys3]BN) or two ([Tyr4,D-Phe12]BN, [D-Phe12,Leu14]BN, and [Leu13-(R)-Leu14]BN) amino acid residues within the peptide chain with a synthetic amino acid, creating antagonists to bombesin, which are useful in the treatment of cancer. It is also used surface enhanced Raman scattering (SERS) to study the differences and changes in the vibrational spectra of BN and its analogs, which were attached to an electrochemically roughened silver surface as these peptides interacted with target proteins. This work explores the use of SERS for molecules anchored to a macroscopic silver surface to interrogate the interaction of these peptides with protein receptors. The results presented here show that all peptides coordinate to the macroscopic silver surface through an indole ring and the methylene group of Trp8, the C==O fragment, and an amide bond; however, the orientation of these fragments on the electrochemically roughened silver surface and the strength of the interactions with this surface is slightly different for each peptide. For example, the interaction of --CH2-- of [D-Phe12]BN, [Tyr4,D-Phe12]BN, [D-Phe12,Leu14]BN, [Leu13-(R)-Leu14]BN, and [Lys3]BN with the silver surface perturbed the vertical orientation of the Trp8 indole ring on this surface. Hence, the indole ring adopted a close to perpendicular orientation on the silver surface for BN and [Tyr4]BN, only.     

Surface-enhanced Raman difference between bombesin and its modified analogues on the colloidal and electrochemically roughen silver surfaces

In this article, surface-enhanced Raman scattering (SERS) spectra of bombesin (BN) and its six modified analogues ([D-Phe(12)]BN, [Tyr(4)]BN, [Tyr(4),D-Phe(12)]BN, [D-Phe(12),Leu(14)]BN, [Leu(13)-(R)-Leu(14)]BN, and [Lys(3)]BN) on a colloidal silver surface are reported and compared with SERS spectra of these species immobilized onto an ellectrochemically roughen silver electrode. Changes in enhancement and wavenumber of proper bands upon adsorption on different silver surfaces are consistent with BN and its analogues adsorption primarily through Trp(8). Slightly different adsorption states of these molecules are observed depending upon natural amino acids substitution. For example, the indole ring in all the peptides interacts with silver nanoparticles in a edge-on orientation. It is additionally coordinated to the silver through the N(1)--H bond for all the peptides, except [Phe(12)]BN. This is in contrary to the results obtained for the silver roughen electrode that show direct but not strong N(1)--H/Ag interaction for all peptides except [D-Phe(12),Leu(14)]BN and [Leu(13)-(R)-Leu(14)]BN. For BN only C==O is not involved in the chemical coordination with the colloidal surface. [Lys(3)]BN and BN also adsorb with the C--N bond of NH(2) group normal and horizontal, respectively, to the colloidal surface, whereas C--NH(2) in other peptides is tilted to this surface. Also, the Trp(8) --CH(2)-- moiety of only [Tyr(4)]BN, [Lys(3)]BN, and [Tyr(4),D-Phe(12)]BN coordinates to Ag, whereas the Phe(12) ring of [Phe(12)]BN, [Tyr(4),D-Phe(12)]BN, and [D-Phe(12),Leu(14)]BN assists in the peptides binding only on the colloidal silver.     

Hairpin ribozyme catalysis: A surface‐enhanced Raman spectroscopy study

The existence of an “RNA world” as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self‐cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface‐enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. On RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus, they propose an interesting alternative to electrophoretic methods. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 384–390, 2009. 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     

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically‐modified [Tyr¹]FQ (1‐6), [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β‐sheet conformation. FQ (1‐11), FQ (7‐17) [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) have an α‐helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ～8.3) was investigated by means of surface‐enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7‐17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1‐11), FQ (1‐6), [Tyr¹]FQ (1‐6), and [His¹]FQ (1‐6) lies almost flat on this surface, while for FQ (1‐13) and FQ (1‐13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1‐6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1‐6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1‐13) and FQ (7‐17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the ? NH₂ group. We also showed that upon adsorption, the secondary structure of these peptides is altered. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1039–1054, 2010. 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     

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.     

Structural analysis of biological aliphatic compounds using surface-enhanced Fourier transform Raman spectroscopy

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids.     

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.     

Theoretical and pH dependent surface enhanced Raman spectroscopy study on caffeine

The surface enhanced Raman spectroscopy (SERS) spectrum of caffeine is recorded on a silver colloid at different pH values. It is discussed on the basis of the SERS "surface selection rules" in order to characterize its vibrational behavior on such a biological artificial model. To improve the previous assignments in the Raman spectrum and for a reliable, detailed analysis of SERS spectra, density functional theory calculations (structural parameters, harmonic vibrational wavenumbers, total electron density, and natural population analysis of the molecule) are performed for the anhydrous form of caffeine and the results are discussed. The predicted geometry and vibrational Raman spectra are in good agreement with the experimental data. The flat orientation of the mainly chemisorbed caffeine attached through the pi electrons and the lone pair of nonmethylated N atoms of the imidazole ring are proposed to occur at neutral and basic pH values. At acid pH values caffeine is probably adsorbed on the Ag surface through one or both oxygen atoms, more probably through the O atom of the conjugated carbonyl group with an end-on orientation. However, the changes in the overall SERS spectral pattern seem to indicate the electromagnetic mechanism as being the dominant one.     

Raman and surface-enhanced Raman spectroscopy investigation of vasopressin analogues containing 1-aminocyclohexane-1-carboxylic acid residue

In this work, Raman spectroscopy (RS) was employed to characterize molecular structures of [Arg8]vasopressin (AVP) and its [Acc2,D-Arg8]AVP, [Acc3]AVP, and [Cpa1, Acc3]AVP analogues. The RS band assignments have been proposed. To determine the mechanism of adsorption of the above-mentioned compounds adsorbed on a colloidal silver surface, surface-enhanced Raman spectra (SERS) were measured. The SERS spectra were used to determine relative proximity of the adsorbed functional groups of [corrected] investigated peptides and their orientation on the silver surface. The AVP and [Acc3]AVP SERS spectra (Acc: 1-aminocyclohexane-1-carboxylic acid) show that the L-tyrosine (Tyr) lies far from the metal surface, whereas the [Cpa1,Acc3]AVP spectrum (Cpa: 1-mercaptocyclohexaneacetic acid) provides evidence that Tyr interacts with the silver surface. These results suggest that [corrected] the binding of the Tyr-ionized phenolic group might be responsible for the selectivity of the analogues. We show that the aromatic ring of L-phenylalanine (Phe) of AVP and [Acc2,D-Arg8]AVP interacts with the silver surface. The strength of this interaction is considerably weaker for [Acc2,D-Arg8]AVP than for AVP. This might be due either to a longer distance between the Phe ring and the silver surface, or to the almost perpendicular orientation of the Phe ring towards the surface. The carbonyl group of the L-glutamine [corrected] (Gln) or L-asparagine [corrected](Asn) of AVP, [Acc2,D-Arg8]AVP, and [Acc3]AVP is strongly bound to the silver surface. We have also found that all peptides adsorb on the silver surface via sulfur atoms of the disulfide bridge, adopting a "GGG" conformation, except [Cpa1,Acc3]AVP, which accepts a "TGG" geometry.     

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.     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

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.     

A de novo designed 11 kDa polypeptide: Model for amyloidogenic intrinsically disordered proteins

A de novo polypeptide GH₆[(GA)₃GY(GA)₃GE]₈GAH₆ (YE8) has a significant number of identical weakly interacting β‐strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that β‐sheet folding of the 11‐kDa amyloidogenic polypeptide is completely aggregation driven. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 607–618, 2010. 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     

Characterization and redox properties of cytochrome c552 from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner

The structure of cytochrome c552 (Cyt-c552) from Thermus thermophilus shows many differences to other c-type cytochromes. The rich lysine domain close to the heme does not exist in this cytochrome, allowing us to postulate that the interaction with its redox partner must be different to the cytochrome c/cytochrome c oxidase interaction. We report a study of Cyt-c552 adsorbed on self-assembled monolayers (SAMs) of functionalized alkanethiols used to mimic the chemical properties of its redox partner (ba3-oxydase). Hydrophilic (-COOH), polar (-OH), hydrophobic (-CH3), and mixed (-OH/-CH3) SAMs grafted on roughened silver electrodes were characterized by X-ray photoelectron spectroscopy. Surface enhanced resonance Raman spectroscopy (SERRS) was employed to determine the structure and the redox properties (E degrees and number of transferred electron) of the heme of Cyt-c552 adsorbed on roughened silver electrodes coated by the different SAMs. The surface that most closely models the environment of the ba3-oxidase is a mixed SAM formed by 50% polar [Ag-(CH2)5-CH2OH] and 50% hydrophobic [Ag-(CH2)5-CH3] alkanethiols. Only the native form B1(6cLS) of Cyt-c552 is detected by SERRS when the protein is adsorbed on such a surface that promotes a protein orientation favorable for the electron transfer (number of transferred electron = 1). We shall discuss the differences and similarities of the electron-transfer mechanism of Cyt-c552 compared to cyt-c.     

Investigations of thermotropic phase behavior of newly developed synthetic PEGylated lipids using Raman spectro-microscopy

In this article, a temperature-controlled Raman spectro-microscopic technique has been utilized to detect and analyze the phase behaviors of two newly developed synthetic PEGylated lipids trademarked as QuSomestrade mark, which spontaneously form liposomes upon hydration in contrast to conventional lipids. The amphiphiles considered in this study differ in their hydrophobic hydrocarbon chain length and contain different units of polyethylene glycol (PEG) hydrophilic headgroups. Raman spectra of these new artificial lipids have been recorded in the spectral range of 500-3100 cm(-1) by using a Raman microscope system in conjunction with a temperature-controlled sample holder. The gel to liquid phase transitions of the sample lipids composed of pure 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) have been revealed by plotting peak intensity ratios in the C--H stretching region as a function of temperature. From this study, we have found that the main phase transitions occur at a temperature of approximately 5.2 and 21.2 degrees C for pure GDM-12 and GDS-23, respectively. Furthermore, the lipid GDS-23 also shows a postphase transition temperature at 33.6 degrees C. To verify our results, differential scanning calorimetry (DSC) experiments have been conducted and the results are found to be in an excellent agreement with Raman scattering data. This important information may find application in various studies including the development of lipid-based novel substances and drug delivery systems. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1012-1020, 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.     

表面增强拉曼光谱技术在微生物鉴定中的应用进展

拉曼光谱自20世纪20年代被发现以来,经过近90年的发展,产生了许多分支。其中表面增强拉曼光谱是利用被测物质与粗糙金属表面的相互作用来提高拉曼光谱的信噪比,从而得到敏感度和精确度更高的图谱,可以将样品在不经过预处理的情况下对其进行快速检测。本文综述了表面增强拉曼光谱技术的原理、分类及鉴定特点,总结了该技术在食品、化学、医药、工业、病原等微生物学科的临床应用,进一步阐述了研究该技术的必要性和应用前景,旨在为从事该领域的科研人员提供参考依据。     

Real‐time detection of single‐living pancreatic β‐cell by laser tweezers Raman spectroscopy: High glucose stimulation

Glucose acts as a β‐cell stimulus factor and leads to cellular responses that involve a large amount of biomolecule formation, relocation, and transformation. We hypothesize that information about these changes can be obtained in real‐time by laser tweezers Raman spectroscopy. To test this hypothesis, repeated measurements designs in accordance with the application of Raman spectroscopy detection were used in the current experiment. Single rat β‐cells were measured by Raman spectroscopy in 2.8 mmol/l glucose culture medium as a basal condition. After stimulation with high glucose (20 mmol/l), the same cells were measured continuously. Each cell was monitored over a total time span of 25 min, in 5 min intervals. During this period of time, cells were maintained at an appropriate temperature controlled by an automatic heater, to provide near‐physiological conditions. It was found that some significant spectral changes induced by glucose were taking place during the stimulation time course. The most noticeable changes were the increase of spectral intensity at the 1002, 1085, 1445, and 1655 cm^?1 peaks, mainly corresponding to protein and lipid. We speculate that these changes might have to do with β‐cell protein and lipid synthesis. Using laser tweezers Raman spectroscopy in combination with glucose stimulation, optical spectral information from rat β‐cells was received and analyzed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 587–594, 2010. 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     

Spectroscopy on the wing: Naturally inspired SERS substrates for biochemical analysis

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface‐enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface‐enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme‐linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large‐scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS‐based bioassays. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy

The structure of a bacterial cell wall may alter during bacterial reproduction. Moreover, these cell wall variations, on a nanoscale resolution, have not yet fully been elucidated. In this work, Raman spectroscopy and atomic force microscopy (AFM) technique are applied to evaluate the culture time‐dependent cell wall structure variations of Pseudomonas putida KT2440 at a quorum and single cell level. The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs till 6 h of cultivation time under our experimental conditions. AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time. This work demonstrates the feasibility of utilizing a combined Raman spectroscopy and AFM technique to investigate the cultivation time dependence of bacterial cellular surface biopolymers at single cell level. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 171–177, 2010. 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