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.     

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.     

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.     

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.     

Raman and surface enhanced Raman spectroscopy of 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide labeled proteins: bovine serum albumin and cytochrome c

2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine serum albumin and cytochrome c at different pH values were prepared and investigated using Raman-resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins in the pH 6.7-11 range were compared to those of the corresponding free species. The SERS spectra were interpreted in terms of the structural changes of the tempyo labeled proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be inactive in the Raman-RR and SERS spectra of the proteins. The alpha-helix conformation was concluded to be more favorable as the SERS binding site of bovine serum albumin. In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.     

Study of ATP binding in the active site of Na,K-ATPase as probed by ultraviolet resonance Raman spectroscopy

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na⁺K⁺-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH₂, group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.     

Rational design of thiolated polyenes as trifunctional Raman reporter molecules in surface‐enhanced Raman scattering nanotags for cytokine detection in a lateral flow assay

The characteristic vibrational spectroscopic fingerprint of Raman reporter molecules adsorbed on noble metal nanoparticles is employed for the identification of target proteins by the corresponding surface‐enhanced Raman scattering (SERS) nanotag‐labeled antibodies. Here, we present the modular synthesis of thiolated polyenes with two to five C═C double bonds introduced via stepwise Wittig reactions. The experimental characterization of their electronic and vibrational properties is complemented by density functional theory calculations. Highly SERS‐active nanotags are generated by using the thiolated polyenes as Raman reporter molecules in Au/Au core/satellite supraparticles with multiple hot spots. The cytokines IL‐1β and IFN‐γ are detected in a duplex SERS‐based lateral flow assay on a nitrocellulose test strip by Raman microscopy. The thiolated polyenes are suitable for use in immuno‐SERS applications such as point‐of‐care testing as well as cellular and tissue imaging.     

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy

A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10^–10 M) solutions of doxorubicin or adriamycin (DOX), and 4O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.Abbreviations DNA deoxyribonucleic acid - DOX doxorubicin - SERS surface-enhanced Raman spectroscopy - THP-ADM 4O-tetrahydropyranyl adriamycin - PBS phosphate buffered saline Offprint requests to: M. Manfait     

Ultra‐low background Raman sensing using a negative‐curvature fibre and no distal optics

Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow‐core negative‐curvature fibres (NCFs) for Raman and surface‐enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785‐nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.      

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.     

Surface enhanced Raman spectroscopy distinguishes amyloid Β‐protein isoforms and conformational states

Amyloid β‐protein (Aβ) self‐association is one process linked to the development of Alzheimer's disease (AD). Aβ peptides, including its most abundant forms, Aβ40 and Aβ42, are associated with the two predominant neuropathologic findings in AD, vascular and parenchymal amyloidosis, respectively. Efforts to develop therapies for AD often have focused on understanding and controlling the assembly of these two peptides. An obligate step in these efforts is the monitoring of assembly state. We show here that surface‐enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) readily distinguishes Aβ40 and Aβ42. We show further, through comparison of assembly dependent changes in secondary structure and morphology, that the SERS/PCA approach unambiguously differentiates closely related assembly stages not readily differentiable by circular dichroism spectroscopy, electron microscopy, or other techniques. The high discriminating power of SERS/PCA is based on the rich structural information present in its spectra, which comprises not only on interatomic resonances between covalently associated atoms and hydrogen bond interactions important in controlling secondary structure, but effects of protein orientation relative to the substrate surface. Coupled with the label‐free, single molecule sensitivity of SERS, the approach should prove useful for determining structure activity relationships, suggesting target sites for drug development, and for testing the effects of such drugs on the assembly process. The approach also could be of value in other systems in which assembly dependent changes in protein structure correlate with the formation of toxic peptide assemblies.     

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.     

SERS观察在Pd OECC薄膜中β-胡萝卜素的基频和高阶拉曼模

利用有极高检测灵敏度的表面增强拉曼散射(SERS)技术,对吸附在银镜表面上的浓度较低的纯化的放氧核心复合物(Pd OECC)薄层进行了频移在250～3 100 cm-1范围内的拉曼光谱测量,除得到β-胡萝卜素分子的基频拉曼振动模外,在高频端还得到了许多弱峰.根据泛音和组合谱带选择定则分析,这些振动模式来自β-胡萝卜素分子的高阶拉曼光谱.还进行了Pd OECC在强光破坏前后的SERS光谱研究.在强光照射下,β-胡萝卜素分子的SERS光谱的散射强度明显降低,且线宽增加,说明强光照射不但改变了β-胡萝卜素的构象,而且也改变了β-胡萝卜素分子所处的微环境.其结果与强光照射前后吸收光谱的变化一致.另外,没有观察到Pd OECC薄层与银镜相互作用的其他新振动峰或Pd OECC中其他振动峰峰型的变化,可见Pd OECC在银镜表面保持原来的状态.这证明SERS技术在光合作用光破坏机理研究中的可行性.     

Feature‐based recognition of Surface‐enhanced Raman spectra for biological targets

We propose and compare multiple approaches to automatically process data measured through surface‐enhanced Raman scattering (SERS), in the context of intracellular molecule probing. It relies on locally detecting the most relevant spectra to retrieve all data independently through indexing, thus avoiding any pre‐filtering which occurs with standard processing methods. We first assess our approach on simulated data of the spectrum of Rhodamine 6G, and then validate high‐performing methods on experimental measurements of this compound. The optimized method is then utilized to extract and classify the complex SERS response behavior of gold nanoparticles taken in live cells. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Interaction of anticancer drug cisplatin with guanine: density functional theory and surface-enhanced Raman spectroscopy study

Surface-enhanced Raman spectroscopy (SERS) in a silver sol assisted by density functional theory (DFT) calculations is shown to be a promising tool in the characterization of platinum complexes and their interaction with nucleic acid bases. This is demonstrated using cisplatin and guanine as a model. The energies and geometric parameters of cisplatin, guanine, and their reaction products are calculated at Becke's nonlocal three parameter exchange and correlation functional and the Lee-Yang-Parr correlation functional level using the 6-31++G(d,p) basis set on the light elements and the effective core potential by Hay and Wadt on platinum. Available X-ray crystallography data are mostly in agreement with predictions within the experimental precision level, although Pt-N bond lengths tend to be systematically overestimated. The normal Raman spectrum of cisplatin is assigned. The SERS spectra of cisplatin and its reaction product with guanine are measured from 10(-6) M aqueous solution. The observed spectral changes in the SERS spectrum of guanine upon cisplatin binding are modeled by DFT calculations. The best agreement between theory and experiment is achieved when the adsorbed reaction product is assumed to be the 1:1 adduct cis-Pt(NH3)2ClG in which Pt is bound to N7 and guanine is deprotonated at N9.     

Highly sensitive SERS analysis of the cyclic Arg–Gly–Asp peptide ligands of cells using nanogap antennas

The cyclic RGD (cRGD) peptide ligands of cells have become widely used for treating several cancers. We report a highly sensitive analysis of c(RGDfC) using surface enhanced Raman spectroscopy (SERS) using single dimer nanogap antennas in aqueous environment. Good agreement between characteristic peaks of the SERS and the Raman spectra of bulk c(RGDfC) with its peptide's constituents were observed. The exhibited blinking of the SERS spectra and synchronization of intensity fluctuations, suggest that the SERS spectra acquired from single dimer nanogap antennas was dominated by the spectrum of single to a few molecules. SERS spectra of c(RGDfC) could be used to detect at the nanoscale, the cells' transmembrane proteins binding to its ligand.

SERS of cyclic RGD on nanogap antenna.     

Surface‐enhanced Raman scattering‐active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity

The tremendous enhancement factors that surface‐enhanced Raman scattering (SERS) possesses coupled with the flexibility of photonic crystal fibers (PCFs) pave the way to a new generation of ultrasensitive biosensors. Thanks to the unique structure of PCFs, which allows direct incorporation of an analyte into the axially aligned air channels, interaction between the analyte and excitation light could be increased many folds leading to flexible, reliable and sensitive probes that can be used in preclinical or clinical biosensing. SERS‐active PCF probes provide unique opportunity to develop an opto‐fluidic liquid biopsy needle sensor that enables one‐step integrated sample collection and testing for disease diagnosis. Specificity being a key parameter to biosensors, the PCF inside the biopsy needle could be functionalized with targeting moieties to detect specific biomarkers. In this review article, we present some of the most promising recent biosensors based on PCFs including hollow‐core PCFs, suspended‐core PCFs and side‐channel PCFs. We provide a wide range of applications of such platform using Raman spectroscopy, label free SERS or labeled SERS detection and analyze some of the main challenges to be addressed for translating it to a clinically viable next generation sensitive biopsy needle sensing probe.     

Bacteriochlorophyll a-protein interactions in a complex from Prosthecochloris aestuarii. A Resonance Raman study

Raman spectra of bacteriochlorophyll a (BChl) bound to the soluble protein complex from Prosthecochloris aestuarii have been obtained at low temperature, using the resonance effect on their Q_x for Soret electronic bands. These spectra show that the acetyl carbonyls of at least four of the seven molecules bound to the monomer subunit of the complex and the ketone carbonyls of at least five of them are oriented close to the mean plane of the conjugated part of the dihydrophorbin macrocycle. Up to three bacteriochlorophyll molecules may have their ketone carbonyls free from hydrogen-bonding and up to two may have their acetyl carbonyls similarly free. Several of the binding sites of the remaining conjugated carbonyls are probably the same as those binding the conjugated carbonyls of bacteriochlorophyll (and of bacteriopheophytin) in reaction centers and in antenna structures of purple bacteria and as those binding chlorophyll in the antenna of higher plants and algae. The present resonance Raman spectra confirm that the magnesium atoms of most of the seven bacteriochlorophylls are pentacoordinated. They also show that polarisation effects from their local environments induce changes in the groundstate structures of the dihydrophorbin skeletons of the complexed molecules with respect to those of isolated, monomeric bacteriochlorophyll. These changes are quasi-identical for the seven molecules. These environmental effects predominate over any structural change brought about by intermolecular bonding of the conjugated carbonyls or of the magnesium atoms. The dihydrophorbin rings of the seven molecules thus appear to be immersed in a nearly homogeneous medium of low permittivity, although specific van der Waals interactions may polarise the free carbonyls to quite different extents. The possible implications of these observations on the interpretation of the electronic spectrum of the set of complexed bacteriochlorophylls are discussed.     

The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy

The pH-dependent structure of calf thymus DNA is analyzed using Raman spectroscopy. The Raman spectra in the acidic region demonstrate that denaturation occurs in several steps. The binding of H⁺ to adenine and cytosine residues is accompanied by a decrease in the percentage of DNA in the B-conformation and a concurrent increase in a conformation most probably related to the C-form. The denaturation of DNA is observed at pH 3.3 and parallels the protonation of guanine bases. The Raman spectra of calf thymus DNA in the basic region (above pH 10) show that guanine residues are deprotonated at a lower pH value than are thymine residues. In addition, Raman spectra in the basic region detect conformational changes of the phosphate backbone different from those found in the acidic region.