INVESTIGATION OF PROTEIN-DRUG INTERACTION USING CAPILLARY ISOELECTRIC FOCUSING WITH WHOLE COLUMN IMAGING DETECTION AND SPECTROSCOPIC TECHNIQUES

In this work, the possibilities of understanding protein-drug interactions and the possible modes of action of the drug have been was explored using capillary isoelectric focusing (CIEF) and spectroscopic techniques. The first study involves the implementation and applications of the new high performance Raman spectrometer with volume phase (VPH) based grating system, which is integrated with a short liquid-core optical waveguide for characterization and analysis of a wide ranges of molecules. The high performance Raman spectrometer has allowed us to measure solid samples with minor differences in chemical compositions. In addition, spectral characteristics of the interactions between therapeutic drugs with human serum albumin have been examined in terms of the cleavage of disulfide (S-S) bonds. The relative intensity of the Raman signal decrease as a complex formation progressed, suggesting perturbation around the disulfide bonds of human serum albumin (HSA). This spectrometer is likely to be broadly applicable for simple to moderately complex samples. Furthermore, the future perspective of combining separation and spectroscopy technologies is a novel approach for capitalizing on the strengths of the two technologies. Capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID) was used to investigate the interaction of platinum-based anticancer drugs, cisplatin and oxaliplatin, with human hemoglobin A0 (Hb) and HSA. This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50 and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein-drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance. Spectroscopic evidence also shows that oxaliplatin causes fluorescence quenching of HSA by formation of on HSA-oxaliplatin complex. The Stern-Volmer equation has been used to calculate the quenching constant in the linear range. The quenching rate constants Kq at three different temperatures indicate the presence of a static quenching mechanism in the interactions of oxaliplatin with HSA. The work describes the validity of the CIEF-WCID technique for the study of protein-drug interactions and provides useful information and insight into the interaction of anti-cancer drugs with HSA concerning their further toxicity of these chemotherapeutic drugs and ways of improving their clinical performance. Finally, a novel two-dimensional (2D) protein characterization using a single one dimensional separation followed by a second characterization in the same instrument was developed. The technique combines capillary isoelectric focusing ( CIEF), which separate proteins based on their isoelectric points, with a dynamic imaging focusing and allows estimates of their molecular weights from measurement of their diffusion coefficients. This techniques applies to simple to moderately complex protein samples

Infrared, Raman and surface-enhanced resonance Raman scattering of N-butylimido, ethyleneamine perylene (PTCD-NH(2)): Single-molecule detection using colloidal silver and silver island films.

The vibrational spectra and the surface-enhanced (resonance) Raman scattering (SERS/SERRS) of a perylene dye containing a terminal NH2 group, n-butylimido, ethyleneamine perylene (PTCD-NH2) have been studied. The infrared, Raman and resonance Raman spectra of the material were recorded and vibrational fundamentals assigned. The vibrational assignments of fundamentals were aided with an ab-initio Hartree-Folk at 6-31G level of theory. Langmuir-Blodgett monolayers of the neat material were fabricated and the pi-A isotherms recorded. Mixed monolayers were also studied for applications in SERBS. The spectra of evaporated thin solid films were obtained and a complete spectroscopic characterization of these films is presented in the thesis. Single molecule detection of PTCD-NH2, adsorbed on colloidal silver and silver island films are investigated using surface enhanced resonance Raman spectroscopy and Langmuir-Blodgett films. (Abstract shortened by UMI.) Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2002 .L46. Source: Masters Abstracts International, Volume: 41-04, page: 1086. Adviser: R. Aroca. Thesis (M.Sc.)--University of Windsor (Canada), 2002

Plasmonic nanostructures through DNA-assisted lithography

Programmable self-assembly of nucleic acids enables the fabrication of custom, precise objects with nanoscale dimensions. These structures can be further harnessed as templates to build novel materials such as metallic nanostructures, which are widely used and explored because of their unique optical properties and their potency to serve as components of novel metamaterials. However, approaches to transfer the spatial information of DNA constructions to metal nanostructures remain a challenge. We report a DNA-assisted lithography (DALI) method that combines the structural versatility of DNA origami with conventional lithography techniques to create discrete, well-defined, and entirely metallic nanostructures with designed plasmonic properties. DALI is a parallel, high-throughput fabrication method compatible with transparent substrates, thus providing an additional advantage for optical measurements, and yields structures with a feature size of ~10 nm. We demonstrate its feasibility by producing metal nanostructures with a chiral plasmonic response and bowtie-shaped nanoantennas for surface-enhanced Raman spectroscopy. We envisage that DALI can be generalized to large substrates, which would subsequently enable scale-up production of diverse metallic nanostructures with tailored plasmonic features.Peer reviewe

Self-Assembly Properties of Amphiphilic Iron(III) Spin Crossover Complexes in Water and at the Air–Water Interface

The assembly properties of three known spin crossover iron(III) complexes 1⁻3, at the air⁻water interface, are reported. All three complexes are amphiphiles, each bearing a pair of Cn alkyl chains on the polyamino Schiff base sal2trien ligand (n = 6, 12, or 18). Complex 1 is water-soluble but complexes 2 and 3 form Langmuir films, and attempts were made to transfer the film of the C18 complex 3 to a glass surface. The nature of the assembly of more concentrated solutions of 3 in water was investigated by light scattering, cryo-SEM (scanning electron microscopy), and TEM (transmission electron microscopy), all of which indicated nanoparticle formation. Lyophilization of the assembly of complex 3 in water yielded a powder with a markedly different magnetic profile from the powder recovered from the initial synthesis, notably, the spin crossover was almost completely quenched, and the thermal behavior was predominantly low spin, suggesting that nanoparticle formation traps the system in one spin state

Identifying yeasts using surface enhanced Raman spectroscopy

The molecular fingerprints of yeasts Saccharomyces cerevisiae, Dekkera bruxellensis, and Wickerhamomyces anomalus (former name Pichia anomala) have been examined using surfaceenhanced Raman spectroscopy (SERS) and helium ion microscopy (HIM). The SERS spectra obtained from cell cultures (lysate and non-treated cells) distinguish between these very closely related fungal species. Highly SERS active silver nano-particles suitable for detecting complex biomolecules were fabricated using a simple synthesis route. The yeast samples mixed with aggregated Ag nanoparticles yielded highly enhanced and reproducible Raman signal owing to the high density of the hot spots at the junctions of two or more Ag nanoparticles and enabled to differentiate the three species based on their unique features (spectral fingerprint). We also collected SERS spectra of the three yeast species in beer medium to demonstrate the potential of the method for industrial application. These findings demonstrate the great potential of SERS for detection and identification of fungi species based on the biochemical compositions, even in a chemically complex sample.peerReviewe

An FT-Raman, FT-IR, and Quantum Chemical Investigation of Stanozolol and Oxandrolone

We have studied the Fourier Transform Infrared (FT-IR) and the Fourier transform Raman (FT-Raman) spectra of stanozolol and oxandrolone, and we have performed quantum chemical calculations based on the density functional theory (DFT) with a B3LYP/6-31G (d, p) level of theory. The FT-IR and FT-Raman spectra were collected in a solid phase. The consistency between the calculated and experimental FT-IR and FT-Raman data indicates that the B3LYP/6-31G (d, p) can generate reliable geometry and related properties of the title compounds. Selected experimental bands were assigned and characterized on the basis of the scaled theoretical wavenumbers by their total energy distribution. The good agreement between the experimental and theoretical spectra allowed positive assignment of the observed vibrational absorption bands. Finally, the calculation results were applied to simulate the Raman and IR spectra of the title compounds, which show agreement with the observed spectra

Synergistic enhancement via plasmonic nanoplate-bacteria-nanorod supercrystals for highly efficient SERS sensing of food-borne bacteria

Bio-sensing techniques utilizing metallic nanoparticles as a probe have gained more and more attention and play today an important role in the detection of bacteria. To date, although several sensing materials have been tested, there is still a long way to go to achieve a fast, low-cost, ultrasensitive and multifunctional substrate suitable for a universal biosensor for detection of bacterial cells. Here, we report a novel probe design based on anisotropic plasmonic nanoparticles organized to a biocompatible 3D bio-inorganic scaffold, i.e., nanoplate-bacteria-nanorod supercrystals (NBNS) with extremely high surface-enhanced Raman spectroscopic (SERS) activity as a model of synergistic plasmonic enhancement from nanoparticles and assembly. This unique structure of nanoparticles incorporated into supercrystal assembly allows efficient detection, identification and classification of cells and bacteria. In this design, the NBNS ensures that the target cells take advantage of the superior multifold increase in Raman scattering signals (electromagnetic enhancement from both types of nanoparticles), due to the geometry of the 3D scaffold. The excellent reproducibility and stability of NBNS substrates were confirmed by comparing the SERS activities of different substrates and analytes. Principal component analysis (PCA) applied to the SERS spectra clearly discriminated the homogeneous bacterial samples and their mixtures. Successful detection and identification of bacteria in model samples consisting of two representative bacteria blends in Fanta soft-drink were demonstrated via plasmonic bio-inorganic scaffold combined with PCA analysis. We believe that this work will greatly facilitate the development of ultrasensitive SERS probes for highly advanced biosensor, pioneering the use of SERS for controlling food safety.peerReviewe