Padilla Jiménez, Amira C.

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2005 - 200912010 - 20141
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    Vibrational spectroscopy studies of biomolecular systems: from amino acids to microorganisms
    (2014) Padilla Jiménez, Amira C.; Hernández Rivera, Samuel P.; College of Arts and Sciences - Sciences; Ríos Velázquez, Carlos; López Moreno, Martha L.; Rivera Portalatin, Nilka M.; Mina Camilde, Nairmen; Department of Chemistry; Estévez De Vidts, L. Antonio
    Vibrational spectroscopy: infrared and Raman, was applied to the study of two amino acids and ten bacterial strains with the purpose of developing rapid methods of identification and discrimination. Use of spectroscopic techniques in characterization of signatures of biological agents has gained considerable attention in recent years, mainly because of the high sensitivity and selectivity that can be attained through their practice. They can also be easily adapted to portable equipment to obtain fingerprint information of biomolecules and microorganisms in the field. In this research, various spectroscopic studies were conducted, both in the lab and in field applications. In the first study the most stable conformations and orientations of Ltryptophan (L-Trp) on silver (Ag) and gold (Au) nanoparticles (NPs) was determined using Raman spectroscopy. The objective of the work was to determine if L-Trp molecules interact with the Ag/Au NPs through the carboxylate end, through the amino group end, or through both using surface enhanced Raman spectroscopy (SERS). The work also focused on how parameters such as analyte concentration, average nanoparticle size and pH affected the binding of L-Trp to the NPs surfaces. In a second related study Ag/Au NPs were synthetized using a laser ablation technique and SERS activity of prepared NPs was evaluated with L-histidine (L-His). In other studies quantum cascade laser spectroscopy (QCLS) was used to identify biochemical components of bacterial cell wall of various microorganism species from vibrational modes of molecular components in the biosamples. Principal component analysis (PCA) and partial least squares analysis coupled to discriminant analysis (PLSDA) of QCL spectra were used to classify and discriminate between gram-positive and gram-negative bacteria at a 95% confidence level. Results demonstrate that the QCLS techniques used: reflection and transmission, accompanied with powerful multivariate analyses techniques were successful in detecting and classifying the microorganisms studied by means of their characteristic spectral information.
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    Detection of flavins exhibiting autofluorescence from "in vivo" bacterial samples using one photon and two photon confocal microscopy
    (2005) Padilla Jiménez, Amira C.; Rivera Montalvo, Luis A.; College of Arts and Sciencies - Sciences; Hernández Rivera, Samuel P.; Mina, Nairmen; Department of Chemistry; Uscian, John
    Studies have shown that certain types of biological agents - bacteria, viruses and fungi - can be observed by their intrinsic fluorescence. These biomolecules include amino acids, coenzymes such as nicotinamide adenine dinucleotide (NADH) and flavins. Recently, it has been shown that confocal microscopy is a very good method for the detection and study of single particles in live organisms or tissues which incorporate natural fluorophores. The purpose of this research project is to develop an analytical methodology for the detection of flavin compounds bacteria aqueous suspension that exhibit intrinsic fluorescence prepared from biological culture media. It is also important to determine the different flavins cofactors present in various species of bacteria; we have focused our attention on three main derivatives of flavins. These include riboflavin (RF) and its two biologically active metabolites: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). One and two photon excited (TPE) fluorescence spectroscopy of intrinsic fluorophores from bacteria aqueous suspensions “in vivo” using confocal microscopy were performed to obtain the TPE spectra of eleven microorganisms: eight bacteria and three fungi. These obtained spectra are the results innovative in the visible region and excited in the near IR at 780nm for to reduce level of photodamage on cells. As expected the emission from all bacteria samples were originated in similar range of emission wavelengths. In addition, we report a reproducible analytical methodology for determining the intrinsic fluorescence spectra of different bacteria aqueous suspension samples by fluorescence correlation spectroscopy (FCS) using Matlab, SimFCS, SPSS, and Minitab programs, to obtain the fluctuations diagrams from confocal microscopy, and then for data manipulation to determine the autocorrelation graph, which contains the dynamic information of the analytes, and later, the fit of this data to a mathematical function. Furthermore, it was found by fluorescence information obtained from Raman spectroscopy instrument in its emission mode, and fluorimeter that the relative concentrations of flavin cofactors at high concentration of bacteria samples (20μL UFC), are present at an approximate concentration of riboflavin 250nM in B. subtilis, FAD 100nM in E. coli and FMN 50nM in B. cereus, but, at low concentration of bacteria samples (10μL UFC), concentration as low as 10.0nM of these flavin cofactors were observed.