Advances in DNA microarray fabrication technologies, expanding probe libraries, and new bioinformatics methods and resources have firmly established array-based techniques as mainstream bioanalytical tools and the application space is proliferating rapidly. However, the capability of these tools to yield truly quantitative information remains limited, primarily due to problems inherent to the use of fluorescence imaging for reading the hybridized arrays. The obvious advantages of fluorescence are the unrivaled sensitivity and simplicity of the instrumentation. There are disadvantages of this approach, however, such as difficulties in achieving optimal labeling of targets and reproducible signals (due to quenching, resonance energy transfer, photobleaching effects, etc.) that undermine precision. We are exploring alternative approaches, based mainly on Raman and resonance Raman spectroscopy, that in principle permit direct analysis of structural differences between hybridized and unhybridized probes, thereby eliminating the need for labeling the target analytes. We report here on the status of efforts to evaluate the potential of these methods based on a combination of measured data and simulated experiments involving short (12-mer) ssDNA oligomer probes with varying degrees of hybridized target DNA. Preliminary results suggest that it may be possible to determine the fraction of duplex probes within a single register on a DNA microarray from 100% down to 10% (or possibly less) with a precision of ±2 5%. Details of the methods used, their implementation, and their potential advantages and limitations are presented, along with discussion of the utility of using 2DCOS methods to emphasize small spectral changes sensitive to interstrand H bonding, backbone flexibility, hypochromicity due to base-stacking in duplex structures and solvation effects.
UV resonance Raman spectroscopy (UVRRS) is becoming a very popular spectroscopic method for bioanalytical investigations due to its high sensitivity, lack of fluorescence, and suitability for use in aqueous solutions. We have made a number of technological advances, especially the development of fiber-optic-based technologies, which permit the performance of remote/in-situ UVRRS measurements. We will be reporting on improved optical fiber probes and demonstrate their benefits in performing UVRRS on neurotransmitters, saliva, and urine.
Many techniques have been developed to investigate the chemistry associated with brain activity. These techniques generally fall into two categories: fast techniques with species restricted sensitivity and slow techniques with generally unrestricted species sensitivity. Therefore, a need exists for a fast non-invasive technique sensitive to a wide array of biologically relevant compounds in order to measure chemical brain events in real time. The work presented here describes the progress made toward the development of a novel neurotransmitter probe. A fiber-optic linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. Probes of several different geometries were constructed and their working curves obtained in aqueous mixtures of methyl orange and potassium nitrate to determine the best probe configuration given particular sample characteristics. Using this system, the ultraviolet resonance Raman spectra of some neurotransmitters were measured with a fiber-optic probe and are reported here for the first time. The probe has also been used to measure neurotransmitter secretions obtained from depolarized rat pheochromocytoma cells.
We have developed fiber-optic probes that facilitate rapid, simultaneous determination of multiple analytes, in situ, over a broad range of concentrations. Theoretical and empirical methods were used to design and characterize prototype probes that comprise a single small-diameter excitation fiber and multiple larger diameter collection fibers for the optical collection of side- and back- scattered or emitted light, depending on the sample characteristics. Prototype were developed for use with pulsed ultra-violet resonance Raman spectroscopy, however, probes of this type are also suitable for use with other spectroscopic techniques such as fluorescence. Materials specifications, modelling methods, fabrication methods, and performance characteristics are described. Probes of our design are at present capable of measuring the aromatic amino acids in the 10 (mu) M range and nM detection limits can be expected. We have also obtained UV Raman and resonance Raman spectra from proteins, DNA, amino acids, steroids, neurotransmitters, and alcohols with these probes.
Resonance Raman spectroscopy, performed via an optical fiber probe, can be used in aqueous samples to detect a wide variety of chemical species in situ. It offers a potentially rapid, on-site alternative to the high performance chromatography/mass spectrometry methods currently considered definitive for the detection of sex steroids in human urine. As a first step in the development of a resonance Raman instrument for the rapid detection of sex steroids in biological samples, it had to be shown that these substances, their analogs, and the major components of human urine can be differentiated on the basis of their resonance Raman spectra. A fiber-optic linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. The ultraviolet absorption spectra of some sex steroids, analogs, and components of human urine were measured in order to determine feasible excitation light frequencies. We present here for the first time the UV resonance Raman spectra of these substances obtained via our novel fiber probes. These results indicate that some of the steroids tested can be differentiated from each other and from the major components of human urine on the basis of their resonance Raman spectra.
Ultraviolet resonance Raman spectroscopy (UVRRS) has the potential to become a sensitive, specific, versatile bioanalytical and biophysical technique for routine investigations of proteins, DNA, and their monomeric components, as well as a variety smaller, physiologically important aromatic molecules. The transition of UVRRS from a complex, specialized spectroscopic method to a common laboratory assay depends upon several developments, including a robust sample introduction method permitting routine, in situ analysis in standard laboratory environments. To this end, we recently reported the first fiber-optic probes suitable for deep-UV pulsed laser UVRRS. In this paper, we extend this work by demonstrating the applicability of such probes to studies of biochemical relevance, including investigations of the resonance enhancement of phosphotyrosine, thermal denaturation of RNase T1, and specific and non-specific protein binding. The advantages and disadvantages of the probes are discussed with reference to sample conditions and probe design considerations.
Silica based optical fibers are in operation for light- guidance in many applications, with wavelengths ranging from 230 nm to 2.2 micrometer. At shorter wavelengths, UV- improved fibers (UVI) can be used, which overcome the significant generation of UV-defects and associated loss in throughput using broadband deuterium-lamps or UV-lasers with fixed wavelengths such as excimer-lasers and quadrupled Nd:YAG lasers. Alternatively, many spectroscopic applications need a tunable and powerful UV-light to carry out remote-measurements, in-situ. In addition, specialized fiber-optics probe may be useful. We report for the first time on the transmission properties of the UVI-fibers with high-power pulsed lasers with wavelengths in the vicinity of the dominant UV-defect at 215 nm using a frequency-doubled dye-laser, tunable from 206 to 245 nm. Due to the high intensity of this pulsed laser system, especially near the fiber frontface, two-photon absorption plays a major role with decreasing wavelength. Therefore, properties such as starting transmission and UV-induced loss were measured as a function of the wavelength and input pulse energy. The envelope of the wavelength-dependent induced losses is comparable with results taken from tests with low-power broadband deuterium-lamps. However, the temporal behavior during recovery is quite different.
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