(Georgia Institute of Technology, 2009-12)
Rizzo, David G.
Desorption electrospray ionization mass spectrometry (DESI MS) has gained significant recognition over the past few years because of its simplicity and rapid throughput capabilities, allowing for the direct analysis of samples with a wide variety of sizes, shapes, and chemistries. Addition of various reagents into the DESI spray solvent enables atmospheric pressure ion molecule reactions between these reagents in the charged micro droplets and analyte molecules on the sample surface affording improved selectivity and sensitivity in some cases. Presented is a rapid screening method for amino acids (aas) based on reactive DESI. Amino acids have been shown to play key roles in the regulation of cellular processes. They are also particularly vital in the determination of metabolic disorders such as phenylketonurea, homocystinuria, and tyrosinemia. The most specific and reliable methods for diagnosing these disorders are based on the determination of aas in body fluids using methodologies such as gas/liquid chromatography, tandem MS, and various combinations thereof. However, these methods are usually time-consuming, increasing the time physicians wait before administering treatment or regulating the diet of diseased infants. The reactive DESI approach presented here is based on the formation of stable noncovalent complexes between alpha-cyclodextrin (dissolved in the spray solvent) and amino acids present in the sample affording a selective method for their detection. However, the selectivity and sensitivity of screening for aas was improved by performing the MS analysis in the multiple reaction monitoring mode when using a quadrupole ion trap or by the precursor ion scan when using a triple quadrupole MS instrument, affording an average twenty-five times sensitivity improvement compared to analysis in full scan mode. The observation of similar complexes with various carboxylic acids including formic acid and acetic acid, and evidence from tandem MS experiments indicate that amino acid:alpha-cyclodextrin complexation reactions occur by hydrogen bonding interaction with carboxyl group of the aas. The specificity and sensitivity provided by this approach seems very promising for applications in the rapid screening of aas directly from body fluids including urine and plasma for amino acid disorders in a clinical setting.
(Georgia Institute of Technology, 2009-05-04)
Siemens, Katherine
Surface enhanced Raman scattering (SERS) is a powerful spectroscopic tool that can be used to identify and characterize compounds at low concentrations. Recent literature reports suggest that SERS may be applicable for the detection and identification of viruses present in biofluids. In this investigation, gold nanorod arrays were evaluated as SERS substrates with the specific purpose of probing spectral differences between single stranded (ssDNA) and hybridized, or double stranded (dsDNA) DNA. This was deemed as the first step in developing a SERS-based hybridization assay for viral identification. Hybridization was carried out both off and on the substrate surface to determine whether there are observable spectral differences from the two different methods of hybridization. Studies were also carried out to determine if it was possible to detect mismatched DNA pairs after hybridization had been attempted. Gold SERS active substrates were utilized instead of silver giving the advantage that these substrate do not oxidize under ambient conditions. It has also been found that ozone cleaning gold substrates before sample application increases the hydrophilicity of the gold, making the use of smaller sample volumes possible. Ozone cleaning the gold substrate after ample binding time has passed increases the SERS signal as well. It is believed that this cleaning immediately prior to SERS acquisition cleans the gold surface to a point where the plasmon being formed is more likely to move up the sample which increases the intensity of the SERS signal. In addition to examining the possibility of using gold instead of silver for the SERS substrates, a method allowing for 36 separate DNA assays to be run at one time was investigated. This is accomplished by creating wells with polymer. Up to 36 wells fit on one glass microscope slide meaning that anywhere from 1 to 36 DNA probes can be attached within individual wells. This allows for either 36 different biological assays for the same virus or 1 biological sample to be tested for 36 different viruses or virus strains.