Browsing by Author "Goodpaster, John"

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    Advancements in forensic DNA-based identification
    (2017) Dembinski, Gina M.; Picard, Christine; Christie, Mark; Walsh, Susan; Randall, Stephen; Goodpaster, John
    Modern DNA profiling techniques have increased in sensitivity allowing for higher success in producing a DNA profile from limited evidence sources. However, this can lead to the amplification of more DNA profiles that do not get a hit on a suspect or DNA database and more mixture profiles. The work here aims to address or improve these consequences of current DNA profiling techniques. Based on allele-specific PCR and quantitative color measurements, a 24-SNP forensic phenotypic profile (FPP) assay was designed to simultaneously predict eye color, hair color, skin color, and ancestry, with the potential for age marker incorporation. Bayesian Networks (BNs) were built for model predictions based on a U.S sample population of 200 individuals. For discrete pigmentation traits using an ancestry influenced pigmentation prediction model, AUC values were greater than 0.65 for the eye, hair, and skin color categories considered. For ancestry using an all SNPs prediction model, AUC values were greater than 0.88 for the 5 continental ancestry categories considered. Quantitative pigmentation models were also built with prediction output as RGB values; the average amount of error was approximately 7% for eye color, 12% for hair color, and 8% for skin color. A novel sequencing method, methyl-RADseq, was developed to aid in the discovery of candidate age-informative CpG sites to incorporate into the FPP assay. There were 491 candidate CpG sites found that either increased or decreased with age in three forensically relevant xii fluids with greater than 70% correlation: blood, semen, and saliva. The effects of exogenous microbial DNA on human DNA profiles were analyzed by spiking human DNA with differing amounts of microbial DNA using the Promega PowerPlex® 16 HS kit. Although there were no significant effects to human DNA quantitation, two microbial species, B. subtilis and M. smegmatis, amplified an allelic artifact that mimics a true allele (‘5’) at the TPOX locus in all samples tested, interfering with the interpretation of the human profile. Lastly, the number of contributors of theoretically generated 2-, 3-, 4-, 5-, and 6-person mixtures were evaluated via allele counting with the Promega PowerPlex® Fusion 6C system, an amplification kit with the newly expanded core STR loci. Maximum allele count in the number of contributors for 2- and 3-person mixtures was correct in 99.99% of mixtures. It was less accurate in the 4-, 5-, and 6-person mixtures at approximately 90%, 57%, and 8%, respectively. This work provides guidance in addressing some of the limitations of current DNA technologies.
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    Analysis of TNT, DNA Methylation, and Hair Pigmentation via Gas Chromatography-Mass Spectrometry and Spectroscopic Techniques
    (2019-08) Ruchti, Jacqueline; Goodpaster, John; Manicke, Nicholas; Picard, Christine
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    Antibacterial Activities of Cannabidiol Against Two Major Oral Pathogens
    (2024-05) Azabi, Asma A.; Windsor, L. Jack; Gregory, Richard L.; Platt, Jeffrey A.; Duarte, Simone; Goodpaster, John
    Despite advances in oral health research, biofilm-mediated oral diseases continue to pose a health challenge. Preventive measures against biofilm-mediated disease aim to inhibit pathogenic biofilm formation on tooth surfaces. Cannabidiol (CBD), a Cannabis sativa L. L. extract, has shown antibacterial properties against various bacterial species, especially gram-positive cocci. This dissertation aimed to evaluate the antibacterial efficacy of CBD against Streptococcus mutans and Porphyromonas gingivalis bacterial growth and virulence factors. The effects of CBD on the planktonic, biofilm, and total growth of S. mutans with or without nicotine and P. gingivalis, were evaluated. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentrations (MBIC), and minimal bactericidal concentrations (MBC) of CBD were assessed. CBD concentrations ≥ 2.5 μg/ml exhibited significant inhibition (p<0.001) against S. mutans biofilm growth, as well as, biofilm metabolic activity, lactate dehydrogenase (LDH) activity, and extracellular polysaccharides (EPS) production. In the presence of nicotine, which upregulates biofilm formation, CBD demonstrated the same inhibitory effects on S. mutans growth and activities. CBD concentrations ≥ 0.47 μg/ml exhibited significant inhibition (p<0.003) against P. gingivalis biofilm growth. CBD reduced the hemagglutination activities of P. gingivalis and reduced the overall proteolytic activity. Concentrations of CBD ≥ 0.63 μg/ml provided an inhibitory effect on lysine-specific gingipain. The results of these studies demonstrate that CBD has antibacterial activities against S. mutans and P. gingivalis growth and virulence factors related to caries and periodontal diseases, respectively.
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    Automated derivatization and identification of controlled substances via total vaporization solid phase microextraction (Tv-Spme) and gas chromatography-mass spectrometry (Gc-Ms)
    (2018) Hickey, Logan D.; Goodpaster, John
    Gas chromatography-mass spectrometry (GC-MS) is one of the most widely used instrumental techniques for chemical analyses in forensic science laboratories around the world due to its versatility and robustness. The most common type of chemical evidence submitted to forensic science laboratories is seized drug evidence, the analysis of which is largely dominated by GC-MS. Despite this, some drugs are difficult or impossible to analyze by GC-MS under normal circumstances. For these drugs, derivatization can be employed to make them more suitable for GC-MS. In Chapter 1, the derivatization of primary amino and zwitterionic drugs with three different derivatization agents, trifluoroacetic anhydride (TFAA); N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS); and dimethylformamide dimethylacetal (DMF-DMA), is discussed. The chromatographic performance was quantified for comparison between the derivatives and their parent drugs. Peak symmetry was compared using the asymmetry factor (As), separation efficiency was measured by the number of theoretical plates (N), and sensitivity was compared by measuring the peak areas. In Chapter 2, derivatization techniques were adapted for an automated on-fiber derivatization procedure using a technique called total vaporization solid phase microextraction (TV-SPME). TV-SPME is a variation of SPME in which a small volume of sample solution is used which can be totally vaporized, removing the need to consider the equilibrium between analytes in the solution and analytes in the headspace. By allowing derivatization agent to adsorb to the SPME fiber prior to introduction to the sample vial, the entire derivatization process can take place on the fiber or in the headspace surrounding it. The use of a robotic sampler made the derivatization procedure completely automated. In Chapter 3, this on-fiber derivatization technique was tested on standards of 14 controlled substances as well as on realistic samples including simulated “street meth”, gamma-hydroxybutyric acid (GHB) in mixed drinks, and hallucinogenic mushrooms, and was also tested on several controlled substances as solid powders. Future work in this area is discussed in Chapter 4, including adapting the method to toxicological analyses both in biological fluids and in hair. Some of the expected difficulties in doing so are discussed, including the endogenous nature of GHB in the human body. The presence of natural GHB in beverages is also discussed, which highlights the need for a quantitative addition to the method. Additional method improvements are also discussed, including proposed solutions for complete derivatization of more of the analytes, and for decreasing analysis time.
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    Chemical Analysis as a Tool in Arson and Explosives Investigations
    (Center for Translating Research Into Practice, IU Indianapolis, 2021-08-27) Goodpaster, John
    In this presentation, Professor John Goodpaster discusses his translational research on “Chemical Analysis as a Tool in Arson and Explosives Investigations.” He explains how his laboratory uses science to design better approaches to identify trace amounts of explosives on post-blast debris from improvised explosive devices or ignitable liquid residues in debris from suspicious fires. He also talks about the best methods for training dogs to detect explosives of all kinds.
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    Chemometric Analysis of Volatile Organic Compound Biomarkers of Disease and Development of Solid Phase Microextraction Fibers to Evaluate Gas Sensing Layers
    (2022-08) Woollam, Mark David; Agarwal, Mangilal; Deiss, Frédérique; Goodpaster, John; Naumann, Christoph
    Canines can detect different diseases simply by smelling different biological sample types, including urine, breath and sweat. This has led researchers to try and discovery unique volatile organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using miniaturized biosensors. However, the identity of the specific VOC biomarkers must be demonstrated before designing and fabricating sensing systems. Through an extensive series of experiments, VOCs in urine are profiled by solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that unique classes of urinary VOCs, primarily terpene/terpenoids and carbonyls, are potential biomarkers of breast cancer. Through implementing chemometric approaches, unique panels of VOCs were identified for breast cancer detection, identifying tumor location, determining the efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples. VOC biomarker identification is an important step toward developing portable gas sensors, but another hurdle that exists is that current sensors lack selectivity toward specific VOCs of interest. Furthermore, testing sensors for sensitivity and selectivity is an extensive process as VOCs must be tested individually because the sensors do not have modes of chromatographic separation or compound identification. Another set of experiments is presented to demonstrate that SPME fibers can be coated with materials, used to extract standard solutions of VOCs, and analyzed by GC-MS to determine the performance of various gas sensing layers. In the first of these experiments, polyetherimide (PEI) was coated onto a SPME fiber and compared to commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity for urinary VOC extraction relative to a polydimethylsiloxane (PDMS) SPME fiber. These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers.
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    Chemometric Comparison Of GC-MS And GC-VUV For The Trace Analysis Of Methamphetamine
    (2024) Lyle, Grant; Goodpaster, John; Sardar, Rajesh; Manicke, Nicholas
    Chemometrics, the application of mathematical or statistical algorithms to make inferences on the state of a chemical system from physical measurements of it, is a powerful tool that can be used to re-read what previously was observed as ‘noise’ in analytical measurements. Instruments such as spectrophotometers can take thousands of measurements over a predefined interval, but the spectra are only of great use when reference libraries exist, or if large trends occur that allow for visual matching, such as with a particular functional group. Application of statistical techniques to these data, such as principal component analysis (PCA) and linear discriminant analysis (LDA), can help to spot underlying variances, and differentiate between similar spectra by using linear combinations of these variables for classification. Methamphetamine (MA) is a member of the phenethylamines, a group of compounds that act as central nervous system stimulants, which are highly addictive and often the subject of law enforcement efforts at the local and federal level. Use of derivatization agents in analysis of seized narcotics is common practice, as it increases volatility/thermal stability of analytes, and improves peak shape for chromatographic resolution. In this analysis, we looked to investigate the difference in instrumental response for MA in its native form, as well as derivatized with two common agents, acetic anhydride and trifluoroacetic anhydride. These three forms were analyzed both on a gas chromatograph- mass spectrometer (GC-MS) and a gas chromatograph- vacuum ultraviolet spectrometer (GC-VUV). The raw GC-MS and GC-VUV data were separately normalized, and the dimensionality of the data was reduced through PCA, which uses orthogonal linear transformations of the data to capture most of the variance between datasets while simultaneously reducing the dimensionality for further analysis. Linear discriminant analysis was utilized to look at the principal components from PCA, and a classification model was built for use in discriminating between forms of methamphetamine from compressed datasets.
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    Critical Comparison of Total Vaporization- Solid Phase Microextraction vs Headspace- Solid Phase Microextraction
    (2021-05) Train, Alexandra; Goodpaster, John; Manicke, Nicholas; Picard, Christine
    Solid Phase Microextraction (SPME) is a popular sampling technique that can be paired with Gas Chromatography/Mass Spectrometry (GC-MS). SPME-GC-MS is used in forensic chemistry due to its simplification of the sample preparation process. Headspace-Solid Phase Microextraction (HS-SPME) is a technique where the sample is heated to generate volatiles in the headspace of the vial. A SPME fiber is then inserted into the vial and the compounds in the headspace will bind to the fiber. Total Vaporization- Solid Phase Microextraction (TV-SPME) is a technique that is derived from the HS-SPME technique. In Chapter 1, the critical comparison of HS-SPME and TV-SPME is discussed. Samples including marijuana, essential oils, and CBD oil were utilized to compare the two techniques. The compounds of interest in marijuana are the three main cannabinoids: cannabinol (CBN), cannabidiol (CBD), and tetrahydrocannabinol (THC). The sample preparation and GC-MS parameters were kept the same for all samples to determine which SPME technique works best for these sample types and yielded the greatest sensitivity. It was found that HS-SPME shows greater sensitivity with CBN and equivalent sensitivity with essential oils, THC and CBD. In Chapter 2, the detection of synthetic cannabinoids utilizing liquid-liquid injection as well as HS-SPME and TV-SPME is discussed. The detection of these compounds is important because this type of drug has become more prevalent in the United States because they can be chemically altered slightly so they still have the effects of a drug but can evade drug legislation. The detection of synthetic cannabinoids using liquid injection was found to be successful but detection using HS-SPME and TV-SPME was found to be unsuccessful. In Chapter 3, the analyses of real and artificial saliva utilizing HS-SPME and TV-SPME is discussed. Determining the compounds present in real saliva and artificial saliva will be of importance for future research into determining if the presence of drugs in saliva can be analyzed with these techniques. The analyses of real and artificial saliva were found to be successful using HS-SPME, without derivatization, and TV-SPME, with and without derivatization. Many of the compounds present in the real saliva were detected and were confirmed to be compounds regularly found in saliva by other scientific literature.
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    Design and Fabrication of Smart SERS Substrates for Forensic Science Applications
    (2023-08) Simas, Maria Vitoria; Sardar, Rajesh; Goodpaster, John; Manicke, Nicholas; Christoph, Naumann
    In the field of forensics and toxicology, it is crucial for analytical techniques to be practical, highly sensitive, and extremely accurate (specific and selective), especially when incorporating acquired data as evidence in a court case. To limit the breadth of this dissertation, the main forensic focuses are to assay (detection and quantification) drugs in patient biofluid specimens and to detect trace explosives. While currently there are a number of analytical tools such as LC/MS, GC/MS, ELISA immunoassays, and electrochemical and aptamer techniques utilized for these two applications, each one introduces its own unique drawback that hinders their accuracy, and therefore applicability, to be used in a legal environment. To overcome these disadvantages, surface enhanced Raman spectroscopy (SERS) has become increasingly popular in the forensic and toxicology field as it provides high sensitivity and specificity data while remaining flexible and efficient in critical situations such as an emergency department and/or an explosion site. In this dissertation, two different, novel SERS substrates are developed, each one designed to tackle a specific forensic application. While the fabrication method and materials of the substrates are significantly different from one another, both display ideal SERS properties due to their unique localized surface plasmon resonance (LSPR) properties at the nanoscale. LSPR is a phenomenon in which the free carriers (electrons or holes) on the surface of nanoparticles collectively oscillate upon light irradiation. In this current dissertation, we selectively focus on two different nanoparticle compositions where LSPR properties originate from the collective oscillation of free electrons. These oscillations of free carriers allow for an electromagnetic (EM) field enhancement of the incident laser which leads to an increase in the SERS enhancement. Particularly, the area in which the SERS signal is the most enhanced is the nanometer-sized gap between nanoparticles called “hot spots.” While primarily noble metal (e.g., Au and Ag) nanoparticles are heavily used for SERS substrate fabrication, this dissertation expands beyond that and focuses on both gold nanorods and oxygen deficient tungsten oxide (metal oxide semiconductor)-based SERS substrates. This dissertation is organized in three chapters, (1) Introduction, (2) Fabrication of a polymer microneedle-based, multimodal SERS and mass spectrometry substrate for the ultrasensitive detection of illicit drugs in human blood plasma, and (3) LSPR active WO3-x-based SERS substrate for the detection of explosives. In chapter 2, a multimodal substrate was strategically designed to serve as a SERS and electrospray ionization- mass spectrometry substrate by using a novel microneedle platform. The microneedles underwent a surface modification and gold nanorods were subsequently adsorbed onto the surface. Illicit drug analytes could then be drop-casted on the tip of the microneedles and left to dry for further SERS and mass spectrometry analysis. This novel platform detected two types of synthetic opioids, alprazolam and fentanyl, down to at least a picomolar limit of detection and successfully distinguished between the two when analyzing 10 patient blood plasma samples. Furthermore, the multimodal approach was confirmed through the detection of both drugs in patient plasma samples down to the ppb limit using mass spectrometry. Chapter 3 introduces an entirely new SERS substrate, which is fabricated using oxygen deficient tungsten oxide (WO3-x) nanoparticles for the detection of the target explosives, e.g., tetryl, TNT, and DNT. The oxygen deficiency in WO3-x nanoparticle lattice introduces free electrons in the conduction band that introduce LSPR properties into the nanoparticles giving rise to the EM field mechanism for SERS enhancement upon incident laser irradiation. Much like with noble metals, the oscillation of these free carriers at nanoparticle hot spots aid in the development of a functional and cheaper SERS substrate. The SERS enhancement factor (EF) of three different morphologies of WO3-x nanoparticles, i.e., nanorods, nanowires, and nanoplatelets are characterized and deemed comparable to noble metal nanoparticles. A Janowsky complex was formed using the explosive, tetryl, as the target analyte. Using our oxygen deficient WO3-x substrate, tetryl was successfully detected down to the nanomolar level. To our knowledge, this is the first time tetryl has been detected utilizing a non-noble metal-based SERS substrate. Taken together, this dissertation presents the unique aspect of nanotechnology for the development of (1) a multimodal MN-based ultrasensitive and (2) an inexpensive noble-metal comparable WO3-x-based SERS substrates, both of which that can be applied to forensic science research, specifically in forensic toxicology and explosive detection to better, and even save, the lives of individuals and improve their quality of life.
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    Detection and Quantitation of Hazardous Chemicals in Environmental Matrices using Paper Spray Mass Spectrometry
    (2019-08) Dowling, Sarah Naciye; Manicke, Nicholas; Goodpaster, John; Sardar, Rajesh
    Paper spray mass spectrometry (PS-MS) is an ambient ionization technique that has been proven useful in many types of investigative analyses. However, the use of this technique with regards to environmental samples has been largely unexplored since the technique’s development. In this work, paper spray mass spectrometry was utilized to detect and quantify compounds for environmental, forensic and chemical defense applications. Due to the sensitive nature of some projects, the work was split into two volumes. Volume 1 focuses on the detection of pharmaceuticals in soil using paper spray (Chapter 2) and the detection of chemical warfare agent (CWA) simulants and CWA hydrolysis products (Chapter 3). Volume 2 focuses on the detection and quantitation of fentanyl analogs in environmental matrices. Chapter 5 focuses on the rapid analysis of fentanyl analogs in soil matrices. The following chapter evaluates the ability of PS-MS to detect low concentrations of fentanyl analogs in water (Chapter 6). Throughout this work, paper spray has proven to be an effective, rapid alternative to chromatography for the analysis of environmental samples.