Research Project Title: Stability of labile organic compounds in small-volume dried blood spheroids studied by hydrophobic paper spray mass spectrometry

Student Presenter: Danyelle Allen

Faculty Mentor: Abraham Badu-Tawiah

Faculty Mentor Department: Chemistry and Biochemistry Department

Research Abstract: Dried blood spots (DBS) are a simple sample collection, transportation, storage, and processing medium well-suited for personalize healthcare. However, DBS must be stored with limited contact with moisture, sunlight, and heat to prevent sample degradation. When sample is instead stored on hydrophobic paper, hydrolytically labile chemicals such as cocaine and diazepam trapped in the three-dimensional dried blood spheroid are stabilized. Hydrophobic paper spray allows direct analysis of both dried and wet blood spots, providing a more sensitive alternative to typical paper spray. Because surface energy of the paper is lowered, target small molecules are less likely to form strong interactions with the paper surface, resulting in a more sensitive analysis when compared to untreated paper strips. When filter paper is exposed to silane vapor, the paper’s surface energy is decreased to approximately 44 mN/m, and aqueous-based samples (surface tension ~72 mN/m) are unable to wet the treated paper, instead resting on the surface as a drop. The advantages and attributes of this approach include blood sample applied on the hydrophobic paper forms a spherical drop due to a mismatch in surface energies, which dries to yield a dried blood spheroid. Experiments have shown that hydrolytically labile chemicals such as cocaine and diazepam trapped in the 3D dried blood spheroid are stabilized, compared with storage done under the porous DBS conditions where a major portion of the sample becomes susceptible to oxidative stress from atmospheric air. Additionally, the hydrophobic paper provides a direct mass spectrometry (MS) detection through paper spray (PS) ionization for sensitive analyte quantification. In-situ extraction of illicit drugs (cocaine, benzoylecgonine, amphetamine and methamphetamine) from the dried blood spheroids resulted in sub-ng/mL limit of detections using ethyl acetate spray solvent. Research Project Title: Thread spray of cytochrome c

Student Presenter: George Durisk

Faculty Mentor: Abraham Badu-Tawiah

Faculty Mentor Department: Department of Chemistry and Biochemistry

Research Abstract: The use of portable Mass Spectrometry to detect disease biomarkers accurately and quickly could be widely applicable as a biological diagnostic test. The study of proteins is a major application in mass spectrometry (MS). This is important because using MS gives an opportunity for detection and characterization of this class of compounds, however, most methods for protein analysis require extensive sample preparation steps. Our lab’s goal is to utilize ambient ionization for this type of analysis via thread spray mass spectrometry. Thread Spray has successfully and accurately detected small molecules and expanding the capabilities of this novel technique to detect macromolecules will provide an efficient way to detect them in the biological samples and in their native states. To do this, we plan on studying Cytochrome C, a small, water-soluble, inner mitochondrial protein. Cytochrome C plays a critical role in oxidative phosphorylation and apoptosis in living organisms and has been successfully detected by methods of Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix-assisted Desorption (MALDI). In Thread Spray, a single thread is placed through a glass capillary and wrapped around the outside of the capillary. An alligator clip is placed on top of the thread wrapped around the outside, then solvent and DC voltage are applied to the thread, ionizing the analyte. The thread is placed directly in front of the instrument and allows for a selective ionization process of the target analyte. It was rationalized that the sharp points of the thread will allow for a better and more conclusive spray efficiency. Different threads will be used to optimize for the best data. Untreated thread, 30-minute gas phase silane treated thread and 60-minute gas phase silane treated thread will be tested with Cytochrome C. Voltage will be optimized and Cytochrome C will be sprayed denatured, natively in a buffer solution and the charge states will be monitored as a function of pH. The benefits of using Thread Spray to analyze Cytochrome C include no sample preparation, quick analysis time, no separation or extraction step needed, and can be done in ambient conditions, native protein form, or conformations. Research Project Title: Rotational parameters from vibronic eigenfunctions of Jahn-Teller active molecules

Student Presenter: Scott Garner

Faculty Mentor: Scott Miller

Faculty Mentor Department: Chemistry and Biochemistry

Research Abstract: The structure in rotational spectra of many free radical molecules is complicated by Jahn-Teller distortions. Understanding the magnitudes of these distortions is vital to determining their equilibrium geometric structure and details of their potential energy surfaces predicted from electronic structure calculations. For example, in recent studies of the NO3 radical, the magnitudes of distortions are yet to be well understood as results from experimental spectroscopic studies of its vibrational and rotational structure disagree with results from electronic structure calculations of the potential energy surface. Using vibronic eigenfunctions and magnitudes of the vibrational Jahn-Teller coupling obtained either from electronic structure calculations or vibrationally resolved spectra, we are able to predict parameters in the rotational Hamiltonian. Here we determine the Watson Jahn-Teller distortion term, h1, and compare with the results from the analysis of rotational experiments. Research Project Title: Color characteristics of gelatin gummies colored with acylated anthocyanins

Student Presenter: Siti Fatimah Binti Khairuddin

Faculty Mentor: M. Monica Giusti

Faculty Mentor Department: Food Science

Research Abstract: Gummies are well-known confectionaries usually dyed by artificial colorants. However, some health concerns about artificial colorants have pushed the industry to produce gummies with naturally derived colors. Anthocyanins are pigments in vegetables and fruits, giving colors from red to purple and blue depending on pH. The objective was to determine the color characteristics and pigment stability of gummies colored with anthocyanins. For this purpose, we prepared gelatin-based gummies colored with acylated anthocyanins from elderberries and compared them with commercially available gummies containing only natural colorants. We also developed a method to extract anthocyanins from gelatine. The color of commercial gummy, Black forest Organic, containing no artificial colorants, was measured with a ColorQuestXE and expressed based on the CIE Lab, chroma, and hue. The gelatine gummies were prepared using published formulations with slight modifications and colored by adding 14.28ml of American elderberry (acylated anthocyanin). The color of the gummy also monitored. Different methods (using a combination of acetone, methanol, and water) were used to recover the anthocyanins from the gummies, for future quantification by the pH differential method. The color of the American elderberry gelatin gummy was in the blue to red (hue angle of 394°) in the L*c*h color scale. It meets the physical color to the observer's eyes. The commercial gelatin gummy was in red to the yellow region (hue angle of 37.7° ) in L*C*h color scale. The method with the highest efficiency for anthocyanin extraction from gelatine was to dissolve it in acidified hot water (40C), precipitate gelatine at pH4.5 (isoelectric point) with sufficient ethanol, centrifuge 10min at 4000rpm to remove solids, and evaporate to recover anthocyanin from ethanol. The anthocyanin content in gelatine gel at Day 0 and Day 3 was 8mg/100g and 6.8mg/100g, respectively, with a 15% decrease in total anthocyanin content. Research Project Title: Molecular recognition of phosphate at the surface of water through synthetic receptors

Student Presenter: Alexander Grooms

Faculty Mentor: Heather Allen

Faculty Mentor Department: Chemistry and Biochemistry

Research Abstract: Phosphate, a common component of fertilizers, is necessary for modern crop growth. Excess phosphate from fertilizers is carried to bodies of water through agricultural runoff, promoting the growth of algal blooms. As algae dies, oxygen levels are lowered, which causes death of aquatic species. The fundamentals of phosphate capture have been explored by use of new biologically inspired synthetic receptors. These receptors are evaluated using surface pressure area isotherms. Where the surface pressure is monitored by the Wilhemy plate method. Phosphate in solution binds to the receptor during the isotherm, which results in an expansion of the receptor monolayer to larger mean molecular areas. The dioctadecyl guanidinium chloride receptor, which was inspired by the phosphate-arginine binding motif, has been thoroughly evaluated with water and phosphate sub phases. Results show that this receptor gives rise to a stable monolayer on both water and phosphate solutions. Further work is required to quantify the binding capabilities of these receptors. Future work will include using surface spectroscopic techniques to quantify the phosphate binding affinities of these receptors. Research Project Title: Generation of vibrationally excited CO in a between carbon vapor and oxygen

Student Presenter: Ilya Gulko

Faculty Mentor: Igor Adamovich

Faculty Mentor Department: MAE and Chemical Physics

Research Abstract: Recent work in the Nonequilibrium Thermodynamics Laboratories demonstrated that a chemical reaction between carbon atoms, generated in a high-temperature arc discharge with graphite electrodes, and molecular oxygen produces highly vibrationally excited CO. In these experiments, absolute among CO vibrational levels has been detected in a collision- dominated environment, suggesting the feasibility of development of a novel CO chemical laser. This laser would be able to operate in a high-speed airflow over a hypersonic vehicle, with carbon vapor generated by surface ablation or by evaporation of amorphous carbon powder injected into the flow. Carbon atoms would then react with molecular oxygen present in the airflow. To achieve optical sufficient for laser power generation, the rate of C atom production needs to be scaled up by at least an order of magnitude, compared to the yield obtained in the arc discharge used in the previous work. The approach used in the present work is to increase the high-temperature plasma volume, by using an inductively coupled volumetric RF discharge sustained in a mixture of micron-size amorphous carbon particles and argon carrier gas, in a custom-design quartz cell. The temperature in the RF discharge, up to T = 2600 K at the discharge power of 1 kW, is inferred from CO emission spectra measured by a Fourier Transform Infrared (FTIR) spectrometer. Carbon vapor generated in the discharge is injected into the low-temperature main flow of argon or nitrogen, and oxygen is added to the flow further downstream. Preliminary experiments indicate that at these conditions, vibrationally excited , up to vibrational level v=6, is produced in the reaction of C atoms with oxygen molecules, C + O2 → CO(v) + O, at low-temperature conditions in the laser , at T = 400 K. The vibrational populations of the CO product generated by this reaction are inferred from CO infrared emission spectra. Further experiments quantifying the yield of vibrationally excited CO, as well as lasing tests are underway. Research Project Title: Interfacial molecular recognition of phosphate by new biologically-inspired receptors

Student Presenter: Brittany Shook

Faculty Mentor: Heather Allen

Faculty Mentor Department: Chemistry

Research Abstract: Modern agricultural practices have increased the concentration of phosphate in fresh waterways, causing disruptions to these ecosystems. Algal blooms initiate hypoxic waterways which leave the water unpotable and the fish populations at risk. This project investigates various synthetic receptors that have high binding affinities to phosphate at the air/aqueous interface. The receptors are biologically-inspired with charged thiouronium and guanidinium headgroups anchored at the surface by long alkyl chains. These receptors can capture phosphate and remove it from the water, which would reduce the negative ecological effects of phosphate. Langmuir isotherms using the Wilhemy plate method were used to study these binding events. The receptor monolayer is compressed and various concentrations of phosphate were tested. High binding affinity, stability, and ion selectivity are the most important characteristics of a promising receptor. The thiouronium functional group was found to have a promisingly high binding affinity to phosphate. Future work will consistent of surface to study the mechanistic binding events of these receptors. Research Project Title: Ionization state of phosphoric and phosphonic acid monolayers at the air-water interface

Student Presenter: Morgan Smeltzer

Faculty Mentor: Heather Allen

Faculty Mentor Department: Chemistry

Research Abstract: Phospholipids are vital to the study of aerosol chemistry and model systems of cell membranes. To better understand the role of phospholipids in these systems, which occur at aqueous interfaces, it is important to determine their ionization state at different pH. The surface pKa for the first deprotonation of the phosphate headgroup at the aqueous interface is still not agreed upon in the literature, and reported pKa values range from 1.7 to 7. The nearly 200,000 fold difference in acidity makes it challenging to study these systems. This work used lysophosphatidic acid, phosphatidic acid, and phosphonic acid to study the first ionization state of the phosphate and phosphonate headgroups. The Wilhemy plate method was used to measure surface pressure using a Langmuir-Blodgett trough at various bulk pH. Surface pressure measurements provided insight into the ionization state of these monolayers since the surface pressure decreased as the headgroups became charged. Brewster Angle Microscopy (BAM) was also employed to visualize the morphology of the monolayer, and the van der Waals interactions between these molecules.

Research Project Title: Thread spray ionization

Student Presenter: Bridget Walsh

Faculty Mentor: Abraham Badu- Tawiah

Faculty Mentor Department: Chemistry and Biochemistry

Research Abstract: Diazepam is a pharmaceutical drug that is used to treat anxiety, muscle spasms, and seizures. This research project utilizes hydrophobic thread spray mass spectrometry to characterize diazepam and other drugs. We will perform stability tests on both diazepam and cocaine over the span of several weeks, and the drugs will be characterized on three different thread types, untreated cotton thread, cotton thread treated with silane vapor for 30 minutes, and cotton thread treated with silane vapor for 60 minutes. This silane treatment converts the surface hydroxyl groups of the thread to a hydrophobic group, increasing its hydrophobicity; the longer the treatment, the larger amount of surface hydroxyl groups removed. These tests will support how well an analyte can be detected overtime, without degradation occurring, and will compare how well different thread types work with the blood samples. We will analyze the drugs in pure ethyl acetate solutions and in blood at 250 ppb concentrations. This project has analytical applications, as detection of illicit and pharmaceutical drugs in biofluid samples is a common procedure. While urine screening, hair screening, and blood screening are currently used for drug detection, they require a lot of sample preparation, and are inefficient. Hydrophobic thread spray requires little to no sample preparation, and the drug can be detected within a matter of seconds. Another advantage of hydrophobic thread spray is that the amount of time the drug has been in the sample can be determined by which metabolite of the drug is most heavily detected. If the cocaine is present in high concentrations as benzoylecgonine, one of its metabolites, it has been in the sample longer than if the mass of cocaine was most heavily detected. The ultimate end goal of this project, however, is point- of- care diagnostics. The idea is to be able to diagnose diseases from a blood or urine sample. Solidifying this method with drug detection is a crucial stepping stone in the process of point- of- care diagnostics because drugs are small molecules, so if they are successfully detectable by this method, this method should also work with immunoassay.

Research Project Title: Relationship between the thermodynamic equilibrium spreading pressure and pH for amphiphilic phosphonic acids

Student Presenter: Mia Lin Zerkle

Faculty Mentor: Heather Allen

Faculty Mentor Department: Chemistry and Biochemistry

Research Abstract: Equilibrium spreading pressure (ESP) is a thermodynamic stability measurement of a monolayer in equilibrium with its crystalline phase. A recent publication of a phosphonic acid discovered that ESP values varied greatly depending on the pH of the bulk solution. This study further explores the ESP of phosphonic acids, and develops a better fundamental understanding of this equilibrium process. The ESP values of hexadecylphosphonic acid and octadecylphosphonic acid were determined by spreading their crystalline form over subphases of water or acidic solution. Surface pressure was recorded on a Langmuir trough using the Wilhelmy plate method with a platinum plate as the probe. Hexadecylphosphonic acid on acidic solution resulted in a higher ESP value than the observed ESP on water. This is attributed to the desorption of the singly deprotonated phosphonic acid into the solution. In contrast with the hexadecylphosphonic acid, the octadecylphosphonic acid spread over an acidic subphase tended towards lower ESP values than on water. These results lend further support to the conjecture that the ionization state of the phosphonic acid, which varies with pH, could result in differing collapse mechanisms that would account for such a variation in ESP.