Robin Turner

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Adsorption of a carboxylated silane on gold : characterization and application to PDMS-based electrochemical cells (2016)

Integrated sensing and biosensing microfluidic systems often require sealing between polydimethylsiloxane (PDMS), glass, and gold interfaces. Studying substances that can self-organize onto glass and gold surfaces may achieve these goals and pave the way for new technological advances. Work presented in this thesis focuses on characterizing the adsorption of N-[(3-trimethoxysilyl)propyl]ethylene-diamine triacetic acid (or TMS-EDTA) on Au and applying this knowledge to construct leak-free PDMS-based electrochemical cells. First, surface analysis of TMS-EDTA-modified Au surfaces was conducted using various techniques. Water contact angle measurements and X-ray photoelectron spectroscopy confirm that the carboxylated silane can chemically modify Au surfaces. Atomic force microscopy studies indicate that a uniform surface coverage with monolayer thickness is formed. Infrared spectroscopy studies indicate that there is little evidence of siloxane cross-linking. Surface plasmon resonance results suggest that the carboxylates on TMS-EDTA-modified Au are available for streptavidin immobilization. Second, electrochemistry was used to determine the Gibbs free energies of adsorption of TMS-EDTA on Au under aqueous conditions. Electrochemical differential capacitance measurements reveal that the potential-dependent free energies of adsorption are ∼ - 20 to - 30 kJ/mol (for potentials between - 0.5 and 0.2 V) in the complex electrolyte solution used. Furthermore, at highly negative potentials ( ∼ - 1.1 V), TMS-EDTA adsorbs minimally onto the Au surface. Third, PDMS surfaces were functionalized to present primary amino groups, and glass or gold slides were functionalized to present carboxyl groups. Strong bonding was achieved by bringing the two surfaces in contact and reacting at room temperature. Shear tests reveal that the novel carboxyl-amine bonding strategy achieved a comparable bond strength as the conventional methods. Subsequently, TMS-EDTA was applied to construct leak-free PDMS-based electrochemical cells. Pressure leak tests were conducted to provide a more realistic measure of the bond strengths under aqueous conditions. A method to electrochemically remove the adsorbed TMS-EDTA layer off of the Au electrode, while maintaining the sealed cell chamber, was also developed. The characterization studies and fabrication strategy presented have led to the development of leak-free PDMS-based electrochemical devices that are suitable for sensing and biosensing applications.

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Investigating biomolecular structure using fiber-optic UV resonance Raman spectroscopy (2011)

The observation of biomolecular structure is critical for the fundamental understanding of biological function. In this work, Fiber-optic UV Resonance Raman Spectroscopy (FO-UVRRS) was employed to study a number of structure-function relationships.A suite of metal-containing dioxygenase enzymes were studied in order to determine the substrate protonation state in the enzyme-substrate complex. Two enzymes with a high degree of sequence identity were studied, where one naturally incorporates a Fe²⁺ metal and the other uses Mn²⁺. Both enzymes react with the same substrate to form an equivalent muconic semialdehyde product. The enzymes are capable of incorporating the non-native metal into the enzymes with no effect on the kinetic properties. The Raman results show that the nature of the metal has no effect on the substrate protonation state. The degradation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) by BphD in the biphenyl catabolic pathway was studied using FO-UVRRS. Previous reports suggest that enol-keto tautomerization was a critical step in the mechanism of the carbon-carbon bond hydrolase. Hydrolytically impaired variants of BphD were used to study the binding mode of HOPDA. The results show that HOPDA binds to these variants in a strained-enolate geometry and does not undergo an enol-keto tautomerization upon enzyme binding. A fundamental FO-UVRRS study of locked nucleic acid (LNA) olgiomers was performed. LNA bases contain a C4’ to O2’ methylene bridge within the furanose ring of the nucleotide. The research results show that incorporation of a LNA induces a conformational change in the glycosyl bond between the backbone and the base. The results also show that incorporation of an LNA base induces changes in the secondary structure of the nucleic acid.In another study, chemical contamination of synthetic DNA oligomers was observed using FO-UVRRS. The data showed that commercially-available oligomers purified using standard desalting conditions are contaminated with residual benzamide. The results show that a previously assigned –NH₂ scissors vibration in the Raman study of oligomers overlaps with a prominent benzamide vibrational band, calling into question these previous assignments. The results also demonstrate that the extent of benzamide contamination varies from sample-to-sample and between different commercial sources.

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New Signal Enhancement Algorithms for One-Dimensional and Two-Dimensional Spectroscopic Data (2011)

In research, the usefulness of the analytical results is crucially dependent upon the quality of the measurements. However, data measured by instruments is always corrupted. The desired information—-the "signal"-—may be distorted by a variety of effects, especially noise. In spectroscopy, there are two additional significant causes of signal corruption—-instrumental blurring and baseline distortion.Consequently, signal processing is required to extract the desired signal from the undesired components. Thus, there is an on-going need for signal enhancement algorithms which collectively can 1) perform high quality signal-to-noise-ratio (SNR) enhancement, especially in very noisy environments, 2) remove instrumental blurring, and 3) correct baseline distortions. Also, there is a growing need for automated versions of these algorithms. Furthermore, the spectral analysis technique, Two-Dimensional Correlation Spectroscopy (2DCoS), needs similar solutions to these same problems.This dissertation presents the design of four new signal enhancement algorithms, plus the application of two others, to address these measurement problems and issues. Firstly, methods for one-dimensional (1D) data are introduced—-beginning with an existing algorithm, the Two-Point Maximum Entropy Method (TPMEM). This regularization-based method is very effective in low-SNR signal enhancement and deconvolution. TPMEM is re-examined to clarify its strengths and weaknesses, and to provide ways to compensate for its limitations. Next, a new regularization method, based on the chi-squared statistic, is introduced and demonstrated in its ability for noise reduction and deconvolution. Then, two new 1D automated algorithms are introduced and demonstrated: a noise filter and a baseline correction scheme.Secondly, a new two-dimensional (2D) regularization method (Matrix-MEM or MxMEM), derived from TPMEM, is introduced and applied to SNR enhancement of images. Lastly, MxMEM and 2D wavelets are applied to 2DCoS noise reduction.The main research contributions of this work are 1) the design of three new high performance signal enhancement algorithms for 1D data which collectively address noise, instrumental blurring, baseline correction, and automation, 2) the design of a new high performance SNR enhancement method for 2D data, and 3) the novel application of 2D methods to 2DCoS.

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Master's Student Supervision (2010 - 2020)
Study of mass transport in electro fluid dynamic devices (2019)

Mass transport in one dimensional (1D) and two-dimensional (2D) electro-fluid dynamic devices for chemical separation is systematically studied. For 1D EFD devices, like capillary electrophoresis (CE), adding external pressure during the process usually results in unwanted band broadening. However, frontal analysis (FA) can potentially benefit from the external pressure by significantly reducing the amount of time needed for analysis. Therefore, the possible impact of the pressure-assisted capillary electrophoresis frontal analysis (PACE-FA) is studied. With a typical CE-FA set-up and a typically used length and internal diameter of the capillary used, it was found that the detected concentrations of analyte will not be significantly affected by an external pressure less than 5 psi in the simulation model. In addition, the measured ligand concentration in PACE-FA was also not affected by common variables such as molecular diffusion coefficient and capillary length within the tested range. By using PACE-FA to study the binding interactions between hydroxypropyl β-cyclodextrin (HP-β-CD) and small ligand molecules, the binding constants determined by CE-FA (18.3±0.8 M-1) and PACE-FA (16.5±0.5 M-1) are found to be similar. Based on the experimental results, it is concluded that PACE-FA can reduce the time of binding analysis while maintaining the accuracy of the measurements. For 2D-EFD device, an EFD desalination chip was designed; the desalting process was then modeled (both in single-element and multiple-element geometric design) and was simulated. The simulation results showed that the ionic components were separated and outflowed to specific channels as designed which suggests a potential alternative way for microscale-desalination. The result of this study also showed that the performance of the device relied on the geometry of the device relatively heavily and can be improved by applying a stronger electric field at the electrodes.

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Raman Spectroscopic Study of Induced Pluripotent Stem Cells: Characterization, Identification, and Discrimination (2012)

Raman microspectroscopy is a non-destructive, label-free technique that offers information-rich molecular analysis of living cells. This work is the first reported Raman spectroscopic study of human induced pluripotent cells (hiPSCs), a very promising new source of non-embryonic pluripotent stem cells for drug screening, toxicity assessment, regenerative therapies, and clinical research. The Raman signatures of hiPSCs and human embryonic stem cells (hESCs) were found to be highly similar, and both distinguishable from differentiated hESCs in terms of relative Raman peak intensities and variances. Principal component analysis (PCA) of the Raman spectra demonstrated a clear distinction between hiPSCs and differentiated hESCs. Additionally, the effects of culture confluencies and cell line differences on Raman spectra of hiPS cells was investigated. It was confirmed that the spectral similarity between hiPSCs and hESCs, along with the dissimilarity between hiPSCs and differentiated hESCs were qualitatively consistent over various cell culture confluencies, and between the two available hiPS cell lines. Therefore, the results suggested that the overall cellular composition of hiPSC was more similar to that of the hESC that these cells were designed to resemble than the somatics cell from which they were derived. It is suggested that the observed spectral differences between hiPSC and hESC may be due to factors relating to reprogramming (rather than cell density difference or cell line artifacts). Attempts were also made to investigate how Raman features of hiPS cells change during their differentiation. The pluripotent and differentiated iPSCs exhibited significantly different Raman spectral profiles; these differences were qualitatively similar to, but less marked, than differences between pluripotent and differentiated hESC. Overall, this work contributed important new data and practical insights into the utility of Raman microspectroscopy for characterization, identification, and discrimination of iPSCs and hESCs.

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Raman Microspectroscopy in the Analysis of Plant Cuticular Membranes (2010)

The outside of plants, including fruit, are covered by a cuticular membrane (CM). This membrane serves as protection from environmental factors. The basic composition of the CM is well understood, however, there are always variations among different species. The CM consists of a cutin matrix, which is the structural component, and cuticular waxes. The study of the CM for both structural and compositional features has been accomplished for a variety of species. The instrumentation methods used to complete these analyses have been mostly destructive and at most been able to provide spatial distribution information along the vertical axis. Raman microspectroscopy has been used previously to quantify and map triterpenoid concentrations in Prunus laurocerasus leaf CMs. This data showed transverse variations in concentration across the CM. The goal of the current work is to explore the viability of using Raman microspectroscopy to analyze other species and classes of compounds. Initially, alkylresorcinols in rye leaf waxes, artificial wax mixtures, and rye leaf CMs were examined. Using the current instrumentation it was not possible to quantify and detect alkylresorcinols in rye CMs, but it may become a viable option in the future. Based on the previous work with triterpenoids, this class of compounds was attempted next. Triterpenoids in tomato CMs were investigated resulting in the discovery that, while triterpenoids are not detectable in the tomato CM due to the presence of polysaccharides, lycopene can be detected. It was found that it is present in the remains of the epidermal cells after digestion of the cell walls (epidermal remnants), but not in the CM itself. The analysis of other species for triterpenoids was attempted including the leaves of Rosa canina, Ligustum vulgare, Kalanchoe daigremontiana, and the fruit of the Prunus avium and Prunus laurocerasus. While quantification of triterpenoids was not successful, a lot was learned about the variables which go into a successful Raman analysis of triterpenoids in plant CMs. These variables include the relative amount of triterpenoids to wax and wax to CM, along with the wax coverage (µg/cm²). These results will aid in future attempts at Raman analysis of triterpenoids in CMs.

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