Christa Colyer | Wake Forest University (original) (raw)

Papers by Christa Colyer

Cell Biochemistry and Biophysics, 2000

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues... more Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent label molecule. To minimize the amount of additional sample handling and time associated with such labeling procedures, not to mention the sometimes-stringent pH and temperature controls they require, noncovalent labeling is presented as a viable alternative. This review article considers established methods for noncovalent labeling of proteins for their subsequent analysis by CE-LIF. Label molecules suitable for excitation and emission in the ultraviolet, visible, and near-infrared regions of the spectrum are enumerated for a variety of protein analytes.

This case provides a brief, factual account of the pioneering work of Ignaz Semmelweis and his ef... more This case provides a brief, factual account of the pioneering work of Ignaz Semmelweis and his efforts to remedy the problem of childbed fever in mid-nineteenth century Europe. The case was designed to be used in a freshman seminar class entitled "Scientific Serendipity " as a concrete example of the "Scientific Method " in practice. It is important for students to understand that although there is no single

Molecules, 2019

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and bi... more Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer ident...

Molecular therapy. Nucleic acids, Jan 15, 2016

Breast cancer (BC) results in ≃40,000 deaths each year in the United States and even among surviv... more Breast cancer (BC) results in ≃40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l), a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox)...

Analytical chemistry, Jan 3, 2015

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with diff... more We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (H-aggregates and J-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with J-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10(1.7) M(-1)), with no such enhancement for SQ-BA dyes with longe...

Proceedings of International Solid State Sensors and Actuators Conference (Transducers '97)

ABSTRACT The challenges and solutions for designing automated microfluidic systems for the entire... more ABSTRACT The challenges and solutions for designing automated microfluidic systems for the entire series of mixing, reaction and separation of proteins and antibodies integrated directly on a chip are presented. Examples of clinically relevant assays such as analysis of human serum proteins for clinical diagnostics and immunoassay of a therapeutic drug for asthma, theophylline, are reported

Journal of Chromatography B, 2015

ABSTRACT The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide... more ABSTRACT The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide (M3G) and morphine-6-beta-d-glucuronide (M6G) is useful for therapeutic drug monitoring and forensic identification of drug use. In particular, capillary electrophoresis with mass spectrometry (CE-MS) represents an attractive tool for opioid analysis. Whereas volatile background electrolytes in CE often improve electrospray ionization for coupled MS detection, such electrolytes may reduce CE separation efficiency and resolution. To better understand the effects of background electrolyte (BGE) composition on separation efficiency and detection sensitivity, this work compares and contrasts method development for both volatile (ammonium formate and acetate) and nonvolatile (ammonium phosphate and borate) buffers. Peak efficiencies and migration times for morphine and morphine metabolites were optimal with a 25mM ammonium borate buffer (pH=9.5) although greater sensitivities were achieved in the ammonium formate buffer. Optimized CE methods allowed for the resolution of the isobaric morphine metabolites prior to high mass accuracy, electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS detection applicable to the analysis of urine samples in under seven minutes. Urine sample preparation required only a 10-fold dilution with BGE prior to analysis. Limits of detection (LOD) in normal human urine were found to be 1.0μg/mL for morphine and 2.5μg/mL for each of M3G and M6G by CE-ESI-QTOF-MS. These LODs were comparable to those for CE-UV analysis of opioid standards in buffer, whereas CE-ESI-QTOF-MS analysis of opioid standards in buffer yielded LODs an order of magnitude lower. Patient urine samples (N=12) were analyzed by this new CE-ESI-QTOF-MS method and no significant difference in total morphine content relative to prior liquid chromatography-mass spectrometry (LC-MS) results was found as per a paired-t test at the 99% confidence level. Whereas the LC-MS method applied to these samples determined only total morphine content, this new CE-ESI-QTOF-MS method allowed for species differentiation in addition to total morphine determination. By this method, it was found that M3G and M6G metabolites were present in a 5:1 concentration ratio, on average, in patient samples. Therefore, the CE-ESI-QTOF-MS method not only allows for total morphine concentration determination comparable to established LC-MS methods, but also allows for differentiation between morphine and its trace glucuronides, yielding additional biochemical information about drug metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

Analytical and bioanalytical chemistry, Jan 13, 2015

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, s... more Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer sy...

Journal of Chromatography A, 1996

... A, 732 (1996) 335-343 JOURNAL OF CHROMATOGRAPHY A Diffusive analyte loss in capillary electro... more ... A, 732 (1996) 335-343 JOURNAL OF CHROMATOGRAPHY A Diffusive analyte loss in capillary electrophoresis caused by delay in field application Christa L. Colyer, Jan C. Myland, Keith B. Oldham ... [11] S. Pleasance and P. Thibault, in P. Camilleri (Editor), Capillary ...

Journal of Chemical Education, 2001

Journal of AOAC International, 2013

New, sensitive, and selective spectrophotometric and spectrofluorometric methods have been develo... more New, sensitive, and selective spectrophotometric and spectrofluorometric methods have been developed for determination of clemastine hydrogen fumarate (Clem), loratadine (Lor), losartan potassium (Los), and ramipril (Ram) in both pure form and pharmaceutical formulations using 4-chloro-7-nitrobenzofurazan (NBD-Cl), which is a highly sensitive chromogenic and fluorogenic reagent. The relation between absorbance at 470, 467, 471, and 469 nm and the concentration was linear over the ranges 5–35, 10–100, 10–90, and 10–120 μg/mL for Clem, Lor, Los, and Ram, respectively. The complexation products were also measured spectrofluorometrically at the emission wavelength 535 nm for Clem, Lor, and Ram and at 538 nm for Los with excitation at 477 and 452 nm for Clem and Lor, respectively, and 460 nm for both Los and Ram. The fluorescence intensity was directly proportional to the drug concentration over the ranges 0.05–0.5, 5–20, 1–6, and 2–15 μg/mL for Clem, Lor, Los, and Ram, respectively. The...

ELECTROPHORESIS, 2002

Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often dif... more Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity and increased selectivity. However, direct LIF detection requires the proteinase analyte to be fluorescently derivatized prior to analysis. A viable alternative is offered by the present work, in which protein substrates are first labeled with BODIPY dye, a relatively pH-insensitive, high-fluorescence quantum yield dye. Upon binding of some 4-10 molecules of dye to a single protein, the dye is effectively fluorescence-quenched. Digestion of the BODIPY--labeled and quenched protein by an unlabeled enzyme yields smaller peptide fragments in which the fluorescence of associated BODIPY tags is restored. We will present how the fragmentation pattern of BODIPY-labeled casein changes as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest.

Canadian Journal of Chemistry, 1994

An aqueous solution containing supporting electrolyte and a small concentration of an electroacti... more An aqueous solution containing supporting electrolyte and a small concentration of an electroactive analyte is allowed to flow, under a gravitational head, through a polarized platinum tube. The tube is narrow enough that, during their few seconds of residence time within the tube, virtually all the analyte molecules are able to diffuse to the tube wall and be electrolytically destroyed. The analyte concentration is accurately calculable, when the applied voltage and head are optimized, from the measured flow rate of the solution and the steady-state electric current, the latter being background-corrected by subtracting the current under no-flow conditions. Though the detailed theory is complicated, the principles of the method and its experimental realization are straightforward.

Analytical Chemistry, 2012

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed an... more A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ ex = 630 nm, λ em = 660 nm) with a high affinity constant (K = ∼10 2.80 M −1) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R s = 6.09, and apparent plate number, N = 2.7−3.3 × 10 5), were successfully achieved.

ANALYSIS BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION: FROM SMALL MOLECULES TO WHOLE CELLS, Dec 1, 2010

Abstract: Manganese (Mn) is an essential trace element, but overexposure is characterized by Park... more Abstract: Manganese (Mn) is an essential trace element, but overexposure is characterized by Parkinson's like symptoms in extreme cases. Previous studies have shown Mn accumulation is exacerbated by dietary iron deficiency (ID) and disturbances in norepinephrine (NE) have been reported. Because behaviors associated with Mn neurotoxicity are complex, the goal of this study was to examine the effects of Mn exposure and ID-associated Mn accumulation on NE uptake in synaptosomes, extracellular NE ...

ELECTROPHORESIS, 2012

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6trimethylbenzene... more A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negativeion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO 4 2− ], thiosulfate [TSFA, S 2 O 3 2− ], and benzenedisulfonate [BZDS, C 6 H 4 (SO 3) 2 2− ], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.

Analytica Chimica Acta, 2003

... 2 to this enantiomer. Carmella et al. ... 559603. Full Text via CrossRef | View Record in Sc... more ... 2 to this enantiomer. Carmella et al. ... 559603. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (429). 3. SE Murphy and DA Spina. Carcinogenesis 15 (1994), p. 2709. View Record in Scopus | Cited By in Scopus (21). 4. PC Gupta, PR Murti and RB Bhonsle. ...

Journal of College Science Teaching, 2000