Darcy Lichlyter - Academia.edu (original) (raw)

Papers by Darcy Lichlyter

Sensors and Actuators B-chemical, 2007

ABSTRACT

Biosensors & Bioelectronics, 2003

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance... more We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab?) 2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody Á/protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor Á/acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots. #

Analytical and Bioanalytical Chemistry, 2005

A novel optical biosensor technique is being developed for the early detection of myocardial infa... more A novel optical biosensor technique is being developed for the early detection of myocardial infarction by utilizing the distance-dependent chemical transduction method of fluorescence resonance energy transfer (FRET). The FRET process requires two fluorophores termed the donor and the acceptor. When in close proximity, the donor absorbs energy from the excitation source and non-radiatively transfers the energy to the acceptor, which in turn emits fluorescent energy. This distance-dependent property was utilized to detect conformational changes when antibodies combine with their respective antigens. The fluorophores were conjugated to an antibody–Protein A complex and then immobilized via silanization to the distal ends of optical fibers. Three different antibody–Protein A complexes were immobilized: generic IgG, cardiac Troponin T (cTnT), and cardiac Troponin I (cTnI). Results showed that upon the addition of the specific antigens, the antibodies underwent a conformational change, reducing the distance between the FRET fluorophores. The generic IgG responded to 233 nM antigens, whereas the cTnT biosensor had a limit of detection of 75 nM, and the cTnI biosensors had a limit of detection of 94 nM.

Biosensors & Bioelectronics, 2005

We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastat... more We have been investigating a fluorescence dual binding biosensor to detect calpastatin. Calpastatin is a protein found in meat and it is a regulator of meat tenderness. The ability to accurately predict the calpastatin concentration of beef with a biological sensor at the time of grading would lead to a more accurate assessment of the overall palatability of beef when it reaches the consumer. Meat can then be labeled as tender or tough, which would greatly enhance meat processors' ability to grade meat, allowing them to recover lost revenue. The biosensor technique utilized the chemical transduction principle of fluorescence resonance energy transfer (FRET). FRET requires the use of two fluorophores, termed a donor and acceptor. In this study, the donor fluorophore was conjugated to the protein, -calpain, while the acceptor fluorophore was conjugated to a monoclonal antibody. The results showed that in the presence of calpastatin, the labeled -calpain and antibody would bind to calpastatin, reducing the distance between the two proteins and eliciting a measurable change in fluorescence. The FRET dual binding technique was tested in heated and unheated meat extract, and a limit of detection for calpastatin was 120 ng/ml in diluted heated meat extract with no significant response in the unheated meat extract. Stable response times were achieved within 5 min. The proof-of-principle of utilizing a FRET dual binding technique to detect calpastatin in heated meat extract has been established.

International Journal of Cancer, 1999

ErbB-2 is overexpressed in several human cancers and conveys a transforming activity that is depe... more ErbB-2 is overexpressed in several human cancers and conveys a transforming activity that is dependent on tyrosine kinase activity. Antibodies and T cells to ErbB-2 have been isolated from cancer patients, indicating ErbB-2 as a potential target of active vaccination. In this study, 3 mutant ErbB-2 DNA constructs encoding full-length, ErbB-2 proteins were tested as tumor vaccines. To eliminate tyrosine kinase activity, the ATP binding lysine residue 753 was substituted with alanine by replacing codon AAA with GCA in mutant ErbB-2A. To direct recombinant ErbB-2 to the cytoplasm where major histocompatibility complex (MHC) I peptide processing takes place, the endoplasmic reticulum (ER) signal sequence was deleted in cyt ErbB-2. The third construct cyt

International Journal of Cancer, 1996

A synthetic peptide E474 SFAVATTAL, derived from the sequence of mouse mammary tumor virus envelo... more A synthetic peptide E474 SFAVATTAL, derived from the sequence of mouse mammary tumor virus envelope protein, was previously shown to bind class I MHC Kd. Immunization of BALB/c mice with E474 in 50% incomplete Freund's adjuvant (IFA) followed by in vitro stimulation of immune cells with E474-coated antigen-presenting cells resulted in peptide-specific cytotoxic T lymphocytes (CTL). Furthermore, anti-E474 CTL lysed mammary tumor cell lines D2F2 and D2A1, derived from a spontaneous tumor that arose in BALB/c pre-neoplastic hyperplastic alveolar nodule (HAN) D2 line. Expression of Kd by D2A1 and D2F2 cells was verified by flow cytometry, and lysis of D2 tumor cells was blocked by monoclonal antibody 31-34-S, which interacted with the peptide-binding region of Kd, supporting the recognition of E474 in Kd by anti-E474 CTL. Immunization of BALB/c mice with E474 before D2F2 tumor challenge resulted in reduced tumor growth. Therefore, E474 is naturally processed and presented by these tumor cells and can induce anti-tumor immunity.

Journal of Immunotherapy, 1995

British Journal of Cancer, 1998

Molecular changes associated with breast cancer progression were characterized using the MCF-1 OF... more Molecular changes associated with breast cancer progression were characterized using the MCF-1 OF cell series. MCF-1 OF was established from fibrous mastectomy tissue of a patient without detectable cancer. In vitro treatment of MCF-1OF cells with benzo(a)pyrene resulted in a transformed subclone MCF-1OF-BP1 (BP1). Transfection of clone BP1 with T24-Hras resulted in the tumorigenic line MCF-1 OF-BP1 -Tras (BP1 -Tras). Using flow cytometry, the expression of HLA I, ERBB-2 and MUC-1 was found to be comparable in 'normal' MCF-1 OF, transformed BP1 and tumorigenic BP1-Tras cells. Glycosylated mucin is elevated in BP1 but reduced in BP1-Tras cells. Using mRNA differential display analysis, cDNA profiles of the 'normal', transformed and tumorigenic cell lines were strikingly similar, yet distinct and elevated expression of several common cDNA fragments was detected in BP1 and BP1-Tras when compared with MCF-1OF cells. These fragments were cloned and sequenced. The sequences of clones Ti-360 and C4-310 are homologous to two reported EST cDNA clones from human fetal tissue and were further characterized. Elevated expression of the genes corresponding to clones Ti -360 and C4-310 was verified using Northern blotting. High-level expression of these genes was also detected in the breast cancer cell line MCF-7 that was derived from the pleural effusion of a patient with advanced breast cancer. Therefore, specific molecular changes associated with breast cancer development were identified and may be indicators of neoplastic progression.

European Journal of Immunology, 1994

Mouse mammary tumor virus MMTV(C4) encodes a Vβ2-specific superantigen. In Vβ2 transgenic (TG2) m... more Mouse mammary tumor virus MMTV(C4) encodes a Vβ2-specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4+ T cells and suppressed thymopoiesis. The number of peripheral CD8+ T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8+ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4+ T cells from MMTV(C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4+ T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.

Bioorganic Chemistry, 2005

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexe... more Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.

Tetrahedron, 2010

It has been shown that compounds containing the p-N,N,-dialkylaminobenzylidene cyanoacetate motif... more It has been shown that compounds containing the p-N,N,-dialkylaminobenzylidene cyanoacetate motif can serve as fluorescent non-mechanical viscosity sensors. These compounds, referred to as molecular rotors, belong to a class of fluorescent probes that are known to form twisted intramolecular charge-transfer complexes in the excited state. In this study we present the synthesis and spectroscopic characterization of these compounds as viscosity sensors. The effects of the molecular structure and electronic density of these rotors to the emission wavelength, fluorescence intensity and viscosity sensitivity are discussed.

Abstract Molecular rotors are fluorescent molecules that are characterized by the ability to form... more Abstract Molecular rotors are fluorescent molecules that are characterized by the ability to form twisted states through the rotation of one segment of the structure with respect to the rest of the molecule. Intramolecular rotation changes the ground-state and excited-state energies, and molecular rotors deexcite from the twisted state either without photon emission or with a different wavelength than from the LE state. Intramolecular rotation is strongly dependent on the solvent. Solvent polarity, hydrogen bond formation, isomerization, excimer formation, and steric hindrance are predominant forms of solvent–fluorophore interaction. Of highest importance is steric hindrance, because it links the solvent's microviscosity to the formation rate of TICT states, which, in turn, determines the spectral emission. For this reason, molecular rotors have found a wide range of applications as fluorescent sensors of microviscosity and solvent free volume. Application examples include bulk viscosity measurement, probing dynamics of polymer formation, protein sensing and probing of protein aggregation, and microviscosity probing in living cells. Graphical Abstract

Journal of The American Chemical Society, 2006

Variations in fluid viscosity are linked to a variety of functions and diseases both at the cellu... more Variations in fluid viscosity are linked to a variety of functions and diseases both at the cellular level (e.g., membrane and cytoplasmic viscosity changes in cell signaling modulation) 1 and at the organismal level (e.g., blood, plasma, or lymphatic fluid viscosity changes in diabetes, hypertension, infarction, and aging). 2 It has been proposed that monitoring of biofluid viscosity could provide a diagnostic tool for the detection of diseases. Since mechanical devices do not provide the spatial and temporal resolution needed, a new type of fluorescent-based viscosity sensors was developed. 4 These sensors are based on a class of environmentsensitive fluorescent dyes that are characterized by a viscositydependent emission quantum yield. 4,5 The chemical structure of these dyes contains an electron donor unit (such as a nitrogen atom) in conjugation with an electron acceptor unit (such as a nitrile). Upon photoexcitation, the two units can rotate relative to each other in a manner that is dependent on the viscosity of their environment. Representative examples of such fluorescent rotors are 9-(dicy-anoVinyl)julolidine (DCVJ, 1) and 2-cyano-3-(4-dimethylaminophenyl)acrylic acid methyl ester (CMAM, 2) ( ). 5 Their viscosity-dependent fluorescent quantum yield is described by the Förster-Hoffmann equation (eq 1). 6 Fluorescent molecular rotors have been used for viscosity studies that are performed by steady-state fluorescence through emission intensity measurements. This method suffers, however, from drawbacks arising from changes of the fluid optical properties and fluctuations in dye concentrations. An additional disadvantage is that a calibration curve is needed for the absolute determination of viscosity. 5 As a consequence, changes in fluid properties and dye concentration may cause erroneous readings.

Sensors and Actuators B-chemical, 2009

a b s t r a c t Molecular rotors are a subgroup of twisted intramolecular charge transfer fluorph... more a b s t r a c t Molecular rotors are a subgroup of twisted intramolecular charge transfer fluorphores with high viscosity sensitivity. These fluorphores enjoy a high and growing popularity as in-solution nonmechanical viscosity sensors. A solid-state sensor with molecular rotor molecules covalently bound to a sensor substrate, such as glass, would be desirable to avoid contaminating the solution with a fluorescent solute and to obtain a more universal sensor platform. Although the proof-of-principle was provided [M.A. Haidekker, W.J. Akers, D. Fischer, E.A. Theodorakis, Optical fiber-based fluorescent viscosity sensor, Opt. Lett. 31 (2006) 2529-2531], a marked loss of sensitivity was observed with rotor immobilization. The influence of immobilization protocol and choice of linker was analyzed more systematically in this study. Among three silane linkers, ureidopropyl-trimethoxysilane was found to provide the highest sensitivity, and a higher immobilization density generally caused higher sensitivity. A glass slide serving as a substrate provided better sensor performance than an optical fiber because the glass-based sensor has a larger surface and no autofluorescence; conversely, an optical fiber is capable of measuring smaller volumes of fluid. Overall, we found that overall sensor performance strongly depends on the details of the immobilization protocol, and without specialized equipment, large variability between sensors exists. Sensitivity was measured as the exponent x in the intensity-viscosity power-law relationship. While in-solution molecular rotors exhibit values of x = 0.6, covalently linked molecular rotors did not exceed x = 0.15. This value is still sufficiently high to produce a sensor with a precision comparable to mechanical rheometers. However, further research is needed to examine the causes of between-sensor variability and to create more reproducible sensors.

Microfluidics and Nanofluidics, 2011

This study describes an analytical model and experimental verifications of transport of non-magne... more This study describes an analytical model and experimental verifications of transport of non-magnetic spherical microparticles in ferrofluids in a microfluidic system that consists of a microchannel and a permanent magnet. The permanent magnet produces a spatially non-uniform magnetic field that gives rise to a magnetic buoyancy force on particles within ferrofluid-filled microchannel. We obtained trajectories of particles in the microchannel by (1) calculating magnetic buoyancy force through the use of an analytical expression of magnetic field distributions and a nonlinear magnetization model of ferrofluids, (2) deriving governing equations of motion for particles through the use of analytical expressions of dominant magnetic buoyancy and hydrodynamic viscous drag forces, (3) solving equations of motion for particles in laminar flow conditions. We studied effects of particle size and flow rate in the microchannel on the trajectories of particles. The analysis indicated that particles were increasingly deflected in the direction that was perpendicular to the flow when size of particles increased, or when flow rate in the microchannel decreased. We also studied “wall effect” on the trajectories of particles in the microchannel when surfaces of particles were in contact with channel wall. Experimentally obtained trajectories of particles were used to confirm the validity of our analytical results. We believe this study forms the theoretical foundation for size-based particle (both synthetic and biological) separation in ferrofluids in a microfluidic device. The simplicity and versatility of our analytical model make it useful for quick optimizations of future separation devices as the model takes into account important design parameters including particle size, property of ferrofluids, magnetic field distribution, dimension of microchannel, and fluid flow rate.

Bioorganic Chemistry, 2004

Recent research shows high potential for some p-N,N-dialkylaminobenzylidenecyanoacetates, part of... more Recent research shows high potential for some p-N,N-dialkylaminobenzylidenecyanoacetates, part of a group known as Xuorescent molecular rotors, to serve as Xuorescent, nonmechanical viscosity sensors. Of particular interest are molecules compatible with aqueous environments. In this study, we present the synthesis and physical characterization of derivatives from 9-(2-carboxy-2-cyanovinyl)-julolidine and related molecules. All compounds show a power-law relationship of Xuorescence emission with the viscosity of the solvent, diVerent mixtures of ethylene glycol and glycerol to modulate viscosity. Compounds with high water solubility exhibit the same behavior in aqueous solutions of dextran, where the dextran concentration was varied to modulate viscosity. In addition, some compounds have been found to have low sensitivity towards changes in the pH in the physiological range. The compounds presented show promise to be used in bioXuids, such as blood plasma or lymphatic Xuid, to rapidly and non-mechanically determine viscosity. (M.A. Haidekker).

Cheminform, 2010

A variety of molecular rotors, mainly p-N,N-dialkylaminobenzylidene compounds, are prepared from ... more A variety of molecular rotors, mainly p-N,N-dialkylaminobenzylidene compounds, are prepared from the aromatic aldehydes and malonic acid derivatives.