A reliable viability assay for Giardia is required for the development of disinfection process de... more A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO®-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight™ was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.
The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central eve... more The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen [1]. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.
We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically ina... more We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO®-9, hexidium and SYTO®-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.
Paralytic shellfish poisoning (PSP) is a major human health issue that occurs worldwide. Species ... more Paralytic shellfish poisoning (PSP) is a major human health issue that occurs worldwide. Species of the marine dinoflagellate genus Alexandrium can produce dangerous amounts of paralytic shellfish toxins responsible for PSP poisoning at extremely low cell densities. Current detection and identification methods for Alexandrium typically used by coastal managers are time-consuming, expensive, require special training, and are typically unable to
The toxigenic O157:H7 Escherichia coli (E. coli) bacterium has been connected with hemorrhagic co... more The toxigenic O157:H7 Escherichia coli (E. coli) bacterium has been connected with hemorrhagic colitis band hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure, and death. On average O157:H7 causes 73,000 illnesses, 2,100 hospitalizations, and 60 deaths annually in the United States alone. Current methods for the detection and identification of pathogenic E. coli typically involve tedious laboratory
Real-time, nondestructive methods for monitoring polymer film properties are increasingly importa... more Real-time, nondestructive methods for monitoring polymer film properties are increasingly important in the development and fabrication of modern polymer-containing products. Online testing of industrial polymer films during preparation and conditioning is required to minimize material and energy consumption, improve the product quality, increase the production rate, and reduce the number of product rejects. It is well-known that shear horizontal surface acoustic wave (SH-SAW) propagation is sensitive to mass changes as well as to the mechanical properties of attached materials. In this work, the SH-SAW was used to monitor polymer property changes primarily dictated by variations in the viscoelasticity. The viscoelastic properties of a negative photoresist film were monitored throughout the ultraviolet (UV) light-induced polymer cross-linking process using SH-SAW delay line devices. Changes in the polymer film mass and viscoelasticity caused by UV exposure produced variations in the phase velocity and attenuation of the SH-SAW propagating in the structure. Based on measured polymer-coated delay line scattering transmission responses (S(21)) and the measured polymer layer thickness and density, the viscoelastic constants c(44) and eta(44) were extracted. The polymer thickness was found to decrease 0.6% during UV curing, while variations in the polymer density were determined to be insignificant. Changes of 6% in c(44) and 22% in eta(44) during the cross-linking process were observed, showing the sensitivity of the SH-SAW phase velocity and attenuation to changes in the polymer film viscoelasticity. These results indicate the potential for SH-SAW devices as online monitoring sensors for polymer film processing.
Proceedings of The National Academy of Sciences, 1988
Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of... more Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of mediators of immediate hypersensitivity by rat basophilic leukemia (RBL) cells, a mast cell line. Digital fluorescence ratio imaging of fura-2 was used to measure [Ca2+]i in individual RBL cells. Changes in [Ca2+]i that occurred in response to crosslinking of IgE receptors on the cell surface or to application of the Ca2+ ionophore ionomycin were studied. Stimulation of RBL cells with antigen resulted in rapid increases in [Ca2+]i following lag times that varied widely from cell to cell. Simple averaging of the Ca2+ responses of many cells yielded a gradual response profile that closely resembled that of suspensions of cells measured in the fluorometer. The results show that single cells can respond much more rapidly to antigen than has previously been suggested by studies on populations of cells. The lag time between addition of antigen and initiation of the increase in [Ca2+]i varied considerably between cells in the same field of view. Both the rise time and the variability and average duration of the lag time increased with decreasing antigen concentration.
RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using... more RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.
A novel method for regenerating biosensors has been developed in which the highly specific detect... more A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) virus. As little as 0.6 fmol of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices.
Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and... more Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.
The monolithic spiral coil acoustic transduction (MSCAT) sensor platform is a novel bulk acoustic... more The monolithic spiral coil acoustic transduction (MSCAT) sensor platform is a novel bulk acoustic wave (BAW) device which is excited by a gold spiral coil antenna photolithographically deposited on one side of an AT-quartz wafer. The MSCAT platform can operate at very high frequencies by efficiently exciting high harmonic transverse shear modes with the application of a high frequency RF signal to the spiral coil. Since one surface of the MSCAT device is bare, this device can be used as a sensing platform upon which one deposits analyte selective chemical or biological films. The bare surface allows the detection of analyte induced mechanical (mass and viscoelasticity) and electrical (conductivity and dielectric constant) property changes in the film. In order to demonstrate the applicability of a MSCAT device as a sensor, the MSCAT platform is coated with biological and chemical films selective to Escherichia coli (E. coli) O157:H7, the E. coli strain most often responsible for serious illnesses in humans, and saxitoxin (STX), the most dangerous neurotoxin associated with shellfish poisoning stemming from red tide, respectively.
The toxigenic E. coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic... more The toxigenic E. coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure, and death. On average, O157:H7 causes 73,000 illnesses, 2,100 hospitalizations, and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies
The innate immune response constitutes the first line of defense against invading pathogens and c... more The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2′,7′-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.
IEEE Transactions on Ultrasonics Ferroelectrics and Frequency Control, 2004
The undetected introduction of pathogens into food or water supplies can produce grave consequenc... more The undetected introduction of pathogens into food or water supplies can produce grave consequences in terms of economic loss and human suffering. Sensitive and selective sensors capable of quickly detecting microbial pathogens are urgently needed to limit the effects of bioterrorist incidents, accidents, or pollution. Shear horizontal surface acoustic wave (SH SAW) devices provide an attractive platform for the design of microbial biosensors that function in liquid media, where Rayleigh-type modes are rapidly attenuated. This paper reports on an exploratory SH SAW delay line designed and fabricated on langasite, La3Ga5SiO14 (LGS), along the novel Euler propagation direction (0 degrees, 22 degrees, 90 degrees). A liquid chamber was fabricated and attached to the top surface, and the device was submitted to liquid and biochemical tests. Moderate (6 dB) additional attenuation of the transmission coefficient, /S21/, was consistently observed when the SH SAW delay line was assembled in the test fixture and submitted to the liquid tests, indicating that LGS is an attractive candidate for liquid sensing. Sensor selectivity can be achieved by integrating the LGS SH SAW delay line with a biochemical recognition layer. A test setup was implemented for the characterization of LGS SH SAW-based biosensors. The delay line response to biomolecule binding was shown by detection of sequential binding of proteins to the SH SAW device delay path. The biotinylated sensor was exposed sequentially to biotin-binding deglycosylated avidin, biotin-modified rabbit IgG, and goat anti-rabbit IgG antibody. As each protein was bound to the sensing surface, marked changes in the delay-line phase were recorded. The reported results demonstrate the capability of these devices to act as biochemical detectors in aqueous solutions, and this work represents the first effort using the novel material LGS in SAW-based biosensor technology.
A reliable viability assay for Giardia is required for the development of disinfection process de... more A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO®-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight™ was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.
The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central eve... more The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen [1]. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.
We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically ina... more We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO®-9, hexidium and SYTO®-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.
Paralytic shellfish poisoning (PSP) is a major human health issue that occurs worldwide. Species ... more Paralytic shellfish poisoning (PSP) is a major human health issue that occurs worldwide. Species of the marine dinoflagellate genus Alexandrium can produce dangerous amounts of paralytic shellfish toxins responsible for PSP poisoning at extremely low cell densities. Current detection and identification methods for Alexandrium typically used by coastal managers are time-consuming, expensive, require special training, and are typically unable to
The toxigenic O157:H7 Escherichia coli (E. coli) bacterium has been connected with hemorrhagic co... more The toxigenic O157:H7 Escherichia coli (E. coli) bacterium has been connected with hemorrhagic colitis band hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure, and death. On average O157:H7 causes 73,000 illnesses, 2,100 hospitalizations, and 60 deaths annually in the United States alone. Current methods for the detection and identification of pathogenic E. coli typically involve tedious laboratory
Real-time, nondestructive methods for monitoring polymer film properties are increasingly importa... more Real-time, nondestructive methods for monitoring polymer film properties are increasingly important in the development and fabrication of modern polymer-containing products. Online testing of industrial polymer films during preparation and conditioning is required to minimize material and energy consumption, improve the product quality, increase the production rate, and reduce the number of product rejects. It is well-known that shear horizontal surface acoustic wave (SH-SAW) propagation is sensitive to mass changes as well as to the mechanical properties of attached materials. In this work, the SH-SAW was used to monitor polymer property changes primarily dictated by variations in the viscoelasticity. The viscoelastic properties of a negative photoresist film were monitored throughout the ultraviolet (UV) light-induced polymer cross-linking process using SH-SAW delay line devices. Changes in the polymer film mass and viscoelasticity caused by UV exposure produced variations in the phase velocity and attenuation of the SH-SAW propagating in the structure. Based on measured polymer-coated delay line scattering transmission responses (S(21)) and the measured polymer layer thickness and density, the viscoelastic constants c(44) and eta(44) were extracted. The polymer thickness was found to decrease 0.6% during UV curing, while variations in the polymer density were determined to be insignificant. Changes of 6% in c(44) and 22% in eta(44) during the cross-linking process were observed, showing the sensitivity of the SH-SAW phase velocity and attenuation to changes in the polymer film viscoelasticity. These results indicate the potential for SH-SAW devices as online monitoring sensors for polymer film processing.
Proceedings of The National Academy of Sciences, 1988
Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of... more Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of mediators of immediate hypersensitivity by rat basophilic leukemia (RBL) cells, a mast cell line. Digital fluorescence ratio imaging of fura-2 was used to measure [Ca2+]i in individual RBL cells. Changes in [Ca2+]i that occurred in response to crosslinking of IgE receptors on the cell surface or to application of the Ca2+ ionophore ionomycin were studied. Stimulation of RBL cells with antigen resulted in rapid increases in [Ca2+]i following lag times that varied widely from cell to cell. Simple averaging of the Ca2+ responses of many cells yielded a gradual response profile that closely resembled that of suspensions of cells measured in the fluorometer. The results show that single cells can respond much more rapidly to antigen than has previously been suggested by studies on populations of cells. The lag time between addition of antigen and initiation of the increase in [Ca2+]i varied considerably between cells in the same field of view. Both the rise time and the variability and average duration of the lag time increased with decreasing antigen concentration.
RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using... more RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.
A novel method for regenerating biosensors has been developed in which the highly specific detect... more A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) virus. As little as 0.6 fmol of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices.
Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and... more Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.
The monolithic spiral coil acoustic transduction (MSCAT) sensor platform is a novel bulk acoustic... more The monolithic spiral coil acoustic transduction (MSCAT) sensor platform is a novel bulk acoustic wave (BAW) device which is excited by a gold spiral coil antenna photolithographically deposited on one side of an AT-quartz wafer. The MSCAT platform can operate at very high frequencies by efficiently exciting high harmonic transverse shear modes with the application of a high frequency RF signal to the spiral coil. Since one surface of the MSCAT device is bare, this device can be used as a sensing platform upon which one deposits analyte selective chemical or biological films. The bare surface allows the detection of analyte induced mechanical (mass and viscoelasticity) and electrical (conductivity and dielectric constant) property changes in the film. In order to demonstrate the applicability of a MSCAT device as a sensor, the MSCAT platform is coated with biological and chemical films selective to Escherichia coli (E. coli) O157:H7, the E. coli strain most often responsible for serious illnesses in humans, and saxitoxin (STX), the most dangerous neurotoxin associated with shellfish poisoning stemming from red tide, respectively.
The toxigenic E. coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic... more The toxigenic E. coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure, and death. On average, O157:H7 causes 73,000 illnesses, 2,100 hospitalizations, and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies
The innate immune response constitutes the first line of defense against invading pathogens and c... more The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2′,7′-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.
IEEE Transactions on Ultrasonics Ferroelectrics and Frequency Control, 2004
The undetected introduction of pathogens into food or water supplies can produce grave consequenc... more The undetected introduction of pathogens into food or water supplies can produce grave consequences in terms of economic loss and human suffering. Sensitive and selective sensors capable of quickly detecting microbial pathogens are urgently needed to limit the effects of bioterrorist incidents, accidents, or pollution. Shear horizontal surface acoustic wave (SH SAW) devices provide an attractive platform for the design of microbial biosensors that function in liquid media, where Rayleigh-type modes are rapidly attenuated. This paper reports on an exploratory SH SAW delay line designed and fabricated on langasite, La3Ga5SiO14 (LGS), along the novel Euler propagation direction (0 degrees, 22 degrees, 90 degrees). A liquid chamber was fabricated and attached to the top surface, and the device was submitted to liquid and biochemical tests. Moderate (6 dB) additional attenuation of the transmission coefficient, /S21/, was consistently observed when the SH SAW delay line was assembled in the test fixture and submitted to the liquid tests, indicating that LGS is an attractive candidate for liquid sensing. Sensor selectivity can be achieved by integrating the LGS SH SAW delay line with a biochemical recognition layer. A test setup was implemented for the characterization of LGS SH SAW-based biosensors. The delay line response to biomolecule binding was shown by detection of sequential binding of proteins to the SH SAW device delay path. The biotinylated sensor was exposed sequentially to biotin-binding deglycosylated avidin, biotin-modified rabbit IgG, and goat anti-rabbit IgG antibody. As each protein was bound to the sensing surface, marked changes in the delay-line phase were recorded. The reported results demonstrate the capability of these devices to act as biochemical detectors in aqueous solutions, and this work represents the first effort using the novel material LGS in SAW-based biosensor technology.
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