PubMed Health⌕ Search

Biomedical subjects

R Kopelman

Publications and source records attributed to R Kopelman.

At least 19 recordsLinked to original sources
PubMed1999

Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors.

Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production of sensors containing any hydrophilic dye or combination of dyes in the matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins, allowing reliable in vivo calibrations of dyes. Sensor response times are less than 1 ms. Cell viability assays indicate that the PEBBLEs are biocompatible, with negligible biological effects compared to control conditions. Several sensor delivery methods have been studied, including liposomal delivery, gene gun bombardment, and picoinjection into single living cells.

Cell Survival↗
PubMed1999

Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.

Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.

Calcium↗
PubMed1999

Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase.

Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its inability to bind oxygen, and its high affinity for nitric oxide make it well-suited for sensor applications. The heme domain has been labeled with a fluorescent reporter dye and changes in this dye's intensity are observed based on the sGC heme domain's characteristic binding of nitric oxide. The current sensors are prepared with 100-microns optical fiber but could also be prepared using submicrometer fiber tips. These sensors have fast, linear, and reversible responses to nitric oxide and are unaffected by numerous common interferents, such as oxygen, nitrite and nitrate. The sensor limit of detection is 1 microM nitric oxide. Glutathione has been shown to decrease the sensitivity of the sensor; however, the sensor response remains linear and can be calibrated on the basis of the glutathione concentration present in the biological environment of interest. The sensors have been used to measure extracellular nitric oxide production by BALB/c mouse macrophages. Minimal nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 microM in a given cell culture.

Animals↗
PubMed1999

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.

Animals↗
PubMed1998

Development and cellular applications of fiber optic nitric oxide sensors based on a gold-adsorbed fluorophore.

A new design for optochemical sensors has been applied to the development of a nitric oxide selective fiber optic sensor. This sensor is composed of a fluorescein derivative dye attached to colloidal gold. The fluorescein dye rearranges as nitric oxide adsorbs onto the gold, inducing a decrease in the fluorescence intensity of the dye. This mechanism has allowed preparation of fiber optic dye-based nitric oxide sensors, which have been made ratio-metric by addition of reference dye microspheres. Previously developed fast, selective optical sensors for detection of aqueous nitric oxide involved a protein, such as cytochrome c'. The new fluorescein derivative chemical sensors have characteristics similar to those of the protein-based biosensors, including fast response times, excellent selectivity, and complete reversibility. In addition, the chemical sensors have advantages such as greater stability and commercially available components. These sensors were utilized to measure nitric oxide production by BALB/c mouse macrophages.

Adsorption↗
PubMed1998

Fiber-optic nitric oxide-selective biosensors and nanosensors.

Fiber-optic biosensors that are selective for nitric oxide and do not respond to most potential interferents have been prepared with cytochromes c'. Both micro- and nanosensors have been prepared, and their response is fast (< 1 s), reversible, and linear up to 1 mM nitric oxide. The detection limit is 20 microM, making the sensor useful for some biological samples, such as the macrophages studied here. While sensors have been prepared based on the fluorescence of the cytochromes c', optodes with greatly enhanced signal-to-noise ratios have been made by labeling the cytochrome c' with a fluorescent dye. Comparisons of cytochromes c' from three species of bacteria as well as of two matrixes were performed and the optimum sensor configuration is described.

Acrylic Resins↗
PubMed1998

Nitrite- and chloride-selective fluorescent nano-optodes and in vitro application to rat conceptuses.

Nitrite- and chloride-selective, ion correlation-based, nano-optodes have been prepared for application in vitro. These fluorescent, liquid polymer based sensors have theoretically predictable responses to anion activities and good selectivity. The nitrite sensor, prepared with a vitamin B12 derivative ionophore, would be useful for determination of oxidized nitric oxide. Chloride nano-optodes, as well as micro-optodes, prepared with an indium porphyrin ionophore, were utilized to determine chloride levels both on the surface and inside the visceral yolk sac of organogenesis-stage rat conceptuses.

Animals↗
PubMed1998

Doing well by doing good. The case for objective feedback in case management.

Social service programs that do not generate sufficient revenues will not survive in a Fee-For-Service (FFS) system. Yet a concern about finances is alien to many social workers' client-centered orientation. This article presents findings from a study that evaluated the effect of an objective feedback intervention on social workers' productivity in an FFS HIV/AIDS case management program. Results showed a substantial increase in billable hours (13.4% year-to-year; 6.4% pre- to-post intervention) which enable the program to reverse its operating deficit and raise staff salaries.

Adult↗
PubMed1997

Utilization of lipophilic ionic additives in liquid polymer film optodes for selective anion activity measurements.

Lipophilic anionic and cationic additives are investigated as components for use in liquid polymer film-based optodes. These ionic additives are known to strongly influence the selectivity behavior of anion-selective electrodes. They also make it possible to construct ion coextraction optodes that can measure anion activities. Theoretical, thermodynamic anion-optode equilibria formalisms were derived to help understand the influence these additives have on the overall optode response and on the selectivity behavior. Neutral and charged anion carrier film configurations are described and tested for two anion ionophores with known modes of action, ruthenium(II) octaethylporphyrin and a vitamin B12 derivative (cyanoaquacobyrinic acid heptakis(2-phenylethyl ester)). These film configurations were further tested using a less well-understood ionophore, indium(III) octaethylporphyrin. Notable is the indium(III) octaethylporphyrin optode, which has a dynamic range appropriate for physiological measurements of chloride at neutral pH values. Also of interest is the optode with the vitamin B12 derivative ionophore, which has a dynamic range appropriate for some physiological measurements of nitrite at neutral pH values.

Anions↗
PubMed1997

High-performance fiber-optic pH microsensors for practical physiological measurements using a dual-emission sensitive dye.

A fast and durable ratiometric pH microoptode that is highly accurate, precise, sensitive, reversible, and reproducible over the physiological ranges of pH, ionic strength, and temperature has been developed. The sensing site consists of 5 (and 6)-carboxynaphthofluorescein (CNF) entrapped in a polyacrylamide gel matrix via photopolymerization at the silanized end of an optical fiber with a diameter of 2 (pulled) or 125 microns (unpulled). The optode's precision for the pH 6.3-8.4 range in rat embryos, sera, or physiological (Earle's and Tyrode's) buffers was found to be better than +/- 0.03 pH unit. The pulled and unpulled optodes have respective upper limit response times of 1 and 400 ms for 1-pH-unit change. Over a 7-week period, they retain sensitivity for 600 and 10,000 measurements, respectively. Ratiometric measurements are made using a pH-sensitive emission peak on each side of an isosbestic point. The CNF microoptode is most suitable for biological applications because of its essentially linear response over the pH 7-8 range, its high sensitivity (slope about 2), and its almost perfect correlation with a pH macroelectrode. Furthermore, errors introduced by photobleaching, leaching, quenching, optode movement, and excitation source fluctuations are minimal.

Animals↗
PubMed1997

Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes.

Previous studies have indicated that the surface (SU) and transmembrane (TM) subunits of the envelope protein (Env) of murine leukemia viruses (MuLVs) are joined by a labile disulfide bond that can be stabilized by treatment of virions with thiol-specific reagents. In the present study this observation was extended to the Envs of additional classes of MuLV, and the cysteines of SU involved in this linkage were mapped by proteolytic fragmentation analyses to the CWLC sequence present at the beginning of the C-terminal domain of SU. This sequence is highly conserved across a broad range of distantly related retroviruses and resembles the CXXC motif present at the active site of thiol-disulfide exchange enzymes. A model is proposed in which rearrangements of the SU-TM intersubunit disulfide linkage, mediated by the CWLC sequence, play roles in the assembly and function of the Env complex.

Animals↗
PubMed1996

Lifetime enhancement of ultrasmall fluorescent liquid polymeric film based optodes by diffusion-induced self-recovery after photobleaching.

The major concern with optodes, especially miniaturized ones, has been their photobleaching limited lifetime. Liquid polymer [highly plasticized poly(vinyl chloride)] films are commonly used to prepare fluorescent optical fiber sensors. A major advantage is the ease of their fabrication. It is demonstrated here that, with proper choice of excitation power and illumination time, the sensor will completely recover itself from photobleaching after each measurement. This self-recovery is demonstrated on single-mode optical fibers with 80 microns diameter (3.1 microns active region) and on near-field scanning optical microscope pulled fiber tips with submicrometer diameter (250 nm active region). The single-mode optode can be used for 30,000 measurements with only a 5% signal loss at a signal/noise of > 66. This opens the way for prolonged ratiometric application of such optodes.

Diffusion↗
PubMed1996

Miniature sodium-selective ion-exchange optode with fluorescent pH chromoionophores and tunable dynamic range.

An extension into the fluorescence mode of ion-exchange optodes is described, allowing miniaturization and its concomitant benefits. A micrometer-size, fluorescent fiber-optic sodium sensor is described, based on a highly sodium-selective, crown ether-capped calix[4]arene ionophore, capable of ratiometric operation. Three sensor configurations are given, employing different lipophilic, fluorescent pH chromoionophores (Nile Blue derivatives), demonstrating the ability to improve the detection limit and tune the dynamic range to the desired region of interest. Two of the sensors are of special interest in that their working ranges lie within those desired for measuring intracellular cytosolic or blood levels of sodium at the respective physiological pH. These optodes have excellent sodium selectivity, with other physiologically relevant cations (e.g., potassium, calcium, and magnesium) being highly discriminated. Three simple mathematical relationships are given for the three experimentally used fluorescent signal mechanisms (intensity, intensity ratios, and inner-filter or energy transfer effects), permitting visualization on a single graph and enabling direct comparison of the different sensors' optical responses on a common platform. Finally, these optodes measure the sample's sodium activity, rather than the concentration, provided that the sample's pH is measured simultaneously by another sensor, such as a glass electrode.

Fiber Optic Technology↗
PubMed1996

Analytical properties and sensor size effects of a micrometer-sized optical fiber glucose biosensor.

A micrometer-sized fiber-optic fluorescence biosensor for glucose has been fabricated. The sensor is 100 times smaller than existing glucose optodes. It is based on the enzymatic reaction of glucose oxidase that catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide while consuming oxygen. Tris(1,10-phenanthroline)ruthenium chloride, an oxygen indicator, is used as a transducer. The ruthenium complex and glucose oxidase are incorporated into acrylamide polymer that is attached covalently to a silanized optical fiber tip surface by photocontrolled polymerization. A study of the dependence of the fluorescence intensity on sensor size shows that, under normal operating conditions, the signal decreases with the sensor diameter rather than its volume. Also, the response of micrometer-sized sensors is improved by about 20% compared to that of larger fiber-optic glucose sensors. Due to its small size and the lack of membrane support, the response time of the sensor is only 2 s. An absolute detection limit of around 1 x 10(-15) mol is achieved. The new glucose sensor is at least 25 times faster and its absolute sensitivity 5-6 orders of magnitude higher than that of current glucose optodes.

Biosensing Techniques↗
PubMed1996

Fluorescent fiber-optic calcium sensor for physiological measurements.

A new optical sensor based on covalent immobilization of a newly synthesized calcium-selective, long-wavelength, fluorescent indicator has been constructed, with a response dynamic range optimal for physiological measurements. Immobilization occurs via photoinitiated copolymerization of the indicator with acrylamide on the distal end of a silanized 125 micrograms diameter multimode optical fiber. The working lifetime of this sensor is limited only by photobleaching of the indicator. Due to the inherent hydrophilic nature of the acrylamide polymer, the response time of this new sensor is governed by simple aqueous diffusion of the ionic calcium. This results in sensor response times fast enough to monitor some concentration fluctuations at physiological rates. The ability to monitor calcium concentration fluctuations in a high background level of magnesium is also demonstrated with a calculated selectivity of 10(-4.5).

Biosensing Techniques↗