Biomedical subjects

George S Wilson

Publications and source records attributed to George S Wilson.

15 recordsLinked to original sources

PubMed2006

Dendrimer FISH detection of single-copy intervals in acute promyelocytic leukemia.

Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14 kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.9-3.6 kb), target-specific, single-copy probes from the human genome sequence. A novel detection approach was evaluated using moieties possessing more fluorophores, DNA dendrimers (up to 375 fluorophores per dendrimer). Two detection approaches were evaluated using the dendrimers: (1) dendrimers modified with anti-biotin antibodies for detection of biotinylated bound probes, and (2) dendrimers modified with 45-base long oligonucleotides designed from the single-copy probes, for direct detection of the target region. The selectivity of the probes was confirmed via indirect labeling with biotin/digoxigenin by nick translation, with detection efficiencies between 50 and 90%. Furthermore, the scFISH probes were successfully detected on metaphase cells with anti-biotin dendrimer conjugates and on interphase cells with 45-base modified dendrimers. Our results bring up the possibility to detect target regions of less than 1 kb, which will be a great contribution to high-resolution analysis of genomic sequences.

Cell Line, Tumor↗

PubMed2005

Protein interactions with subcutaneously implanted biosensors.

Biofouling of in vivo glucose sensors has been indicated as the primary reason for sensitivity losses observed during the first 24 h after implant [Wisniewski N, Moussy F, Reichert WM. Characterization of implantable biosensor membrane biofouling. Fresen J Anal Chem 2000; 366(6-7): 611-621]. Identification of the biomolecules that contribute to these sensitivity perturbations is the primary objective of the research presented. Active needle-type glucose sensors were implanted in Sprague-Dawley rats for 24h, and then a proteomics approach was used to identify the substances absorbed to the sensors. MALDI-TOF mass spectrometry was the primary tool utilized to identify the biomolecules in sensor leachate samples and species absorbed directly on sensor membranes excised from explanted in vivo sensors. Not surprisingly serum albumin was identified as the primary biomolecule present, however, predominantly as endogenous fragments of the protein. In addition, several other biomolecule fragments, mainly less than 15 kD, were identified. Based on these findings, it is concluded that fragments of larger biomolecules infiltrate the sensor membranes causing diminished glucose diffusivity, thus decreasing in vivo sensitivity.

Adsorption↗

PubMed2005

Mediation of in vivo glucose sensor inflammatory response via nitric oxide release.

In vivo glucose sensor nitric oxide (NO) release is a means of mediating the inflammatory response that may cause sensor/tissue interactions and degraded sensor performance. The NO release (NOr) sensors were prepared by doping the outer polymeric membrane coating of previously reported needle-type electrochemical sensors with suitable lipophilic diazeniumdiolate species. The Clarke error grid correlation of sensor glycemia estimates versus blood glucose measured in Sprague-Dawley rats yielded 99.7% of the points for NOr sensors and 96.3% of points for the control within zones A and B (clinically acceptable) on Day 1, with a similar correlation for Day 3. Histological examination of the implant site demonstrated that the inflammatory response was significantly decreased for 100% of the NOr sensors at 24 h. The NOr sensors also showed a reduced run-in time of minutes versus hours for control sensors. NO evolution does increase protein nitration in tissue surrounding the sensor, which may be linked to the suppression of inflammation. This study further emphasizes the importance of NO as an electroactive species that can potentially interfere with glucose (peroxide) detection. The NOr sensor offers a viable option for in vivo glucose sensor development.

Animals↗

PubMed2005

Fractionation of chromosome 15 with an affinity-based approach using magnetic beads.

The two main shortcomings of the state-of-the-art method of sorting chromosomes, specificity and the efficiency of fractionating a significant amount of chromosomes, are addressed by this work in the design of a massively parallel approach using magnetic beads binding to a chromosome-specific DNA probe. In an attempt to isolate human chromosome 15 from a lymphoblastoid cell line, a chromosome 15 centromere-specific DNA probe with a fluorescent tag attached was reacted with the chromosomes. Magnetic beads bound to anti-FITC antibody were reacted with the labeled pool of chromosomes and separated by exposure to a magnetic field. The specificity of the fractionated pool was verified by performing fluorescence in situ hybridization on the isolated pool. The chromosome of interest could be enriched to about 75% within a maximum of 3-4 days, regardless of the amount of material.

Cell Line, Tumor↗

PubMed2005

DNA aptamer-based bioanalysis of IgE by fluorescence anisotropy.

A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5'-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. The results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.

Aptamers, Nucleotide↗

PubMed2005

Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer.

We report the picosecond and nanosecond timescale rotational dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to immunoglobulin E. Rotational dynamics in combination with fluorescence lifetime measurements provide information about dye-DNA interactions. Comparison of Texas Red (TR), fluorescein, and tetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for biophysical studies employing such conjugates. Time-resolved anisotropy studies demonstrate that the TR- and TAMRA-aptamer anisotropy decays are dominated by the overall rotation of the aptamer, whereas the fluorescein-aptamer anisotropy decay displays a subnanosecond rotational correlation time much shorter than that expected for the overall rotation of the aptamer. Docking and molecular dynamics simulations suggest that the low mobility of TR is a result of binding in the groove of the DNA helix. Additionally, associated anisotropy analysis of the TAMRA-aptamer reveals both quenched and unquenched states that experience significant coupling to the DNA motion. Therefore, quenching of TAMRA by guanosine must depend on the configuration of the dye bound to the DNA. The strong coupling of TR to the rotational dynamics of the DNA aptamer, together with the absence of quenching of its fluorescence by DNA, makes it a good probe of DNA orientational dynamics. The understanding of the nature of dye-DNA interactions provides the basis for the development of bioconjugates optimized for specific biophysical measurements and is important for the sensitivity of anisotropy-based DNA-protein interaction studies employing such conjugates.

Anisotropy↗

PubMed2005

Biosensors for real-time in vivo measurements.

The current status of sensors capable of continuous measurement of analytes in biological media is reviewed. This review containing 173 references deals with devices whose use in single cells, tissue slices, animal models and humans has been demonstrated. In addition to sensors specific for glucose, lactate, glutamate, pyruvate, choline and acetylcholine, insights obtained from monitoring nitric oxide, Na(+), K(+), Ca(2+), and dopamine are presented. Performance criteria for sensor performance are described as is the subject of biosensor calibration. Biocompatibility issues are dealt with in some detail as is the status of continuous blood glucose monitoring in humans.

Biosensing Techniques↗

PubMed2004

Electrochemical and spectroscopic characterization of surface sol-gel processes.

(3-Mercaptopropyl)trimethoxysilane (MTS) forms a unique film on a platinum substrate by self-assembly and sol-gel cross-linking. The gelating and drying states of the self-assembled MTS sol-gel films were probed by use of electrochemical and spectroscopic methods. The thiol moiety was the only active group within the sol-gel network. Gold nanoparticles were employed to detect the availability of the thiol group and their interaction further indicated the physicochemical states of the sol-gel inner structure. It was found that the thiol groups in the open porous MTS aerogel matrix were accessible to the gold nanoparticles while thiol groups in the compact MTS xerogel network were not accessible to the gold nanoparticles. The characteristics of the sol-gel matrix change with time because of its own irreversible gelating and drying process. The present work provides direct evidence of gold nanoparticle binding with thiol groups within the sol-gel structures and explains the different permeability of "aerogel" and "xerogel" films of MTS on the basis of electrochemical and spectroscopic results. Two endogenous species, hydrogen peroxide and ascorbic acid, were used to test the permeability of the self-assembled sol-gel film in different states. The MTS xerogel film on the platinum electrode was extremely selective against ascorbic acid while maintaining high sensitivity to hydrogen peroxide in contrast to the relatively high permeability of ascorbic acid in the MTS aerogel film. This study showed the potential of the MTS sol-gel film as a nanoporous material in biosensor development.

Electrochemistry↗

PubMed2004

Selective site-specific fenton oxidation of methionine in model peptides: evidence for a metal-bound oxidant.

The metal-catalyzed oxidation (MCO) of proteins represents an important pathway for protein degradation. Although many mechanistic details of MCO are currently unknown, such mechanistic information would greatly benefit formulation scientists in the rational design and analysis of protein formulations. Here, we describe the Fenton oxidation (by Fe(2+)/H(2)O(2)) of several Met-, Tyr-, and His containing model peptides, including one derivative containing a conformationally restricted norbornyl Met analogue (Nor), Nor-Gly-His-Met-NH(2). Our results will provide evidence for a metal-bound reactive oxygen species selectively oxidizing Met to Met sulfoxide, indicating a Met-specific oxidant and arguing against the involvement of freely diffusible hydroxyl radicals. The Fenton oxidation of Nor-Gly-His-Met-NH(2) yields a 2:1 preference for sulfoxide formation at the C-terminal Met versus the N-terminal Nor residue, respectively, while incubation of the peptide with H(2)O(2) alone results in a 1:1 ratio. These results are rationalized by the better access of the thioether side chain of the flexible C-terminal Met residue to the peptide-bound iron compared with the conformationally restricted Nor residue. It is commonly believed that Fenton oxidation reactions involve hydroxyl radicals, and that Met oxidation in proteins is predominantly controlled by the surface-accessibility of the respective Met residues. However, occasionally protein oxidation in formulations shows selectivities, which are not consistent with these paradigms. Our results demonstrate additional features of the Fenton reaction such as the formation of a metal-bound oxidant specific for Met (and not Tyr or His), which may assist formulation scientists in the rationalization of unexpected oxidation selectivities.

Binding Sites↗

PubMed2002

Fundamental studies of glucose oxidase deposition on a Pt electrode.

The direct electrodeposition of glucose oxidase (EC 1.1.3.4) on a platinum electrode was investigated as a means for controlled immobilization. The presence of a nonionic detergent, Triton X-100, was found essential to produce a multilayered deposit. Moreover, to work properly, the detergent must be present above its critical micelle concentration. Under these conditions, a deposit of approximately 50 enzyme layers (480 nm), with surface uniformity of +/-20 nm, was verified using an electrochemical quartz crystal microbalance and by atomic force microscopy. In the absence of detergent, a layer of 25 nm is formed. Contrary to most previous claims, the deposition, which is potential dependent but optimal at 1.3 V versus AgCl/Ag electrode, is not electrophoretically driven, but is instead controlled by a lowering of the pH at the electrode surface due to concomitant oxygen evolution.

Biosensing Techniques↗

PubMed2002

Electrochemically mediated electrodeposition/electropolymerization to yield a glucose microbiosensor with improved characteristics.

A procedure is described that provides for electrochemically mediated deposition of enzyme and a polymer layer permselective for endogenous electroactive species. Electrodeposition was first employed for the direct immobilization of glucose oxidase to produce a uniform, thin, and compact film on a Pt electrode. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)trimethoxysilane was constructed by electrochemically assisted cross-linking. This hybrid film outside the enzyme layer contributed to the improved stability and permselectivity. The resulting glucose sensor was characterized by a short response time (<4 s), high sensitivity (1200 nA/mM x cm2), low interference from endogenous electroactive species, and working lifetime of more than 50 days.

Biosensing Techniques↗

PubMed2002

A new amperometric glucose microsensor: in vitro and short-term in vivo evaluation.

For biosensor fabrication, it is important to optimize materials and methods in order to create predictable function in vitro and in vivo. For this reason, we designed a new glucose sensor ('revised protocol') that utilized an outer permselective membrane made of amphiphobic polyurethane which allows glucose passage through hydrophilic segments. An inner polyethersulfone membrane, stabilized with a trimethoxysilane, provided specificity. Before application of the inner membrane, it was necessary to etch the platinum electrode with a radio frequency oxygen plasma. The revised protocol sensors (n=185) were compared with sensors fabricated with an earlier ('original') protocol (n=204) which used an outer polyurethane without hydrophilic segments and a complex inner membrane of cellulose acetate and Nafion. The function of revised protocol sensors was more predictable in vitro as evidenced by a much lower variation of glucose sensitivity than the original protocol sensors. Revised and original protocol sensors were nearly linear up to a glucose concentration of 20 mM. In vitro interference from 0.1 mM acetaminophen was minimal in both groups of sensors and would be expected to represent about 2% of the total sensor response at normal glucose levels for revised protocol sensors. Prolonged testing of the revised protocol sensors for 11 days during immersion in buffer revealed stable sensitivities (day 1: 6.12+/-1.34 nA/mM; day 3: 6.33+/-1.40; day 8: 7.13+/-1.39; and day 11: 7.56+/-1.47; sensitivity for day 1 vs. each other day: not significant) and no critical loss of glucose oxidase activity. The response of the revised protocol sensors (n=7) to intraperitoneal glucose was tested in rats approximately one day after subcutaneous implantation and the sensors tracked glucose closely with a slight lag of 3-6 min.

Animals↗

PubMed2002

Glucose microbiosensor based on alumina sol-gel matrix/electropolymerized composite membrane.

A procedure is described that provides co-immobilization of enzyme and bovine serum albumin (BSA) within an alumina sol-gel matrix and a polyphenol layer permselective for endogenous electroactive species. BSA has first been employed for the immobilization of glucose oxidase (GOx) on a Pt electrode in a sol-gel to produce a uniform, thin and compact film with enhanced enzyme activity. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)-trimethoxysilane was constructed by electrochemically-assisted crosslinking. This hybrid film outside the enzyme layer contributed both to the improved stability and to permselectivity. The resulting glucose sensor was characterized by a short response time (<10 s), high sensitivity (10.4 nA/mM mm(2)), low interference from endogenous electroactive species, and a working lifetime of at least 60 days.

Aluminum Oxide↗

PubMed2002

Prevention of hypoglycemia using risk assessment with a continuous glucose monitoring system.

Due to the lag between sugar intake and the beginning of recovery from hypoglycemia, it is necessary to intervene in an anticipatory way if one wants to prevent, not only detect, hypoglycemia. This article presents the principle of a hypoglycemia prevention system based on risk assessment. The risk situation can be defined as the moment when the system estimates that the glucose concentration is expected to reach a hypoglycemia threshold in less than a given time (e.g., 20 min). Since there are well-known discrepancies between blood and interstitial glucose concentrations, the aim of this experimental study performed in nondiabetic rats was first to validate this strategy, and second to determine whether it can work when the glucose concentration is estimated by a glucose sensor in subcutaneous tissue rather than in blood. We used a model of controlled decrease in blood glucose concentration. A glucose infusion, the profile of which mimicked the appearance of glucose from an intragastric load, was administered either when hypoglycemia was detected or on the basis of risk recognition. Despite the lag between the beginning of the load and that of the increase in blood glucose concentration, which was in all experiments 15-20 min, hypoglycemia was fully prevented without overshoot hyperglycemia in the groups of rats in which the glucose load was started when the hypoglycemia risk was detected, on the basis of either blood or interstitial glucose concentration. This was, of course, not the case when the same glucose load was infused at the detection of the hypoglycemia threshold.

Animals↗

PubMedDate not supplied

Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer.

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5'-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.

DNA↗