Biomedical subjects

Libby G Puckett

Publications and source records attributed to Libby G Puckett.

6 recordsLinked to original sources

PubMed2005

Magnetoelastic transducers for monitoring coagulation, clot inhibition, and fibrinolysis.

Magnetoelastic transduction has been used to detect and monitor the viscosity changes that occur during the biological reactions of coagulation and fibrinolysis. Magnetoelastic sensors can be used, because the characteristic resonance frequency of the magnetoelastic strip shifts in response to the changes in fluid viscosity. At a set frequency, the output signal can be obtained over time to develop a coagulation and/or dissolution profile, which display the change in viscosity of a plasma sample that has undergone either coagulation or fibrinolysis. For coagulation screening, an exogenous tissue factor is added to an anticoagulated plasma sample to initiate coagulation. Further studies were performed to investigate fibrinolysis through the addition of plasmin. Plasmin is used in two different ways-as a competitive inhibitor before the initiation of clotting and also as a protease to dissolve the previously formed clot. This method is a viable option for the monitoring of processes that are paramount to maintaining hemostasis.

Anticoagulants↗

PubMed2004

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Antidepressive Agents↗

PubMed2004

Whole-cell-reporter-gene-based biosensing systems on a compact disk microfluidics platform.

Biosensing systems such as reporter-gene-based whole-cell assays are increasingly finding applications in biological and environmental screening. A whole-cell approach to such analyses can provide valuable information about the bioavailable level of a compound of interest. These biosensing systems rely on the molecular recognition of a specific analyte by a regulatory protein and, therefore, can detect low levels of the target analyte. In this study, Escherichia coli cells containing plasmid pSD10 were engineered to sense the model target analytes arsenite and antimonite, the target analytes in this study. The biosensing system takes advantage of the recognition of the regulatory protein, ArsR, for arsenite and antimonite to produce the reporter protein, which in this case is GFPuv. The fluorescence emitted by the GFPuv in the cells can be directly related to the concentration of the analyte in the cell, making this biosensing system useful in the detection of arsenite and/or antimonite in a variety of samples. Miniaturization of biosensing systems can further enhance their utility by decreasing reagent consumption and analysis time and by allowing for the high-throughput screening of samples. To that end, we employed a microcentrifugal microfluidics platform that has low power, space, and reagent requirements, increased speed of detection, and the potential for portability. Herein, we demonstrate for the first time the adaptation of a whole-cell sensing system to a microcentrifugal microfluidics platform. Moreover, we were able to detect our target analytes in a rapid and sensitive manner compared to conventional sensing methods.

Antimony↗

PubMed2003

Covalent immobilization of beta-galactosidase onto a gold-coated magnetoelastic transducer via a self-assembled monolayer: toward a magnetoelastic biosensor.

The enzyme beta-galactosidase has been covalently immobilized onto a gold-coated magnetoelastic film via a self-assembled monolayer (SAM) of omega-carboxylic acid alkylthiol. Use of magnetoelastic transduction allows for the wireless monitoring of enzymatic activity through the associated change in the frequency and amplitude of magnetic fields. The formations of SAMs of 3-mercaptopropanoic acid and thioctic acid were monitored by magnetoelastic transduction. After coupling of beta-galactosidase to the SAMs, the enzyme activity was monitored by using a substrate that forms an insoluble product upon action of the enzyme. Specifically, an indolyl galactopyranoside substrate was employed in conjunction with an azo dye as the precipitating system. The immobilized enzyme was evaluated and found to have an apparent Michaelis-Menten constant (KM) of 1.2 mM for the indolyl galactopyranoside. Calibration plots for both substrates and inhibitors were generated to establish the versatility of this sensing system. Kinetic parameters for nonprecipitating substrates were determined in conjunction with a precipitating enzymatic substrate by way of a competitive inhibition study using beta-galactosidase attached to magnetoelastic strips. The methods developed within this work allow for the fabrication of wireless enzyme sensing systems, which can also be used as another means of screening for enzyme inhibitors.

Biosensing Techniques↗

PubMed2003

Monitoring blood coagulation with magnetoelastic sensors.

The determination of blood coagulation time is an essential part of monitoring therapeutic anticoagulants. Standard methodologies for the measurement of blood clotting time require dedicated personnel and involve blood sampling procedures. A new method based on magnetoelastic sensors has been employed for the monitoring of blood coagulation. The ribbon-like magnetoelastic sensor oscillates at a fundamental frequency, which shifts linearly in response to applied mass loads or a fixed mass load of changing elasticity. The magnetoelastic sensors emit magnetic flux, which can be detected by a remotely located pick-up coil, so that no direct physical connections are required. During blood coagulation, the viscosity of blood changes due to the formation of a soft fibrin clot. In turn, this change in viscosity shifts the characteristic resonance frequency of the magnetoelastic sensor enabling real-time continuous monitoring of this biological event. By monitoring the signal output as a function of time, a distinct blood clotting profile can be seen. The relatively low cost of the magnetoelastic ribbons enables their use as disposable sensors. This, along with the reduced volume of blood required, make the magnetoelastic sensors well suited for at-home and point-of-care testing devices.

Animals↗

PubMed2002

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein.

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.

Aequorin↗