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Biomedical subjects

P Vadgama

Publications and source records attributed to P Vadgama.

At least 19 recordsLinked to original sources

Polypyrrole-based conducting polymers and interactions with biological tissues.

Polypyrrole (PPy) is a conjugated polymer that displays particular electronic properties including conductivity. In biomedical applications, it is usually electrochemically generated with the incorporation of any anionic species including also negatively charged biological macromolecules such as proteins and polysaccharides to give composite materials. In biomedical research, it has mainly been assessed for its role as a reporting interface in biosensors. However, there is an increasing literature on the application of PPy as a potentially electrically addressable tissue/cell support substrate. Here, we review studies that have considered such PPy based conducting polymers in direct contact with biological tissues and conclude that due to its versatile functional properties, it could contribute to a new generation of biomaterials.

Biocompatible Materials↗

Characterization of a laminar flow cell for the prevention of biosensor fouling.

A flow cell using dual parallel laminar flows was designed and fabricated with the aim to prevent biosensor fouling. Y-shaped entrances allowed the introduction of sample and electrolyte streams, with the electrolyte serving as a mobile protective layer near the biosensor. Potassium permanganate was used to quantify the diffusion in the flow cell. Optical intensity analysis of potassium permanganate along a series of transverse lines across the flow cell was carried out under different flow conditions. It was found that the error function, erf(y/[square root](Dt)), where y was the position along the transverse line, D the diffusion coefficient of the solute and t was the time, gave reasonable approximation to the diffusion of potassium permanganate in the cell. The diffusion coefficient of potassium permanganate was determined in stop-flow measurements and the value, D=4.0 x 10(-5)cm(2)/s, agreed to previously reported values. Velocity distribution in the flow cell was simulated numerically to reveal the development of two inflows into one single laminar flow. Results from the study provided preliminary data on solute diffusion characteristics in the flow cell and supported the working principle of laminar flow cells in preventing biosensor fouling.

Biosensing Techniques↗

O2 microsensors for minimally invasive tissue monitoring.

Tissue oxygenation is a key factor ensuring normal tissue functions and viability. Continuous real-time monitoring of the partial pressure of oxygen, pO(2), in tissues gives insight into the dynamic fluctuations of O(2) supplies to tissues by blood circulation. Small oxygen sensors enable investigations of the spatial variation of pO(2) in tissues at different locations in relation to local microvessels. In this paper, pO(2) measurement using microelectrodes and biocompatible sensorsv is discussed and recent progress of their application in human skin is reviewed. Emphasis is given to working principles of a number of existing oxygen sensors and their potential application in vivo and in tissue engineering. Results on spatial and temporal variations of the pO(2) in human skin introduced by localized ischaemia-reperfusion are presented when the surface of the skin is covered by an oxygen-free paraffin oil layer and the range of the tissue pO(2) is deduced to be between 0 and 60 mmHg. In the study, pO(2) increases from 8.0 +/- 3.2 mmHg (n = 6) at the surface of the skin to 35.2 +/- 8.0 mmHg (n = 9) at a depth just above the subpapillary plexus. Temporal decay in pO(2) following tissue compression and rise in pO(2) following pressure release can be described using mono-exponential functions. The time constant for the exponential decay, tau = 8.44 +/- 1.53 s (n = 7) is consistently greater than that for the exponential rises, tau' = 4.75 +/- 0.82 s (n = 6). The difference in pO2 change with the time following tissue compression and pressure release reveals different dynamic mechanisms involved in the two transient phases. The elevated steady state pO(2) following reperfusion, which is approximately 20% higher than the pre-occlusion value, indicates localized reactive hyperaemia. Possible applications of O(2) microsensors in diseases, e.g. tumours, pressure ulcers, are also discussed.

Biosensing Techniques↗

Hybridization of DNA at the surface of phospholipid monolayers. Effect of orientation of oligonucleotide chains.

We studied the properties of lipid monolayers formed at the air-water interface composed of dioleoylphosphatidylcholine (DOPC) with incorporated short (19-mer) oligonucleotides. These oligonucleotides were modified by oleylamine at both (3' and 5') terminals or only at one (3') terminal. Interaction of single-stranded (19-mer) oligonucleotides without oleylamine with DOPC monolayers resulted only in slight increase of surface pressure and the area per phospholipid molecule, while more substantial and significant increase of these values were observed following incorporation of oligonucelotides modified by oleylamine. This influence is similar for both types of oligonucleotide modifications. However, considerable differences in changes of monolayer properties took place after hybridization with complementary oligonucleotides. The hybridization of oligonucleotides with the DNA modified by oleic acid at both 3' and 5' terminals at the surface of lipid monolayer resulted in further increase of surface pressure and in the increase of the area per phospholipid molecule, while decrease of both the surface pressure and the area per phospholipid molecules were observed for hybridization with DNA modified by oleic acid at 3' terminal. It is possible that in latter case, the hybridization caused the loss of hybridized molecules from monolayers. Interaction of noncomplementary chains with DOPC monolayers with incorporated oleyl acid-modified DNA also influenced the properties of monolayers, but the effect was weaker in comparison with that observed for complementary chains.

DNA↗

Strategic issues in reliable sensing.

Amperometric enzyme biosensors must possess two important characteristics if they are to be successfully utilised as reliable monitoring devices. They must exhibit linearity over concentrations relevant to the target analyte, and they must avoid contamination, adverse reactions with the sample matrix response to interferents that react directly at the polarised working electrode surface. Covering polymeric membranes have provided a useful route to overcoming these problems. This report summarises successful modulation of membrane bulk as well as surface properties using surfactant-loaded diffusion limiting PVC and phenolic membranes, and the possible exploitation of direct response conducting poly (pyrrole) membrane loaded with affinity molecules through impedance spectroscopy.

Biosensing Techniques↗

Reagentless biosensing using electrochemical impedance spectroscopy.

The use of electrochemical impedance spectroscopy (EIS) and the conducting polymer, poly (pyrrole), as an integrated recognition and transduction system for reagentless biosensor systems was demonstrated with two different systems. The first system being an immunoassay for detection of luteinising hormone (LH) with the antibody being entrapped with in the poly (pyrrole) matrix and the second, a construct for DNA hybridisation discrimination able to differentiate single- and double-stranded DNA based on the interaction of the DNA with poly (pyrrole).

Biosensing Techniques↗

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains.

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.

Base Sequence↗

A versatile biosensor device for continuous biomedical monitoring.

Although biosensors are by means suitable for continuous biomedical monitoring, due to fouling and blood clotting, in vivo performance is far from optimal. For this reason, ultrafiltration, microdialysis or open tubular flow is frequently used as interface. To secure quantitative recoveries of the analyte of interest, sampling at submicrolitre level will be necessary which in turn necessitates the development of small and versatile biosensor devices. Here, a miniaturised biosensor device, which directly can be connected to various interfaces will be presented. The biosensor device consists of a pulsefree pump and a biosensor with an internal volume of 10-20 nl. In this article, the production as well as the construction of the flow-through cell of the biosensor will be discussed. The advantages and disadvantages of several production processes will be demonstrated and a detailed protocol for the production of such a nanoliter flow-through cell will be presented. With respect to the bio-selector, several permselective membranes have been tested on their performance characteristics. Results obtained with these biosensors will be presented and discussed. Finally, a protocol based upon in situ electropolymerisation for the immobilisation of the biological component was defined and several biosensors based upon this principle have been produced and tested for the monitoring of glucose respectively lactate. To demonstrate, data obtained during a variety of in vivo studies at different clinical relevant applications will be presented.

Animals↗

Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays.

Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) were then captured on this surface in the correct orientation for binding. LH (500 IU/l) was injected over the surface and the subsequent binding and dissociation events were monitored. The resulting optical response curves allowed fast analysis of the binding interactions of eight selected MAbs. It was possible to develop a two-site immunometric assay for LH using a pair of these MAbs. The effect of biotinylating the MAbs, using various biotin:antibody coupling ratios, on their subsequent binding to both LH and avidin conjugated alkaline phosphatase was also investigated. This approach has allowed rapid evaluation of the effect of changes in both reagent and reaction conditions on immunoassay performance and appears to be a valuable adjunct to immunosensor and immunoassay development.

Alkaline Phosphatase↗

Minimal-Fouling enzyme electrode for continuous flow measurement of whole blood lactate.

A new membrane system evaluated in an enzyme electrode for monitoring of whole blood lactate under continuous flow conditions has been developed. The membrane confers functional haemocompatibility and has been coupled to an electrode based on amperometric detection of hydrogen peroxide with lactate oxidase interposed between the haemocompatible membrane acting as diffusion barrier and an inner membrane excluding interfering electroactive species. Here, the enzyme electrode is combined with a flow-through measuring chamber and a double lumen catheter permitting continuous sampling and heparinization of blood. The blood contacting membranes have unique haemocompatibility properties and were produced through modification of poly(vinyl chloride), with the nonionic surfactant Pluronic F-68. On-line monitoring of whole blood samples with different amounts of added lactate was carried out to simulate fluctuations in blood lactate levels during extracorporeal monitoring and results were compared with a standard lactate analyser.

Biocompatible Materials↗

Comparison of lactate and bicarbonate buffered haemofiltration fluids: use in critically ill patients.

OBJECTIVE: To compare acid-base balance, lactate concentration, and haemodynamic and O2 transport variables during haemofiltration with replacement fluid containing 44.5 mmol/l Na+ lactate or 40 mmol/l Na+ HCO3- and 3 mmol/l lactic acid. DESIGN: A prospective, randomized trial. SETTING: A multidisciplinary, adult intensive care unit in a university hospital. PATIENTS: Forty acidotic patients who required haemofiltration, were dependent on mechanical ventilation, and had PA catheters in situ. INTERVENTIONS: During haemofiltration patients received lactate or bicarbonate replacement fluid at a mean rate of 1.7 l/h (SD 0.3). Arterial blood gases, plasma lactate, and haemodynamic and O2 transport variables were measured before and after 12 and 24 h haemofiltration. Ultrafiltrate was collected for lactate estimation. MEASUREMENTS AND MAIN RESULTS: As means (SD). The net gain of lactate was 63 mmol/h (12 mmol) with Na+ lactate and 0 mmol/h (0.3 mmol) with Na+ HCO3-. There was a significant increase in pH and [lactate] in both groups, but [lactate] was higher in patients receiving lactate. Twenty-one patients survived to ICU discharge, these patients were significantly less acidotic after filtration (lactate group: 0 h: pH 7.23 (0.09), [lactate] 2.4 mmol/l (1.7); 12 h: pH 7.34 (0.09), [lactate] 4.7 mmol/l (2.4); 24 h: pH 7.36 (0.07), [lactate] 4.7 mmol (2.7). HCO3 group: 0 h: pH 7.23 (0.09), [lactate] 2.3 (1.3); 12 h: pH 7.32 (0.06), [lactate] 2.9 mmol/l (1.8); 24 h: pH 7.35 (0.08), [lactate] 2.8 mmol/l (2.0). Base deficit: survivors: 0 h: 9 mmol/l (4); 12 h: 2 mmol/l (3). Non-survivors: 0 h: 10 mmol/l (3); 12 h: 6 mmol/l (3)). Haemodynamic and O2 transport variables were not significantly affected by treatment group or outcome. CONCLUSIONS: The degree of correction of acidosis during the first 24 h of haemofiltration was determined by patients outcome but was not affected by the substitution of bicarbonate- for lactate-containing replacement fluids.

Acidosis↗

Infrared analysis in clinical chemistry: its use in the laboratory and in non-invasive near patient testing.

Laboratory based NIR analysers have been available for some time. The recent development of more portable equipment such as the commercially available Futrex-9000 NIR transmittance blood chemistry analyser, which can be used to analyse relatively opaque samples for a mixture of components, shows promise but requires further evaluation for routine clinical use. NIR equipment for general use has only recently become available and is therefore relatively expensive. However, as the development of new applications occurs the instrumentation will become more widely used, which will inevitably result in reduced capital cost. The advantages of NIR systems are speed, portability, lack of consumables, dry chemistry, non-invasive, modest running costs, virtually no moving parts and almost infinite applications in clinical biochemical analysis. It is likely that the first applications of NIR will be where there is a requirement for multiple assays such as glucose, urea and bilirubin and where sample size is a limitation. Thus non-invasive near patient testing may become common in the future in settings such as neonatal units, renal units, diabetic clinics and intensive care units.

Chemistry, Clinical↗

Stabilized needle electrode system for in vivo glucose monitoring based on open flow microperfusion.

Preliminary in vitro studies and in vivo performance of amperometric glucose needle enzyme electrodes incorporating an open microflow technique, in which the sensor surface in subjected to a flow of fluid, are reported. Initially using a slow flow (60 microliters h-1) of isotonic phosphate buffer over the enzyme electrode tip, an interface was created which reduced cellular/protein fouling for electrode measurements in whole blood. Here a minor reduction in electrode response (apparent only at high glucose concentration) occurred which was not cumulative and therefore not associated with fouling. The protection afforded by the moving aqueous film was independent of fluid composition; the use of isotonic/hypertonic buffer, addition of anticoagulant (1% m/v heparin) or enhanced fluid viscosity (addition of 1% v/v glycerol) did not affect the system. Implantation of the electrode and its microflow cannula into subcutaneous tissue in rats was associated with a decreased buffer flow (30 microliters h(-1)) and had the effect of (i) reducing electrode stabilization (30 min), (ii) accelerating 'pick up' of tissue glucose changes after intravenous glucose (1-2 min lag) or insulin (3-7 min lag) and (iii) achieving a correlation between tissue and blood glucose values under dynamic conditions (r2 = 0.98, y = 0.99x + 0.23). Reassessment of the electrode response in vitro, following a 4 h monitoring period, provided a sensor response within 3% of the original electrode sensitivity, indicating little or no surface fouling and avoiding the requirement for repeated in vivo calibrations at least over the initial implantation period.

Animals↗

Open flow microperfusion: approach to in vivo glucose monitoring.

A slow flow of liquid over the working tip of a classical glucose needle electrode allows a significant reduction in surface fouling with minimal sample dilution. The fluid flow technique enables relatively drift-free in vivo operation for up to four hours (electrode responses in vitro following explanation of the device after a 4 h monitoring period are within +/- 5% of original values), exhibits a close correlation with blood glucose levels and is unique in requiring no in vivo calibration.

Animals↗

Bio-/haemocompatibility: implications and outcomes for sensors?

Sensors are a sample contacting technology, and when exposed to biological matrices tend to suffer from the problem of poor biocompatibility and surface fouling. The effects are evidenced by time dependent signal drift (particularly for those devices which are implanted intravascularly). Practical methods to reduce such effects require an understanding of the interface between the electrode and its environment prior to assessment of the potential areas for improvement. Current procedures employed to overcome the observed losses in electrode sensitivity (following exposure to whole blood) include surface modification of the outer diffusion limiting membrane (via variation in film porosity) or even biomimicry of the fluid cell membrane. At amperometric electrodes incorporation of additional inner perm-selective membranes has achieved a reduction in electrode passivation from undesirable surface active compounds as has the use of low polarisation potentials. Other studies have attempted to induce an aqueous barrier between the matrix and the electrode tip which physically prevents passage of cellular components from the sensor surface. Careful choice of the system and materials will ultimately lead to biocompatible non-fouling devices capable of functioning in an array of bio-environments suitable for clinical monitoring of the critically ill patient.

Biocompatible Materials↗