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Chemical Sensors for In Vivo Monitoring

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The research has focused on new materials for in vivo sensors according 2 lines, novel experimental materials and treated materials. For the first line of research some biopolymers of polysaccharidic nature were tested as component materials for in vivo biosensors, with particular attention to their stability and biocompatibility. Some determinations of ammonia, creatinine and cholic acids were attempted in biological fluids, with satisfying results for precision and accuracy, based on enzyme biosensors with potentiometric and amperometric detection. For the second line of research treatments of polyvinyl chloride (PVC) surfaces to achieve thromboresistance by heparinization were performed. This method involves two steps: impregnating PVC with an organic soluble quaternary ammonium salt, tridodecylmethylammonium chloride (TDMAC), and then drying under vacuum until any trace of organic solvent is removed; heparinzing using an aqueous methanol solution of heparin sodium salt. The antithrombogenenicity of heparinized PVC is evaluated by measurement of the Activated Partial Thromboblastin Time (APTT). The influence of the amount of organic solvent and of contact time between PVC and TDMAC on the APTT value of heparinized PVC has been studied, and hence the optimum conditions for the preparation of heparinized PVC have been established. The results obtained show that by this method it is possible to heparinize PVC with good reproducibility and excellent haemocompatibility (APTT value tending to infinite).
A newly developed sensor of oxygen flux measurements in vivo has been prepared for a clinical trial in the Dermatological Hospital of the University Bochum (Universitaetshautklinik Bochum). A new programme for reflectance measurements has been developed using the Kubelka-Munk algorithm which has been applied for in vivo monitoring of the metabolic state of biological tissue. It has been shown that biological tissues are quite transparent in the wavelength range 650 nm to 1200 nm. This optical window will be used to develop sensors by which interesting parameters (as for example pH) can be measured non-invasively in the unopened tissue).
Optical methods were used to measure the oxygen concentration in human blood. The sensor was based on spectral measurements of blood colour and applied as a pulse generator for cardiac pacemakers. The system was successfully tested under real conditions in cooperation with the medical department of the University of Munich. A special sensor system as developed on the basis of conductivity measurements for the determination of lung volume and breath moisture. The sensor is applied for diagnostic and therapeutic control in medicine.
In terms of in vivo sensing, in a joint programme between DCU, St Vincent's Hospital (Dublin), Ciba-Geigy (Basle), Max-Delbruch Centre (Berlin) and University of Vienna, novel instrumentation has been developed which couples microdialysis to microflow systems and biosensor array detection for glucose and lactate. The ultimate aim is to develop a bedside monitor system for critical care situations. Work is currently being done on test animals, before moving on to human subjects. A prototype device now exists for online monitoring of glucose and lactate, however it needs new investment to develop a product which can be marketed worldwide.
The main field of research is the in vivo noninvasive investigation and assessment of the autonomic nervous system and the cardiovascular system. The group has conducted extensive studies related to investigating the possibility of the early detection of physiological changes associated with diabetes neuropathy by making measurements on juvenile diabetics. On the basis of data from hundreds of patients, the group has designed and built 5 novel pieces of experimental computerized electronic equipment. In addition, the group is collaborating with other researchers and clinical investigators to investigate the possibility of developing a sensitive technique and method of in vivo monitoring for the onset of hypoglycaemia, by extracting hidden information both from the real time skin transient pulse blood (STPB), detected absorption, and from the response waveform as a whole of the transient signal of the skin sympathetic sudomotor activity, detected as skin electrical impedance. This project involves the application of bioelectronics and information technology in the in vivo sensing of glycaemia. The personal computer (PC) based system developed by the group supports a variety of analysis programs which have been instrumental to the success of its research efforts. Because of this, the results obtained have indicated the suitability of the use of the fast Fourier transform (FFT) analysis of STPB to the assessment of the autonomic cardiac dysfunction, and that it is possible to easily, rapidly and non-invasively distinguish between the sympathetic and parasympathetic cardiac activity significantly improving cardiovascular diagnosis by lowering both the cost of diagnosis and the discomfort to the patient. With the development of a new PC based system it has been possible to study the reaction of the autonomic nervous system to blood sugar concentration experienced in hypoglycaemia. Preliminary in vivo evidence has confirmed that insulin can have a direct effect on cardiovascular dynamics and particularly on blood pressure, heart rate, sudomotor activity, vasomotor activity etc.
Devices for the measurement of oxygen, pH, carbon dioxide, glucose, lactate, glutamine and glutamate are already in a mature state. A nonlinear optically transduced pH sensor for microphysiometry and immunosensor application is being investigated. The first generation device has a resolution of 0.001 pH units. The glucose, lactate, glutamine, glutamate and lactose devices are based on the electrochemical detection of enzymatically produced hydrogen peroxide. The enzymes are physically entrapped in hydrogel membranes covering a modified thin film platinum electrode. The key technology developed is a thin film process for the formation of miniaturized hydrogel membranes. In gas and ion sensors, these hydrogel membranes serve as electrolyte compartments. In biosensors, they serve as a matrix for the entrapment of enzymes and as well as a diffusion barrier. The hydrogels consist basically of poly(HEMA) and they are prepared by crosslinking a liquid precursor solution under ultraviolet irradiation. Performing this irradiation on a mask aligner allows microfabrication of such membranes. Sensors comprising multiple closely spaced enzyme membranes can be realized without cross-contamination of enzymatic precursor is a gentle method of immobilization, the immobilized enzymes showing dramatically increased stability. The enzyme membrane precursors can be applied to the substrate comprising the electrodes either by a spinon process or by simply dispensing it. Depending on the desired application different technologies for the fabrication of the transducing electrode arrangement are applied. Devices intended for in vivo application should be small and flexible in order to avoid excessive tissue damage. Therefore a highly flexible polymeric base material onto which an electrode arrangement is deposited using thin film technology was employed. This transducer comprises the necessary counter and reference electrodes in addition to 2 working electrodes and is typically configured as an integrated glucose/lactate sensing device. Many medical monitoring applications require sensing elements incorporated in an extracorporeal flow through arrangement. A microfluidic liquid handling part with an integrated sensor array was developed. A thin or thick film electrode arrangement was used comprising 1 reference electrode and 4 individually configurable working electrodes and printed circuit board comprising a counter electrode, drilled holes for inlet and outlet of analyte, and a photo-patterned seal. These 2 parts are assembled to give a 4 parameter (eg glucose/lactate/glutamine/glutamate array) flow through device with an internal volume of 1 ul.
Advances have been made in in vivo glucose sensor design and development of chemically modified electrodes. This approach avoids use of mediators and instead employs electrocatalytic films on the electrode surface. These electrodes have been fabricated in a needle-type format for potential in vivo implantation. The patent on this technology (PCT/GB95/00265) is available for license or sale. A key element in the commercial introduction of in vivo sensors is their automated production. Cranfield has developed a number of technologies suitable for the rapid production of reproducible sensor structures. Screen printers are used to produce multilayered structures on planar substrates and nondelicate surfaces. Ink jet printing is suitable for deposition of material onto nonplanar and delicate surfaces, and is economical with reagents. Air brush printing produces a very fine spray which is deposited as a printed pattern. Cranfield Biotechnology Centre offers these and other deposition technologies as a service to industry. Monitoring of lactate can be used to assess either fitness or well being especially under stress. A miniature screen printed amperometric electrode has been developed for determining in vivo lactic acid concentrations in the horse. The flexibility and small size of the electrodes linked to the ability to detect a wide range of concentrations offer major advantages for in vivo lactate monitoring. The approach is being extended and compared to alternative strategies using direct electron transfer from lactate oxidase, with a view to application in critical care monitoring in man. Surfaces showing reduced protein adsorption are usually the most biocompatible. This can have a great impact on the stability and performance of in vivo sensors which may be required to function inside the body for days or weeks. Cranfield has developed new methods for evaluating the biocompatibility of a material in real time. Optical sensors have been developed based on the interaction of molecules with exponentially decaying electromagnetic fields. This permits real time monitoring of biomolecule surface interactions.
Research has focussed on needle type enzyme electrodes utilizing immobilized oxidase enzymes with detection of hydrogen peroxide. In recent years, the basic structure has been optimized to achieve sensor responses that are independent of sample variables such as viscosity and stirring. Enhanced stability in blood has been achieved by controlling the properties both of external and internal barrier membranes of the device, using, respectively, polyurethane and a sulphonated polyether ether sulphone; such inner membrane protection appears to be due to rejection of electroinactive passivating species. Some form of inner membrane appears vital for stability quite beyond any selectivity criteria. Irrespective of the membrane materials used, some loss of sensitivity in blood or tissue is inevitable and in tissue response does not correspond to blood values. Accordingly, an Open Microflow system has been devised, whereby an ultrafine flow of solution over the sensor tip maintains separation of the sample and sensor while allowing a substrate (eg glucose) to diffuse to the detector surface. During in vivo use, the procedure allows reliable continuous determination of body glucose levels following a short 30 min to 60 min stabilization period with no apparent requirement for in vivo calibration or evident drift (explantation response within 3% of original). The latter feature is of particular relevance to the ease of use of a device, giving clinical reliability, during least short term monitoring. Open microflow in tissue implanted devices does not require the use of a pump, the fluid requirement is minimal and the external cannula for channelling the fluid flow is of the diameter of standard needles used for venepuncture. In tissue, the novel fluid interface helps to minimize the tissue inflammatory response and to maintain tissue hydration for good local solute mass transfer characteristics. Of possible relevance is the use of a stainless steel pseudo reference electrode to avoid the local toxic effects due to silver/silver chloride.

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