Isabella Moser

Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine

For simultaneous measurement of glucose, lactate, glutamine, and glutamate a biosensor array is implemented in a micro flow-system thus giving a microsystem. The microsystem consists of a glass chip with the integrated biosensor array and a bottom part, which comprises a gold counter electrode, a 300 microm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 microl when integrated with a mixer on chip. The biosensors with no crosstalking and high long term stability were produced by modifying the electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producable devices for in vivo and ex vivo applications in whole blood and fermentation broth. Due to a novel glutaminase with an activity optimum in the neutral pH range direct and simultaneous monitoring of glutamine together with glucose, lactate, and glutamate could be performed.

Design and development of a miniaturised total chemical analysis system for on-line lactate and glucose monitoring in biological samples

A miniaturised Total chemical Analysis System (μTAS) for glucose and lactate measurement in biological samples constructed based on an integrated microdialysis sampling and detection system. The complete system incorporates a microdialysis probe for intravascular monitoring in an ex vivo mini-shunt arrangement, and a silicon micromachined stack with incorporated miniaturised flow cell/sensor array. The prototype device has been developed based on state-of-the-art membrane and printed circuit board technology. The flow-through detection system is based on a three-dimensional flow circuit incorporating silicon chips with stacked micromachined channels. An integrated biosensor array (comprising enzyme sensors specific for glucose and lactate) is placed at the base of the stack allowing the detector to be incorporated within the μTAS assembly. These glucose and lactate biosensors are prepared using photolithographic techniques, with measurement based on the detection of hydrogen peroxide at glucose oxidase and lactate oxidase modified platinum electrodes. The resulting amperometric current (at 500 mV vs, Ag/AgCl) is proportional to the concentration of analyte in the sample. All instrumentation is under computer control and the complete unit allows continuous on-line monitoring of glucose and lactate, with fast stable signals over the relevant physiological range for both analytes. The microdialysis system provides 100% sampling efficiency. Sensor performance studies undertaken include optimisation of sensitivity, linearity, operational stability, background current, storage stability and hydration time. The total system (sampling and detection) response time is of the order of 4 min, with sensor sensitivity 1-5 nA mM-1 for glucose and lactate over the range 0.1-33 and 0.05-15 mM, respectively.

Mass producible miniaturized flow through a device with a biosensor array

A mass producible miniaturized device for the simultaneous monitoring of different metabolites was realized by assembling of a biosensor array produced by thin film technology with a flow through cell produced by printed circuit board technology. The biosensor array comprises four working electrodes which can be individually configured. Glucose-lactate devices were made for human whole blood monitoring. Ex vivo experiments, performed with human volunteers, where the device was continuously operated in an extra corporeal undiluted heparinized blood stream for 6 h, gave close tracing to laboratory techniques by using in vitro calibration without loss in sensitivity.

Targeting tumour hypoxia to prevent cancer metastasis: from biology, biosensing and technology to drug development : the METOXIA consortium

Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation–deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009–2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [18F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Microdevice with integrated dialysis probe and biosensor array for continuous multi-analyte monitoring

The simultaneous on-line determination of glucose and lactate using a microdevice that consisted of a dialysis sampling system incorporated to the flow-through cell of a microfabricated biosensor array is presented. The fluidic connections between the different devices components were realized by subsequent processing of stacked dry resist layers on a plastic support that provided also the means for electric connections. The performance of the device was evaluated in vitro. The cross-talk effect on the downstream sensor was investigated and found to be negligible. Recoveries of over 95% for both analytes were achieved when flow rates of the perfusion fluid </=0.5 microl/min were used. At this flow rate, the response time of the device was 2.4 min, which is acceptable for on-line analysis. The linear response concentration range extended up to 30 mM for glucose and 15 mM for lactate. Interference from electroactive species such as ascorbic acid, 2-acetamidophenol and uric acid, was minimal (less than 5% increase in biosensors signal for all substances tested). In addition, the device presented long-term run stability both in buffer and serum samples.

Rapid liver enzyme assay with miniaturized liquid handling system comprising thin film biosensor array

A miniaturized analysis system for the rapid assay of liver transaminases activities was produced by means of hybrid technology. Microfluidics was realized with printed circuit board technology and combined with thin film glutamate biosensors. The function of this system was demonstrated in buffer solutions with GOT (AST) and GPT (ALT). Sample and reagent volumes required are 70 μl each and assay time is 4.5 min. The miniaturized system has an outer dimensions of 42×22×1.5 mm3 and a total internal volume of 11 μl.

Novel instrumentation for real-time monitoring using miniaturized flow systems with integrated biosensors

A prototype miniaturized Total Chemical Analysis System (μTAS) has been developed and applied to on-line monitoring of glucose and lactate in the core blood of anaesthetized dogs. The system consists of a highly efficient microdialysis sampling interface sited in a small-scale extracorporeal shunt circuit (‘MiniShunt’), a silicon machined microflow manifold and integrated biosensor array for glucose and lactate detection with associated computer software for analytical process control. During in-vivo testing the device allowed real-time on-screen monitoring of glucose and lactate with system response times of less than 5 min, made possible by the small dead volume of the microflow system. On-line glucose and lactate measurements were made in the basal state as well as during intravenous infusion of glucose or lactate. The prototype μTAS is currently suitable for trend monitoring but refinements are necessary before application of the system for determination of individual lactate values.

Integrated optical pH sensor using replicated chirped grating coupler sensor chips

Integrated optical sensor chips suitable for high-resolution pH measurements are presented. The pH-sensitive swelling of a polymer membrane is detected by refractometry using a compact multi-channel sensor module. The signal transduction is achieved by means of chirped grating couplers which allow simple yet high functionality sensor modules to be built. The experiments have been performed with high sensitivity replicated polycarbonate TiO2 waveguide sensor chips coated with an ultrathin photopatterned hydrogel membrane having functional groups which reversibly change from the neutral state to a charged state upon acidification. A resolution δpH <±1.1×10−4 in terms of the pH (at pH 7.5) has been obtained in a dual-channel module with size 10×10×10 cm3.

BioMEMS device with integrated microdialysis probe and biosensor array

The fabrication of a microdevice for continuous sampling and on-line monitoring of glucose is described. The device comprised a microdialysis sampling system integrated on the flow through channel of a microfabricated enzyme sensor. The sensor was produced by thin film technology and was assembled to a printed circuit board (PCB) that provided the means for both electrical and fluidic connections. A polyacrilonitrile fibre, with a cut-off of 50 kDa, was used in the fabrication of the sampling probe. The performance of the device was evaluated in-vitro. High sampling efficiency of the microdialysis probe was achieved by appropriate selection of the perfusion fluid flow rate. Response times varying from 1.5 to 3.0 min were determined for flow rates ranging between 1 and 0.2 micro l/min. The linear response range was up to 30 mM glucose and interference from other electroactive substances was almost negligible. The device showed excellent stability under continuous operation for at least 5 days and sensitivity variation less than 3% over a period of 15 days.

Continuous Measurement of Subcutaneous Lactate Concentration During Exercise by Combining Open-Flow Microperfusion and Thin-Film Lactate Sensors

The present study was carried out to investigate in vivo in healthy humans the method of open-flow microperfusion for monitoring of the subcutaneous (s.c.) lactate concentration during rest and cycle ergometer exercise. Using open-flow microperfusion, a perforated double lumen catheter with an inflow and an outflow connection is inserted into the s.c. adipose tissue and perfused with a sterile, isotonic, ionfree fluid. Due to the low flow rate, the fluid partially equilibrates with the surrounding tissue. The equilibrated perfusate passes a sensor flow chamber where the substance of interest and the rate of recovery (i.e. the ratio of sampled concentration to interstitial concentration) are continuously monitored. Within this study, the method was evaluated in four healthy volunteers during cycle ergometer exercise. The relative increase of the lactate concentration was approximately a third in the s.c. tissue compared to the capillary blood and the peak time was delayed on average by 10 min. The correlation coefficient between blood and s.c. tissue lactate concentration ranged from r = 0.41 to r = 0.90 (n = 29) in the individual experiments. The combination of open-flow microperfusion and lactate and conductivity sensors enables on-line monitoring of the s.c. lactate concentration without in vivo calibration during steady-state and cycle ergometer exercise.