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

ANALYTICA CHIMICA ACTA

ELSEVIER

Analytica Chimica Acta 346 (1997) 341-349

Design and development of a miniaturised total chemical analysis system for on-line lactate and glucose monitoring in biological samples Eithne Dempsey a'*, Dermot Diamond b, Malcolm R. Smyth b, Gerald Urban c, Gerhard Jobst c, Isabella Moser c, Elisabeth M.J. Verpoorte d, Andreas Manz e, H. Michael Widmer f, Kai Rabenstein g, Rosemarie Freaney g aDepartment of Chemistry, Regional Technical College, Tallaght, Dublin 24, Ireland bSchool of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland ¢Institut fiir Allgemeine Elektrotechnik und Elektronik, Technische Universitiit Wien, Gusshausstrasse 27, A-1040 Wien, Vienna, Austria dlnstitute of Microtechnology, University of Neuch~tel, Rue Jaquet-Droz 1, C-2007 Neuch~tel, Switzerland eDepartment of Chemistry, Imperial College, London SW7 2AY, UK fCorporate Analytical Research, CIBA AG, FO 3. K-12Z1.56, CH-4002 Basel, Switzerland gst. Vincents Hospital Elm Park, Ballsbridge, Dublin 4, Ireland

Received 8 September 1996; received in revised form 21 January 1997; accepted 30 January 1997

Abstract

A miniaturised Total chemical Analysis System (~tTAS) 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 btTAS 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/AgC1) 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 0.1-35 and 0.05-15 mM, respectively. Keywords: laTAS; Microdialysis; Biosensors; Lactate; Glucose; On-line monitoring

*Corresponding author. 0003-2670/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. P l l S 0 0 0 3 - 2 6 7 0 ( 9 7 ) 0 0 1 0 1-3

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1. Introduction Continuous on-line monitoring of biochemical analytes such as lactate and glucose in critically ill patients would be of great benefit for therapeutic decisions and disease prognosis classification [1]. In order to achieve this goal a reliable sampling and analysis system must be developed which meets the necessary clinical requirements, e.g. size, response time, specificity, sensitivity, reliability and biocompatibility. The concept of a miniaturised Total chemical Analysis System (~tTAS) was first proposed by Manz et al. [2], and numerous systems have since been developed based on miniaturised analytical techniques. Practical benefits to be derived from such a miniaturised analysis system include the ability to analyse small volume samples, with increased speed of analysis, reduction in reagent consumption, and consequent reduction in waste disposal. The reduction in device size also allows for increased flexibility resulting in location of instruments at or near the site of use. This allows for continuous analyte monitoring, which is of great benefit in biomedical applications, where elimination of dependence on external laboratory analysers should have an enormous impact on the way in which chemical and biochemical processes are controlled, as the relevant chemical information may be provided to the ~tTAS user in the form of a continuous electronic readout, over the monitoring period. Since high analyte recoveries in microdialysis sampling requires very low ~tl min -1 and even nl min -1 flow rates, avoidance of dead volume becomes an important issue in system design. Miniaturisation of the flow manifold may be achieved by reducing both the length and the radius of the flow channels, with subsequent integration into complex channel networks. The application of photolithographic techniques [3], using a standard double-sided single mask procedure, to design and fabricate flow manifolds on planar substrates such as silicon and glass, allows structures with ~tm dimensions to be fabricated, and was the approach implemented here. These structures can incorporate liquid handling features for sample preparation, injection and post-column reactions and detector cells in microchromatography and capillary electrophoresis systems [4-9]. The microflow stack employed is capable of providing the flexibility of

sample processing (e.g. dilution or reagent mixing), allowing a built-in calibration capability to compensate for changes in sensor sensitivity. Integrated biosensors specific for glucose and lactate were employed in combination with the silicon structures, and were fabricated using thin film technology to deposit electrodes on glass. The sensors are based on the measurement at a platinum electrode of H20 2 produced by lactate oxidase and glucose oxidase, which were entrapped in photopatterned poly (hydroxyethylmethacrylate) (pHEMA) hydrogel membranes [10,11]. A very small volume measuring cell integrated with the sensor array was developed, and incorporated into the base of the silicon microstructure stack, allowing a closed microflow manifold with incorporated detector. A prototype ~tTAS device for continuous monitoring of glucose and lactate has been developed and subsequently employed in vivo [ 12] using a novel high efficiency microdialysis sampling system [13]. This paper addresses the development and optimisation of the microflow manifold/biosensor component of this prototype IxTAS.

2. Experimental 2.1. Materials

Electrochemical flow measurements were conducted at room temperature in a buffered heparinised modified Dulbeccos solution of pH 7.6, osmolality 300 mOs kg -1 H20, containing 7.54 g 1-1 NaC1, 0.2 g 1-1 KC1, 0.2 g 1-1 KH2PO4, 1.15 g 1-1 KzHPO4 2.1 g 1-1 Na(HCO3)2 and 60 mg 1-1 Enoxaparin. Glucose and lactic acid were purchased from GPR (London, UK) and Sigma (Poole, Dorset, UK), respectively. KH2PO4, K2HPO4 and NaHCO3 of AnalaR grade were purchased from BDH (Poole, Dorset, UK) and NaC1 and KC1 from Merck (Darmstadt, Germany). Microdialysis probes (CMA/10) and the microperfusion pump (CMA/100) were purchased from Carnegie Medicin AB (Stockholm, Sweden). Dialysis membranes employing aminocellulose or polyacrylonitrile fibres were used in a mini-shunt arrangement described previously [13]. Batches of silicon chips were fabricated at the Centre Suisse d'Electronique et de Microtechnique

E. Dempsey et al./Analytica Chimica Acta 346 (1997) 341-349

,~--~ I reservoir _n ~[.~ ~ minishunt ~ n

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Fig. 1. Experimental setup showing a simulated vascular circuit incorporating a peristaltic pump for pumping blood/serum through the minishunt (3 ml min-1); microdialysis perfusion pump pumps perfusion buffer through the inner fibre of the mini-shunt at 3.0 Ixl min 1 in the countercurrent direction; 3x3 way valves; biosensor with signal acquisition under computer control. Reservoirs 1, 2 and 3 contain buffer, low (0.2 and 1 mM) lactate and glucose standard and high (2 and 10 mM) lactate and glucose standard, respectively, for sensor calibration purposes.

SA (CSEM), Neuchatel, Switzerland. The biosensor array was designed and fabricated at the Technische Universit~it Wien, Vienna, Austria. Three way solenoid diverter valves (LFYA) were purchased from Lee (Bucks, UK) and the DAQPad-1200 interface module from National Instruments, Austin, TX.

2.2. A p p a r a t u s

The microdialysis sampling and electrochemical analysis system consists of a microperfusion syringe pump (CMA/100) for delivery of the microdialysis buffer (modified Dulbeccos), 3 x 3 way valves for diversion of flow during calibration or microdialysis, a simulated vascular circuit incorporating a peristaltic pump, and the microflow stack/integrated biosensor with interface to a LapTop PC (Fig. 1). Three digital input/output signals (0-5 V) from the computer control the three-way diverter valves with switching achieved via a power circuit, built in-house. During normal microdialysis sampling and subsequent dialysate analysis, valve 1, which is connected to syringe 1 of the microdialysis perfusion pump (refilled by buffer reservoir 1) is open. This allows

continuous perfusion of the microdialysis probe (minishunt [13]) with at a constant flow rate of 3 pl min -1 while valves 2 and 3, connected to syringes 2 and 3 of the perfusion pump (refilled by calibrant reservoirs 2 and 3), are closed to allow flow to waste (Fig. 2). This allows the microdialysate sample, containing the analyte of interest, to be pumped continuously to the biosensor for analysis. The biosensor array may be calibrated independently of microdialysis, before and after dialysis experiments, by switching diverter valves 1 and either 2 or 3 to waste (Fig. 2). This allows the calibration solution from reservoir 2 (low calibrant glucose (1 mM) and lactate (0.2 mM)) or reservoir 3 (high calibrant glucose (10 mM) and lactate (2 mM)) to reach the biosensor detection system via the microdialysis perfusion pump (3 ~tl min-1). A two point calibration is achieved with sensor slope calculated and stored via the LabView software. The detection system includes the microflow unit consisting of a stack of silicon wafers (22 × 22 mm, 380 ~tm thick), with etched flow channels. At the base of the silicon stack (Fig. 3) is the amperometric biosensor array comprising in total six electrodes, two active glucose sensors, two active lactate sensors,

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E. Dempsey et al./Analytica Chimica Acta 346 (1997) 341-349

3

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Fig. 3. Arrangement of the microflow stack for microdialysate sample analysis. Silicon wafers are arranged to form a closed flow manifold allowing dialysate sample or calibrants to reach the sensor at the base of the stack.

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waste bioseneor array

Fig. 2. Diagram showing valve fluidics. Perfusion pump syringes 1-3 contain buffer, low standard (glucose and lactate) and high standard (glucose and lactate), respectively, refilled by reservoirs 1-3 (Fig. 1). During microdialysis valve 1 is open (full black lines) and buffer is pumped (at 3 Hml rain-l) from syringe 1 on the microdialysis perfusion pump through the mini-shunt system and on to the sensor. During calibation, valve 2 or 3 is open (dashed lines) for low or high standard, respectively, to be pumped (3 lal min-1) from syringe 2 or 3 directly to the sensor.

an Ag/AgC1 reference electrode and a gold counter electrode. A four channel potentiostat [11] with analog outputs was employed for application of the voltage (500 mV vs. Ag/AgC1) to the sensors. The sensor array output signals were monitored using a DAQPad-1200 module via four differential analog input channels which read the voltage signals from each of the biosensors vs. a c o m m o n reference (Ag/AgC1), with subsequent display on the LapTop screen. The DAQPad-1200 module also enables complete control o f system fluid handling operations via control of the microdialysis pump (CMA/100) and switching of diverter valves (allowing either normal microdialysis and analysis, or alternatively independent sensor calibration).

The peristaltic pump (Fig. 1) was used to pump dialysis calibrants, plasma or serum samples through the mini-shunt microdialysis probe (at 3 ml min -1) while the microdialysis pump perfused the inner fibre with modified Dulbeccos buffer in the countercurrent direction (at 3 ~tl min 1). The resultant microdialysate sample could be collected for serial discrete analysis using the YSI STAT automatic analyser or presented to the on-line biosensor for quantitative analysis. A complete in vitro characterisation of the microdialysis system has been carried out and is the subject of a separate report [15]. A YSI 2300 STAT-PLUS glucose and lactate analyser (Yellow Springs Instruments, OH) [14] was employed for some of the microdialysis in vitro evaluation studies and used as a comparison method for serum analysis. 2.3. Procedures 2.3.1. Integrated ~tflow stack and biosensor array Fabrication and assembly The biosensor array comprises a platinum layer of 60 nm thickness, sandwiched between two layers of 100 nm titanium, which was evaporated under high vacuum conditions onto a glass substrate (300 ~tm thickness and 6 × 6 cm2). This metal layer was patterned in a lift-off process, where a negative photoresist pattern - made on the glass wafer before evaporation of the metal layer - was removed with

E. Dempsey et al./Analytica Chimica Acta 346 (1997) 341-349

solvent. A 1 ~tm thick silicon nitride insulation layer was deposited in a plasma enhanced chemical vapour deposition process. This insulation was removed from the desired electrode and contact areas by a plasma etch with O2/CF4. The formed electrode pattern comprises five 0.5×0.5 m m 2 electrodes per device. One wafer of 5 ×5 cm a dimension comprises 45 of thesis devices with a size of 4 × 7 m m 2. Four of these five electrodes are intended as working electrodes with the fifth as a reference. All working and all reference electrodes are electrically interconnected on the wafer to allow galvanic processing at wafer level. A 10 ~tm thick layer of silver was galvanically deposited on the reference electrode which was subsequently chlorinated galvanically. The platinum working electrodes were platinised in 2% aqueous hexachloroplatinic acid and modified with a potentiodynamically electropolymerised 1,3-diaminobenzene [16,17] semipermeable membrane of less than 0.1 g m thickness. A photocrosslinkable hydrogel membrane precursor solution, typically consisting of 47% hydroxyethyl methacrylate (HEMA) (Merck) as reactive monomer, 47% of polyHEMA (Polyscience) as polymeric binder, 5% of tetraethylene glycol dimethacrylate (Aldrich) as crosslinker, and 1% of a~,aJ-dimethoxyco-phenylacetophenone (Aldrich) as photoinitiator dissolved in a water/ethylene glycol mixture was used to immobilise the enzymes by physical entrapment. 50 nl of the enzyme membrane precursors was deposited on the corresponding working electrodes by a nanolitre dispenser on a X - Y stage and subsequently crosslinked under UV flood light for 1 min. After development in water/ethylene glycol the membranes were 6 g m thick. A 8 ~tm enzyme-free hydrogel membrane acting as diffusion barrier was applied on top of the five electrodes. Finally a catalase membrane 5 g m thick was applied as the top layer to prevent crosstalk and to reduce flow sensitivity. The printed circuit board (PCB) for the assembly of the thin film biosensor array comprises conducting pads for the thin film device, the plug connection to the potentiostat, a 5 × 1 m m 2 gold counter electrode, drilled-through holes (0.5 m m diameter) for liquid inlet and outlet, and a photopatterned spacer made from the dry film resist used for insulation of the PCB. Assembly of the thin film sensor array with the PCB (Fig. 4) using conductive adhesive and subsequent dye bonding gives an analytical micro flow system with a

345

Fig. 4. Photomicrographillustrating the assembling of the thin film sensor array with the printed circuit board: (a) sensor array; (b) counter electrode; (c) spacer; (d) channel for conductive adhesive; (e) inlet hole for conductiveadhesive; (f) outlet hole for conductive adhesive; (g) bonding pads.

total internal volume of 2.1 pl. The dimensions of the flow chamber are 5 x l x0.3 mm 3 (see cross section, Fig. 5). The fabrication process involved the following stages. First the laminated copper layer of a PCB was patterned to give the electrical connections to the biosensor array and a large counter electrode in the flow cell. Through holes of 0.5 m m diameter for sample inlet and outlet were drilled through the PCB. The entire structure was galvanically coated with a 2 p m layer of gold. Insulation of the contact lines and formation of the seal forming the flow cell walls was achieved by application of a dry film solder mask resist of 100~tm thickness. The resist was laminated by a heated roll onto the PCB. After exposure to UV radiation through a mask on a PCB illuminator the unexposed areas were removed with sodium hydroxide solution. This process was repeated three times to give a flow chamber height of 300 ~tm. The flow cell was formed by assembling this PCB part with the miniaturised biosensor array and subsequent dye bonding. The large gold counter electrode on the PCB, which forms the top of the measuring cell, saves space on the thin film device and is very efficient in shielding the sensors from electrical noise. The electrical contact between the sensor array and the PCB was achieved by dispensing a two component conductive adhesive into the inlet holes from where it is guided by the channel made with dry film resist over

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E. Dempsey et al./Analytica Chimica Acta 346 (1997) 341-349

Ag/AgCI Reference Electrode

Glass/Chip Insulat~\l\ ndividua/E/nzyme Electrodes/ SPa//c~mplInle etAuCounte!Ele~;~ieOutletp°xy~Sea' Fig. 5. Schematic showing a cross section of the micro flow cell and sensors. Dimensions of flow cell 5x 1x0.1 mm3, electrode dimensions 0.5×0.5mm 2. Reproduced with permission of the editor, Annals of Clinical Biochem.

the contact pads of the PCB and finally through the outlet hole to the bonding pads of the sensor array (Fig. 4). Since the conductive adhesive is thixotropic, the flow of the conductive adhesive stops sharply after reaching the bonding pads. After room temperature curing of the conductive adhesive is completed, the sensor array is encapsulated with a two component epoxy resin. Prior to use, assembly of the silicon stack and integrated biosensor array involved firstly cleaning each silicon chip by sonication in distilled water,

followed by methanol, trichloroethylene, methanol and finally water again. The chips were then dried under nitrogen and the stack assembled, allowing the correct flow channels to be formed, within a screwdown holder, with the integrated biosensor array at the bottom (Fig. 6). This procedure was carried out in a dust free clean-room environment or a laminar flow hood. An adapter clip containing a shallow cavity corresponding to the biosensor unit is required under the sensor chip to ensure a snug fit in the stack. Once assembled in the appropriate configuration the chips are pressed together in the holder to achieve a leaktight arrangement. The unit is held together by screwing a PTFE cover on top of the silicon devices. Solution connections to the top of the stack were made using stainless steel capillaries pressed into Plexiglass plates. 2.4. Software

Virtual instrument (VI) software (LabView Version 3.0, National Instruments) was developed in-house to provide a user-friendly graphical interface for accessing all system functions. The microdialysis control system is the main application VI which allows control of CMA/100 pump O N / O F F actuation, fast syringe refill/empty, slow syringe refill, flow rate (pl min -1) from virtual on/off switches (high/low output) on the VI front panel. The acquisition period

to waste micmdialysis sample inlet (screwed down)

iosensor

~adaptor

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Fig. 6. Diagram of holder and stack assembly, with integrated sensor at the base. An adaptor clip allows for connection to the potentiostat. Solution inlets at the top of the stack allow either perfusion buffer through valve 1 (during microdialysis), or high/low standards (valves 2/3) to be pumped through the stack. Reproduced with permission of the editor, Annals of Clinical Biochem.

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control enables the user to select the data acquisition rate, which is particularly useful as the monitoring process may be continued for long periods of time without generating excessively large data files. The VI software can calculate calibration slopes and intercepts for each of the sensors individually. Post-run data analysis may be performed using EXCEL with data files in text (ASCII) format.

3. Results and discussion Sensor performance studies undertaken included optimisation of sensitivity, linearity, operational stability, background current, storage stability and hydration time. A summary of the sensor characteristics is given in Table 1. The integrated flow cell/biosensor array employed had six electrodes. Electrodes 1 and 2 were glucose oxidase modified sensors, electrodes 3 and 4 were modified with lactate oxidase, electrode 5 was a Ag/ AgC1 reference electrode and electrode 6 was a gold counter electrode. The second sensor for each analyte provides confirmation of the analytical determination

and allows more reliable system fault diagnosis, thus leading to an inherently more robust analytical system. It also allows the option of a redundant (blank electrode) layout for background correction. As the Ag/AgC1 reference electrode has no internal reference solution, it is essential that the chloride ion concentration be held constant in the sampling solution. During clinical microdialysis this is easily achieved by maintaining a consistent chloride concentration in the physiological buffer with which the microdialysis probe is perfused. This matches the biological fluid (serum or plasma) with respect to ionic strength, pH and osmolality. In the development of an on-line system for continuous monitoring, calibration of sensor response is mandatory. The capability to calibrate the sensor quickly is required such that the on-line monitoring process will not be interrupted for longer than can be tolerated. Simple two-point calibrations in the range 0.2-2 m M lactate and 1-10 m M glucose were sufficient to reliably indicate sensor drift over several weeks. Two-point calibrations were achieved by switching the appropriate valves (Figs. 1 and 2) and the resulting model current-to-concentration conver-

Table 1 Features of the integratedbiosensors Feature

Glucose sensor

Lactate sensor

Linear range Background current (nA mm 2)a 95% Rising time Sensitivity(nA mM-1 mm-2) Storage stability at 4°C Storage stability at 40°C Operational stability at 22°C Operational stability at 37°C Within run reporducibilityd Production reproducibilitye Hydration timef Interferencesg Flow sensitivityh

0.1-35 mM 1-5 <25 s 3 >1 year >1 weak >2 weeksb >1 weekc <1% r.s.d. <3% r.s.d. <10 min <0.5 mM -3.1%

0.05-15 mM 1-5 <15 s 8 >1 year >1 weak >2 weeksb >1 weekc <1% r.s.d. <3% r.s.d. <10 min <0.2 mM -3.0%

r.s.d.=relative standard deviation. aCurrent in buffer solution after 30 min at first operation of the device. bContinuousoperation in undiluted bovine serum. COperated in a commercialanalyser and stored in the refrigerator. dTested with the electrodes in a commercialclinical analyser. eSensitivityvariation of devices derived from the same wafer. fTime to reach 98% of final response after exposure of the dry device to the analyte. gReading error due to the presence of 2.0 mM acetaminophen. hChange in reading by reducing the flow speed from 230 to 0.23 mm min-1 .

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sion factors were stored by the VI to facilitate switching between current/concentration scaling of the ' Y ' axis in the VI-recorder display. A calibration study for the integrated biosensor over the range 0-15 m M lactate and 0-35 m M glucose showed a linear relationship with a response time below 15 s. The sensitivity of the biosensors over a four week period ranged from 2.2 to 3.2 nA m M 1 for glucose and from 3.4 to 4.1 nA raM-~ for lactate. The lactate calibration slope relative standard deviations over one week were 5.3%, 6.7% and 2.6% at 2, 4 and 10 mM, respectively. The 3or detection limit of the system for lactate was evaluated at a flow rate of 10 gl min -1 and found to be 45 gM. The lifetime of the biosensor array is ca. four weeks when operated at room temperature, and calibration linearity is not influenced by lifetime. The influence of interferents is low. Addition of potential interferents to a glucose solution resulted in a relative sensitivity of the glucose electrode to ascorbic acid and acetaminophen of 0.3 and 0.1 n A m M -1, respectively. Upon exposure of the microdialysis mini-shunt to human serum [12,15], using the microflow stack/integrated biosensor a fast dynamic response to glucose and lactate was obtained as a steady state signal (Fig. 7). Microdialysis efficiency was determined by the relative recovery of lactate and glucose using the microdialysis shunt dialysis probe. This was found to be 97.7+6,1% and 93.84-5.3% ( m e a n + s t a n d a r d deviation, both n = 14) for lactate and glucose, respec5045 --

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Fig. 8. Inter-method correlation study for serum lactate determination; dialysate samples following microdialysis (Fig. 1) were measured by the YSI analyser and the on-line integrated biosensor (Int BS). Each point represents the mean of two measurements by both methods.

tively, as compared to lactate and glucose measurements in the collected dialysate using the reference YSI analyser. A correlation study to determine the difference between the results from the YSI STAT analyser and the integrated biosensor array was performed by use of microdialysis and subsequent analysis of a series of serum samples (n=19) by both methods. The samples covered the physiological lactate range 0.83-3.08 m M (Fig. 8). The average recovery was calculated as the integrated biosensor signal as a percentage of the YSI direct serum measurement. This was 9 8 . 3 i 5 . 3 % for glucose and 109.7±9.8% for lactate. Lactate resulted in a slope of 1.2 with intercept - 0 . 2 3 m M and correlation coefficient 0.9562 while glucose gave a slope of 0.92, an intercept 0.6 m M and correlation coefficient 0.8469. Each point represents the mean of duplicate measurements (by each method) for each serum sample.

50

Time (rains)

Fig. 7. Biosensor amperometric response (current vs. time), to spiked plasma following microdialysis (experimental setup Fig. 1) Eapp=500 mV vs. Ag/AgC1. Mini-shunt probe was exposed to plasma sample at t=6 min. The two signals for each analyte represent the response from each of the two lactate and glucose electrodes over the time period shown.

4. Conclusions A gTAS system incorporating sampling and analysis has been designed and evaluated for application in lactate and glucose monitoring. Future advances and

E. Dempsey et al./Analytica Chimica Acta 346 (1997) 341-349

developments in membrane technology may in the future replace the functions of the silicon stack and even provide microdialysis with a short time delay in a fully integrated ~tflow device. As each device consists of five electrodes which can be coated individually, this allows for the fabrication of devices with a redundant layout or with the capacity to measure other analytes such as oxygen, carbon dioxide, glutamate or glutamine [11]. Therefore the system may be applied to the simultaneous determination of a wide variety of analytes, with potential application in the biomedical, biotechnological, industrial process control and environmental monitoring areas. Practical benefits to be derived from a miniaturised system include the ability to analyse small volume samples with increased speed of analysis. Further miniaturisation will require acquisition and performance testing of miniaturised pumps and valves for direction of calibrant or dialysate flow, together with the redesign of the individual fluidic elements for smaller volumes.

Acknowledgements The authors acknowledge funding from the Measurements and Testing Programme of the European Commission (Proj ect No. 4014) and facilities afforded at the Education and Research Centre, St. Vincent's Hospital, Dublin.

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