Multisensor for Clinical Analysis with Impact on Public Health Evaluation

of the body. There are clinical situations, for example during cardiac surgery and intensive care, where knowledge of blood p H and K concentration ( p K + ) could warn the clinician of

Potassium is the principal intercellular anion (98% of the potassium is intercellular and only 2% is intracellular), in the adult body is an average of 150 g potassium [2].The potassium has many metabolic roles.Together with the sodium the hydro balance of the body is maintained.While the sodium favoring the retention of the water in the body the potassium is favoring the renal removal of the sodium [3].The potassium has a role in the proteins and lipids metabolism, in harmonic synthesis has a role in the hepatic and muscular synthesis of the glycogen from the glucose, as well as in the muscular fiber synthesis.Potassium is necessary for the transmission of the excitation to the nerve endings of the effectors organs adjusting the neuronmuscular activity.At the same time the potassium is adjusting the cardiac rhythm.At the cardiac muscle level it has an antagonistic action in relation with the calcium; the potassium ions are accelerating the cardiac rhythm and the calcium ions are decelerating it.When it is in excess it can determine muscular paralysis, modification of respiratory and cardiac rhythm until the heart is stopping in the systole [4].
Calcium is the mineral element found in the biggest quantity in the human body (a kilo -a kilo and a half).Almost the whole calcium quantity from the body is fixed in the bones and teeth and the rest is distributed in the tissues and in the biological liquids.Reported to 100 g of tissue in the muscles are 70 g of calcium, in the nerves 15 g, in the cephalorachidian liquid 4, 5 g, and in the plasma between 9 and 10 mg of calcium [5].The concentration of calcium in the blood is maintained between 9-11 %.The calcium has an important role in transmitting nervous impulses, being a tonic of the nervous system and strengthening the functional balance of it.In this way the muscular contraction is dependent on the presence of calcium, because the phenomenon which couples the nervous impulse to the actual muscular contraction is made only by the calcium ions.Together with the magnesium it is responsible for the health of the cardiovascular system.The calcium ions are stimulating the enzymatic equipment of the body.
There are clinical situations, for example during cardiac surgery and intensive care, where knowledge of blood pH and K concentration (pK + ) could warn the clinician of impeding problems.Blood pH is an useful indicator of respiratory efficiency, and pK + levels could both change very rapidly, but presently known methods chemical analysis are not capable of monitoring these vital parameters continuously.The extracellular potassium concentration level (which could vary rapidly) affects both heart rate and contractility, and can be critical in some cases.Knowledge of the activities of other ions, such as calcium and bicarbonate, is also useful in the clinical assessment of patients, and knowledge of the simultaneous activities of a plurality of ions would be especially valuable as their concerted physiological action and interaction may permit more accurate diagnosis.In order to provide the clinician with this information, a number of novel microelectronic chemical sensors have been successfully developed for the measurement of pH, pK + , pCa 2+ , pNa + , pO 2 , and which are readily modified for the detection and measurement of other ionic species and chemical substances [6].Lately it was possible to develop four-channel ChemFET devices, which could be successfully used for the on-line analysis of ions of biological interest in human blood [7].A multiple function chemical transducer for this application would allow more precise computation of ionic activities because compensation could be made for imperfections in the selectivity of individual sensors by processing data from the transducers array in parallel.
The measurement of ionic species in blood has traditionally been achieved by flame photometry, but the information provided by this technique relates to the total species concentration, and not the ionic activity.The latter is more appropriate for clinical diagnosis as the activity of a species is an index of its availability to participate in a chemical reaction, whereas the total concentration may not be related simply to ionic activity [1].For example, plasma proteins bind significant amounts of electrolytes, particularly Ca 2+ which has a normal fasting venous plasma level of 2.4 mmol.L -1 , of which 1.2 mmol.L -1 is complex bound.In this important respect, electrochemical methods of ion analysis are preferable to the use of a flame photometer which gives the total calcium concentration.Furthermore, the flame photometer is large and expensive, and is not suited to use in the operating theatre or at the bedside.
The potential revolutionary improvements in clinical monitoring offered by the integration of ion-specific electroactive materials with the appropriate electronic signal processing circuitry at the microelectronic level justifies a significant effort in the development, investigation and appraisal of chemical-sensitive semiconductor devices for clinical monitoring.
The considerations regarding the ex-vivo monitoring advantages were presented in our previous work [1], where also the various techniques applied in clinical analysis were discussed.
The active component of any ion-responsive sensor is an electroactive material which, ideally, interacts with one particular ionic species in the solution to which the sensor is exposed [8].In practice, no electrode is perfect, and interference from other ions occurs.

Experimental Part
We propose a ChemFET structure using the known Eµ146 for these experiments.The dimension of the channel determines the overall transconductance of the device (dI D / dpX) [9].The Eµ146 device is geometrically similar to the well known Utah University UU-O3 device, with a slightly smaller size [10].The Utah chip uses a 400 mm wide channel which is 20 mm in length, as is the Eµ146 device used in this work.The chips were given n-type channel ion implants (depletion mode).Depletion mode devices had an arsenic implant of 2 .1011 ions.cm -2 by using a 90 keV ion beam; this gave an approximate projected range of ions into silicon of approximately 0.03µ and a projected straggle of approximately 0.02 µ.The devices used were dual-dielectric structures having a 50 nm SiO 2 / 90 nm Si 3 N 4 gate insulator.The Si 3 N 4 acts as an ion-barrier and is an essential feature for stable and reproducible device operation.In figure 1, the Eµ146 device which was manufactured within the Microfabrication Facility at Edinburgh University is presented.
The E µ146 chip (similar to E µ145) features folded gate geometry which allows three active channels.In fact, there are 3 ChemFETs and a single IGFET on this 1.25 mm .2.00 mm chip.In order to use the space efficiently, folded gated are used in all devices.The diffused source and drain regions have low aspect ratios and the metallization tracks extended as far to the channel as effective encapsulation allows, providing minimal serial parasitic resistance.A common drain connection for all devices minimizes the number of lead-out wires (7).
The initial working ISFETs were obtained by bonding the Eµ146 devices on printed circuit board.A transparent polythene sheet, having the design of the circuit board required was placed on top of a circuit board, one side of which was plated with copper and covered with thin film of cross-linked positive photoresist.This was exposed to UV light for 7 min.The areas covered by the printed circuit board patter remain cross-linked, while the area exposed was degraded.The polythene transparency was then removed from the board surface and the latter was placed in a solution of 0.5 mol.dm -3 NaOH for 15s to develop and remove the degraded photoresist and reveal the printed circuit board.The board was then washed in distilled water and placed in an etch solution of 2 mol.dm -3 FeCl 3 at 40 0 C.This removed the copper unprotected by cross-linked photoresist.After washing in distilled water, the crosslinked positive photoresist was removed with acetone to reveal the printed circuit below.The quality of the obtained printed circuit board depends on the initial copper deposition as shown in our previous studies [11].After drying, the board was cut and finally the printed circuit board had the design shown in figure 2. Eµ146 device was bonded to printed circuit boards with an ultrasonic wirebonder (Kulicke and Soffa) using 25 mm gold wire.With the Eµ146 only seven connections were necessary, or possibly only five if the IGFET source and gate were left unconnected.It is necessary that only the chemosensitive gates of devices are exposed to solution.An encapsulant is required that will flow around the bond wires without breaking them but having sufficient low viscosity so that the encapsulant will not run over the chemosensitive gate areas [12].The encapsulant must be electrically inert in such way not to compromise the device performance.It was used as encapsulant Epo-Tek H54 -Alpha Metals Ltd., Surrey.All devices were encapsulated with this epoxy under 95 0 C for 60 minutes, and then to each printed circuit board an edge connector -RS Components, was soldered.Before the chemical-sensitive membranes were deposited upon device gate areas, the latter were cleaned ultrasonically in distilled water for 60s and in isopropyl alcohol for 120s and then dried in a stream of nitrogen.The solutions of the appropriate membranes were applied to ISFET gates by placing a drop from a syringe over the gates [12] and several layers were successively cast.Between each casting, a period of 2-3 h was allowed for evaporation of the solvent.
With regard to the sensor preparation we applied the presently known methods of encapsulation and gate membrane deposition [12].The physical characteristics of the encapsulated devices render them ideal for flowthrough sensor application.
Potassium -responsive devices were obtained by casting valinomycin -doped films from THF solution over the first gate area of suitably encapsulated device.The following chemicals were used for preparing the PVC selective solutions: PVC high molecular weight -Breon (165 mg), valinomycin -Fluka (5 mg), dioctyl phthalate -Fluka (330 mg), potassium tetraphenyl borate -Fluka (1.2 mg), tetrahydrofuran (THF) -Fluka (4 mL).As the previous works proved that the purity of THF used as the PVC solvent affected the sensor response-time and detection limit, then double-distilled THF was used exclusively.The PVC-films were applied to the first gate (the upper one) of encapsulated Eµ 146 device by adding under microscope a single drop of PVC / valinomycin solution.The devices were appraised using a series of calibration solutions of potassium chloride (Analar grade), ranging between 10 -1 mol.L -1 and 10 -6 mol.L -1 , made up with: distilled water, and then with 0.15 mol.L -1 sodium chloride, the later providing a level of sodium interference similar to that of whole blood and establishing a constant activity coefficient for the calibration solutions [11].All appraisals were carried out in a thermostatic bath at 25 0 C. The calcium responsive devices were prepared by dissolving commercially available PVC electrodes membranes (Pye Ltd.) in double distilled tetrahydrofuran.The calcium selective membrane solution was casted over the third gate area (lowest) of suitable encapsulated device.The calibration solutions were made under dry conditions using CaCl 2 .6H 2 O (Fluka) in distilled water and also isotonic saline (150 mmol.L -1 NaCl + 4.5 mmol.L -1 KCl) to provide a background solution similar to whole blood and with an invariant activity coefficient.The membranes were deposed applying strictly the procedure describe in our previous work [1,11].
The obtained devices were calibrated using the dip method with correspondent calibration solutions as mentioned above and also using the constant volume dilution method.The reference electrode used for the determinations was a porous plug saturated calomel electrode -Russell pH Plc, with a 10 -1 mol.dm -3 NH 4 NO 3 salt bridge.
All devices were operated in the constant current mode [13] with I D (drain current) set at 100 µA and V D -(drain potential) set at 1V.The determinations were run on a digital multimeter -Thandar TM451 with precision of 0.1 µV and chart recorder -Linseis 0.5.80Lfitted with back off facilities and incorporating a suitable capacitor across the input to smooth the signal.The devices were tested to ensure that none was liable to electrical leakage.No leakage current should be observed as the bias potential is increased.The devices with a leakage current greater than 1 nA were rejected [14].
For sensitive devices in flow through cell, 16 pin DIL ceramic headers -Shinko, SHK -CA-P160035, Dage Intersem Ltd., were used.These headers are compatible with 16 pin PIL socket adaptors -types 103 -218, Farnell Electronic Components Ltd.Flow through sensors require Perspex flow caps to be fitted (fig.3).
These were attached to the device using silicone rubber -Dow-Corning, RTV-3145, ensuring that no contamination of the gate regions occurs and requiring 12 h curing.The assembly was then screwed into the main body of the Bellhouse dialysis cell-Bellhouse Medical Products.This was then connected via thin, flexible PVC tubing to the sample and the peristaltic pump.The cell could be used with Bellhouse's vortex mixing system.This cell was intended for use in the analysis of blood and serum, where the concentrations of specific ions as sodium, potassium, calcium are of critical importance.It was considered advisable to use a cellophane dialysis membrane, integrated in the flow cell, to separate the ISFET sensing membrane from the sample solution.The dialysis membrane, thereby, avoids sterilization problems associated with protein and anti-coagulant adhesion on the FET.Evaluations of the potassium and calcium ISFETs mounted into a Bellhouse cell for use in clinical chemistry were made.

Results and Discussions
Many factors influence the overall response-time of the ChemFET analysis system, but most significant has proven to be the flow-rate of analyte through the ChemFET flowthrough cell, the distance which separates the input port inner orifice from the ChemFET gate(s) and the dead-space of the flow-through cell.The optimal solution flow-rate through the system for this work was of approximately 1.5 mL.min -1 .Fig. 3.The Perspex flow cap as used to realize the flow-cell assembly

Table 1 TYPICAL CHARACTERISTICS OF ChemFET DEVICE FOR K + AND Ca 2+
The K + -responsive devices were functional as soon as they were exposed to solution although the output signal was prone to drift slightly during the first 12 h of use, after which the device stability was improved and response-time was fast -typically 100 ms.This could be attributed to the gradual hydration of the PVC membrane [15], which is not a symmetrical process for the ChemFET and requires that frequent calibration is carried out during the initial working life of the device if accurate results are required.
A sensitive device to potassium incorporating valinomycin in PVC has been described [16].This gave a linear response to potassium between 10 -1 -10 -5 mol.dm -3 in a background solution of 1.08 .10 -1 mol.dm -3 sodium chloride and the magnitude of the response was approximately 58-59 mV per decade over its linear region during the devices working lifetime which was of 20 days.
The response characteristics of a typical K + ChemFET are shown in figure 4 and summarized in table 1.The devices were not flow-sensitive; the flow-through cell Eµ146 proved to be remarkably stable during use, even when used with a remote, down-stream reference electrode.
The lifetime of the potassium device is limited by the thin membrane which could not provide a larger reservoir of electroactive material as in the case of the corresponding ion selective electrodes [17].The leakage of valinomycin aided by the leakage of plasticizers from the membrane is probably the most important factor in limiting devices' lifetime.
approximately 20 days the magnitude of the response has dropped to 17 mV / decade change in concentration.Device lifetime was most likely to be limited by leakage of the ionophore and plasticizer [20] from the membrane although some devices had their lifetimes shortened by lifting of the PVC membrane from the silicon nitride gate which would infer a certain incompatibility between the two [21].
The manipulation of the ISFET into the holder was awkward and there was a high probability of physical damage to the devices if frequent insertion and removal should be necessary.This could be consider as a pitfall of the flow cell design, as the handling of the devices should be minimized in order to reduce the likelihood of static charging from the operator to the integrated circuit.
Nevertheless, the results obtained show that it might be possible to use the flow cell to monitor the response of both potassium and calcium ChemFETs against a calomel reference electrode, when solutions of differing analyte concentrations were placed either side of the dialysis membrane.
In order to provide a multi-channel on-line monitoring of various electrolytes in a clinical environment there were done some attempts to realize a multiplexing ChemFET interface which met the legal requirements for electrical isolation.Much more, it must be both functionally efficient and aesthetically acceptable to the clinician [22].A central multiplexing interface was designed and is intended for use with a maximum four ChemFET sensors, supporting even a pair of two channel devices as Eµ146 is.Specific software was developed in order to permit the device transfer characteristics to be analyzed under software control, which significantly improves the quality and speed of data collection.All the work developed in this direction will be presented in a following paper after the validation of the data obtained will be also performed.

Conclusions
It was shown that microelectronic engineering techniques can also be used to provide other novel chemical sensors which are well-suited to biomedical applications.Such devices possess unique advantages over their conventional counterparts, in terms of robustness, size, mass-fabrication potential, cost, utility and the superiority of signal quality conferred by the integrated signal-processing elements.
The results obtained for the structure Eµ146 substantiated our earlier work [12,13,21] that the devices are suitable as ex-vivo clinical sensors.The further work The Ca 2+ devices were stored for 24 h in 10 -3 mol.L -1 CaCl 2 solution before testing, as usual practice for commercially available liquid-filled electrodes [18].The response characteristics, summarized in table 1, of a typical Ca 2+ -responsive Eµ device are shown in figure 5.The dynamic response recorded for the calcium selective device proved that the Ca 2+ Eµ146 has a 95% response time of approximately of 400 ms, much faster than its liquidfilled counterpart which has a typical response time of 10s.
Towards the end of the lifetime (18-20 days), the calcium ISFET response time steadily increased to 2 -3 min.At supposes to obtain a multi-sensor by applying the two selective membranes on the same device and the monitoring of the potassium and calcium sensitive devices simultaneously in a flow cell.Also, the calibration solutions for the examination of biological samples should contain protein, thus resembling the sample matrix as closely as possible.In this sense, following our work [15] impedance studies should be carried out using albumin, as the interferent protein in most investigations, but also other proteins as globulin in order to establish if the diffusion of ions at the membrane surface could be affected by the protein.Both selective ISFETs gave good Nernst response, though the calcium device lifetime is shorter when compared with the potassium device, maybe to a faster leakage of the ionophore incorporated in the calcium membrane which is an ion-exchanger in fact.The lifetimes of these polymeric devices appear to be determined by leaching of the electroactive material / plasticizer from the membrane.Owing the ver y thin nature of these membranes and open polymeric structure, it is likely that they are hydrated almost up to the gate dielectric surface, consequently leaching will be enhanced.
The behavior of the devices realized for calcium and potassium presenting a Nernstian answer allowed us to go further in developing a specific interface and software to be used for a completely integrated on-line system.In the immediate future the present work will extend to provide such continuous monitoring of ions of biological interest in a variety of clinical situations following the strict requirements of the public health sector.
Much more, it is likely that our future work will be possible to provide a knowledge and understanding of blood electrolyte kinetics during surgical procedures and post-operative care.Also, it is realistic to think that such research will lead to important improvements in patient care by providing new devices for on-line analysis of the biological fluids.