Microsystem Technologies z (1995) 75-83 �9 Springer-Verlag 1995

Biosensors for analytical microsystems U. Wollenberger, R. Hintsche, F. Scheller

Abstract A modular concept for analytical microsystems with separately developed and optimized components is proposed, where parts can be easily substituted. Since the system should be universal hybrid integration is preferred over monolithic fabrication technology.

The biosensor as an essential part of an analytical microsystem is commonly restricted in its functional stability due to the lifetime of the biological component and the adhesion of the sensitive layer at the transducer. Optimum design requirements have to be derived from the particular system to be measured. For the optimization of the (bio)sensor the polymer matrix carrying or covering the enzyme has been modified, the transducer design itself has been varied, and enzymes have been coupled.

1 Introduction Miniaturized biosensors are receiving increasing interest for medical, biochemical, and environmental applications. The miniaturization can offer diminished production costs, reliability, small size and weight, and therefore can open new fields of application, such as portable devices for field measurements and personal control. However, their application is restricted by their reliability and the limited measuring range. Functional stability of the biocomponent, electrochemical interferences, and adsorbtion of various molecules on the sensor membrane are serious drawbacks. Numerous efforts have been made in the field of modification of polymers to reach the desired performance characteristics, such as selectivity, sensitivity and stability (for overview see: Scheller and Schubert (1989), Edelman and Wang (1992), Buck et al. (199o)).

For the construction of microsystems the transducer fabrication as well as application of the biological compound should be done with microelectronic production facilities. However, the introduction of biocomponents into the sensor fabrication process generates additional problems. For these sensors not only the enzymatic and diffusion characteristics in

Received: 26 May 19941Accepted: 17 June 1994

U. Wollenberger, Rainer Hintsche Fraunhofer Institute for Silicon Technology, Dillenburger Str. 53, D-14199 Berlin, Germany

F. Scheller University of Potsdam, c/o MDC, D-13122 Berlin, Germany

Correspondence to: U. Wollenberger

the membrane are of critical importance, but also the adhesion to the detector surface and the patterning capabilities. In general, new methods of immobilization compatible with the fabrication process of the basic transducer have been developed. Those microelectronic fabrication compatible immobilization matrices include: photopolymers (e.g. Kimura et al. (1986), van den Berg et al. (1992)), plasmapolymers (Kampfrath and Hintsche (1989)), conductive polymers (recently reviewed by Bartlett and Cooper (1993)), Langmuir-Blodgett films (Anzai et al. (1989) Tatsuma et al. (1991), Zaitsev et aL (1991)), and self-assembling layers (Witlner et al. (t992)).

With photolithography and electron beam lithography high structural resolution of the electrode pattern can be realized. Arrays of identical or different sensors fabricated in close vicinity are also feasible. Thus new devices with novel characteristics will extend the applicability of the sensors. Furthermore micromachining techniques permit the formation of three dimensional micromechanical structures for injection, separation, and measurement. With respect to the analytical objection these elements are combined to functional units of a microsystem (Heuberger 1989). The total analytical microsystem is completed with separately developed signal processor and interfaces.

This papers reviews attempts to develop parts of modular analytical microsystems with special emphasis given to the combination of enzymes and microfabricated sensors and describes some results and procedures for optimizing the functional parameters of biosensors.

2 Microsystems Common for all modules of a microsystem is their fabrication in a semiconductor compatible process. From draft design to an optimized microsystem module extensive work is required. Separate development and optimization of the micromachined elements is an important advantage of the hybrid integration technique. Nowadays various types of micromechanical devices, such as valves, pumps, switches, motors, constructive elements like grooves and channels, as well as sensors have been realized. In addition, the use of silicon offers the unique possibility to integrate transducers with standard microelectronics. The range of integrated circuits may extend from amplifiers over signal converter and computer interfaces to local intelligence to create autonomous smart sensor system (Benecke (199o), Heuberger (1989), Menz and Bley (1993)).

Among the first examples of partially developed analytical microsystems are a high pressure liquid chromatographic device,

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were structures for injection and detection, such as a split injector, a packed column, flits and an optical detector cell were integrated onto a silicon chip (Manz et al. (199o)), the integration of an electrophoresis capillary and a sample injector together on a planar device for capillary electrophoresis (Harrison et al. (1992)), and the recently commercialized micro gas chromatograph M2oo (MIT Analytical instruments, Microsensor Technology, Inc., Fremont, CA, USA). The miniaturized components of M2oo include the injector with micromachined capillaries, silicon microvalves, and flow resistors and the solid state detector (Bruns (1992)).

Smart electrochemical sensors have been proposed which are assembled of a CMOS interface circuit and electrode on a silicon substrate, and a micromachined top structure induding a micro-sieve and a cavity (Lambrechts and Sansen (1992)). The important problems of package, bonding, or sealing are still restricting its use. The hybrid construction of two silicon (piezoelectric) micropumps and an ISFET-based flow through cell is the basis of modular fia-system for chemical analysis (van der Schoot et al. (1993)).

A few attempts have been made to fabricate parts of flow systems for biochemical analysis by micromachining techniques. A flow-type biosensing system has been described, where a glucose oxidase-immobilized column was integrated with an electrochemical flow cell on a silicon wafer (Murakami et al. (1993)). Gold electrodes fabricated on Pyrex glass, were anodically bonded to the structured silicon chip before enzyme immobilization. The electrochemical flow cell was used in a fia system for the determination of glucose concentration.

Other efforts include the combination of carrier structures such as micromachined wells and planar sensors (Parce et al. (1989), Suzuki et al. (1991)). Those structures have been constructed in order to improve the attachment of bio- components, which introduce additional specificity to the sensor probe.

3 Biosensors Biosensors are devices which can detect or respond to environmental chemicals through specific interaction with a biological sensing component that is in intimate contact with a signal transducer. The biological component translates the specific molecular recognition of the analyte into a change of an easily measurable chemical or physical parameter. This change is sensed by the transducer which generates an electrical output signal.

Analyte recognition is usually performed by immobilized biocomponents, such an enzymes, cells, organelles, tissue, receptors, nucleic acids, antibodies, and antigens. The most frequently used transducers are electrochemical, optical and thermal detectors, but piezoelectric and surface acoustic wave methods may also be used (Scheller and Schubert (1989), Turner et al. (1987), Hall (199o)). Most of the working principles have been evaluated using enzymes, but with increasing number immunosensors are being developed.

Electrochemical enzyme sensors for about 14o different analytes have been described, among them amperometric biosensors clearly dominate. In amperometric electrodes besides the natural electron acceptors also artificial redox mediators have been included (e.g., Cass et al. (1984), Green and Hill

(1986)). When the redox potential of the artificial mediator is low electrochemical measurements can be performed with diminished interferences. Integration of auxiliary enzymes or cofactors and mediators in the enzyme layer improves the analytical performance and simplifies the handling (e.g., Albery et al. (1987), Hale et al. (1989), Bremle et al. (1991), Yabuki et al. (1991), Schuhmann et al. (1993)). An alternative to the use of mediators is the direct electron transfer between the prosthetic group of enzyme and the electrode (e.g., Armstrong et al. (1988), Guo et al. (1989), Ikeda et al. (1991), Gorton et al. (1992), Wollenberger et al. (199o)). A symbiosis of mediated and direct electron transfer is obtained by covalent fixation of electron- channeling relays to the enzyme protein close to the active center of oxidoreductases (Degani and tteller (1988)). The dense network of such polymeric wires facilitates a very fast electron shuttling from enzyme to the electrode (Ye et al. 1993). Since a molecular wired oxidase biosensor can produce a high charge density, glucose and lactate sensors with gm diameters are being developed based on this approach (Heller et al. (1992), Ohara et al. (1993)).

Nature, however, provides only a limited number of enzymes that are able to generate or consume electrochemicaly active species. Thus the number of analytes that can be measured with usual monoenzymatic amperometric electrodes is limited. One way of overcoming this problem is to couple different enzymes either in sequences, in competing pathways, and cycles. In this way not only does a much wider range of analytes become accessible to measurement, but also the selectivity and sensitivity of the biosensor may be enhanced (Scheller and Schubert (1989), Wollenberger et al. (1993)).

Future prospects are expected from the integration of biosensors in microsystems including other chemical and physical sensors, actuators, fluidic components as well as controller and processors. Therefore presently much emphasis is put on the miniaturization of the biosensor as an evident prerequisite for their integration. To construct microbiosensors in general the same enzymes and coupling principles can be employed, as long as a sufficient activity can be bound.

4 Transducer Benefiting both from the simple principle of measurement and their low price, biosensors based on electrochemical devices are the most developed to date. Ion-sensitive field-effect transistors (ISFETs) (e.g., Caras and ]anata (198o), van der Schoot and Bergveld (1988)) gas sensitive MOS capacitors and thin-film metal electrodes (TFME) (e.g., Koudelka et al. (1989), Hintsche et al. (1991)) are the basic types of microsensors used so far (Lambrechts and Sansen (1992)). The latter can be ampero- metric, potentiometric or conductivity sensors. The major advantages of planar sensors resulting from their micro- electronic production technique are low cost per sensor (if mass- produced in standard process steps), small dimension (high structure resolution possible), high reproducibility (batch processing), and potential for development of smart sensor systems (integration of interface electronics in CMOS process or in multichip module technique), multiple sensor arrays, and microsystems (scaled down analytical systems).

With photolithography and electron beam lithography high structural resolution of the electrode pattern in nanometer

dimensions with various shapes and designs can be realized. Arrays of identical or different sensors fabricated in close vicinity on one sensor chip are also feasible. Thus new devices with novel characteristics will extend the applicability of the sensors.

An array of submicrometer (30o nm-l.5 gm) spaced inter- digitated microbandelectrodes has been presented recently (Hintsche et al. 1993). Using photolithography and an advanced lift-off process these nobel metal electrodes could be buried into the silicon substrate with the result of a planar sensor chip. The small dimensions of the electrodes permit a multielectrode arrangement on one chip.

5 Immobilization In order to create planar biosensors the biological components have to be immobilized directly onto the surfaces. The sensor covering layer or multilayer must provide a maximum activity Of biocomponents and include in dependence on the analytical question appropriate semipermeable, hydrophobic, hydrophilic, and conducting properties.

All procedures used to construct biosensing layers are based either on direct covalent chemical bonding of the bioactive components, or immobilization in a polymer by entrapment, covalent bonding, and adsorption, or combinations thereof.

5.1 Dispensing The simplest way to immobilize an enzyme membrane on a sensor surface is to cast an enzyme gel or dip a mounted sensor into a polymer solution. Immobilization methods using aqueous as well as nonaqueous polymer dispersions such as biopolymers, polyester urethane, polyhydroxymethacrylate, polyvinyl alcohol, polyvinyt pyrrolidone, polyethylene oxide, silicons, and polyacrylamide have been described.

For example enzymes such an urease and fi-lactamase have been entrapped on the sensitive gate area of an pH-ISFET for measurement of urea and penicillin (Caras and Janata (198o), Anzai et al. (1987)).

Other examples include maltose and lactate probes on the basis of polyurethane entrapped enzymes in combination with pF-FETs (Hintsche et al. (1989) and (199o)). In order to eliminate erroneous readings difference ENFETs have been developed which use an enzyme loaded and a blank reference sensor. Furthermore, with the same type of polyester urethane planar thin-film metal biosensors for glucose, lactate, and lysine have been constructed (Hintsche et al. (1989). Because the diffusion of the indicated hydrogen peroxide is only slightly influenced these sensors show a rapid response.

To improve adhesion between the surface and the bio- component containing layer, surface silanization (Colowick and Kaplan (1976)) is often used. Methacryl functionalities are covalently anchored to silicon dioxide surfaces (Sudh61ter et al. (199o)). The resulting polyhydroxymethacrylate (pHEMA) or polysiloxane layers show an improved adhesion to ISFETs or electrodes.

The methacryl modified surface provides the site for a photo initiated radical polymerisation of methacrylate derivatives (Reinhoudt and Cobben (1992)). Microstructuring of enzyme

layers on the wafer plays an important role for the development of multifunctional miniaturized sensors (van der Schoot and Bergveld (1988)).

5.2 Photolithographic patterning of enzyme membranes A number of techniques for deposition and patterning of biochemical membranes for the mass production of sensors have been developed. Most of these techniques, such as photo- lithography, plasma etching, ink-jet-printing, spin coating, screen printing, and lift-off derived from standard CMOS procedures. The most important method is the photolitho- graphy.

For the patterned immobilization of enzyme membranes in a multi-ISFET enzyme sensor for glucose and urea Kimura et al. (1989), developed a lift-off method. In order to obtain thickness and characteristics controllable membrane positive photoresist was used, which consisted of phenol resin and a photosensitive reagent (Nakamoto et al. (1988)). Urease and glucose oxidase membranes were precisely deposited lOO txm wide and 400 pm long on each ISFET-gate. The precision of the membrane thickness (o.1 pro) results from the spincoating step. Negative photoresists have been employed alternatively (Nakako et al. (1986), Hanazato et al. (1987)).

Enzyme-free reference sensors and counter electrodes can be produced by the above procedure, or more easily by inactivation of enzyme spots with X-ray or UV irradiation.

A nonaqueous and an aqueous photopolymer has been used with acetylcholinesterase on a thin-film platinum electrode and urease on the gate of a pH-ISFET to achieve sensors for the respective enzyme inhibitors (Zuern and Mfiller (1993)). Taking advantage from the fabrication technology an amperometric biosensor with glucose oxidase immobilized into stacked (pHEMA) membranes and a potentiometric pH sensor have been integrated on one substrate (Urban et al. (1992)). In this way not only the enzyme layer has been covered by an additional protective membrane but also differential measurements can be performed in order to eliminate unspecific response.

5.3 Conducting polymers Preparation of conducting polymer has become an attractive method for the spatially precise immobilization of enzyme molecules onto planar amperometric and conductimetric sensors. The process can be determined by the electrode potential, and therefore allows control of the film thickness and amount of enzyme entrapped. Basic materials such as pyrrote and anilin have been used extensively (Foul& and Lowe (1986), Bartlett and Whitaker (1987), Fortier et al. (1988), Strike et al. (1993)). The far most examples concern with immobilization of glucose oxidase in polypyrrole for the development of glucose sensors. For example, enzyme immobilization on the sensing micropattern of the chip containing two working electrodes has been performed successively for glucose oxidase and galactose oxidase resulting in a microbiosensor for glucose and galactose. Besides, a variety of oxidoreductases and other conducting and non-conducting electropolymers have been investigated. Glucose oxidase immobilized in conducting polymer which is located within the pores of track etched membrane gave rise to a stable reagentless glucose sensor (Koopal et al. (1992)).

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Furthermore, the functionalization of conducting polypyrrole films has been used for formation of surfaces suitable to link enzymes or other proteins covalently (Schumann et al. (199o), Pittner et al. (1992)).

5.4 Plasmapolymerization Radio-frequency plasma methods have become popular for the surface modification of different materials (Yasuda et al. (1987)). In the case of plasma polymerization, a highly adhesive thin film is deposited from an organic monomer target plasma onto the surface of anorganic and organic substrates.

For example, plasma polymers with amino- or carboxyl- groups have been deposited onto TFME and ISFETs (Kampfrath and Hintsche (1989)).

Figure 1 illustrates the compatibility of this method with the semiconductor technology. By a low temperature glow discharge amino- or carboxyl- containing monomers are deposited onto the sensor active areas using open stencil masks. To the resulting functional groups at the plasmapolymer glucose oxidase has been covalently bound by means of common bifunctional reagents. This procedure could be performed with the whole wafer. In case of more weak biocomponents, the activated single sensor can be loaded with biocomponents immediately prior to use, which is of special interest for sensor constructions with immunoproteins.

6 Optimization of sensor performance For the optimization of the (bio)sensor performance with respect to practical application the polymer matrix carrying or covering the enzyme has been modified, the transducer design itself has been varied, and enzymes have been coupled.

6.1 Increase of sensitivity: Enzymatic recycling The most elegant way to improve the sensor performance is the coupling of enzymes (Scheller et al. (199o)).

Step 1 : grow discharge

Monomers: acryt acidi aminobenzotrif[uoride

Low temperature pl.asma V V V / \ Mask

\ Etectrode or ISFET

I Ste# 2: or NH3 ptasma treatment 02

I Step 3: Covatent coupting of protein side chains to-COOH or-NH2 of ptasmopotymer

Fig. 1. Scheme of immobilization of proteins at plasmapolymers

Substances disturbing either the enzymatic reaction or the electrochemical indication can be eliminated by additional enzyme layers, thus leading to improved selectivity. Preconcentration of intermediates of enzyme reactions leads to amplification between ~ and 60 (Schubert et al. 1991).

A tremendous increase of biosensor sensitivity has been obtained when cycling enzyme reaction have been applied to the electrode (Schubert et al. (1985), Wollenberger et al. (1987), Mizutani et al. (1985)).

The sensitivity enhancement is provided by shuttling the analyte between enzymes acting in cyclic series of reactions under cosubstrate consumption and accumulation of byproducts. In a bienzyme electrode the analyzed species is converted in the reaction of one enzyme to a product which is the substrate of the other enzyme. The latter catalyzes the reformation of the analyzed species which thus becomes available for the first enzyme again, and so forth. Assuming a sufficiently high activity of one enzyme in the presence of its cosubstrate (Q) and an analyte (the concentration of which is far below its Michaelis constant), an amplification is achieved by switching on the second enzyme by addition of its cosubstrate, C2. The analyte is then shuttled between the two enzymes. Consequently a much higher quantity of cosubstrates will be converted to the respective products (P1 and Pz) than analyte is present in the membrane, with the overall reaction of both coreactants: C1 + Cz--+P1 + P2

By measuring the concentration change of one of the coreactants directly or via additional analytical steps, the recycling system is used as a biochemical amplifier for the analyte. The recycling principle has been achieved for about 15 pairs of enzymes such as dehydrogenases and oxidases or transaminases, and kinases couples (Table 1). The limit of detection could be reduced to nanomolar concentrations for some substrates.

The copper enzyme laccase, which catalyzes the oxidation of a wide range of substances including catecholamines, phenols, and ferrocyanide by dissolved oxygen has been used in combination with haem and pyrroloquinoline quinone (PQQ) containing (NADH independent) dehydrogenases. An ultrasensitive biosensor has been created when PQQ glucose dehydrogenase and laccase from Coriolus hirsutus were coentrapped in a lo Mm layer of polyvinyl alcohol in front of a Clark-type oxygen electrode. Owing to the broad spectrum of cosubstrates for both enzymes, the sensor responds to various catecholamines, aminophenol, and ferrocene derivatives. The highest sensitivity were obtained for aminophenol and epinephrine where the lower limit of detection (S/N 3 : 1) is 70 p real/1 and I nmol/1, respectively. The extraordinary efficiency of the amplification sensor is based on the excess of enzyme molecules compared with the concentration of the analyte molecule within the reaction layer. The current density of the membrane covered sensor is almost three orders of magnitude higher than the bare electrode. In this respect the enzyme cycle represents a chemical signal amplifier coupled to transducer by oxygen diffusion. The sequential fixation of laccase and glucose dehydrogenase to a modified carbon fibre permits analyte recycling with mediatorless electron transfer. By analogy to the recently described diode-like electrochemical behaviour of succinate dehydrogenase (Sncheta et al. 1992), direct electron transfer between a cycling enzyme and a redox electrode mimics a biochemical transistor.

Table 1. Substrate recycling in biosensors

Analyte Enzyme couple Transducer

Glucose glucose oxidase/glucose DH oxygen electrode Lactate/pyruvate lactate oxidase/ oxygen electrode

lactate DH

Lactate/pyruvate cytochrome bz/lactate DH NADH/NAD + peroxidase/glucose DH Glutamate glutamate DH/

alanine aminotransferase glutamate oxidase/ glutamate DH glutamate oxidase/ alanine aminotransferase

ADP/ATP hexokinase/pyruvate kinase

Epinephrine glucose DH/laccase Aminophenol Epinephrine oligosaccharide DH/ Aminophenol taccase Benzoquinone/ cytochrome bz/laccase hydroquinone Malate/ malate DH/ oxalacetate lactate monooxygenase L-Leucine leucine DH/

amino acid oxidase Phosphate nucleoside phosphorylase/

alkaline phosphatase

Pt-electrode oxygen electrode modified carbon electrode oxygen electrode

hydrogen peroxide electrode oxygen electrode: lactate DH/lactate monooxygenase; glucose-6-phosphate DH modified carbon electrode: oxygen electrode

oxygen electrode

oxygen electrode

oxygen electrode

oxygen electrode

oxygen electrode with xanthine oxidase

Amplification factor

10 48000; ** 4100; 250 10 60 15

20

500

220

1200"

3900 16700 1000 2000 50O

3

40

2O

79

DH dehydrogenase, * - in combination with intermediate accumulation; ** - different immobilization

Electrochemical recycling The principle of cycling amplification has been also employed at microstructured planar electrodes as part of a modular analytical microsystem (Wollenberger et al. (1994)).

Here, the particular potential of high structural resolution of planar electrode fabrication is exploited that allows novel measurement methods to be created (Aoki (1993)).

The measurement is based on the redox cyclization (Bard et al. (1986), Nishihara et al. (1991), Aoki et al. (1988), Niwa et al. (199o)) between the adjacent microband electrodes of an interdigitated electrode array (IDA). The process occurs when both the oxidation and the reduction potential of the reversible redox species are applied to pairs of interdigitated electrodes. The species is detected at one microband electrode, while the generated product is collected at the other electrode, where it is converted to its initial state. The multiple oxidation and reduction result in an increased anodic and cathodic current generation. Short average diffusion length and the large number of alternating electrodes are necessary for effective current amplification by recycling of reversible redox species. For this purpose, multielectrode arrays of four IDA pairs have been fabricated where pairs of interdigitated thin-film nobel metal electrodes in ~tm and sub-~tm geometries have been arranged on a silicon substrate. The planarized array has been fabricated using photolithography, electron-beam lithography and lift-off technique. A custom made multipotentiostat was used to control the potential of up to 16 electrodes independently (Paeschke unpublished). The current response of the individual electrodes

were separately preamplified after current to voltage conversion and drawn into a data acquisition board.

At the interelectrode spacing of < 1.5 pro, each electrode finger lies within the diffusion layer of the closest neighbouring electrode band.

For the detection of p-aminophenol the anode, which serves as generator electrode, is potentiostated to + 250 mV and the cathode (collector) to - 5 0 mV.

The addition of lO pmol/1 p-aminophenol results in an anodic current of 16o nA within 1 s. A response in the same extent is indicated simultaneously at the cathode. The simultaneously measured response of both electrodes can be summarized. A concentration dependence for aminophenol illustrates the increase of sensitivity (Fig. z). In comparison, the single anode shows a poor response at this low substrate concentration.

The steady state response is enhanced by a factor of lo-15 by the electrochemical cycling, summarized anodic and cathodic currents yield a factor of about 25. Thus the detection limit could be reduced to lOO nmol/1 with reasonable reproducibility. The signal enhancement by redox recycling has been studied for a number of reversible species (Table 2). Obviously collection efficiencies around o.go have been achieved.

The results compare well with theoretical predictions and experimental studies of IDA with large number of alternating microband electrodes of 2 pm width and 1 pm gap (Niwa et al. (1993)).

The combination with enzymes releasing a reversible redox species imports the specificity to the already established

8o

1000 �9 209#` ~ Sum anode +

soo -Oo 500 1000~ ~

_jJJ -~ / ~ Anode w'ffhout recycling ~_ 0, .S~...-.-~ . . . . . . o

V . . . . . . . . :-7 0 25 50

p-aminophenoI (pmot/[)

Fig. 2. Dependence of the response of collector (cathode), generator (anode) and its sum (amplified with redox recycling) and anode (with- out redox recycling) of an IDA on p-aminophenol concentration, poten- tials: anode: 25o mV, cathode -5o mV, gap between microband 1.2 gm

sensitivity. Additional improvement can be drawn from the redundance of the measurements at the other electrode pairs of the array. This results in a better reproducibility.

The IDA-sensor has been applied to the determination of alkaline phosphatase and fl-galactosidase activity by detecting enzyme generated p-aminophenol for electrochemical enzyme immunoassays with this enzyme label (Wollenberger 1994). The activity measurement is based on the quantification of the p-aminophenol formation from electrochemically at + 0.25 V inactive p-aminophenyl phosphate or aminophenylated pyranose derivative.

6.2 Flow arrangement This electrode array has been developed as part of a modular analytical microsystem. Further modules of the microsystem include a flow chamber, a separation element and a multi- potentiostat with a microcontroller (Fig. 3).

REF!

A U X w ~ . • j_UI Microcontrot[er

W ~ i ~ ~ - Potential. control

U 2 - Data aquisition

- Flow control

Multipotentiostat

- - ~ ' /~ ~ ~ ]Sensor chip

~ tz [] (Si flow cett

h

Fig. 3- Illustration of the measurement device with electrode chip and flow-through cell

The flow cell has been fabricated using double side anisotropic silicon etching. The etched channel which can be reaction chamber and measuring cell has the dimensions of the active electrode area of the sensor chip. A window at each end serves as in- and outlet for the sample flow. One of the windows is designed as a filter element by etching a grate. The cell has been stacked onto the electrode chip, equipped with tubings, and connected to the multipotentiostat with a microcontroller. The micropotentiostat unit applies the desired potential to each of

Table 2. List of reversible mediators studied with redox recycling

Mediator Ea, mV Ec, mV S e n s i t i v i t y , Amplification Collection efficiency nA l/gmol factor

Fe(CN)~- 350 -- 120 2.8 8 0.98 o-HQ/o-BQ 600 - 200 7.2 9 0.92 p-HQ/p-BQ 400 - 75 5.7 14 0.94 p-Aminophenol 250 - 50 16.4 12 0.92 Dimethylferrocene* 600 0 4 n.d. 0.96 Ferrocene 600 0 2.5 8 0.93 dicarboxylic acid* Ferrocene lysine* 400 0 6 13 0.96 *** PEG-ferrocene** 400 100 7 0.94/0.83 Dopamine* 800 100 7 3.5 0.82 PMS 0 -- 200 18.6 12 0.95

*Au IDA; **PEG MW: to/2o T, Pt; ***flow, PMS: phenazine methosulfate, HQ: hydroquinone, BQ: benzoquinone, average values, ampl. factor in quiscent solution

the electrodes; the microcontroller enables various electro- chemical measurement procedures to be performed.

6.3 Adjusted selectivity and sensitivity The measuring setup has been employed also for glucose and lactate biosensing. Here a glucose oxidase or lactate oxidase immobilized planar sensor chip replaces the interdigitated electrode array.

A platinum area of o.5 x o.5 mm 2 has been covered by a layer of polyurethane containing glucose oxidase. Additional crosslinking with isothiocyanate prevents enzyme leaching of the biopolymers and produces a very thin diffusion limiting layer (I-Iintsche et al. (1989)). This sensor responses linearily on addition of glucose between o.ol and 2.5 retool/1 within a few seconds. The excellent mechanical stability is caused by the glue like adhesion of the polyurethane and its reduced swelling after cross linking. The surface of the polyurethane-glucose oxidase layer, chemically modified by the polar urethane groups, suppresses the responses of interferences such as ascorbic acid and paracetamol to higher extent than that of the analyte.

A stepwise adjustment to the appropriate analytical per- formance has been achieved by additional cellulose acetate layer. Along with dispensed polymer concentration and number of layers this characteristics can be achieved by wet-etching of selective pores and additional chemical derivatization of the isocyanate crosslinker (Hintsche to be punished). Typically for film formation an organic solution of cellulose acetate containing water soluble additives such as polyethylene glycol is dispensed onto a TFME and air dried. When the dry film is inserted into an aqueous solution the additive is leached and hence pores are obtained. Both, the pore size and the polarity of the etched cellulose acetate enable selection and separation of the low molecular weight compounds. Thus hydrogen peroxide and ascorbic acid are separated from each other.

The introduction of the additional diffusion barrier reduces electrochemical interferences and at the same time extends the linear measuring range of the sensor to 3o retool/1 (Fig. 4). The excellent working stability (Fig. 5) is the result of the

300

200

(.3

100

GOD in potyurethene

GOD in potyurethcme+ diffusion barr ier

10 20 30 Glucose (minor/ t )

Fig. 4- Concentration dependence of a planar platinum electrode with polyurethane immobilized glucose oxidase with and without additional diffusion barrier

4.0

42

~ 2o {_ c_

(_1

I I i I I I

20 L,0 60 80 100 120 %0 81 Time (d)

Fig. 5. Long term stability of the glucose chip sensor under discontinuous use

particular good adhesion of the urethane and optimum pro- tection of the enzyme. An analytical system for lactate is under development which uses the same system components and a similar immobilization procedure. Optimization is performed with respect to reduction of interferences and improved stability in undiluted real samples.

7 Conclusions The study of the current state of the biosensor based analytical microsystems reveals, that the electrochemical transducers are at well developed stage. Single and multiple sensors have been manufactured on a single surface to which the integration of enzymes is demonstrated in many variation. Also the modification of the biosensitive layer in order to adjust measurement performance is shown to be a known tool. But there is still a lack of technologically compatible processes for the application of these methods for the production of large quantities.

The combination of sensor chips and microfluidic elements serves to be an excellent way to create cost effective and improved modules of the analytical microsystem. These constructive parts of ~m dimension and nl volume can be handled using common production and mounting techniques. However, it appears that the development of mounting and packaging technology is of paramount importance for the hybrid system integration of sensoric, actoric, fluidic, and electronic elements. Therefore intensive research is still required, not only on the engineering, production, and combination of enzymes but also on their sensor integration, on interfacing circuits and packaging techniques, before the first biosensor based analytical microsystems will appear on the market.

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