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Microfabrication of Amperometric Biosensor for                  Glucose Monitoring                      Aminuddin Debatara...
proportional to the concentration of that electroactive           stoichiometric relationship with its substrate or target...
such as enzymes, antibodies, antigens, mediators, andcofactors. In amperometric electrochemical processesare generally com...
0.1 M Potassium chloride (KCl) solution. The                 the cathode for electrolysis, with the copper wire silverfoll...
electrode, in order to make a valid assessment of an              electrodes is less than 1 mV. Temperature of the setupin...
[3]    E.J. Calvo, C. Danilowicz, (1997), “Amperometric       Enzyme Electrodes”, J. Braz. Chem. Soc., Vol. 8,       No. 6...
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  1. 1. Microfabrication of Amperometric Biosensor for Glucose Monitoring Aminuddin Debataraja, Nur Fauzi Soelaiman, Latif mawardi. Electrical Engineering State Polytechnic of Jakarta University of Indonesia, Kampus Baru UI DEPOK, 16424, Phone: (021)7863531; Fax: (021)7863531 E-mail: Abstract— The objectives of this research are to sensor research is a relative mature and well workeddesign and fabricate glucose sensor using enzyme research field. The majorities of sensors are based onbased amperometric sensor on glass substrate. electrochemical principles and employ enzymes asMicrofabrication technology was used to fabricate the biological components for molecular recognition.electrode. The biosensor developed initially to Several new techniques for glucose sensing have beendetermine glucose in aqueous solutions, with later developed in clinical practice as well as inapplication to others analytes. The glucose electrode biotechnology and the food industry. Glucose sensorsitself consists of a silver silver chloride reference can be broadly classified in three main categorieselectrode, a working electrode and a counter electrode. depending on the number of applications underCharacterization of the reference electrode has been investigation: (1). The first and by far the largestevaluated. The output potential and noises are category consist of the enzyme-based needle-typeobserved at random times and the potential variation electrochemical glucose sensors. The detectionof the two reference electrodes is less then 1 mV. It is principle of these sensors is based on the monitoringdue to copper wire silver paste coated was used but of the enzyme-catalyzed oxidation of glucose. Thewith unknown purity. category includes glucose sensors using amperometric or potentiometric operating principles (hydrogen- I. INTRODUCTION peroxide electrode based, oxygen-electrode based, mediator-based and potentiometric-electrode based). A glucose test is one of many tests performed in a (2). The second category consists of glucose sensorsclinical laboratory. The concept of a glucose sensor based on the direct electro-oxidation of glucose onwas first introduced by Clark & Lyons in 1962 [1]. In noble metal electrodes (electrocatalytic glucosetheir article dealing with continuous monitoring of sensors). (3). The third category consists of glucoseblood chemistry, they suggested that a thin layer of sensors based on a number of different detection orsoluble enzyme might be retained at the surface of an glucose extraction techniques. This category includesoxygen electrode using a dialysis membrane. Glucose affinity-based glucose sensors, coated wire glucoseand oxygen would diffuse into the enzyme layer from electrodes, reverse ionophoresis based glucosethe sample site and the consequent depletion of sensors, suction effusion fluid based glucose sensorsoxygen would provide a measurement of the glucose and microdialysis based glucose sensors.concentration. Since this pioneer work in the 1960s,reasonable research effort has been devoted to the The objective of this present research is to designdevelopment of glucose sensors by a number of and fabricate glucose sensor using enzyme basedprocess for fabrication of the electrode is also amperometric sensor on glass substrate. The aim ofexplained. this paper is to describe the design and fabrication of Starting from publication of Clark and Lyons in amperometric glucose sensor. The fabrication of the1962, the amperometric biosensors became one of the silver-silver chloride reference electrode is presentedpopular and perspective trends in biosensor. The and the microfabrication configuration as shown inamperometric biosensors measure the changes of the figure 1. To the working electrode (cathode, negative)current of indicator electrode by direct is applied a potential which is constant relative to theelectrochemically oxidations or reduction of the reference electrode, which itself serves as a referenceproducts of the biochemical reaction [2]. In potential. As this potential is applied, the currentamperometric biosensors the potential at the electrode between the working electrode and counter electrodeis held constant while the current is measured. A is monitored. If there is no electroactive substance insimple circuit involves a two-electrode configuration the electrolyte solution then no current occuralthough more precise control of the applied potential (essentially). Cottrell equation indicates in thecan be achieved by using a potentiostat and a three- presence of an electroactive substance a currentelectrode research groups worldwide. Today, glucose ensues. The magnitude of the current is directly
  2. 2. proportional to the concentration of that electroactive stoichiometric relationship with its substrate or targetsubstance if all else, such area, diffusion coefficient analytes. Amperometric biosensors are typicallyand the underlying assumptions in deriving the inexpensive and less critically dependent of anequation, remain constant. The amperometric accurate reference electrode [6]. The amperometricbiosensors are known to be reliable, less critically biosensor allows the electrochemical reactiondependent of an accurate reference electrode, typically (oxidation or reduction) to proceed at the electrodeinexpensive and highly sensitive for environment, surface, giving rise to a current. Amperometricclinical and industrial purposes [3]. biosensors operate at a fixed potential with respect to a reference electrode and the current generated by the oxidation or reduction of the substrate at the surface of the working electrode is measured. This current is directly related to the bulk substrate concentration. It is important that electron transfer between the biological molecules and electrode material be facilitated. The design of amperometric biosensors involves several strategies. These can be divided into two main groups: those, which require a soluble enzyme and those where the enzyme is immobilized at the electrode surface [7]. The enzyme layer can be sandwiched between two permeable membranes. A layer-by-layer deposition technique may be used to optimize enzyme loading in bi-enzyme systems [8]. An Figure 1. Electrode configuration (a) two electrode (b) three outer cellophane, polycarbonate, or cellulose acetate electrode. membrane served to exclude larger molecules or molecules like ascorbate. The most common enzymes used in monoenzymatic systems are oxidases, which catalyze the model oxidation eraction Oxidases are usually flavoproteins that use O2, the natural electron acceptor, to regenerate the reducedWhere, i = current (A), D = Diffusion Coefficient,A = Area, C = Analyte concentration, n = number ofelectrons involved in the electrode reaction, F =Faraday’s constant (96485 C/mol). enzyme during the reaction. New systems have A reference electrode is used in measuring the recently been developed in which a chemical mediatorworking electrode potential of an electrochemical cell replaces oxygen. Among them, ferrocene-ferricinium[4]. The precision and accuracy of the measurement ion couples or derivatives are the most frequentlydepend strongly on the effective performance of the employed [8, 9]. Glucose oxidase is widely used forreference electrode, which affects the results through the determination of glucose in body fluids and influctuations in its own potential and through the removing residual glucose and oxygen from beveragesliquid-junction potential at the interface with the test and foodstuffs. Furthermore, Glucose oxidase-solution. Therefore, a reference electrode should have producing moulds such as aspergillus and penicilluma stable electrochemical potential as long as no current species are used for the biological production offlows through it. The purpose of the reference gluconic acid.electrode is to complete the measuring circuit andprovide a stable and reproducible potential against II. EXPERIMENTALwhich the indicator electrode is compared [5]. The Design and construction technology and materialscontact is made through a liquid junction that allows science are intimately linked in biosensorthe reference electrolyte to contact the sample. The development. Sensor design, including materials, sizesilver-silver chloride electrode is the most common shape and methods of construction, are largelydue to its ease of manufacture or simple, inexpensive, dependent upon the principle of operation of thenon-toxic, very stable and superior temperature range, transducer, the parameters to be detected and theactually usable even above 130oC. The electrode is a working environment [2, 8, 10]. Materials used insilver wire that is coated with a thin layer of silver electrochemical biosensors are classified as (1).chloride either by electroplating or by dipping the wire Materials for the electrode and supporting substrate,in molten silver chloride. In an amperometric enzyme (2) materials for the immobilization of biologicalelectrode the function of the enzyme is to generate (or recognition elements, (3) materials for the fabricationto consume) an electroactive species in a of the outer membrane and (4) biological elements,
  3. 3. such as enzymes, antibodies, antigens, mediators, andcofactors. In amperometric electrochemical processesare generally complex and may be considered asuccession of electron transfers and chemical events.Amperometric biosensors function by the productionof a current when a potential is applied between twoelectrodes. For example, the overall oxygen reductioninvolves different steps (such as oxygen reduction tohydrogen peroxide, hydrogen peroxide reduction toH2O, and dismutation of hydrogen peroxide), and Figure 2. Glucose sensor layoutnumerous parameters influence the rates of thesereactions (the potential, the nature of electrode metal,and the operating conditions). The overall sensorcurrent is thus dependent on many factors; includingcharge transfer, adsorption, chemical kinetics,diffusion, convection, and substrate mass transport[11-13]. The understanding of the kinetic peculiarities Figure 3. Glucose sensor structureof the biosensors is of crucial importance for theirdesign. One of the most critical characteristics of using IC technology, has been extremely challenging,biosensors is their stability. The operational stability mainly due to process incompatibility issues,of a biosensor response may vary considerably packaging problems, failure to incorporate a truedepending on geometry and method of sensor reference electrode and the difficulties involvingpreparation, a transducer use and some other some patterning relatively thick organic layers such as ionother parameters [2]. Furthermore it is strongly depend selective membrane and hydrogel [11]. In addition,on the response rate-limiting factor, i.e. substrate microfabrication techniques can also be used to eitherdiffusion and enzymatic reaction rate [13]. significantly improve sensor characteristics (with In this research, the layout of the glucose sensor respect to conventionally fabricated devices) or toto be constructed is shown in figure 2. Three different develop devices with new functionality, which cannotpatterns and sizes of glucose electrode are used to be realised in conventional fabrication technology.evaluate the sensor response rate (A1= 9.63 mm2, A2= Chemical sensors or biosensors usually consist of a4.60 mm2, A3= 3.97 mm2). The base transducer sensitive layer or coating and a transducer orconsists of H2O2 sensor, which is essentially the same electrode. Various enzyme materials serve asas the oxygen sensor. The enzyme glucose oxidase is biologically sensitive layers that can be coated ontoimmobilized in front of the H2O2 sensor between two the different transducers. The set of microfabricationmembranes. The inner membrane is a permeselective processes used for coating a sensitive layer formembrane that allows passage of H2O2 where as the biosensors is completed by various depositionouter membrane separates the biosensor from techniques for biologically sensitive layers such asmeasurement medium (figure 3). dispensing or spray coating, polymerisation, a sol-gel In order to coat a thin sensing membrane onto agold electrode, appropriate membrane materials have process, printing and by sputtering. For exampleto choose. The requirements of the membrane are electrochemical polymerisation is an attractivegood adhesion to the electrode surface, good approach for enzyme immobilisation. This is a simplepermeability of the membrane are good adhesion to procedure where a suitable monomer is oxidised in thethe electrode surface, good permeability of glucose presence of an enzyme. The enzyme is confined to aand adequate mechanical strength. Albumin layer adjacent to the electrode. The techniques usedcrosslinked by glutaraldehyde and cover with a for the production of electrode can be roughlycellulose acetate adhesion layer may be use in this classified as: (1) printing, (2) deposition, (3)research. Microfabrication processes are used to polymerisation, (4) plasma induced polymerisation,produce devices with dimensions in the micrometer to (5) photolithography and (6) nano technology. In thismillimetre range [10]. Over the past few years, research project, deposition and photolithographymicrofabrication technology has emerged as a techniques were used to fabricate and pattern thepromising technology for miniaturising and integrating glucose electrode. Flow diagram and fabrication stepschemical or biosensor systems. Microfabrication of for fabrication of biosensor glucose sensor are shownelectrochemical sensors, in figures 4(a) and (b). The next steps are manufacturing the reference electrode (Ag|AgCl) and enzyme coating using spray coating. The silver/silver chloride reference electrode is produced by chloridising the gold wire coated with silver paste in
  4. 4. 0.1 M Potassium chloride (KCl) solution. The the cathode for electrolysis, with the copper wire silverfollowing are the fabrication steps involved for paste coated electrode as the anode. A current of ± 50amperometric glucose electrode on glass substrate: µA was passed through the electrode for  Deposition of chrome/gold electrode using approximately 120 mins in 0.1 M KCl, where a sputtering system magnetic stirrer was employed to keep homogeneity of  Patterning of gold electrode using the solution. The electrode was then immediately photolithography rinsed in deionised water and subsequently stored in  Etching (wet or dry) the plastic bottle. Before potential measurements, the  Enzyme coating using spy coating or sol-gel reference electrode was immersed in the test solution process for about 1 hour to establish a stable electrode/electrolyte interface. The reversible electrode reaction consists of silver ions going into solution and then combining with the chloride ions to form silver chloride. Thus its potential is determined by the following reactions: Ag  Ag + e- (3.5) Ag+ + Cl-  AgCl(s) (3.6) Ag + Cl  AgCl(s) + e - - (3.7) Figure 4. (a) Flow diagram of the fabrication of glucose sensor used. (b) Glucose sensors fabrication steps. Figure 5. (a) Schematic Diagram reference electrode Different approaches of fabrication of silver- experimental set-up (b) Photograph of reference electrodesilver chloride reference electrode have been cited in experimental set-upliteratures [14-18]. In this research, the referenceelectrode used was a Ag|AgCl electrode. The The key feature of a reference electrode is itssilver/silver chloride reference electrode was produced reproducible and stable potential that is not influencedby chloridising the copper wire coated with silver by the measuring solution. The fabricated referencepaste but to minimise the contamination hazard and to electrode developed during this research was of aachieve good surface contact, the copper wire was first Ag|AgCl type. The simplest way to test an unknownrinsed with 10% HNO3 solution followed by acetone reference electrode is to compare its potential with a(CH3COCH3) solution. The copper wire coated with known good reference electrode in a beaker containingsilver paste (drying at ± 150 0C for 120 mins) was an electrolyte and connect the two electrodes to thechemically chloridized in 0.1 M potassium chloride inputs of a good quality voltmeter. Ideally the(KCl) solution. The experimental set-up is illustrated voltmeter’s input impedance should be 100 MW orin figure 5(a) & (b). A gold coated wire was used as greater. Since we don’t have a good reference
  5. 5. electrode, in order to make a valid assessment of an electrodes is less than 1 mV. Temperature of the setupintegrated reference electrode, two identical reference during the experiment was not taken into account.electrodes (Ref. A and Ref. B) were fabricated andthen immersed in electrolyte. Read the open circuitvoltage of the system. This is a measure of thepotential difference between the two referenceelectrodes. This enables a comparison to be madebetween electrodes of the same type as a consistencycheck. III. RESULTS AND DISCUSSIONS The quality of the AgCl layer depends on the Figure 8. The response potential of two identical referencecurrent density and length of time during its growth. electrodesThe length of time the electrode is submerged wasobserved to be directly proportional to the thickness ofthe AgCl layer. Figure 6 shows the fabricated of IV. CONCLUSIONSreference electrode. In this present research glucose sensor using enzyme-based amperometric sensor was designed and fabricated. Microfabrication technology was used to fabricate the electrode. The biosensor developed initially to determine glucose in aqueous solutions, with later application to others analytes. The glucose electrode itself consists of a silver silver chloride reference electrode, a working electrode and a counter electrode. The intention during this research was to Figure 6. Images of two reference electrodes made of develop a miniature reference electrode that was fully copper wire coated with silver paste (I = 50 A, T=120 integrated with the working and counter electrode. mins) However, since silver coating cannot be made in PIDC Figure 7 shows a schematic diagram of the – Hsinchu Taiwan and silver wire (with purityexperimental set-up for stability testing of the 99.99%) is not available also, copper wire silver pastefabricated reference electrode. The measurement coated was used but with unknown purity. This raisesapparatus consisted of a multimeter (BRYMEN question on the integrity of the results. FromBM859CF) with a resolution of 0.01 mV was selected preliminary test the output potential and noises areto measure the voltage generated by the electrode and observed at random times. The potential variation ofwas interfaced with a PC. The test solution was 0.1 M the two reference electrodes is less then 1 mV.KCl with magnetic stirrer at the bottom. Both Temperature of the setup during the experiment wasfabricated reference electrodes Ref. A and Ref. B were not taken into account. Due to limitation of time theimmersed in test solution at room temperature for glucose sensor developed cannot be completed. Themore than 2.5 hours and the output signal was enzyme coating and the performance or response raterecorded. of the glucose sensor cannot be done during this time. AKNOWLEDMENT Multimeter The authors would like to thank Dr. Hiskia for …. mV theoretical calculations and technical support. Part of Ref. A Ref. B the research was supported by DP2M-Dikti (Hibah Bersaing). 0.1 M KCl Magnetic stirrer IBM Compatible REFERENCESFigure 7. Schematic diagram of set-up to evaluate fabricated [1] Winncy Du, (2003), “State-of-the-Art of reference electrode Biosensors”, Mechanical & Aerospace Figure 8 shows of the potential output of two Engineering, San José Sate University, Powerreference electrodes in 0.1 M KCl solution for about point file, June 6, 2003.2.5 hours. Homogeneous concentration of the solution [2] Romas Baronas, Feliksas, Juozas Kulys, (2003), “The Influence of the Enzyme Membrane Thickness onused was achieved by the use of magnetic stirrer. The the Response of Amperometric”, Sensors 2003, Vol. 3,output potential and noises are observed at random pp. 248-262.times. The potential variation of the two reference
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