A microbioreactor based on interfacial polymerisation and application to flow injection analysis of glucose

ELSEVIER

Sensors and ActuatorsB 34 (1996) 422-428

A microbioreactor based on interfacial polymerisation and flow injection analysis of glucose

Hication to

Muntak Son”, Frank Peddie a-*,Dennis Mulcahya, David Daveya , Malcolm R. Haskardb BSckool

of Chemical Technology, bMicr&?cfronics

Univtwity of Soul Centre, Univar.@

Australia,

Level.~ Campus. SA 5095, Allslrufia SA 5095, Austrulia

cfSoulhAustralia,

Accepted24 June1996

Abstrnct A miniature bioreactor is described that uses micromachining fabrication techniques. The bioreactor chamber is created by anisotropic etching of a silicon wafer and attached to a microelectronic sensorby epoxy resin. Any biocatalyst such as an enzyme, a combination of enzymes, or intact viable cells can be contained within the microscopic chamber by an t&ra-thir. nylon membrane. Tight sealing of the membrane to the silicon wafer surface is achieved by pretreating the anisotropically etched wafer surface with a silylating reagent and then creating the membrane by interfacial polymerisation. By appropriate selection of aqueous and organic phase constituents, the nylon membrane can be covalently linked to the wafer surface by amide bonds. As a simple demonstration for the usefulness of this concept, the microbioreactor has been configured as a glucose sensorin a flow injection cell. Initial results are encouraging and demonstratethat many other applications are feasible. Keywonls: Micromachining; Miniaturisation; Bioreactor; Interfacial polymerisation; Glucose sensor

1. Intmiuction A bioreactor is an enclosed c

chemical reactions occur and which is eqMi~~~~with sensors to measure biochemical variables such as temperature, dissolved oxygen level, pH or sugar concentration. In addition, the bioreactor has to be operated with a mass transfer system to transport material in and out. Bioreactors are used for industrial manufacture of biological compounds, for the characterisation of biocatalysts such as enzymes or microorganisms, or as waste treatment systems. If a bioreactor could be miniaturised to the micrometer scale, numerous new applications would become possible, such as biosensors, micro-fermentor arrays or microbiological assay kits. To allow such miniaturisation to be achieved, at least three requirements must be achieved. Firstly. the reaction chamber itself should be reduced to micrometer dimensions. Secondly, the biocatalyst must be entrapped in the chamber without any damage or leakage. Finally, novel mass transfer mechanisms must be devised * Comsponding author.

09254O696R315.00 Q 19% Elsevier Science S.A. All rights reserved P if s0925-4005(96)01944-2

to deliver material to the tiny chamber and remove it again. The rapid development of micromachining technology has provided valuable tools which realise the first requirement. Anisotropic etching enables the creation of precise three-dimensional shapes on a silicon wafer. Laurell and Rosengren [ 1] made a zigzag shaped microcapillary structure on a wafer by anisotropic etching and anodie bonding of a thin glass plate over the groove. Glucose oxidase was immobilised on the wall of the microcapillary. They used a pump and fine tubing to deliver reactant to the capillary. This is a good example of miniaturisation of an enzyme column reactor that is commonly used in biochemistry laboratories and biotechnology production facilities. Our approach is quite novel and we believe has many advantages. The microbioreactor shown in Fig. 1 has a simple structure which is conceptually similar to a conventional bioreactor or a living microbial cell. The fact that the biocatalysts exist in free aqueous solution is an advantage since many physicochemical parameters such as the K,,, or V,, values of enzymes, diffusion coefficients for gases and solutes and specific growth rate con-

M. Son et al. / Sensors and Actuators B34 (1996) 422-428

423

Then, at the end of a polymer chain, residual carboxyl chloride groups react with the surface amines to give

Membrane

Z

-['CO'(CH:~)R-CO-NH-(CH 2)6-NH-]n-CO-(CH2)B-CO-NH-(CH2) ~.SiiO.SillWafer Polymer chain

Silicon oxide

surface 2.1. Mass transport

Scmo s) Wafer Fig. I. Conceptualdesign of a microbioreactor.The bottomwaferwith electrochemical sensor(s) attached is created by conventional techniques. stants of microorganisms ,nay be employed in modelling the system without major wodification. The bottom wafer with electrochemical sensor(s) is bonded to an anisotropically etched wafer using epoxy adhesives. Whole cells or enzymes in an aqueous solution are then entrapped by a nylon membrane created by interfacial polymerisation. During this process, strong amide bonds are formed between the ends of polymer chains in the membrane and amine groups on the silanised wafer surface. This ensures tight sealing at the top of the chamber and is the key innovation of this system. This Nylon membrane fulfils the second requirement for entrapment of the biocatalyst. 2. M e m b r a n e p r e p a r a t i o n

The synthesis of Nylon is a polycondensation reaction between dichloride and diamine compounds [2]. What has made the Nylon polymeric membrane useful is its mode of synthesis:- interfacial polymerisation. Because sebacoyl chloride dissolves only in organic solvents while hexamethylene diamine dissolves in both water and organic solvent, the polycondensation reaction occurs at the interface of the two phases. ClCO(Cll2)~COCI

+

lt2N(CH~)~qli~

~

Mass transport through the Nylon membrane relies on the size of its pores. The effective pore size of this type of membrane has been reported to be 18 A,, based on studies [3] using radioactively labelled compounds. This is large enough to permit passage of relatively small molecules such as glucose, but small enough to hold back macromolecules such as enzymes. This porosity satisfies the third requirement for an effective microbioreactor, i.e., the feasibility of efficient mass transport of material into and out of the reactor chamber. This paper outlines the methods used to fabricate the microbioreactor. In addition, as a simple demonstration of its capabilities, application of the microbioreactor as a glucose sensor is described. 3. Experimental

3.1. Preparation of reactor chamber Several pyramidal holes (upper square, 500/~m x 500/tm; bottom square, 154.6 x 154.6/~m; depth, 206/zm) were etched anisotropically onto a silicon wafer ({100}, 250/~m thickness). The wafer was cut into individual pieces, each containing a single reactor chamber (Fig. 2). by sonication. A silicon dioxide layer was then grown on the surface of reactor chambers by heating them at I I00°C in a furnace under wet oxygen for 3 h. The

-~CO(CH2)RCO.NH(CH2hNti-']-n

The chemistry of the bond joining the membrane to the silanised wafer surface may be described as follows. y-Aminopropyl triethoxysilane (APTES) is a widely used surface modifying reagent in silicon chemistry. I -- Si-OHt

I I -~/i-O-~i-CH2CHzCH2NH2

9

-si-oa, + n [ (C.,CH20~-S,-(CH~),N,~] ~

9

9 9

- Si-OH I

9

Silicon dioxide surface

9 9

-~i-O-~i-CII2CH2CH2NHa "~i-O-~I-CH 2CH2CH2Nlt2

APTES

Silanised surface

Fig. 2. A pyramidal hole etched into a silicon wafer and viewed by scanning electron microscopy.

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M. Son et aL / Sensors and Actuators B34 (1996) 422--428 .

resulting silicon dioxide thickness was estimated to be about 1.5/~m. A silanised surface was then created by dipping the chips into dry toluene containing 10% y-APTES and refluxing for 24 h. The quality of the modified surface was investigated by observing its wetting properties. The modified surface was highly hydrophobic, so that the contact angle of water was large compared to a normal silicon oxide surface.

f

3.2. Preparation of a membrane on glass

A 6,10-Nylon membrane was created on a thin glass cover slip (i.e. without a reactor chamber), in order to provide a sample membrane suitable for microscopic examination. The glass cover slip was first washed with nitric acid and then silanised by heating at 120°C for 4 h in a Petri dish with a drop of ~,.APTES. The aqueous amine reagent was prepared by dissolving hexamethylene diamine (40 mg) in 1 mi of distilled water. The organic reagent was prepared by adding 4/zl of sebacoyl chloride to ! ml of a cyclohexane/chloroform mixture (83:!7). Because of the high pga value of hexamethylene diamine, the aqueous solution was initially quite alkaline. By adjustment with hydrochloric acid, reagents with pHs of 9.5, 10.5, 11.5 and 12.5 respectively were achieved. With a micropipette, four drops of each aqueous solution were placed on the glass cover slip and the organic solution spread over the droplets. Aftei 30 s, the reaction was stopped by washing the glass with a stream of demineralised water. The resulting membranes were observed and photographed using a compound ~ight microscope (Fig. 3).

Fig. 4. Membrane created on the square hole. An air bubble trapped on the underside during rehydration because of a sudden rush of water into the chamber, disappeared after a few seconds.

3.3. Preparation of a membrane over a reactor chamber

In the case presented here, a gold microelectrode functioning as an amperometric oxygen electrode was attached to the base of the microchamber and a 6,10 Nylon membrane attached to the upper surface. A similar technique can be used however for membrane formation over a microchamber attached either to a glass slide, a silicon chip electrochemical sensor such as a pH-ISFET, or any other electrochemical device. Using suction applied to a capillary tube, a piece of wafer containing a reactor chamber was placed onto its base (glass slide, silicon chip or gold electrode), and the two bonded together by epoxy resin, followed by drying at 90°C in an oven overnight. A clean capillary tube containing the aqueoL,~ hexamethylene diamine solution (supplemented with a biocatalyst if required) was positioned over the reactor chamber and gently touched to the inside edge of the wall. The solution was permitted to drain into the chamber, creating a domeshaped droplet (constrained to a hemispherical shape by surface tension) above the square hole. An overlay of organic solvent was then created by touching the tip of another capillary tube containing the organic solution near one edge of the hole. After waiting 30 s to allow for interracial polymerisation, the newly created membrane was washed with demineralised water, quickly followed by dipping in a solution of 0.2 M HCI and 0. l M glycine. The resulting membrane-enclosed chamber or microbioreactor was stored in a phosphate buffered saline buffer (PBS, 50 mM, pH 6,5, 0.85% NaCI) at room temperature, Membrane covered chambers prepared on glass were observed using optical and scanning electron microscopes (Figs. 3 and 4). 4. Results and discussion 4.1. Physical properties of the membrane

Fig. 3. A membrane created on a glass cover slip and vlewecl by a compound light microscope. The seal around the circumference can be seen as a track about 30/~ru wide. The dome has shrunk due to dehydration.

The membranes created on a glass cover slip followed the contours of the droplet and appeared dome-shaped

114.Son et aL /Sensors and Actuators B34 (1996)422--428

Fig. 5. A membrane created with an excess of aqueous solution. The dia,ne|er was about 245/~m. (Fig. 4). Around the circumference of the circle, the edge of the membrane was bound tightly to surface amine groups. The strength of the bonding was such that the membrane could be washed in a vigorous stream of water without any damage. By comparison, membranes formed on unsilanised normal glass were amorphous and easily removed. The membrane of the microbioreactor covered the square hole completely. On microscopic inspection, no gaps were visible even at the corners of the square (Fig. 4). The thickness of the membrane was dependent on various parameters, but in a typical case was 0.5/~m (measured by scanning electron microscopy). The amount of aqueous solution applied determined the shape of the membrane. With 67 nl, the profile of the membrane was fiat, whereas with a smaller amount, the membrane was concave. Excess aqueous reagent created a bulbous, highly convex shape.(Fig. 5). The dome shaped structure remained stable as long as the membranes were stored in an aqueous PB9 buffer solution, showing no visual damage or loss of shape after three weeks. If the membranes were allowed to dry out, the shape collapsed within a few minutes. The conditions of membrane formation were carefully optimised. Variables influencing the strength of the membrane included the pH of the aqueous solution, the concentration of acid acceptor, the specific gravity of the organic solvent, the partition coefficient of the diamine between the organic and aqueous phases, the silanisation method and the concentration of the two monomers.

425

When the pH of the aqueous solution was higher than I 1.5, solvolysis [4] of the silane bonds between the membrane and the glass apparently occurred. As a result, the surface lost its hydrophobicity. The droplet then flowed more extensively over the surface~ causing weak bonding between the membrane and the surface even though the membrane was thick. After an extensive review of condensation polymerisation methods, Morgan [2] concluded that the polymerisation of Nylon 6,10 is heavily dependent on the presence of an acid acceptor in the aqueous phase, to remove the acid by-product of polymerisation. Adding more diamine than the amount needed for membrane formation was recommended so that the excess could serve as an acid acceptor by protonation of the amine groups. However, this procedure increased the pH to a large extent and bicarbonate was suggested as an alternative acid acceptor. Our results are consistent with the suggestion that if the pH is too high, solvolysis of the silane bond occurs resulting in poor membrane adhesion. Another problem of high pH is the loss of biocompatibility. A pH near 10 appears to be the best choice even though it is still quite alkaline 4.3. Selection criteria for organic solvent

Since the solubility of sebacoyl chloride is negligible in water but high in the organic solvent, the diamine must diffuse across the interfacial boundary to react with the acid chloride. Therefore interfacial polymerisation occurs and the reaction site is in the organic phase [2]. The diamine must be able to dissolve in both the aqueous solution and the organic solvent, with a low partition coefficient, K (CH2o/Cor~ani¢). Hexamethylene diamine has high solubility in chlorinated hydrocarbons such as chloroform (K = 0.7). In addition, since the solution is to be placed on top of the aqueous solution, its specific gravity (s.g.) had to be smaller than that of water. A cyclohexane (s.g. -- 0.78, K= 180) and chloroform (s.g. = 1.47) pair was selected to make the specific gravity smaller than unity and keep the partition coefficient value low. A mixture ratio of 83:17 was selected and the concentration of diamine was chosen by following the principle explained by Morgan [4]. 4.4. Effect of silanisation methods

Two other silanisation methods, namely vapour phase and aqueous phase silanisation, were tested, but neither was better than the toluene refluxing method in terms of resultant membrane adhesion.

4.2. Effect of pH 4.5. Sealing quality of membrane over reactor chamber

On glass, membrane hemispheres with different properties were created by changing the pH of the aqueous solution.

A drop of aqueous solution placed in a square hole with hydrophobic surfaces aligns itself to fit symmetri-

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M. Son et al. /Sensors and Actuators B34 (1996) 422.-428

• R.E.

Sample

I

Because of the likely involvement of surface amine groups, the proteins would be copolymerised underneath the membrane. We believe that this effect enhanced the membrane strength [3] and also made the membrane more flexible. 4. 7. Flow injection analysis of glucose

Fig. 6. Flow cell of glucose biosensor. R.E., Ag/AgCI reference electrode; C.E., platinum foil counter electrode; W.E., microdeetrode (10/~m diameter, gold). The mierobioreactor was positioned above the mictoclectrode.

tally in the hole. The high surface tension of water causes the gaps at the four corners of the square to remain untitled. When the organic liquid is layered over the droplet and tile hole, all four gaps become filled with organic solvent and polymerisation commences. The hydrophobicity of the silanised surface allows the organic solvent to spread and fill the gaps quickly. In addition, the fact that the polymerisation site is in the organic phase makes the edge of the membrane bind to the layer of amine groups on the silicon dioxide surface surrounding the hole. 4.6. Application as a glucose sensor

For an electrochemical sensor, a microelectrode (gold, diameter lO/~m, Bioanalytical Systems) was chosen because the current from it is so small that the amount of oxygen reduced at the electrode surface is negligible. Nation was coated onto the electrode surface by dipping in 0.1% Nation solution and drying. It was found that noise was greatly reduced with the Nation coating. The microehamber was placed on the electrode and bonded by epoxy resin to the electrode housing. The electrode-microreactor combination was dried at 90°C overnight. As a biocatalyst 5% glucose oxidase containing catalas¢ (Sigma, G6641) and 5% bovine serum albumin (BSA) was mixed in 1 ml of bicarbonate buffer (pH 8, 0.5 M) containing hexamethylene diamine (0.3 M). The Reactor chamber was tilled with this aqueous solution. bovine serum albumin was included to stabilise the glucose oxidase and lessen the amount of active enzyme involved in the polymerisation reaction. After the membrane was created by following the method outlined above, the microbioreactor was washed with PBS buffer and placed in the flow cell. PBS buffer containing 0.1% Triton X-IO0 was flowed through the cell for a few minutes to wash the remaining organic solvent from the membrane surface.

To evaluate the microbioreactor as a glucose sensor, a flow injection analysis system was implemented. A diagram of the flow cell is shown in Fig. 6 and Fig. 7 shows the chronoamperogram of the microbioreactor under airsaturated operation. With a potentiostat (BASIOOB/W, Bioanalytical Systems, with a low current module, EFI069) a constant reduction potential was applied and the cathodic current gathered by an IBM PC using the equipment software (BASI00B/W in a Microsoft Windows environment). Whenever the potential was applied to reduce oxygen, a reductive peak appeared. This confirmed the effective wash out of remaining hexamethylerie diamine inside the reactor chamber. The time needed for the wash-out process could be reduced if the flow rate of the carrier wag increased. During stabilisation of the biosensor (Fig. 7), the initial current decreased rapidly as reducible material at the surface became exhausted. Thereafter, however, the current increased for approximately 30 rain and then declined gradually to reach an equilibrium after 40 rain. A second run demonstrates that a baseline for the biosensor has been reached. When a glucose bolus was passed through the flow cell, glucose diffused rapidly through the membrane and was consumed by the glucose oxidase. Concomitant reduction of oxygen in the chamber caused a decrease in the current. 4.0

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. . . . . . .

.

3.5 3,0

i

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80

100

\

2.5

0

t /" "x.

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1,5

~/'/ First run 1,0

I 0.0

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r 40 Time(min)

Fig. 7. Stabilisation uf a microbioreactor configured as a glucose biosensor. -800 mV versus Ag/AgCi was applied to microelectrode (diameter t0#m). Air saturated PBS buffer was pumped with a flow rate of 0.5 ml/min.

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M. Son et al. /Sensors and Actuators B34 (1996) 422-.428

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3

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2mM

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(I IG)-oriented silicon, Sensors and Actuawrs B, 18-19 (1994) 614--617. [2] W.P. Morgan, Condensation Polymers by Inte~,:cial and Solution Methods, lnterscience, 1965, pp. 19-63. [3] T.M.S. Chang and M.J. Poznansky, Semipermeable aqueous microcapsule (Artificial cells). V. Permeability characteristics, Journal of Biomed. Mat., 2 (1968), 187-199. [4] Fluka, SilylatingAgents, FlukaChemie AG, 1988, p. 15.

Biographies 25ra M

0

5

10

15

2O

2~

T im e(m i n )

Fig. 8. Flow injection analysis of glucose. Samples of 2, 5, 10 and 25 mM glucose in PBS (50 raM, pH 6.5) buffer were injected. The injection volume was 0.5 ml and the flow rate was 0.5 ml/min.

Glucose + 02

c;°D--->Gluconate+ 1-1202

To explain the fast recovery of the biosensor, we suggest the following mechanism. As soon as hydrogen peroxide is formed, catalase begins to act, increasing the oxygen level again. Therefore, this system may involve a competition between the two enzymes. 5. Conclusion Using micromachining, interracial polymerisation and silanisation techniques, a microbioreactor was fabricated and applied to the flow injection analysis of glucose, employing a gold microelectrode as an oxygen sensor. This concept has a number of advantages in terms of simplicity, analogy to a conventional bioreactor and the possible use of accumulated data for analysis of bioreactor dynamics. However, the problem of the initial alkaline conditions during membrane polymerisation could be a problem for some biocatalysts.

Acknowledgements The authors gratefully acknowledge the Targeted Institutional Links (TIL) grant provided by the Australian Government Department of Employment, Education and Training. This supports collaboration with the Sensor Technology Research Center at Kyungpook National University, Korea. We also acknowledge Terence Yeow of the Microelectronics Centre, University of South Australia for advice on micromachining techniques, Dr. Rob Hayes (Ian Wark Research Institute, University of South Australia) and Dr Don Barnett (CSIRO Sensory Research Centre, Sydney) for valuable discussions.

References [11 T. Laurell and L. Rosengren, A micromachined enzyme reactor in

Mr. Muntak Son [B.Sc. (Seoul, 1989); M.Eng. (Seoul, 1994)] is a postgraduate student in the School of Chemical Technology, University of South Australia, undertaking a Ph.D. degree with funding from the Australian Government Targeted Institutional Links Program. Collaborators in the program are the University of South Australia and tile Sensor Science Research Centre, Kyungpook National University, Taegu, Korea. His research project is to develop an electrochemical biosensor for free sugars and his long term goal is to design and construct an embedded controller for bioprocess control using the principles of biosensors and flow -injection analysis. Prior to undertaking postgraduate research in Australia he was employed as a bioprocess control engineer for the Lucky Central Research Institute, Seoul, Korea and as a firmware developer for Youngma Electronics Co. Ltd., Korea. Mr Frank Peddie [B.Se. (Syd, 1965); M.Sc. (UNSW, 1975); M.Sc.Biotech~(UNSW, 198 I)1, is the Leader of the Microbiology and Process Chemistry Group within the School of Chemical Technology and Director of the Food Science and Technology Centre, University of South Australia. He has a broad training in the biological sciences and a total of 25 years industrial, academic and consultancy experience in the fields of Biochemistry, Pharmacology, Microbiology, Food Technology, Biotechnology and Environmental Sciences. His professional career has included roles as a Medical Services Departmerit Manager, Academic, Research Supervisor and Project Manager, Industrial L ,:ms~:ltant and Environmental Auditor. He has co-authorea eight technical publications and over forty commercial consultancy reports (19831996) prepared on a confidential basis for clients of the Food Science and Technology Centre. As a consequence of his broad background and interests, he is a member of four professional societies. Specialist interests include the physiology of yeasts and their application to brewing and winemaking, food technology and quality management in the food industry, biodegradation and bioremediation, electronic biosensors and other applications of enzymes and microbes. Dr David Davey [B.Sc.(Hons), (Melb, 1965); M.Sc. (Melb, 1968); Ph.D., (La Trobe, 1975)1, is the Leader of the Analysis and Sensors Group within the School of Chemical Technology. He is also a Board Member of the Sensor Science and Engineering Group (SSEG). Dr Davey has wide experience in applying analytical chemis-

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M. Son et al. /Sensors and Actuators B34 (1996) 422-428

try to industrial problems. He has strong industrial involvement over the I~t 10 years, including 12 months industrial leave in 1983, and a further 12 month's project in 1988. A major 3-year APRA(I) project with an Australian multi-national company in 1991 has been followed by a further ARC Collaborative stody with three companies. Dr Davey is the author and co-author of more than 40 publications. His research interests include analytical chemistry, sensor response mechanisms, atomic spectroscopy, flow injection analysis and on-line sample pretreatment. Associate Professor Dennis Mulcahy [B.Sc.(Hons), (Adel, 1963); Ph.D., (Ariel, 1968)], has been an active member of the University of South Australia since 1967 and is the current Chairman of the Sensor Science and Engineering Group (SSEG) and Head of the School of Chemical Technology. He is also the Education and Training Program Leader in the Cooperative Research Centre (CRC) in Water Quality and Water Treatment and a Board Member of the University's Urban Water Resources Centre. Professor Mulcahy is a member of the Australian Scientific Industry Association and a Fellow of

the Royal Australian Chemical Institute. His principal research interests are in the fields of chemical sensor development and flow-analytical systems development. He is author of more than 70 publications and has two patents to his credit - both for sensor innovations. Professor Malcolm Haskard [B.Eng.(Hons); (Adel, 1959), M.Eng. (Adel, 1964)], is the Director of the Microelectronics Centre and Head of the School of Electronic Engineering at the University of South Australia. He is a Fellow of three professional bodies, The Institute of Engineers, Australia, The Institution of Radio and Electronics Engineering, The Institution of Electrical Engineers, London. He has been responsible for the development of the University's Microelectronics Centre and its linkage to the Sensor Science and Engineering Group. He has made significant contributions in the areas of micromachining, VLSI design, CMOS technology, artificial neural networks and smart sensor systems. He has authored nine books, been an invited lecturer in six countries, conducted courses on behalf of UNESCO in India and Malaysia and a Government Adviser in the field of microelectronics.