Needle-Shaped Glucose Sensor with Multi-Cell Electrode Fabricated bySurface Micromachining

Youn Tae Kim, Young -Yong Kim and Chi-Hoon Jun

Electronics and Telecommunications Research Institute, P.O.Box 1 06 Yusong,Taejon 305-600, Korea

*D of Ceramic Engineering, Chonnam National University, 300 Yongbong-dong,Kwangju 500-757, Korea.

ABSTRACT

A needle-shaped glucose sensor for in-situ glucose monitoring has been fabricated by surface micromachining and itscharacteristics were examined. The sensor consists ofthe needle-shaped sensing part (length: 5OOOjaii, width: 17Otni, depth:

5OtllI), metal lines and pads to supply a bias voltage and measure the cell current. The sensing part is seated at the end of theneedle, and it has three or four working electrodes, a counter electrode, and a reference electrode. A SO! (Silicon OnInsulator) wafer was used as a substrate and a metal layer was deposited to improve bending characteristics ofthe sensor asa reinforced layer. A Ti/Pt layer is deposited on the thermally oxidized layer and patterned to form cells, electrodes, andmetal lines. A Ag/AgCl layer was added to form the reference electrode. And then, the edge of the sensor structure wasdefined and etched to form the needle shape, and the windows ofthe cells and the electrodes were opened using wet and dryetching. Finally, a sacrificial oxide layer was removed using wet and gas phase etching and the apparent shape ofthe sensorwas accomplished. The needle shaped microelectrode for the glucose sensor exhibits chemically stable characteristics, andthe glucose concentration-dependent oxidation current of hydrogen peroxide produced by the conversion of glucose andoxygen at the working electrode, were measured.

Keywords: glucose sensor, surface m icromachining, multi-cell electrode

1. INTRODUCTION

Glucoses are the source of metabolism which supply energy to muscle and cell, and long time higher level of glucosethan normal makes abnomal metabolism like as artherosclerosis, stroke, angina pectoris, renal failure, retinopathy, and thisdiabetic complication end to death. For this reason, it is important to the DM (diabetic milletus) patient that aquisition ofaccurate glucose level in the respect of prevention of aggravation and treatment. Now, the widely used glucose sensor is adisposable sensor possessing a small finger size surfaced area composed of fixed glucose oxidase or glucosedehydrogenase and HRP measuring the glucose level by way of changing color or current. These sensor have a shortmeasuring time and small size can easily measure the glucose level, but on the other side have a defect that sampling ablood in every measuring time and in these much measuring time lead to rise the cost of sensor. Still more, these sensorcan reflect the glucose level at that time and don't help diagnos hypoglycemia and hyperglycemia in sleeping that arefatal to the DM patient. In these reason, sensors are required can always wear and at the level of much reliability andaccuracy that can differentiate the normal level glucose and hyper or hypoglycemia in a least.

The conventional glucose sensors have been used ion or gas electrodes fabricated on platinum tips, but have someproblems of poor reliability, large volumes, and slow response time. Micromachined biosensors have been under extensiveinvestigation during the last decade, because they have a number of advantages: cost-cutting through miniaturization andmass production, fast response time, reliability and so on.'2 Especially, on the glucose sensor, the microelectrode fabricatedby micrornachining fit in with application to in situ and in vivo monitoring of glucose. However, the microelectrode have

* Correspondence: Email: ytkimcadvax.etri.re.kr; Telephone: +82-42-860-5305; Fax: +82-42-860-6836

Part of the Symposium on Design. Test, and Microfabrication of MEMS and MOEMSParis, France. March-April 1999

924 SPIE Vol. 3680 • 0277-786X199/$10.00

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disadantages: the output current is ser low because of their small size and it is difficult to immobilize enzyme on thesurface of it. Further in estigation of enhancement of the output current and new method of immobilization is required.

In this stud\. a microelectrode for a glucose sensor \as fabricated by surface micromachinmg We have measured theelectrocheniical characteristics of themicroelectrode and its feasibility for realization of the microglucose sensor.

SOl(Silicon on Insulator) wafer deposited ith both 3,aa sacrificial oxide aver and a 6.5,aui silicon aver, was used as asubstrate. First, a thermally oxidized layer was deposited as a passivation layer, and then cells, electrodes and metal laserere tbrmed through deposition of Ti Pt. Secondly, a 5m silicon oxidized layer was deposited as a insulating layeL Thegeometries of the sensor were patterned using the lift-off technique and etched by wet and dry etching. Wells and electrodewere opened through dr etching using a photoresist as a masking layer. Finally, the sacrificial oxide layer was removedthrough wet etching and GPE(Gas Phase Itching) processing using SiN as a protecting layer. The structure of the sensorwas accomplished after the protecting layer of surface was removed.

Figure I shows the schematic and cross section of the glucose sensor. Figure 2 shows the SEM photograph oIthe sensor.

PEOX 5Si02 1 uiSi 6.5

Si02 3 ;niSi substrate

2. FABRICATION

Metal linePad (6 ea) Bridge (3 ea)

Well (6 ea) III

Etched\lot (4 ea)

A'Sen edge

30 Well Pt/Ti: 0.3/0.03 ni

Fig. 1. Schematic and cross section of the microelectrode.

925

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The fabricated sensor consists of sensing cells: sorking electrode(W) . counter electrode(C) and reference electrode(R).and the metal lines and pads s hich suppl a voltage and measure the current in cells, the metal aver was deposited as anintermediar adhesion la er. 'lo improve sensitivity of the electrode, electrodes were consisted of multi sites. the referenceelectrode is preferably made from Ti Pt deposition on the insulating layer, and deposition and chloridize of silver for theformation of silver chloride on the surface. The fixed trim voltage is applied between the reference electrode and theworking electrode. The reference electrode is made from Ti Pt deposition on the insulating la er. and formation of hiola\ er.the counter electrode is also made from Ti Pt deposition for the measuring the current variation on the orking electrode.When a trim '.oltage is placed across the reference electrode and working electrode, glucose react oh sensing la er andgenerate peroxide in the enzymatic reaction. The peroxide is oxidized electrochemically and the measured current is direetlproportional to the glucose concentration. Figure 3 sho s the mask Ia\ out of the glucose sensor chip. l'here were It) sensors

are located in the I Ox 0 nuit chip area.Flectrochemical experiments were performed with a windows-driven electrochemical analyzer (CR660. CH Instruments

Inc.) using positive feedback routines to compensate for resistance. Cyclic Voltammetrv. Chronoeoulornetry. and ACImpedance measureiient ' were measured. The working electrode sites on silicon sensor body were rinsed with plenty of'deionized water. The Pt wire counter electrode and Ag AgCI (in KCI 3 M) reference electrode were used for voltammetricexperiments. A home-made electrode holder grabbed the sensor body and accurately controlled the length of themicroelectrode array that was immersed in the solution with a micrometer and a magnifying glass.. ll experiments werecarried out in the faradaic cage at room temperature. The bare electrode surfaces of the microelectrode array were firstdegreased h\ washing with acetone. It was then rinsed with deionized water and dried in a cold air stream beforemodification. A small volume of GOx solution (20 mg mL in 0.1 M PBS) was held at the tip of a micropipet (maximumvolume I mL. ) in the form of a droplet and was transferred to the sensor both' simply by moving it through the droplet. theenzyme solution was allowed to dr for S mm at room temperature while holding the sensor body horizontally. loimmobilize the enz\me. the sensor as exposed to a small volume of glutaraldehde solution in the same way as exerted

ith GOx arid dried in air for tO mm. Deposition of GOx and glutaraldehyde was repeated in this way three times. Beforethe deposition of enz me. all working electrode sites were pretreated by cyclic voltaminetry in 0.5 M H:S04 as follows: fours\\eeps at 50 mV'sec vere made. ith the first two and final one being made over the range -0.25- l. V. while for the thirdsseep the anodic limit was increased to 2.0 V. The final sweep was halted at 0.2 V. The sensor body was dipped into a cell

containing 7 mL of stirred PBS. pH 7.4 (air-saturated). and a potential of --600 mV (for hydrogen peroxide detection) asapplied between the working and the reference 'counter electrodes. The background current was allowed to stabilize for at

least O mm. 'The calibration of the sensor was carried out by adding increasing amounts of glucose to the stirred buffer. l'hecurrent as measured at the plateau (stead -state response) and was related to the concentration of the analvte.

926

Fig. 2. SFM photograph of the microelectrode.

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3. R

ESU

LT

S

AN

D

DISC

USSIO

N

Figure

4 shows

the

Cyclic

Voltam

mogram

ofthe

electrode

at various

scan

speed

in O

.5M

KN

O1

When

the

electrode

was

tested

in electrolyte

solution

without

electroactive

species,

there

was

no

difference

in

characteristics

compared

to

conventional

Pt electrodes.

It show

s

that

the

fabricated

electrode

was

chemically

stable,

when

O.2V

-1

.2V

applied

to

it.

Considering

that

O.5-O

.7V

potential

difference

was

applied

to m

ost

of the

glucose

sensors

based

on

measuring

H202

concentration,

the

electrode

was

chemically

stable

over

the

wide

range.

C a)

0

Fig.

4. C

yclic

Voltam

mogram

s

of

in O

.5M

KN

O3

927

Fig.

3. M

ask

layout

of the

glucose

sensor

chip.

0.9

0.8

0.7 E

(V,

vs.

AgIA

gCI)

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928

Figure 5 shows the Nyquist plot of the electrode from I to 100kHz, 10mM K3Fe(CN)6 in O.5M KNO3,EdC=O.2V vs.Ag/AgCI. Measured impedance by the impedance spectroscopy was rather lower than that of array for measuring neuralsignal and stimulation.°'7 This is why that the characteristic of electric double layer formed on the surface of electrode andits capacitance was largely changed according to component of used solution when the potential was applied to theelectrode. The capacitance of electric double Iayer(C) that was obtained through AC impedance measurement in O.5MKNO3 with 10mM K3Fe(CN)6 as the electroactive species was I 94jiF/cm2. This value was larger than that of theconventional metal electrode (1 O-4OjiF/cm2), however, it was so good, compared with capacitance of the reported thin filmelectrodes.68 Generally, it is difficult to apply governing model for conventional wide electrode to the microelectrode, and itis limited to get a quantitative information from behavior ofpotential vs. current.9

20 -

15 - U

- UR • U

N • U

5.0 f 60

Z'(kc)

Fig. 5. Nyquist plot in O.5M KNO3 with 10mM K3Fe(CN)6

Figure 6 shows the Cyclic Votammogram of the electrode at various scan speed in O.5M KNO3 with 10mM K3Fe(CN)6.Redox current responses and scan rate effects were verified. To get similar behavior of conventional large electrode, themicroelectrode was designed so that wells was deeply formed so as to occur one-dimensional diffusion of analyte. Figure 6clearly shows its effect. In the case of the non-well type microelectrode in same solution, the current was not decreased atthe high potential area.

Figure 7 shows the Chronocoulometric response of the electrode. The effective area of microelectrode calculated byChronocoulometry was 6.7 x 104cm2. This value was rather smaller than that of an apparent area (7.2 X 104cm2), but it isfound that the effective area of the microelectrode was no less than the apparent area, taking into consideration of surfaceroughness. From the preliminary experiments on the current response vs. glucose concentration, we can detect the currentin the range of pA - nA/mM on the stepwise addition of glucose. These values are so small, however if we consider thearea of the electrode on the inicrostructure, the microelectrode could be applicable to realize the microglucose sensor.

4. CONCLUSIONS

In conclusion, the needle-shaped microelectrode for the glucose sensor exhibits chemically stable characteristics, and canbe utilized as a good alternative to a conventional glucose sensor. It may be also possible to form high performance glucosesensor with cost-cutting through miniaturization, mass production and reliability. Further investigations are in progress toenhance the sensor performance, by improving the sensitivity and development of a new method for immobilization ofenzymes to a metal electrodes.

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0.000006

0.000004

0.000002

0.000000

0-0.000002

-0.000004

0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2

E (V, vs AgIAgCI)

Fig. 6. Cyclic Votammogram in OSM KNO3 with 10mM K3Fe(CN)6

8.0

7.0

6.0

5.0

4.0

3.0-

2.0

1.0

0

-1.0

1

1rD

0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

Time I sec

Fig. 7. Chronocoulometric response of the electrode.

0.55

929

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930

5. REFERENCES

1 . Q. Wu, K. M. Lee, and C. C. Liu, "Development of chemical sensors using microfabrication and micromachiningtechniques," Sens. andActuators B 13-14, pp. 1-6, 1993.

2. M. Koudelka-Hep, F. Rohner-Jeanrenaud, E. Bobbioni-Harsch, J. Terrettaz, N.F. de Rooij and B. Jeanrenaud "Arnicrofabricated glucose sensor fabrication, characteriztion, in vitro and in vivo performances, and related problems"Advances in Biosensors, 2, pp. 13 1-149, 1992.

3. M. P. Nagale and I. Fritsch "Individually addressable, submicrometer band electrode arrays. 1 . Fabrication frommultilayered materials" Anal. Chein. 70, pp.2902-2907, 1998.

4. M. P. Nagale and I. Fritsch "Individually addressable, submicrometer band electrode arrays. 2. Electrochemicalcharacterization" Anal. Chem. 70, pp. 2908-2913, 1998.

5. M. Wittkampf, K. Cammann, M. Amrein and R. Reichelt "Characterization of microelectrode arrays by means ofelectrochemical and surface analysis methods" Sens. andActuators B 40, pp. 79-84, 1997.

6. U. M. Twardoch "Integrity of ultramicro-stimulation electrodes determined from electrochemical measurements" I Appi.Electrochem. 24, pp. 835-857, 1994.

7. 0. J. Prohaska, F. Olcaytug, P. Pfundner and H. Dragaun "Thin film multiple eletrode probes: possibilities andlimitations" IEEE Transactions on Biomedical Engineering BME-33, pp. 223-229, 1986.

8. S. L. Bement, K. D. Wise, D. J. Anderson, K. Najafi and K. L. Drake "Solid-state electrodes for multichannel multiplexedintracortical neuronal recording" IEEE Transactions on Biomedical Engineering BME-33, pp. 230-24 1, 1986.

9. R. M. Wightman and D. 0. Wipf "Voltammetry at ultramicroelectrodes" in Electroanalytical Chemistry, edited by A. J.Bard, Vol. 15, Marcel Dekker, New York, 1989.

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