Sensors and Actuators B 106 (2005) 591–601

A silicon-based bulk micromachined amperometric microelectrodebiosensor with consecutive platinization and polymerization of pyrrole

Jingwei Liu, Chao Bian, Jinghong Han, Shaofeng Chen, Shanhong Xia∗

State Key Laboratory of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, Beijing 100080, China

Received 26 April 2004; received in revised form 8 July 2004; accepted 24 July 2004Available online 22 September 2004

Abstract

A new silicon-based amperometric microelectrode biosensor with consecutive platinization and polymerization of pyrrole is designedand fabricated with bulk micromachining technology. The biosensor consists of a p-type silicon substrate, two respective Au microelectrodesfabricated in two respective in-device micropools formed by anisotropic silicon wet etching, and an SU-8 microreaction pool. To our knowledge,consecutive platinization and polymerization of pyrrole is the first such use for surface modification. The biosensor aims for low unit cost, smalldimensions and compatibility with CMOS technology. Successful experimental results have been achieved for glucose detection. Comparedt chnology, ith 1× nds ature for am©

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o conventional amperometric biosensors and amperometric microelectrode biosensors made with surface micromachining teas several advantages, such as smaller sensitive surface area (1 mm× 1 mm), lower detection limit (1× 10−4 M), larger linear range (

10−4–1 × 10−2 M), larger sensitivity per unit area (39.640 nA mM−1 mm−2), better reproducibility (3.2% RSD for five detections) atability (relative remaining sensitivity per unit area kept well above 95% after being stored in a clean container at room temperonth), easier to be made into arrays and to be integrated with processing circuitry, etc.2004 Elsevier B.V. All rights reserved.

eywords:Biosensor; Anisotropic silicon wet etching; SU-8 microreaction pool; Surface modification

. Introduction

Enzyme electrode analysis (EEA) was first used in 1962,hen Clark and Lyons[1] succeeded in detecting glucoseoncentration by circum-embracing solvable glucose oxi-ase (GOD) around pH-sensitive electrode (glass ball withentimeters in diameter). Updike and Hicks[2] successfullyetected glucose concentration in plasma serum by immo-ilizing GOD on the surface of Pt electrode (amperometriclectrode) in 1967, and first used the termenzyme electrode.ensitive surface areas of these amperometric electrodeensors were usually of the order of 1 cm2 or even larger.ompared with current amperometric microelectrode sen-ors (usually of the order of 1 mm2 or even 0.1 mm2), they

∗ Corresponding author. Tel.: +86 106 255 2279; fax: +86 106 263 6516.E-mail addresses:jingweil@andrew.cmu.edu (J. Liu),

hxia@mail.ie.ac.cn (S. Xia).

were classified as macro-electrode sensors. The so-amperometric microelectrode sensors made of small dision carbon pastes[3–6] or slim metal rods/slices[7–9] withsensitive surface areas of the order of 10 mm2 or even largewere widely used between 1960s and 1990s[7,9–13].

With the rapid research and development of microetronics technologies, especially MEMS and lab-on-a-technologies, disadvantages of either conventional melectrode sensors or so-called microelectrode sensorsas large sensitive surface areas, large volume in reagendifficulty in being integrated with processing circuitry, mathem lose their intriguing prospects. Research and apptions of amperometric microelectrode biosensors withsitive surface areas of the order of 10, 1, 0.1 mm2 or evensmaller have been widely carried out in recent yearspecially in clinical and biochemical research[7,14–26], inview of their special characteristics, such as small volumreagents, quick response time, easiness to be made in

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592 J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601

rays, easiness to be integrated with processing circuitry, etc.[27–29].

Microelectrode fabrication technology still remains an im-portant element of the commercial success of microelec-trode biosensors. For commercial application, biosensorsmust have extremely low unit costs and be capable of un-dergoing mass production, while still maintaining adequatelevels of reproducibility[26]. Several fabrication systems areavailable, most of which have borrowed from the microelec-tronics industry, such as photolithography systems, thin-filmtechnologies and screen-printing technology[30]. In viewof above considerations, microelectrode biosensors reportedwere often made using silicon surface micromachining tech-nology. Recently, a number of studies have been reportedon these surface micromachined microelectrode biosensors[18–25]. Even microelectrode biosensor fabricated by dryprocess that fully compatible with CMOS process was re-ported[26]. Such technologies have advantages of precisecontrol of dimensions, excellent uniformity and reproducibil-ity, integrability of circuitry, mass production, etc. At thesame time, this micromachined and miniaturized biosensoritself has very attractive advantages of the small IR drop, thefast establishment of steady-state mass transfer, and smallcapacitive charging currents[18].

In fact, with specific coating, wet etching and exposuretechniques, bulk micromachining technology can alsoa rfacea conw ch-n rfacea rodeb sur-f cingt tom

alld gy,w cesso kingm cost,a ma-c nces rizeb ass ogyp

2

2

inh outi -t

GOD is known to be an enzyme which can catalyze theelectro-oxidation of glucose:

Glucose+ O2GOD−→ gluconic acid+ H2O2

When H2O2 comes further to anode surface, the followingreaction is carried out:

H2O2+0.7 V−→ O2 + 2H+ + 2e

When dipped into glucose solution (using PBS as a sol-vent), the microelectrode immobilized with GOD can be usedas an amperometric glucose-sensitive system. The redox re-action of H2O2 near the microelectrode surface results in acurrent increase, which corresponds to glucose concentra-tion. Thus by reading anodic current increase in this course,glucose concentration in the solution can be detected[16].

In different fields, such as microelectronics, etc., differ-ent surface modification methods were introduced to acti-vate sensitive surfaces[31–33]. Lots of methods were used tocarry out electrode surface modification[9,10,12,30,34–40],among which, platinization and polymerization of pyrrolewere two methods often used[9,34,35].

Platinization can form a porous film with platinum parti-cles of the order of 10 nm[14]. On one hand, platinum par-ticles of the order of 10 nm have better activity than metalplatinum, and they can speed up the electron transfer rate( hep film,t ul fore

ex-c ofs

edoxp (orP ucedss tate,f te, orv

P

P ee y. Inl n,w rc

huss p thee

er-i ion.T andp fol-l

chieve these advantages. Specially, taking lateral sureas of in-device micropools formed by anisotropic siliet etching into consideration, bulk micromachining teology here can not only enlarge actual sensitive sureas, which are crucial in amperometric microelectiosensors, but also keep the microelectrode sensitive

aces from environmental interference by circum-embrahem, thus enhancing their sensitivity and helpinginiaturize their dimensions.This new biosensor aims for low unit cost, sm

imensions and compatibility with CMOS technolohich are important elements of the commercial sucf microelectrode biosensors. SU-8 is used for maicroreaction pools to reduce reagent volume and unitnd to miniaturize biosensor dimensions. Bulk microhining, platinization and polymerization of pyrrole enhaensitivity per unit area, thus also helping to miniatuiosensor dimensions. Using p-type silicon wafersubstrates make compatibility with CMOS technolossible.

. Experimental

.1. Theories

In view of the important role of glucose concentrationuman life activities, lots of studies have been carried

n glucose sensors[7–11,14,16,17,26,29], with enzyme elecrode glucose sensors as vanguards.

VePt > Ve

WE, seeFig. 1a and b). On the other hand, torous film has larger surface area than planar platinum

hus enhancing the sensitive area. They are both helpfnhancing sensor activity.

Polymerization of pyrrole forms an electroactive ionhange polymer (polypyrrole, PPy) film on the surfaceubstrate.

Previous studies have reported that PPy film has rroperties[41]. PPy has one oxidized states, i.e. PPyPy0), and two possible reduced states, i.e. the lower redtate, PPy•+, and the upper reduced state, PPy•2+. It can bewitched from the oxidized state to the lower reduced srom the lower reduced state to the upper reduced staice versa.

Py0E1−→ PPy•+ + e, PPy•+ E2−→ PPy•2+ + e

E1 andE2 stand for the voltages for forming PPy•+ andPy•2+, respectively. PPy•+ and PPy•2+ play roles on thlectron transfer process in the PPy film synchronousl

ow PPy concentration, PPy•+ makes the major contributiohile in high PPy concentration, PPy•2+ makes the majoontribution[42].

PPy film helps to form lots of electroactive centers, thortening each electro-transfer paths, and speeding ulectron transfer rate (Ve

PPy > VeWE, seeFig. 1c and d).

To our knowledge, consecutive platinization and polymzation of pyrrole is the first such use in surface modificathe electron transfer process of consecutive platinizationolymerization of pyrrole enhances sensor activity is as

ows (seeFig. 1e and f):

J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601 593

Fig. 1. Schematic electron transfer processes of anodic reaction: (a) no surface modification, (b) platinization, (c) polymerization of pyrrole (PPy0 → PPy•+),(d) polymerization of pyrrole (PPy•+ → PPy•2+), (e) consecutive platinization and polymerization of pyrrole (PPy0 → PPy•+), (f) consecutive platinizationand polymerization of pyrrole (PPy•+ → PPy•2+).

A. Platinization forms an electroactive porous nanometerplatinum film (porous Pt film) on the surface of work-ing electrode (District A), thus enhancing the sensitivesurface area and speeding up the electron transfer rate.

B. Polymerization of pyrrole deposits an electroactive PPyfilm on the surface of porous Pt film (District B), thusenhancing the electron transfer rate.

C. When enzyme solution is deposited on the surface of PPyfilm (District C), a consecutive electron transfer channelis formed from GOD film to PPy film, porous Pt film, andto working electrode, i.e. electrons are transferred fromDistrict C to B, then to A consecutively.

From the process, we can see that consecutive platinizationand polymerization of pyrrole has a better performance thaneither platinization or polymerization of pyrrole.

2.2. Apparatus, materials and reagents

The equipments used to characterize the microelectrodebiosensors included a homemade voltage-scanning module,a potentiostat CH-1 (Jiangsu Jiangfen ElectroanalyticalInstrument, Co., Ltd., China) used to measure currentand a PC with a data acquisition and a plotting program.The microelectrodes used were a working electrode

594 J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601

Fig. 2. Fabrication process of bulk micromachined microelectrodes (Au).

Fig. 3. Fabrication process of surface micromachined microelectrodes (Pt).

Fig. 4. Fabrication process of surface micromachined microelectrodes (Au).

(WE, made of Au or Pt) and a counter-electrode (CE,made of the same metal as WE). The microelectrodeswere fabricated following the procedures reported in thiswork.

Materials and regents used were listed below: glucoseoxidase (GOD) from aspergillus niger (57.4 IU/mg), bovineserum albumin (BSA), 25% glutaraldehyde, phosphate-buffered saline (PBS) tablets (pH 7.4 at 25◦C), chloroplatinicacid were from Sigma, lead tetra acetate was from Aldrich,and pyrrole from Fluka. All other materials and regents, suchas glucose, potassium chloride, etc., were from China. Allthe materials and regents were of analytical grade. Except

Table 1Type denomination of microelectrodes

Type denomination Description of material, fabrication technology and surface modification

Type A Au, bulk micromachining; consecutive platinization and polymerization of pyrroleType B Au, bulk micromachining; polymerization of pyrroleType C Au, bulk micromachining; platinizationType D Au, bulk micromachining; no surface modificationType E Au, surface micromachining; consecutive platinization and polymerization of pyrroleType F Au, surface micromachining; polymerization of pyrroleType G Au, surface micromachining; platinizationType H Au, surface micromachining; no surface modificationType I Pt, surface micromachining; consecutive platinization and polymerization of pyrroleType J Pt, surface micromachining; polymerization of pyrroleType K Pt, surface micromachining; platinizationT surfac

for specific description, all the water used in our experimentsis DI water.

2.3. Microelectrode fabrication

Two different configurations were used to build the micro-electrode construction: planar multiple-layer (surface micro-machining, microelectrode made in Pt and Au, respectively)and in-device micropool (bulk micromachining, anisotropicsilicon wet etching, only made in Au). Using p-type sili-con wafers as substrates, fabrication process of each con-struction was presented inFigs. 2–4, respectively.Fig. 5

ype L Pt,

e micromachining; no surface modification

J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601 595

Fig. 5. Configurations and dimensions of microelectrode biosensors: (a) top view; (b) cross view (part); (c) dimensions of microelectrodes.

showed the configurations and dimensions of microelectrodebiosensors.

Notes:

1. SU-8 insulating layer inFig. 5a is painted in dark gray todistinguish it from the color of SU-8 microreaction pool.Its true color should be light blue, the same as SU-8 mi-croreaction pool (seeFig. 5b).

2. Working electrodes and counter-electrodes are all formedby anisotropic silicon etching.

3. Enzyme immobilization sites are kept for surface modifi-cation and immobilization.

4. Blank sites are kept only for forming a complete cur-rent circuit between working electrodes and counter-electrodes.

5. Welding pads are kept for welding anodes and cathodes.

Detailed description of fabrication process of bulk micro-machined microelectrodes is as follows:

(a) Wafer cleaning;(b) A thin Si3N4 layer (1000A) by LPCVD is deposited

(Fig. 2a), which will be patterned and micromachinedto serve as a mask for the followed anisotropic siliconwet etching;

(c) PR (photo resist) BP212 (positive) is spin-coated andpatterned (Fig. 2b and c);

-

(e) PR BP212 is removed;(f) Anisotropic silicon wet etching is carried out at 80◦C

with an etching of KOH:IPA:H2O at 24:16:60 (w:v:v).The etching rate is about 1�m/min. With depth at100�m and upper surface area at 1 mm× 1 mm, in-device micropools are formed at a lateral angle of 54.7◦(Fig. 2e);

(g) Mask Si3N4 layer is SF6 plasma-etched (Fig. 2f);(h) For our lab fabrication technology, SiO2 has a better ad-

herence with Si and a better electric insulating functionthan Si3N4, while Si3N4 has a better adherence withAu/Pt than SiO2. A thick SiO2 layer (1�m) formedby thermal oxidation and a thin Si3N4 layer (3000A)formed by LPCVD are deposited for electric and chem-ical insulating layers (Fig. 2g and h);

(i) A thin Cr/Au layer (200/3000A) (Cr layer used as seedlayer) is deposited by evaporation, respectively, on thesurface for microelectrodes (Fig. 2i);

(j) PR BN303 (negative) is spin-coated and patterned;(k) Cr/Au layer uncoated with PR BN303 is released with

gold and then chromium etchings (Fig. 2j);(l) PR BN303 is removed;

(m) A light oxygen plasma etching is followed to eliminateany residues;

(n) A thick SU-8 insulating layer (20�m) is coated andpatterned to cover the Au connecting tracks between en-

ads

(d) Si3N4 layer uncoated with PR BP212 is SF6 plasmaetched (Fig. 2d);

zyme immobilization sites/blank sites and welding p(Fig. 2k);

596 J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601

(o) Microreaction pools (2 mm in length× 2.5 mm in width× 100�m in depth) are formed by coating and pattern-ing of a thick SU-8 layer (100�m) (Fig. 2l).

Sensitive surface area of microelectrode for eitherconfiguration was 1 mm× 1 mm. For planar multiple-layerconfiguration, two SU-8 microreaction pools were made,the smaller one (1 mm in length× 1 mm in width× 100�min depth) for enzyme immobilization, and the bigger one(2 mm in length× 2.5 mm in width× 100�m in depth)for containing analyte (dissolved in buffered solution).For in-device micropool configuration, only one SU-8microreaction pool (2 mm in length× 2.5 mm in width×100�m in depth) was made to contain analyte (dissolvedin buffered solution), and an in-device micropool (1 mm inlength× 1 mm in width× 100�m in depth) by anisotropicsilicon etching was for enzyme immobilization.

2.4. Surface modification

Platinization and polymerization of pyrrole were twomethods often used. In this work, we tried these two methods.At the same time, a novel method, consecutive platinizationand polymerization of pyrrole was used. To our knowledge,this was the first such use. Microelectrodes with differentfabrication technologies and different surface modificationmethods are denominated as Types A, B, C, and so on, as inT

en-z ce,a bout1

tionm

23%

c adt n,a

s Ptfi ensi-t gapb 8 nm.F im-m %.

ula-t akea tions es orc withp rfacea s thes

2.4.2. Polymerization of pyrroleWith pyrrole concentration at 0.25 M (w/v, in 1 M KCl

solution) and current intensity at 0.2 mA/cm2, it was elec-trolyzed for 30 min, and then left at room temperature to dry.

With a stylus profilometer, we could see that a PPy film(445 nm in average thickness) was formed on the sensitivesurface of working electrode (made of Au here). For simulta-neous polymerization of pyrrole in five different enzyme im-mobilization sites, RSD in average PPy thickness was 4.3%.

2.4.3. Consecutive platinization and polymerization ofpyrrole

After dipped into 0.3% chloroplatinic acid (w/v, in DIwater, containing 50 ppm lead tetra acetate) with potential−0.3 V (versus SCE) for 30 min, it was rinsed with DI watertwice. Then with pyrrole concentration at 0.25 M (w/v, in1 M KCl solution) and current intensity at 0.2 mA/cm2, itwas electrolyzed for 30 min, and left at room temperature todry.

Consecutive platinization and polymerization of pyrrolewas actually the direct combination of platinization and poly-merization of pyrrole. After platinization, a porous Pt film(213 nm in average thickness) was formed on the sensitivesurface of working electrode (made of Au here). After con-secutive platinization and polymerization of pyrrole, the totalaverage thickness was 703 nm (larger than 213 + 445), andR % +4 ationo

2

en-t kingw tion[ odp .

en-z mgB -h andsz ensi-t new .1e an-o ighta er atr

2

m-m atedb glu-c

able 1.Before surface modification and after fabrication, the

yme immobilization site was rinsed with DI water twind then activated by light oxygen plasma etching for a0 s to eliminate any residues.

Detailed descriptions of the three surface modificaethods are as follows.

.4.1. PlatinizationIt (the enzyme immobilization site) was dipped into 0.

hloroplatinic acid (w/v, in DI water, containing 50 ppm leetra acetate) with potential−0.3 V (versus SCE) for 30 mind then left at room temperature to dry.

With a stylus profilometer, we could see that a poroulm (213 nm in average thickness) was formed on the sive surface of working electrode (made of Au here). Theetween the thickest part and the thinnest part was 4or simultaneous platinization in five different enzymeobilization sites, RSD in average Pt thickness was 5.4It was difficult, even impossible to make a clear calc

ion on the total surface area of porous Pt film. Let us msimple supposition that 80% of enzyme immobiliza

urface area was covered with concave Pt half-spheronvex Pt half-spheres, the other 20% was coveredlanar Pt. Calculation 1 demonstrated that the total surea of porous Pt film was about five times as large aurface area of planar Pt film.

Calculation 1 :

Sporous

Splanar= (2πr2 × 80%+ r2 × 20%)

r2= 5.224

SD in total average thickness was 6.7% (lower than 5.4.3%). These digital data demonstrated that polymerizf pyrrole could benefit from aforehand platinization.

.5. Enzyme immobilization

Four kinds of enzyme immobilization methods, i.e.rapment, adsorption, covalent binding, and cross-linere used in enzyme or antibody/antigen immobiliza

7–11,14,34,43–45]. Owning to its easy operation and goerformance, cross-linking was the method used mostly

Using cross-linking method, detailed description ofyme immobilization was as follows: 5.0 mg GOD, 5.0SA, 40�l PBS (pH 7.4 at 25◦C) and 2�l 25% glutaraldeyde (v/v, directly from Sigma) were merged togethertirred to get uniform enzyme solution. 0.1�l uniform en-yme solution was then deposited on microelectrode sive surface (1 mm× 1 mm). After a thin enzyme membraas formed (usually took several minutes), another 0�lnzyme solution was deposited on its surface to formther membrane (enhancing membrane). After left overnt room temperature, it was stored in a clean containoom temperature until its usage in detection.

.6. Cyclic voltammetry

To access proper working potential, the cyclic voltaetric experiment was carried out. The potential vibretween 0.0 and 1.2 V at a scanning rate 50 mV/s. Withose concentration at 1× 10−3 M, PBS (pH 7.4 at 25◦C,

J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601 597

Fig. 6. C–V curve.

dissolved from phosphate-buffered saline tablets, Sigma) asbuffer, a high oxidation current peak, corresponding to thereaction H2O2 → O2 + 2H+ + 2e, existed at approximately0.7 V (seeFig. 6), which we chose as the static working po-tential in our experiments. The static working potential, i.e.0.7 V, was in good agreement with literatures and independentof the electrode material, fabrication technology and surfacemodification[8,10,11,17]. According to the Nernst equation,two branches (cathodic wave and anodic wave) were asym-metric, thus proving that reaction was irreversible[13].

2.7. Amperometric measurement

Each glucose concentration measurement was done atroom temperature, in the SU-8 microreaction pool (2 mmin length× 2.5 mm in width× 100�m in depth) contain-ing 0.5�l analyte (glucose dissolved in PBS, concentrationwaiting to be detected, pH 7.4 at 25◦C), where two micro-electrodes (WE and CE, made of the same metal) were intro-duced. The microelectrodes were connected to a potentiostat(CH-1) that defined the static working potential (0.7 V versusCE) and provided a current signal reading. A PC acquired andrecorded the data. Thus by reading anodic current increasein this course, glucose concentration in the solution can bedetected. All detection solution was used only once and thend

3

s ofd

, an-o horsd ordi-n as mi-c ea nol-o mMa inc et as

Fig. 7. Response of Type A microelectrode sensor to glucose concentration.

3.1. PH and temperature

Both pH and temperature have great influence on enzymeefficiency [40]. We used phosphate-buffered saline tabletsfrom Sigma, which kept pH at 7.4 at 25◦C to keep pH sta-ble in detection process. As to temperature influence, withthe glucose concentration at 3× 10−3 M, Type C microelec-trode biosensor (with sensitive surface area 1 mm× 2 mm)was used at 15◦C (with outcome current 285 nA) and 20◦C(with outcome current 313 nA), respectively. The outcomeshows that the relative current change is about 10%, thusdemonstrating that temperature is crucial in detection. Wecarried out our experiments at room temperature to minimizethe temperature influence.

3.2. Sensitivity per unit area and linear range

From Table 2and Fig. 7, we can see that of all types,Type A microelectrode biosensor has the largest sensitivityper unit area of 39.640 nA mM−1 mm−2 and the largest linearrange of 1× 10−4–1 × 10−2 M (with a pretty good corre-lation coefficientr, i.e.R2, at 0.9916), thus proving Type Amicroelectrode biosensor has the best performance and bulkmicromachining technology is really a good technology formicroelectrode biosensors. It mainly owns to two reasons:t vicem sen-s entali poly-m ithg ofTa onalg ea,l ther

3

ple,R tiono

isposed.

. Results and discussion

Table 2shows the denominations and characteristicifferent microelectrode biosensors.

Besides above types of microelectrode biosensorsther Type M microelectrode biosensor, which the autesigned and fabricated before, was introduced. Usingary glass plate (3 mm in thickness) as substrate and Ptroelectrode material, with 4 mm× 5 mm in sensitive surfacrea, it was fabricated with surface micromachining techgy. Its total sensitivity and linear range were 223.38 nA/nd 3× 10−4–6× 10−3 M, respectively. For convenienceomparison, its total sensitivity was divided by 20 to gensitivity per unit area of 11.169 nA mM−1 mm−2.

he first, taking its lateral surface area into account, in-deicropool formed by silicon wet etching enlarges actual

itive surface area and keeps surface area from environmnterference; the second, consecutive platinization and

erization of pyrrole greatly activate sensitive surface. Wlucose concentration at 1× 10−3 M, the response timeype A microelectrode biosensor is about 50 s (seeFig. 8),little longer than 20 s, the response time of conventi

lucose sensors[10,11]. With smaller sensitive surface aress GOD existing in the GOD film, it is reasonable thatesponse time is longer.

.3. Reproducibility

Using Type A microelectrode biosensor as an examSD for five detections is only 3.2% at glucose concentraf 1 × 10−3 M. It is endurable for practical applications.

598 J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601

Table 2Denominations and characteristics of different microelectrode biosensors

Type denomination ofmicroelectrode biosensor

Correspondingmicroelectrode type

Characteristics

Sensitivity per unit area(nA mM−1 mm−2)

Linear range (M)

Type A Type A 39.640 1× 10−4–1× 10−2

Type B Type B 34.723 3× 10−4–6× 10−3

Type C Type C 33.334 5× 10−4–6× 10−3

Type D Type D 3.500 1× 10−3–6× 10−3

Type E Type E 2.892 1× 10−3–6× 10−3

Type F Type F 2.342 3× 10−4–6× 10−3

Type G Type G 1.266 1× 10−3–6× 10−3

Type H Type H 0.141 1× 10−3–6× 10−3

Type I Type I 19.926 1× 10−3–6× 10−3

Type J Type J 14.033 5× 10−4–6× 10−3

Type K Type K 7.732 5× 10−4–6× 10−3

Type L Type L 0.390 5× 10−4–6× 10−3

Fig. 8. Response of current to time.Note: glucose concentration is at 1× 10−3 M.

3.4. Stability

Using Type A microelectrode biosensor as an example,RSD for 10 detections is only 4.1% at glucose concentra-tion of 1 × 10−3 M. Table 3 demonstrates that after be-ing stored in a clean container at room temperature for amonth, these microelectrode biosensors kept their enzymeefficiency well above 90%.Table 3also demonstrates thatbiosensors with consecutive platinization and polymeriza-tion of pyrrole have better stability than biosensors with onlyplatinization.

Table 3Sensitivity per unit area stability research

Description

Sensitivity per unit area of new-made microelectrode biosensor (nA mM−1 mm−2)Sensitivity per unit area of microelectrode biosensor stored at RT for a montLoss of sensitivity per unit area (nA mM−1 mm−2)Relative loss of sensitivity per unit area (%)Relative remaining sensitivity per unit area (%)

Note: RT stands for room temperature.

3.5. Fabrication technologies

Table 2shows that, with the same microelectrode materialand surface modification method, biosensors made with bulkmicromachining technology have better performance thancorresponding biosensors made with surface micromachin-ing technology in sensitivity per unit area (at least 10 timeslarger) and linear range, i.e. Type A better than Type E, TypeB than Type F, Type C than Type G, and Type D than Type H.It mainly owns to two reasons: the first, lateral surface areasof in-device micropools formed by anisotropic silicon wetetching (bulk micromachining) help to enlarge the sensitivesurface areas (about 45% larger than surface micromachin-ing), and the second, in-device micropools circum-embracethe sensitive microelectrodes, thus keeping them from envi-ronmental interference[10].

3.6. Surface modification methods

Table 2shows that Type A, B and C microelectrode biosen-sors are much more sensitive than Type D microelectrodebiosensor, Type E, F and G than Type H, and Type I, J and Kthan Type L, demonstrating that surface modification (eitherpolymerization of pyrrole or platinization) is greatly helpfulin promoting sensitiveness. It mainly owns to two reasons: thefirst, polymerization of pyrrole enhances the electron transferr -t on

Type A Type C

39.640 33.334h (nA mM−1 mm−2) 38.198 31.295

1.442 2.0393.637 6.117

96.363 93.883

ate between enzyme film and anode[46], the second, plainization electroplates a porous nanometer platinum film

J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601 599

Fig. 9. Response of different microelectrode biosensors to glucose concen-tration.

the microelectrode surface, thus helping to enlarge the sensi-tive surface area of microelectrode (about five times as largeas planar Pt film) and speed up the electron transfer rate (seeSection 2.3).

Table 2also shows that, with the same microelectrode ma-terial and fabrication configuration, microelectrode biosen-sors with consecutive platinization and polymerization ofpyrrole are more sensitive than microelectrode biosensorswith platinization or polymerization of pyrrole (i.e. TypeA more sensitive than Type B or C, Type E than Type For G, and Type I than Type J or K), and microelectrodebiosensors with polymerization of pyrrole are more sensi-tive than microelectrode biosensors with platinization (i.e.Type B more sensitive than Type C, Type F than TypeG, and Type J than Type K), demonstrating that consec-utive platinization and polymerization of pyrrole is betterthan platinization or polymerization of pyrrole, and poly-merization of pyrrole is better than platinization for surfacemodification.

3.7. Microelectrode materials

Owning to different redox properties of Au and Pt, dis-sociation rates of H2O2 on Au and Pt electrode surfacesare different. We can see fromFig. 9 and Table 2 that,w icro-m lec-t thanb mi-c etterp iple-l f Au,d odemA mi-c each.S cro-e andt

Fig. 10. Vivid photograph of microelectrode biosensors.Notes: coin in themiddle is about 19 mm in diameter; sensitive surface area: 1 mm× 1 mm (theleft one), 1 mm× 2 mm (the top one and the right one); SU-8 microreactionpool: 2 mm in length× 2.5 mm in width× 100�m in depth (the left one),3 mm in length× 2.5 mm in width× 100�m in depth (the top one and theright one); total surface area: less than 5 mm× 8 mm).

3.8. Miniaturization

Fig. 9 demonstrates that, if transferred to the same sen-sitive surface area of 1 mm× 1 mm, Type J microelectrodebiosensors (p-type silicon wafers as substrates, surface ma-chining, polymerization of pyrrole, 1 mm× 1 mm, made inPt) are more sensitive than Type M microelectrode biosensors(ordinary glass plates as substrates, surface machining, poly-merization of pyrrole, 4 mm× 5 mm, made in Pt), demon-strating that miniaturization benefits to sensitivity.

3.9. Prospects

From Fig. 10, we can see that this new microelectrodebiosensor is very small in dimensions (sensitive surface area1 mm× 1 mm, less than 5 mm× 8 mm in total) and portable.Its good performance and portability prove its excellence inthis field. Most important, in view of its usage of p-type sili-con wafer as substrate and small dimensions, it is possible forthis microelectrode biosensor to be integrated with ICs[26].With more and more emphases being paid on miniaturizationand system integration, such as microneedle-based glucosemonitor [29] and electrochemical microanalysis system forheavy metal ions[48] reported on Transducer 2003, stud-ies on carbon nanotube glucose sensors reported on MEMS2 is-i ionsa n-b secu-t dea hipr

4

olei ion.T ates

ith the same surface modification method, surface machined (planar multiple-layer configuration) microe

rode biosensors made of Pt have worse performanceulk micromachined (in-device micropool configuration)roelectrode biosensors made of Au, but have much berformance than surface micromachined (planar mult

ayer configuration) microelectrode biosensors made oemonstrating that Pt is better than Au for microelectraterial (for detailed explanation, please refer to Ref.[47]).ccording to our current experimental conditions, bulkromachined microelectrodes made of Pt are hard to rome resolutions for fabricating bulk micromachined milectrode biosensors made of Pt are still under design

rial.

004[49,50], etc., lab-on-a-chip will be definitely a promng research hotspot. With low unit cost, small dimensnd compatibility with CMOS technology, this new silicoased amperometric microelectrode biosensor with con

ive platinization and polymerization of pyrrole will provipromising platform for clinical detection and lab-on-a-c

esearch.

. Conclusions

Consecutive platinization and polymerization of pyrrs a novel and promising method for surface modificathis paper theoretically and experimentally demonstr

600 J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601

that consecutive platinization and polymerization of pyrrolehas a better performance than either platinization or polymer-ization of pyrrole.

Successful experimental results have been achieved onglucose detection. Compared to other amperometric elec-trode biosensors mentioned above, including conventionalmacroelectrode biosensors, so-called microelectrode biosen-sors, surface micromachined microelectrode biosensors[7–11,18,45], and even bulk micromachined microelectrodebiosensors with either platinizaiton or polymerization ofpyrrole, though a little complicated in fabrication technology,this new bulk micromachined amperometric microelectrodebiosensor with consecutive platinization and polymerizationof pyrrole has several advantages, such as smaller sensitivesurface area (1 mm× 1 mm), lower detection limit (1×10−4 M), larger linear range (1× 10−4–1 × 10−2 M),larger sensitivity per unit area (39.640 nA mM−1 mm−2),better reproducibility (3.2% RSD for five detections) andstability (relative remaining sensitivity per unit area keptwell above 95% after being stored in a clean container atroom temperature for a month), easier to be made into arraysand to be integrated with processing circuitry, etc.

Acknowledgements

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[ 279.[[ 772.[[ 190.[

[21] G. Jobst, et al., Sens. Actuators B 43 (1997) 121–125.[22] T. Matsumoto, et al., Sens. Actuators B 49 (1998) 68–72.[23] C. Tian, et al., Sens. Actuators B 52 (1998) 119–124.[24] H. Suzuki, et al., Anal. Chim. Acta 387 (1999) 103–112.[25] A. Hiratsuka, et al., Analyst 126 (2001) 658–663.[26] S.-I. Park, et al., A new well-shaped micromachined glucose sen-

sor using Cl-plasma treated Ag/AgCl reference electrode, Biologi-cal MEMS, 2002, Advances in medical and analytical applications,Royal Sonesta Hotel, Cambridge, MA, USA, April 25–26, 2002.

[27] Q. Li, S. Zhang, et al., Appl. Biochem. Biotechnol. 59 (1) (1996)53–61.

[28] B. Ye, Q. Li, et al., J. Biotechnol. 42 (1995) 45–52.[29] S. Zimmermann, et al., A microneedle-based glucose monitor: fab-

ricated on a wafer-level using in-device enzyme immobilization, in:Proceedings of the 12th International Conference on Solid State Sen-sors, Actuators, and Microsystems, Boston, June 8–12, 2003.

[30] A.J. Killard, S. Zhang, et al., Anal. Chim. Acta 400 (1999) 109–119.[31] L. Yue, Z. Xu, Chin. J. Chem. 9 (1998) 28–31.[32] Z. Chang, B. Li, et al., Chin. J. Appl. Chem. 20 (4) (2003) 360–364.[33] Z. Zhang, X. Wu, Chem. Sens. 21 (3) (2001) 13–14.[34] Z. Liu, B. Liu, et al., Anal. Chim. Acta 392 (1999) 135–141.[35] Q. Gao, C. Sun, et al., Chin. J. Anal. Chem. 20 (7) (1992) 828–830.[36] J. Wang, P.V.A. Pamidi, et al., Anal. Chim. Acta 330 (1996)

151–158.[37] R.W. Keay, C.J. McNeil, Biosens. Bioelectron. 13 (1998) 963–970.[38] B. Lu, E.I. Iwuoha, et al., Anal. Chim. Acta 345 (1997) 59–66.[39] R. Krishnan, P. Atanasov, et al., Biosens. Bioelectron. 11 (8) (1996)

811–822.[40] E.I. Iwuoha, M.R. Smyth, Biosens. Bioelectron. 12 (1) (1997) 53–75.[41] S. Dong, J. Ding, Synth. Met. 20 (1987) 119.[42] S. Dong, G. Che, Y. Xie, Chemical Modified Electrodes, 2nd ed.,

[ 57.[ (8)

[[[[ eavy

ence, June

[ T)IEEEand2004.

[ ingle-icro-gress

B

J ina,o 2004.H uterE g int MOSi

C ina.S ratoryo y ofS s anda

The authors greatly acknowledge the financial suprom the National Natural Science Foundation of ChGrant No. 90307014). We gratefully thank Prof. Xinxia Cs. Yuanyuan Xu, Ms. Hong Zhang, and Mr. Huaqing Li

heir successive help with our experiments.

eferences

[1] L.C. Clark, C. Lyons, Ann. NY Acad. Sci. 29 (1962) 102.[2] S.J. Updike, G.P. Hicks, Nature 214 (1967) 986.[3] E.J. Kim, T. Haruyama, et al., Anal. Chim. Acta 394 (1999) 225–[4] P.V.A. Pamidi, et al., Concepcion Parrado Talanta 44 (1

1929–1934.[5] E. Crowley, C. O’Sullivan, et al., Anal. Chim. Acta 389 (199

171–178.[6] V. Rajendran, E. Csoregi, et al., Anal. Chim. Acta 373 (19

241–251.[7] S. Sun, Z. Li, Acta Nutrimenta Sinica 20 (2) (1998) 229–233.[8] X. Yu, D. Zhou, et al., Chin. J. Appl. Chem. 12 (1) (1995) 108–1[9] B. Liu, Q. Li, et al., Chin. J. Sensor Technol. 4 (1998) 93–96.10] X. Yu, D. Zhou, Chem. Sensors 19 (2) (1999) 48–52.11] L. Guo, J. Li, et al., Chin. J. Anal. Chem. 20 (7) (1992) 828–812] I. Pankratov, O. Lev, J. Electroanal. Chem. 393 (1995) 35–41.13] S. Alegret, F. Cespedes, et al., Biosensors Bioelectron. 11

(1996) 35–44.14] X. Cai, H. Li, et al., Micro and Nanoeletron. Technol. 40 (314/3

(2003) 359–361.15] K. Aoki, K. Honda, et al., J. Electroanal. Chem. 182 (1985) 267–16] J. Zhu, X. Liu, et al., Chin. J. Chem. 4 (1994) 48–50.17] J. Wu, J. Zhu, et al., Chin. J. Anal. Chem. 24 (7) (1996) 768–18] O. Niwa, et al., Anal. Chem. 62 (1990) 447–452.19] R. Steinkuhl, et al., Biosens. Bioelectron. 11 (1/2) (1996) 187–20] H. Frebel, et al., Sens. Actuators B 43 (1997) 87–93.

Science Press, Beijing, China, 2003.43] M.A. Sirvent, A. Merkoci, et al., Sens. Actuators B 79 (2001) 48–44] E.I. Iwuoha, D.S. de Villaverde, et al., Biosens. Bioelectron. 12

(1997) 749–761.45] S. Andreescu, T. Noguer, et al., Talanta 57 (2002) 169–176.46] M. Umana, J. Waller, Anal. Chem. 58 (1986) 2979–2983.47] J. Liu, Master Dissertation, Chapter 3.8.48] H. Suzuki, et al., Electrochemical micro analysis system for h

metal ions, in: Proceedings of the 12th International Conferon Solid State Sensors, Actuators, and Microsystems, Boston8–12, 2003.

49] K.S. Teh, L. Lin, A polypyrrole-carbon-nanotube (PPY-MWNnanocomposite glucose sensor, in: The 17th InternationalMicroelectromechanical Conference, Maastricht ExhibitionCongress Centre, Maastricht, The Netherlands, January 25–29,

50] J. Chung, et al., Microfabricated glucose sensor based on swalled carbon nanotubes, in: The 17th International IEEE Melectromechanical Conference, Maastricht Exhibition and ConCentre, Maastricht, The Netherlands, January 25-29, 2004.

iographies

ingwei Liu graduated in 2001 from Tsinghua University (BE), Chbtained his ME degree from Chinese Academy of Sciences ine is now pursuing his PhD degree in Electrical and Compngineering Department, Carnegie Mellon University. He is workin

he development of microsensors and actuators, and MEMS and Cntegration.

hao Biangraduated in 2003 from Zhengzhou University (ME), Chhe is currently pursuing a doctor degree in the State Key Labof Transducer Technology, Institute of Electronics, Chinese Academciences. She is now working in the development of microsensorctuators such as amperometric microelectrode biosensors.

J. Liu et al. / Sensors and Actuators B 106 (2005) 591–601 601

Jinghong Hangraduated in 1966 from Peking University, China. Sheis now working as a research fellow in the State Key Laboratory ofTransducer Technology, Institute of Electronics, Chinese Academy ofSciences. Her research interests include microsensors and MEMS, SOC,BioMEMS, biosensors and bioactuators.

Shaofeng Chengraduated in 1975 from Fudan University, China. Sheis now working as a senior engineer in the State Key Laboratory ofTransducer Technology, Institute of Electronics, Chinese Academy of

Sciences. Her research interests include microsensors and MEMS, SOC,BioMEMS, biosensors and bioactuators.

Shanhong Xiagraduated in 1983 from Tsinghua University, China, ob-tained her PhD in electrical engineering from University of Cambridge,UK, in 1994. She is now working as a research fellow in the State KeyLaboratory of Transducer Technology, Institute of Electronics, ChineseAcademy of Sciences. Her research interests include microsensors andMEMS, SOC, BioMEMS, biosensors and bioactuators, etc.