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Analytica Chimica Acta 387 (1999) 103112

An integrated three-electrode system with a micromachined


liquid-junction Ag/AgCl reference electrode
Hiroaki Suzukia,*, Taishi Hirakawaa, Satoshi Sasakib, Isao Karubeb
a
Institute of Materials Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba Science City, 305-8573 Japan
b
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904 Japan

Received 17 July 1998; received in revised form 4 January 1999; accepted 9 January 1999

Abstract

A micromachined liquid-junction Ag/AgCl reference electrode was employed to fabricate an integrated three-electrode
system. The reference electrode features a durable thin-lm Ag/AgCl element whose entire surface was coated with a
hydrophobic membrane and AgCl layer grown from its periphery. On the other side of the chip were formed a platinum
working electrode and a platinum counter electrode. The cleanliness of the thin-lm platinum working electrode was
satisfactory to conduct electrochemical analyses including cyclic voltammetry. No substantial difference was observed in the
cyclic voltammograms between the integrated system and the macroscopic three-electrode system in terms of their peak
current and half-wave potential. The integrated electrode system was also applied to fabricate one-chip bio/chemical sensors.
The miniature glucose sensor showed a distinct response against step changes of glucose concentration. The one-chip
hydrogen electrode was used to conduct potentiometric titration. A good coincidence was again conrmed between the
integrated system and the macroscopic system. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: Ag/AgCl; Liquid junction; Platinum; Cyclic voltammogram; Glucose; Potentiometric titration

1. Introduction [1] and was applied to a micromachined liquid-junc-


tion Ag/AgCl reference electrode [2]. Although there
One of the most troublesome problems in the is still room for its performance to be improved
development of micro electrochemical sensors has further, its realization made the integration of all
been the unavailability of a miniature reference elec- the electrochemical system possible. In this paper
trode with a liquid junction. In realizing such a we will show a device on which the working, counter,
reference electrode the foremost issue has been the and reference electrodes are integrated.
development of a durable thin-lm Ag/AgCl element There are some potential applications for the
which functions even in a solution saturated with KCl. device. Chemical analysis on samples of minute
In our previous study a novel thin-lm Ag/AgCl quantities is attracting attention in medicine and biol-
element with satisfactory durability was developed ogy. Electrochemistry is one of the most powerful
techniques for selective and sensitive detection in such
*Corresponding author. Fax: +81-298-55-7440; e-mail: an environment. In basic researches the cost and/or
hsuzuki@ims.tsukuba.ac.jp difculty in obtaining puried analyte sometimes pose

0003-2670/99/$ see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S0003-2670(99)00063-X
104 H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112

a serious problem and the analysis in a small sample Japan Silicon. A photo-patternable polyimide prepo-
volume is imperative. An extreme case is the chemical lymer (Photoneece 3100) was a gift from Toray. A
analysis of a single cell [3,4]. In order to achieve such positive photoresist (S1400-31) used in fabrication
an electroanalysis in microvolume, several micro was purchased from Shipley Far East, Tokyo. A
systems have been proposed [3,511], some of which negative photoresist, OMR-83, used in fabrication
have been fabricated using thin-lm technologies and and the electrode structure was purchased from Tokyo
micromachining [3,911]. By using these systems not Ohka Kogyo, Kawasaki, Japan. A photocurable adhe-
only can the dilution of the analyte due to the diffusion sive (BENEFIX VL) was purchased from Adell,
to the bulk solution be alleviated to maintain the low Tokyo. The reagents used in fabricating the electrode
analyte levels (in the order of femtomolezeptomole) were of semiconductor grade and purchased from
present in the single cell, but also a fast response is Kanto Chemicals, Tokyo, and Wako Pure Chemicals,
obtained because the diffusion length (or the distance Osaka. All reagents used to examine the electrode
between the cell and the working electrode) is reduced performance were purchased from Wako Pure Che-
[3]. With the recent advent of mTAS (micro total micals. They were of analytical reagent grade and
analysis system) technology the integration of the used without further purication. Glucose oxidase
three-electrode system in a small space is becoming (type X-S, from Aspergillus niger) and bovine serum
more and more important. Once a reliable miniature albumin (BSA) were used as supplied from Sigma.
integrated system is realized, it would not be difcult Distilled water was used throughout the experiments.
to modify its design and use it as a detector in other
microanalytical devices such as a microfabricated 2.2. Structure and fabrication of the integrated
capillary electrophoresis system [12]. three-electrode system
Another important application of the integrated
device is in vivo, in situ measurements. Hundreds The integrated three-electrode system was fabri-
of papers have been published which deal with micro cated on both sides of a glass substrate (Fig. 1).
electrochemical sensors including ion sensitive eld Ordinary semiconductor and micromachining pro-
effect transistors (ISFETs). However, in almost all cesses were followed, which will not be described
cases the miniaturization is limited to the working or in detail here. Metals were deposited by sputtering.
indicator electrode and a macroscopic reference elec- The dimensions of the chip were approximately
trode (and a macroscopic counter electrode for 1.5 mm wide, 13 mm long and 0.9 mm thick. On
amperometric sensors) are used. It might be enough one side were formed a working electrode and a
for a basic research but it is actually the cause which counter electrode both of which were made from
hinders further practical application of these miniature 300-nm-thick platinum (Fig. 1(a)). A 40-nm-thick
sensors to many potential elds. For in vivo and in situ chromium layer was used as an adhesive layer. In
measurements, all electrode elements must be inte- order to delineate the electrode areas and insulate the
grated on a chip. In order to show a possibility of lead patterns, a 600-nm-thick polyimide layer was
application of this integrated device to one-chip formed. The exposed areas on the working and counter
microsensors, a glucose sensor and a hydrogen elec- electrodes were 0.5 mm0.5 mm and 0.5 mm
trode were fabricated. The performance of the inte- 6.4 mm, respectively. In curing the polyimide patterns
grated electrode system and the one-chip sensors will the devices were subjected to bakings for 15 min at
be presented in this report. 1508C, 15 min at 2008C, and 30 min at 3008C.
The miniature liquid-junction Ag/AgCl reference
2. Experimental electrode was formed on the other side of the glass
substrate (Fig. 1(b)), the structure and fabrication of
2.1. Materials which were the same as reported elsewhere [1,2]. In
brief, the thin-lm Ag/AgCl element used in this
Glass wafers (No. 7740, 3 in., 500 mm thick) were device consisted of a silver layer (300 nm thick)
purchased from Corning. (1 0 0)-oriented silicon formed on a gold backbone layer (200 nm thick)
wafers (3 in., 400 mm thick) were purchased from which made partial contact with the silver layer. A
H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112 105

Fig. 1. Structure of the integrated three-electrode system: (a) glass substrate on which the working and counter electrodes are formed; (b) the
other side of (a) on which the novel thin-film Ag/AgCl element is formed; (c) silicon substrate on which the container for the electrolyte
solution and the liquid-junction are formed. The surfaces of (b) and (c) are bonded to make (d); (d) completed liquid-junction reference
electrode. The other side of the silicon substrate shown in (c) can be seen; (e) cross section of the currently-used thin-film Ag/AgCl element;
(f) cross section of the thin-film Ag/AgCl element shown in (a) along the line xx0 .

hydrophobic negative photoresist layer (2.7 mm thick) for the internal electrolyte solution was formed by
covers the entire silver layer. AgCl layers were formed anisotropically etching a silicon substrate in a KOH
not from the surface but from the periphery of the solution at 808C (Fig. 1(c) and (d)). The glass and
silver pattern as shown in Fig. 1(b) and (f). The silver silicon substrates were bonded with a photocurable
layer was transformed to AgCl by immersing the chip adhesive and the end of the liquid junction was closed
in 1.0 M FeCl3 for 50 min at 308C. The microcavity with cellulose acetate as described elsewhere [2]. In
106 H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112

performing the work, the working and counter elec- AgCl reference electrode (Horiba 2080A-06T with a
trodes and their leads were protected with a poly- ceramic-plug junction), and a macroscopic platinum
(vinylalcohol) (PVA) coating to avoid mechanical plate counter electrode. The internal lling solution
damage, which was removed just before the introduc- for the macroscopic reference electrode was saturated
tion of the electrolyte solution. The internal electrolyte with KCl and AgCl. The second type used the work-
solution for the reference electrode was a solution ing, reference, and counter electrodes on the chip.
saturated with KCl and AgCl, which was introduced Most of the experiments were conducted in a 100 ml
into the microcavity by immersing the chip in the solution lled in a 100 ml beaker. The latter type was
electrolyte solution in a beaker, placing it in a cham- also used to take cyclic voltammograms in a 100 ml
ber, and evacuating it [2,13]. Air bubbles often trapped solution lled in a plastic tip of a 100 ml micropipette
at the liquid junction were removed thoroughly by whose end was closed by silicone rubber. In conduct-
centrifuging the chip. Ninety devices were batch- ing these experiments the platinum working electrode
fabricated on a 3 in. wafer and were diced into chips. site was cleaned by repeatedly cycling its potential
In fabricating the one-chip glucose sensor, the between 0.2 and 1.3 V (vs. Ag/AgCl) at 1 V/s. The
working electrode area of the chip was immersed in sample solutions to be analyzed were bubbled with
10% 3-aminopropyltriethoxysilane for 1 h, rinsed pure nitrogen beforehand.
with distilled water, and dried. One mg of glucose The performance of the glucose sensor was eval-
oxidase was dissolved in 25 ml of 2% BSA solution, uated in a 20 mM KH2PO4-NaOH buffer solution (pH
which was subsequently mixed with 15 ml of 10% 7.0) containing 0.1 M KCl. Applied potential to the
glutaraldehyde solution with a small rod. The working working electrode was 0.7 V (vs. Ag/AgCl). 1.0 M
electrode area of the chip was immediately immersed glucose sample solution was prepared and a prede-
in the mixture to coat it, which was allowed to react for termined amount of the solution was injected into the
about 10 min, then the chip was rinsed with distilled buffer solution to check the sensor response.
water and was immersed in 0.1 M glycine solution for In conducting the potentiometric titration the inte-
30 min. The whole immobilization procedure was grated electrode chip was immersed in 100 ml solution
carried out at room temperature. of 20 mM HCl and pure hydrogen gas (1 atm) was
In forming the hydrogen electrode, the platinum bubbled into the solution. After the response of the
working electrode site and approximately half of the hydrogen electrode was allowed to stabilize, 2.0 M
platinum counter electrode site were platinized in a NaOH solution was added progressively to conduct
solution containing 66 mM H2PtCl6 and 0.88 mM the titration. In one experiment the site of the platinum
(CH3COO)2Pb at 30 mA/cm2 for 60 min. Current of black and the miniature reference electrode on the
the opposite polarity was own for a brief period every chip were used. The same experiment was repeated
10 min. The reference electrode site was protected using the same platinum black site and the macro-
with the positive photoresist during the platinization. scopic Ag/AgCl reference electrode.
The platinum working and counter electrode sites and All pH measurements were made using a TOA
the obtained platinum black were activated before and Electronics HM-20S pH meter, with a combination
after the procedure in a 0.1 M H2SO4 solution by pH glass electrode previously calibrated in precision
polarizing it repeatedly between 0.5 and 0.5 V buffers of pH 4.01 and 6.86 (TOA Electronics). All
(vs. Ag/AgCl). The reference electrode was formed experiments were conducted at a temperature of
and assembled after the procedure. 25.00.18C.

2.3. Procedure 3. Results and discussion

In order to evaluate some electrochemical charac- 3.1. Cleanliness of the thin-film platinum working
teristics two sets of three-electrode system were exam- electrode
ined in combination with a Hokuto-Denko HA-151
potentiostat/galvanostat. The rst type uses the plati- Generally speaking, cleanliness of the surface of a
num working electrode on the chip, a macroscopic Ag/ thin-lm electrode is preserved relatively well in
H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112 107

the potential of the working electrode in the 0.5 M


H2SO4 solution. This indicates that the cleaning pro-
cedure is effective even if some organic residues were
left during the fabrication process. During the poten-
tial scan neither dissolution of the chromium layer nor
corresponding waves in the voltammogram were
observed judging from Fig. 2 and visual inspection.

3.2. Application to electrochemical analysis

In examining the applicability of the integrated


micro system to electroanalysis, cyclic voltammetry
is a good test. Fig. 3 shows cyclic voltammograms of
3 =4
FeCN6 redox couple. Curve (a) is a voltammo-
gram taken in a plastic tip of a 100 ml pipette using the
integrated working, reference, and counter electrodes
Fig. 2. Cyclic voltammogram in 100 ml solution of 0.5 M H2SO4
at 258C obtained using the platinum working electrode on the chip. on the chip. Curve (b) was taken in a 100 ml beaker
A macroscopic reference electrode and a macroscopic platinum using the same working electrode on the chip and the
plate counter electrode were used. Scan rate: 50 mV/s. macroscopic Ag/AgCl reference electrode and the
macroscopic platinum counter electrode. The shape
and the positions of the peaks of the voltammogram
microfabrication processes as can be imagined from obtained in the plastic tip are identical with those
the quality of the cleanliness which the current semi- obtained in the beaker and coincide with the fre-
conductor process requires. However, depending on quently described curves in related literature [15].
the facility and materials used, residues of various Although a liquid-junction potential of several mV
polymers used in fabrication might remain on the had been measured for the present miniature liquid-
surface and affect electrochemical characteristics of junction reference electrode [2], the difference was not
the system. It is known that a residue of polyimide clear on the scale shown in the gure. The peak-
always persists on the surface of a thin-lm metal shaped curves indicate that planar diffusion perpen-
pattern. In order to obtain a clean surface which is free dicular to the working electrode dominates.
from such a contaminant, a nickelchromium sacri- The difference between the two cases was examined
cial layer was used and was removed by chemical in more detail (Table 1). Overall, a good coincidence
etching [10]. Because a photopatternable polyimide between the integrated system and the macroscopic
was used to delineate the working and counter elec- system was conrmed. The difference in the cathodic
trodes in our device, the cleanliness of the platinum peak current between the integrated system and the
working electrode was checked. macroscopic system or among chips is possibly due to
Fig. 2 shows a cyclic voltammogram using the thin- the slight difference in cleanliness of the working
lm platinum working electrode in 0.5 M H2SO4. A electrode surface. Although a couple of E1/2 values
well-known shape of the voltammogram was obtained deviate from the average, this is due to the unsatis-
[14]. The peaks corresponding to the adsorption and factory function of the cellulose acetate liquid
desorption of hydrogen, formation and reduction of junction. Furthermore, the dependence of the peak
oxide are clearly distinguished, which indicates that currents on the square root of the scan rate was also
the cleanliness of the surface is preserved well for use conrmed in both systems. Note that the overall
in electroanalysis. Although there were chips which coincidence of the results could be achieved only
did not show the cyclic voltammograms as in Fig. 2 by the liquid-junction reference electrode. In this
possibly due to the polyimide residue on the working sense, our study is essentially different from the other
electrode, the clean surface and the clear cyclic vol- related ones using pseudo-reference electrodes. The
tammograms could be obtained by repeatedly cycling results obtained in the experiments show that not only
108 H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112

3 =4
Fig. 3. Cyclic voltammograms of FeCN6 couple. The experiments were conducted in a solution containing 5 mM K3Fe(CN)6 and 0.2 M
Na2SO4. (a) The working, counter, and reference electrodes on the chip were used and the cyclic voltammogram was taken in a plastic tip of a
100 ml micropipette. (b) The working electrode on the chip and macroscopic reference and counter electrodes were used in the solution filled
in a 100 ml beaker. Scan rate: 50 mV/s. Insets show the image of each three-electrode system.

Table 1
Distribution of the cathodic peak current ipc and the half-wave potential E1/2 obtained from the cyclic voltammograms of 10 mM K3Fe(CN)6 in
0.2 M Na2SO4

Chip no. Micro Macro

ipc (mA) E1/2 (V) ipc (mA) E1/2 (V)

1 3.98 0.24 3.25 0.27


2 3.86 0.30 3.45 0.26
3 3.63 0.27 3.85 0.26
4 3.56 0.21 3.30 0.26
5 3.49 0.30 3.58 0.28
6 3.44 0.28 3.81 0.28
7 3.33 0.24 3.53 0.27
8 3.76 0.27 3.87 0.27

Average (SD) 3.63 (0.22) 0.26 (0.03) 3.58 (0.24) 0.27 (0.01)
The columns noted as ``Micro'' are the results obtained with the working, reference, and counter electrodes on the chip in the 100 ml solution.
The columns noted as ``Macro'' are the results obtained with the working electrode on the chip, the macroscopic reference electrode, and the
macroscopic counter electrode in the 100 ml solution. Scan rate: 50 mV/s.

the miniature liquid-junction reference electrode but a micropipette, which would actually be fabricated by
also our integrated device as a whole can be used like some technical improvements. The micropipette sys-
the currently-used macroscopic three-electrode sys- tem makes transfer of the sample solution to a con-
tem. ventional analysis system (e.g., ow injection analysis
Although the curve (a) was taken in a tip of a 100 ml (FIA)) unnecessary. Sensing or electrochemical ana-
pipette, there was still enough space for the integrated lysis can be nished on the spot following suction of
chip and even 50 ml of the sample solution would have the sample solution. Otherwise, the integrated system
been enough. A possible application of this study could be incorporated in a small ow cell, which will
would be a micro analytical system incorporated in contribute to the miniaturization of the existing ana-
H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112 109

lytical equipment. Although the present integrated The performance of the integrated systems has been
device is a needle-type one, the design can be modied evaluated mainly by cyclic voltammograms, some
to a planar-type and the miniaturized electrode system of which have been described with the potential
can be incorporated in a microvial as reported else- measured against the pseudo-reference electrode.
where [3,10]. Furthermore, the present planar working Although it would be satisfactory as far as the shape
electrode pattern can be modied to a microelectrode and/or the peak height of the voltammograms are
conguration for more suitable analytical measure- concerned, reliable information could not be provided
ments. in terms of its potentials. The potential of the pseudo-
As the size of the electrode system is reduced, reference electrode can be determined with consider-
electrochemical reaction or equilibrium becomes able accuracy if the activity of the related primary
adversely inuenced by reaction products from other ion is given. However, such a case will be rare in
electrodes located in the vicinity [16]. This electro- real electroanalysis and the sample solution to be
chemical crosstalk is not specic to the microsystem analyzed might contain some redox compounds which
but has also been observed with conventional rotating adversely affect the pseudo-reference electrode.
ring-disk electrodes. Electrodes from an interdigitated Therefore, if reliability is required, a liquid-junction
electrode array studied by several groups use this reference electrode will be indispensable as in macro-
crosstalk advantageously [17,18]. Given this fact, it scopic electrochemical experiments.
is highly possible, in extremely miniaturized systems, An inevitable problem in using a pseudo-Ag/AgCl
that the reaction products from the counter electrode reference electrode is its poorly xed potential which
diffuse to the working and reference electrodes (or varies according to Cl ion activity, which manifests
from the working electrode to the reference electrode) itself as a shift of the cyclic voltammogram along
located in the vicinity, which either generate a residual abscissa. The tendency is well described in Fig. 4
current or might shift the reference electrode potential. which shows the cyclic voltammograms taken against
Therefore, each electrode must be separated as far as the pseudo-Ag/AgCl reference electrode on the chip
possible in a limited microspace. A solution will be to (i.e., the Ag/AgCl element directly exposed to the
store each electrode in isolated compartments and solution without the microcontainer). Five solutions
connect them with a liquid junction [13]. Fabrication were used each containing 5 mM K3Fe(CN)6, 0.1 M
of such a device is easy, which will be reported in the KNO3, and various concentration of KCl. The Ag/
future. AgCl electrode can be regarded as a Cl ion sensitive
electrode. Its potential varies by approximately 59 mV
3.3. Effect of using a pseudo-reference electrode at 258C as predicted by the Nernst equation, although
it becomes insensitive to Cl activity near the lower
As mentioned before, some microfabricated elec- detection limit [19]. The effect is clearly shown as the
trochemical cells used a thin-lm pseudo-reference shift of the voltammogram. The same experiment was
electrode which is in direct contact with the test conducted in solutions using 0.2 M Na2SO4 as a
solution [3,9,10]. Otherwise a microcapillary type supporting electrolyte instead of KNO3. However,
liquid-junction reference electrode was used [11]. the cyclic voltammograms for 10 mM and 0.1 M
These are a compromise due to the unavailability of KCl, settled virtually the same position. The discre-
a reliable microfabricated liquid-junction reference pancy between the two kinds of solution with different
electrode. Needless to say, the potential of the former supporting electrolyte indicates that the linear range of
pseudo-reference electrode varies depending upon the the calibration curve of the Ag/AgCl element regarded
activity of the primary ion which contributes most to as the Cl ion electrode depends on the ionic envir-
the equilibrium potential. Unlike macroscopic elec- onment.
trodes a thin-lm reference electrode often behaves In Fig. 4 the shift of the potential is approximately
unexpectedly and utmost care must be paid in using it 100 mV for KCl concentrations between 1 mM and
in real measurements. The latter microcapillary refer- 1 M. Signicance of this shift will depend upon the
ence electrode requires some skilled techniques and is sample solution to be analyzed and the required
not easy for unskilled persons to handle. accuracy. If more than two redox species are contained
110 H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112

3 =4
Fig. 4. Cyclic voltammograms of FeCN6 couple obtained using the pseudo-Ag/AgCl reference electrode and the working and counter
electrodes on the chip. The experiments were conducted in 100 ml solutions containing 5 mM K3Fe(CN)6, 0.1 M KNO3, and various
concentration of KCl: (a) no KCl, (b) 1 mM, (c) 10 mM, (d) 0.1 M, and (e) 1.0 M KCl. Scan rate: 50 mV/s.

in the sample solution or multipotential electroche- response curve. Despite small dimensions of the chip,
mical detection is desired [12], more reliable informa- a very distinct response curve was obtained. The 90%
tion about the potential will be required. It will not be response time was within 5 s. Fig. 5(b) shows the
denied that a liquid-junction reference electrode is calibration curve. A linear relationship was obtained
more suitable for that purpose. in the range of glucose concentration less than about
1.2 mM and the slope gradually decreased above this
3.4. Application to a one-chip biosensor concentration.
The data showed that the miniature liquid-junction
In order to test the applicability of the integrated reference electrode can be successfully used to con-
system to chemical sensors, a one-chip miniature struct miniature amperometric sensors. In real situa-
glucose sensor was fabricated and its performance tions, however, a pseudo-reference electrode might be
evaluated. Its detection was based upon the measure- enough depending on the accuracy and the samples to
ment of H2O2 produced by the enzymatic oxidation of be treated. In some commercialized disposable glu-
glucose by glucose oxidase. Fig. 5(a) shows its cose sensors, a pseudo-reference electrode without a

Fig. 5. Application to a miniature one-chip glucose sensor. (a) Response curve against step changes of glucose concentration. Two
hundred mM concentration of glucose was added to the buffer solution stepwise at times indicated by arrows. (b) Calibration curve. Applied
voltage: 0.7 V vs. Ag/AgCl.
H. Suzuki et al. / Analytica Chimica Acta 387 (1999) 103112 111

liquid-junction or even one which does not based on a does not mean that the hydrogen electrode itself is
high exchange current density reaction is used. This applicable to a micro pH sensor. It was just used as an
design is allowable when the limiting-current region in example of a potentiometric sensor using platinum as
the voltammogram is at enough within the accuracy an indicator electrode.) The titration curves for both
of the experiment and the shift of the reference cases showed a good coincidence in terms of their
electrode potential does not cause serious variation potential values in particular (Fig. 6). Notice that the
in the output current. Actually, repetition of the coincidence can be obtained only using a liquid-
experiment, using the pseudo-Ag/AgCl reference junction reference electrode, which exemplies the
electrode mentioned before, conrmed no noticeable applicability of the miniature liquid-junction refer-
difference. However, it should be borne in mind that ence electrode to potentiometric sensing.
reliable data cannot be obtained if the residual current
from interfering materials is affected due to the shift of
the reference electrode potential. 4. Conclusions

3.5. Application to potentiometric sensing A miniature liquid-junction reference electrode was


employed to integrate the three-electrode system on a
More stringent stability of the reference electrode chip. Evaluation of the system by cyclic voltammetry
potential is required in potentiometric sensing, and as an amperometric glucose sensor conrmed that
because the shift of the potential immediately man- the system can actually be used in real electrochemical
ifests itself as an error in the measurement. The sensing. Our present study showed a possibility that a
applicability of the miniature reference electrode to miniature disposable three-electrode system with a
one-chip potentiometric sensing was examined using a clean working electrode can be provided by mass
hydrogen electrode formed on the back of the liquid- production. The results given in Figs. 3 and 5 showed
junction Ag/AgCl reference electrode. (Note that it another new possibility. Namely, incorporation of an
electrode system or microsensors in a micropipette.
Measurements could be made immediately on the spot
after suction of the sample solution and no further
procedure such as an injection of the sample solution
into an analytical system is necessary. Also, it was
shown that the miniature liquid-junction reference
electrode was applicable to one-chip potentiometric
sensors. If the lifetime of the reference electrode is
elongated, the sensors will be used for practical use.

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