8
Sensors and Actuators B 178 (2013) 465–472 Contents lists available at SciVerse ScienceDirect Sensors and Actuators B: Chemical journa l h o mepage: www.elsevier.com/locate/snb An array configuration to increase the performance of a biosensor Congo Tak-Shing Ching a,b,c,∗∗ , Kang-Ming Chang c,d , Yu-Lung Hung e,, Chin-Kang Chen e , Kok-Khun Yong e , Yue-Xiu Jiang f , Tai-Ping Sun a,b,∗∗ , Hsiu-Li Shieh a a Department of Electrical Engineering, National Chi Nan University, Nantou, Taiwan, ROC b Department of Applied Materials and Optoelectronic Engineering, National Chi Nan University, Nantou, Taiwan, ROC c Department of Photonics and Communication Engineering, Asia University, Taichung, Taiwan, ROC d Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, ROC e Department of Internal Medicine, Puli Christian Hospital, Nantou, Taiwan, ROC f Department of Nursing, Puli Christian Hospital, Nantou, Taiwan, ROC a r t i c l e i n f o Article history: Received 13 June 2012 Received in revised form 19 December 2012 Accepted 2 January 2013 Available online 11 January 2013 Keywords: Uric acid Biosensors Array Amperometric Signal-to-noise ratio a b s t r a c t This study aims to investigate whether an amperometric biosensor in an array configuration can increase its signal-to-noise ratio (SNR) so as to increase its performance, like sensitivity. Uric acid biosensor (UAB) was used as an example in this study. Both single UAB (UAB-1 × 1) and UAB array (UAB-1 × 2 and UAB- 1 × 3) were fabricated with uricase immobilized on commercial graphite working electrodes. The UAB showed an excellent (r 2 > 0.96) linear response range (0–0.8 mM) and moderate response time (20 s). Results showed that the sensitivity and SNR of UAB can be significantly increased by the use of an array configuration. It was found that the sensitivity and SNR of the UAB-1 × 3 is significantly higher (p < 0.05 in all cases) than that of the UAB-1 × 1 (sensitivity: 3.1-fold; SNR: 4.4-fold) and UAB-1 × 2 (sensitivity: 1.9-fold; SNR: 3.4-fold). Moreover, an excellent linear relationship (r 2 = 0.96) was found between the sen- sitivity and SNR. Finally, the UAB showed good repeatability (maximal relative standard deviation = 4.9%) and long-term stability (retained 92% of its initial value of current response after storing 60 days). In conclusion, the use of an array configuration can result in the increase of SNR, subsequently resulting in the increase of sensitivity. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Research and development in biosensors as analytical tools, e.g. clinical diagnosis [1–4], food safety monitoring [5–7], envi- ronmental surveillance [5,7–9] and so on, have gained increasing importance in the past decade because of their beneficial proper- ties such as simple, reliable, portable and low-cost. On the other hand, many researches have been focusing on the improvement of biosensor performance, for example sensitivity enhancement. The enhancements of amperometric biosensor sensitivity can be achieved by various ways and have been reported notably in recent literatures. For example, some researchers used electron-transfer mediators to improve the biosensor sen- sitivity [10–13]. In 2010, Zheng et al. [10] reported the use of Corresponding author at: Puli Christian Hospital, Teh-Shan Road, Puli, Nantou County 54546, Taiwan, ROC. ∗∗ Corresponding authors at: Department of Electrical Engineering, National Chi Nan University, No. 1, University Road, Puli, Nantou County 54561, Taiwan, ROC. Tel.: +886 492910960x4774; fax: +886 492917810. E-mail addresses: [email protected] (C.T.-S. Ching), [email protected] (Y.-L. Hung), [email protected] (T.-P. Sun). ferrocene-carbonyl-b-cyclodextrin inclusive complex, an electron- transfer mediator, and carbon nanotubes to build a glucose biosensor with low working potential (<0.2 V), fast current response time (<5 s), and excellent sensitivity to avoid interfer- ence from interferents (>5-times). A number of researchers used bimetallic materials [14–16]. Chen et al. [14] reported the use of bimetallic PtPd nanoparticle and multiwalled carbon nanotube to construct a hydrogen peroxide sensor with low working poten- tial (0 V), fast current response time (<5 s), and high sensitivity (414.8 A mM 1 cm 2 ). Several researchers used polymer or elec- tropolymerized films for the sensitivity enhancement [17–23]. For instance, Razola et al. [18] demonstrated the entrapment of horseradish peroxidase onto a platinum electrode coated with a polypyrrole film layer in order to fabricate a hydrogen peroxide sensor with highly sensitive to very low hydrogen peroxide con- centration (0.1 M). Some studies showed the use of nanoparticles for the sensitivity improvement [24–26]. In 2008, Loh et al. [25] demonstrated an electrochemical biosensor based on immobilized alkaline phosphatase and Fe 3 O 4 nanoparticles. They found that the use of the magnetic Fe 3 O 4 nanoparticles can two-fold increase the sensitivity of the biosensor toward 2,4-dichlorophenoxyacetic acid. Some researches used carbon nanotubes for sensitivity enhance- ment [27–30]. For example, Tasca et al. [27] reported the use of 0925-4005/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.01.001

An array configuration to increase the performance of a biosensor

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Sensors and Actuators B 178 (2013) 465– 472

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

n array configuration to increase the performance of a biosensor

ongo Tak-Shing Chinga,b,c,∗∗, Kang-Ming Changc,d, Yu-Lung Hunge,∗, Chin-Kang Chene,ok-Khun Yonge, Yue-Xiu Jiangf, Tai-Ping Suna,b,∗∗, Hsiu-Li Shieha

Department of Electrical Engineering, National Chi Nan University, Nantou, Taiwan, ROCDepartment of Applied Materials and Optoelectronic Engineering, National Chi Nan University, Nantou, Taiwan, ROCDepartment of Photonics and Communication Engineering, Asia University, Taichung, Taiwan, ROCGraduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, ROCDepartment of Internal Medicine, Puli Christian Hospital, Nantou, Taiwan, ROCDepartment of Nursing, Puli Christian Hospital, Nantou, Taiwan, ROC

r t i c l e i n f o

rticle history:eceived 13 June 2012eceived in revised form9 December 2012ccepted 2 January 2013vailable online 11 January 2013

eywords:

a b s t r a c t

This study aims to investigate whether an amperometric biosensor in an array configuration can increaseits signal-to-noise ratio (SNR) so as to increase its performance, like sensitivity. Uric acid biosensor (UAB)was used as an example in this study. Both single UAB (UAB-1 × 1) and UAB array (UAB-1 × 2 and UAB-1 × 3) were fabricated with uricase immobilized on commercial graphite working electrodes. The UABshowed an excellent (r2 > 0.96) linear response range (0–0.8 mM) and moderate response time (∼20 s).Results showed that the sensitivity and SNR of UAB can be significantly increased by the use of an arrayconfiguration. It was found that the sensitivity and SNR of the UAB-1 × 3 is significantly higher (p < 0.05

ric acidiosensorsrraymperometricignal-to-noise ratio

in all cases) than that of the UAB-1 × 1 (sensitivity: 3.1-fold; SNR: 4.4-fold) and UAB-1 × 2 (sensitivity:1.9-fold; SNR: 3.4-fold). Moreover, an excellent linear relationship (r2 = 0.96) was found between the sen-sitivity and SNR. Finally, the UAB showed good repeatability (maximal relative standard deviation = 4.9%)and long-term stability (retained 92% of its initial value of current response after storing 60 days). Inconclusion, the use of an array configuration can result in the increase of SNR, subsequently resulting inthe increase of sensitivity.

. Introduction

Research and development in biosensors as analytical tools,.g. clinical diagnosis [1–4], food safety monitoring [5–7], envi-onmental surveillance [5,7–9] and so on, have gained increasingmportance in the past decade because of their beneficial proper-ies such as simple, reliable, portable and low-cost. On the otherand, many researches have been focusing on the improvement ofiosensor performance, for example sensitivity enhancement.

The enhancements of amperometric biosensor sensitivityan be achieved by various ways and have been reported

otably in recent literatures. For example, some researcherssed electron-transfer mediators to improve the biosensor sen-itivity [10–13]. In 2010, Zheng et al. [10] reported the use of

∗ Corresponding author at: Puli Christian Hospital, Teh-Shan Road, Puli, Nantouounty 54546, Taiwan, ROC.∗∗ Corresponding authors at: Department of Electrical Engineering, National Chian University, No. 1, University Road, Puli, Nantou County 54561, Taiwan, ROC.el.: +886 492910960x4774; fax: +886 492917810.

E-mail addresses: [email protected] (C.T.-S. Ching), [email protected]. Hung), [email protected] (T.-P. Sun).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.01.001

© 2013 Elsevier B.V. All rights reserved.

ferrocene-carbonyl-b-cyclodextrin inclusive complex, an electron-transfer mediator, and carbon nanotubes to build a glucosebiosensor with low working potential (<0.2 V), fast currentresponse time (<5 s), and excellent sensitivity to avoid interfer-ence from interferents (>5-times). A number of researchers usedbimetallic materials [14–16]. Chen et al. [14] reported the use ofbimetallic PtPd nanoparticle and multiwalled carbon nanotube toconstruct a hydrogen peroxide sensor with low working poten-tial (0 V), fast current response time (<5 s), and high sensitivity(414.8 �A mM−1 cm−2). Several researchers used polymer or elec-tropolymerized films for the sensitivity enhancement [17–23].For instance, Razola et al. [18] demonstrated the entrapment ofhorseradish peroxidase onto a platinum electrode coated with apolypyrrole film layer in order to fabricate a hydrogen peroxidesensor with highly sensitive to very low hydrogen peroxide con-centration (0.1 �M). Some studies showed the use of nanoparticlesfor the sensitivity improvement [24–26]. In 2008, Loh et al. [25]demonstrated an electrochemical biosensor based on immobilizedalkaline phosphatase and Fe3O4 nanoparticles. They found that the

use of the magnetic Fe3O4 nanoparticles can two-fold increase thesensitivity of the biosensor toward 2,4-dichlorophenoxyacetic acid.Some researches used carbon nanotubes for sensitivity enhance-ment [27–30]. For example, Tasca et al. [27] reported the use of

466 C.T.-S. Ching et al. / Sensors and Actuators B 178 (2013) 465– 472

F , i.e. Ua

sfsdzWpgs

oelbtaia

2

2

aub((teeZ

2o

bswwoat

tc

ig. 1. The single UAB and UAB array. (a) UAB-1 × 1, i.e. single UAB. (b) UAB-1 × 2rray fabricated by connecting 3 single UAB in parallel.

ingle-walled carbon nanotubes with medium length can five-old increase the sensitivity of an amperometric biosensor modelystem toward the oxidation of 1,4-dihydronicotinamide adenineinucleotide. Last but not least, a number of researchers used bien-ymes [29,30] and special electrode materials [31,32]. For instance,

ang et al. [29] demonstrated the use of bienzyme, horseradisheroxidase and glucose oxidase, to fabricate an amperometriclucose biosensor with low working potential (−0.1 V) and highensitivity (113 mA M−1cm−2).

In spite of the big amount of literatures on the improvementf biosensor sensitivity, no paper has been found on studying thenhancement of biosensor signal-to-noise ratio (SNR). In particu-ar, no research was found on investigating the relationship amongiosensor configuration, SNR and sensitivity. Therefore, the aim ofhis study is to investigate whether an amperometric biosensor inn array configuration can increase its SNR in an attempt to increasets sensitivity. An amperometric uric acid biosensor (UAB) was useds an example in this study.

. Materials and methods

.1. Chemicals and reagents

In this study, all chemicals and reagents were commerciallyvailable and they were used with no further purification. Uricase,ric acid, bovine serum albumin (BSA), glutaraldehyde, phosphateuffered saline (PBS) were purchased from Sigma Chemical Co.St. Louis, MO). De-ionized water purified by a Millipore SystemMilli-Q UFplus; Bedford, MA) was used to prepare all solu-ions. Three-electrodes screen-print sensor, consisted of workinglectrode (graphite), counter electrode (graphite) and referencelectrode (silver–silver chloride, Ag/AgCl), was purchased fromensor R&D Co., Ltd. (Taichung, TW).

.2. Immobilization of uricase onto the sensor for the fabricationf UAB

The fabrication steps of a single UAB (UAB-1 × 1) is describedelow. By the use of the commercial three-electrodes screen-printensor, glutaraldehyde (2.5%, 4 �L) was initially pipetted onto theorking electrode of the sensor and then kept at 4 ◦C for 1 h. After-ard, the enzyme (uricase, 0.5 U/mL, 4 �L) was pipetted again

nto the working electrode of the sensor, followed immediately bydding BSA (0.1 mM, 4 �L) for the purpose of cross-linking. Lastly,

he sensor was kept in the dark at 4 ◦C before use.

After the fabrication of the single UAB (UAB-1 × 1), the fabrica-ion of UAB array in the configuration of 1 × 2 (UAB-1 × 2) is simplyonstructed by connecting 2 pieces of UAB-1 × 1 in parallel, while

AB array fabricated by connecting 2 single UAB in parallel. (c) UAB-1 × 3, i.e. UAB

the fabrication of UAB array in the configuration of 1 × 3 (UAB-1 × 3)is fabricated by connecting 3 pieces of UAB-1 × 1 in parallel (Fig. 1).

2.3. Equipments

All measurements were conducted by the use of an electrochem-ical analyzer (CHI 627C, CH Instrument, USA) and the measurementsetup was shown in Fig. 2. In brief, the UAB was connected to theelectrochemical analyzer, which was connected to a computer viaa RS232 cable. A CHI electrochemical analyzer software (version10.04) was installed in the computer and the software was used fordata collection during measurements.

2.4. Hydrodynamic and cyclic voltammetry measurements of theUAB

The measurement setup was shown in Fig. 2. Hydrodynamicvoltammetry measurements were conducted, using the electro-chemical analyzer, with the UAB immersing in a solution of 0.8 mMuric acid solution subjected to steady stirring at 25 ◦C. Incrementalpotentials (0–1 V, 0.1 V increments) versus Ag/AgCl electrode of thesensor were applied to the working electrode of the UAB and thecurrent responses of the UAB to uric acid were measured.

For cyclic voltammetry measurements, the UAB was also con-nected to the electrochemical analyzer. A solution (1 mL) of 0.8 mMuric acid was pipetted onto the surface of the UAB. The cyclicvoltammetry was recorded between −2.1 V and +1.4 V using a scanrate of 100 mV/s.

2.5. Measurements of the UAB current response to uric acid

The measurement setup was shown in Fig. 2. Electrochemicalanalyzer was used and measurements were carried out at 25 ◦C.An applied potential of +700 mV versus Ag/AgCl pad was applied tothe working electrodes of the UAB. PBS was firstly pipetted onto theUAB. After waiting for 150 s to achieve a steady background current,uric acid solution in different concentrations was then added ontothe UAB and the current response of the UAB to uric acid was thenmeasured.

2.6. Hysteresis test of the UAB

The measurement setup was shown in Fig. 2. Measurements

were carried out at room temperature (25 ◦C). The UAB was con-nected to the electrochemical analyzer, with an applied voltage of+700 mV versus Ag/AgCl pad being applied to the working elec-trodes of the UAB. Uric acid solutions was applied to the UAB in

C.T.-S. Ching et al. / Sensors and Actuators B 178 (2013) 465– 472 467

F electrv talled

(

ta

2

21otnwAasco

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3

Ut+a

the linear range were found to be at +700 mV potential. A moderateseparation between the oxidation peak and reduction peak repre-sented a moderate electron transfer being happening. Therefore,

0

1

2

3

4

5

6

Cu

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UBA-1x1

UBA-1x2

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ig. 2. The experimental setup for all UAB measurements. UAB was connected to ania a RS232 cable. A CHI electrochemical analyzer software version 10.04 has been insa) The photo and (b) the schematic diagram of the experimental setup.

he loops of 0.4 mM → 0.8 mM → 0.4 mM → 0.1 mM → 0.4 mM over period of 300 s.

.7. Life-span measurements of the UAB

The long-term stability of the UAB was evaluated at Day 0 (i.e.4 h after enzyme immobilization), Day 30 and Day 60. Only UAB-

× 1 was used for evaluation in order to represent the life-spanf the UAB. The UAB-1 × 1 was placed inside a dark sealed plas-ic box containing ordinary atmospheric air only (in other words,o preservation with special air like pure nitrogen) and the boxas kept at a 4 ◦C fridge over 0-day, 30-day and 60-day periods.t the specific days, the UAB-1 × 1 was taken out from the fridgend placed at room temperature (about 25 ◦C) for 5 min before mea-urement. The measurement setup and procedures of the UAB-1 × 1urrent response to uric acid was identical to Section 2.5, exceptnly 0.8 mM uric acid solution was used for evaluation.

. Results and discussion

.1. Hydrodynamic and cyclic voltammogram of the UAB

Fig. 3 demonstrates the hydrodynamic voltammogram of the

AB at a regularly-stirred 0.8 mM uric acid solution. Reduc-

ive currents generated by the UAB were found to begin at600 mV potential, gradually increase with the applied potential,nd become plateau starting from +800 mV potential afterward.

ochemical analyzer and the electrochemical analyzer was connected to a computerin the computer and the software was used for data collection during measurements.

Therefore, +700 mV potential (the mean of +600 mV and +800 mV)versus the Ag/AgCl reference electrode was used for all measure-ments. On the other hand, an array configuration was found tobe able to increase the reductive current and this means that thesensitivity of the UAB can be increased by using array configuration.

Fig. 4 demonstrates the cyclic voltammogram of the UAB at a0.8 mM uric acid solution between −2.1 V and +1.4 V, using a scanrate of 100 mV/s. It was found that the UAB array (UAB-1 × 2 andUAB-1 × 3) had a higher response current as compared to the sin-gle UAB (UAB-1 × 1). Again, the optimum reductive currents within

0 100 200 300 400 500 600 700 800 900 1000 1100

Applied Potential vs. Ag/AgCl Electrode (mV)

Fig. 3. Hydrodynamic voltammogram of the single UAB and UAB array in constantly-stirred solution of 0.8 mM uric acid.

468 C.T.-S. Ching et al. / Sensors and Actuators B 178 (2013) 465– 472

Fig. 4. Cyclic voltammogram of the single UAB and UAB array. Uric acid (0.8 mM)was used for evaluation with the scan rate of 100 mV/s between −2.1 V and +1.4 V.

-1.60

-1.40

-1.20

-1.00

-0.80

-0.60

-0.40

-0.20

0.00130 230 330 430 530 630

Time (s)

Cu

rre

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esp

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(u

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Addition of 0.8 mM

uric acid solution

Ft

tr

3

amartsw4r(

c

Fbs

0

1

2

3

4

5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Uric Acid Concentration (mM)

Curr

ent R

esponse (

uA

)

UAB-1x1

UAB-1x2

UAB--1x3

(y = 1.55x; r2=0.987)

a

(y = 2.47x; r2=0.964) b

(y = 4.73x; r2=0.984)

Fig. 7. Calibration curves of the single UAB and UAB array to uric acid concentration(0–0.8 mM). The line is that of best fit found by linear regression (the correlation coef-ficient, r2, of UAB-1 × 1, UAB-1 × 2 and UAB-1 × 3 is 0.99, 0.96 and 0.98, respectively).The sensitivity of the UAB (1.55, 2.47 and 4.73 �A/mM for UAB-1 × 1, UAB-1 × 2 andUAB-1 × 3, respectively) is the slope of the linear regression line. Remarks: (a) sta-tistically significant differences (independent-samples t test: p < 0.05) was found onits sensitivity as compared to UAB-1 × 2 and UAB-1 × 3. (b) Statistically significant

ig. 5. Current–time curve obtained by the addition of 0.8 mM uric acid solution tohe UAB-1 × 1. The response time of the biosensor is about 20 s.

his can explain the phenomenon that the UAB have a moderateesponse time, and this agreed with the finding as shown in Fig. 5.

.2. Response behavior of the UAB

Fig. 5 shows a current–time curve for the addition of 0.8 mM uriccid solution, where the current response of the UAB-1 × 1 beingeasured at the applied potential of +700 mV. After adding the uric

cid solution onto the UAB-1 × 1, the reductive current raised andeached 95% of the steady state current at 20 s approximately andhis was in agreement with the cyclic voltammogram finding ashown in Fig. 4. UAB-1 × 1 has a faster response time as comparedith that reported by Arora et al. (about 60 s) [33], Luo et al. (about

5 s) [34] and Zhang et al. (about 50 s) [35], but it has a sloweresponse as compared with that reported by Chauhan and Pundir

about 7 s) [36].

Fig. 6 shows a calibration curve of the UAB-1 × 1 to uric acidoncentration. The sensitivity of the UAB-1 × 1 was determined

y = 1..5456x

R2

= 0..9867

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Uric Acid Concentration (mM)

Cu

rre

nt R

esp

on

se

(u

A)

ig. 6. Calibration curve of the UAB-1 × 1 to uric acid concentration. The lines areest fit found by linear regression. Sensitivity of the biosensor is indicated by thelope of the linear regression line and it is 1.55 �A/mM.

differences (independent-samples t test: p < 0.05) were found on its sensitivity ascompared to UAB-1 × 3.

by the slope of linear regression of this current response versusuric acid concentration. It was found that the current response isdirectly proportional to the uric acid concentration over a widerange of concentration (0–0.8 mM) sufficiently covering the nor-mal physiological and pathological blood uric acid levels, wherecorrelation coefficient (r2) is 0.99 and sensitivity is 1.55 �A/mM.The range of concentration measurement of the UAB-1 × 1 is com-parable to the findings reported by other researchers [34,36] andis slightly narrower as compared with that reported by Zhang et al.(0.2–1 mM) [35]. And, the sensitivity of the UAB-1 × 1 is lower thanLuo’s findings (16.6 �A/mM) [34] but is higher than Zhang’s find-ings (5.5 nA/mM) [35].

Fig. 7 shows the calibration curve of the UAB-1 × 1, UAB-1 × 2 and UAB-1 × 3 to uric acid concentration. Again, the currentresponses of the three UAB (all have r2 > 0.96) were found to bedirectly proportional to the uric acid concentration over the con-centration range of 0–0.8 mM. It was observed that the use of arrayconfiguration can result in the increase of the sensitivity of the UABin general. Specifically, the sensitivity of the UAB-1 × 3 was found tobe significantly higher (independent-samples t test: p < 0.05 in bothcases) than that of the UAB-1 × 1 (3.1-fold) and UAB-1 × 2 (1.9-fold),while UAB-1 × 2 also has a significant higher (independent-samplest test: p < 0.05) sensitivity as compare to UAB-1 × 1 (1.6-fold). Apossible explanation for these phenomena is that the improvedsensitivity is due to the higher signal-to-noise ratio (SNR) achievedwith the larger reductive current signals observed at the UAB in ahigher array configuration and this will be explained later in thissection.

Fig. 8 shows the plot of UAB sensitivity versus the type ofUAB array configuration. The sensitivity of the UAB exponentiallyincreases with the UAB in higher array configuration (ExponentialRegression Equation: UAB sensitivity = 0.86e0.56 × array configuration;r2 = 0.99). Based on this finding, it can be understood that the UABsensitivity can be significantly increased (independent-samples ttest: p < 0.05 in all cases; UAB-1 × 1 < UAB-1 × 2 (1.9-fold) and UAB-1 × 3 (3.1-fold); UAB-1 × 2 < UAB-1 × 3 (1.6-fold)) by using UABin array configuration. However, this method cannot unlimitedlyincrease the UAB sensitivity as it has an exponential increase phe-nomenon. Therefore, it can be expected that the UAB sensitivity canbe increased and reach its maximum steady state at a particulararray configuration.

Fig. 9 shows the plot of UAB SNR versus the type of UAB array

configuration. The SNR of the UAB exponentially increases with theUAB in higher array configuration (Exponential Regression Equa-tion: UAB SNR = 5.51e0.74 × array configuration; r2 = 0.88). It was found

C.T.-S. Ching et al. / Sensors and Actu

y = 0..8578

8

e0.559x

R2

= 0..9915

0

1

2

3

4

5

6

UAB-1x1 UAB-1x2 UAB-1x3

Sensitiv

ity (

uA

/mM

)

a

b

Fig. 8. The UAB sensitivity-type of array curve. The line is that of best fit found byregression (r2 = 0.99). The sensitivity of the UAB exponentially increases as the typeof array changing from single biosensor (i.e. UAB-1 × 1) to array biosensor (i.e. UAB-1 × 2 or UAB-1 × 3). Remarks: (a) statistically significant differences (independent-samples t test: p < 0.05) was found as compared to UAB-1 × 2 and UAB-1 × 3. (b)Statistically significant differences (independent-samples t test: p < 0.05) was foundas compared to UAB-1 × 3.

y = 5.5077e0.7448x

R2

= 0.8809

0

10

20

30

40

50

60

70

UAB-1x1 UAB-1x2 UAB-1x3

Sig

na

l-to

-No

ise

Ra

tio

a

Fig. 9. The UAB signal-to-noise (SNR)-type of array curve. Data is obtained by using0.8 mM uric acid solution. The line is that of best fit found by regression (r2 = 0.88).The SNR of the UAB exponentially increases as the type of array changing fromsingle biosensor (i.e. UAB- × 1) to array biosensor (i.e. UAB-1 × 2 or UAB-1 × 3).Remarks: (a) statistically significant differences was found as compared to UAB-1t

tgtii1do(tchh

Ft

achieved.

× 2 (independent-samples t test: p < 0.05) and UAB-1 × 1 (independent-samples test: p < 0.001).

hat the higher the array configuration, the greater the UAB SNR ineneral. To be more specifically, the SNR of the UAB-1 × 3 was foundo be significantly greater than that of the UAB-1 × 1 (4.4-fold;ndependent-samples t test: p < 0.001) and UAB-1 × 2 (3.4-fold;ndependent-samples t test: p < 0.05). Although the SNR of the UAB-

× 2 is greater (1.3-fold) than that of UAB-1 × 1, no significantifference (independent-samples t test: p = 0.228) is found. On thether hand, as shown in Fig. 10, an excellent linear relationshipr2 = 0.96) was found between the sensitivity and SNR. It was foundhat the greater the SNR, the larger the sensitivity. Therefore, this

an explain the phenomenon found in Fig. 7 that UAB-1 × 3 has aigher sensitivity as compared to UAB-1 × 1 as a result of UAB-1 × 3aving a higher SNR as compared to UAB-1 × 1.

y = 0.0621x + 1.0186

R = 0.9585

0

1

2

3

4

5

6

0 10 20 30 40 50 60 70

Signal-to-Noise Ratio

Se

nsitiv

ity (

uA

/mM

)

ig. 10. The sensitivity–SNR curve. Data is obtained by using 0.8 mM uric acid solu-ion. The line is that of best fit found by linear regression.

ators B 178 (2013) 465– 472 469

The definition of SNR is defined as the ratio of the power of thewanted signal (i.e. sensed signal) to the power of the unwantedsignal (i.e. noise). In a biosensor, SNR increases as the sensed sig-nal is increased, assuming that the unwanted noise signal remainsrather constant. Since UAB-1 × 3 had a higher current response (i.e.sensed signal) than UAB-1 × 1 and UAB-1 × 2 at various concentra-tion of uric acid (Fig. 7), therefore UAB-1 × 3 had a higher SNR thanUAB-1 × 1 and UAB-1 × 2 with the explanation mentioned above.

As shown in Table 1, three different sensors (Sensor-X, Sensor-Y and Sensor-Z) have been fabricated by screen-printing methodin order to make comparisons with the UAB-1 × 3 in this study.The screen-printing procedures and methods were similar to previ-ously described [37]. In brief, each sensor has 3 electrodes: graphiteworking, graphite counter and silver–silver chloride reference elec-trodes. In this study, the ratio of counter electrode area (AC) toworking electrode area (AW), AC/AW, was equal to 1 for all UAB-1 × 3, Sensor-X, Sensor-Y and Sensor-Z. The optimum ratio of AC/AWhas been reported in literatures and it is between 1 and 12 [38–42].Also, for an inactivated graphite counter electrode, its AC/AW ratioshould be at least of 0.125 [43]. Therefore, UAB-1 × 3, Sensor-X,Sensor-Y and Sensor-Z has the optimum ratio of AC/AW. On theother hand, the enzyme loading (EL) per working electrode area,EL/AW, of UAB-1 × 3 (0.029 unit/cm2) was the same as Sensor-X(0.029 unit/cm2), and was smaller than that of Sensor-Y (0.086unit/cm2) but was higher than that of Sensor-Z (0.010 unit/cm2).Interestingly, it was found that the current response, sensitivity andSNR of UAB-1 × 3 was significantly (independent-samples t test:p < 0.05) higher than that of Sensor-X although they both had thesame EL/AW (0.029 unit/cm2). This might be due to the functionof the array configuration of UAB-1 × 3 to enhance its SNR so as toenhance its sensitivity and current response. Surprisingly, althoughthe EL/AW of UAB-1 × 3 (0.029 unit/cm2) was lesser than Sensor-Y(0.086 unit/cm2), the current response, sensitivity and SNR of UAB-1 × 3 was found to be significantly (independent-samples t test:p < 0.05) higher than that of Sensor-Y. Again, this might be due tothe function of the array configuration of UAB-1 × 3 to enhance itssensing performance.

In fact, all the UAB-1 × 1 (AW = 0.07 cm2, AC = 0.07 cm2, EL = 0.002unit), UAB-1 × 2 (AW = 0.14 cm2, AC = 0.14 cm2, EL = 0.004 unit) andUAB-1 × 3 (AW = 0.21 cm2, AC = 0.21 cm2, EL = 0.006 unit) have thesame ratio of EL/AW (0.029 unit/cm2) and AC/AW (1). However, itwas found that the current response (Fig. 7), sensitivity (Fig. 8) andSNR (Fig. 9) of UAB-1 × 3 was significantly (independent-samplest test: p < 0.05) higher than that of UAB-1 × 1 and UAB-1 × 2.Once more, it can be understood that array configuration helps toenhance sensing performance. And, the higher the array configu-ration used the more enhancement of sensing performance can be

Fig. 11 shows the hysteresis test of the UAB and no hysteresisphenomenon was observed. This represented that UAB have goodimmobilization of enzyme and fast enough electron transfer rate

0

1

2

3

4

5

0 50 100 150 200 250 300 350

Time (s)

Re

sp

on

se

Cu

ure

nt (u

A)

UAB-1x1

UAB-1x2

UAB-1x3

Fig. 11. Hysteresis evaluations of the UAB. Current response of UAB during the uricacid solution loop: 0.4 mM → 0.8 mM → 0.4 mM → 0.1 mM → 0.4 mM.

470 C.T.-S. Ching et al. / Sensors and Actuators B 178 (2013) 465– 472

Table 1The enzyme loading per working electrode area (EL/AW), ratio of counter electrode area to working electrode area (AC/AW), current response, sensitivity and signal-to-noiseratio (SNR) of various sensors (UAB-1 × 3, Sensor-X, Sensor-Y and Sensor-Z). The Sensor-X, Sensor-Y and Sensor-Z were fabricated by the authors.

This study (UAB-1 × 3) Comparison studiesconducted by the authorsSensor-X Sensor-Y Sensor-Z

Sensor patterns andfeatures, and enzymeloading

C = counter electrodeW = working electrodeR = reference electrode

Before dot lines(representing conductivewires) connection, i.e.individual sensorAW = 0.07 cm2

AC = 0.07 cm2

EL = 0.002 unit

AW = 0.21 cm2

AC = 0.21 cm2

EL = 0.006 unit

AW = 0.07 cm2

AC = 0.07 cm2

EL = 0.006 unit

AW = 0.21 cm2

AC = 0.21 cmEL = 0.002 unit

After dot lines connection,i.e. sensor connected inparallel and used in thisstudyAW = 0.21 cm2

AC = 0.21 cm2

EL = 0.006 unit

EL/AW (unit/cm2) 0.029 0.029 0.086 0.010AC/AW 1 1 1 1Current response (�A) 3.84 ± 0.17 3.01 ± 0.12* 2.11 ± 0.25* 1.83 ± 1.05*

Sensitivity (�A/mM) 4.73 ± 0.09 3.9 5 ± 0.17* 2.87 ± 0.53* 2.51 ± 0.24*

SNR 60.31 ± 2.17 32.18 ± 3 * * *

Remark: AW: area of working electrode; AC: area of counter electrode; EL: enzyme loading* A significant difference was found as compared with this study (UAB-1 × 3), with p < 0

Table 2The sensitivity and repeatability of the UAB on measuring uric acid.

UAB Sensitivity (�A/mM) Relative standard deviation (%)

UAB-1 × 1 1.55 ± 0.07 4.7

ba

osTtd

3

eT

TEo

storage should be higher than that after 60-day storage. One possi-ble explanation for this abnormal observation might be that therewere large variations (owing to the small sample size, n = 3) on the

140nse

UAB-1 × 2 2.47 ± 0.12 4.9UAB-1 × 3 4.73 ± 0.22 4.6

etween the oxidation and reduction of uric acid (Fig. 4), leading to moderate response time.

The repeatability of the UAB-1 × 1, UAB-1 × 2 and UAB-1 × 3n measuring uric acid concentrations was evaluated from thetandard deviation of the sensitivity of the UAB. As shown inable 2, the three different UAB demonstrate a reasonably attrac-ive analytical feature of repeatability (maximal relative standardeviation = 4.9%).

.3. Life-span of the UAB

The long-term stability of the UAB-1 × 1, kept at 4 ◦C, wasvaluated over 0-day, 30-day and 60-day periods (Fig. 12 andable 3). It was found that the current response of the UAB-1 × 1

able 3valuations of the sensing performance of UAB after 60-day storage at 4 ◦C. Data isbtained from the UAB-1 × 1 tested at 0.8 mM uric acid solution.

UAB-1x1 Day 0 Day 30 Day 60

Current response (�A) 1.21 ± 0.05 1.09 ± 0.15 1.11 ± 0.29Percentage current response

retaining (%)100 ± 0 90 ± 12 92 ± 24

.54 39.72 ± 2.54 27 ± 3.72

on working electrode..05.

to 0.8 mM uric acid solution retained 92% approximately of its ini-tial value after 60-day storage. The percentage current responseretaining at a specific day (PCRRDayX) is calculated by 1 plus thesubtraction of the current response value at Day 0 (CRVDay0) fromthe current response value at the specific day (CRVDayX), andsuch calculated value is divided by the CRVDay0 and then follow-ing by the multiplication of 100% (i.e. PCRRDayX = 1 + ((CRVDayX −CRVDay0)/CRVDay0) × 100). It was observed that the life-span of theUAB-1 × 1 after 30-day storage (∼90% retained of its initial value)was slightly lower (but no statistical significant difference beingfound) than that after 60-day storage (∼92% retained of its initialvalue). Theoretically, the life-span of the UAB-1 × 1 after 30-day

100 9290

0

20

40

60

80

100

120

0 30 60

Days

Pe

rce

nta

ge

Cu

rre

nt R

esp

o

Re

tain

ing

(%

)

Fig. 12. The current response of the UAB was found to be about 92% retained ofits initial value after a 2-month storage at 4 ◦C, showing a good long-term stability.Data is obtained from the UAB-1 × 1 tested at 0.8 mM uric acid solution.

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C.T.-S. Ching et al. / Sensors an

ife-span measurements of the UAB-1 × 1 at a specific day and thisould be seen from the standard deviation being so high (Fig. 12).n the other hand, the life-span of our UAB-1 × 1 is longer than

hat reported by Hoshi et al. [44] and their uric acid sensor can onlyetain 60% activity after 30 days storage. A possible explanation forhe good life-span of the UAB-1 × 1 is that the enzyme is immobi-ized well onto the sensor and maintains its enzymatic activity. Inhis study, bovine serum albumin and glutaraldehyde was used torosslink with the enzyme onto the sensor. This crosslink structurerobably provides a superior micro-environment for the enzymeo stay alive and keep up its enzymatic activity.

. Conclusion

Biosensor in an array configuration can increase its SNR, whichubsequently results in the increase of its sensitivity. Also, an excel-ent positive linear relationship is found between the sensitivitynd SNR. This method is reliable, simple and inexpensive to fab-icate a highly-sensitive biosensor. In this study, UAB array wassed as an example and it has a linear working range (0–0.8 mM),

moderate response time (∼20 s), and good repeatability.

onflict of interest statement

None declared.

cknowledgements

This work was supported by grants (NSC 100-2221-E-260-007-nd NSC 98-2221-E-260-024-MY3) from National Science Council,aiwan, Republic of China. This work was also supported by grantrom the National Chi Nan University and Puli Christian Hospital,aiwan, Republic of China.

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iographies

ongo Tak-Shing Ching received the PhD degree in bioengineering from the Uni-ersity of Strathclyde, Glasgow, UK, in 2005. He is currently working as an associaterofessor in the Department of Electrical Engineering, National Chi Nan University,antou, Taiwan. His main research interests include biosensors, biomedical instru-entation design, noninvasive medical diagnostics/monitoring/therapeutics, point

f care diagnostics and bioimpedance.

ang-Ming Chang is currently working as an associate professor in the Departmentf Photonics and Communication Engineering, Asia University, Taichung, Taiwan.is main research interests are biosensors and biomedical signal processing.

ators B 178 (2013) 465– 472

Yu-Lung Hung is currently working as a medical doctor in the Department of Inter-nal Medicine, Puli Christian Hospital, Nantou, Taiwan. His main research interest isbiosensors.

Chin-Kang Chen is currently working as a medical doctor in the Department of Inter-nal Medicine, Puli Christian Hospital, Nantou, Taiwan. His main research interest isbiosensors.

Kok-Khun Yong is currently working as a medical doctor in the Department of Inter-nal Medicine, Puli Christian Hospital, Nantou, Taiwan. His main research interest isbiosensors.

Yue-Xiu Jiang is currently working as a nurse in the Department of Nurs-ing, Puli Christian Hospital, Nantou, Taiwan. Her main research interest isbiosensors.

Tai-Ping Sun was born in Taiwan on 20 March 1950. He received the BS degreein electrical engineering from Chung Cheng Institute of Technology, Taiwan, in1974, the MS degree in material science engineering from National Tsing Hua Uni-versity, Taiwan, in 1977, and PhD degree in electrical engineering from NationalTaiwan University, Taiwan, in 1990. From 1977 to 1997, he worked at Instituteof Science and Technology, Republic of China, concerning the development ofinfrared device, circuit and system. He joined the Department of ManagementInformation System, Chung-Yu College of Business Administration since 1997 asan associated professor. Since 1999 he has joined the Department of ElectricalEngineering, National Chi Nan University as a professor. From 2001 to 2005, hewas secretary-general at the National Chi Nan University. Since 2006, he has beenchairman of the Department of Electrical Engineering at National Chi Nan Univer-sity. And his research interests are infrared detector and system, analog/digital

applications.

Hsiu-Li Shieh is currently pursuing the PhD degree in the Department of Electri-cal Engineering, National Chi Nan University, Nantou, Taiwan. Her main researchinterest is biosensors.