Biosensors and Bioelectronics 22 (2007) 1694–1699

A portable potentiostat for the bilirubin-specific

sensor prepared from molecular imprinting
Chun-Yueh Huang a , Mei-Jywan Syu b,∗ , Yong-Shuen Chang b ,
Chih-Hung Chang c , Tse-Chuan Chou b , Bin-Da Liu d
a Institute of Communication Engineering, National University of Tainan, Taiwan, ROC
b Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101, ROC
c Department of Electronic Engineering, Kun Shan University of Technology, Taiwan, ROC
d Department of Electrical Engineering, National Cheng Kung University, Taiwan, ROC

Received 25 May 2006; accepted 28 July 2006

Available online 8 September 2006

Abstract
A portable amperometric potentiostat was designed and implemented in this work. It was developed to acquisit the current signals produced from
bilirubin by an electrochemical sensor. Based on an SOC-based chip, this potentiostat has the merits of moderate accuracy, small size, low cost, and
high portability. The bilirubin electrode was prepared by synthesizing a thin layer of bilirubin imprinted poly(methacrylic acid-co-ethylene glycol
dimethacrylate) onto the Au layer. With the molecularly imprinted polymer (MIP) film, specific detection of bilirubin was successfully achieved.
The cyclic voltammogram of the electrode was measured from this assembled potentiostat. The performance from a commercial potentiostat was
considered rather stable and was used as a reference to examine and evaluate the performance of the assembled potentiostat. The detected current
signals by the bilirubin sensing were obtained. Linear calibration with a sensitivity of 1.344 ± 0.38 ␮A/mg dl was achieved. Our experimental
results showed that the proposed potentiostat’s performance could achieve sufficient performance. The evaluation was also made from the aspects
such as reset time and steady-response time. The self-assembled potentiostat thus demonstrated its ability in precise detection of bilirubin from an
electrode layered with the imprinted polymer film.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Potentiostat; Amperometric; SOC-based chip; Bilirubin; Imprinted poly(methacrylic acid-co-ethylene glycol methacrylate)

1. Introduction al., 1997). A review on the application of molecularly imprinted

Bilirubin is an end product from hemoglobin metabolism
(Wang et al., 2006). Total and direct bilirubins are usually mea-
sured to screen or to monitor liver and gall bladder disfunctions.
The bilirubin test can be used to judge a liver disease or a
blocked bile duct. Owing to the physiological importance of
bilirubin, it was chosen as the target molecule to be investigated
in this work. The electrochemical sensor for bilirubin was pre-
pared from molecular imprinting in this work. The molecular
imprinting technique is an important tool for the measurements
particularly for biosensors (Blanco-López et al., 2004) because
it has the advantages of high selectivity, sensitivity, and low cost.
In 1997, the MIP for sensor technology was discussed (Kriz et
∗ Corresponding author. Tel.: +886 6 2757575x62631; fax: +886 6 2344496.
E-mail address: syumj@mail.ncku.edu.tw (M.-J. Syu).
polymer to the biomimetic sensors was published (Haupt and
Mosbach, 2000).
Based on molecularly imprinted polymers (MIP), the elec-
trochemical sensors for a variety of applications can be made
(Sergeyeva et al., 1999; Piletsky and Turner, 2002). Theo-
phylline was used as the template to form an MIP film on
indium tin oxide (ITO) and silicon platforms. The cyclic voltam-
mogram of theophylline measured by the MIP–ITO electrode
was compared with those obtained from different electrode and
blank solution (Kindschy and Alocijia, 2005). Gate effect of
thin layer of MIP, imprinted poly(methacrylic acid-co-ethylene
glycol methacrylate), was investigated (Yoshimi et al., 2001).
The diffusive permeability of the thin MIP layer was sensitive
to the target template molecule. As a result, by the presence of
the template, the electrode performance was enhanced.
Electrochemical biosensors, which can show the detected
data by means of potential or current, are commonly applied in

0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2006.07.036
 C.-Y. Huang et al. / Biosensors and Bioelectronics 22 (2007) 1694–1699
DNA identification, protein classification, neural recording, glu-
cose determination, or pH variation detection. In 2000, a review
paper describing and discussing the interaction and functional-
ization of the electrode surface with the immobilized enzyme
(or protein) layer was proposed (Willner and Katz, 2000). By
immobilizing pyruvate oxidase on a screen-printed electrode,
the amperometric biosensor for the analysis of human sali-
vary phosphate was constructed (Kwan et al., 2005). In the
signal processing, each electrochemical sensor needs a poten-
tiostat to maintain the electrochemical stability for the sensor
and to convert the sensor’s output into an analog signal. So, the
potentiostat is an indispensable device for the electrochemical
sensors.
Many researchers devoted themselves to develop the single-
chip potentiostat so that the chip size and cost can be both
reduced. Fidler (Fidler et al., 1992) designed a potentiostat based
on a voltage-controlled current source for the amperometric
gas sensors. The circuit maintained electrochemical stability
in the sensor as well as buffered the current output. There
are many other research reports, for instance, Turner (Turner
et al., 1987) presented a basic CMOS integrated potentiostat,
Kakerow (Kakerow et al., 1995) used a monolithic potentiostat,
Bandyopadhyay (Bandyopadhyay et al., 2002) proposed a multi-
channel potentiostat, and Frey (Frey et al., 2003) reported an
integrated potentiostat for biosensor chips. Murari (Murari et al.,
2005) also developed an integrated potentiostat for neurotrans-
mitter sensing. The emphasis from these researchers was merely
focused on the integration of potentiostats and sensors, there is
no further effort on the remote transmission of potentiostats.
Nevertheless, in 2005, a miniature implantable in vivo teleme-
try monitoring system for biosensing was proposed (Beach et
al., 2005). In authors’ previous work (Huang et al., 2004a,b),
a potentiostat designed with portability would be described and
implemented in this work. Additionally, an SOC-based chip was
further used for the portable potentiostat to improve the system
performance as well as to reduce the cost. As a result, the mea-
surement of the proposed potentiostat can be carried out in daily
life.
Consequently, a potentiostat was designed and assembled
for the electrochemical detection of bilirubin by the electrode
coated with imprinted poly(methacrylic acid-co-ethylene glycol
Fig. 1. The circuit diagram of the portable potentiostat.
1695
dimethacrylate) thin film. The performance from the proposed
potentiostat was investigated from the cyclic voltammogram,
current signals, calibration sensitivity, reset time and steady-
response time.
2. Material and methods
2.1. A SOC-based portable potentiostat
Basically, a potentiostat is an electronic device that controls
the voltage difference between a working electrode and a ref-
erence electrode. The two electrodes are basic components of
an electrochemical sensor. By injecting currents into the sen-
sor through a counter electrode, the potentiostat controls the
electrochemical reaction. In other words, the potentiostat has
two tasks. Firstly, it measures the potential difference between a
working electrode and a reference electrode without polarizing
the reference electrode, and compares the potential difference
with a preset voltage. Secondly, it injects a current flowing from
a counter electrode to a working electrode in order to counter-
act the difference between the preset voltage and the existing
working electrode potential. After completing the two tasks, the
outputs of the potentiostat are the currents flowing from a counter
electrode to a working electrode. The controlled variable in the
potentiostat is the sensor’s preset voltage. Such an approach is
usually used to measure the amperometric sensor’s signals.
Fig. 1 shows the circuit diagram of a portable potentio-
stat. The proposed potentiostat is mainly constructed by a
C8051F020 mixed-signal microprocessor, a current-to-voltage
converter, three inverter amplifier, two low-pass filters, and an
RS232 serial data transfer interface. The C8051F020 chip is
an 8051 based CPU with 12-bit digital-to-analog converters,
12-bit analog-to-digital converters, and digital peripherals. A
personal computer (PC) is used to control the experimental
process and collect the experimental data of potentiostat. The
proposed potentiostat design specifications are described as fol-
lows: the programmable counter voltage ranges between −3 and
0 V under the resolution 1 mV, the measured reference voltage
ranges between 0 and 1 V under the resolution 1 mV, and the
measured current ranges between 0 and 100 ␮A under the reso-
lution 0.1 ␮A.
1696 C.-Y. Huang et al. / Biosensors and Bioelectronics 22 (2007) 1694–1699

In this portable potentiostat, the control component is real-

ized by a 12-bit ADC (ch2), a 12-bit ADC (ch7), and a 12-bit
DAC in the C8051F020 (SOC-chip). An 8051 microprocessor
is used for signal generation, data acquisition, and experimen-
tal management. The ch2-ADC measures the voltage difference
between a reference electrode and a working electrode and mon-
itors whether the potential of the reference electrode reached
the preset voltage or not. According to the output of ch2-ADC,
the microprocessor generates a corresponding control signal to
DAC. Then, DAC produces a bias voltage to control the injected
current into the counter electrode. This feedback control pro-
cedure continues until the voltage measured by the reference
electrode is close to the preset polarization voltage. The control
algorithm of the amperometric potentiostat is shown as below,
Algorithm (Amperometric potentiostat). Given: the set poten-
tial (or voltage difference) between the working electrode and
the reference electrode (Vset )
Fig. 2. The circuit diagrams of (a) the current-to-voltage converter; (b) the
Step 1:
inverter amplifier.
Initialize System Clock, Port I/O and PCA
Calibrate ADC for Measurement
Start ADC Sampling at 47.85 kHz dropping across the resistor represents a corresponding cur-
Establish the initial counter electrode voltage (VC) rent of the sensor. The circuit diagram of a current-to-voltage
Step 2: While (1) converter is shown in Fig. 2(a). The conversion ratio of the
{
current-to-voltage converter (Franco, 1988) can be expressed
Set up ADC2’s and ADC7’s AMUX, Throughput, Gain
Monitor the potential between the working electrode and the reference as
  
electrode: VWR R3 R2
Monitor the current of the counter electrode: ICW Vo = − 1 + 2 Rf Ii (1)
Verror = Vset − VWR RG R1
If (|Verror | < 1 mV)
Subsequently, an inverter amplifier as shown in Fig. 2(b)
VC (t + 1) = VC (t)
Else is required to optimize the output voltage level of current-to-
sign = sign (Verror ) voltage converter with respect to the maximum resolution of the
case of |Verror | { ADC. Then, the output voltage of the inverter amplifier, propor-
(1 mV < |Verror | ≤3 mV): tional to the electrochemical sensor current, is measured by the
VC (t + 1) = VC (t)–sign * 1 mV, break;
ch7-ADC. A low-pass filter is used to cancel the electronic noise
(3 mV <|Verror | ≤9 mV):
VC (t + 1) = VC (t)–sign * 2 mV, break; from the measured signal.
(9 mV < |Verror | ≤33 mV):
VC (t + 1) = VC (t)–sign * 3 mV, break; 2.2. Chemicals
(33 mV < |Verror |):
VC (t + 1) = VC (t)–sign * 9 mV, break;
}
The Al2 O3 plates were purchased from Laser Tek,
} Inc., Taiwan. They were cleaned with H2 O2 /ammonia,
H2 O2 /concentrated sulfuric acid, and H2 O2 /concentrated HCl
sequentially. The ammonia solution was purchased from Showa,
In the adjustment strategy of the counter voltage, a fuzzy-like
Inc., Japan. Sulfuric acid and HCl were from Union Chemi-
modification method is used to speed up the time for the adjust-
cal Works Ltd, Inc., Taiwan. Methacrylic acid (MAA) used as
ment. The adjustment algorithm can be expressed as follows:
the monomer was obtained from Acros, Inc. Ethylene glycol
dimethacrylate (EGDMA) was from Fluka, Inc. ␣-Bilirubin and
IF the error of VWR is tiny, THEN the adjustment is zero. benzophenone were obtained from Sigma, Inc. and Lancaster,
IF the error of VWR is small, THEN the adjustment is small. Inc., respectively. All chemicals were used as received.
IF the error of VWR is medium, THEN the adjustment is
medium. 2.3. Fabrication of MIP-modified electrode
IF the error of VWR is large, THEN the adjustment is large.
A square surface area of either 0.5 cm × 0.5 cm or
In the above feedback control procedure, a current-to-voltage 1.0 cm × 1.0 cm or 2.0 cm × 2.0 cm sputtered with platinum (Pt)
converter measures the sensor current. The sensor current was formed on the Al2 O3 plate. The Pt electrode was soaked in
between a counter electrode and a working electrode pass 10 mM allyl mercaptan solution for 24 h. After drying, 0.5 ␮l
through a current measurement resistor (10 k). The voltage of 0.15 M benzophenone was dropped onto the allyl mercaptan-
 C.-Y. Huang et al. / Biosensors and Bioelectronics 22 (2007) 1694–1699
Fig. 3. Schematic illustration of the fabrication of the MIP film onto the electrode.
coated Pt electrode. The treated electrode was then spinned at
1000 rpm and dried at 40 ◦ C. Then, 0.5 ␮l of pre-polymerization
mixture comprised of methacrylic acid (MAA), ethylene glycol
methacrylate (EGDMA) and bilirubin in solvent was spin-coated
onto the electrode at 1000 rpm. The bezophenone solution and
the pre-polymerization solution were both purged with nitrogen
flow before the polymerization. The UV light was used for the
irradiation polymerization of the above solution onto the elec-
trode. The template, bilirubin, was removed from the polymer
layer by placing the electrode in 10 mM NaOH with intensive
stirring and it was repeated 10 times. The fabrication of the MIP
onto the substrate is illustrated in Fig. 3.
2.4. Cyclic voltammetry and amperometric measurements
The cyclic voltammetry measurements were performed in
10 mg/ml bilirubin at a scan rate of 7.5 mV/s and the scanned
voltage was between −0.4 and +0.8 V. The electrodes for cyclic
voltammetry measurements all had the same surface areas of
0.5 cm × 0.5 cm or 2.0 cm × 2.0 cm. Amperometric measure-
ments were performed at the potential of 0.55 V with the injec-
tion of bilirubin solution at an interval of 5 min. The reference
electrode is the standard calomel electrode (SCE) and the counter
electrode is the Pt wire electrode.
3. Results and discussions
With the assembled potentiostat, the calibration of current
signal with respect to bilirubin concentration was carried out.
The gain of the IC was also tuned to achieve optimal cur-
rent signals. The preliminary test was accomplished before the
experiments. Then, the detection experiments were carried out
using this potentiostat. Basically, there were two modes designed
for this potentiostat, one was in AD (analog-to-digital) mode
and the other was in scan-mode. The potentiostat in AD-mode
was used to measure the current signal of the bilirubin con-
centration and the one in scan-mode was used for the cyclic
voltammetry measurement of the solution. The working elec-
trode used for the current detection was the MIP film coated Pt
electrode.
3.1. Cyclic voltammetry measurement from the scan-mode
potentiostat
The cyclic voltammetry profile of the MIP electrode obtained
from the bilirubin solution by the proposed scan-mode potentio-
stat is shown in Fig. 4. The profile shows a significant oxidation
current around the potential of 0.4 V. Hence, the profile already
1697
confirmed the feasibility of the electrochemical detection on
bilirubin by the MIP modified electrode. The scan rate was set to
be 7.5 mV/s. In fact, even without the MIP film, bilirubin can be
oxidized into other compound. However, the binding specificity
of bilirubin toward this MIP film can be further helpful to reduce
interference problem.
3.2. The current signals of bilirubin from the MIP modified
electrode
The measured results from the proposed potentiostat show
that the current responses increase with the bilirubin concen-
tration. The detected signals are given in Fig. 5. Very good
current signals corresponding to the injection of bilirubin solu-
tions were obtained from the potentiostat in A-D mode. The
current signal profiles were then used to produce the calibration
curves as shown in Fig. 6(a)–(c). Fig. 6(a) is the calibration curve
obtained from the MIP modified electrodes with surface areas
of 1.0 cm × 1.0 cm. The detection sensitivity from the proposed
potentiostat was 1.344 ± 0.383 ␮A/mg dl with a relative stan-
dard deviation (R.S.D.) of 28.48% while the one obtained from
the commercial potentiostat was 1.600 ± 0.194 ␮A/mg dl with a
R.S.D. of 11.68%. It should be noticed that the deviation might
be a combined result from either the potentiostat or the MIP
modified electrode. Evaluation of the detection performance on
the assembled potentiostat could not actually be distinguished
from the deviation caused by the irreproducibility of the MIP
electrode.
Fig. 4. Cyclic voltammograms measured from the proposed potentiostat in scan-
mode.
1698 C.-Y. Huang et al. / Biosensors and Bioelectronics 22 (2007) 1694–1699

Fig. 5. The detected current signal profiles of the bilirubin concentration from
the proposed potentiostat.

Further examination of the current response from single injec-

tion of bilirubin solution was made. The response signals by the
AD-mode potentiostat were evaluated from the aspects such as
the reset time and steady-response time. Gathered the data from
several batches (not shown here), the times required to reach a
steady state before the measurement were 1,983 ± 126 s (6.34%)
from the proposed potentiostat. Although the proposed potentio-
stat was slightly slower than the commercial potentiostat, they
could reach similar performance. For the steady-response time, it
was 41.33 ± 2.15 s (5.21%) from the proposed potentiostat. Sim-
ilarly, the deviation on the reproducibility of the MIP electrode
also contributed to the standard deviation of the comparison
made between the potentiostats.

3.3. Calibration of bilirubin concentration against current

signal

The calibration curves were further compared to those

obtained from a 2.0 cm × 2.0 cm MIP electrode, the coincidence
among the data was rather high. In Fig. 6(a), the detection was
performed by an MIP electrode of 1.0 cm × 1.0 cm. The detec-
tion area of 1.0 cm × 1.0 cm achieved a detection sensitivity of
1.344 ␮A/mg dl while 5.685 ␮A/mg dl (with a standard devia-
tion (S.D.) of 0.393 and a relative standard deviation (R.S.D.) of
6.91%) for a corresponding area of 2.0 cm × 2.0 cm as shown in
Fig. 6(b). The detection sensitivity was 4.23-fold with respect
to a detection area increment of four-fold. Therefore, within the
experimental range of detection, the sensitivity is almost lin- Fig. 6. Calibration curves of the bilirubin concentrations from the proposed
early proportional to the electrode surface area. In conclusion, potentiostat. (a) With a detection area of 1.0 cm × 1.0 cm; (b) with a detection
the detection precision performance was rather good. area of 2.0 cm × 2.0 cm; (c) with the bilirubin concentration in the range of
To further achieve the limit of detection (LOD), the current 0.1–1.0 mg/dl and a detection area of 1.0 cm × 1.0 cm.
signals were measured at a lower bilirubin concentration range
within 1.0 mg/dl. Fig. 6(c) shows the calibration result. The sur- confirmed. With this LOD, the detection of bilirubin concentra-
face area of the MIP modified electrode was 1.0 cm × 1.0 cm. A tion from the serum of a normal adult can be expected.
calibration parameter of 0.950 ± 0.041 ␮A/mg dl with an R.S.D. In addition, the noises of the detected current signals were
of 4.26% was obtained. Therefore, the LOD at this range was calculated. Therefore, the signal-to-noise ratio can be obtained
 C.-Y. Huang et al. / Biosensors and Bioelectronics 22 (2007) 1694–1699
Table 1
The signal-to-noise-ratio data calculated from the detection current signals
Bilirubin (mg/dl) N (␮A) S (␮A)
1 0.096 3.680
2 0.151 5.240
3 0.372 6.186
4 0.328 7.276
5 0.797 9.040
6 0.141 10.942
7 0.159 12.287
Average 0.278 7.807
as shown in Table 1. Obviously, judging from the S/N ratio in
the table, the detected current signal was significant enough for
measuring the bilirubin concentration.
4. Conclusions
In this paper, we propose a portable potentiostat and success-
fully verify its performance by using the MIP film fabricated Pt
sensor for the detection of bilirubin. According to the experimen-
tal results, the proposed potentiostat’s performance can achieve
almost the same accuracy as the commercial potentiostat’s.
Nice current response corresponding to the change of bilirubin
concentration obtained from the proposed potentiostat in AD-
mode was achieved. Accordingly, a linear calibration curve was
obtained. The performance of the proposed potentiostat for the
electrochemical sensing of bilirubin was made from the aspects
such as the reset time and steady-response time. The calibration
of bilirubin concentration from 0.1 to 1.0 mg/dl is also excellent,
which is then feasible for the detection of the bilirubin con-
centration for a normal adult. Although the performance from
the assembled potentiostat may not be as equally good as the
commercial one, the ability to achieve precisely amperometric
detection of bilirubin by the MIP modified electrode was con-
firmed. The cyclic voltammetry measurement from the proposed
scan-mode poentiostat was available as well. Besides, being
different from the commercial potentiostat, the proposed poten-
tiostat is even suitable for processing the electrochemical signals
of the amperometric sensor outdoors. Furthermore, the porta-
bility and convenience of the proposed potentiostat are quite
obvious. In conclusion, the proposed potentiostat has the merits
of moderate accuracy, small size, low cost, and high portability.
1699
Acknowledgments
S/N The grant supported from ROC Ministry of Education Ex-
38.140
91-E-FA09-5-4 on this work is appreciated. The discussion of
34.702 the work with the team members of this project is helpful and
16.638 highly appreciated.
22.194
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