Optics and Photonics Journal, 2013, 3, 305-307
doi:10.4236/opj.2013.32B071 Published Online June 2013 (http://www.scirp.org/journal/opj)
Effect of Electrode Surface Modification by Sulfide on
QCM Based Protein Biosensor
Yu-Cheng Lin1, Yi-Chi Chen1, Liang-Yu Chen2
1Department and Graduate Institute of Electronic Eng, Ming Chung University, Taoyuan, Chinese Taipei
2Department and Graduate Institute of Biological Technol, Ming Chung University, Taoyuan, Chinese Taipei
Email: yclin@mail.mcu.edu.tw, ioknath@mail.mcu.edu.tw
Received 2013
The rapid development of surface sensitive biosensor technologies requires optimum control of surface modification to
provide reliable and reproducible results. With the aim to assemble a quartz crystal microbalance (QCM)-based protein
biosensor, we focus our attention on sulfide receptor and its integration with the surface of the electrode. Here, we pre-
sent different surface modification processing time to allow sulfide molecules to be immobilized to gold coated sensor
for QCM sensing. The optimum surface modification processing time is also obtained by bovine serum albumin (BSA)
binding measurement.
Keywords: Biosensor; QCM; Protein Binding; Surface Modification
1. Introduction
An important advance in biosensor was done during the
last two decades. Especially, protein biosensors are now
intensely developed for diagnostic applications [1], en-
vironmental monitoring [2] and food controls [3]. Mass-
sensitive biosensor systems have attracted considerable
attention in recent years since many important physical
and chemical processes can be followed by observing the
associated mass changes. The QCM system is based on
the principle that the resonant frequency shifts of a pie-
zoelectric crystal are directly proportional to the ad-
sorbed mass [4]. QCM is suitable for several applications
especially in biosensors [5]. QCM biosensors are time
resolved, enough sensitive to detect non-labeled DNA [6],
enough selective to detect single mismatch DNA [7], and
renewable [3]. QCM protein biosensors may be designed
by a wide choice of immobilization techniques of short
receptor on the quartz microbalance electrode surface. As
immobilization techniques of the protein probe on the
surface are essential and critical for biosensor, we evalu-
ate in this work different receptor immobilization time
with adsorption of a disulfide on a gold-covered quartz
surface of a microbalance. The disulfide immobilization
process has several advantages: the adsorption of the
receptor by binding between sulfur atom of disulfide and
a gold atom of the surface is irreversible and stable. It
takes less than one hour [8] and gold is very stable versus
oxidation. And then, BSA is used to verify the sensing
processing and to obtain the optimum receptor immobi-
lization time.
2. Operation Principle of QCM
A flat quartz disc with electrodes on both surfaces can be
forced to oscillate in a transverse acoustic mode (motion
parallel to the surface) by an RF voltage applied at the
acoustical resonance frequency of the plate. This device
is called a transverse shear mode (TSM) quartz plate
resonator. The frequency of the fundamental mode is
inversely proportional to plate thickness and mass. TSM
quartz plate resonators have been used as sensitive
microbalances for thin adherent films since the late
1950's, following the pioneering work of Sauerbrey [9].
The frequency of TSM resonator is continuously moni-
tored when a sample is deposited on its surface. The shift
in frequency due to deposition of a film of the same
acoustic impedance as quartz is proportional to the de-
posited mass per unit area of the film,
 (1)
where μq, ρq and f0 are the shear modulus, density and the
resonant frequency of bare quartz crystal and A and m
are the electrode area and sampling mass difference, re-
spectively. QCMs have been used as film thickness
monitors in vacuum deposition of metals and inorganic
solids. QCMs are useful because of their sub-nanogram
Copyright © 2013 SciRes. OPJ
3. Experiment
3.1. QCM Apparatus
The microbalance resonators, provided by Ya-Shin
company, are AT-cut planar quartz crystals, 7mm in di-
ameter, with a 9 M Hz nominal resonance frequency.
Two identical gold electrodes, 500 Å in thickness and 3.5
mm in diameter, were deposited on both sides of the
quartz. The QCM chip structure is shown in Figure 1.
The crystal is mounted between two O-ring seals inserted
in a glass cell to form an experimental cell. The gold side
of the quartz used in the experiments wass cleaned with a
pickling solution for 5 minutes, the sulfuric acid and hy-
drogen peroxide in a 1:3 ratio, and then rinsed with de-
ionized water. We use 4,4'-dithiodibutyric acid to modify
electrode surface. First, the sulfide powder was diluted
with aqueous ethanol. The concentration of sulfide solu-
tion was 500 ppm in the experiment. Subsequently, sul-
fide solution was dropped on four QCM chip surface full
and lasting for 15, 30, 45 and 60 minutes at 25, respec-
tively, to immobilize protein receptor on gold electrode
3.2. Protein Detection
The ADS Plus instrument offers a platform to measure
equilibrium binding affinity and kinectics using unmodi-
fied molecules in solution phase. The system allowing
affinity measurement when reach equilibrium and kinet-
ics measurement under pre-equilibrium conditions was
used in our experiment. QCM chip was inserted into
ADS PLUS and a computer was connected to the instru-
ment for data acquisition. Deionized water or BSA solu-
tion via a peristaltic pump sent to the QCM chip, the
system architecture is shown in Figure 2.
Initially, the average resonant frequency of the deion-
ized water on QCM was recorded for 10 minutes after
stable state. The stable frequency is as a benchmark.
Then, a BSA solution of 50 ppm was injected through
Figure 1. QCM chip structure.
peristaltic pump. In order to observe the BSA binding
kinetics and find the binding time, we use a QCM chip
with 60 minutes sulfide modification to monitor the
binding process for a long time. In terms of the QCM
resonant frequency variation, we could observe the BSA
binding saturation time. The injection process has three
periods. The first one is 1600 seconds for water, the sec-
ond one is 7200 seconds for BSA solution of 50 ppm and
the last one is 1800 seconds for water again. The QCM
resonant frequency during injection process is shown in
Figure 3. After injection BSA about 1800 seconds, which
occurred at 3400 seconds in Figure 3, the resonant fre-
quency largely degraded to -3093 Hz. Approximately
after 10,400 seconds, the resonant frequency was almost
stable. In other words, the BSA binding was completed
and saturated. Then, deionized water was injected again
to verify BSA strong binding. There was only slight in-
crease in frequency owing to some BSA molecule weak
binding escape. Since most of the sulfides for protein
binding strength are very strong, it is belong to strong
binding. For the binding time observation, we could fine
that 86% frequency degradation occurred at about 30
minutes after BSA injection. So, the BSA binding time is
set as 30 minutes (1800 seconds) for our work.
Figure 2. ADS PLUS and QCM for BSA binding detec t ion.
Figu re 3. Re so nan t fr eq uenc y va ria ti on d uri ng pr ot ein bin din g
Copyright © 2013 SciRes. OPJ
Copyright © 2013 SciRes. OPJ
Table 1. Surface modification and protein binding effect.
chip # 1 # 2 # 3 # 4
Surface modification time (minute) 15 30 45 60
BSA binding time ( minute) 30 30 30 30
BSA frequency shift (Hz) 117.3 136.8 137.8138.0
Efficiency (%) 3.75 4.37 4.354.65
Adsorption mass (ng) 314 366 368369
4. Results and Discussion
The frequency difference after BSA injection for 4 QCM
chips with modification time of 15, 30, 45 and 60 min-
utes, respectively, is list in Table 1. Since sulfide could
bind BSA and adsorb the mass on the surface of the chip,
the resonant frequency would degrade. More BSA be
binded, larger frequency difference will be. The fre-
quency difference were measured as 117.3 Hz, 136.8 Hz,
137.8 Hz and 138.0 Hz for the 15, 30, 45 and 60 minutes,
respectively. The efficiency is defined as the frequency
difference divided with the initial deionized water reso-
nant frequency. The results are 3.75%, 4.37%, 4.35% and
4.65%, respectively. So, the optimum sulfide modifica-
tion time for BSA protein QCM biosensor is about 30
5. Conclusions
In this study, we present different surface modification
processing time to allow sulfide molecules to be immobi-
lized to golden electrode for QCM protein sensor. The
surface modification was also tested by BSA binding
measurement. The optimum surface modification proc-
essing time is detected as 30 minutes.
[1] M. Campàs and I. Katakis, “DNA Biochip Arraying, De-
tection and Amplification Strategies Trend,” Analytical
Chemistry, Vol. 23, 2004, pp. 49-62.
[2] S. Rodriguez-Mozaz, M. J. López de Alda, M.-P. Marco
and D. Barceló, “Biosensors for Environmental
Monitoring A Global Perspective,” Talanta, Vol. 65,
2005, pp. 291-297, “Title of Paper If Known,” unpub-
lished. doi:10.1016/S0039-9140(04)00381-9
[3] I. Mannelli, M. Minunni, S. Tombelli and M. Mascini
“Quartz Crystal Microbalance (QCM) Affinity Biosensor
for Genetically Modified Organism (GMOs) Detection,”
Biosensors and Bioelectronics, Vol. 18, 2003, pp.
[4] J. Rickert, A. Brecht and W. Gopel, “Quartz Crystal
Microbalances for Quantitative Biosensing and Character-
izing Protein Multilayers,” Biosensors and Bioelectronics,
Vol.12, pp. 567-575, Mill Valley, CA: University Science,
[5] S. Lin, C. C. Lu, H. F. Chien and S. M. Hsu. “An On-line
Quantitative Immunoassay System Based on A Quartz
Crystal Crobalance,” Journal of Immunological Methods,
Vol. 239, No.1-2, 2000, pp. 121-124.
[6] Y. Okahata, Y. Matsunobu, K. Ijiro, M. Mukae, A. Mu-
rakami and K. Makino, “Hybridization of Nucleic Acids
Immobilized on A Quartz Crystal Microbalance, Journal
of the American Chemical Society,Vol.114,1992,pp.
[7] F. Höök, A. Ray, B. Nordén and B. Kasemo, “Charac-
terization of PNA and DNA Immobilization and Subse-
quent Hybridization with DNA Using Acous-
tic-Shear-Wave Attenuation Measurements,” Langmuir,
Vol. 17, 2001, pp. 8305-8312.doi:10.1021/la0107704
[8] E. Huang, M. Satjapipat, S. Han and F. F Zhou, “Surface
Structure and Coverage of An Oligonucleotide Probe Teth-
ered onto A Gold Substrate and Its Hybridization Effi-
ciency for A Polynucleotide Target,” Langmuir, Vol. 17
2001, pp. 1215-1224.doi:10.1021/la001019i
[9] G. Sauerbrey, Z. Physik, Vol. 155, 1959, p.