IEEE SENSORS JOURNAL, VOL. 8, NO.

5, MAY 2008 501

Phase Detection of the Two-Port FPW Sensor

for Biosensing
Wen-Yang Chang, Po-Hsun Sung, Chun-Hsun Chu, Ching-Jui Shih, and Yu-Cheng Lin

Abstract—This study reports the phase detection of the two-port chemical warfare agents, and biological species [1]–[4]. They
flexural plate wave (FPW) sensor for designing and integrating the are used mostly to detect the adsorption or attachment of pro-
miniature system and provides a comprehensive methodology for tein molecules [5], cells [6], bacteria [7], or the immunological
portable using in the biosensor applications of severe acute respi-
ratory syndrome coronavirus (SARS-CoV). The miniature system properties of cytokines conjugated to organisms on the plate sur-
mainly utilizes the concept of the frequency divider that involves a face of two-port IDTs [8].
divider, a time-based oscillator and a gate to reduce the high fre- There has been a notable development in biosensing detec-
quency, and the FPW sensor is fabricated using microelectrome- tions in recent years, such as the quartz crystal microbalance
chanical systems (MEMS) technologies for producing a potable (QCM) [9], [10], the thickness shear mode (TSM) [1], the
biosensing detector. The results demonstrate that the insertion loss
decreased about 1 15% dB/ C, and the phase delay was about acoustic plate mode (APM) [11], the flexural plate wave (FPW)
2.05 /(1000 cP). The phaseshift resolution was about 10 mV per [12]–[16], the love mode [17], the shear horizontal acoustic
degree, and the original frequency of 4.2 MHz was divided by 100 plate mode (SH-APM) [18], and the surface acoustic wave
to reduce the frequency to 42 kHz. The SARS-CoV could be de- (SAW) [19]. In general, the FPW has a higher sensitivity with
tected via the S protein binds to the human angiotensin-converting the loading changes at lower operating frequencies among
enzyme 2 (hACE2) as a functional receptor, which would cause
the phase delay due to the combining of the antibody with the all these detecting methods [20], [21]. However, the FPW
antigen. Therefore, the feasibility studies provide the information sensors in the most studies can only detect at a low level of
that phase detection is an appropriate low-cost technology via fre- frequency change during the different loadings [5], [22]. It is a
quency divider for fabricating of the miniature biosensors. well-known hard task to deal with a signal of only a few Hertz
Index Terms—Biosensing, flexural plate wave, integrated comparing to the original resonant frequency of several mega-
system, phase detection, readout concept, severe acute respiratory hertz, especially since this biosensor is commonly measured
syndrome coronavirus (SARS-CoV). by a network analyzer instrument [10], [23] or fluorescent
microscope [24]–[26]. As a portable device, measuring the
frequency shift by a network analyzer is a very difficult and
I. INTRODUCTION
unpractical way. In addition, it is also very hard to assemble a
N GENERAL, the biosensing signals that is based on an control device for acoustics waves, which could be easily read
I acoustic wave sensors could be done in two methods, using
a one-port interdigitated transducer (IDT) or a two-port IDT.
out and mastered by users.
Therefore, we presented an adaptive readout system using
One-port IDTs are arranged to conduct surface to propagate in the frequency divider, phase detector, and microelectromechan-
one direction, and then turn back to create a resonant cavity ical systems (MEMS) technologies for miniature system and
to measure the resonant frequency shift. Thus, it is very dif- portable using. Furthermore, the miniature system was veri-
ficult to design a critical resonator with a higher bandwidth fied by the varied viscosity solutions, standard products of the
for biosensing detection and gravimetric detection ability of Brookfield laboratories, to test the frequency amplitude decay
an added mass in a liquid. However, the two-port IDTs placed and phase delay. In the meantime, the phaseshifts could be de-
at opposite ends of a slice can easily measure the attenuation, tected in the SARS virus due to adding S protein and employing
delay, or frequency shift of signals in liquid sensing such as human angiotensin-converting enzyme 2 (hACE2) to the func-
tionalized FPW biosensor.
Manuscript received May 4, 2007; accepted July 13,2007. This study was II. EXPERIMENTAL SETUP
supported in part by the Ministry of Economic Affairs as part of an environ-
mental construction project by the government of Taiwan, R.O.C. The associate A. Flexural Plate Wave Device
editor coordinating the review of this paper and approving it for publication was
Dr. Richard Fair. The two-port FPW sensor was designed and fabricated, as
W.-Y. Chang is with the Department of Engineering Science, National Cheng
Kung University,Tainan 701, Taiwan, and also with the Microsystems Tech-
shown in Fig. 1(a). We started with a standard silicon wafer
nology Center, Industrial Technology Research Institute, Tainan 709, Taiwan. of 4 inches in diameter. The design and fabrication sequences
P.-H. Sung, C.-H. Chu, and C.-J. Shih are with the Microsystems Technology were described as follows. First, a 0.4 m thick film of low
Center, Industrial Technology Research Institute, Tainan 709, Taiwan.
Y.-C. Lin is with the Department of Engineering Science, National Cheng
stress silicon nitride was deposited on the silicon surface, the
Kung University, Tainan 701, Taiwan, and also with the Center for MicroNano average tensile stress of which was about 110–130 MPa. The
Technology, National Cheng Kung University, Tainan 701, Taiwan (e-mail: next step was to grow the ground electrode and the piezoelectric
yuclin@mail.ncku.edu.tw). thin film of aluminum nitride (AlN), with a thickness of about
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.ieee.org. 0.1 and 1 m, respectively. Generally speaking, the thickness
Digital Object Identifier 10.1109/JSEN.2008.918728 of the AlN thin film is larger than that of a silicon nitride thin
1530-437X/$25.00 © 2008 IEEE
502 IEEE SENSORS JOURNAL, VOL. 8, NO. 5, MAY 2008

Fig. 1. FPW sensor chip. (a) Microfabrication process using microelectrome-

chanical systems technologies. (b) The photo of FPW from top view that the
period of IDT is 200 m with 13 pairs and total thickness is about 1.6 m.

film. Finally, the top electrodes with a thickness of 0.1 m were

patterned onto the surface, followed by the etching of the AlN
film. Consequently, the total thickness of the FPW thin film was
about 1.6 m.
The last process step was to etch the backside of the silicon
wafer in order to release an unsupported resonant diaphragm.
Since it is hard to dice a wafer when the backside is etched in
one step, the backside etching process was done in two steps.
First, we placed the wafer in the special holder and used KOH to
partially etch the backside. After dicing, the tetramethylammo-
nium hydroxide (TMAH) was used for the last step of etching.
The recipe to etch silicon on the silicon substrate is 35% KOH
plus 10% IPA prior to dicing and 5 wt% TMAH plus 1.65 wt%
silicon at 80 C after dicing. AlN film was etched by the Al
standard etchant of eSolv EA-855 for 100 Å/s at 60 C. The
FPW chip after the MEMS fabrication was shown in Fig. 1(b).
The width and spacing of the IDTs each was both 100 m and
comprising of 13 pairs. During the experiment testing, the FPW
chip device which measured 12 8 0.5 mm was fixed on the
Fig. 2. The FPW control process of the integrated miniature system for
PCB board. The impedance of the input and output signals of biosensing. (a) The control block diagram that includes a WBO, a voltage
the FPW device were matched by tuning test, where was follower, a FPW device, a PLL, a PD, a MCU, and a LCM. (b) The schematic
52.3 . circuit of the WBO. (c) The model of the PLL circuit design.

B. Miniaturized System Design

and a liquid crystal display module (LCM) display. First, we de-
The control procedure of the miniature system was shown in signed the 4.2 MHz WBO to excite the resonant frequency of the
Fig. 2(a). The system framework of the FPW sensor was built of FPW thin film. It is a well-known basic form of the WBO which
a Wien-Bridge oscillator (WBO), a voltage follower, a FPW de- was made up of an operational amplifier, resistances, and capac-
vice, a phase locked loop (PLL) that comprised a phase detector itances, as shown in Fig. 2(b). The resonant frequency control
(PD), loss pass filter (LPF) and voltage controlled oscillator part is composed of , and with the oscillation fre-
(VCO), a microprocessor (MCU), a frequency phase detector, quency defined as and the resistance of
CHANG et al.: PHASE DETECTION OF THE TWO-PORT FPW SENSOR FOR BIOSENSING 503

and controlling the operational amplitude of the resonant

frequency.
After passing through the voltage follower, the WBO signal
is input into two FPW devices; one is a reference chip with no
mass load that provides zero phases in a liquid, and the other
is a sensing device with sensing load. Then, the output signals
travel through the closed-loop feedback control system of PLL,
and then the phase of the two outputs of the and , which are
the PLL output signals of a reference and a sensing chip, respec-
tively, are compared using an ANALOG DEVICES AD8302.
Finally, we employ the MCU and LCM to complete the minia-
ture system measuring 11 10 7 cm , as shown in the left
corner above of Fig. 2(a).

C. Control System Analysis

The schematic control circuit of the PLL was designed, as
shown in Fig. 2(c). The PD is the XOR gate or high-speed per-
Fig. 3. The immobilization process diagram is that an anti-SARS drug trans-
formance dual D flip-flop. The D flip-flop output produces a mits into sensor system with immobilization by S-hACE2 hybrid protein, which
regular oscillation when the input CLK and the have would cause the phaseshifts due to adding S protein and employing hACE2 to
one quarter of period shift. The LPF was used to transform the the functionalized FPW biosensor.
instantaneous phase difference into analog voltage which
is equivalent to the average voltage . The current oscillator
can be used as a VCO for PLL, with a modification of its voltage Therefore, the hACE2 could be used as a functional receptor of
control circuit so that the center frequency can stay at 4.2 MHz. the antigen for the immobilization process in the clinical exper-
The equations for the output of the PD and PLL might be iment, and its binding site on the SARS-CoV glycoprotein is
derived as follows. Let the input to the PD be , which was localized between amino acid residues 303 and 537 [28].
the output via the sensor chip, and the output of the VCO be The immobilization process diagram of the antibodies
in the feedback path of the PLL, then the output of on FPW sensing area was shown in Fig. 3. The Au and
PD was given by (1) Cr film were first deposited about 1500 and 160 A on the
sensing area, respectively, and then the cross-linker ithiolbi
[N-(5-amnno-5-carboxypentyl)—propionmide—N’, N’ di-
acetic acid] dihydrochloride (disulfide-NTA) as the antibody
were patterned for 15 min on the sensing area by the plasma-en-
hanced chemical vapor deposition (PECVD), washed by DI
water for two times, and then immersed in the 0.1 M NiSO
for 10 min. In addition, S proteins and hACEs—Histidine were
(1) incubated together to form the effective affinity via protein–pro-
tein interaction at 4 C and 5% CO for 1 h in the incubator.
where and are the output frequency and phase of the All experiments of biosensing were performed at temperature
sensor chip, and and are the feedback frequency and phase of 4 C. For the biosensing measurement, the miniature FPW
of the PLL output signal. system detected the phase change due to the mass changes
According to the output of the closed-loop feedback, the occurring on progressive deposition of the antibody–antigen
output of the reference chip via the PLL can be analyzed as a binding.
control system below (2)
III. SYSTEM CHARACTERIZATION AND VERIFICATION

A. FPW Chip
After the MEMS fabrication of the FPW chips, we first ver-
(2) ified the character of FPW using the network analyzer, Agi-
lent model 8753ES/ET. The scattering parameters of the for-
where is the transformation of a low-pass filter. ward or reverse transmission, or , could be determined
In the same deriving way, the output of the sensing chip, by calculating the difference between the input and the output
, can be acquired and compared with . power level response for the two-port IDT, i.e.,
. Typically, the insertion loss of the two-port
D. FPW Immobilization Process FPW device is at its resonant frequency. The peak of the reso-
The severe acute respiratory syndrome coronavirus (SARS- nant frequency was almost at 4.2 MHz, as shown Fig. 4. These
CoV) infects the ciliated airway epithelial cells via an inter- three curves were measured from different samples, and the re-
action with ACE2 on the apical surface of ciliated cells [27]. sults showed a good uniformity property of fabrication.
504 IEEE SENSORS JOURNAL, VOL. 8, NO. 5, MAY 2008

Fig. 4. The transmission insertion loss of the FPW device was measured only
considering the S or S parameter. These three curves are measured from
different samples; the result showed the resonant frequency was about 4.2 MHz.

Fig. 6. The flowchart block of the frequency divider. (a) A time-based oscillator
and a gate to reduce frequency. (b) Using trigger signal to sample the sinusoidal
signal for a square wave.

Fig. 5. The experimental testing of the FPW characteristics with different tem-
0
peratures presented the insertion loss decreases of about 1:15% (dB/ C).
during 1 s of the gate time in the measure-
ment period. Thus, the resolution of 100 MHz at the count
of will be 100 Hz. The original resonant frequency
Corresponding with intermediate frequency signals, the of the sinusoidal signal was 4.2 MHz, then the square wave
insertion losses were calibrated to different temperatures, as was obtained using a trigger signal, followed by being counted
shown in Fig. 5. The results demonstrated that the charac- down to 1/100 with a divider of 100 times by each counter
teristic of the insertion loss was a linear curve and the slope for MCU processing, as shown in Fig. 6(b). Consequently,
was decreased about % dB/ C. The effect is the mem- the frequency was counted down to 42 kHz. Therefore, it was
brane tension caused by thermal expansion. To improve the adequate to directly count the frequency of the FPW sensor
temperature stability of the FPW, it suggests using a heater using MCU when the trigger button was started.
and a temperature sensor to compensate the temperature shift
phenomenon. Therefore, the employing advance processing C. MCU Control
based on the phase delay effect, the small relative transmission
The MCU is the detecting kernel of the FPW sensing system
loss caused by the temperature variation could be evaluated at in integration of the miniature system for portable using of the
a high accuracy.
biosensor applications. Here, we used the MCU of TI’s MSP430
products, the main function of which was to integrate multifunc-
B. Frequency Divider Counting
tions into a single chip. In order to fit the frequency range of the
Generally speaking, it is very difficult to sample the high-fre- sampling, the MCU needs an extra crystal oscillator to enlarge
quency signal by an MCU for portable use in an integrated the frequency range. After modification, the instruction cycle
miniature system. Thus, we proposed a frequency divider which time and the highest frequency range could reach 125 ns and
used a divider, a time-based oscillator, and a gate to reduce 8 MHz, respectively. During the measurement operation, the
the high frequency. The flowchart block of reduced frequency function of the detecting pin was enabled to sense the negative
counting was shown in Fig. 6(a). The counting numbers of edge of the periodic square wave when the MCU detected the
the periodic square wave can be transferred to frequency data trigger from the button. To the counter register, we added one
related to the duration and the decade counter was used as automatic timer function that enabled us to calculate the fre-
a frequency divider to cut down the frequency. The count is quency at the same time. After the timer stopped, the MCU first
CHANG et al.: PHASE DETECTION OF THE TWO-PORT FPW SENSOR FOR BIOSENSING 505

Fig. 7. The phase delay and amplitude decay of the FPW propagation were Fig. 8. The relationship between phaseshifts and output voltages was tested by
measured by the different viscosity standard solutions. The nonlinear regression dropping the different viscosity standard solutions. The system resolution of the
curves presented an exponential rising. phase delay is about 10 mV per degree.

disabled the function of the detecting pin and the timer. The mi-
croprocessor enabled the timer and set the gate time to 100 s at
the same time. Based on the register of the counter at 10 counts
per 100 s, the microprocessor transferred the counts to 42 kHz
and displayed the phaseshifts on the LCM.
The operation DC power of the FPW sensor system included
three levels, 5 V, V, and 3.3 V. For a stable DC power sup-
plied, the AC power was rectified to DC 12 V, and then trans-
ferred to a regulator for further adjustment.
IV. RESULTS AND DISCUSSIONS
A. Propagation Testing Using Viscosity Materials
Fig. 9. The phase delay and amplitude decay was comparing the reference de-
During the FPW propagation measurement, we used variant vice to another sensing device of the FPW in frequency response. The f ; f ,
viscosity standard cP (m.Pa.s) products from the Brookfield lab- and f are output signals of the reference chip, sensing chip without loading
oratories to test the amplitude reduction and phaseshift at en- and loading, respectively.
vironment temperature of 25 C, as shown in Fig. 7. For the
measurement process, the FPW system inputted a resonant fre-
quency to the reference and sensing chip, and used a small tube degree. The approximate function is ,
by hand to drop the viscous liquids into the sensing area at the where is the output voltage, and is the phase delay. There-
top of the backside cavity. The phase detector monitored the fore, the detection sensitivity is based on the relative phase delay
changes of phaseshift in the acoustic wave surface between the of the membrane instead of the frequency of operation like other
two FPW transducers. Therefore, experiment results showed the acoustic devices.
amplitude reducing and phase delay with respect to a reference
signal at different viscosity standard solutions presenting the ex- B. Practical Biological Testing
ponential satisfaction, which had larger changes due to the vis- Antibodies were first immobilized on the gold surface of a
cously liquid mass coupled to the acoustic wave surface plate at FPW among IDTs and the hACE2 binding with S protein be-
low viscosity and had fewer changes due to the FPW chip would came a functional receptor for the SARS corona virus which
largely shift from the mechanical resonance when loading a high led to the unique combination of the antibodies and the antigens
viscosity material. materials. Therefore, the sinusoidal waves would delay and at-
According to the descriptive measuring method above, we tenuate within the propagation region of the sensing chip due
measured the relationship between the phase delay and output to the mass increase on the FPW surface. A phase delay was
voltage of the FPW system as shown in Fig. 8. The curve of detected between two signals, one is reference and the other is
the FPW phase variation presented the triangular waveform and the sensing chip with adding mass loading of the antigen in a
was almost linear; however, it had a nonlinear property at below liquid, as shown in Fig. 9. The phase delays of the reference
or above than 134 when the negative phase is the and sensing without loading chip of the FPWs caused by im-
mirror of the positive phase. The FPW characteristics can be mobilizing of the antibody on FPW sensing surface, and ,
predicted to be satisfied and shifted from the mechanical reso- respectively, were initially about degree. When loading
nance response at nonlinear curve regions when loading a high the cells per microliter on the sensing region that output
viscosity material. The sign is based on which output of the ref- signal is , the phase delay was about compared with
erence or sensing chip was input to the positive detection pin the reference chip. The S protein bound to the hACE2 as a func-
of the AD6302. The phaseshift resolution of the minimum de- tional receptor with different cell numbers per microliter was
tectable change in the measurement system is about 10 mV per shown in Fig. 10(a), which would cause the phaseshifts due to
506 IEEE SENSORS JOURNAL, VOL. 8, NO. 5, MAY 2008

display into the FPW readout system. In addition, the system

used the frequency divider counting concept and MCU pro-
cessor to reduce the frequency for portable use. In the practical
biology testing, we used antibodies to be immobilized on the
gold surface of a FPW among IDTs. Then, the interaction be-
tween the antibodies and the antigens biomaterials will bind to-
gether caused by specific recognition results in mass increase.
Thus, the FPW waves attenuated within the propagation range of
the device, and the phaseshift would change due to the mass in-
crease on the surface of FPW, that is the FPW waves attenuated
within the propagation range of the device, the phaseshift would
change when the different numbers of cells loaded. Therefore,
we believed that with the packaging improvements, this minia-
ture applications system is quite feasible, and through these ex-
periments, we have described a prototype flow of a cell design
that addresses some of the problems, this study was more com-
plete, and the miniature system could provide quantity detection
for biosensing applications.

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[25] J. W. Kim, Y. Yamagata, M. Takasaki, B. H. Lee, H. Ohmori, and T. Currently, he is with the Microsystems Technology Center, Industrial Tech-
Higuchi, “A device for fabricating protein chips by using a surface nology Research Institute, Tainan, Taiwan, studying the MEMS sensor. His
acoustic wave atomizer and electrostatic deposition,” Sens. Actuators main research interests in auto-fusion and asymmetric division in HL-CZ cells.
B, vol. 107, pp. 535–545, 2005.
[26] J. P. Black, R. M. White, and J. W. Grate, “Microsphere capture and
perfusion in microchannels using flexural plate wave structures,” in
Proc. IEEE Ultrasonics Symp., 2002, vol. 1, pp. 475–479.
[27] W. Li, M. J. Moore, N. Vasilieva, J. Sui, and S. K. Wong, “Angiotensin Yu-Cheng Lin received the B.S. and M.S. degrees in mechanical engineering
- Converting enzyme 2 is a functional receptor for the SARS coron- from the National Cheng Kung University, Tainan, Taiwan, in 1985 and 1987, re-
avirus,” Nature, vol. 426, pp. 450–454, 2003. spectively, and the M.S. degree and Ph.D. degree in electrical engineering from
[28] S. K. Wong, W. Li, M. J. Moore, H. Choe, and M. Farzan, “A the University of Illinois at Chicago, Chicago, in 1994 and 1996, respectively.
193-amino acid fragment of the SARS coronavirus S protein effi- He is a faculty member with the Department of Engineering Science, Na-
ciently binds angiotensin-Converting enzyme 2,” J. Biological Chem., tional Cheng Kung University. His main research interests include bio-MEMS,
vol. 279, pp. 3197–3201, 2004. microfluidic systems and nanotechnology in biomedical applications. He cur-
[29] J. W. Grate, S. W. Wenzel, and R. M. White, “Flexural plate wave de- rently serves as a member in the International Advisory Board of the interna-
vices for chemical analysis,” Anal. Chem., vol. 63, pp. 112–123, 1991. tional journal Lab-on-a-Chip.