AC impedance sensing is an important method for biological cell analysis in flow cytometry. For micro impedance cell sensors, downsizing electrodes increases the double layer impedance of the metal-electrolyte interface, thus leaves no sensing zone in frequency domain and reduces the sensitivity significantly. We proposed using platinum black electroplated electrodes to solve the problem. In this paper, using this technique we demonstrated human blood cell sensing with high signal to noise ratio

Nandra, Mandheerej S.

Tai, Yu-Chong

Zheng, Siyang

English

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Human Blood Cell Sensing with Platinum Black Electroplated Impedance Sensor

Caltech Authors - Main

Proceedings of the 2nd IEEE International
Conference on Nano/Micro Engineered and Molecular Systems
January 16 - 19, 2007, Bangkok, Thailand
Human Blood Cell Sensing with Platinum Black
Electroplated Impedance Sensor
Siyang Zheng, Student Member, IEEE, Mandheerej S. Nandra and Yu-Chong Tai, Fellow, IEEE
Caltech Micromachining Laboratory, California Institute ofTechnology, Pasadena, CA, U.S.A.
Abstract-AC impedance sensing is an important method for
biological cell analysis in flow cytometry. For micro impedance
cell sensors, downsizing electrodes increases the double layer
impedance of the metal-electrolyte interface, thus leaves no
sensing zone in frequency domain and reduces the sensitivity
significantly. We proposed using platinum black electroplated
electrodes to solve the problem. In this paper, using this
technique we demonstrated human blood cell sensing with high
signal to noise ratio.
Keywords-impedance sensor; Coulter counter; blood cells;
platinum black electroplating
I. INTRODUCTION
Electrical impedance sensing has been one of the preferred
technologies for particle sensing and counting. Since the
invention of Coulter counter by Wallace H. Coulter in 1940s
[1], it has been used in bulk blood counters and flow
cytometers. The initial DC resistance sensing also extended to
AC impedance sensing [2]. For electrical impedance sensing
in a flow system, cells flow by sensing electrodes one by one.
This creates changes of the electrical impedance between the
electrodes. Therefore, the existence of a biological cell and
some of its properties can be determined. At low AC
frequency (under 1OOkHz), the signal is determined mainly by
the cell volume. At higher frequency (1OOkHz to 1OMHz), the
intracellular structures also contribute to the overall measured
impedance and become explorable measurands [3].
Traditional electrical impedance sensors require large
electrode size to reduce the metal-electrolyte interface
impedance (i.e. double layer impedance) and allow the particle
impedance to dominate the overall system impedance. Bulk
blood counters, for example, use macro-electrodes (such as
Platinum wire) across the aperture whose size is in the range of
hundreds of micron. Recently, there's a push to shrink the
sensor size as miniaturization enables better sensitivity, lower
coincidence error rate, smaller sample volume and faster
detection with parallelization. However, there is a major
difficulty associated with micro impedance sensing. The series
metal-electrolyte impedance is inversely proportional to the
electrode surface area so the downsizing unavoidably reduces
the sensor sensitivity [4-7]. For examples, recent works on
micro DC Coulter sensors were forced to stay with very large
thin film electrode [8], macroscopic gold pin electrodes [9] and
salt bridge with nonpolarizing Ag/AgCl electrodes [10].
Another approach is to use microelectrodes in high-frequency
AC impedance sensing mode because high frequency reduces
the double-layer impedance. Unfortunately there is an upper
limit of the operation frequency imposed by the stray
capacitance between sensing electrodes, which is resulted from
coupling and non-ideal isolation. So to study particles with
AC impedance sensing, the device is limited to a frequency
range, which is high enough to bypass electrode double layer
impedance and low enough that the stray capacitance does not
play a significant role in the overall system impedance [7].
Micro AC impedance sensors have been attempted to sense
cell suspensions and single cells. The problem of large double
layer impedance was overcome by electroplating very thick
gold micro electrodes [5] or reducing the stray capacitance to
shift the sensing to high frequency range [11]. Generally
speaking, high frequency AC impedance sensing requires more
consideration in electrode design, chip isolation, signal
conditioning and processing. In this study, we demonstrated
Platinum black electroplated electrodes as an effective way to
extend AC impedance sensing to lower frequency range. This
not only expands the sensing frequency range of micro
impedance sensors, but also avoided some of the complexities
associated with the high frequency sensing.
Theoretically, electrodes with high effective surface area
are ideal to overcome the problem with double layer
impedance. Platinum black electroplated electrodes have
porous surface structures and thus increase the effective
electrode surface area. It can reduce the electrode surface
double layer impedance by two orders of magnitude.
Consequently the channel impedance becomes the dominant
system impedance in some intermediate frequency range and
the device become sensitive to what is flowing inside.
Platinum black electroplated micro electrodes have been used
for extracellular neural signal recording [12-14], gas sensing
[15], thermal infrared detection [16]. Specifically, we report
here the first use of Platinum black electroplated
microelectrodes as AC impedance sensors for human blood
cell sensing.
II. THEORY
The device can be modeled electrically as the channel
impedance Z in series with a constant phase element (CPE)
and then in parallel with the stray capacitance C5t (Fig. 1). Z
includes both the electrolyte impedance and impedance of
particle to be sensed. In this paper, the particles were sensed at
lOkHz, so the channel impedance was effectively a resistance
R. C5t is the unwanted coupling capacitance between the two
electrodes. CPE is contributed by the double layer impedance
of the metal-electrode interface. Impedance of CPE is ZO/00),
with constant Zo, angular frequency co and exponent a. The
phase of CPE is a constant of -a;TI2. Normal metal electrodes
have a close to one and the CPE behaves like a capacitor with
capacitance 1 ZO and phase -p/2. There are at least two effects
that can make a deviate from one. The surface roughness can
1-4244-0610-2/07/$20.00 C)2007 IEEE 520
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change a from 1 in perfect flat 2D electrodes to 0.5 in 3D
porous cubic electrodes [17]. Platinum black electroplating
can greatly change the surface roughness of metal electrodes to
transform them from flat 2D surface toward 3D structures.
Another effect that is likely to change a in micro domain is the
transport of electrochemical active molecules on the electrode
surfaces. In micro devices, the ratio of electrode surface area
to the volume of the sensing zone is so large that the
electrochemical reaction on the electrode surface is diffusion
limited. This also reduces a.
CPE In
-1-
Figure 1. System Model
From the system model, the overall measured impedance
can be written as:
z
(1)1
R+z
(j)a
For channel resistance to dominate in some frequency range, it
requires:
R > Cst/l-lzoy-a (2)
Clearly from equation (2), lowering the stray capacitance and
double layer impedance are favored for particle sensing. If the
equation (2) is satisfied, the system shows some typical
asymptotic behaviors. When co approaches zero, the system is
reduced to the channel impedance in series with the CPE.
When co approaches infinity, the system approaches the
channel impedance in parallel with the stray capacitance.
These asymptotic behaviors are illustrated in the Nyquist plot
of Fig.2, which can be used to extract parameters from
measurement.
30k X 7
CPE R
E20k
0E
0 10k 20k 30k 40k 50k 60k
ZRO()
Figure 2. Asymptotic behaviors in Nyquist plot. coo is the angular
frequency when ZRe is R/2.
In order for the system to sense flowing-by particles, the
particle impedance should be the major component in overall
system impedance at the excitation frequency. This is difficult
to achieve in micro systems because the miniaturization of the
electrodes. In low frequency range, impedance of CPE
dominates the system impedance, while at high frequency the
stray capacitance in parallel to the system dominates. To
enable particle sensing in low frequency domain, an effective
way is to lower impedance of CPE by platinum black
electroplating. It can increase surface area by two orders of
magnitude and thus reduce electrical impedance proportionally.
III. DESIGN AND FABRICATION
The chip layout is illustrated as Fig.3(A). Two fluidic
chambers are separated by an aperture. The width of the
aperture is 15pim. The length of the aperture is 30pim. One
pair of metal electrodes senses the electrical impedance across
the aperture. The size of the chamber is very large compared
with the aperture so that the measured channel impedance is
dominated by the impedance of the aperture region. The
distance of the electrodes is 70pm. At the inlet and outlet,
some filter structures are designed to mechanically block
contaminants and particle aggregates and preventing them
from clogging the aperture. Channel height is optimized to be
around 15pm so that all blood cells can pass while keeping
signal magnitude as high as possible.
The device fabrication was started with molding channel
structure on PDMS with DRIE etched Si mold. Ti/Pt
(200A/2500A) electrodes were patterned on soda lime glass
substrate with lift-off process. Platinum black was then
electroplated to the selected areas Fig.3(B). Electroplating
solution contains 10% chloroplatinic acid, 0.0025%
hydrochloric acid, and 0.01% lead acetate in water [13].
Electrodes were plated with 5V DC through a 10MQ series
resistor for up to four minutes to achieve maximal double layer
impedance reduction. The thickness of the electroplated
platinum black layers was normally larger than 1Rm. Finally
the PDMS block was bonded to glass surface at 80°C
overnight. After fabrication, the chip was glued and wire
bonded onto a printed circuit board (PCB).
Figure 3. Device design layout (A) and SEM of Platinum black
electroplated electrodes (B).
IV. RESULTS
The effectiveness of platinum black electroplating was
studied by measuring the impedance spectra with LF
impedance analyzer (HP 4192A) before and after platinum
black electroplating on devices filled with electrolyte
phosphate buffered saline (PBS). Fig.4 and Fig.5 are the Bode
and the Nyquist plot of simulated and measured system
impedance data. Before platinum black electroplating, the
system impedance response shows capacitive characteristics in
all the frequency range. And the impedance responses in air,
521
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DI water and PBS have no difference (data no shown here).
After Platinum black electroplating, resistive characteristic in
the frequency range from 100Hz to 3MHz (the plateau in the
figure) showed up. This is where the channel resistance
dominates and it is the frequency range suitable for cell
sensing. CPE parameter oc was determined to be around 0.5
from fitting the data to both plots, which indicated extreme
surface roughness after Platinum black electroplating and the
surface seemed to be 3D in electrochemical sense.
60k
50k
0-
a
C)
0)
4ok :
30k F
20k _
-10
-20
-30
-40
560
-60
-70
-80
-90
0,
0
.0
"0
0.
E
1k 10k 100k 1 N lOM
Frequency (Hz)
Figure 4. Bode plot of simulated and measured system impedance
magnitude (solid lines) and phase (dash lines) response in electrolyte PBS.
Data were fitted by least-squares curve-fitting, with parameters Zo=2.24M.Q,
COt=0.5pF and oc=0.5.
30k
Linear: circular, R C, CL
|Z-A 5B. (z a)2, (Z bb)2 r (k( ) (pF)
A (k() B R2 a b (() r Rz
| ~~~~(kQ) (kQ} le
-35.22 1.074 ± 0.99 16.9 -281 17.9 0.99 35.8 3.6 0.52
0.25 0.006 86 0.2 197 + 0.2 67
E
lok
0 10k 20k 30k 40k 50k 60k
ZRe(Q)
Figure 5. Nyquist plot of measured impedance data. The table contains
parameters obtained by fitting Nyquist plots to asymptotes. R2 is the multiple
correlation coefficient.
For particle sensing, the sample was pumped into device
with a syringe pump. The system was excited with 100mV
sinusoidal wave at 10kHz. Output signal was amplified with a
lock-in amplifier. Data was recorded with a deep memory
oscilloscope and then transferred into a computer. The peaks
were detected with a Matlab program.
The particle sensing was first calibrated with polystyrene
beads with different sizes. The flow rate was lnLmin. The
particle concentrations were in the range of 104/pL. Fig.6
shows a time trace and a typical peak of 8ptm polystyrene
beads. The peak height was 0.2608V+0.0127V, which meant
under 5% standard deviation. The duration of the peaks was
17.21ms±0.67ms, which corresponded well with the expected
time that the beads passed the sensing zone. We also
measured signals from 5ptm and 10ptm polystyrene beads as
plotted in Fig.7. Theoretical channel resistance change under
DC excitation was calculated and plotted as the dash line. The
measured bead resistance changes fit well on the curve. It is
well know as Coulter principle that under DC excitation the
signal magnitude is determined by the size of the particles.
Our measurement results confirmed that for 10kHz excitation
frequency, the magnitude of resistance change was still
determined by the size of the particles.
(A) (B)
Tine (Se)} Tme SecI
Figure 6. Sensing of 8ptm beads tested at lOkHz. (A) a time trace. (B) a
typical peak. The solid line is the least-squares curve-fitting to parabola.
14.Ok
12.Ok
a
10 10.0k
m 8Ok
0
c 6.Ok
0 40Ok
0
2.Ok
0.0
2 4 6 8
Particle Diameter (gtm)
10
Figure 7. Sensing signal depends on the size of polystyrene beads. Dash
line is theoretical relationship at DC.
Human blood cell sensing was performed with either
diluted whole blood or diluted leukocyte rich plasma. Whole
blood samples were obtained from healthy donors and used
within 48 hours. Normal whole blood has an erythrocyte to
leukocyte ratio about one thousand to one. So it is good for
erythrocyte characterization. Leukocyte rich plasma was
prepared with Wintrobe method. Erythrocyte to leukocyte
ratio can be reduced by two orders of magnitude. So it is
better for leukocyte testing. Fig.8 shows a time trace and some
example peaks for human whole blood diluted by one
thousand times. The signal to noise ratio was not as good as
the 8ptm beads and slowly drifting of baseline was sometimes
experienced. The distribution of pulse height matched well
with the known volume distribution of erythrocytes (Fig.9).
Leukocyte rich plasma was used for leukocyte sensing. The
tail part of the peak height distribution was contributed by
leukocytes. It resembles the known leukocytes volume
distribution (Fig. 10).
V. CONCLUSION AND DISCUSSION
We demonstrated sensing of human erythrocytes and
leukocytes with platinum black electroplated micro impedance
sensors with high signal to noise ratio. The advantages of
downsizing electrodes and the sensing zone in micro devices
522
-Measurembent after Electroplating
- Simulation after Electroplating
Measurement before Electroplating
Simulation before Electroplating
[UK _ t. . . .. . ,, -l A Ev
I i
7
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-
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0
12-1
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include increased sensitivity, reduced coincidence errors,
lowered sample dilution factor and thus increased system
throughput.
detail of the tail of the distribution. Top inset is leukocyte volume
distribution [18].
ACKNOWLEDGMENT
This work is supported by the National Space Biomedical
Research Institute through NASA NCC 9-58. The authors
would like to thank other members of the Caltech
Micromachining Laboratory for their valuable assistance.
, >
m
(A) Time (Sec)
.(C) (m
Figure 8. Sensing cells in diluted human whole blood. (A) A time trace.
(B) A typical peak. (C) Two close peaks. (D) Two peaks too close to be
seperated. The solid lines are least-squares curve-fittings to parabola.
40 _
30 k
0
20 _
10k
0
U.U0 U.04 U.0b U0U0 U.1U
Pulse Height (V)
Figure 9. Pulse height distribution of diluted human whole blood, in
which leukocyte to erythrocyte ratio is about one to a thousand. Inset is
erythrocyte volume distribution from [18].
500
400
= 300
0
0
200
100
0 I
0.0 0.1 0.2 0.3 .4 0.5 0.6 0.7
Pulse Height (V)
Figure 10. Pulse height distribution of leukocyte rich plasma, in which
leukocyte to erythrocyte ratio is about one to ten. Middle inset shows the
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Clinical Laboratory Hematology:

ClinicalLaboratory Hematology: Prentice Hall,

Patent 2656508,

Progress In

Sensors and actuators.

https://authors.library.caltech.edu/18105/1/Zheng2007p91842007_2Nd_Ieee_International_Conference_On_NanoMicro_Engineered_And_Molecular_Systems_Vols_1-3.pdf

Human Blood Cell Sensing with Platinum Black Electroplated Impedance Sensor

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