Mini-electrochemical detector for microchip electrophoresis.

Abstract

This paper presents the development of a mini-electrochemical detector for microchip electrophoresis. The small size (3.6 x 5.0 cm2, W x L) of the detector is compatible with the dimension of the microchip. The use of universal serial bus (USB) ports facilitates installation and use of the detector, miniaturizes the detector, and makes it ideal for lab-on-a-chip applications. A fixed 10 M ohm feedback resistance was chosen to convert current of the working electrode to voltage with second gain of 1, 2, 4, 8, 16, 32, 64 and 128 for small signal detection instead of adopting selectable feedback resistance. Special attention has been paid to the power support circuitry and printed circuit board (PCB) design in order to obtain good performance in such a miniature size. The working electrode potential could be varied over a range of +/-2.5 V with a resolution of 0.01 mV. The detection current ranges from -0.3 x 10(-7) A to 2.5 x 10(-7) A and the noise is lower than 1 pA. The analytical performance of the new system was demonstrated by the detection of epinephrine using an integrated PDMS/glass microchip with detection limit of 2.1 microM (S/N = 3).

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Mini-electrochemical detector for microchip electrophoresis
Lei Jiang,abYao Lu,abZhongpeng Dai,aMinhao Xiecand Bingcheng Lin*a
Received 19th April 2005, Accepted 13th June 2005
First published as an Advance Article on the web 13th July 2005
DOI: 10.1039/b505467b
This paper presents the development of a mini-electrochemical detector for microchip
electrophoresis. The small size (3.6 6 5.0 cm2, W 6 L) of the detector is compatible with the
dimension of the microchip. The use of universal serial bus (USB) ports facilitates installation and
use of the detector, miniaturizes the detector, and makes it ideal for lab-on-a-chip applications. A
fixed 10 MV feedback resistance was chosen to convert current of the working electrode to
voltage with second gain of 1, 2, 4, 8, 16, 32, 64 and 128 for small signal detection instead of
adopting selectable feedback resistance. Special attention has been paid to the power support
circuitry and printed circuit board (PCB) design in order to obtain good performance in such a
miniature size. The working electrode potential could be varied over a range of ¡2.5 V with a
resolution of 0.01 mV. The detection current ranges from 20.3 6 1027A to 2.5 6 1027A and
the noise is lower than 1 pA. The analytical performance of the new system was demonstrated
by the detection of epinephrine using an integrated PDMS/glass microchip with detection limit of
2.1 mM (S/N 5 3).
1. Introduction
Miniaturized total analysis systems (m-TAS), also called lab-
on-a-chip, open up new possibilities for the miniaturization of
chemical and biochemical analysis systems.1One of the most
significant advantages associated with these devices is the
increased potential for automation and portability,2thereby
reducing the amount of hands-on labor and enabling more
immediate on-site analytical testing.
Microchip is an emerging technology that has generated a
great deal of interest. Many processing steps such as stacking,
PCR and protein digestion have been integrated on the
microchip. However, while microchip devices are small and
require little sample and reagent consumption, they are
generally connected to large, bulky detection systems.1
Without developing and integrating miniature microchip
systems, the ultimate effectiveness, flexibility, and potential
applications of microchip systems are limited.3
Recently there have been a few reports involving the
development of miniaturized or portable microchip detection
systems. LIF and other optical approaches do present
significant opportunities for ‘‘whole system’’ miniaturization,
but the electrochemical detection methodologies, in which the
detectionelectrodesareintegrated
microchip during microfabrication, would seem to offer an
alternative.4
Many researchers are devoted to applying
electrochemical detection on microchip CE, which includes
electrode integrations,5–8
sensitivity improvement9–12
application expansion.13–15
directlyonto the
and
A few researchers are devoted to developing the portable
electrochemical system for the microchip. Martin et al.16
reported a battery powered and electrically isolated potentio-
stat circuit for electrochemical detection in the microchip CE
system. The more impressive electrochemical devices to date
are Baldwin’s4mini-microchip electrophoresis system with a
portable high-voltage power supply and electrochemical
detection circuits, but an additional DAQ card is needed to
provide data acquirement and relay controlling.
In this paper, we describe a mini-electrochemical detector
(3.6 6 5.0 cm2, W 6 L), only slightly larger than a microscope
slide. The most attractive feature of this system is the
utilization of universal serial bus (USB) ports to communicate
with the PC and to get power for the electrochemical detection
circuit, which facilitates installation and the use of the
detection system, miniaturizes the detector and is convenient
for portability and in-site detection. A fixed 10 MV feedback
resistance was chosen to convert the current of the working
electrode to voltage instead of adopting selectable feedback
resistance. Special attention has been paid to the power
support circuitry and printed circuit board (PCB) design in
order to obtain good performance in such a miniature size.
The analytical performance of the new USB-based detection
system was demonstrated by the detection of epinephrine using
our integrated PDMS/glass microchip with detection limit of
2.1 mM (S/N 5 3).
2. Materials and methods
2.1 Chemicals
SU-8 2035 photoresist was purchased from MicroChem and
propylene glycol methyl ether acetate (PGMEA) from Aldrich
was used as developer. Sylgard 184 silicone elastomer and
curing agent were obtained from Dow Corning. Epinephrine
was purchased from Sigma Chemical Co. (St. Louis, MO) and
aDalian Institute of Chemical Physics, Chinese Academy of Sciences,
Dalian,116023,China. E-mail: bclin@dicp.ac.cn; Fax: 86 411 84379065;
Tel: 86 411 84379059
bGraduate School of the Chinese Academy of Sciences, Dalian, 116023,
China
cDepartment of Human Sport Science, Beijing Sport University, Beijing,
100084, China
PAPERwww.rsc.org/loc | Lab on a Chip
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was used as received without further purification. Aqueous
solutions were prepared using analytical grade reagents and
double-distilled water.
2.2 Apparatus and electrical components
The electronic diagram of the electrochemical detector is given
in Fig. 1. Electrical components building the system include
USB to UART BRIDGE CP2101 (from Silicon Laboratories,
Austin, Texas), microcontroller MSC1211Y5 and operational
amplifier OPA129 (from Texas Instruments Inc., Dallas,
Texas),DC–DCconverter
Devices Inc., Norwood), as well as other elements such as
resistors, potentiometers and capacitors purchased from a
local electrical market.
ADM660(from Analog
The high voltage power supply (HVPS), designed and
fabricatedin-house, provides
electrical nodes for performance of microchip separation.
The HVPS was built by one positive (C40) and one negative
(C20N) DC–HVDC converters using an input voltage of
11–15 V and the output voltage of C40 and C20N was
0–4000 V and 22000–0 V respectively. The microchip
electrophoresis system is schematically shown in Fig. 2.
independently-controlled
2.3 Microchip fabrication
The method used to create channels in PDMS is based on
previously published procedures.17A 3 inch silicon wafer was
cleaned and oxidized with piranha solution (3 : 1 H2SO4/H2O2)
(Caution! Piranha solution is a powerful oxidizing agent that
reacts violently with organic compounds; it should be handled
with extreme care) and was then coated with a SU-8 2035
negative photoresist by use of a spin coater. The coated wafer
was baked at 65 uC and 95 uC for 3 min and 6 min respectively
and a digitally produced mask was then placed on the
photoresist. After the system was exposed to a near-UV light
source for 3 min and post baked at 95 uC for 6 min, the wafer
was developed in propylene glycol methyl ether acetate,
leaving a positive relief patter on the wafer. Once the master
was completed, replica molding was used to create channels in
the PDMS. A degassed mixture of PDMS and Sygard 184
elastomer curing agent (10 : 1) was poured onto the silicon
wafer and cured at 80 uC for 2 h. The PDMS was then peeled
off the silicon wafer, leaving a negative relief of the channels in
the PDMS. The electrode plates were constructed according to
Lunte’s18method and not stated here. Then the PDMS layer
and the electrode plate were reversibly bonded together by
aligning the working electrode on the electrode plate close to
the end of the separation channel (50 mm) with the aid of a
light microscope. The channel is 50 mm wide and 25 mm deep.
Fig. 1
of the mini-electrochemical detector (3.6 6 5.0 cm2, W 6 L).
(A) Schematic of the mini-electrochemical detector; (B) picture
Fig. 2
reservoir; B—buffer reservoir; BW—buffer waste reservoir; SW—
sample waste reservoir; W(1,2,3)—working electrode; R—reference
electrode; C—counter electrode; G—ground; HV1—high voltage 1
(positive high voltage); HV2—high voltage 2 (negative high voltage));
effective separation length is 4.0 cm.
Schematic of the microchip electrophoresis system (S—sample
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2.4 Microchip elecrophoresis procedure and electrochemical
detection
10 mM stock solutions of epinephrine were prepared in water
and appropriate dilutions were made with the corresponding
CE runing buffer prior to use. Channels of the chip were
treated before use by rinsing with 0.1 M NaOH and deionized
water for 10 min and 5 min respectively. The channels, buffer
reservoir, buffer waste reservoir and sample waste reservoir
were first filled with the corresponding CE running buffer and
the sample reservoir with the sample. Injection was then
carried out by applying the desired negative potential to the
sample waste reservoir and an output voltage of zero to the
buffer reservoir for 20 s with both the sample reservoir and
the buffer waste reservoir grounded. Once the injection was
completed, separation was performed by applying the corres-
ponding voltages to the buffer reservoir with the buffer waste
reservoir grounded and both the sample and sample waste
reserviors floating.The potential was applied to the working
electrode during the electrophoretic separations. The injection
was performed after a baseline stabilization.
2.5 Software and data treatment
The electrochemical detector and the HVPS were controlled by
software written using Microsoft Visual C++6.0 on an external
PC under the windows environment. The firmware was
developed in C language and stored in the flash memory of
the microcontroller. The experimental controlling parameters,
including running time and electrochemical potential, were
initialised in the parameter setting at the beginning of the
experiment. The running time was separated into six steps in
this experiment and all parameters could be set differently in
each step (six steps can satisfy the requirement for most
detection experiments; if not, the user can set the desired
number of steps by modifying the program). The parameters
weresimultaneouslytransferred
(MSC1211Y5) while the experiment was running. The time
of the steps was controlled by a windows multimedia timer
with a high resolution of 1 ms, which was essential to
microchip electrophoresis because the analysis time was very
short. The experiment stopped when the running time expired
and all parameters were automatically set to zero. Meanwhile
it could also be interrupted at any time if so desired by the user
by pushing the ‘‘stop’’ button on the software tool bar. The
data were transferred to the PC at the rate of 10 samples per
second, and were displayed on the PC screen simultaneously.
The data were stored on the PC using the special format we
defined in the VC++ and could also be easily converted into
TXT format for a different choice of data treatment.
tothe microcontroller
3. Results and discussion
3.1 Construction and design of the detector
The overall physical dimensions of our electrochemical
detector are 3.6 6 5.0 cm2(W 6 L), only slightly larger than
a microscope slide. This miniature detector was constructed by
three parts: controlling unit, communication unit and analog
unit. The circuit of the detector is schematically shown in
Fig. 1.
The controlling unit is based on a fully integrated mixed-
signal system-on-a-chip 8-bit microcontroller MSC1211Y5
operating with a 24 MHz system clock. Its high integration
of functions in a 12 mm 6 12 mm small size of 64-pin TQFP
suffices for the electrochemical detection used in microchip
electrophoresis while remarkably miniaturizing the controlling
unit of the system.
One of the most attractive features of this miniature
electrochemical detector is the use of USB ports in the
communication unit. USB ports, with fast data transport rates
and supporting plug-and-play installation and hot swapping,
can thereby realize fast and reliable communication with the
PC and facilitate installation and use of the detector. Also,
USB has the function of distributing electrical power to the
peripherals, which enhances the miniaturization of the detec-
tion system by eliminating those clunky power supply boxes.
Therefore, the USB-based miniaturized detector system is
convenient for portability and in-site detection and ideal for
potential lab-on-a-chip applications. In the detector, CP2101,
a highly integrated USB-to-UART Bridge Controller provid-
ing a simple solution for USB design using a minimum of
components and PCB space (5 6 5 mm), was chosen to
implement the USB functions.
On the EC detection circuit, one DA of MSC1211Y5 was
used to convert the digital representation of the electrochemi-
cal potential function into an analog signal, which was then
applied to the input of the potentiostat. Because of the
uniploar characteristics of the DAs, an operational amplifier
was used to shift the level of an output (22.50 to +2.50 V) from
a D/A internal (0 to +2.50V) of the microcontroller. An ultra-
low bias current (100 fA max.) operational amplifier OPA129
was used to ground the working electrode potential and acted
as a current converter. Because the current of electrochemical
detection in microchip electrophoresis is usually in the order of
nA, the fixed 10 MV feedback resistance was chosen to convert
the current of the working electrode to voltage instead of
adopting selectable feedback resistance. For small signal
detection, the MSC1211Y5 internal programmable gain
amplifier (PGA) was used to provide a second gain of 1, 2,
4, 8, 16, 32, 64 and 128. This simplified the design and reduced
the size of the electrochemical detector while providing
sufficient functions for microchip electrophoresis.
To enable optimal performance of an electrochemical
detector requires much attention to its power support circuitry
and printed circuit board (PCB) design. As shown in Fig. 1(B),
separation of the digital parts (communication unit and
controlling unit) and the analog parts (AD, DA and
electrochemical detection unit) helps to avoid their reciprocal
interference during system operation. Special attention has
also been paid to the analog inputs in order to improve their
signal to noise (S/N). First, low-pass filters were placed at both
the input end and the output end in the OPA129 circuit to
minimize high-frequency electronic noise. Second, the OPA129
input pad is designed as short as possible and shielded with
ground pads. Accordingly, the working electrode potential
could be varied over a range of ¡2.5 V with a resolution of
0.01 mV by using an MSC1211Y5 internal 16 bit DAC. The
detection current ranges from 20.3 6 1027A to 2.5 6 1027A
and the noise is lower than 1 pA. In the miniaturized
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electrochemical detector, consideration has also been given to
the electrode treatment by combining with sin and square
electrode treatment procedures for the user’s selection in case
electrode fouling occurs.
3.2 Microchip CE/EC
The
detection system was characterized for the anodic detection
of epinephrine.
Previous studies with CE/EC have shown that an end-
channel detection scheme can lead to positive or negative shifts
in the half-wave potentials of oxidizable compounds,19and it is
also true for microchip-CE/EC. The optimum potentials
applied for microchip-CE/EC are shifted with both the
separation voltage and the distance of the electrode from
the end of the channel. In order to find the potentials with the
maximum response, a series of potentials should be tested for
each analyte of interest. In the mini-electrochemical detector
we have developed, the potentials could be easily set in the
program. The electrode potential vs. the signal is shown in
Fig. 3. It can be seen that the response rises gradually between
+0.3 V and +0.6 V and remains almost constant from +0.6 V to
+1.0 V, after which it rises again and levels off at +1.1 V. A
dramatic increasein the
corresponding noise was observed at potentials higher than
+1.0 V. Therefore, a potential of +0.8 V was finally selected as
the optimum detection potential because it can provide
good recurrenceaswell
characteristics. Fig. 3 indicates that our mini-electrochemical
detector provides effective
electrophoresis.
As illustrated in Fig. 4, five times detection of 0.1 mM
epinephrine is used to show the recurrence of the electro-
chemical detector. Because of the high diffusibility of
epinephrine, the sample could easily leak into the separation
channel before the injection, leading to imperfect recurrence of
the result. In our experiment, the injection was carried out by
applying the desired negative potential to the sample waste
reservoir and an output voltage of zero to the buffer reservoir
for 20 s with both the sample reservoir and the buffer waste
reservoir grounded. Both the sample in the sample reservoir
analyticalperformanceofthe electrochemical
baselinecurrent and the
as thebest signal-to-noise
detection formicrochip
and the buffer in the buffer reservoir and buffer waste
reservoir are pumped by electro-osmotic flow (EOF) to the
sample waste reservoir. Thus, even if leakage of epinephrine
into the separation channel occurs before the injection, the
leaked epinephrine can be effectively drawn back to the
injection channel during the injection process. A long injection
time (20 s) could also help to form a well-defined sample
plug at the channel intersection during injection. Therefore,
recurrence of the result was achieved in our experiment as
shown in Fig. 4. The relative standard deviation (RSD) for the
migration time and the peak height is 3.37% and 3.61% (n 5 5)
respectively. The detection limit of epinephrine using our
integrated PDMS microchip is 2.1 mM (S/N 5 3), which is
comparable to that of the fully integrated electrode glass
microchip.
Summary
The goal of the present work is to show the USB-based mini-
electrochemical detector in a move towards the development of
complete miniaturized electrochemical devices. The mini-
electrochemical detector (3.6 6 5.0 cm2, W 6 L) we have
developed is compatible with the dimension of the microchip
with USB ports to communicate with the PC and to obtain
power for an electrochemical detection circuit. A fixed 10 MV
feedback resistance was chosen to convert the current of the
working electrode to voltage with a second gain of 1, 2, 4, 8,
16, 32, 64 and 128 for small signal detection instead of
adopting suitable feedback resistance. Special attention has
been paid to the power support circuitry and printed circuit
board (PCB) design in order to obtain good performance on
such a miniature scale. The analytical performance of the new
USB-based detection system was demonstrated by the detec-
tion of epinephrine using an integrated PDMS/glass microchip
with a detection limit of 2.1 mM (S/N 5 3).
Acknowledgements
This project was supported by the National Natural Science
Foundation of China (29975030), (20035010), (20299030) and
the Olympic project (2002BA904B04-04).
Fig. 3
Conditions: 20 mmol L21phosphate buffer, pH 8.0; injection voltage
600 V, 20 s; separation voltage, 800 V; detection potential (vs. Pt).
Hydrodynamic voltammogram for 0.1 mM epinephrine.
Fig. 4
2nd time, (c) 3rd time, (d) 4th time, (e) 5th time. Conditions:
20 mmol L21phosphate buffer, pH 8.0; injection voltage 600 V, 20 s;
separation voltage, 800 V; detection at 0.8 V (vs. Pt).
Five times detection of 0.1 mM epinephrine. (a) 1st time, (b)
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References
1 D. R. Reyes, D. Iossifidis, P. A. Auroux and A. Manz, Anal.
Chem., 2002, 74, 2623.
2 J. Wang, Trends Anal. Chem., 2002, 21, 226.
3 R. F. Renzi, J. Stamps, B. A. Horn, S. Ferko, V. A. VanderNoot,
J. A. A. West, R. Crocker, B. Wiedenman, D. Yee and J. A. Fruete,
Anal. Chem., 2005, 77, 2, 435.
4 D. J. Jackson, J. F. Naber, T. J. Roussel, Jr., M. M. Crain,
K. M. Walsh, R. S. Keynton and R. P. Baldwin, Anal. Chem.,
2003, 75, 14, 3643.
5 A. Hilmi and J. H. T. Luong, Anal. Chem., 2000, 72, 19, 4677.
6 J. Yan, Y. Du, J. Liu, W. Cao, X. Sun, W. Zhou, X. Yang and
E. Wang, Anal. Chem., 2003, 75, 20, 5406.
7 R. S. Martin, A. J. Gawron, S. M. Lunte and C. S. Henry, Anal.
Chem., 2000, 72, 14, 3196.
8 R. P.Baldwin, T. J. Roussel, Jr., M. M. Crain, V. Bathlagunda,
D. J. Jackson, J. Gullapalli, J. A. Conklin, R. Pai, J. F. Naber,
K. M. Walsh and R. S. Keynton, Anal. Chem., 2002, 74, 15, 3690.
9 N. A. Lacher, S. M. Lunte and R. S. Martin, Anal. Chem., 2004,
76, 9, 2482.
10 D. M. Osbourn and C. E. Lunte, Anal. Chem., 2001, 73, 24,
5961.
11 C. C.Wu, R. G. Wu, J. G. Huang, Y. C. Lin and H. C. Chang,
Anal. Chem., 2003, 75, 4, 947.
12 N. E. Hebert, W. G. Kuhr and S. A. Brazill, Anal. Chem., 2003, 75,
14, 3301.
13 J. Wang, A. Ibanez, M. P. Chatrathi and A. Escarpa, Anal. Chem.,
2001, 73, 5323.
14 J. Wang, A. Escarpa, M. Pumera and J. Feldman, J. Chromatogr.
A, 2002, 952, 249.
15 J. Wang, M. Pumera, M. P. Chatrathi, A. Escarpa, M. Musameh,
G. Collins, A. Mulchandani, Y. H. Lin and K. Olsen, Anal. Chem.,
2002, 74, 1187.
16 R. S. Martin, K. L. Ratzlaf, B. H. Huynh and S. M. Lunte, Anal.
Chem., 2002, 74, 5, 1136.
17 D. C. Duffy, J. C. McDonald, O. J. A. Schueller and
G. M. Whitesides, Anal. Chem., 1998, 70, 4974.
18 R. S. Martin, A. J. Gawron and S. M. Lunte, Anal. Chem., 2000,
72, 3196.
19 S. R. Wallenborg, L. Nyholm and C. E. Lunte, Anal. Chem., 1999,
71, 544.
934 | Lab Chip, 2005, 5, 930–934 This journal is ? The Royal Society of Chemistry 2005