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CHINESE JOURNAL OF ANALYTICAL CHEMISTRY

Volume 42, Issue 1, January 2014


Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2014, 42(1), 2127.

RESEARCH PAPER

MicroreagentDispensingMethodBasedonPulseDriving
&ControllingofMicrofluidsTechnologyandApplication
Research
ZHENG Yue*, HOU Li-Ya, ZHU Li, WANG Hong-Cheng, HE Jia-Qiao, ZHANG Wei-Yi
School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China

Abstract: With the development of bioanalytical technique and high throughput screening techniques, the amount of reagent was
decreased to nanoliter and even picoliter level. To achieve high precision of micro-reagent dispensing, a two-channel micro-reagent
dispensing system based on pulse driving & controlling technique of micro-fluids was developed. Then, taking glycerine solution as
dispensing reagent, influences of viscosities, inner diameter of the micro-nozzle, driving frequency, and voltage amplitude on the
dispensing volume were investigated. With the voltage amplitude of 70 V, driving frequency of 4 Hz, and 100 m inner diameter of
the micro-nozzle, different ratios of Na2HPO4 to KH2PO4 solution were prepared to study the mixing-reaction. In the experiment, a 3
3 phosphate buffer arrays solution with a pH gradient was first prepared. pH indicator was then added to detect the acidity and
alkalinity of the mixed solution. The results showed that the relative standard deviation (n = 9) of spots diameter of the prepared pH
gradient arrays was 0.8%; the spots were fully reacted, and uniform colors and significantly changed gradients were achieved. By the
micro-reagent dispensing method based on pulse driving & controlling of micro-fluids technique, parallel, automated and micro
volume (picoliter) dispensing with higher dispensing precision and higher repeated accuracy could be realized, and especially, micro
reaction with different ratios could be achieved without separate reaction pools.
Key Words:

Micro-reagent dispensing; Two-channel; Pulse driving and controlling of micro-fluids technology

Introduction

High-speed and accurate micro-reagent dispensing which is


an indispensable technology has been widely used in the fields
of life science such as protein crystallization, drug screening,
gene sequencing and so on[15]. To realize micro reaction and
analysis, different kinds of reagents are usually needed to be
transferred into the same reaction pools. With the
development of bio-analysis technique and high-throughput
screening technique, the sample volumes should be reduced to
increase the screening chance with a given amount of sample.
Therefore, high dispensing speed and accuracy are required in
a single experiment. It is of great significance to develop

novel method of automatic liquid dispensing for transferring


micro-reagent, which can improve the dispensing accuracy,
decrease the time involved and reagent consuming, thus
enhance experimental techniques in biomedical areas[69].
Currently, automatic liquid dispensing mainly includes
contact and non-contact methods. In the contact dispensing
method, the nozzle contacts with the substrate, and reagent is
transferred to the substrate due to interface external force.
Although this method is simple and flexible, it may bring
pollution while the nozzle contacts the substrate and it is
difficult to achieve accurate dispensing when reagent volume
reduces to nanoliter scale[10].
In non-contact dispensing method, the nozzle needs not to

Received 7 August 2013; accepted 5 Sepember 2013


* Corresponding author. Email: yuezzz@126.com
This work was supported by the National Natural Science Foundation of China (Nos. 51175268, 11102090), and the Specialized Research Fund for the Doctoral
Program of Ministry of Education, China (No. 20113219110004).
Copyright 2014, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
DOI: 10.1016/S1872-2040(13)60703-3

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

contact with the substrate, an external force is used to provide


enough energy to overcome liquid surface tension and
viscosity, and then the droplet could be ejected out of the
nozzle. Compared with the contact dispensing method,
non-contact dispensing method is a mainstream way for
micro-reagent dispensing due to its high-speed, non-pollution
and micro volume. The current non-contact micro-dispensing
technology varies on the basis of the driving types such as
piezoelectric, thermal and pneumatical driving. Piezoelectric
dispensing is realized by the displacement of piezoelectric
elements applied with a pulse voltage signal, with which the
volume of the chamber can be changed and a droplet is jetted
out of the orifice. It has several advantages such as fast
response, wide frequency range and picoliter dispensing
volume. However, the piezoelectric head is structural
complexity, high cost and difficult to disassemble and
repair[11]. The thermal driving type is simple and can be used
in high-density integration of nozzle arrays. However, it may
affect the activity of the biological sample as it needs to be
heated while the droplet is ejected from the nozzle[12].The
pneumatic driving type, with a simple structure, is widely used
and can work at higher temperature environment. However,
unsteady ejection may occur with the gradual decline of liquid
level in the chamber, which will result in poor droplets
controllability[13].
Pulse driving and controlling of micro-fluids technique,
also called micro fluidic digitalization technique, could realize
micro-reagent (picoliter level) dispensing with simple,
pyrogen-free and low-cost micro dispensing system[14]. In
order to solve the problems mentioned above in the micro
reagent dispensing technology, a novel precision dispensing
system based on pulse driving and controlling technique of
micro-fluids was developed, and automated two-channel
dispensing of micro-reagent was realized in this study. The
impacts of system parameters on the dispensing amount were

experimentally investigated. Moreover, a pH gradient


microarray of phosphate buffer solution was prepared by
mixing Na2HPO4 and KH2PO4 solution in different ratios with
the two-channel dispensing system.

Materials and methods

Pulse inertia force generated by various impact or vibration


is the driving source to produce droplets in the pulse driving
and controlling of micro-fluids system. In this study,
piezoelectric actuator was used as the driving source to
provide micro-fluidic pulse inertia force, since the pulse
inertial force generated by piezoelectric devices had many
merits, such as high response frequency and excellent
dynamic response of voltage-displacement. The parameters of
pulse driving and controlling of micro-fluids system were as
follows: driving voltage waveform (driving waveform),
driving frequency f, voltage amplitude U and micro-nozzle
inner diameter d. As shown in Fig.1, by applying a driving
waveform, piezoelectric actuators generated a pulse inertia
force[15]. When the amplitude of the voltage rose instantly
from 0 V to the maximum, the direction of the acceleration
generated by piezoelectric actuator would be changed, which
provided pulse inertia force to the micro-nozzle. The
acceleration of the piezoelectric actuator could be adjusted by
changing the voltage amplitude, which was used to control the
pulse inertia force, and thus to accurately control the volume
of a single injection of the liquid. The devices with neither
micro movable parts nor embedded microcircuits have the
advantages of high reliability, resisting solid particles jam and
bubbles block, simple structure, low-cost, and appropriate
working conditions which is of benefit to maintain the
biological activity[16].
The schematic diagram of the two-channel micro-reagent
dispensing system is shown in Fig.1.

Fig.1 Schematic diagram of two-channel micro-reagent dispensing system

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

The micro-nozzle was connected with the movable ends of


a piezoelectric actuator. Influenced by the piezoelectric
actuator, micro droplets were jetted out of the micro-nozzle. A
pressure adjusting device was fixed at the end of the
micro-nozzle, and it generated positive or negative pressure to
realize the regent providing, discharging, and the cleaning of
the micro-nozzle. A digital microscope was also used for
real-time monitoring the dispensing status.
The control circuit of this system was constructed on the
basis of FPGA (Field Programmable Gate Array) built-in
32-bit soft core processor, which could provide two-channel
driving waveform. Upper computer sent control word and data,
and communicated with the lower computer through RS232
interfaces. The voltage amplitude and frequency of the driving
waveform were adjustable with the voltage amplitude from 0
to 80 V and driving frequency ranging from 1 Hz to 256 Hz.
After being amplified by the power amplifier, the waveform
could drive the piezoelectric actuator. Planar worktable moved
along with the driving waveform with the following manner:
when planar worktable arrived at one position, single or two
channels would generate single or multiple driving waveforms
to drive piezoelectric actuator, then micro droplet would be
injected and deposited onto the substrate. After the above
process, the planar worktable moved to the next position. The
steps are repeated to realize the accurate dispensing.
Micro-nozzle is a key device of the system. Borosilicate
glass capillary with good chemical resistance was chosen to
make the micro-nozzle. In the fabrication process, the laser
beam was projected on the central part of a glass capillary to
heat it. When the temperature exceeded a certain value, the
glass capillary would be elongated under the tension action on
both sides. The parameters, such as heating temperature, time
and position were adjusted to control the morphology of
micro-nozzle. Then the glass nozzle could be forged to obtain
micro-nozzles with different diameters by a micro forger
(MF-900, Narishige Co., Ltd.). The micrograph of the forged
micro-nozzle is shown in Fig.2. The micro-nozzle with an
inner diameter of 100 m, a front contract angle 1 of 35 and
a rear contract angle 2 of 3.9 was used in the present study.

Results and discussion

In the micro-reagent dispensing method based on pulse


driving & controlling technology of micro-fluids, the single
dispensing volume is influenced by parameters of the system,

Fig.2 Microphotograph of micro-nozzle

the viscosity of reagents, the inclined angle (the included


angle between micro-nozzle and vertical direction) and so on.
Among them, the viscosity of reagents, the driving frequency,
the voltage amplitude and the inner diameter of micro-nozzle
have significant influences[15]. Therefore, the basic
experiments were carried out under the conditions with
inclined angle of 15, micro-nozzles of various inner
diameters, reagents with different viscosities and driving
signals with different frequencies and voltage amplitudes.
Based on the basic experiments, Na2HPO4 and KH2PO4 solutions
were used to prepare the microarray with a pH gradient.
In order to make the solutions to be mixed and reacted
sufficiently and improve the contact angle and the roundness
of the micro droplet, the glass or ceramic substrates were first
treated with a hydrophobization process. As is known, the
surface hydrophobization mainly contains two methods. In the
first method, the rough surface is modified by the materials
with low surface energy. In another method, roughened
surfaces are fabricated on the substrates with low surface
energy. In this study, the hydrophobic film was prepared by a
sol-gel process with amino acrylic resin as the forming film
resin and xylene as the solvent. They were stirred and mixed
with the SiO2 nanoparticles in a high speed shearing mode.
After letting it sat for 510 min, the sol suspension was
formed. The obtained sol suspension was then sprayed on the
glass substrate uniformly. The glass substrate covered with
hydrophobic film was finally obtained after keeping the
substrate in a hot oven (approximately 150 C) for 20 min[17].
3.1

Measurement of micro droplet volume

To the best of our knowledge, none of the existing


dispensing systems with picoliter level resolution are equipped
with the function to detect the real transferred amount of
liquid. The system in the present study was also open-loop
controlled. To precisely control the volume of the dispensed
liquid, the droplet volume of single jetting needed to be
measured. Since the size of the droplet was quite small, direct
measurements of quality or volume in a traditional way could
not meet the requirement to measure the volume precisely. In
this study, the method adopted was as follows: firstly the
liquid was ejected into cedar oil, and the droplets stayed in the
form of separated spheres as shown in Fig.3 due to the
immiscibility of the two kinds of liquid. Then, the collected

Fig.3 Microphotograph of spherical micro-droplets

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

data were transferred to computer for image processing


through a microscopic image processing system with image
capture card. Finally, the dispensing volume was obtained by
measuring the mean diameter of 50 droplets.

with the inner diameter of the micro-nozzle. When the


diameter of the nozzle was 200 m, the dispensing volume
was less than that dispensed by the 175 m nozzle. Thus,
increasing the micro-nozzle inner diameter would not
necessarily improve the dispensing volume. Under the same
amplitude of the voltage, the dispensing volume was
determined by both the inner diameter of the nozzle and the
viscosity of the reagent.

3.2 Relation between the viscosity and dispensing volume


In biochemistry field, reagents with different viscosities are
often needed to be dispensed. To obtain micro-reagent
dispensing medium with a certain viscosity gradient, here,
glycerine solution was used as the micro-reagent dispensing
medium. The glycerine solution was prepared by glycerine
and deionized water in different volume ratios, thus the
viscosity of the solution dependented on the glycerol
concentration. The viscosity value was measured by rotational
viscometer (NDJ-79, Shanghai Pingxuan Scientific Instrument
Co., Ltd.) at room temperature (25C).
Experimental conditions: the micro-reagents were glycerine
solution with different viscosities, f = 4 Hz, U = 80 V (the
maximum voltage that the piezoelectric actuator could bear to
improve the micro dispensing ability), d = 100 m and room
temperature (25 C). The mean dispensing volume was
obtained by detecting the diameters of 50 droplets, and then
the RSD (Relative standard deviation) of dispensing volume
was calculated. The results are shown in Table 1.
As shown in Table 1, with the micro-nozzle of d = 100 m,
the largest reagent viscosity was 35 mPa s. All the volume
RSDs of the reagent with different viscosities were less than
2.5%. Therefore, the micro-reagent dispensing with high
accuracy and good repeatability were achieved by the method
even when the viscosity of reagent was changed.
3.3

3.4

Relation between driving frequency and dispensing


volume

Experimental conditions: the micro-reagent is glycerine


solution with 30% volume fraction, f wih a range from 1 Hz to
30 Hz, U = 70 V, d = 100 m and the temperature was 25 C.
Micro dispensing experiment was carried out to study the
effect of driving frequency on the dispensing volume. With
the same driving frequency, the diameters of 50 samples were
measured to obtain the mean dispensing volume, and the
results are shown in Table 3.
As shown in Table 3, under the above experimental
conditions, the dispensing volume decreased with the
increasing of driving frequency. Within the driving frequency
range from 1 to 30 Hz, uniform and stable dispensing could be
realized. When the driving frequency was 4 Hz, the minimum
volume RSD was obtained. When the frequency was larger
than 30 Hz, the dispensing began to be unstable and some
bubbles emerged in the tip of the nozzle. Therefore, 4 Hz was
taken as the driving frequency in the following experiments.
3.5

Relation between inner diameter of micro-nozzle and


dispensing volume

Relation between voltage amplitude and dispensing


volume

Experimental conditions: the reagent was glycerine solution


with 30% volume fraction, f = 4 Hz, U ranged from 20 V to

Experimental conditions: glycerine solution with 30%


volume fraction was used as the micro-reagent (the viscosity
was 12.5 mPa s), f = 4 Hz, U = 80 V, d = 25, 50, 75, 100, 125,
150, 175 and 200 m, at 25 C. With the same size of
micro-nozzle, the average diameters of 50-droplets were
sampled to obtain the mean dispensing volume. The results
are shown in Table 2.
As shown in Table 2, with the inner diameter of
micro-nozzle d  175 m, the dispensing volume increased

Table 1 Relation between different viscosities and dispensing volumes


Viscosity of reagent
(mPa s)

Dispensing volume
(pL)

RSD
(%, n = 50)

810
746
696
628
533
0

2.5
2.1
2.0
1.9
1.7
-

1
5
15
25
35
> 35

Table 2 Relation between different micro-nozzle inner diameters and dispensing volumes
Micro-nozzle inner diameter (m)

25

50

75

100

125

150

175

200

Dispensing volume (pL)

24

105

242

715

1210

2146

3343

2041

Table 3 Relation between different driving frequencies and dispensing volume


Driving frequency (Hz)
Dispensing volume (pL)
RSD (%, n = 50)

10

15

20

25

30

> 30

564
1.9

554
1.6

536
1.4

521
2.2

502
2.1

486
3.2

470
3.5

458
4.1

443
5.8

Unstable
-

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

Table 4 Dispensing volumes and RSD of different voltage amplitudes


Voltage amplitude (V)
Dispensing volume (pL)
RSD(%, n=50)

< 20

20

30

40

50

60

70

80

0


122
1.3

159
1.6

214
2.2

280
1.8

395
2.1

536
1.4

708
2.0

80 V, d = 100 m, at 25 C. Micro dispensing experiment was


taken to study the influence of voltage amplitude on the
dispensing volume. Under the same voltage amplitude, the
diameters of 50 droplets were measured to obtain the mean
dispensing volume. The obtained results are shown in Fig.4,
and the calculated RSDs are listed in Table 4.
The glycerine solution of 30% volume fraction was
dispensed on hydrophobized glass substrates to prepare 7 7
microarray with voltage amplitudes ranged from 20 V to 80 V.
As shown in Fig.5, the voltage amplitudes were 20, 30, 40, 50,
60, 70 and 80 V from left to right, and each column of spots
were prepared with the same voltage amplitude. The distance
between the spots center was 330 m. The 10 10 microarray
prepared with the voltage amplitude of 20 V is shown in Fig.6.
The distance between the spot center was 200 m, the average
diameter of the microarray was 142 m, the diameter RSD (n
= 100) was less than 0.7%, and the spot density was around
2500 spot cm2.

Fig.4 Relation between different voltage amplitudes and dispensing


volumes (d = 100 m)

Fig.6 Microphotograph of microarray (voltage amplitude: 20 V)

Based on the above results, when the voltage amplitude was


less than 20 V, the inertial force obtained by the liquid reagent
in the micro-nozzle could not overcome the viscous force and
the surface tension, and thus the normal dispensing could not
be realized. When the voltage amplitude was greater than 20
V, the reagent could be jetted out. The higher the voltage
amplitude was, the larger force of inertia would be obtained
by the reagent. From Fig. 4, single dispensing volume ranged
from 120 pL to 715 pL could be obtained by adjusting the
voltage amplitude.
Therefore, this method could dispense various amount of
reagent in each sample spot and the volume could be changed
in two ways: (a) changing the single dispensing volume
according to the size of micro-nozzle, driving frequency and
voltage amplitude; (b) changing the dispensing times.
3.6

Fig.5 Microphotograph of microarray (voltage amplitude: 2080 V)

Preparation of phosphate buffer solution array with


gradient pH value

Phosphate buffer solution (PBS) is usually used in


biological research to maintain the pH of experimental system.
The changes of pH value in biochemical analysis solution
often have direct impact on the analysis results[18]. Based on
the above experimental results, PBS microarray with gradient
of pH value was prepared by dispensing phosphate buffer
solution with different ratios of Na2HPO4 and KH2PO4, and
then the pH reagent was added in each spot of the microarray.
The change of pH was reflected by the chromogenic reaction.
The two-channel reagent dispensing system had two
separately controlled micro-channels, which were used for
dispensing two kinds of reagent with any proportion. The two
channels worked under the same experimental conditions with

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

U = 70 V, f = 4 Hz, d = 100 m, at 25 C. Two reagents A


(0.1 M Na2HPO4) and B (0.1 M KH2PO4) were used. To
prepare the PBS, the two reagents were dispensed on a white
hydrophobized ceramic substrate which made it easy to read
the color of the chromogenic reaction.
Glycerine solution of 30% volume concentration was added
into the Reagent A and B to change the viscosity
characteristics of the reagents and made the reagents less
volatile and thus made it easy to observe the results. Note that
the addition of glycerine solution had no appreciable effect on
the reaction between the reagents[7,19]. It should be pointed that
the low concentrations of Na2HPO4 and KH2PO4 had no
obvious effect on the reagent characteristics, and the reagent
characteristics of reagent A and B were almost same with that
of the glycerine solution, the curve in Fig.4 was used to
calibrate experimental results. 3 3 array with gradient pH
value was prepared by dispensing 10 droplets with the ratio of
the two reagents in the range from 1:9 to 9:1, and the volume
of each droplet was 536 pL. The micro-nozzle filled with
Reagent A was cleaned up and the pH reagent with 30% of
glycerine solution was then sucked in. Finally, one droplet of
the pH reagent was respectively dispensed into the pH buffer
solution spots under the conditions of U = 70 V, f = 4 Hz, and
d = 100 m. Because the droplet was jetted out of the
micro-nozzle with a constant speed and collided with the
solution on the substrate, the mixing process and reaction
between the reagents were promoted and thus the reaction
time was shortened. The spots of the microarray showed
different colors from yellow, green to blue immediately after
the pH reagent was added. As shown in Fig.7, the average
diameter of the samples was 443.2 m, and the RSD (n = 9) of
diameter was 0.8%. Moreover, the regularity and uniformity
were both fine, and the spots were fully reacted and exhibited
uniform colors, indicating that the proportion and reaction
between Na2HPO4 and KH2PO4 had been realized. The ratios
of Na2HPO4 and KH2PO4, the spot diameters, the
corresponding pH and the changing of the reaction color are
shown in Table 5.

inner diameter of the micro-nozzle, driving frequency and


voltage amplitude on the dispensing volume were investigated.
The results indicated that the viscosity of less than 35 mPa s
was suitable for the dispensing system and the dispensing
volume could be as small as picoliter. The volume RSDs (n =
50) of reagent with different viscosities were all less than
2.5%. A 10 10 microarray of glycerine solution of 30%
volume fraction was prepared on a hydrophobic glass under
the voltage amplitude of 20 V. The prepared microarray spots
were uniform (the RSD (n = 100) of diameter was 0.7%) and
the density was calculated to be 2500 spot cm2.
(2) A mixing-reaction experiment was conducted with
different ratios of Na2HPO4 and KH2PO4 solution by a
two-channel micro-reagent dispensing system. The microarray
of phosphate buffer with PH gradient was prepared on a
ceramic substrate. The microarray spots were uniform (the
RSD (n = 9) was 0.8%). The spots fully reacted, resulting in
uniform color and obvious gradient variation. The micro
proportion and reaction between the two solutions were
realized.
(3) High dispensing precision and repeatability were
realized by this method. It could be used for the micro
dispensing of solutions with different properties and the
parallel accurate dispensing of two or more solutions. By
adjusting the ratio of different reagents, micro solution with
different ratios could be realized without reaction pools. The
parallel, automated and micro volume (picoliter) dispensing
could be realized in the process of high-throughput biological
experiments. The method may have potential applications in
the fields of biochip, micro drug dispensing, biosensor,
combined materials, drug screening, microchemical analysis
and so on.

Conclusions

(1) A two-channel micro-reagent dispensing system based


on pulse driving and controlling technique of micro-fluids was
developed. The influences of viscosity of glycerine solution,

Fig.7 Microphotograph of pH gradient microarray

Table 5 Spot diameters, ratios, the corresponding pH and the color change rule
No.
Spot diameter(m)
Ratio
pH
Color change rule

A1

A2

A3

B1

438
1:9

440
2:8

439
3:7

444
4:6

B2
444
5:5
Acidity
Yellow

B3

C1

C2

C3

RSD (%, n = 9)

444
6:4

448
7:3

444
8:2
Basicity
Blue

448
9:1

0.8

Green

ZHENG Yue et al. / Chinese Journal of Analytical Chemistry, 2014, 42(1): 2127

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