DNA Detection Method based on the Microbead Velocity under

Traveling Wave Dielectrophoresis

Zhenhao Ding

, Michihiko Nakano

and Junya Suehiro

Graduate School of Information Science and Electrical Engineering, Kyushu University, 744 Motooka, Fukuoka, Japan

Faculty of Information Science and Electrical Engineering, Kyushu University, 744 Motooka, Fukuoka, Japan

Keywords: DNA Detection, Traveling Wave Dielectrophoresis, Microbeads.

Abstract: Polymerase chain reaction (PCR) is a highly specific and sensitive detection method for bacterial and viral

infections by amplifying the specific regions of DNA or RNA via enzymatic reaction. The authors have

developed a rapid DNA detection method based on the dielectrophoresis (DEP) characteristic of DNA

labeled microbeads for the rapid detection of the DNA that amplified by PCR. This method is based on the

change of the Clausius-Mossotti (CM) factor K of DEP when DNA is attached onto microbeads. In former

studies, we developed a new DNA detection method based on the change of the real part of K (Re[K]).

However, this method requires a large amount of DNA attaching to a microbead to alter the microbead DEP

behaviour for DNA detection. In this study, we focus on the imaginary part of K (Im[K]), which

theoretically will change more dramatically than Re[K] when DNA is attached onto the microbeads. Since

the traveling wave dielectrophoresis behaviour is based on the Im[K], we propose a new method for DNA

detection based on the traveling wave dielectrophoresis (twDEP) of microbeads.

1 INTRODUCTION

There are various diseases caused by bacterial or

viral infections. In order to select an appropriate

treatment for an infectious disease, the early

detection and identification are extremely important.

A nucleic acids amplification assay is a highly

sensitive and specific method among various

diagnosis methods for infectious diseases. The

polymerase chain reaction (PCR) is a type of nucleic

acids amplification assay, which is well developed

for clinical applications. The PCR is used to amplify

specific regions of DNA or RNA of a target

pathogen via enzymatic reaction. The amplicons

amplified by PCR are generally detected by the

agarose gel electrophoresis, which is well

established and reliable. However, the agarose gel

electrophoresis requires time-consuming manual

operation by experts.

The authors develop and demonstrate a novel

electrical method for rapid detection of amplicons by

dielectrophoresis (DEP) of microbeads (Nakano et

al. 2014). In the method, the amplicons are

chemically attached to dielectric microbeads so that

the DNA attaching alters the surface conductance of

microbeads and result in the change of Clausius–

Mossotti (CM) factor K of DEP. When enough

amount of DNA is attached to the microbeads, the

real part of K (Re[K]) will change from negative to

positive, which means the DNA-labeled microbeads

will be trapped on a microelectrode under the action

of positive DEP, whereas pristine ones are not

trapped. Combining this dramatic alteration in DEP

phenomena with impedance measurement allows

rapid and quantitative detection of the amplicons,

and can be used for bacterial detection (Ding et al.

2016). However, this method requires a large

amount of DNA attaching to a microbead to alter the

microbead DEP behaviour for DNA detection.

In this study, we propose a new DNA detection

method based on the traveling wave

dielectrophoresis (twDEP), which is a phenomenon

affected by the imaginary part of K (Im[K]). Since

the Im[K] will change more dramatically than Re[K]

against DNA attachment on the microbeads when

microbeads surface conductance is small, the twDEP

can lead to a more sensitive detection of DNA. Since

the velocity of microbeads will change due to

different twDEP forces, we measured the velocity of

DNA labeled microbeads under twDEP force using

computer-based image analysis.

Ding, Z., Nakano, M. and Suehiro, J.

DNA Detection Method based on the Microbead Velocity under Traveling Wave Dielectrophoresis.

DOI: 10.5220/0007341800210025

In Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2019), pages 21-25

ISBN: 978-989-758-353-7

2 THEORY

DEP is the electrokinetic motion of dielectrically

polarized materials in non-uniform electric fields,

and it is currently an active area of research for

manipulation of biological particles and

nanomaterials, including bacterial cells and DNA

molecules (Pethig, 2010, Hughes, 2000, Washizu

and Kurosawa, 1990). The DEP force acting on a

spherical dielectric particle of radius r suspended in

a medium of absolute permittivity ε

is given by as

follows





2





















(1)

where E is the magnitude of the applied field.

Re[K(ω)] is the real component of the Clausius–

Mossotti (CM) factor, given by















∗





∗





∗

2



∗

(2)

where ε

and ε

are the complex permittivities of

the particle and the surrounding medium,

respectively. For a real dielectric, the complex

permittivity is defined as ε

= ε-j(σ/ω), where ε is the

permittivity, σ is the conductivity of the dielectric,

and ω is the angular frequency of the applied electric

field. When Re[K(ω)] has a positive value, the

particle is propelled toward the high field region

(positive DEP, p-DEP). With a negative value of

Re[K(ω)], the particle is repelled from the high field

region (negative DEP, n-DEP).

The conductivity of a solid dielectric particle, σ

can be expressed by the following equation (Zhou

al.

1995, Ermolina and Morgan, 2005).











2





(3)

where σ

and K

are the bulk conductivity and the

surface conductance of the particle. Equations 1–3

imply that the dielectric properties and the resultant

DEP force acting on a smaller particle should be

more dependent on the surface conductance K

Hughes et al. reported that antibody (protein)

coating of submicrometer latex spheres altered the

surface conductance and DEP spectrum of the

particles, enabling the separation of unlabeled and

protein-labeled particles (Hughes and Morgan,

1999).

Figure 1: The twDEP of microbeads under four voltage

waveforms with shifted phase.

Figure 2: Calculation results of relationship between

microbeads surface conductance and Re[K], Im[K].

The twDEP will be generated when the electric field

has a spatially varying phase. When the electrodes

are equally spaced, the phase angle change across

each electrode will be the same. The twDEP force is

given by (Cheng et al. 2009, Fathy et al. 2017):







4



























(4)

where  is the distance between every four

electrodes. Microbeads will experience twDEP force

against or along the direction of field travel when the

Im[K] is positive or negative as shown in Fig. 1. The

theoretical calculation results of relationship

between microbeads surface conductance and Re[K]

as well as Im[K] are shown as Fig. 2. The voltage

frequency used in the calculation is 100 kHz. The

Im[K] changed more dramatically than Re[K] when

microbeads surface conductance is small (less than

0.2 nS), which means Im[K] will change more

dramatically when less DNA was attached on the

microbeads. Since the twDEP force is associated

with the Im[K] and change the velocity of the

BIODEVICES 2019 - 12th International Conference on Biomedical Electronics and Devices

microbeads, the target DNA can be detected more

sensitively by measuring the velocity of microbeads

under the twDEP.

3 EXPERIMENTS

We used pUC 19 DNA as the template for PCR. The

5’ end of forward primers were labeled with biotin.

As the results of PCR, 391 bp DNA were amplified

and the amplicons were confirmed by standard

agarose gel electrophoresis.

Magnetic microbeads (Dynabeads

M-280, Life

Technologies 2.8 μm in diameter) were used in this

experiment. The surface of the microbead was

coated with streptavidin, which binds specifically to

biotin. Microbeads (3x10

beads/μl) were mixed

with the reaction solution (5 mM Tris-HCl (pH 7.5),

0.5 mM EDTA, 1 M NaCl), including the

amplicons. The amplicon concentrations in the

solution were 3.5x10

copies/μl and 3.5x10

copies/μl, which make the ratio of DNA to

microbeads as 10

: 1 and 10

: 1, respectively. The

mixtures of the amplicons and microbeads were

incubated at room temperature for 30 minutes.

Hence, the microbeads were labeled with amplicons

via biotin-streptavidin interaction. Then, the DNA

labeled microbeads were suspended in deionized

water (conductivity 4 × 10

−4

S/m).

In order to manufacture the Indium Tin Oxide

(ITO) microelectrode with 30 fingers, the glass

substrate with ITO thin film (Narika, Inc.) was used.

The microelectrode was fabricated by

photolithography and wet etching. The schematic

diagram and microscope image of the micro

electrode were show as figure 3.a. The ITO

microelectrode, which is transparent, was chosen in

this study in order to simplify the process of velocity

analysis by computer-based image analysis.

20 μl of the solution containing the DNA-labeled

microbeads was placed on the microelectrode and

covered with a cover slip. The microscope image of

the microbeads solution at the detection region was

shown as figure 3. b. Then the voltage (6 Vp-p, 100

kHz) was applied on the microelectrode with 90-

degree phase shift between each electrode fingers to

generate twDEP and the twDEP was confirmed by

microscope observation. As a result, the microbeads,

which were shown in figure 3.b. moved horizontally

to the left. The movements of microbeads were

recorded by a CCD camera at 100 fps and used for

velocity analysis.

Figure 3: a. The schematic diagram and microscope image

of the microelectrode; b. The photo of the electrode and

the microscope image of detection region after the

microbeads solution was placed on the microelectrode.

The velocities of microbeads were analysed by

image analysis software Image-Pro (Media

Cybermetics Inc). The trajectories of microbeads

DNA Detection Method based on the Microbead Velocity under Traveling Wave Dielectrophoresis

inside the detection region were automatically

tracked and analysed.

4 RESULTS AND DISCUSSION

The velocity of bare microbeads and DNA labeled

microbeads at ratio of 10

and 10

copies DNA to 1

microbead was shown in figure 4.a. b. and c.

respectively. The velocity of microbeads distributed

in a range of velocities. The main reason was that

the amount of DNA attached to each microbead was

not perfect uniform.

The average velocity of bare microbeads and

DNA labeled microbeads at ratio of 10

and 10

copies DNA to 1 microbead was calculated and

shown in figure 5. The average velocity of

microbeads increased as the labeled DNA amount

increased. This is because the increase of labeled

DNA will result in the increase of the surface

conductance of microbeads. As shown in figure 2,

the Im[K] will increase against the increase of

microbead surface conductance when the surface

conductance is small. Hence, the twDEP force that

microbeads experienced would increase along with

the increase of labeled DNA and result in the

increase of the average velocity as shown in the

figure 5.

As shown in figure 5, the velocity of microbeads

under twDEP force can only be measured when less

than 10

copies of DNA attached on 1 microbead.

This was because the twDEP would only occur

when the Re[K] was negative. When the Re[K] was

positive, the microbeads would experience positive

DEP and be trapped to the gap of microelectrode.

Hence, the microbeads would not be able to move

upon the electrode. Since the microbeads would

experience positive DEP when the ratio of DNA to

microbeads was above 10

:1, the twDEP would

only occur when the labeled DNA was less than 10

copies on 1 microbead. Hence, the velocity of

microbeads under twDEP force can be measured

when less than 10

copies of DNA were labeled on 1

microbead as shown in figure 5. However, when the

Re[K] was positive, which means the microbeads

would be trapped between the microelectrode and

induce the impedance change of the microelectrode

as former proposed method (Nakano et al. 2014).

Hence, the detection sensitivity can be increase by

applying the twDEP and the detection range can be

increase by combine the former method and twDEP

based method.

Figure 4: The velocity of a. bare microbeads; b. DNA

labeled microbeads at ratio of 10

copies DNA to 1

microbead; c. DNA labeled microbeads at ratio of 10

copies DNA to 1 microbead.

BIODEVICES 2019 - 12th International Conference on Biomedical Electronics and Devices

Figure 5: The velocity of bare microbeads and DNA

labeled microbeads at ratio of 10

and 10

copies DNA to

1 microbead.

5 CONCLUSION

The velocity of DNA labeled microbeads under

twDEP was measured and analysed by image

analysis. The average velocity of DNA labeled

microbeads would increase along with the increase

of the amount of the labeled DNA when the ratio of

DNA to microbead is above 10

: 1. Since the former

proposed method required the amount of DNA to

alter the DEP force from negative to positive, which

requires the amount of DNA to achieve the ratio of

DNA to microbeads above 10

: 1, this method can

increase the sensitivity of rapid DNA detection

based on the twDEP. Furthermore, by combing this

method with the former proposed method, the

detection range of DNA can be increased as well.

For example, by measuring the impedance change of

the electrode as well as the velocity of the

microbeads after unknown amount of DNA labeled

microbeads placed on the microelectrode, the DNA

can be detected as long as there were more than 10

copies of DNA labeled on one microbeads.

ACKNOWLEDGEMENTS

This work was partly supported by JSPS KAKENHI

Grant number JP17H03277.

REFERENCES

Cheng, I.-F., Froude, V. E., Zhu, Y., Chang, H.-C. and

Chang, H.-C., 2009, A continuous high-throughput

bioparticle sorter based on 3D travelling-wave

dielectrophoresis, Lab on chip, 9, 3193-3201.

Ding, Z., Kasahara, H., Nakano, M. and Suehiro, J., 2017.

Bacterial detection based on polymerase chain reaction

and microbead dielectrophoresis characteristics. IET

Nanobiotechnology, 11, 562-567.

Ermolina, I. and Morgan, H., 2005. The electrokinetic

properties of latex particles: comparison of

electrophoresis and dielectrophoresis. Journal of

Colloid and Interface Science, 285, 419-428.

Fathy, J., Pourmand, A. and Ghavifekr, H. B., 2017,

Design and simulation of a MEMS based separator

utilizing 3D travelling-wave dielectrophoresis,

Microsystem Technologies, 23, 1351-1360.

Hughes, M. P. and Morgan, H., 1999. Dielectrophoretic

characterization and separation of antibody-coated

submicrometer latex spheres. Analytical Chemistry,

71, 3441-3445

Hughes, M. P., 2000. AC electrokinetics: applications for

nanotechnology. Nanotechnology, 11, 124-132.

Nakano, M., Ding, Z., Obara. R., Kasahara, H. and

Suehiro, J., 2014. Rapid DNA detection based on

direction reversing of dielectrophoresis of DNA-

attached microbeads. Biosensor 2014.

Pethig, R., 2010. Review article-dielectrophoresis: status

of the theory, technology and applications.

Biomicrofluidics, 4, 022811.

Washizu, M. and Kurosawa, O., 1990. Electrostatic

manipulation of DNA in microfabricated structures.

IEEE Transactions on Industry Applications, 26,

1165-1172.

Zhou, X. F., Markx, G. H., Pethig, R. and Eastwood, I.M.,

1996. Effect of biocide concentration on

electrorotation spectra of yeast cells. Biochimica et

Biophysica Acta, 1281, 60-64.

DNA Detection Method based on the Microbead Velocity under Traveling Wave Dielectrophoresis