Dielectrophoretic Characteristics of Microbeads Labeled with DNA

of Various Lengths

Zhenhao Ding

, Hiromichi Kasahara

, 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 Labelling, Dielectrophoresis, Rapid DNA Detection, Crossover Frequency.

Abstract: Polymerase chain reaction (PCR) is one of the most sensitive and specific detection methods of bacterial

and viral infections. The authors proposed a new electrical technique for rapid detection of DNA amplified

by PCR using dielectrophoresis (DEP) of microbeads. The method is based on dramatic alteration of DEP

characteristics of microbeads caused by DNA labelling. DNA labeled microbeads are trapped on a

microelectrode under the action of positive DEP, whereas pristine microbeads are not. DEP-trapped

microbeads can be measured impedimetrically to realize rapid and quantitative detection of the amplified

DNA. In this study, it was aimed to reveal how DNA length affects DEP characteristic of DNA-labeled

microbeads. Dielectrophoretic crossover from the negative to the positive was measured for microbeads

labeled with DNA length in 204 bp, 391 bp and 796 bp. After theoretical fitting of DEP crossover data, it

was revealed that the surface conductance increased when the length of labeled DNA increased.

1 INTRODUCTION

There are several methods to diagnose bacterial or

viral infections in human and animals. A nucleic

acid amplification test (NAT) is a highly sensitive

and specific method for detecting DNA or RNA of a

target pathogen. Polymerase chain reaction (PCR) is

a type of NAT used to amplify specific regions of

DNA or RNA via enzymatic reaction. PCR is a

widespread application in several areas of genetic

analysis (Storch, 2000; Malorny, 2003; Chung et al.,

2006).

DNAs amplified by PCR, amplicons, are

generally separated by size and detected by agarose

gel electrophoresis. Although this method is well

established and reliable, it requires rather

complicated and time-consuming manual operations

by experts. To overcome this drawback, real-time

PCR has emerged as an improvement for rapid

analysis. Real-time PCR optically detects amplicons

during PCR using a fluorescent probes that bind to

DNA. The fluorescence intensity increases with the

number of amplicons during the amplification.

However, the apparatus for real-time PCR is

expensive, almost 10 times the cost of a general

PCR equipment. Moreover, special knowledge and

experience are required to design fluorescent DNA

probes for optical detection (Mackay, 2002). Hence,

rapid, simple and economical amplicons detection

method was required.

The authors develop and demonstrate a novel

electrical method for detection of amplicons by

dielectrophoresis (DEP) of microbeads (Nakano et

al., 2014). In the method (Figure 1), the amplicons

are chemically immobilized on dielectric microbeads

so that DNA immobilization alters the DEP

characteristics of the microbeads. DNA-labeled

microbeads are 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. An electrical detection technique called

dielectrophoretic impedance measurement (DEPIM),

which was originally developed by the authors’

group for bacterial and viral inspection, can be used

for the impedance measurement (Suehiro et al.,

1999). It was demonstrated that DNA-labeled

microbeads were trapped in the electrode gap, which

caused a detectable change in the electrode

impedance in a few seconds, whereas pristine

microbeads would repelled from the electrode gap,

which resulted in no impedance change (Nakano et

185

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

Dielectrophoretic Characteristics of Microbeads Labeled with DNA of Various Lengths.

DOI: 10.5220/0005280701850189

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2015), pages 185-189

ISBN: 978-989-758-071-0

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

al., 2014). Hence, this method would provide rapid

detection of DNAs amplified by PCR, which may be

applicable to rapid, quantitative, and automated

diagnosis of bacterial and viral infections. However,

this method still required separation and selective

detection of PCR amplified DNA of different length.

Figure 1: Schematic illustration of the microbeads-based

detection of amplicons. After PCR amplification,

amplicons (amplified DNA) are chemically immobilized

on the microbeads. The DNA-labeled microbeads behaves

positive DEP, whereas the pristine microbeads behaves

negative DEP. The DEP-trapped microbeads can be

detected by DEPIM

This study aim to reveal how the length of

labelling DNA affects DEP characteristics of DNA

labeled microbeads. Alteration of DNA-labeled

microbeads DEP characteristics, which is affected

by the length of labeled DNA, can lead to rapid

separation and selective detection of PCR amplified

DNA of different length by impedance

measurement. Crossover frequency of the DEP for

microbeads labeled with DNA of different length

were measured at different suspending medium

conductivities. The alteration of DEP charateristics

was analysed using theoritical fitting of measured

data.

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, Ausbury et

al., 2002,). 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



σω

⁄

, 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 crossover

frequency f

is defined as the value of the applied

frequency which results in the cessation of particle

motion. Therefore, measurement of the crossover

frequency can used to characterize the dielectric

properties of single particle.

The conductivity of a solid dielectric particle, σ

can be expressed by the following equation

(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). Zhou et al. found that the dielectric properties

of microbeads were modified by coating with

bacterial biofilms, resulting in an altered

electrorotation spectrum (Zhou et al., 1995).

3 EXPERIMENTS

We used pUC 19 DNA as template for PCR. The 5’

end of forward primers, which were designed for

amplifications of 204 bp DNA, 391 bp DNA and

796 bp DNA from pUC 19, were tagged with biotin.

As the results of PCR, 204 bp DNA, 391 bp DNA

and 796 bp DNA were amplified and these

amplicons were confirmed by standard agarose gel

electrophoresis.

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186

Magnetic microbeads (Dynabeads

M-280, Life

Technologies 2.8 μm in diameter) were used in this

experiment. The surface of the microbeads is 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 of 204

bp, 391 bp and 796 bp separately. The amplicon

concentrations in the solution were approximately

3.5x10

DNA/μl. The mixtures of the amplicons

and microbeads were incubated at room temperature

for 15 minutes. Hence, the microbeads were

functionalized with amplicons via biotin-streptavidin

interaction. Then, the DNA labeled microbeads were

suspended in deionized water (conductivity 2 × 10

−4

S/m).

A castle-walled microelectrode having the

narrowest gap of 5 μm, which is shown as black part

in Figure 2, was used. DEP behaviors of microbeads

labeled with DNA of different length were observed

with an inverted microscope equipped with a CCD

camera. The DNA-labeled microbeads were

suspended in NaCl solution of concentration range

from 5 μM to 1 mM. Then, 5μl of the solution

containing the DNA-labeled microbeads was placed

on the microelectrode and covered with a cover slip.

An AC voltage of 20 V

peak-to-peak

was applied to the

microelectrode to generate DEP force. The DEP

crossover frequency was measured by observing

DNA-labeled microbeads motion with varying

applied voltage frequency. The DEP force changed

from negative DEP, where microbeads were repelled

from the electrode gap, which is the high electric

field (Figure 2. a.) to positive DEP, where

microbeads were trapped in the electrode gap

(Figure 2. b.) along with the decreasing of applied

voltage frequency. The DEP crossover frequency

was determined as the frequency when the DEP

force changed from n-DEP to zero (Figure 2. c.).

4 RESULTS AND DISCUSSION

The crossover data for microbeads labeld by DNA

of 204 bp, 391 bp and 796 bp in length are shown in

Figure 3. The data are plotted for DNA-labeled

microbeads suspended in NaCl solution of different

conductivities range from 10

-4

to 10

-1

S/m. At high

suspending medium conductivities the DNA labeled

microbeads experienced only negative DEP. At

suspending medium conductivities below 10

-3

S/m,

the crossover frequency was clearly dependent on

the length of labeled DNA. For example, Figure 4

shows that at suspending medium conductivity of

2x10

-4

S/m, the crossover frequency became higher

when the length of labeled DNA increased. This

suggests that applying voltage of appropriate

frequency can separate microbeads labeled by DNA

of different length. For example, as shown in Figure

3, in suspending medium conductivity of 2x10

-4

S/m, if the frequency between 3.1 x 10

Hz and 3.8 x

Hz is applied, 796 bp-DNA-labeled microbeads

would experience positive DEP, while 391 bp-DNA-

labeled microbeads and 204 bp-DNA-labeled

microbeads would experience negative DEP.

a. Negative DEP

b. Positive DEP

c. No DEP force

Figure 2: Optical images showing DEP behaviors of

DNA-labeled microbeads.

The solid lines in Figure 3 are the best fit to

model described by Equations 1 - 3 and the fitting

data are summarised in Table 1.

DielectrophoreticCharacteristicsofMicrobeadsLabeledwithDNAofVariousLengths

187

Figure 3: Crossover frequency for microbeads labeled by

DNA length in 204 bp, 391 bp and 796 bp plotted as a

function of suspending medium conductivity. Solid lines

are the best fir to the model described by Equation 1- 3.

The fitting data are summarized in Table 1.

Figure 4: Crossover frequency plotted as a function of

labeled DNA length.

Table 1: Fitting data of surface conductance of microbeads

labeled by DNA length in 204 bp, 391 bp and 796 bp.

Labeled DNA length (bp) 204 391 796

Surface conductance (nS) 9.45 10.29 11.28

Table 1 shows that the surface conductance of

DNA labeled microbeads increased with the DNA

length. This was because of the negative electrical

charges of DNA. Longer DNA has lager negative

electrical charges, therefore, the surface conductance

of DNA labeled microbeads will increase when the

length of labeled DNA increases, which will cause

the alteration of crossover frequency of DNA-

labeled microbeads.

5 CONCLUSIONS

Dielectrophoretic crossover frequency of

microbeads labeled by DNA of virous lengths were

measured at different suspending medium

conductivities. At suspending medium conductivities

below 10

-3

S/m, the crossover frequency was clearly

dependent on the length of labeled DNA that the

crossover frequency became higher when the length

of labeled DNA increased. After theoretical fitting

of DEP crossover data, it was revealed that the

surface conductance increased when the length of

labeled DNA increased. Hence, DEP characteristics

of DNA-labeled microbeads altered with DNA

length. Therefore, if voltage of appropriate

frequency were applied, longer DNA would

experience positive DEP while shorter DNA would

experience negative DEP, which could lead to rapid

separation and selective detection of PCR amplified

DNA of different length by impedance

measurement. Hence the proposed microbead-based

assay may provide rapid detection of DNAs

amplified from multiplex PCR, which may be

applicable to rapid, quantitative, and automated

diagnosis of bacterial and viral infections.

ACKNOWLEDGEMENTS

This work was partly supported by JSPS KAKENHI

Grant numbers 25820174, and 26289125.

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