MICROCHIP CAPILLARY ELECTROPHORESIS DEVICE FOR

AMPEROMETRIC DETECTION OF DNA WITH REDOX

INTERCALATION

Rohit Chand, You-Cheol Jang, Sandeep Kumar Jha

Dept. of Nanoscience and Engineering, Myongji University, Yongin, 449728, Republic of Korea

Yong-Sang Kim

Dept. of Nanoscience and Engineering, Dept. of Electrical Engineering, Myongji University

Yongin, 449728, Republic of Korea

Keywords: Capillary electrophoresis, Genomic DNA, Methylene Blue, Amperometry.

Abstract: Microfabricated biochips are very efficient platforms for analysis of biologically important molecules such

as DNA, RNA, enzymes, antibodies etc. These devices requires sample in micro/nano volume and produces

faster and better results. For these reasons, we fabricated a simple, disposable microfluidic device for

amperometric detection of DNA intercalated with methylene blue redox dye. The devices were fabricated

using conventional photolithographic method. The microchannels were laid in PDMS using negative

molding. The microchannel was 2 cm in length while the height and thickness were 250 µm and 200 µm

respectively. The electrodes used for electrophoretic separation and amperometric detection were made of

gold and were deposited by thermal evaporation on glass substrate. For the detection of DNA, fish sperm

DNA was intercalated with methylene blue as an analyte. The cyclic voltammograms of free methylene blue

and those of different concentrations of DNA intercalated with same amount of methylene blue was

obtained in this study. The intercalated DNA was then injected in the sample reservoir of fabricated device

and subjected to a separation electric field. The i-t curve was monitored for this process. The

electropherograms thus obtained suggested a possibility of rapid detection of DNA with high sensitivity and

reproducibility.

1 INTRODUCTION

Ever since the publication of DNA’s double helix

structure, electrophoresis has been a standard,

indispensible analytical tool in modern biochemistry

and molecular biology; electrophoretic procedures

are used in almost every aspect of basic or applied

biomedical and clinical research.

Traditional techniques as performed today in the

majority of laboratories, is still typically a manual

process which makes electrophoretic procedures

often time consuming and labour intensive.

Capillary electrophoresis (CE) on the other hand, is

a relatively new separation technique that is ideally

suited for handling small amounts of sample

material. The advent of microfabricated fluidic

devices in the past decade promises to address some

of these issues by miniaturizing and automate these

devices including the CE process (In-Je Yi, 2006).

More recently, electrochemical detection (ED)

has been reported for microchip (MC) CE (Ju-Ho

Kim, 2004). This mode of detection is ideally suited

for miniaturization to the microchip format. If the

power supply and electrochemical analysers are also

miniaturized, it is possible to envision a complete

µTAS (Dolnik, 2000; Woolley, 1998).

A typical MC-CE device has channel widths

varying from 50 to 200 µm with typical straight

separation channels between 1 and 5 cm in length. A

serpentine or semi-circular design can be

implemented to increase the separation channel

length up to 15 cm (Jacobson, 1994; Culbertson,

2000).

DNA-binding or intercalating dyes have been

284

Chand R., Jang Y., Jha S. and Kim Y..

MICROCHIP CAPILLARY ELECTROPHORESIS DEVICE FOR AMPEROMETRIC DETECTION OF DNA WITH REDOX INTERCALATION.

DOI: 10.5220/0003290802840287

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2011), pages 284-287

ISBN: 978-989-8425-37-9

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

used for fluorometric and amperometric DNA assays

and in flow cytometry applications. Ethidium

bromide (EtBr) was the first of such intercalators to

be used for DNA assays. Interestingly, the resolution

of dsDNA separations in CE can be improved by

using intercalating dyes. This is usually done by

adding dye to the running buffer (and/or sample) in

concentrations of 0.5 to 5 mg/mL. The dye molecule

inserts itself (“intercalates”) between the base pairs

of DNA, changing the molecular persistence length,

conformation, and charge of the DNA.

Amperometric CE detection was first reported as

a detection technique for CE by Wallingford and

Ewing in 1987 for the quantitation of catechol and

catecholamines. Amperometric detection is based on

electron transfer to or from the analyte of interest at

an electrode surface that is under the influence of an

applied DC voltage. The result of electron transfer is

a redox reaction at the electrode that produces a

current that is directly related to the analyte

concentration. Thus, by analysing the DNA

intercalated redox-active dye by amperometric

method; it should be possible to analyse the

concentration of DNA after its capillary

electrophoretic separation. In the present work, we

fabricated a CE-AD device for CE separation

followed by amperometric analysis of DNA-

intercalated methylene blue (MB) dye, thus

providing the basis for detection of DNA fragments

by indirect means.

2 MATERIALS AND METHOD

2.1 Reagents and chemicals

DNA Sodium salt fish sperm (Ultra-Pure) was

obtained from Bio Basic Inc., Korea. Methylene

blue (MB) (Reagent grade) was purchased from

Biopure, Canada. PDMS Sylgard 184 was from Dow

Corning Corp. (Midland, MI, USA). SU-8 2000

negative photoresist and XP SU-8 developer were

from Micro-Chem Co. USA and AZ 1512 positive

photoresist and AZ developer was from AZ

electronics material, Korea. The other chemicals of

ACS grade were purchased from Sigma-Aldrich,

Korea. All solutions were prepared afresh, stock

solution were made using double-distilled deionized

water (DI) and further diluted to required

concentration using the supporting electrolyte.

2.2 DNA Precipitation

Fish sperm DNA obtained was precipitated using

ethanol precipitation. After precipitation, the

mixtures were then centrifuged to collect the

precipitate. The pellet was washed twice with 1 ml

of 70 % Ethanol. The pellet was air dried. Later, the

pellet was dissolved in 800 µl of DI water and used

for spectrophotometric study. For electrochemical

study the samples were further diluted with 200 µl

of 1 M KCl.

2.3 Fabrication of Microchip

A three-electrode detection system was used for CE-

AD. The simple process flow for the fabrication of

the CE-AD device is shown in Figure 1. We can

divide the procedure in two parts: fabrication of

microchannel in PDMS mold and laying gold

electrodes on glass substrate. The electrodes were

200 µM each in width. Gold electrode was choice

for the detection and separation electrodes due to its

inertness to redox reaction. The microchannels, each

Figure 1: Fabrication of CE-AD device.

200 µm in width, 250 µm in height and 2 cm in

length were fabricated using negative molding

method (Gi-Sung Joo, 2009). Finally the PDMS was

bonded on glass wafer using UV ozone bonding.

MICROCHIP CAPILLARY ELECTROPHORESIS DEVICE FOR AMPEROMETRIC DETECTION OF DNA WITH

REDOX INTERCALATION

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2.4 Electrochemical Detection

Electrochemical detection was performed using

Electrochemical analyser, CHI 800B (CH

Instruments, USA). The three-electrode

electrochemical system was used for cyclic

voltammetry, which consisted of an Ag/AgCl

reference electrode (RE-5B, BASi), a Platinum wire

counter electrode (CHI 115) and a gold working

electrode (CHI101). Prior to voltammetry, the gold

and platinum electrodes were cleaned using chromic

acid, polished using electrode polishing kit (CHI

120) and cyclic sweep was performed in the range of

2 V to -2 V at a scan rate of 100 mV/sec in 0.1 M

Sulphuric acid until a stable curve was obtained.

Voltammetric sweep in sulphuric acid was repeated

before every voltammetric study. Cyclic

voltammograms of 200 mM Potassium chloride, 100

mM MB in 200 mM KCl, intercalated MB-DNA

complex sample and the other two precipitated

negative control were recorded at various potential

range and scan rate.

3 RESULTS AND DISCUSSION

Spectrophotometric study of MB-DNA complex

revealed that approximately 0.03 µM of MB binds to

1 mg/mL. Further electrochemical studies were

carried out with the free MB and MB-DNA

complex.

3.1 Electrochemistry of MB and DNA

A reversible redox cycle was obtained at 100 µM

concentration (Figure 2) in scan range 0.2 V to -0.6

Figure 2: CV of methylene blue, Conc.: 100 µM, in KCl

200 mM, scan rate 0.1 V/s.

V with scan rate 100 mV/sec. The cyclic

voltammograms shows a cathodic process of MB

(Ep

) at -0.280 V.The precipitated MB-DNA

complex was studied in the same range as that of

free MB. Figure 3 shows the cyclic voltammograms

of different concentration of intercalated MB-DNA

complex. The cyclic voltammograms of the complex

shows the similar redox process as that of free MB

with a cathodic process (Ep

) at -0.28 V.

Figure 3: CV showing peaks for different concentrations

of MB-DNA complex in 200 mM KCl, Scan rate: 0.1 V/s.

Figure 4: Shows a correlation between various

concentration of intercalated MB and peak current

produced by it using cyclic voltammetry. Suitable controls

without DNA and without MB did not produce any

corresponding peak, suggesting that the peak obtained in

the DNA-MB complex is only due to the MB bound to

DNA and not due to any other free MB.

3.2 Microchip CE-AD

Figure 5 shows the image of microchip CE-AD

device. The microchannel in the device was filled

with 200 mM KCl as a separation medium and

support analyte. A blank i-t curve was observed

without addition of any test analyte for the control

purpose. The i-t curve of free MB was also

monitored which showed no peak as free MB is

positive in charge and will not migrate in the

channel along the separation field for DNA.

The electrophoretic separation for MB-DNA

complex was initiated by adding 2 µL of complex

into the sample reservoir and a separation voltage of

100 V was applied. DNA-MB complex as migrating

towards anode is detected amperometrically using

in-channel gold working electrode. Figure 6 shows

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the i-t curve of MB-DNA detection through CE-AD.

Different peaks resemble MB attached to various

bands of the DNA. The peak was detected after 2

min of sample injection. The detection sensitivity

and LOD for this reaction were 640 nA/μg of DNA

and 140 ng of DNA respectively.

Figure 5: CE-AD microchip.

Figure 6: i-t curve of MB-DNA complex in KCl 200 mM.

Therefore, it would be possible to first determine

peak current for MB from CE-AD experiment and

then decipher its concentration using standard curve

obtained under control conditions (Figure 4).

Thereafter, DNA concentration can be calculated

from the standard value obtained. This shows the

potential of present study towards successful

detection of DNA of any sizes and deciphers its

concentration on our microchip. Further studies are

underway to enhance the reproducibility and

sensitivity of these devices and for detection of a

mixture of short DNA fragments such as found in

DNA molecular weight markers.

4 CONCLUSIONS

It was concluded in the present study that disposable

CE-AD microchips with gold electrodes and PDMS

channels can be used to prepare effective DNA

detector in place of existing gel electrophoresis

based system. The disposable electrochemical

detector fabricated in this study displayed good

performance in terms of sensitivity, stability,

resolution and peak density. This type of microchip

can also be used for detection of various other

organic or inorganic compounds, or can be

integrated with microfluidic modules on a µTAS.

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REDOX INTERCALATION

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