μ-eLCR: a microfabricated device for electrochemical detection of DNA base changes in breast cancer...

Registered Charity Number 207890

Accepted Manuscript

This is an Accepted Manuscript, which has been through the RSC Publishing peer review process and has been accepted for publication.

Accepted Manuscripts are published online shortly after acceptance, which is prior to technical editing, formatting and proof reading. This free service from RSC Publishing allows authors to make their results available to the community, in citable form, before publication of the edited article. This Accepted Manuscript will be replaced by the edited and formatted Advance Article as soon as this is available.

To cite this manuscript please use its permanent Digital Object Identifier (DOI®), which is identical for all formats of publication.

More information about Accepted Manuscripts can be found in the Information for Authors.

Please note that technical editing may introduce minor changes to the text and/or graphics contained in the manuscript submitted by the author(s) which may alter content, and that the standard Terms & Conditions and the ethical guidelines that apply to the journal are still applicable. In no event shall the RSC be held responsible for any errors or omissions in these Accepted Manuscript manuscripts or any consequences arising from the use of any information contained in them.

www.rsc.org/loc

Lab on a ChipView Article OnlineView Journal

This article can be cited before page numbers have been issued, to do this please use: E. J. H. Wee, S. Rauf, K. Koo, M. J. A.Shiddiky and M. Trau, Lab Chip, 2013, DOI: 10.1039/C3LC50528F.

http://www.rsc.org/Publishing/Journals/guidelines/AuthorGuidelines/JournalPolicy/accepted_manuscripts.asp

http://www.rsc.org/help/termsconditions.asp

http://www.rsc.org/publishing/journals/guidelines/

http://dx.doi.org/10.1039/c3lc50528f

http://pubs.rsc.org/en/journals/journal/LC

µ-eLCR: A Microfabricated Device for Electrochemical Detection

of DNA Base Changes in Breast Cancer Cell Lines

Eugene JH Wee,¶,1 Sakandar Rauf, ¶,1 Kevin M. Koo,1 Muhammad J. A. Shiddiky,1,*

and Matt Trau1,2*

Centre for Biomarker Research and Development, Australian Institute for Bioengineering and

Nanotechnology (AIBN) , and School of Chemistry and Molecular Biosciences, The

University of Queensland, Brisbane, QLD 4072, Australia

Emails: [email protected] (MJAS); [email protected] (MT)

Tel: +61-7-33464178; Fax: +61-7-33463973

1 Centre for Biomarker Research and Development, Australian Institute for Bioengineering

and Nanotechnology (AIBN) 2 School of Chemistry and Molecular Biosciences ¶ Authors contributed equally

Page 1 of 19 Lab on a Chip

Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.

View Article OnlineDOI: 10.1039/C3LC50528F


Abstract

Microfabricated devices for the electrochemical detection of single DNA base changes in

cancer cell lines are highly desirable due to the inherent advantages such as portability,

simplicity, and the rapid and inexpensive nature of electrochemical readout methods.

Moreover, molecular sensors that use microscale-footprint working electrodes have shown

high signal-to-noise ratio. Herein we report a microdevice-based electrochemical assay (µ-

eLCR) measuring ligase chain reaction (LCR)-amplified long and short "knife" motifs which

reflect the presence or absence of a DNA base change of interest in a target sequence. This µ-

eLCR approach has higher sensitivity (4.4 to 10 fold improvement over macro-disk

electrodes) and good reproducibility (%RSD 6.5%, n = 12) for the detection of LCR-

amplified DNA bases. The devices also exhibited excellent sensitivity for the detection of

DNA methylation (C to T base change in a locus associated with cancer metastasis) in two

cell lines and serum derived DNA samples. We believe that the µ-eLCR device may be a

useful diagnostic tool for inexpensive and rapid detection of single DNA bases change

applications such as DNA methylation and single nucleotide polymorphism (SNP) detection.

Page 2 of 19Lab on a Chip

Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Introduction

Single DNA base changes are common events in many diseases including cancer.1-3 Such

genetic anomalies can include single nucleotide polymorphisms (SNPs), and point mutations,

as well as DNA methylation events where a C to T base change occurs after bisulfite

treatment of DNA.4-5 Screening for these changes is potentially useful for identifying

individuals at risk, and can allow clinicians to prescribe personalized preventive measures

based on their detection.1-3 Current screening methods for such changes usually involve some

form of DNA amplification (e.g. PCR) followed by optical or mass spectrometric detection.

Such readout methods are poorly suited for routine diagnostic due to high running costs, long

assay times, and a lack of simplicity. Therefore, there is a need for convenient, rapid,

inexpensive, and sensitive methodologies that can readily detect single DNA base changes in

routine clinical applications.

Electrochemical detection methods are one potentially rapid, portable, and cost-effective

alternative to conventional detection methods, and their usefulness in detecting single DNA

base changes is well established.6-9 We have previously demonstrated a method for the

electrochemical detection of single DNA base changes based on ligase chain reaction (LCR)-

amplification method (referred to as eLCR).10 In this method, LCR exponentially generates

long and short "knife" motifs that represent the presence or absence of a DNA base change of

interest respectively (Fig. 1D). Base changes are then electrochemically detected by labelling

only the long "knife" motifs with horse radish peroxidase (HRP) via a streptavidin/biotin

coupling. The generated electrochemical signal is then proportional to the amount of the

DNA base change in a sample. This method uses conventional macro-disk electrodes and

hence exhibits low sensitivity (e.g., low signal-to-noise ratio) and struggles to discriminate

between long and short “knife” motifs at lower target concentrations. However, a special

feature of this method is that because it uses electrochemical detection, it can be transformed

into a microdevice-based sensor platform. Since the detection limit of an electrochemical

biosensor can be improved by decreasing the effective working area of the sensor11,12, we

believe this method can be useful for the quantification of low level DNA bases change in

biological samples (e.g., the detection of the C to T base change in DNA methylation) or as a

means of reducing assay running costs since less reagents are required.


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Aberrant DNA methylations frequently occur in cancer and are potential cancer

biomarkers.13-16 However, to the best of our knowledge, there are no approaches available for

detecting DNA methylation in routine clinical practice. To help address this issue, we herein

fabricate a microdevice-based arrayed microbiosensor platform for the detection of DNA

methylation in two breast cancer cell lines and serum derived DNA samples. To demonstrate

the utility of this platform, the methylation event in a DNA sequence was first specifically

amplified by LCR, hybridized to capture probes preimmobilized on gold (Au)

microelectrodes, and detected via electrocatalytic reduction of hydrogen peroxide (H2O2)

(only "long motifs" will be HRP tagged). Using this approach, we could distinguish between

a high (C base) and low (T base) methylation simultaneously at a locus that may be

associated with cancer metastasis.13 We believe that the current µ-eLCR approach may be

useful for sensitive, rapid and low cost detection of clinically important single DNA base

changes including DNA methylation and somatic mutation detection at specific loci.

Experimental

Reagents

All reagents were of analytical grade and purchased from Sigma Aldrich (Australia). All

buffers unless stated otherwise were Phosphate Buffer Saline (PBS) at various

concentrations. A stock 200mM PBS (200mM phosphate buffer, 2640 mM NaCl, 54 mM

KCl, pH7.4) was made and diluted with water as stated. DNA oligonucleotides were

purchased from Integrated DNA Technologies (USA) and sequences are provided in Table 1.

Fabrication of Microdevices

Microdevices were fabricated at the Queensland node of Australian National Fabrication

Facility (Q-ANFF) (See Fig S1† and text in †ESI for details). Initially, they were designed

using Layout Editor (L-Edit) and printed on a chrome mask. The Pyrex glass wafers were

first covered with a 2 µm thick photoresist layer. After the photolithography step, a 200 nm

of gold with 10 nm titanium adhesion layer was then deposited on the wafers using an e-beam

evaporation method. The patterned electrodes were obtained with a lift off process. The gold

electrodes were then passivated with a 5µm thick insulating resist layer to achieve recessed

disk working electrode. The working electrodes and a 1×1 mm connecting pad were then


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



fabricated using the second photolithography. The wafers were then cut into individual chip.

Each chip consists of five sets of electrode. In order to get four replicates from a single

experiment, each set contains four independent electrodes. To complete the device, the

PDMS cover containing five reservoirs connected to inlet and outlet reservoirs by

microchannel was bonded permanently onto the chip. The PDMS microchannel were

fabricated from silicon master prepared using standard photolithography process (see †EIS

for details). This design could facilitate either five simultaneous sample measurements for a

given biomarker or addressing each well individually and simultaneously measure five

biomarkers for a given sample.

Optimization of the Insulating Resist Layer

As microelectrodes with the lower aspect ratio (diameter/height; height = thickness of the

insulating resist layer used to cover the gold surface) allowed faster kinetic regimes due to the

three-dimensional diffusion of species, aspect ratio of the electrode was optimized using one

electron reduction of 2.5 mM [Fe(CN)6]3-/4- in 100 mM PBS solution containing 0.1 M KCl.

Lowering the thickness of the insulating layer (aspect ratio <4) increases the reductive

responses but gives highly irreproducible data. The aspect ratio of >4 resulted highly

reproducible cyclic voltammetric profiles (see Figure S2†). Therefore, a 20 µm diameter

recessed disk electrode with the aspect ratio of 4 was used throughout the study.

LCR Protocol

LCR was performed as described previously.10 Briefly, the Taq ligase (NEB) kit was used

with 100nM of each DNA probe in a 25 µL LCR reaction. 50ng of salmon sperm DNA was

used as a blocking agent with various concentrations of synthetic targets for sensitivity

experiments. LCR generates long and short "knife" motifs which represent the presence (i.e.,

long motif) or absence (i.e., short motif) of a DNA base change. Long "knife" motifs are

generated by the enzymatic ligation of DNA probes pairs P1/P4 to P2/P3 in the presence of a

matching template (see Fig.1D and ref 10). For DNA methylation studies, C and T base

changes in a duplex reaction (methylated and unmethylated respectively) were amplified by

LCR using the probes outlined in Table 1 (i.e., P1 to P6) targeting CpG12 of the miR200b P2

promoter. For MDA MB 231 and MDA MB 468 breast cancer cell lines (ATCC, USA) and

serum purified DNA (Red Cross, Australia) experiments, methylation event (CpG12 of the

miR200b P2 promoter) were amplified by LCR using 1 pg of bisulfite PCR samples


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



generated via the previously described protocol.13 LCR thermocycling adopted was 95°C for

3min, 15 cycles of 95°C for 30s followed by 63°C for 2 min. 5 µL of the LCR reaction was

then analysed by standard ethidium bromide gel electrophoresis to verify amplification (data

not shown).

For LCR optimization experiments, Cy5 fluorophore-tagged probe sequences were used. This

was done because the initial starting probe concentration was too low for conventional

ethidium bromide staining. Each 25 µL LCR reaction was performed using Taq Ligase kit

(NEB) with 40U of ligase at designated probe concentrations (10 - 100 nM),

annealing/ligation temperatures (63°C, 65°C), and cycle numbers (15 - 40 cycles). 5 µL of

each reaction was then analysed on a 3% agarose gel and imaged with a Typhoon 9100

scanner (GE Healthcare). Densitometry was then performed using ImageJ software to

quantify amplified products. Signal to noise (S/N) ratio was determined by the following

equation:

S/N ratio = Densitymatch /DensityNoT ... ... (1)

where NoT is the no template control.

Functionalization of the Devices

Devices were cleaned by rinsing with water, acetone, and isopropyl alcohol and dried under a

flow of nitrogen. In order to form self-assembled monolayer (SAM), a 20 µL aqueous

solution containing 4 µM thiolated DNA probes in 100 mM PBS (pH 7.4) was placed in each

well for 16 h at room temperature. Each well was then back filled with 0.5 mM 6-mercarpto-

1-hexanol in 100 mM PBS containing 5% glycerol solution for 30 mins at room temperature.

After washing with 100 mM PBS buffer, the wells were then filled with 20 µL LCR products

mixed with equal volume of 200 mM PBS at 40°C for 2 h, and sealed with adhesive film to

prevent evaporation during the hybridization. For cell lines and serum DNA experiments,

wells 1 and 2 were modified with Capture 1 probes while wells 3 and 4 were modified with

Capture 2. Capture 1 probes specifically recognized P1 (methylated event) while Capture 2

probes recognized P5 (unmethylated event). The diluted LCR product was then loaded into

the inlet well and directed to individual wells by suction. The wells were then washed with

100 mM PBS several times to remove excess LCR products. Long "knife" motifs were made

electrochemically active via a streptavidin/biotin coupling by incubating 20 µL of

streptavidin-HRP solution (1/500 dilution in 10 mM phosphate buffer) at room temperature


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



for 15 min. Unbound HRP was then washed off with 100 mM PBS before conducting

electrochemical measurements.

Electrochemical Experiments

All electrochemical experiments were carried out using a CH1040C potentiostat (CH

Instruments) with a three-electrode system containing Ag Quasi reference electrode (QRE),

Pt counter electrode, and DNA-modified gold microelectrode as working electrode.

Electrocatalytic reduction of H2O2 has been carried out in 100 mM PBS containing 1 mM

H2O2 using cyclic voltametry (CV) from 100 mV to -600 mV at a scan rate of 50 mVs-1. The

increase of the electrocatalytic response corresponding to the HRP-conjugated duplex DNA

was calculated as follows:

% increase in CV response = [(jfinal – jini)/ jini] ×100% ... ... (3)

where jini = the mean current density at 0 M template (no template control) concentration,

jfinal = mean current density at any concentration of templates. The mean current density was

calculated by averaging the current density from four independent electrodes present in the

same well of the device (current density = electrocatalytic response/effective area of the

electrode).

The effective working area of the electrode was estimated from the limiting current (ilim)

obtained under cyclic voltammetry conditions for the one electron redox reaction of

[Fe(CN)6]3-/4- (CV profiles shown in Fig. S2†) and the used of the following relationship18:

ilim = 4nFDCreffe ... ... ... ... ... ... (2)

where ilim is the limiting current (A), n ( = 1) is the number of electrons transferred, D (cm2s-

1) is the diffusing coefficient of [Fe(CN)6] 3- or [Fe(CN)6] 4-, C is the concentration of

[Fe(CN)6] 3-/4-, F is the Faraday constant, reffe is the effective radius of the electrode. The

estimated area was again validated by scanning in 50 mM H2SO4 and measuring the area of

gold oxide reduction peak.19

Results and Discussion

To enable the use of µ-eLCR based microbiosensors (Fig. 1) as a potential platform for the

future point-of-care devices, the current report describes a new platform for single DNA base


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



changes, which includes the following steps. First, the LCR assay was performed to generate

amplified DNA sequences with single-base DNA mismatches. Second, an electrocatalytic

reduction reaction was used to discriminate LCR-amplified long and short “knife” motifs in

synthetic, cancer cell line, and serum DNA samples. Fig. 1D outlines the electrochemical

detection of LCR- amplified products. After LCR amplification (see Ref 10 for details), long

and short "knife’’ motifs were then hybridized with the surface bound capture probes. Only

long "knife" motifs are labelled with biotin, to which streptavidin-HRP can be coupled for

specific electrochemical tagging. Under electrocatalytic conditions, HRP (Fe3+) reduces to

HRP (Fe2+) by accepting electrons from the electrode surface. One possible mechanism of

this charge transfer could be the direct contact of DNA-coupled redox species diffusing to

regions in the surface with little or no DNA. This electrochemically generated HRP (Fe2+)

can reduce H2O2 to water and regenerate HRP (Fe3+) which will continue in the catalytic

cycle.10 Since HRP is specifically coupled by streptavidin/biotin to only DNA sequences with

long “knife” motifs, it is a powerful means of discriminating single DNA base changes. The

level of electrocatalytic current represents amount the single DNA base change present in the

sample, such as a C-to-T base change which distinguishes a methylated and unmethylated

cytosine in DNA samples.

The parameters that determine the specificity and efficiency of the amplification can directly

affect the analytical performance of the µ-eLCR devices. Therefore, optimization of the LCR

parameters such as probe concentration, LCR cycle numbers, and annealing temperatures

were conducted to maximize the specificity and efficiency of the LCR assay. The specificity

in LCR reactions is controlled by adjusting the annealing/ligation temperature. Since the

theoretical melting temperature (Tm) of the probes was 65°C, we examined the ligation

specificity at 63°C and 65°C (Fig. S3A†); using 10 nM of probes and after 50 cycles at 63°C,

we were able to detect 40 pM of matching template. No detectable amplification in the 40 pM

mismatch target and no template control (0 M template) indicated good specificity of the

LCR reaction. However, when probe concentration was increased to 40 nM, specific LCR

products could be detected after 40 cycles (Fig. S3B†), which suggested that probe

concentration affects total cycle numbers of the reaction, a parameter that relates to assay run

times. Encouraged by this, we further increased probe concentration to 100 nM to further

minimize assay run times (i.e., the lower cycle numbers the shorter running times). To

evaluate the optimal probe concentration and cycle conditions, we compared the S/N ratio

(noise is defined as non-specific amplification) over a range of LCR cycle numbers for both


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



40 nM and 100 nM probes (Fig. S3C†). The best S/N was observed for 100 nM at 30 cycles.

However, since we expect more than 40 pM of target in practice, a lower cycle condition of

20 cycle is preferred to prevent signal saturation. As reactions using 100 nM probes have

sufficient DNA for standard ethidium bromide visualization by gel electrophoresis,

subsequent experiments were verified using ethidium bromide staining. However, when we

prepared products for LCR using biotin-tagged DNA sequences, we found that 15 cycles was

sufficient to generate products for electrochemical detection. The exact mechanism for this

was not clear, but could be related to the size of the biotin-tagged probe. It is possible that

biotin, being a smaller molecule, may have better diffusion rates than fluorophores and hence

results in faster LCR kinetics.

In our previous report,10 we used a conventional three-electrode electrochemical cell where 3

mm diameter (effective working area = 0.071 cm2) gold electrode as working, Ag/AgCl as

reference, and Pt wire as counter electrodes to detect LCR-amplified products. While this

approach was a simple and inexpensive alternative to existing fluorescence and mass

spectroscopic single DNA-base detection methodologies, it was limited by the poor

sensitivity, and was not suitable for multiplexed detection and automation. In the µ-eLCR

devices, the diameter of the recessed disk microbiosensors was 20 µm. This size was

optimized based on the reproducible cyclic voltammetric responses for the reduction of

[Fe(CN)6]3-/4- system (See Experimental). The electrode with the aspect ratio of >4 (i.e.,

diameter, >20 µm; thickness of the insulating layer, 5 µm) resulted highly reproducible

voltammetric profiles (see Figure S2†). Further lowering the thickness of the insulating layer

results in lower aspect ratio which gives rise to irreproducible voltammetric data (not shown).

This might be due to the difficulty in mass transport of reactants for diffusion-controlled

Faradaic current (i.e., three-dimensional diffusion) on the recessed disk microelectrode

surface.

These microelectrodes were initially characterized by optical imaging and cyclic

voltammetric measurements. From the optical image measurement (a typical image shown in

Fig. 1 inset), the average diameter of the fabricated electrodes was 20 ± 0.4 µm with the

relative standard deviation (% RSD) of 1.5% n = 4. This diameter was verified with the

electrochemical measurements where the effective surface area of the electrode was found to

be 20 ± 0.7 µm with the %RSD of < 5% (n = 5) by measuring the ilim and use of eqn 2 (see


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Fig. S2†). This value was further verified using the gold reductive peak area measurements

in 50 mM H2SO4. By taking the literature value of 450 µC cm−2 for the charge passed per

unit area on the surface of bulk gold19-20 the effective diameter of the electrodes was found to

be 19.5 ± 0.7 µm with the %RSD of < 5% (n = 5).

To demonstrate the impact of microelectrode-based arrayed microbiosensors on the

electrochemical detection of LCR-amplified products (e.g., single DNA base changes), the

electrocatalytic responses at the microelectrodes in µ-eLCR devices were compared with the

response obtained previously on gold macro-disk electrodes. Fig. 2 shows the comparison

data obtained for the LCR-amplified samples produced at a 0.04 - 40 pM concentration range

of input templates. As expected, a higher relative signal change as input template increased

was observed. For instance, at 0.04 pM and 40 pM, the responses at the microelectrodes were

4.4- and 10-fold higher than those of the macro-disk electrodes respectively, clearly

indicating the improved sensitivity on the microbiosensor compared to the macro-electrodes.

A possible explanation for the enhanced sensitivity could be that HRP-tagged DNA

immobilized on microelectrodes appear more accessible to H2O2 in the surrounding solution

and therefore react more rapidly, thus leading to a higher relative electrocatalytic response18

and consistent with other studies on micro- and nanosized electrodes.11-12 This level of the

sensitivity could be further increased by using microelectrode with smaller effective area.11-

12,17-19 However, in the current design, the optimal diameter of the recessed disk

microbiosensors was 20 µm (See above). While eLCR is already comparable to florescent-

tagged probe detection (0.05 pM)21 under current conditions, µ-eLCR is likely able to detect

lower than 0.04 pM of starting material based on the 4.4-fold sensitivity improvement at 0.04

pM template. However, due the limitations of fluorescent-based detection methods, it was not

possible to visually verify LCR amplification with less than 0.04 pM starting template.

Another possible advantage of improved sensitivity with microelectrodes is that lesser

reagents may be required to illicit a detectable response, which in turn can reduce running

costs.

The reproducibility (%RSD) of the devices for the analysis of LCR-amplified product was

examined by comparing electrocatalytic responses over three independent devices (i.e., 60

electrodes) and was found to be 10.5%. This value between three wells (i.e., 12 electrodes) at


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



40 pM was 6.5%. While still not ideal for diagnostic purposes, future improvements to device

design and workflow could help to improve RSD values to 5% or less.

Detecting DNA methylation can involve bisulfite treatment of DNA followed by PCR

amplification to convert all unmethylated C present in any DNA sequence to T.4-5 The µ-

eLCR approach can thus be employed to detect the C to T base change in complex biological

systems such as that of cell lines. The methylation state at CpG12 of the miR200b P2

promoter, a potential biomarker of metastasis13, was chosen as a model system in this report.

Ideally, both C (methylated) and T (unmethylated) base changes in any DNA sequence

should be amplified and detected simultaneously by our method. To demonstrate the

multiplexing capability of our devices, we performed duplexed LCR reaction for MDA-MB-

231 and MDA-MB-468 cell line samples (i.e., simultaneous amplification of both methylated

and unmethylated events) and detected the amplified products simultaneously by µ-eLCR

devices. Methylated and unmethylated products were differentiated by their different barcode

sequences encoded on probes P1 and P5 respectively and recognised by the pre-immobilized

Capture 1 and Capture 2 probes respectively (See Table 1). Initially, two capture probes were

immobilized on two separate wells in the device (see Experimental for details). 20µL of LCR

products generated from MDA-MB-231 cells were then diluted with equal volume of

phosphate buffer (40 uL total) and loaded into the inlet of the device. The sample was then

directed to capture probe-immobilized reaction reservoirs by suction, and hybridized for 2 h.

Fig. 3 (left) shows the response from the MDA-MB-231 cells from capture probe-modified

wells. A high response was observed in wells 1 and 2 (Capture probe 1 modified) but not in

wells 3 and 4 (Capture probe 2 modified, %RSD = 17, n = 8) indicating high levels of

methylation in this cell line sample. This experiment was then repeated with the MDA-MB-

468 cell line where a reciprocal response was detected (%RSD = 6, n = 8), indicating low

levels of methylation (Fig 3 centre). These results were concordant with a previous study

using PCR and fluorescence.13 In addition, distinguishing the two cell lines based on DNA

methylation also demonstrates, to some extent, good specificity of duplexed LCR reaction for

DNA methylation applications. Moreover, the miR200 family is implicated in cancer

metastasis, and aberrant methylation of the miR200b is associated with poor breast cancer

prognosis.13, 22-23 As such, a method such as the µ-eLCR that can rapidly and simultaneously

assess the methylation states of multiple CpG sites at low cost may be clinically useful.


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



We next extended the approach to detect the methylation events in DNA purified from serum.

The response from serum DNA is shown in Fig 3 (right panel). Unlike the cell lines,

electrocatalytic currents were detected for both the methylated and unmethylated probes in

the serum DNA sample (with slightly higher currents at electrodes with methylated probes),

indicating a heterogeneous methylation state (% RSD = 7, n = 8). The higher response for

methylated probe was also confirmed using standard gel electrophoresis where methylated

and unmethylated probes sets were used individually in the LCR reaction (Fig S4). This data

clearly demonstrates that eLCR is applicable for detecting DNA methylation in complex

serum sample.

Conclusions

We have demonstrated a simple, sensitive approach to detect single DNA base changes using

eLCR on a microdevice. The sensitivity of µ-eLCR approach has been significantly improved

by 4.4 – 10 fold compared to macro-disk electrodes while maintaining good reproducibility.

Most importantly, the successful application of the µ-eLCR approach to detecting the

methylation states in two differentially methylated cell lines and a serum derived DNA

sample at a potential cancer biomarker site demonstrates its potential application on complex

biological systems, and thus may be very useful for routine clinical practice. The use of

microdevices also paves the way for massively high throughput eLCR for economical

diagnostics by leveraging on the fabrication of miniaturized micro electrode arrays. A future

µ-eLCR application may include high throughput, multiplexed, simultaneous detection of

SNPs, point mutations and DNA methylation at several clinically important loci.

Acknowledgements

This work was supported by the UQ Post-doctoral Research Fellowship (RM # 2009001818),

ARC DECRA Fellowship (DE120102503), and the National Breast Cancer Foundation (CG-

08-07). The fabrication work was performed at Queensland node of the Australian National

Fabrication Facility (Q-ANFF). We also thank Darren Korbie and Erica Lin for supplying the

purified serum DNA sample.


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Notes and References †Electronic Supplementary Information (ESI) available (1) Pleasance, E. D., Cheetham, R. K., Stephens, P. J., et al., Nature 2010, 463. 191-196 (2) Whibley, C., Pharoah, P. D., Hollstein, M., Nat. Rev. Cancer 2009, 9. 95-107 (3) Komar, A. A., Science 2007, 315. 466-467 (4) Frommer, M., Mcdonald, L. E., Millar, D. S., et al., Proc. Natl. Acad. Sci. USA 1992, 89. 1827-1831 (5) Clark, S. J., Harrison, J., Paul, C. L., et al., Nucleic Acids Res. 1994, 22. 2990-2997 (6) Wong, E. L., Gooding, J. J., Anal. Chem. 2006, 78. 2138-2144 (7) Kelley, S. O., Boon, E. M., Barton, J. K., et al., Nucleic Acids Res. 1999, 27. 4830-4837 (8) Walter, A., Wu, J., Flechsig, G. U., et al., Anal. Chim. Acta 2011, 689. 29-33 (9) Shiddiky, M. J. A., Torriero, A. A. J., Zeng, Z. H., et al., J. Am. Chem. Soc. 2010, 132. 10053-10063 (10) Wee, E. J., Shiddiky, M. J., Brown, M. A., et al., Chem. Commun. 2012, 48. 12014-12016 (11) Gasparac, R., Taft, B. J., Lapierre-Devlin, M. A., et al., J. Am. Chem. Soc. 2004, 126. 12270-12271 (12) Soleymani, L., Fang, Z., Sun, X., et al., Angew. Chem. 2009, 48. 8457-8460 (13) Wee, E. J., Peters, K., Nair, S. S., et al., Oncogene 2012, 31. 4182-4195 (14) Lister, R., Pelizzola, M., Kida, Y. S., et al., Nature 2011, 471. 68-73 (15) Hill, V. K., Ricketts, C., Bieche, I., et al., Cancer Res. 2011. (16) Hansen, K. D., Timp, W., Bravo, H. C., et al., Nat. Genet. 2011, 43. 768-775 (17) Rauf, S., Shiddiky, M. J. A., Trau, M., et al., Rsc Adv 2013, 3. 4189-4192 (18) Arrigan, D. W. M., Analyst 2004, 129. 1157-1165 (19) Rauf, S., Shiddiky, M. J. A., Asthana, A., et al., Sensor Actuat B-Chem 2012, 173. 491-496 (20) Ulman, A., An introduction to ultrathin organic films: From Langmuir-Blodgett to self-assembly. Academic Press: Boston, 1991 (21) Benjamin, W. H., Jr., Smith, K. R., Waites, K. B., Methods Mol. Biol. 2003, 226. 135-150 (22) Bracken, C. P., Gregory, P. A., Kolesnikoff, N., et al., Cancer Res. 2008, 68. 7846-7854 (23) Korpal, M., Lee, E. S., Hu, G., et al., J. Biol. Chem. 2008, 283. 14910-14914


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Figure Captions

Fig. 1 Optical microscopy images of a (A) microdevice featuring five independent sample

well connected by a michochannel, (B) a single well containing four independently wired

electrodes (demarcated by arrow heads), and (C) a single electrode. (D) Schematic of µ-

eLCR. A capture probe is immobilized on the surface of the microelectrodes and back-filled

with 6-mecarpto-1-hexenol (MCH). LCR generates long and short ‘knife’ motifs representing

the presence or absence of single DNA base change respectively. Both ‘knife’ motifs are then

hybridized to capture probes. Long ‘knife’ species are then labelled with HRP via a

streptavidin/biotin and in the presence of H2O2, electrocatalytic response is detected,

signalling the presence of a DNA base change.

Fig. 2 (A) Comparison of the sensitivities of the µ-eLCR device (green bar) and gold macro-

disk electrodes (red bar) over the range of 0 – 40 pM input template concentration. Inset

figure is the magnified view of the group obtained for the 0.4 and 0.04 pM template. (B)

Comparison of the exponential increase of the change of the response (e.g., sensitivity

enhancement) over the range of 0 – 40 pM input template concentration. (C) Corresponding

electrocatalytic responses obtained at a microelectrode in the µ-eLCR device.

Fig. 3 Detection of DNA methylation in breast cancer cell lines MDA-MB-231 (left panel),

MDA-MB-468 (middle panel), and a serum derived DNA sample (right panel) using

methylated (M, blue) and unmethylated (UM, red) probes. % Response changes are

expressed relative to a no template (0 M template) control. Inset shows the corresponding

CV profiles.


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Table 1 Sequences of DNA oligonucleotides used in 5' to 3' orientation. Modifications are as indicated. DNA base being interrogated is highlighted in bold font and underlined. Oligos 5'-Sequence-3'

Capture 1 GAACATGACGATCTGTAACTGG-C3-SH Capture 2 GTACATTGGTGCAGACAGAATT-C3-SH

P1 CCAGTTACAGATCGTCATGTTCAACGCCCCTCGACGCACCTAACCG

P2-biotin Phos-CCCTACCCGCCTACCTAACCGA-C6-biotin P2-Cy5 Phos-CCCTACCCGCCTACCTAACCGA-C6-Cy5 P3 TCGGTTAGGTAGGCGGGTAGGGC P4 Phos-GGTTAGGTGCGTCGAGGGGCGTT

P5 AATTCTGTCTGCACCAATGTACAACGCCCCTCGACGCACCTAACCA

P6 TCGGTTAGGTAGGCGGGTAGGGT Matched Target

GGGATTCGGTTAGGTAGGCGGGTAGGGCGGTTAGGTGCGTCGAGGGGCGTTTTTA

Unmatched Target

GGGATTCGGTTAGGTAGGCGGGTAGGGTGGTTAGGTGCGTCGAGGGGCGTTTTTA


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Fig. 1


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Fig. 2


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



Fig. 3


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



TOC for

µ-eLCR: A Microfabricated Device for Electrochemical Detection of

DNA Base Changes in Breast Cancer Cell Lines

Eugene JH Wee,¶,1 Sakandar Rauf, ¶,1 Kevin M. Koo,1 Muhammad J. A. Shiddiky,1,* and

Matt Trau1,2*


Lab

on

a C

hip

Acc

epte

d M

anu

scri

pt

Publ

ishe

d on

23

Aug

ust 2

013.

Dow

nloa

ded

on 0

5/09

/201

3 21

:03:

59.



μ-eLCR: a microfabricated device for electrochemical detection of DNA base changes in breast cancer...

Documents

Transcript of μ-eLCR: a microfabricated device for electrochemical detection of DNA base changes in breast cancer...