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SECM Imaging of DNA Arrays


sites. As a control experiment, a positively charged mediator,

[Ru(NH3)6]3+ gave no such behavior. They explained that the response was caused by the electrostatic repulsion between the mediator ion and the negatively charged DNA molecules (repelling

mode). This mode of SECM successfully developed 3D images for

the DNA dots including their hybridization.

This type of SECM imaging has been extended for single-nucleotide mismatch detection by Diakowski and Kraatz.41

They prepared spotted-type DNA arrays with 50-μm-diameter dots

with 200 μm spacing through the standard 5'-modified chemisorption on gold-covered silicon wafers. The oligonucleotide sequences (25-mer), including the probe DNA are shown below, and the

DNA array prepared was examined by SECM imaging using

[Fe(CN)6]3Ѹ as a mediator. As shown in Fig. 10, each DNA spot

that was clearly visualized in the current mapping, gave a small

but reproducible difference of the tip current, varying with their

mismatched structure. This sort of SECM responses was in agreement with previous work.42 Interestingly, the results of this study

showed that the mismatch response could be detected with certain

enhancements by examination in the presence of Zn2+. Based on

these findings, this research team concluded that the differences in

the tip current were due to the variability of the Fe(CN)63-/4- redox

probe in penetrating the film, as previously reported.43 Mathematical simulations based on the finite element method determined

and further discussed the apparent electron transfer coefficients.

1: HO–(CH2)6–S–S–(CH2)6–




(full match)







(The underlined base corresponds to the location of the mismatch.)


K. Nakano

Figure 10. Left: Typical SECM image and current profile recorded over a ds-DNA

microarray on a Au substrate in the absence of Zn2+ (a) Each spot follows the order,

1 + 2, 1 + 3, 1 + 4, and 1 + 5 from left to right. Typical SECM image and current

profile recorded above DNA microarray in the presence of Zn2+ (b) Data were

obtained for the same sample after incubation in Zn(ClO4)2 solution. Experiment

carried out in 1 mM K4Fe(CN)6 (50 mM NaClO4–20 mM Tris–ClO4, pH 8.6) using

25-μm Pt tip. During measurements, the electrode potential of the tip was kept at

+0.5 V (Ag/AgCl) for ferrocyanide oxidation. Right: Typical normalized approach

curves observed above individual ds-DNA spots for strands 1 + 2 (ì), 1 + 3 (á), 1 +

4 (o) and 1 + 5 (ă) measured in the absence (a) and presence (b) of Zn2+. Solid lines

represent simulated approach curves. All pictures taken from Ref. 41–Copyright

(2009) Reproduced by permission of the Royal Society of Chemistry.

SECM Imaging of DNA Arrays


As an alternative for DNA microarrays with electrochemical

configurations, physisorption of salmon sperm DNA on

screen-printed carbon electrodes having dotted polyethylenimine

cast membranes on the surfaces was investigated using SECM.44


Examples of Positive Feedback Mode Imaging

SECM imaging of DNA arrays in the positive feedback mode was

also established in an early stage of the research. For the particular

mode of imaging to be feasible, each DNA spots must possess a

certain degree of electrical conductivity to form a concentration

cell. Wang et al. developed a specific property in which electroinactive DNA spots were activated by the application of silver staining: with biotinylated DNA as the target DNA each spot was first

treated with a streptavidin-gold conjugate and subsequently subjected to silver staining.45 The resulting silver nanoparticle-covered

DNA spots were highly practical for use in positive mode

SECM-type operation. A higher level of sensitivity was achieved

for detecting a 17-mer DNA target at only 30 amol per spot, which

was comparable to what is usually achievable by fluorometry detection.

The earlier example of SECM experiments using a DNA intercalative agent as a redox-active binder has been revived in recent years. Wain and Zhou reported a detailed study of SECM imaging of DNA arrays using methylene blue (MB) as a redox–active

intercalator (Fig. 11).42 For imaging experiments, they prepared

spotted-type DNA arrays with 100-μm-diameter dots and 100 μm

spacing on a polycrystalline gold disk. The base sequences of the

oligonucleotides (15-mer) are shown below,

1: 5' HS–(CH2)6–AGT-ACA-GTC-ATC-GCG-3',









(The underlined base corresponds to the location of the mismatch).


K. Nakano



Figure 11. Left: SECM approach curves obtained at a ds-DNA-modified gold substrate in pH 7.2 PBS containing 2 mM K4Fe(CN)6. The SECM tip was a

25-μm-diameter Pt disk held at a potential of 0.5 V. (A) Unbiased substrate in the

absence of MB (—), substrate biased at –0.4 V in the absence of MB (---), and

substrate biased at –0.4 V in the presence of 2 μM MB (···). (B) Effect of substrate

potential (Es) in the presence of 2 μM MB (Es = –0.1, –0.2, –0.3, and –0.4 V). Circles show theoretical curves for finite electron-transfer kinetics. Right: (A) SECM

image of a DNA microarray immobilized on a gold disk substrate biased at –0.4V

in pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 and 2 μM MB. Both rows of

spots correspond to (a) ss-DNA, (b) ds-DNA (complementary), (c) ds-DNA (one

base mismatch), (d) ds-DNA (two base mismatches), and (e) ds-DNA (three base

mismatches). The spots are surrounded by 6-mercaptohexanol. The section profile

reveals negative-feedback-mode imaging. (B) SECM image of a DNA microarray

immobilized on a gold disk substrate biased at –0.4 V in pH 7.2 PBS solution containing 2 mM K4Fe(CN)6 and 2 μM MB. Both rows of spots correspond to (a)

ss-DNA, (b) ds-DNA (complementary), (c) ds-DNA (one base mismatch), (d)

ds-DNA (two base mismatches), and (e) ds-DNA (three base mismatches). The









mono-11-mercaptoundecyl ether. The section profile describes that the tip current

enhances at the DNA spot showing positive-feedback response. Reprinted with

permission from Ref. 42. Copyright (2008) American Chemical Society.

SECM Imaging of DNA Arrays


As standard use, immobilization of the probe DNA was made

through normal 5'-thiol modification chemistry. A Fe(CN)63–/4–

redox pair was chosen for the mediator and SECM experiments

were made in the presence of MB. Initially, the approach curve

was measured with the electrode potential of the DNA array controlled. They found that, with the electrode potential cathodically

polarized, the feedback behavior of the Fe(CN)63–/4– turned positive even though it was initially in the negative feedback mode.

This was due to the cathodic atmosphere providing the reduced

form of MB (leucomethylene blue, LB), which could reduce the

mediator ion for the positive feedback functions at the tip, Eqs.


SECM tip (0.5 V):

Fe(CN)64– – e– ĺ Fe(CN)63–


DNA surface (–0.4V):

MB+ + 2e– + H+ ĺ LB



2 Fe(CN)63– + LB ĺ 2 Fe(CN)64– + MB+ + H+


SECM experiments were achieved with varying visualization

conditions from the negative to the positive feedback mode. The

DNA microarray was imaged at a detection level of 14 fmol per

spot for the particular dsDNA consisting of 15 base pairs. More

importantly, the feasibility of detecting base pair mismatches was

demonstrated. Later, Barton's group independently reported a similar type of SECM experiment using Nile Blue as the mediator.46

Previously, we reported the surface-grafting immobilization of

capture-probe (CP) DNA sequences on carbon electrodes coated

with poly(1,4-benzoquinone), PQ, that were oxidatively polymerized using peroxidase.47 The DNA-polymer conjugate film was

found to be electrochemically responsive, allowing the label-free

detection of hybridization. Furthermore, the potentially electroconductive nature of the covalently linked, redox-active conjugate

molecule was expected to be suitable for the robust, positive-feedback detection in SECM. From these viewpoints, we

adopted the surface modification, as a DNA array model, towards

a carbon fiber (CF) electrode that was 33 μm in diameter and examined the material by SECM characterization.48 The CP DNA


K. Nakano

(12-mer) was a 5'-NH2 modified synthetic oligonucleotide with the

sequence, 5'-GCC-ACC-AGC-TCC-3'. The DNA probe was covalently grafted into the PQ matrices developed on CF surfaces. The

target DNA was the K-ras12 gene, 5'-GGA-GCT GGT GGC-3',

one of the human oncogenes found at high rates in colon cancer,

pancreatic cancer and lung cancer. We employed the positive

feedback mode of operation using Fe(CN)63–/4– as the mediator, Eq.

(9)–(11). The results are summarized in Fig 12.

SECM tip (+0.6 V):

Fe(CN)64– – e– ĺ Fe(CN)63–

CF substrate (–0.4V):

Q + 2 H+ + 2 e– ĺ H2Q



Overall: 2 Fe(CN)63– + H2Q ĺ 2 Fe(CN)64– + Q + 2H+ + 2 e–


From the SECM approach curves, we found that the polymer

matrices, even after conjugation with CP DNA, possess a certain

degree of charge-transfer capability and thus allow for positive

feedback mode imaging. We have successfully obtained

well-resolved micrometer-sized dot images (diameter 60–100 μm)

of the microelectrodes: they generate a considerable magnitude of

current rise over 10 nA while they gave a current decrease, typically 1 nA, in response to the hybridization event at the CP DNA.

The sensor response was found to fall slightly more than the background current (0.6–0.8 nA). However, the particular SECM

measurement system gave a good signal-to-noise ratio, thereby

reliably allowing the detection of DNA hybridization.


Examples of Enzymic-Reaction-Coupled Imaging

The use of SECM shows promise in characterizing a variety of

biomacromolecules and biomacromolecular reactions at surfaces

including enzymic reaction kinetics. The achievements in this area

have been applied in DNA biosensors and DNA arrays. Initially,

the substrate generation, tip collection mode (SG/TC) imaging was

used for such kinds of measurements, and Gyuurcsányi et al. reported a preliminary study on detecting DNA hybridization using

SECM Imaging of DNA Arrays






Figure 12. (A) Schematic illustration for the SECM imaging at the CP-DNA/PQ

microdots (a) and the surface state after hybridization (b). (B) Detection of hybridization of CP-DNA confined in the substrate electrode with the tip CV measurements. CVs were obtained with a potential scan rate of 50 mV s-1 and the substrate

was fixed at –0.2 V. During the measurements, the electrodes were hold adjacent at

20 μm-distance. The substrate surface was varied form the CP-DNA-attached (a) to

the hybridized state (b). The mediator solution was 10 mM Fe(CN)64– containing

0.1 M KCl.

50 Pm

13 nA


0.5 nA


Figure 12. Continuation. (C) Representative SECM images (raw data) and their 3D expressions for the CP DNA/PQ/CF electrodes (a), and after

hybridization (b). All data were obtained at 19-μm s–1 scan rate and +0.6 V for the Pt tip electrode, whereas the electrode potential of the substrate was fixed at 0 V.



K. Nakano

SECM Imaging of DNA Arrays


glucose oxidase to SG.49 In SG/TC mode imaging, the use of mediator ions is not required and measurements are not limited to

conductive surfaces, in contrast to the particular cases of positive

feedback. With these potential advantages, SG/TC imaging has

become particularly important in studying biological entities. Examination of the SG/TC mode through DNA array developments

indicates that the method suffers from lower resolution and often

requires a very active enzyme to be used and/or high enzyme

loading. Recently, Fortin's and Palchetti's groups independently

reported an enzyme-linked method for feedback moderation for


Fortin and his colleagues described a patterning / immobilization method for a pyrrole-oligonucleotide (ODN) conjugate (Fig.

13).50 They used the direct mode SECM, in which the electrical

field is established between the tip and the substrate (gold) to deposit the poly(pyrrole/pyrrole-ODN) copolymer in the vicinity of

the tip through electro-oxidative polymerization. For the detection

of the hybridization reaction, they combined a subsequent reaction

with streptavidin and biotinylated horseradish peroxidase after

hybridization with the target ODN covalently modified with biotin.

With the resulting DNA-peroxidase conjugate at the substrate surface, catalytic oxidation of 4-chloro-1-naphthol in the presence of

H2O2 led to the formation of 4-chloro-3,4-dihydronaphthalen-1

(2H)- one as the product. Accumulation of the precipitate at the

substrate surface caused a local alteration of the conductivity,

which was detected with sufficiently high sensitivity using the

negative feedback operation. With SPR measurements, they determined the film thickness of the precipitate to be 22 nm. The

results of this study should be interesting for the possible application of nanometer-sized film detection using formation SECM.

Palchetti's group has extended this sort of enzyme-linked precipitation method to a sandwich assay.51 First, they prepared spotted-type DNA microarray using a capture probe DNA (12-mer)

through the standard 5'-SH modification method on evaporated

gold films. The DNA arrays were next treated with the target,

35-mer DNA in the presence of the biotinylated signaling probe.

Finally, the DNA array was reacted with streptavidin–alkaline

phosphate to form the surface conjugates. The DNA array was

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