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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


K. Nakano

Figure 13. (A) Schematic representation of the direct mode of SECM for the localized electro-oxidative polymerization of pyrrole-oligonucleotide (ODN) probes. For

the copolymerization of pyrrole/pyrrole-ODN, solutions of 200 mM pyrrole/10 μM

pyrrole-ODN in LiClO4 (0.1M in water) were used. The potential applied was +0.7

V vs. Ag/AgCl and the polymerization time was 20 ms. The distance between the

substrate (gold) and the microelectrode used here as the counter electrode was 60

μm. (B) Schematic of the assembly process. The horseradish peroxidase

(HRP)-biocatalyzed oxidation of (1) in the presence of H2O2, and produces precipitates of the insoluble product (2). (C) Molecular Assembly process. The detection of

the hybridization reaction of the ODN probes with their biotinylated complementary strands using SECM was possible after subsequent reactions with streptavidin

and biotinylated HRP. The insoluble product (2) precipitated on the Au film caused

a local alteration of conductivity, which can be sensitively detected by the SECM

tip and allowed imaging of DNA arrays in a fast and straightforward way. Reprinted

from Ref. 50. Copyright (2006). Reproduced by permission of the Royal Society of


SECM Imaging of DNA Arrays


Figure 13. Continuation.

incubated in a 5-bromo-4-chloro-3-indoyl phosphate/nitro blue

tetrazolium mixture to precipitate the insoluble reaction product.

Simple negative feedback mode SECM gave surface images as

shown Fig. 14. As a possible application to real samples, they

demonstrated the SECM detection of a long PCR amplicon (255

bp) at a very low concentration (60 nM).


The Human Genome Project has altered the mindset and approach

in biomedical research and medicine. The sequencing of genomes

has become a central research tool. With faster, yet cheaper DNA

sequencing technologies anticipated, the burgeoning field of personal genome analysis will soon be routine practice.52 Currently,

dye-terminating methods with capillary electrophoresis separation

is the primary DNA sequencing tool used; however, significant

efforts are being explored to develop sequencing technologies with

improvements in miniaturization, parallelism and simplification,

e.g., the parallel bead array (pyrosequencing),53 nanopore devices

and biochips. DNA-array-based methods include shotgun sequencing by hybridization.13 An individual addressable microelec-


K. Nakano


Figure 14. (a) Scheme of the enzyme-linked precipitation method to a sandwich

assay. A capture probe DNA (12-mer) formed spotted-type DNA microarray based

on the standard 5'-SH modification chemistry on evaporated gold films. The DNA

arrays were next treated with the target, 35-mer DNA in the presence of the biotinylated signaling probe and subsequently reacted with streptavidin–alkaline phosphate. Precipitation of the insoluble reaction product deteriorates the conductivity

of the substrate. Simple negative feedback mode SECM report that to give surface

images. (b) SECM images of DNA spots. (A) Area scan of array of spots of 2.5u

2.0 mm, with the capture probes arrayed in spots in three rows at a distance of 1

mm, center to center. (B) A cross-section line scan reporting the current values vs.

relative horizontal distance of a part of the area described by the white line depicted

in (A). The concentration of target was 40 nM. Imaging measurements were carried

out using 2 mM ferrocenemonocarboxylic acid in 0.1 M phosphate buffer, pH 7.4,

0.1 M NaCl as mediator, Etip +0.6 V, Esubstrate -0.1 V (Ag/AgCl). The tip was a disk

Pt microelectrode with a diameter of 10 μm and scanned at a constant speed of 10

μm s-1. Reprinted with permission from Ref. 51. Copyright (2007) American

Chemical Society.

SECM Imaging of DNA Arrays



Figure 14. Continuation.

trode array represents an interesting option, but multiplexing of

high-density arrays remains a major instrumental challenge.

SECM represents a potential option; however, to meet the criteria

of the next generation of instruments, SECM-based approaches

will require improvements in

(a) the lateral resolution for massive analysis

high-density arrays;

(b) flexible detection that is inexpensive; and

(c) shortened operation times and high throughput.


As a stand-alone technique, SECM is a powerful tool for particular applications, but when combined with other imaging tools,

its power of resolution, in terms of the quality of information provided, is greatly enhanced. Hyphenation or hybridization with

scanning force microscopy30 and surface plasmon resonance imaging54 should represent an interesting approach. Current sequencing technologies are too expensive and labor intensive. Prospects seem to indicate that SECM-based detection methods lessen

the cost, to a considerable extent, as they allow diverse modes of

measurements with label-free detection in some cases. As seen in

the examples of impedance-based DNA biosensors, combined detection with impedance measurements would offer a versatile way

to readout hybridization events. Recent activities in microelectromechanical systems (MEMS) have been generating various types

of micro-fluidic devices and lab-on-chip sensing technologies.


K. Nakano

Microfabrication of each recognition electrode equipped with a

near-field, cantilever-type tip electrode may streamline and accelerate current SECM operations.

Although electrochemical methods are often instrumentally

simple and cost-effective, they remain under-developed and their

applications are not as widespread as standard fluorescence based

techniques. With current advances, electroanalytical methods may

soon rival other detection methods.


The author acknowledges the financial support by a Grant-in-Aid

for Scientific Research from the Ministry of Education, Culture,

Sports, Science and Technology (MEXT), Japan.



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SECM Imaging of DNA Arrays


Professor Koji Nakano

Professor Koji Nakano is with the Faculty of Engineering at Kyushu University

since 1994. He received his BSc and MSc degrees from Tohoku University, and his

PhD from Kyushu University in 1993. In 1996 he received the Japan Society for

Analytical Chemistry Commendation as Distinguished Young Chemists thanks to

his pioneering work on chemical analysis using molecular organizates involving

synthetic bilayer membrane, self-assembled monolayer, and DNA self-assembly.

Currently, his scientific interest is focused on DNA nano-bio-technology, with a

central focus on single-molecule measurements and molecular-chemical device



Electrochemistry of Biomimetic


Rolando Guidelli and Lucia Becucci

Dept. of Chemistry, Florence University, Via della Lastruccia 3

50019 Sesto Fiorentino, Firenze, Italy



Biological membranes are by far the most important electrified

interfaces in living systems. They consist of a bimolecular layer of

lipids (the lipid bilayer) incorporating proteins. Lipid molecules

are amphiphilic, i.e., consist of a hydrophobic section (the hydrocarbon tail) and a hydrophilic section (the polar head). In biological membranes the two lipid monolayers are oriented with the hydrocarbon tails directed toward each other and the polar heads

turned toward the aqueous solutions that bath the two sides of the

membrane. The resulting lipid bilayer is a matrix that incorporates

different proteins performing a variety of functions. Biomembranes form a highly selective barrier between the inside and the

outside of living cells. They are highly insulating to inorganic ions,

and large electrochemical potentialdifferences can be maintained

across them. The permeability and structural properties of biological membranes are sensitive to the chemical nature of the mem-

N. Eliaz (ed.), Applications of Electrochemistry and Nanotechnology


in Biology and Medicine II, Modern Aspects of Electrochemistry 53,

DOI 10.1007/978-1-4614-2137-5_4, © Springer Science+Business Media, LLC 2012


R. Guidelli and L. Becucci

brane components and to events that occur at the interface or within the bilayer. For example, biomembranes provide the environmental matrix for proteins that specifically transport certain ions

and other molecules, for receptor proteins and for signal transduction molecules. The lipid and protein portions of biomembranes

are also sensitive to the presence of lipophilic perturbants. Anaesthetics, for example, readily partition into lipid membranes, altering their electrical and permeability characteristics, thus providing

an indicator for these agents. The various responses observed in

biomembranes are concentration-dependent, usually very rapid and

reversible, and frequently voltage-dependent.



In view of the complexity and diversity of the functions performed

by the different proteins embedded in a biomembrane (the integral

proteins), it has been found convenient to incorporate single integral proteins or smaller lipophilic biomolecules into experimental

models of biological membranes, so as to isolate and investigate

their functions. This serves to reduce complex membrane processes to well-defined interactions between selected proteins, lipids

and ligands. There is great potential for application of experimental models of biomembranes (the so-called biomimetic membranes) for the elucidation of structure-function relationships of

many biologically important membrane proteins. These proteins

are the key factors in cell metabolism, e.g., in cell-cell interactions,

signal transduction, and transport of ions and nutrients. Thanks to

this important function, membrane proteins are a preferred target

for pharmaceuticals, with about 60% of consumed drugs addressing them. Biomimetic membranes are also useful for the investigation of phase stability (e.g., lipid-lipid phase separation, lipid raft

formation, lateral diffusion), protein-membrane interactions (e.g.,

receptor clustering and co-localization), and membrane-membrane

processes such as fusion, electroporation and intercellular recognitions. They are also relevant to the design of membrane-based biosensors and devices, and to analytical platforms for assaying

membrane-based processes.

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