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II. THE BIOMIMETIC MEMBRANES: SCOPE AND REQUIREMENTS
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
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).
IV. CONCLUSIONS AND FUTURE OUTLOOK
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-
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
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
(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.
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.
II. THE BIOMIMETIC MEMBRANES:
SCOPE AND REQUIREMENTS
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