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Scanning Electrochemical Microscopy

Imaging of DNA Arrays for High

Throughput Analysis Applications

Koji Nakano

Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744

Motooka, Nishi-ku, Fukuoka 819-0395, Japan.



With the completion of the human genome sequencing efforts,

functional genomics, which aims to understand the relationship

between an organism's genome and its phenotype, has become a

primary research field.1 Various kinds of biochips, a bio-microarray device, are extensively used for substantial analysis purposes

by promoting the effective use of the vast wealth of data produced

by genomic projects.2 The strength of biochips lies in the spatial

grid of miniaturized specific binding sites, at which unique binding

events can be analyzed simultaneously. Moreover, by introduction

into a microfluidic device, the next generation of biochips has been

found to possess both biochemical reaction capabilities, including

amplification, and chemical analysis. Such a device will be used

on a single platform with multiple reactions being carried out on

individual sites.

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_3, © Springer Science+Business Media, LLC 2012


K. Nakano

The earliest DNA arrays were developed as filter arrays that

are capable of the Southern blot technique and the many derivatives thereof. Subsequent improvements in laboratories led to the

creation of high-density macroarrays, in which two different microarray-based technologies arose; one was the DNA microarrays

from the Stanford University group of Davis and Brown,3 and the

other was the GeneChip® from Affymetrix (Santa Clara, CA,

USA).4 Currently, a wide selection of DNA microarrays offers

researchers a high throughput method for simultaneously evaluating large numbers of genes. Some of these arrays have been accumulating real results in gene expression analysis, and these are

useful for disease diagnosis, drug discovery studies and toxicological research. Additionally, with a DNA array, it is possible to discover any single nucleotide polymorphisms (SNPs); the inter-individual differences in the genome. Moreover, a DNA array

has been employed to determine the expression level of RNA and

the abundance of genes that cause this expression of RNA. On one

single DNA array, scientist can simultaneously perform tens of

thousands of bioaffinity reactions, including hybridizations.

To an electrochemist, the microelectronic array, the most recent entry in this class of device, has been an appealing feature for

the last decade. The widget was originally developed to place biomolecules arbitrarily at the test sites through the application of an

electric field.5 The electronic biochip is typically composed of

arrayed pairs of working electrodes and counter electrodes. Thus,

with any electronically addressable electrode design, electrochemical detection also becomes feasible. In particular, the intimate

combination of this tool with the electrochemical DNA biosensor

has led to a dramatic increase in research using this technology;

the electrochemical detection-based DNA arrays are anticipated to

provide many advantages over radioisotope- or fluorophore-based

detection systems.

Various kinds of scanning probe microscopy (SPM) approaches have served as engines for a variety of nanotechnology

achievements. The scanning electrochemical microscope (SECM)

that falls under this category has also produced successful results

in a wide range of electrochemical studies, from the fundamental

analysis of the electrode reaction to electrochemical fabrication of

SECM Imaging of DNA Arrays


functional surfaces.6 Due to the high spatial resolution of SECM,

this technology has been demonstrated as a readout method for

locally immobilized, micrometer-sized biological recognition elements, including a variety of DNA arrays with different formats

and detection modes.

This review examines how a SECM can facilitate DNA array

analysis and provides the underlying electrochemistry facets of

SECM. We also introduce some of our latest achievements in

SECM imaging of DNA microdots that respond toward the target

DNA through hybridization. Several comprehensive reviews, including of electrochemical DNA sensors, have been published

recently.7-11 Given the pace of advancement in this field, the development of any high-throughput device, even point-of-care DNA

diagnostics that takes the full, latent strength of electrochemical

measurements, appears to be a realistic goal.


Completed in 2003, the Human Genome Project has identified all

the 20,000–25,000 genes present in human DNA. The task has also

determined the sequence of the 3 billion chemical base pairs that

make up human DNA. With this knowledge, how transcription

occurs and is regulated has become a challenge for biologists. Genetic alterations in tumor cells which are recognized often lead to

the emergence of growth stimulatory autocrine and paracrine signals. Single-nucleotide polymorphisms (SNPs) are a common form

of genome variations where alternation of a single nucleotide occurs within the base sequence. Current methods for DNA testing

are restricted because they need extensive sample treatment undertaken by skilled researchers. DNA arrays should aid scientists

to screen for SNPs throughout genomes, as these arrays will permit

the parallel processing of human DNA for a number of genetic



K. Nakano


Types and Manufacture Methods of DNA Arrays

The earliest DNA arrays may be classed as filter arrays that were

able to accommodate the Southern blot technique and the many

derivatives thereof. Subsequent improvements led to the creation

of high-density filter arrays, termed macroarrays. Subsequently,

two different microarray-based technologies arose: the DNA microarray, better known as the spotted microarray, developed at

Stanford University,3 and the oligonucleotide arrays synthesized in

situ, such as Affymetrix GeneChips®.4

In preparing a DNA array, gene-specific probes are created

and immobilized on a solid support. When the DNA sequence information is available, oligonucleotides that hybridize with each

gene can be synthesized. This approach precludes the need to

manage large clone libraries as it is guided by sequence information. Moreover, this strategy is particularly well suited to the

expression profile analysis of organisms with completely sequenced genomes, as the focused custom array can analyze all

predicted genes in a single experiment.

In the case of the DNA microarray, the method starts by synthesizing cDNA from a cell's messenger RNA using reverse transcriptase polymerase chain reaction. Robotically operated, small

pipettes or a piezoelectric device similar to an inkjet printer produces the microarrays by accurately placing large numbers of spots

of the cDNA onto a substrate, usually glass (Fig. 1). This type of

DNA array can either be homemade on a microscope glass slide,

or custom made with 1,000 to 10,000 different spots per slide. Spot

sizes of 20 to 50 μm are commonly used and each spot can be analyzed with a fluorometry scanner with a conventional design. This

type of DNA array is particularly suitable for in-house fabrication

of each experiment with its relative ease of custom array preparation by choosing the probes, the printing and the array settings.

The light-directed, combinatorial solid-phase chemistry

method can directly synthesize complete sets of oligonucleotide

probes on a substrate, as developed by Affymetrix Corp (Fig. 2). In

this method, a fused silica substrate is coated with a hydroxyl terminated, silane coupling agent to render the surface active. The

treatment is followed by the reaction with the DMT-hexa-ethyl-

SECM Imaging of DNA Arrays

Figure 1. (Top) An example of microarrayer and (middle two

panels) its printhead assembled with spotting pins. In the microarray picture, 20 samples were printed in quintuplicate at

200-μm spacing on a glass substrate.(bottom) Samples were

mixtures of green- and yellow-dye in arbitrary compositions.

Photographs provided with permission from Arrayit Corporation.



K. Nakano


Figure 2. (A) The GeneChip® from Affymetrix (http://www.

affymetrix.com / about_affymetrix / media / image-library.affx).

Courtesy of Affymetrix. (B) Procedures of photolithography

process for on-chip oligonucleotide synthesis. The lithographic

exposure (a) converts the protective group on the terminal nucleotide into a hydroxyl group (b) for further coupling reaction

with a nucleoside phosphoramidite with a light-sensitive protecting group becomes feasible (c). Another type of photomask

can direct the reaction to occur at appropriate regions for the

next extension reaction (d), (e). Finally, an oligonucleotide array

whose surface is modified arbitrary with different oligonucleotide probes sequences in full length, usually 25 nucleotides is

available (f).

oxy-O-cyanoethyl phosphoramidite linker that is protected with

(Į-methyl-2-nitropiperonyl oxycarbonyl) (MeNPOC) at the 5'-OH

function. Deprotection is achieved upon irradiation with UV light,

in which the subsequent phosphoramidite coupling reaction becomes feasible. Therefore, the deprotection–extension reaction

cycle can be repeated as necessary to obtain the DNA array with

the intended oligonucleotide probes prepared at a specific length.

Moreover, by using a lithography mask with a segmented,

blocked/unblocked regions for UV light, and by suitable use of the

A-, T-, G-, C-base reactant, a DNA array that is composed of every

oligonucleotide probe attached can be prepared with unique base





Figure 2. Continuation.




SECM Imaging of DNA Arrays



K. Nakano

sequences. The oligonucleotide probe has a chain length, typically

a 25-mer, or possibly longer (60-mer). Although longer oligonucleotide probes are more expensive, they can provide several advantages over shorter sequences. For fluorometry readout, the

smaller size of the target site (20 u 20 μm or less) requires a

high-resolution fluorescence scanner with laser excitation.

Besides these two types of DNA arrays, macroarrays and microelectronic arrays with a variety of formats have also been investigated. They are summarized in Table 1 and some of these are

commercially available. Here, only the spot density distinguishes

the macro- and micro-array: the spot size is 300 μm or larger for

the macroarrays. Choice of any one of these DNA arrays would be

dependent on the research applications and budget. The microelectronic arrays can be considered as a new entry in this field and will

be discussed in the following section.


Gene Expression Profiling

Hybridization of DNA, the process whereby complementary nucleic acid sequences pair by forming hydrogen bonds between

complementary bases (e.g., A-T and G-C), is the core principle

behind microarrays. The noncovalent binding between the two

strands becomes stronger with increasing sequence length; as such,

only strongly paired strands remain hybridized even after some

nonspecific bonding sequences are removed by exhaustive washing. Most labeling for DNA microarray analysis involves the use

of fluorescence, which possesses the particular benefit of allowing

the simultaneous reading of several experimental parameters in

samples, otherwise known as multiplexing.

Figure 3 shows a typical dual-color microarray experiment

with a cDNA-pair prepared from two specimen materials to be

compared, such as a particular type of cancer cells vs. normal cells.

Commonly used fluorescent dyes include Cy3, which shows fluorescence at 570 nm (green-colored emission) and Cy5 with fluorescence at 670 nm (red-colored emission). To convert the mRNA

samples to the labeled cDNA, reverse transcription is carried out in

the presence of the fluorescent-labeled nucleotide precursors.

These cDNA products are then combined to hybridize on a corre-

Robotic printing or

piezoelectric inkjet

printing of synthesized DNA probes

or PCR products.

In-situ synthesis by

light-directed, combinatorial solidphase chemistry.

Probes are spotted

onto nylon membrane, plastic or

nitrocellulose solid


Electrical field or

current delivers

DNA probes to chip








Probe DNA preparation method

Affymetrix, NimbleGen (Roche),

Febit, CombiMatrix.

Clontech Laboratories, Research Genetics.

ELI Tech Molecular


The densest array.

The most sensitive


Active hybridization;

electrical or electrochemical leadout of hybridization..

Fluorescent labeling

prior to hybridization.

Radioactive labeling;

use of phosphorimager detector.

Fluorescent labeling

prior to hybridization.

1-cm by 1-cm fused

silica with approximately 40,000


8-cm by 12-cm with

approximately 200

to 5,000 genes.

Dependent on the

number of ultramicroelectrode array

that can be formed

on substrate surface.

Agilent Technologies,

GE Healthcare Life

Science, and others.

Fluorescent labeling

prior to hybridization; fluorophore is

added after hybridization.

2.5-cm by 7.5-cm

glass slide with approximately 10,000



Labeling and

detection method

Array size and probe

DNA density

Functionality and



Complimentary DNA

(cDNA) library.

Table 1. A Summary for the Types and the Features of DNA Microarrays in Different Formats.

SECM Imaging of DNA Arrays



K. Nakano

Figure 3. Diagram of typical dual-color microarray experiment. The cDNA prepared from two samples to be compared (e.g. cancer cells versus normal cells) and

that are labeled with two different fluorescent dyes, namely Cy3 (Oem 570 nm) and

Cy5 (Oem 670 nm). The two Cy-labeled cDNA samples are mixed and hybridized to

a single microarray that is then scanned in a microarray scanner. Relative intensities

of each fluorophore may then be used in ratio-based analysis to identify

up-regulated and down-regulated genes.

sponding DNA array. After hybridization, a scanning fluorescence

microscope illuminates each DNA feature or spot and measures the

fluorescence from each dye separately. The yellow-colored spot

means that the particular gene can be equally expressed in both

disease and normal cells, as indicated by the additive color mixing

caused by the labeled cDNA binding. Other coloring observations

represent the presence of either downregulation or upregulation in

the corresponding cells. Ratio-based analysis of the relative intensities of each fluorophore quantifies this.


Sequencing by Hybridization

Current advanced modern instrumental analytical techniques can

sequence genome DNAs most efficiently. However, sequencing of

megabase to gigabase quantities of DNA still requires significant

SECM Imaging of DNA Arrays


Figure 4. Top: An array consisting of tetranucleotides with 44 = 256 spots. Although each spots is depicted only one strand per spot, it contains many copies

of the same oligonucleotide. Bottom: Summary of the SHB experiment. With

treating by a partially digested DNA strand, hybridization occurred in six different spots, colored in black. After identifying the sequence of these spots and

sorting them, the sequence of the sample DNA strand can be reconstructed.

improvement in the methods used. Normally employed methods of

sequencing (direct methods such as gel electrophoresis and pyrosequencing) determine each consecutive base position in the

DNA chain individually. In contrast, an indirect method assembles

the DNA sequence based on experimental determination of the

oligonucleotide content of the DNA chain. Sequencing by hybridization (SBH) is a promising indirect method in which sets of oligonucleotides are hybridized under conditions that allow detection

of complementary sequences in the target nucleic acid.12

Figure 4 shows the procedure of base sequence determination

exemplifying a tetramer DNA sample. An array consisting of 256

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