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gaotiger

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Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays
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1楼2011-05-08 10:19:28
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dhd997(金币+6, EPI+1): 热心啊 2011-05-08 13:04:36
Ultrasensitive DNA sequence detection
using nanoscale ZnO sensor arrays
Nitin Kumar, Adam Dorfman and Jong-in Hahm1
Department of Chemical Engineering, The Pennsylvania State University, 160 Fenske
Laboratory, University Park, PA 16802, USA
E-mail: jhahm@engr.psu.edu
Received 21 February 2006
Published 26 May 2006
Online at stacks.iop.org/Nano/17/2875
Abstract
We report that engineered nanoscale zinc oxide structures can be effectively
used for the identification of the biothreat agent, Bacillus anthracis by
successfully discriminating its DNA sequence from other genetically related
species. We explore both covalent and non-covalent linking schemes in order
to couple probe DNA strands to the zinc oxide nanostructures. Hybridization
reactions are performed with various concentrations of target DNA strands
whose sequence is unique to Bacillus anthracis. The use of zinc oxide
nanomaterials greatly enhances the fluorescence signal collected after
carrying out duplex formation reaction. Specifically, the covalent strategy
allows detection of the target species at sample concentrations at a level as
low as a few femtomolar as compared to the detection sensitivity in the tens
of nanomolar range when using the non-covalent scheme. The presence of
the underlying zinc oxide nanomaterials is critical in achieving increased
fluorescence detection of hybridized DNA and, therefore, accomplishing
rapid and extremely sensitive identification of the biothreat agent. We also
demonstrate the easy integration potential of nanoscale zinc oxide into high
density arrays by using various types of zinc oxide sensor prototypes in the
DNA sequence detection. When combined with conventional automatic
sample handling apparatus and computerized fluorescence detection
equipment, our approach can greatly promote the use of zinc oxide
nanomaterials as signal enhancing platforms for rapid, multiplexed,
high-throughput, highly sensitive, DNA sensor arrays.
DNA sequence analysis is widely applied to the areas of
mapping genes, determining genetic variations, detecting
genetic diseases, and identifying pathogenic micro-organisms.
The rapidly increasing numbers of sequencing data have
revealed a large number of single nucleotide polymorphisms
and other mutations in the human genome and in the
genomes of other organisms [1–5]. Subtle differences
in DNA sequence due to these polymorphic sites can
lead to considerable changes in disease susceptibility and
drug response in humans [1, 2, 6–8]. Similarly, small
disparity in genetic code can cause significant variations
in phenotypes and biological activities of micro-organisms.
Therefore, the development of improved DNA sequencing
technologies is critical in correlating specific DNA sequences
1 Author to whom any correspondence should be addressed.
with the particular biological function of an organism. Novel
techniques which can perform rapid and accurate genetic
sequence analyses on a large scale are specially warranted
as the need for fast, inexpensive, ultrasensitive, and highthroughput
DNA detection escalates in the areas of medicine,
public health, forensic studies, and national security.
Biomolecular fluorescence is the most widely used
detection mechanism in both laboratory-scale and highthroughput
genomics research. Fluorescence detection is the
dominant mechanism and extensively utilized in state-of-theart
DNA sensors such as DNA arrays and gene chips [5–12].
The emerging need for high-throughput genetic detection will
continue to push the limit of fluorescence detection sensitivity.
These sequencing assays require the use of lower DNA
concentrations as well as smaller amounts of fluorophores in
order to cope better with the increasing demands for effectively
0957-4484/
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3楼2011-05-08 12:57:34
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appleXYJ

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2楼2011-05-08 12:54:18
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appleXYJ

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screening human genes or biological agents at large scale. At
the same time, these DNA sensor platforms need to eliminate
high costs associated with large numbers of samples and
biomedical reaction steps. Therefore, novel techniques are
currently warranted in order to facilitate cataloguing genetic
variants and enhance the fluorescence detection sensitivity of
DNA beyond the limits that current technologies offer.
Innovative assembly and fabrication of nanomaterials for
use as advanced biosensor substrates can be greatly beneficial
in increasing the detection sensitivity of biomolecular
fluorescence. Zinc oxide (ZnO) nanostructures have received
considerable attention, particularly due to their desirable
optical properties, which include a wide bandgap of 3.37 eV
and a large exciton binding energy of 60 meV at room
temperature. ZnO has been previously demonstrated
as a candidate material for use in a broad range of
technological applications. Examples of ZnO materials in
these areas include short-wavelength light-emitters [13, 14],
field-emitters [15], luminescence devices [16], UV lasers [17],
and solar cells [18–24]. Nanometre scale ZnO has
very good potential for aiding optical detection of target
bioconstituents, as ZnO nanomaterials are stable in typical
biomolecular detection environments, have attractive optical
properties, and can be easily processed throughmany synthetic
routes [25–30]. Despite its demonstrated functions in
broad areas and suitability for advanced optical detection,
biosensing applications of wide bandgap ZnO have not yet
been extensively realized.
Herein, we report the use of nanoscale ZnO materials
in the enhanced fluorescence detection of genetic materials.
We demonstrate that ZnO nanomaterials exhibit an optical
property useful in fostering the fluorescence signal from
fluorophore-linked DNA molecules and promoting detection
at ultratrace concentrations. Specifically, we show that
ZnO nanomaterials can serve as excellent signal-enhancing
substrates for hybridization reactions of model DNA systems
which involve genetically related Bacillus bacteria. Enhanced
detection limits of ZnO nanoplatforms in the identification
of a harmful Bacillus species were explored using both
covalent and non-covalent schemes of DNA immobilization.
In addition, in order to facilitate high-throughput screening
of genetic variants, we establish simple and straightforward
assembly routes which yield successful growth and fabrication
of these useful nanomaterials in a dense array format
directly upon their synthesis. Lastly, we show that arrayed
ZnO nanomaterials allow unambiguous detection of the
presence/absence of fluorescence signal from duplex-formed
DNA, which, in turn, enables rapid and accurate identification
of genetic mutation sites and discrimination of genetically
similar bacterial species.
Gram-positive Bacillus bacteria are commonly found in
soil, water, and airborne dust. Although most species of
Bacillus are harmless saprophytes, two species are considered
medically significant: Bacillus anthracis (B. anthracis) and
Bacillus cereus (B. cereus) [4, 31, 32]. B. anthracis is an
endospore-forming bacterium that causes inhalational anthrax.
It is considered to be one of the most potent biological
weapons because the spores are highly pathogenic, easily
transmissive, and very resistant to environmental stress. In the
suspected case of a biological attack, the accurate detection
of a biological agent such as B. anthracis will provide the
most direct and effective pathway in devising appropriate
treatment and containment plans in a timely manner since
the first appearance of noticeable anthrax symptoms can take
up to two months in humans. B. cereus, a genetically
closely related bacterium to B. anthracis, is motile and it can
cause toxin-mediated food poisoning. Health risks associated
with B. cereus are non-lethal, whereas B. anthracis can
potentially prompt a widespread fatal threat to public health.
When assessing impending health risks and threats, effective
DNA sequence analysis targeting specifically the genetically
differentiating regions of B. anthracis from its closely related
species is imperative in accurate identification of B. anthracis
among many Bacillus species with similar genetic sequences.
Three types of ZnO nanoplatform were used as needed
in our experiments: individual ZnO nanorods, striped ZnO
arrays, and open square ZnO arrays. Individual ZnO nanorods
were produced by using Ag colloids as catalysts. In order
to assemble striped ZnO nanoplatforms, microcontact printing
was used to deliver catalysts to predetermined locations of
substrates. The open square ZnO platforms were obtained by
first inking catalysts onto an elastomer stamp which contained
square arrays of desired dimensions and then transferring
the catalysts onto growth wafers via overpressure contact
printing [33]. Subsequently, ZnO nanomaterials were grown
from the patterned catalytic sites.
Three, custom-synthesized, probe oligonucleotides were
used in our experiments. The three oligonucleotides are
5-AGTGCGCGAGGAGCCT-3 (bas), 5-GTTACGGAAA
GAACCA-3 (bce), and 5-AGTGCGCGAGGAGCCT-C6-
NH2-3 (basa). The sequences of bas and basa probes
are specific to B. anthracis, whereas the sequence of
bce probes is specific to B. cereus. In addition, 6-
carboxyfluorescein modified oligonucleotide that is fully
complementary to the DNA sequence of bas as well as
that of basa was synthesized, 5-TCACGCGCTCCTCGGA-3
(basr). Upon covalently or non-covalently linking the three
probe oligonucleotides on ZnO nanoplatforms, hybridization
reactions were carried out using various concentrations of basr
in order to detect duplex formation of fully matching DNA
pairs and, therefore, to discriminate the biothreat agent of B.
anthracis from its genetically closely related but non-fatal B.
cereus. A commercially available confocal microscope was
used for fluorescence detection. The excitation and detection
wavelengths were chosen according to the specific emission
properties of the fluorophore that was employed in our proofof-
concept experiments.
Figure 1(a) displays our experimental design in order
to synthesize and assemble simultaneously nanoscale ZnO
materials into various platforms. Low-density synthesis on Ag
catalysts led to the growth of individual ZnO nanorods whose
average size is 4.1± 0.3 μm in length and 313.3± 68.3 nm in
width. Regularly spaced, stripe or square, platforms consisting
of nanoscale ZnO materials were constructed directly upon
their synthesis by microcontact printing catalyst particles on
the selective locations of growth substrates with the help of
pre-fabricated polydimethylsiloxane (PDMS) stamps. These
ZnO nanorods in the striped array platforms were grown lyingdown
parallel to the growth substrate, as shown in the left panel
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4楼2011-05-08 12:58:00
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appleXYJ

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of figure 1(b). The diameter of ZnO nanorods is larger than the
2876
Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays
Figure 1. (a) Schematic illustrations showing simultaneous synthesis and assembly of ZnO nanoplatforms consisting of (left) individual ZnO
nanorods and (right) periodically patterned ZnO nanostructures. (b) (Left) SEM image of a patterned ZnO platform with the stripe width and
repeat spacing of 50 μm. The inserted SEM image at the bottom left corner shows the lying-down arrangement of ZnO nanostructures inside
the patterned stripes. (Right) Confocal fluorescence image taken from the as-synthesized, striped, ZnO nanoplatform where no fluorescence
emission was detected. (c) Detection scheme to identify B. anthracis from B. cereus using ZnO nanoplatforms: PDMS chambers were used in
order to carry out simultaneous hybridization reactions on the same ZnO nanoplatform. The ZnO nanoplatform contained regularly patterned
ZnO stripes with a repeat spacing of 20 μm. Oligonucleotide probes of bce and bas were first introduced to the reaction chambers 1 and 2,
respectively. Subsequently, fluorescein modified basr strands were added to both chambers and allowed to form DNA duplex under the same
hybridization conditions. Confocal images taken from these samples showed clear fluorescence emission from chamber 2, in contrast to no
discernable fluorescence signal from chamber 1. The insets in the upper left corners of the confocal images are the corresponding bright field
images taken from each chamber after the duplex formation reaction. Distinctive fluorescence emission monitored from chamber 2 is due to
DNA duplex formation between fully complementary strands of bas and basr, whereas the lack of duplex formation between mismatching
sequences of bce and basr led to no observable fluorescence in chamber 1. The striped patterns of fluorescence emission observed from
chamber 2 faithfully mimic the underlying geometry of the ZnO nanoplatform.
diameter of the Ag catalyst particles. This effect is likely due to
high mobility of the Ag catalyst at our growth temperature of
900 ◦C, leading to the formation of catalyst aggregates, which,
in turn, serve as catalytic clusters for ZnO nanorod growth.
Apatterned substrate comprised of striped ZnO nanostructures,
20 μm inwidth and 20 μm in repeat spacing, was used as
a test bed in order to discriminate B. anthracis from B. cereus.
The as-synthesized ZnO platforms do not show any fluorescence
in the visible range as shown in figure 1(b). A PDMS
vessel containing two reaction chambers was placed on top of
the ZnO test bed (figure 1(c)). Two solutions of 20 μM bce
and 20 μM bas in TE buffer were subsequently introduced
to the chambers 1 and 2, respectively. After incubating the
two oligonucleotide strands on the exposed substrate surface in
each chamber, unbound bas and bce were removed from their
chambers by thoroughly rinsing with TE buffer. 20 μM basr
solution was then added to both chambers for a possible duplex
DNA interaction to take place. After the hybridization reaction,
the sample was rinsed carefully and thoroughly with the buffer
solution and unreactedDNA moleculeswere removed from the
substrate. The confocal fluorescence data of the two reaction
chambers are displayed in figure 1(c). No discernable fluorescence
signal was observed from chamber 1 due to lack of
DNA hybridization between the sequence mismatching strands
of bce and basr. In contrast, strong green emission was identified
from ZnO nanostructures in chamber 2 owing to bas/basr
duplex formation between the fully complementary pairs of
bas and basr. As the above experimental scheme involves nonspecific
adsorption of oligonucleotide probe molecules, single
stranded DNA probe molecules of bce or bas were randomly
distributed over the entire exposed surface area in each chamber
upon deposition. Despite the homogeneous distribution
of the DNA on both the exposed silicon oxide and patterned
ZnO regions in chamber 2, hybridization reactions between bas
and basr always led to fluorescence emission only from the
surface areas where ZnO is present. Therefore, the presence
of the underlying ZnO nanomaterials is evident in achieving
enhanced fluorescence detection of hybridized DNA. This effect
is clearly seen in the fluorescence patterns observed from
chamber 2 in figure 1(c). The striped patterns of fluorescence
emission monitored from chamber 2 faithfully mimic the underlying
geometry of the ZnO nanoplatform. On the other
hand, sample chambers containing bce showed no observable
fluorescence emission after hybridization reactions, regardless
of the chemical composition of the exposed surface area. All
oligonucleotides used in our experiments contain similar ratios
of pyrimidines to purines in their DNA sequences and, thus,
they exhibit similar adsorption behaviour to the underlying surfaces.
Therefore, the results shown in figure 1 are not due to
possible differences in the adsorption behaviour of the oligonucleotides.
Rather, the results in figure 1 clearly suggest that the
two critical factors leading to successful fluorescence emission
are DNA duplex formation between the fully complementary
strands and the presence of ZnO nanoplatforms.
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