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gaotiger

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Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays
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appleXYJ

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detection discussed above, we have also explored a covalent
strategy in immobilizing oligonucleotide probes and compared
the fluorescence detection capability of ZnO nanoplatforms
systematically at various target DNA concentrations. Figures 2
and 3 summarize our results gathered from fluorescence
emission of DNA duplexes of bas/basr and basa/basr when
the oligonucleotide probes were non-covalently and covalently
linked to the underlying ZnO nanoplatforms. The specific
DNA sequences of bas and basa are identical to each other. In
comparison to the bas probe containing non-modified 5 end,
the basa probe has an amine group at the 3 end linked through
a short spacer. This primary amine group of basa was used to
couple basa strands directly and covalently to an epoxy group
of a silane-treated ZnO nanoplatforms [9, 34–37]
Figure 2(a) displays the result from the non-covalent
linking method where bas was nonspecifically deposited onto
the underlying ZnO nanoplatform before introducing basr.
Figure 2(a)-1 represents a typical SEM image of a striped
ZnO sensor array before coupling any biomolecules. Probe
oligonucleotide strands were adsorbed onto the nanoplatform
by incubating a 20 μM solution of bas for 5 min. Then,
loosely bound strands were removed by multiple washing
with TE buffer. A 20 μM solution, containing the target
strands of basr, was added to bas-ZnO stripe arrays in
order to carry out DNA hybridization for 1 h at room
temperature. After the hybridization reaction, the sample
was rinsed with an ample amount of TE buffer and gently
blow dried before imaging with a confocal microscope.
Figure 2(a)-2 shows the fluorescence emission monitored
from the sample after duplex DNA formation of bas/basr.
Figure 2(b) displays fluorescence emission monitored by
first covalently linking basa through an epoxy terminus on
the glycidoxylpropyltrimethoxysilane-(GOPS-) modified ZnO
surface and then performing hybridization reactions with a
2 μM solution of basr. The targetDNA concentration used in
this covalent coupling scheme is an order of magnitude lower
than that of the previously described non-covalent detection. In
addition, the PMT setting for the confocal measurements was
20% lower for figure 2(b)-2 than the value used to collect the
fluorescence image of figure 2(a)-2. Despite the reduced DNA
concentration and detection setting, fluorescence signal from
the covalently conjugated basa/basr pairs was much stronger
than physically adsorbed bas/basr pairs. Figure 2(c) displays
fluorescence emission monitored on an open square ZnO array
after carrying out a hybridization reaction between covalently
bound basa strands and the fully sequence matching basr
strands. The underlying ZnO nanostructures exhibit squares of
10 μm in length with a repeat spacing of 10 μm. In the series
of confocal images of figure 2(c) taken after the hybridization
reaction of 20 μM basa/20 μM basr, open square patterns
of fluorescence emission are clearly visible, which closely
follows the underlying ZnO square sensor array.
As our covalent attachment scheme is effective for
derivatizing both silicon oxide and ZnO surfaces, basa strands
are present not only on the ZnO surface but also on silicon
oxide after the covalent linking procedure. However, we
monitored fluorescence only from the surface areas where
nanoscale ZnO materials are present, i.e., DNA fluorescence
images closely mimic the underlying ZnO patterns. This
1) 2)
1) 2)
Figure 2. (a) Fluorescence emission monitored using a non-covalent
DNA immobilization scheme. (1) SEM image of as-grown, patterned
ZnO nanoplatforms consisting of stripes with a repeat spacing of
20 μm. (2) Oligonucleotide probe strands of bas were
nonspecifically adsorbed onto the ZnO nanoplatform shown in (1)
and subsequently reacted with 20 μM basr to form double stranded
DNA. (b) Fluorescence emission monitored using a covalent strategy
in order to link oligonucleotide probe molecules to striped ZnO
arrays. (1) SEM image of as-grown, patterned ZnO nanoplatforms
consisting of stripes with a repeat spacing of 20 μm. (2)
Amine-terminated oligonucleotide strands of basa were used to
covalently link the probe molecules to the ZnO nanoplatform shown
in (1) and they were subsequently reacted with 2 μM basr to form
DNA duplex. Confocal images taken from these samples indicate
that covalent derivatization of probe DNA strands to the
nanoplatform, when compared to physical adsorption of the probe
strands, leads to higher fluorescence emission even when using a
lower target DNA concentration. (c) Fluorescence emission
monitored from covalently bound basa strands and the fully
sequence matching basr strands on an open square ZnO array. The
concentrations of the probe and target strands were the same, 20 μM.
The underlying ZnO nanostructures as well as confocal fluorescence
patterns exhibit open squares of 10 μm in length with a repeat
spacing of 10 μm.
observation again demonstrates the importance of ZnO
nanoplatforms in the enhanced fluorescence detection resulting
from DNA duplex formation.
We performed a control experiment using silicon nanorods
as substrates. These silicon nanorods exhibit similar
dimensions as ZnO nanorods used in our experiments and,
thus, present similar amounts of surface area as ZnO
nanoplatforms. Yet, after hybridization reactions between the
strands of bce/basr as well as bas/basr, the silicon nanorod
samples did not yield any fluorescence emission even though
higher DNA concentrations than 20 μM were used to carry
out the duplex formation reactions on silicon nanorod surfaces.
Therefore, we do not believe that the observed fluorescence
enhancement is due to possible variations in exposed surface
area.
The fluorescence effect is likely to be related to the
inherent optical property of ZnO. Enhanced fluorescence
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6楼2011-05-08 12:59:09
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appleXYJ

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我有，怎么发给你呀！           
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2楼2011-05-08 12:54:18
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appleXYJ

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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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appleXYJ

银虫 (小有名气)

【答案】应助回帖

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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