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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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appleXYJ

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basa/basr at 20 μM basr concentration. Data points corresponding
to the covalent basa/basr and non-covalent bas/basr
are shown in red and blue, respectively. Figure 3(b) displays
typical confocal images taken from (1) 20 μMbasa/2 μMbasr
and (2) 20 μM bas/20 μM basr on striped ZnO sensor arrays
and figure 3(c) shows representative images obtained from (1)
20 μM basa/20 μM basr and (2) 20 μM bas/20 μM basr on
individual ZnO nanorods. Data provided in figure 3(a) clearly
indicate that the covalent linking scheme of DNA to the underlying
ZnO nanoplatforms led to much higher fluorescence
intensity of the duplex DNA regardless of the target concentration
or the type of ZnO nanoplatforms. The combined use of
ZnO nanoplatforms and the covalent linking scheme allowed
ultrasensitive genetic sequence detection at DNA concentration
levels down to 2 fM when using a conventional confocal
microscope equipped with a 40 mW Ar laser. On the other
hand, the detection limit of ZnO nanoplatforms coupled with
DNA through the non-covalent linking scheme was 20 nM. The
lowest detection limit is defined by the DNA concentration for
which the observed fluorescence signal exceeds the baseline
noise by a factor of three.
In summary, we demonstrated for the first time that
engineered nanoscale zinc oxide structures can be effectively
used for screening genetic variants of closely related Bacillus
species and identifying the biothreat agent B. anthracis. We
used both covalent and non-covalent linking schemes in order
to attach probe DNA strands to various ZnO nanoplatforms.
After carrying out duplex formation reaction with target DNA
strands, fluorescence intensity was measured and compared.
The presence of the underlying ZnO nanomaterialswas critical
in achieving increased fluorescence detection of hybridized
DNA. When coupled with the covalent attachment strategy
of DNA to these nanomaterials, the inherently increased
fluorescence detection capability of ZnO nanoplatforms was
enhanced even more significantly. This collective approach
allowed detection of the target species B. anthracis at sample
concentrations as low as a few femtomolar level and, therefore,
permitted highly sensitive identification of the biothreat agent.
We also demonstrated the easy integration potential of the
nanoscale ZnO materials into high-density arrays directly upon
their synthesis. When combined with conventional automatic
sample handling apparatus and computerized fluorescence
detection equipment, our approach can greatly promote the
use of ZnO nanomaterials as signal enhancing substrates
for multiplexed, high-throughput optical DNA sensor arrays.
These ZnO nanoplatforms will be extremely beneficial in
accomplishing rapid, multiplexed, high-throughput, highly
sensitive detection of genetic variations.
Methods
Individual and patterned ZnO growth
Silicon wafers (resistivity < 1  cm, thickness 0.017 inch)
were obtained from Silicon Inc. Poly-L-lysine (PLL) in H2O
(0.1% w/v) and Ag colloids (40 nm in diameter) were obtained
from Ted Pella, Inc. Zinc oxide (99.999%) and graphite (99%)
powders were obtained from Alfa Aesar. 100 μl of Ag colloid
was deposited on a PLL treated Si wafer for 30 min. The
substrate was then rinsed with deionized water and gently
blow dried with nitrogen. The growth wafer was placed
approximately 5–6 inches downstream from a 2:1 mixture of
graphite powder and zinc oxide, which was kept at the centre of
a horizontal resistance furnace. The sample was subsequently
heated to 900 ◦C for 30 min under a constant flow of 100
standard cubic centimetres per minute (sccm) of Ar.
In order to pattern the substrates with catalysts
at predetermined locations, polydimethylsiloxane (PDMS)
stamps containing periodic stripe or square patterns of 10, 20,
or 50 μm in width were constructed by casting and curing
an elastomeric polymer, Sylgard 184 (Dow Corning), against
a photoresist micropatterned Si master, which was fabricated
using standard photolithography procedures [42]. 50 μl of
PLL placed on the PDMS stamp was gently blow dried with
nitrogen and was then transferred onto clean growth wafers
for 30 s. Following elastomeric stamping, the samples were
treated with 100 μl of Ag colloid (40 nm in diameter) for
30 min. ZnO nanomaterials were synthesized at 900 ◦C for
1–2 h under a constant flow of 100 standard cubic centimetres
per minute (sccm) of Ar.
Preparation of DNA
Three custom synthesized probe oligonucleotides were used
in our experiments. The three oligonucleotides are 5-
AGTGCGCGAGGAGCCT-3 (bas), 5-GTTACGGAAAGAA
CCA-3 (bce), and 5-AGTGCGCGAGGAGCCT-C6-NH2-
3 (basa). 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). All oligonucleotides
were reconstituted in TE buffer solution (10 mM Tris, 1 mM
EDTA, pH 8.0).
When using the covalent detection scheme, ZnO
nanoplatforms were first silanized through the following
process. The ZnO platforms were first submerged in a
0.1% (v/v) solution of 3-glycidoxylpropyltrimethoxysilane
(GOPS) in 95% ethanol for 1 h. Following the GOPS
incubation, the nanoplatforms were rinsed with ethanol in
order to remove excess silane and then gently blow dried. A
mixture of 0.01% (v/v) of poly-L-lysine (PLL, 0.1% solution)
was deposited onto the ZnO substrates and allowed to sit
for 1 h. The samples were then rinsed thoroughly with deionized
water and dried. Probe oligonucleotides, basa, of
desired concentrations were prepared in a reaction mixture
(10 mM Tris, 1 mM EDTA, 10 mM MgCl2, 50 mM NaCl,
and 0.1 M KOH) and deposited on the ZnO nanoplatforms.
After basa deposition, the ZnO substrates were placed in a
humidity chamber at 37 ◦C and incubated for 6 h. Upon
covalent attachment of basa, the sampleswere then rinsed with
de-ionized water and dried. Complementary strands of DNA
were then added to the probe strands. Varying concentrations
of basr in hybridization buffer (10 mM Tris, 1 mM EDTA,
10 mM MgCl2, 50 mM NaCl, pH 8.0) were deposited onto
the modified ZnO platforms for 1 h at room temperature.
The concentration of basr used in our experiments ranged
from 2 fM to 20 μM. After duplex formation reaction, the
samples were then rinsed multiple times with TE and dried
with a gentle stream of nitrogen. For a noncovalent detection
scheme, 50 μl of 20 μM bas were deposited onto ZnO striped
2880
Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays
arrays or nanorods for 5 min and then rinsed with TE. The
compliment strand, basr, of varying concentrations in reaction
buffer (10 mM Tris, 1 mM EDTA, 10 mM MgCl2, 50 mM
NaCl, pH 8.0) was deposited onto the platforms for 1 h at room
temperature. The samples were rinsed with ample TE.
Simultaneous monitoring of DNA interactions were
carried out on the same ZnO striped substrate by using a PDMS
elastomer that contained two reaction chambers. The PDMS
piece conformed to the underlying ZnO substrate and provided
isolated compartments for conducting two independent DNA
hybridization reactions. 5 μl of bas and bce at a concentration
of 20 μMwere first introduced into the two separate chambers.
After 5 min of deposition, the solutions were removed and then
rinsed with TE solution. Subsequently, 5 μl of 20 μMbasr was
added to both chambers and the oligonucleotides were allowed
to hybridize for 1 h at room temperature. Before disengaging
the PDMS piece from the ZnO substrate, excess DNA was
carefully removed and unbound oligonucleotides were rinsed
with an ample amount of TE.
Sample characterization
FEI/Philips XL 20 operated at 20 kV was used in the
SEM characterization of as-grown nanomaterials of ZnO. The
confocal microscope data were collected using a conventional
confocal laser scanning microscope (Olympus Fluoview 300).
Samples were excited with the 488 nm line of a 40 mW
Ar laser. The fluorescence emission from biosamples was
separated from the excitation light by a dichroic beam splitter
and a long-pass filter with cut-off wavelengths of 510 nm.
Scanned fluorescence signals from samples were collected at
a resolution of 512 pixels. The confocal microscope was also
equipped with a 100 W mercury arc lamp (Osram), which
allowed overall inspection of a large sample area in a single
view frame.
Acknowledgments
We acknowledge Dr C Reddy for the use of the confocal and
fluorescence microscope and thank E Kunze and S Magargee
for helpful discussions regarding fluorescence microscopy
measurements. J H acknowledges partial support of this work
by the Huck Institutes of the Life Sciences and the Materials
Research Institute at the Pennsylvania State University.
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8楼2011-05-08 12:59:48
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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

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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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