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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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Figure 3. Fluorescence intensity comparison between covalent and non-covalent DNA detection schemes. Striped ZnO sensor arrays and
individual ZnO nanorods were utilized as enhanced platforms for identifying DNA sequence. The ZnO sensor arrays consisted of periodically
spaced, striped patterns of 20 μm in both width and repeat spacing. The average length and width of the individual ZnO nanorods are
4.1 ± 0.3 μm and 313.3 ± 68.3 nm, respectively. After covalently or non-covalently attaching the oligonucleotide probes,
fluorescein-conjugated target DNA whose sequence is fully complementary to the probe strands was annealed onto the probe strands. Probe
strands used for the covalent and non-covalent attachment schemes were 20 μM solutions of basa and bas, respectively. Concentrations of the
target basr, used in each hybridization reaction, ranged from 2 fM to 20 μM. (a) Fluorescence intensity was measured from the two
hybridized DNA duplexes, one involving covalently bound basa/basr and the other involving non-covalently attached bas/basr. The relative
fluorescence intensity is then plotted versus the concentration of basr. Data shown in red represent the results from the covalent strategy
whereas data indicated in blue correspond to those from the non-covalent linking scheme. The two insets display rescaled data points at low
concentration and clearly show the lowest detection limits of the two DNA linking schemes observed from the ZnO stripe array and individual
ZnO nanorods. (left) Relative fluorescence intensity observed from basa/basr versus bas/basr duplexes on striped ZnO sensor arrays is
plotted against basr concentration. (right) Relative fluorescence intensity observed from basa/basr versus bas/basr duplexes on individual
ZnO nanorods is plotted against basr concentration. The dashed lines are inserted as a guide to the eye to follow data points. (b) Confocal
images taken from (1) 20 μM basa/2 μM basr and (2) 20 μM bas/20 μM basr on striped ZnO sensor arrays. (c) Confocal images obtained
from (1) 20 μM basa/20 μM basr and (2) 20 μM bas/20 μM basr on individual ZnO nanorods. The covalent linking scheme to the
underlying ZnO nanoplatforms led to much higher fluorescence from duplex DNA in all cases. When using the covalent linking scheme on
ZnO nanoplatforms, fluorescence signal was detectable even at as low as 2 fM of the target DNA concentration. 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.
emission in the presence of ZnO nanorods may be explained
by changes in photonic mode density and/or reduction in selfquenching
of fluorophores. Changes in photonic mode density
and subsequent alterations in radiative decay rates have been
previously observed in metal enhanced fluorescence [38–40].
The presence of ZnO nanorods may lead to modifications
in the decay rates of radiative and non-radiative pathways,
leading to dominantly fast radiative decay. The fluorophores
used in our experiment display a self-quenching property
due to the presence of traps in their energy levels [40, 41].
The presence of ZnO nanorods may disable these traps and
reduce self-quenching, resulting in enhanced fluorescence. The
exact mechanisms governing the observed ZnO nanoplatformenabled
fluorescence need to be explored further and are
currently under our investigation.
The ZnO nanoarrayed substrates displayed in figure 2(c)
can be seamlessly combined with conventional robotic sample
deposition apparatus in order to handle many DNA samples
for simultaneous screening. Combined with the easy synthetic
and integration routes of the materials demonstrated in this
paper, our results suggest that ZnO nanoplatforms can be
efficiently used for rapid identification of a large number
of biologically threatening subjects and bioagents from their
genetically similar species.
In order to substantiate the increased fluorescence intensity
monitored when using the covalent linking strategy, we
measured fluorescence intensity of the duplex forming strands
of basa/basr and bas/basr at various basr concentrations. The
observed fluorescence intensity values between the covalent
and non-covalent DNA attachment schemes were then systematically
compared against one another (figure 3). Striped ZnO
sensor arrays and individual ZnO nanorods were utilized as
duplex detection platforms. The sensor arrays consisted of
periodically spaced ZnO stripes of 20 μm in width and repeat
spacing. 20 μM solutions of basa and bas were used for
the covalent and non-covalent attachment of oligonucleotides
to underlying ZnO platforms, respectively. After covalently
or non-covalently attaching the oligonucleotide probes to desired
ZnO nanoplatforms, fluorescein-conjugated basr whose
sequence is fully complementary to the probe strands were annealed
onto basa or bas strands. Solutions ranging from 2 fM
to 20 μM basr were used in these hybridization experiments.
Fluorescence intensity was measured and relative fluorescence
intensity is then plotted versus the concentration of the target
strand, basr. Figure 3(a) summarizes the results of fluorescence
intensity difference of the DNA duplexes formed on
striped ZnO sensor arrays (left) and individual ZnO nanorods
(right). All fluorescence signals of both basa/basr and bas/basr
were normalized to the fluorescence intensity measured from
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7楼2011-05-08 12:59:24
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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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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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