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dhd997(金幣+6, EPI+1): 熱心啊 2011-05-08 13:04:36
dhd997(金幣+6, EPI+1): 熱心啊 2011-05-08 13:04:36
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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/ |
3樓2011-05-08 12:57:34
2樓2011-05-08 12:54:18
【答案】應(yīng)助回帖
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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 |
4樓2011-05-08 12:58:00
【答案】應(yīng)助回帖
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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. |
5樓2011-05-08 12:58:43
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