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| Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays |
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【答案】應(yīng)助回帖
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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 |
7樓2011-05-08 12:59:24
2樓2011-05-08 12:54:18
【答案】應(yīng)助回帖
★ ★ ★ ★ ★ ★
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
【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
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