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| Ultrasensitive DNA sequence detection using nanoscale ZnO sensor arrays |
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2樓2011-05-08 12:54:18
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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
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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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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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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 |
6樓2011-05-08 12:59:09
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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
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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. |
8樓2011-05-08 12:59:48
9樓2011-05-08 13:00:43
10樓2011-05-08 13:15:36
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