Immobilization of Oligonucleotides onto Zirconia-Modified Filter Paper

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Immobilization of Oligonucleotides onto Zirconia-Modified Filter Paper and Specific Molecular Recognition Wei Xiao and Jianguo Huang* Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 310027, China

bS Supporting Information ABSTRACT: A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile solgel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminalphosphate probe DNA was abundantly immobilized on the zirconiamodified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.

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NA detection has attracted considerable attention because of its wide applications in various areas such as biomedical diagnostics,1 gene expression,2 disease prevention,3 reaction discovery,4 forensic determination5 and so on. A large variety of DNA biosensors and microarrays based on optical, electrochemical, and mass-sensitive signals have been exploited;68 these regularly depend on the immobilization of a DNA probe on a solid substrate and the following hybridization assay with a target DNA in a homogeneous solution. Consequently, to manufacture a sensitive, reusable DNA device, the dense, stable immobilization of DNA probes on solid supports is essential. A great number of strategies including chemical bonding, the specific binding of biotinylated oligonucleotides to (strept)avidin, and physical adsorption for the attachment of DNA probes onto solid supports have been developed.816 For instance, interactions between functionalized surfaces and DNA probes such as activated carboxylic acidamine,9 thiolthiol,10 streptavidinbiotin,11 and electrostatic interactions12 are routinely utilized. However, most of the substrates are often made of glass,13,14 silicon wafers,15 mica,16 and so forth, whose surfaces are rigid and planar. By contrast, increasing interest is focused on morphologically complicated substrates because of their higher surface area, which furnishes more binding sites to DNA probes, thus effectively promoting the sensitivity of DNA recognition. In related work, the immobilization of DNA probes on diverse semiconducting nanowires,1720 metal/ metal oxide particles,21,22 quantum dots,23 and carbon nanotubes/ nanofibers24,25 has been reported. However, these nonflat supports are usually either expensive or toxic; therefore, it is necessary to r 2011 American Chemical Society

pursue low-cost and environmentally friendly substrates to which to attach DNA probes. Cellulose, which is abundant in nature, is a linear polymer of β-D-glucose that assembles into fibers with diameters ranging from nanometers to micrometers through multiple hydrogen bonds to form crossed 3D network structures. Such fibers have numerous hydroxyl groups on their surfaces by virtue of the sugar backbone and hence can be easily modified by an ultrathin zirconia gel layer.26 Meanwhile, zirconia is regarded to be a promising inorganic platform for anchoring biomolecules such as DNA and proteins,27 owing to its good biocompatibility and strong affinity for oxygen atoms. Herein, we combine the advantages of the two substances by coating cellulose fibers of ordinary filter paper with an ultrathin zirconia gel layer and then use the morphologically complex substrate to immobilize oligonucleotides directly so as to convert the composite into a novel biofunctional material. Because it has a high surface area, the oligonucleotide-functionalized paper achieved sensitive, selective, repetitive recognition for the corresponding complementary target DNA as illustrated in Figure 1. By means of a surface solgel approach, an ultrathin zirconia film was deposited onto the surface of each cellulose nanofiber of common commercial filter paper using Zr(OnBu)4 as a precursor (details in the Supporting Information). The calcination of the as-prepared cellulose/zirconia composite sheet results in a bulk Received: August 12, 2011 Revised: September 10, 2011 Published: September 11, 2011 12284

dx.doi.org/10.1021/la203150f | Langmuir 2011, 27, 12284–12288

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hierarchical nanotubular-structured zirconia sheet with a little shrinkage that is composed of zirconia nanotubes as replicas of the initial cellulose nanofibers. The tube wall thickness is ca. 20 nm for 10 deposition cycles of the zirconia gel film (Supporting Information, Figure S1). These results confirmed that the whole filter paper was uniformly and precisely deposited with the zirconia gel layer with nanoprecision. The as-prepared filter paper/zirconia composite sheet was used to capture oligonucleotides via a coordination effect between zirconia and the phosphate group of probe DNAs, which are listed in Table 1. To this end, a small patch of filter paper (8 4 mm2) deposited with an ultrathin zirconia gel film, denoted as [cellulose/(ZrO2)10] paper, was immersed in a solution of probe 1 (phosphate-functionalized at the 50 end and FITC-labeled at the 30 end, Table 1) in 1 M Tris-HCl buffer solution (pH 7.5) for 48 h in the dark at room temperature, followed by washing with copious amounts of buffer solution and pure water and drying in a stream of nitrogen. Upon observing the probe 1-modified filter paper (denoted as [cellulose/(ZrO2)10/probe 1] paper in this work; when other probe DNAs or target DNAs were captured on the filter paper or quartz plate, they were analogously denoted

Figure 1. Schematic representation of DNA immobilization and recognition on the surface of cellulose fibers.

hereafter) under a fluorescence microscope, it gave a characteristic green fluorescence of the FITC label (Figure 2a). The inset of Figure 2a is a magnified fluorescent image of a single microfiber, which produced clear green fluorescence throughout its body, indicating the viability of the intensive immobilization of probe 1 on the cellulose fibers. The self-assembly process of probe 1 was further verified by UVvis absorbance at around 495 nm for the FITC label and at 257 nm for DNA bases (Figure 2c) and by fluorescence emission at around 518 nm for the FITC fluorophore (Figure 2d). After attaching probe 1 from solutions with a series of concentrations, the zirconia-coated filter paper turned strongly fluorescent at high concentrations and gradually got weaker and weaker with decreasing concentration but remained visible enough even at a concentration of 1011 M (Figure 3), which is ascribed to the high surface area of the filter paper and the intense conjugation property of the zirconia gel layer for

Figure 2. Immobilization of oligonucleotide onto the surfaces of zirconia-coated cellulose fibers of filter paper. (a) Fluorescence and (b) transmitted bright-field micrographs of the [cellulose/(ZrO2)10/ probe 1] paper obtained by capturing probe 1 at a concentration of 107 M; the insets are enlarged images of individual microfibers. (c) UVvis spectra of the [cellulose/(ZrO2)10/probe 1] paper (red curve) and the [cellulose/(ZrO2)10] paper (black curve). (d) Fluorescence emission (λex = 490 nm) spectra for [cellulose/(ZrO2)10/probe 1] paper (red curve) and [cellulose/(ZrO2)10] paper (black curve). The [cellulose/ (ZrO2)10/probe 1] paper in plots c and d was obtained by capturing probe 1 at a concentration of 10 μM.

Table 1. Oligonucleotides Employed in This Work DNA denotation

base sequence

modification

probe 1

50 -TTT TCG ATG TCC GTA TGC-30 b

phosphate-functionalized at the 50 end and FITC-labeled at the 30 end

probe 2


FITC-labeled at the 30 end

probe 3


phosphate-functionalized at the 50 end

probe 4 FMTa

50 -TTT TCG ATG TCC GTA TGC-30 b 50 -GCA TAC GGA CAT CGA-30

N/A FITC-labeled at the 50 end

SMTa

50 -GCA TAC GCA CAT CGA-30 c


a

DMT

50 -GCA TAC GCA CTT CGA-30 c


TMTa

50 -GCA TCC GCA CTT CGA-30 c


NCTa

50 CGT ATG CCT GTA GCT-30


a

FMT, SMT, DMT, TMT, and NCT are abbreviations for fully matched, single-base-mismatched, double-base-mismatched, triple-base-mismatched and noncomplementary target DNA, respectively. b Italic letters serve as the 3-T spacer, which does not take part in hybridization but is capable of minimizing the interferences between the probe DNA and the solid surface.28 c Underlined letters represent the mismatched positions. 12285

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Figure 3. Fluorescence micrographs of a series of [cellulose/(ZrO2)10/ probe 1] paper obtained by immobilizing probe 1 in a 1 M Tris-HCl buffer solution (pH 7.5) at different concentrations. In each image, the sample and a piece of contrasting bare filter paper were placed on the left and right sides for comparison, respectively. Scale bars are 200 μm.

phosphate-functionalized probe 1. Porous solid substrates such as silicon,29,30 alumina oxide,31,32 silica,33 and conducting polymers34 were reported to be employed as matrices for DNA immobilization. Compared to these artificial substrates, the naturally occurring mechanical strength and flexibility of cellulose substances offers prior potentials of the biomolecular functional materials that were fabricated. Oligonucleotides contain a phosphate ester backbone, so terminally nonphosphate-functionalized oligonucleotides are also able to bind to the zirconia-modified cellulose fiber surface. For probe 2 (merely FITC-labeled at the 30 end, Table 1), whose sequence was the same as that of probe 1, the [cellulose/(ZrO2)10/probe 2] paper imparted evident fluorescence only after it was captured from solutions with concentrations higher than 109 M (Supporting Information, Figure S2). Therefore, compared with nonphosphatefunctionalized oligonucleotides, the affinity of the zirconia gel layer for terminally phosphate-functionalized ones is stronger. This effect could provide a possible pathway to separate oligonucleotides with terminal phosphates from a solution containing mixtures. Prior to recognition with complementary target DNA, a nonlabeled but terminally phosphate-functionalized oligonucleotide (probe 3, Table 1) was first immobilized on zirconiamodified filter paper at a concentration of 10 μM. Then the [cellulose/(ZrO2)10/probe 3] paper was placed in Denhardt’s solution (1% w/v BSA (bovine serum albumin), 2% Ficoll 400, and 2% polyvinylpyrollidone (PVP)) for 5 h to adsorb BSA sufficiently to block the nonspecific adsorption of the target DNA. Actually, the nonspecific adsorption of FMT (Table 1) mainly arising from its phosphate ester backbone could thus be effectively avoided because only a totally black fluorescence micrograph of [cellulose/(ZrO2)10/BSA] paper was acquired after it was soaked in FMT solution at a concentration of 106 M (Supporting Information, Figure S3). The resulting [cellulose/ (ZrO2)10/probe 3/BSA] paper was rinsed and then immersed into FMT solution in a 1 M Tris-HCl buffer solution (pH 7.5), followed by dark incubation for 24 h. Afterward, the DNArecognized filter paper was thoroughly washed, dried, and visually inspected under the fluorescence microscope. Figure 4 exhibits the DNA recognition results of [cellulose/(ZrO2)10/ probe 3/BSA] paper for FMT. It is seen that both bulk paper and individual microfiber emitted bright fluorescence, testifying to a successful recognition event (Figure 4a,b). The deposition of a [zirconia/probe 3/BSA/FMT] multilayer on the cellulose fiber surface was carefully examined by field-emission scanning

Figure 4. Recognition of oligonucleotide-functionalized filter paper for corresponding complementary target DNA. (a) Fluorescence and (b) transmitted bright-field micrographs of [cellulose/(ZrO2)10/probe 3/BSA/FMT] paper obtained by recognizing FMT at a concentration of 107 M, where the insets are enlarged images of an individual microfiber. (c) Fluorescence micrographs of a set of [cellulose/(ZrO2)10/probe 3/BSA/FMT] paper obtained by recognizing FMT at different concentrations. In each image in c, the sample and a piece of contrasting bare filter paper were placed on the left and right sides for comparison, respectively.

electron microscopy (FE-SEM), but no obvious morphological change was found after all of the modification steps, illustrating that each substance was uniformly deposited as a monolayer, and the situation was the same for the deposition of the [zirconia/ probe 3/BSA/FMT] multilayer on a planar quartz plate (Supporting Information, Figure S4). Moreover, the fluorescence intensity of the [cellulose/(ZrO2)10/probe 3/BSA/FMT] paper gradually decreased as the concentration of FMT diminished, and the detection limit for FMT emerged at 109 M (Figure 4c). The lowest detectable concentration for the same target DNA recognized by [cellulose/(ZrO2)10/probe 4/BSA] paper was only 107 M (Supporting Information, Figure S5), which was 2 orders of magnitude higher than the former, implying that the terminal phosphate group in probe DNA was crucial to the high-sensitivity recognition. These phenomena can be attributed to two reasons. On the one hand, in contrast with probe 4, more probe 3 molecules were captured on cellulose fibers to provide a higher surface concentration to react with target DNA (Supporting Information, Figure S6); on the other hand, because of the stronger affinity of zirconia for the terminal phosphate group, probe 3 preferred to bind to cellulose fibers through coordination between zirconia and its terminal phosphate group instead of that between zirconia and its phosphate ester backbone. Therefore, a majority of probe 3 molecules were likely to stand on the surface, adopting a perpendicular conformation, which favored hybridization. Nevertheless, probe 4 was obliged to rely on its phosphate ester backbone to be anchored and thus was inclined to lie on the surface, whose prostrate conformation was less hybridizable, leading to insensitive recognition behavior. Similarly, for single-base-, doublebase-, and triple-base-mismatched target DNAs (Table 1), only 12286


Langmuir their concentrations were respectively higher than 108, 107, and 106 M, and [cellulose/(ZrO2)10/probe 3/BSA] paper could be fluorescent after it was soaked in the corresponding mismatched target DNA solution (Supporting Information, Figure S7ac). In spite of the high concentration of NCT (Table 1) reaching to 106 M, there was still no green fluorescence for [cellulose/(ZrO2)10/probe 3/BSA] paper after immersion in an NCT solution (Supporting Information, Figure S7d). These results suggested that such oligonucleotide-functionalized cellulosic material possessed high selectivity for the recognition of a sequence-specific target DNA. The nanomolar sensitivity achieved by the current cellulose-based system for the detection of oligonucleotides is comparable to those of the fluorescence-based fiber-optic biosensors35,36 and the biotinylated molecular beacon (MB) probes,3739 and the present device fabrication method is much more facile and low-cost. The deposited zirconia gel film rendered an activated surface to bind oligonucleotides. Without it, the direct immobilization of DNA on cellulose fibers was not feasible. Control experiments on bare filter paper for capturing probe 1 or determining FMT at a concentration of 10 μM were conducted, but no fluorescence response was found for the resulting paper, reflecting that the zirconia nanocoating was essential for probe DNA immobilization and subsequent target DNA recognition. Benefiting from the high surface area, which arose from the hierarchically fibrous structures of filter paper, highly effective DNA immobilization and sensitive recognition were accomplished on such a morphologically complex substrate. However, this was not the case with respect to the flat quartz support. The detection limits for the immobilization of probe 1 with the [quartz/(ZrO2)10] plate and the recognition of FMT with the [quartz/(ZrO2)10/probe 3/BSA] plate were 109 and 107 M, respectively (Supporting Information, Figure S8), both of which were 2 orders of magnitude higher than the corresponding detection limits based on the aforementioned cellulose substance system. To make an intuitive comparison, the two substrates, filter paper and quartz plate, which were immobilized with probe 1 at concentrations of 107 and 105 M, respectively, were put together and subjected to fluorescence observation at the same time. The cellulose specimen was so fluorescent that the quartz one was totally black (Supporting Information, Figure S9a), although it surely emitted fluorescence; likewise, filter paper recognized with FMT at a concentration of 107 M showed much stronger fluorescence so that the fluorescent quartz plate, which was recognized with FMT at a concentration of 105 M, was seen as dark (Supporting Information, Figure S9b). Namely, thanks to the high surface area, a larger amount of probe or target DNA was immobilized or recognized on the morphologically complex surface of the filter paper. Reproducibility is one of the most important parameters for DNA recognition or sensing, which could be evaluated by an HDR (hybridizationdehybridizationrehybridization) experiment.19 The [cellulose/(ZrO2)10/probe 3/BSA] paper gave a fluorescence response after hybridizing with FMT, then the fluorescence disappeared under treatment with an aqueous solution of 0.1% (w/v) sodium dodecyl sulfate (SDS) for 1.5 h at 100 °C and the fluorescence signal was revived by the adsorption of BSA and rehybridization with FMT, symbolizing the occurrence of repetitive recognition event. Cycles of HDR were repeated seven times until no fluorescence was detected (Supporting Information, Figure S10). According to the fact that the fluorescent [cellulose/(ZrO2)10/probe 1] paper no longer

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emitted fluorescence after a heat treatment in 0.1% SDS solution for 60 h (Supporting Information, Figure S11), it is assumed that the gradual removal and loss of activity of probe 3 took place during dehybridization procedures,40 which would be responsible for the eventual disappearance of the recognition ability. Even so, the oligonucleotide-functionalized filter paper remained quite reusable for sequence-specific DNA recognition. In summary, a new platform based on a morphologically complicated cellulose substance has been proposed for the immobilization of oligonucleotides, which is first modified by a zirconia nanocoating through a facile surface solgel process. The oligonucleotide-functionalized paper realizes a sensitive and duplicated recognition effect for corresponding complementary target DNA on a nanomolar level by taking advantage of the high surface area of filter paper and the extensive concentration of probe DNA on the zirconia-coated cellulose fiber surface. The deposition of a metal oxide gel film onto cellulose fibers could afford an activated surface with a large area over which to immobilize different biomolecules and endow cellulosic materials with various biological functions. Moreover, cellulose substances such as filter paper have been demonstrated to be an ideal platform for the development of unique sensitive probes and detectors for biomolecules and inorganic ions because of their naturally produced hierarchical structures with a large surface area.41,42 With the intrinsic properties of nontoxicity, degradability, and flexibility, biologically functionalized cellulosic materials will be potentially applicable to many practical and challenging fields such as biomolecular enrichment, separation, sensing, and immunoassay.

’ ASSOCIATED CONTENT

bS

Supporting Information. Experimental details. Digital and fluorescent photographs. SEM and TEM images and fluorescence emission and UVvis adsorption spectra of the corresponding cellulose- and quartz-based samples. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel and Fax: +86-571-8795-1202. E-mail: [email protected].

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Immobilization of Oligonucleotides onto Zirconia-Modified Filter Paper

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