DNA Hybrids: Functional Modules for the Detection of

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Ag Nanocluster/DNA Hybrids: Functional Modules for the Detection of Nitroaromatic and RDX Explosives Natalie Enkin,‡ Etery Sharon,‡ Eyal Golub, and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: Luminescent Ag nanoclusters (NCs) stabilized by nucleic acids are implemented as optical labels for the detection of the explosives picric acid, trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The sensing modules consist of two parts, a nucleic acid with the nucleic acid-stabilized Ag NCs and a nucleic acid functionalized with electron-donating units, including L-DOPA, L-tyrosine and 6-hydroxy-L-DOPA, self-assembled on a nucleic acid scaffold. The formation of donor−acceptor complexes between the nitro-substituted explosives, exhibiting electronacceptor properties, and the electron-donating sites, associated with the sensing modules, concentrates the explosives in close proximity to the Ag NCs. This leads to the electrontransfer quenching of the luminescence of the Ag NCs by the explosive molecule. The quenching of the luminescence of the Ag NCs provides a readout signal for the sensing process. The sensitivities of the analytical platforms are controlled by the electrondonating properties of the donor substituents, and 6-hydroxy-L-DOPA was found to be the most sensitive donor. Picric acid, TNT, and RDX are analyzed with detection limits corresponding to 5.2 × 10−12 M, 1.0 × 10−12 M, and 3.0 × 10−12 M, respectively, using the 6-hydroxy-L-DOPA-modified Ag NCs sensing module. KEYWORDS: Sensor, nanoparticle, luminescence, TNT, picric acid, RDX

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luminescence of the QDs, thus providing a readout signal for the sensing process. The synthesis of fluorescent metal nanoclusters (NCs), for example, Ag NCs, and their application for developing sensing platforms attracts substantial research activities.25−27 Specifically, it was found that programmed cytosine-rich singlestranded nucleic acids or duplex nucleic acid structures stabilize the luminescent Ag NCs.28−32 The nucleic acid-stabilized Ag NCs exhibit unique photophysical properties reflected by their high-luminescence quantum yields, photostability, and tunable luminescence features that are governed both by the sizes of the different Ag NCs, and the specific sequence of the protecting DNA coating. The unique optical properties of Ag NCs were recently implemented to develop different sensing platforms. These included the detection of DNA analytes,33 single nucleotide mutations,34,35 micro-RNA,36 aptamer-substrate complexes,37−39 and for the analysis of enzymatic activities,40 such as of glucose oxidase or tyrosinase. Also, Ag NCs have been applied as optical probes for the detection of metal ions,41 such as Hg2+ or Cu2+, and low molecular weight substrates such as cysteine or glutathione.42 Nonetheless, the application of the Ag NCs for the sensing of low molecular weight analytes was limited to thiol-containing substrates that directly interact with

he rapid and sensitive detection of nitroaromatic explosives attracts recent research efforts,1,2 and different optical3−8 or electrochemical9−14 sensors for trinitrotoluene (TNT) or hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were reported. Other sensors for explosives include surface plasmon resonance (SPR)15 and surface acoustic wave (SAW)-based piezoelectric crystals modified with organic coatings.16,17 Antibody-based surface plasmon resonance (SPR) biosensors for the detection of TNT were reported, through the displacement of surface-bound antibodies by the target explosive molecules.18 Also, the aggregation of electron donor-functionalized Au NPs by the electron acceptor TNT molecule was applied for the optical detection of the TNT analyte.19 Semiconductor quantum dots (QDs) exhibit unique optical and photophysical properties, and these were extensively applied for the development of various luminescent sensors20 and biosensors.21,22 Specifically, semiconductor QDs were implemented for the optical detection of explosives.23 CdSe/ZnS QDs modified with electron-donating functionalities were used as luminescent probes for the binding and sensing of either TNT or RDX via an electron-transfer quenching mechanism of the QDs. Alternatively, semiconductor QDs were modified with anti-TNT antibodies that bind a dyelabeled TNT molecule probe that stimulates a FRET quenching of the QDs.24 The displacement of the dye-labeled probe by the unmodified TNT analyte triggered-on the © 2014 American Chemical Society

Received: July 17, 2014 Revised: July 28, 2014 Published: July 29, 2014 4918

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Figure 1. Schematic nucleic acid−based sensing module consisting of Ag nanocluster- and electron donor-functionalized nucleic acids. The quenching of the luminescence of the Ag NCs by the explosive substrates associated with the sensing module via donor−acceptor interactions provides the readout signal.

that lacks the L-DOPA donor units (only the amino-modified nucleic acid (3) is hybridized with the scaffold) with diffusional picric acid (even at a high concentration, 1.5 μM), does not lead to an efficient luminescence quenching of the NCs. These results imply that the concentration of (7) on the sensing module, via donor−acceptor interactions, brings (7) into a spatial proximity with the Ag NCs, thus leading to the effective quenching of the NCs. Figure 2B depicts the resulting calibration curve corresponding to the luminescence changes of the sensing module in the presence of different concentrations of picric acid, (7). The detection limit for sensing picric acid by the system corresponded to 1.2 × 10−11 M. Assuming that only the picric acid bound to the L-DOPA donor units leads to the quenching of the luminescence of the Ag NCs, and that a 1:1 donor−acceptor ratio between L-DOPA and picric acid is formed, the calibration curve shown in Figure 2B was analyzed in terms of the Langmuir-type binding model to evaluate the dissociation constant, Supporting Information Figure S1. The derived dissociation constant corresponds to Kd = 1.7 × 10−11 M. It should be noted that the stated concentration of the sensing module (1 × 10−6 M, see Supporting Information) is needed to generate a detectable luminescence change upon analyzing (7). This module concentration is substantially higher than the value required to reach the stated detection limit. This is attributed to the fact that the synthesis of the nucleic acid-stabilized Ag NCs leads only to a fraction of luminescent Ag NCs. That is, the nonluminescent nucleic acid-stabilized Ag NCs occupy the sensing module, yet they lack the photophysical properties for the sensing process. Realizing that the donor−acceptor complex between the donor units associated with the sensing module and picric acid provides the mechanism for the successful analysis of picric acid by the Ag NCs, tuning the electron-donating properties of the electron-donor unit would control and improve the sensing performance of the system. Toward this goal, we compared the sensing performance of different sensing modules that included

the Ag NCs. In fact, the potential use of the Ag NCs/DNA hybrids as functional scaffolds to construct versatile sensing modules for analytical applications is yet unexplored. In the present study, we demonstrate the assembly of supramolecular nanostructures of Ag NCs/nucleic acid hybrids coupled to electron donor-modified nucleic acids, and the use of these systems as functional modules for sensing substrates exhibiting electron-acceptor properties, particularly, explosives modified by nitro substituents. The sensing module for the analysis of explosives by Ag NCs is depicted in Figure 1. The nucleic acid (1) acts as a scaffold for organizing the different components of the system and contains two hybridization domains for nucleic acids (2) and (3). The nucleic acid (2) includes two domains, I and II, where domain I stabilizes the Ag NCs (λex = 520 nm, λem = 615 nm), and domain II hybridizes with the complementary domain of the scaffold (1). The amino-modified nucleic acid (3) is functionalized with the π-donor units, L-DOPA (4), L-tyrosine (5), or 6-hydroxy-L-DOPA (6), and hybridizes with the complementary domain of the scaffold (1). The resulting structure between nucleic acids (2) and (3), in the presence of the scaffold (1), leads to a spatial proximity between the luminescent Ag NCs and the π-donor sites. Upon introducing the explosive molecules to the system, the nitro-substituted electron-acceptor substrates bind to the π-donor sites via donor−acceptor interactions, and the concentration of the acceptor substrates on the sensing module leads to an effective electron-transfer quenching of the luminescence of the Ag NCs. The quenching of the Ag NCs then provides the readout signal for the sensing process. Figure 2A depicts the luminescence spectra of the Ag NCs associated with the L-DOPA (4)-functionalized module, upon the addition of variable concentrations of the nitroaromatic picric acid molecule (7). As the concentration of (7) increases, the luminescence intensity of the Ag NCs decreases, consistent with the improved quenching of the NCs. Control experiments reveal that the treatment of the Ag NCs-functionalized module 4919

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Figure 3. Calibration curves corresponding to the luminescence changes of the Ag NCs upon analyzing picric acid, (7) by (a) the LDOPA, (4)-modified sensing module; (b) by the L-tyrosine, (5)functionalized sensing module; and (c) by the 6-hydroxy-L-DOPA, (6)-modified sensing module. L-tyrosine, (5) and 6-hydroxy-L-DOPA, (6) to be Kd = 6.1 × 10−11 M and Kd = 7.2 × 10−12 M (for the analyses, see Supporting Information Figure S1). These dissociation constants are in agreement with the observed quenching efficiencies of the luminescence of Ag NCs. The detection limits for analyzing picric acid by the (5)- and (6)-sensing modules correspond to 5.2 × 10−11 M and 5.2 × 10−12 M, respectively. We, then, implemented the (4)- and (6)-modified sensing modules for the analysis of TNT, (8), and of RDX, (9). The luminescence spectra of the (4)- and (6)-modified sensing modules, in the presence of variable concentrations of TNT (8), are depicted in Figures S2 and S3, Supporting Information. As the concentration of TNT increases, the degree of quenching of the luminescence of the Ag NCs is intensified. Control experiments reveal that the exclusion of the electrondonating groups (4) or (6) from the sensing module does not lead to any quenching of the luminescence of the Ag NCs by diffusional TNT. Figure 4 depicts the calibration curves corresponding to the luminescence changes of the Ag NCs upon the analysis of different concentrations of TNT by the (4)-modified sensing module, curve (a), and by the (6)functionalized sensing module, curve (b). The luminescence changes, in the presence of the (6)-functionalized sensing module, are higher than with the (4)-modified system. These results are consistent with the higher electron-donating features of (6) as compared to (4), a property that enables the improved binding of the TNT electron-acceptor to (6), as compared to (4), and to the enhanced quenching of the Ag NCs in the (6)-modified sensing module. Indeed, this is reflected in the calculated detection limits for analyzing (8) by the (4)- and (6)-modified sensing modules corresponding to 2.5 × 10−12 M and 1.0 × 10−12 M, respectively. The Ag NCs sensing modules modified with either the (4)or (6)-electron-donating groups were further applied to sense

Figure 2. (A) Luminescence spectra of the Ag NCs/L-DOPA, (4), functionalized sensing module, subjected to variable concentrations of picric acid, (7): (a) 0 M, (b) 0.01 × 10−9 M, (c) 0.08 × 10−9 M, (d) 0.14 × 10−9 M, (e) 1.5 × 10−9 M, (f) 8 × 10−9 M, (g) 20 × 10−9 M, (h) 80 × 10−9 M, (i) 200 × 10−9 M. (B) Derived calibration curve upon analysis of (7) according to (A).

either L-tyrosine (5), L-DOPA (4) or 6-hydroxyl-L-DOPA (6)functionalized (3), Cf. Figure 1. The electron-donating properties of these units follow the order (6) > (4) > (5). Figure 3 depicts the resulting calibration curves that compare the luminescence changes observed upon subjecting the (4)modified sensing module to different concentrations of picric acid, (7), curve (a), to the luminescence changes upon interacting the (5)-modified and the (6)-functionalized sensing modules with variable concentrations of picric acid, (7), curves (b) and (c), respectively. As the electron-donating properties of the electron-donor sites improve, the luminescence quenching of the Ag NCs is intensified, consistent with the higher binding strength of picric acid, (7), to the donor units possessing enhanced electron-donating properties. By analyzing the calibration curves shown in Figure 3, curves (b) and (c), we estimate the dissociation constants between picric acid (7) and 4920

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Figure 4. Calibration curves corresponding to the luminescence changes of the Ag NCs in the presence of different concentrations of TNT, (8), using (a) the L-DOPA, (4)-functionalized sensing module and (b) by the 6-hydroxy-L-DOPA, (6)-modified sensing module. (For the respective fluorescence spectra, see Figure S2 and S3, Supporting Information.)

Figure 5. Calibration curves corresponding to the luminescence changes of the Ag NCs in the presence of different concentrations of RDX, (9), using (a) the L-DOPA, (4)-functionalized sensing module and (b) by the 6-hydroxy-L-DOPA, (6)-modified sensing module. (For the respective fluorescence spectra, see Figure S4 and S5, Supporting Information.)

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the RDX, (9), explosive. Supporting Information Figures S4 and S5 depict the luminescence changes upon treatment of the Ag NCs sensing modules modified with either the (4) or (6) electron-donating groups. As the concentration of RDX increases, the luminescence quenching of the Ag NCs is enhanced. Figure 5 shows the calibration curves corresponding to the quenching of the luminescence of the (4)-modified Ag NCs sensing module and of the (6)-functionalized sensing module in the presence of variable concentrations of RDX, curves (a) and (b), respectively. The detection limits for analyzing RDX by the (4)- and (6)-modified sensing platforms correspond to 3.1 × 10−11 M and 3.0 × 10−12 M, respectively. The higher sensitivity observed with the (6)-modified sensing module is consistent with the enhanced electron-donating properties of (6) that leads to the improved binding of RDX to the electron-donating site, and to the more efficient quenching of the Ag NCs. In conclusion, the present study has introduced the novel application of luminescent Ag NCs for the sensing of lowmolecular-weight analytes, for example, explosives. We introduced the assembly of a hybrid sensing module composed of a nucleic acid framework that includes luminescent DNAstabilized Ag NCs and π-donor functional units organized on a DNA scaffold. The π-donor components preconcentrate the electron acceptor analytes on the sensing modules in close proximity to the luminescent Ag NCs, resulting in the effective quenching of the luminescence. We demonstrated that the electron-donating properties of the donor units control the sensitivity of the sensing platforms and highlighted that the formation of the donor−acceptor complexes between the explosive analytes and the functional sensing modules is a key step in the analytical platforms. The sensing platforms were successfully applied to detect picric acid, TNT, and RDX.

ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental methods, Langmuir-type binding model calculation for the picric acid-π-donor complex and luminescence spectra of TNT- and RDX-induced quenching of the Ag NCs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 972-2-6585272. Fax: 972-2-6527715. Author Contributions ‡

N.E and E.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Office of Naval Research, USA, and by the NanoSensoMach ERC Advanced Grant No. 267574 under the EC FP7/2007-2013 program.

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