NANO LETTERS
NAD+/NADH-Sensitive Quantum Dots: Applications To Probe NAD+-Dependent Enzymes and To Sense the RDX Explosive
2009 Vol. 9, No. 1 322-326
Ronit Freeman and Itamar Willner* Institute of Chemistry and the Farkas Center for Light-Induced Processes, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel Received October 8, 2008; Revised Manuscript Received November 1, 2008
ABSTRACT Phenyl boronic acid-functionalized CdSe/ZnS quantum dots (QDs) were synthesized. The modified particles bind nicotinamide adenine dinucleotide (NAD+) or 1,4-dihydronicotinamide adenine dinucleotide (NADH). The NAD+-functionalized QDs are effectively quenched by an electron transfer process, while the NADH-modified QDs are inefficiently quenched by the reduced cofactor. These properties enable the implementation of the QDs for the fluorescence analysis of ethanol in the presence of alcohol dehydrogenase. The NADH-functionalized QDs were used for the optical analysis of the 1,3,5-trinitrotriazine, RDX explosive, with a detection limit that corresponded to 1 × 10-10 M. We demonstrate cooperative optical and catalytic functions of the core-shell components of the QDs in the analysis of RDX.
Surface-modified semiconductor quantum dots (QDs) are extensively used as optical labels for sensing events. The semiconductor QDs were used as fluorescence labels for biorecognition processes,1 and the dynamics of biorecognition events or biocatalytic reactions was followed by fluorescence resonance energy transfer (FRET) or electron transfer (ET) processes stimulated by the excited QDs.2 For example, the hybridization and replication of DNA on CdSe/ ZnS QDs were probed by a FRET process,3 and the activity of tyrosinase was followed by an ET quenching process occurring upon the biocatalyzed oxidation of the tyrosinemethyl ester capping layer associated with the QDs to the dopaquinone derivative.4 Also, the activities of different proteases were followed by QD-stimulated FRET that used fluorophore-modified peptides associated with the QDs.5 In the present study we introduce the functionalization of CdSeZnS core-shell QDs with a phenyl boronic acid ligand. The modified QDs bind 1,4-nicotinamide adenine dinucleotide, NAD+, or 1,4-dihydronicotinamide adenine dinucleotide, NADH cofactors. The NAD+ units quench the luminescence of the QDs by an ET mechanism. This yields NAD+/NADHsensitive QDs that enable us to follow the activities of NAD+-dependent enzymes, and to develop a unique optical sensor for the detection of the 1,3,5-trinitrotriazine, RDX, explosive. Furthermore, with RDX-sensing NADH-modified * Corresponding author: e-mail,
[email protected]; tel, 972-26585272; fax, 972-2-6527715. 10.1021/nl8030532 CCC: $40.75 Published on Web 11/24/2008
2009 American Chemical Society
Figure 1. Fluorescence spectra of (a) The NADH-modified CdSe/ ZnS QDs and (b) The NAD+-functionalized CdSe/ZnS QDs. The spectra indicate that the NAD+ reveals superior luminescence quenching properties.
QDs, we reveal cooperative optical and catalytic functions of the core and shell components of the QDs. (3-Aminophenyl)boronic acid was covalently linked to glutathione (GSH)-capped CdSe/ZnS QDs. The boronic acid ligand forms boronic esters with vicinal diols, and specifically with saccharides.6 Accordingly, the NAD+ or the NADH cofactors were linked to the boronic acid ligands (for detailed procedures, see Supporting Information). The loading of the NAD+ or NADH cofactors on the QDs was estimated to be ca. 40 ( 10 units per particle for the two cofactors. The loading was determined by recording the absorbance spectra of the QDs before and after functionalization with the NAD+
Scheme 1. Schematic Sensing of Ethanol by the NAD+-Functionalized CdSe/ZnS QDs, in the Presence of Alcohol Dehydrogenase (AlcDH)a
a The enzyme AlcDH catalyzes the oxidation of ethanol to acetaldehyde with the concomitant reduction of the NAD+ units associated with the QDs to NADH.
or NADH cofactors, and subtraction of the respective spectra to generate the pure absorbance of the NAD+/NADH cofactors. Knowing the extinction coefficients of NAD+ (16900 M-1 cm-1 at λ ) 260 nm), and of NADH (6220 M-1 cm-1 at λ ) 340 nm), and knowing the concentration of the QDs, the average loading of the cofactor units per particle was determined. Figure 1 shows the fluorescence spectra of the NAD+- and the NADH-functionalized QDs. The quenching of the luminescence of the QDs by the NAD+ cofactor, presumably by an ET quenching route, is substantially more efficient than the quenching of the QDs by the reduced cofactor, NADH. This difference enables us the use of the NAD+/NADH-sensitive QDs as luminescent probes that follow NAD+-dependent enzyme activities as well as their substrates, such as the NAD+-dependent alcohol dehydrogenase (AlcDH)/ethanol system, Scheme 1. The biocatalytic reduction of the NAD+ capping layer by ethanol in the presence of AlcDH is anticipated to yield the NADHfunctionalized QDs and, thus, to switch-on the luminescence of the QDs. The time-dependent luminescence changes upon the reaction of the NAD+-modified QDs with ethanol/AlcDH are depicted in Figure 2A. The luminescence of the system is intensified as the reaction time is prolonged, and it levels off to a saturation value after ca. 12 min. These results are consistent with the time-dependent reduction of the capping NAD+ cofactor layer to the NADH state that yields QDs of enhanced luminescence. Figure 2B depicts the fluorescence intensities of the QDs upon interaction with variable concentrations of ethanol for a fixed time interval of 12 min, in the presence of AlcDH. As the concentration of ethanol increases, the luminescence of the QDs is intensified. This Nano Lett., Vol. 9, No. 1, 2009
Figure 2. (A) Time-dependent fluorescence changes upon the interaction of the NAD+-functionalized QDs with ethanol, 5 mM, in the presence of AlcDH, 5 units. Spectra were recorded at time intervals of 3 min. (B) Calibration curve corresponding to the fluorescence analysis of variable concentrations of ethanol by the NAD+-functionalized QDs. Each sample was analyzed by reacting the functionalized QDs with a different concentration of ethanol in the presence of AlcDH, 5 units, for a fixed time interval of 12 min. All measurements were performed in a 10 mM phosphate buffer solution, pH ) 8.8. 323
Scheme 2. Sensing of the RDX Explosive by the NADH-Functionalized CdSe/ZnS QDsa
a
The Zn2+ ions associated with the shell act as Lewis acid that activates the NO2-functionalities toward reduction by the NADH cofactor.
is consistent with the formation of higher contents of the NADH-modified QDs as the concentration of ethanol increases. A control experiment revealed that in the absence of ethanol, the added AlcDH had no effect on the luminescence of the QDs. The use of the NAD+-modified QDs for the analysis of ethanol in the presence of AlcDH represents a generic approach to apply the QDs for analyzing the activity of any NAD+-dependent enzymes and their substrates. We searched, however, for possibilities to implement the NAD+/NADH-sensitive QDs for other analytical applications and found that the NADH-modified CdSe/ ZnS QDs act as an effective optical label for the fluorescent analysis of the 1,3,5-trinitrotriazine, RDX, explosive material. The analysis of explosives attracts substantial recent research efforts because of broad homeland security needs. While numerous optical7,8 or electrochemical9,10 sensors for nitroaromatic compounds exist, the sensing methods for the problematic RDX explosive are scarce. Optical sensors for RDX include the fluorescence changes upon the photochemical hydride transfer from a hydroacridinium dye to RDX11 or a competitive immunoassay that implemented CdSe QDs and a quencher-modified RDX derivative as a FRET acceptor.12 Other methods included fluorescence detection of RDX using a microcapillary competitive immunosensor.13 In a series of previous studies, NADH was used for the biocatalyzed reduction of RDX in the presence of different enzymes,14 and the process was suggested as a method to degrade the RDX pollutant. We attempted to use the NADHmodified CdSe/ZnS QDs as a reducing agent for the reduction of RDX and the fluorescent detection of RDX 324
through the formation of the quenched NAD+-modified QDs, Scheme 2. Reaction of the NADH-modified QDs with RDX resulted in the decrease of the luminescence of the QDs. Figure 3A shows the time-dependent fluorescence changes of the QDs upon reaction with RDX. The fluorescence intensity of the QDs decreases with time, consistent with the transformation of the NADH-capped QDs to the quenched NAD+-modified QDs. In order to prove that the NADHcapped layer was indeed transformed to the NAD+-modified particles, the QDs that resulted upon reaction with RDX were subsequently reacted with ethanol/AlcDH. The fluorescence of the QDs was almost fully restored, Figure 3A, curve h, indicating that the capping layer was regenerated to the fluorescent NADH-modified QDs. The NADH-functionalized QDs were then reacted with variable concentrations of RDX for fixed time intervals of 21 min. Figure 3B depicts the quenching degree of the system analyzing different concentrations of RDX. As the concentration of RDX is higher, the luminescence of the QDs is quenched to a higher extent, consistent with the formation of a higher coverage of the NAD+-modified QDs. The system enabled the detection of RDX with a detection limit corresponding to 10-10 M. It should be noted that the reduction of RDX by the NADHfunctionalized QDs proceeds effectively even at low concentrations of RDX. The fact that NADH acts as an electron donor while RDX is an electron acceptor may suggests that π donor-acceptor interactions concentrate RDX at the QDs surface, leading to enhanced reduction of the explosive. In fact, such π donor-acceptor interactions were previously demonstrated to concentrate the TNT explosive at Au nanoparticle surfaces, giving rise to the sensitive electrochemical detection of TNT.15 The detection limit for the Nano Lett., Vol. 9, No. 1, 2009
Table 1. Analysis of RDX by Different Sensing Methods method fluorescence detection of RDX by NADH-modified CdSe/ZnS QDs fluorescence detection of RDX by photoreaction of an acridinium dye QDs conjugate fluoroimmunoassay microcapillary competitive immunosensor voltammetric sensing
detection limit (M) 1×
ref
10-10
7 × 10-5
11
9 × 10-8 5 × 10-10
12 13
2.7 × 10-7
10d
analysis of RDX is impressive and seems to be the most sensitive available sensing procedure for RDX. Table 1 compares the detection limit of the present method to other sensing procedures. The successful analysis of RDX by the reduction of the explosive with the NADH-modified QDs still raises some fundamental questions. All previously reported NADHmediated reduction processes of RDX required enzyme catalysts that were not present in our system. Furthermore, a control experiment revealed, to our surprise, that in a homogeneous solution NADH does not reduce RDX. These two points suggest that some unique catalytic features exist in the CdSe/ZnS core-shell QD system. To resolve this enigma, we realized that numerous biomimetic studies reported that the reduction of different substrates (i.e., ketones or NO2 groups) by NADH model systems required the coaddition of metal ions such as Zn2+ or
Figure 3. (A) Curves a-g represent the time-dependent fluorescence changes upon treatment of the NADH-functionalized QDs with RDX, 1 × 10-4 M. Spectra were recorded at time intervals of 3 min. Curve h depicts the restoration of the fluorescence of the QDs upon the interaction of the QDs generated in curve g with ethanol, 5 mM and AlcDH, 5 units. (B) Calibration curve corresponding to the analysis of different concentrations of RDX by the NADH-functionalized QDs for a fixed time interval of 21 min. Nano Lett., Vol. 9, No. 1, 2009
Figure 4. Time-dependent absorbance changes of NADH at λ ) 340 nm, upon interaction with RDX in the absence of Zn2+, curve a, and in the presence of Zn2+, 1 × 10-7 M, curves b-d. Spectra were recorded at time intervals of 15 min.
Mg2+.16 It was demonstrated that these metal ions act as Lewis acids that bind the reducible functionality and activate it toward hydride transfer from the NADH-model compound. The fact that our QDs consist of a CdSe/ZnS core-shell structure suggests that the Zn2+ surface states might activate the nitro functionalities on the RDX toward hydride transfer, Scheme 2. To support this unexpected observation, we reacted NADH with RDX in a homogeneous solution in the presence of Zn2+, 1 × 10-7 M, Figure 4. Under these conditions, the reduction of RDX proceeded effectively, as evident by the depletion of the characteristic NADH absorbance at λ ) 340 nm. It should be noted that although the surface coverage of the QDs by Zn2+ ion may be quite small, the formation of the π donor-acceptor complex between RDX and NADH might cooperatively assist the activation of the NO2 groups by the Zn2+ Lewis acid-base complex. In conclusion, the present study has introduced NAD+/ NADH-modified QDs as fluorescent labels for different sensing events. The NAD+-functionalized QDs provide a versatile tool to analyze the activities of NAD+-dependent enzymes and their substrates. The NADH-modified QDs were successfully applied to analyze the RDX explosive. We have demonstrated that the reduction of the RDX explosive by NADH is activated by Zn2+ ions associated with the shell structure of the QDs. The fact that the biocatalyzed transformation of the NAD+ cofactor to NADH is controlled by the specific cofactor-dependent enzyme turns the functionalized QDs to selective and specific reporters for the substrates of the enzymes. The NADH-functionalized QDs could, however, reduce other NO2-activated functionalities and specifically detect other explosives. Besides the importance of this feature to construct the fluorescent RDX sensor, our results suggest that appropriate tailoring of the shell layer of the QDs may lead to new material functions that cooperatively couple the optical properties of the core with catalytic functions of the shell. Acknowledgment. This research is supported by the Israel Science Foundation, Converging Technologies Project, and by the Israel Ministry of Defense. 325
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NL8030532
Nano Lett., Vol. 9, No. 1, 2009
