Article pubs.acs.org/ac
Nonexclusive Fluorescent Sensing for L/D Enantiomers Enabled by Dynamic Nanoparticle-Nanorod Assemblies Lei Song,† Sufan Wang,† Nicholas A. Kotov,‡ and Yunsheng Xia*,† †
College of Chemistry and Materials Science, Anhui Normal University, Wuhu, 241000, China Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States
‡
S Supporting Information *
ABSTRACT: Fluorescence sensing of enantiomers is a much needed yet very challenging task due to nearly identical chemical and physical properties of the chiral isomers also known as chiral equivalence. In this study, we propose a novel strategy for fluorescence sensing of enantiomers using chiral nanoparticles and their ability to form dynamic assemblies. Fluorescence resonance energy transfer (FRET) in nanoscale assemblies consisting of either L-cysteine- or D-cysteine-modified quantum dots (QDs) and gold nanorods (GNRs) was found to be strongly dependent on traces of cysteine. This occurs due to high sensitivity of dynamic assemblies to the weak internanoparticle interactions that can exponentially increase energy transfer efficiencies from QDs to GNRs. Comprehensive analysis of the fluorescence responses in the two types of chiral nanoscale assemblies enables accurate determination of both concentration and enantiomeric composition of the analyte, i.e., cysteine. The described method can quantify the composition of a chiral sample, even the content of one enantiomer is as low as 10% in the mixture. Exceptional selectivity in respect to D/L-cysteine in comparison to analogous small molecules was observed. Versatility of nanoparticle−nanorod assemblies and tunability of intermolecular interactions in them open the road to adaptation of this sensing platform to other chiral analytes. distinguished by fluorescence signal, will be significant for both fundamental research and applications. Such platform would greatly simplify chiral sensing, increase the speed, and enhance the efficiency for high-throughput screening of drugs and catalysts. Herein, we report a new sensing platform for comprehensive analysis of D- and L-cysteine in mixtures and alone, used here as a model chiral analyte. This platform is based on recently developed chiral semiconductor nanoparticles, aka quantum dots (QDs)6 and their ability to form dynamic nanoscale assemblies.7 Two types of assemblies enabling fluorescence resonance energy transfer (FRET) consisting of either Lcysteine- or D-cysteine-modified CdTe quantum dots denoted here as L-QDs or D-QDs, respectively, and gold nanorods (GNRs)8 were investigated (Scheme 1). It was found that even small traces of cysteine trigger the formation of tight assemblies of QDs around GNRs, where efficient energy transfer from the QDs (donors) to the GNRs (acceptors) can take place. The chiral discrimination occurs due to strong dependence of ability of nanoparticles and nanorods to form such assemblies on a manifold of weak multibody interactions sensitive to the chirality of analyte. Comprehensive analysis of the fluorescence responses in the two individual FRET systems, both concentration and enantiomeric composition, of the analyte can be quantified.
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apid, selective, and accurate detection of chiral isomers, aka enantiomers, is of critical importance in many areas of catalysis, medicine, and biotechnology, particularly for drug design and metabolomics.1 The task of enantiomeric analysis is quite challenging due to small differences in binding energies or reactivity between different enantiomers. Enantioselective fluorescent sensors offer the potential for real-time, highsensitivity techniques for determining enantiomeric data in chiral assays.2 Determination of both concentration and enantiomeric composition of a substrate often relies on separation-based techniques such as gas chromatography, high-performance liquid chromatography, and capillary electrophoresis equipped with chirality-specific columns.3 Up to now, complete quantification of a chiral substrate using fluorescence has been only demonstrated in a very limited number of cases. Wolf et al. successfully determined both the concentration and enantiomeric composition of carboxylic acids and amino acid derivatives using racemic and enantiopure sensors, respectively.4 Independently, Pu et al. developed an analytical method for mandelic acid based on pseudoenantiomeric fluorescent sensors. 5 Generally speaking, the current methods of fluorescence sensing of chiral isomers are complicated by the interference and crosstalk between the two probes, which would be avoided if the two enantiomers can be determined independently. Unfortunately, design and realization of exclusive sensors for enantiomeric determination have been an extreme challenge because of their virtual identity in respect to physical/chemical properties and fundamental difficulty of breaking chiral equivalence. So, a conceptual demonstration of a sensing platform, in which enantiomers can be selectively © 2012 American Chemical Society
Received: February 16, 2012 Accepted: August 6, 2012 Published: August 7, 2012 7330
dx.doi.org/10.1021/ac300437v | Anal. Chem. 2012, 84, 7330−7335
Analytical Chemistry
Article
QD solution. Then, the mixture solution was heated and was maintained at 80 °C in an oil-bath for 6 h to form CdTe@CdS core@shell structure. For the purification of CdTe@CdS core@shell QDs, ethanol was added dropwise until the QD solution became turbid. Then, the turbid dispersion was left stirring for 15 min, and the precipitate containing CdTe@CdS QDs was isolated from the supernatant by centrifugation (6000 r/min), which was dried 3 h under vacuum at 28 °C. Finally, the dried precipitate of CdTe@CdS QDs was redispersed in 5 mL of water as stock solution for fluorescent titration experiments. The concentration of CdTe@CdS QDs was 1.10 × 10−6 M, estimated according to the concentration of CdTe core using Peng’s method.11 Synthesis and Purification of GNRs. GNRs were prepared using a seeded growth protocol.12 Specifically, the seed solution was first made by the addition of 0.01 M HAuCl4 solution (0.25 mL) into 0.1 M CTAB (9.75 mL) in a 15-mL plastic tube. The solution was gently mixed by inversion. A freshly prepared, ice-cold 0.01 M NaBH4 solution (0.6 mL) was then injected quickly into the mixture solution, followed by rapid inversion for 2 min. The resultant seed solution was kept at 30 °C for 2 h before use. To grow GNRs, 0.01 M HAuCl4 (2 mL) and 0.01 M (0.32 mL) AgNO3 were first mixed with 0.1 M CTAB (40 mL) in a 50 mL plastic tube. 1.0 M HCl (0.8 mL) was then added for adjusting the pH of the growth solution, followed by the addition of 0.1 M ascorbic acid (0.32 mL). After the growth solution was mixed by inversion, the CTABstabilized seed solution (0.096 mL) was rapidly injected. The resultant solution was gently mixed for 10 s and left undisturbed for 12 h in a 30 °C water-bath. For the purification of the GNRs, 5 mL of GNR sample was collected by centrifugation (7000 r/min for 15 min), and it was further washed two times by water. It should be noted that a sufficient wash can effectively decrease the ξ-potential value of the GNRs. The obtained precipitate was redispersed in water (5 mL), and its concentration was estimated by the Lambert−Beer Law.13 Procedures for Cysteine Detection. 50 μL of Tris−HCl buffer (50 mM, pH 6.6), 6 μL of purified L-QDs (or D-QDs) solution (1.10 × 10−6 M), and 100 μL of GNRs (7.91 × 10−10 M) were placed in a series of 5-mL colorimetric tubes. Then, Lcysteine, D-cysteine, or some analogues were added, respectively. The mixtures were diluted to 5 mL with water and mixed thoroughly. Twenty min later, the fluorescent spectra were recorded (λEx = 400 nm) at 25 ± 1 °C. Both excitation and emission were performed with a slit width of 10 nm. Procedures for Complete Quantification of Cysteine. A series of cysteine samples with different enantiomeric compositions (L/D = 0:100, 25:75, 50:50, 75:25, 100:0) were prepared first. Then, various amounts of the prepared samples were added to the systems of L-QDs/GNRs and D-QDs/GNRs, respectively. Finally, the fluorescence spectra were recorded. All the experimental conditions were the same with those of the above recognition procedures. Procedures for Complete Quantification of Cysteine in an “Unknown” Sample. 50 μL of Tris−HCl buffer (50 mM, pH 6.6), 6 μL of purified L-QD (or D-QDs) solution (1.10 × 10−6 M), and 100 μL of GNRs (7.91 × 10−10 M) were placed in a series of 5-mL colorimetric tubes. For the samples with higher concentrations (>35 nM), the samples were gradually diluted and added to the above systems. The mixtures were diluted to 5 mL with water and mixed thoroughly. Twenty min
Scheme 1. Schematic Illustration of Nonexclusive QD/GNR Based FRET Sensors for Chiral Assays in Two Individual Systems
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EXPERIMENTAL SECTION Materials. D-Cysteine hydrochloride monohydrate (99%), L-cysteine hydrochloride monohydrate (99%), 3-mercaptopropionic acid (MPA, 99%), cysteamine, and Te powder (−60 mesh, 99.999%) were purchased from Alfa Aesar. Other thiolfree amino acids were obtained from Shanghai HuiXing Biochemistry Co., Ltd. HAuCl4·3H2O was obtained from Sigma-Aldrich. Tris (hydroxymethyl) aminomethane (Tris), cetyltrimethylammonium bromide (CTAB), ascorbic acid, NaBH4, CdCl2·2.5H2O, AgNO3, HCl, NaOH, and other routine chemicals were acquired from Shanghai Reagent Company and used as received without further purification. All solutions were prepared with double deionized water. Instruments and Characterizations. A Hitachi-U-3010 spectrometer was used to record the UV−vis spectra. Fluorescence measurements were performed using a Hitachi F-4500 spectrofluorometer equipped with a R3896 redsensitive multiplier and a 1 cm quartz cell. Characterizations of transmission electron microscopy (TEM) were carried out on Tecnai G2 20 ST (FEI) under the accelerating voltage of 200 kV. The samples for TEM measurements were prepared by the deposition of one drop of dilute aqueous dispersion on a copper grid coated with thin films of carbon, and the solvent was removed by evaporation in air. The solutions containing GNRs or QDs were analyzed for ξ-potential values using dynamic light scattering (Zetasizer Nano ZS series, Malvern Instruments) with 633 nm laser wavelength and a measurement angle of 173° (backscatter detection) at 25 °C. Synthesis and Purification of CdTe@CdS Core@shell QDs. L-Cysteine (D-cysteine) modified CdTe QDs were prepared using the method described previously.9 Briefly, 1.25 × 10−3 mol of CdCl2·2.5H2O was dissolved in 100 mL of water, and 3.0 × 10−3 mol of L-cysteine (or D-cysteine) was added under stirring, followed by adjusting the pH to 11.0 by adding dropwise 1.0 mol·L−1 NaOH. The solution was deaerated by N2 bubbling for 40 min. Under vigorous stirring, 1.6 × 10−4 mol of freshly prepared oxygen-free NaHTe was injected to the above solution. Afterward, the resulting solution mixture was heated to 100 °C and refluxed for 8 h. We used previous reports for CdTe@CdS core@shell QDs.9b,10 A 50 mL crude CdTe solution was cooled to room temperature and deaerated by N2 bubbling for 30 min. Under vigorous stirring, 8.0 × 10−5 mol of thioacetamide (dissolved in 1 mL of water), which acted as sulfur source, was added to the 7331
dx.doi.org/10.1021/ac300437v | Anal. Chem. 2012, 84, 7330−7335
Analytical Chemistry
Article
later, the fluorescent spectra were recorded (λEx = 400 nm) at 25 ± 1 °C. Both excitation and emission were performed with a slit width of 10 nm. For samples with lower concentrations (
