Luminescent Core–Shell Imprinted Nanoparticles ... - ACS Publications

Letter

Luminescent Core−Shell Imprinted Nanoparticles Engineered for Targeted Förster Resonance Energy Transfer-Based Sensing Ana B. Descalzo,*,† Clara Somoza,† María C. Moreno-Bondi,‡ and Guillermo Orellana*,† Optical Chemosensors and Applied Photochemistry Group (GSOLFA), Departments of †Organic Chemistry and ‡Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, E-28040 Madrid, Spain S Supporting Information *

ABSTRACT: Red-luminescent 200 nm silica nanoparticles have been designed and prepared as a versatile platform for developing FRET (Förster resonance energy transfer) biomimetic assays. Ru(phen)32+ dye molecules embedded off-center in the silica core provide the long-lived donor emission, and a near-infrared labeled analyte serves as fluorescent acceptor (the measured R0 of this D−A pair is 4.3 nm). A thin surface-grafted molecularly imprinted polymer (MIP) shell intervenes as selective enrofloxacin-binding element. These nanoparticles have been tested for photochemical detection of enrofloxacin by using a competitive scheme that can be readily performed in MeCN−HEPES (pH 7.5) 7:3 (v/v) mixtures and allows for the antibiotic detection in the μM range (LOD = 2 μM) without optimization of the assay. Given the well-known difficulties of coupling the target-binding-to-MIP and the transducing events, the novel photochemical approach tuned up here will be valuable in future developments of MIP-based assays and optosensors that capitalize also on the advantages of nanomaterials for (bio)analysis.

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medicine. Its widespread use causes contamination of food and aquatic systems, triggering the appearance of antibiotic-resistant bacteria strains. Our group has developed successful MIPs for selective sorption of this fluoroquinolone from aqueous media and applied them as stationary phases in chromatography.5 These polymers, however, are optically silent. In order to report the analyte binding, we have now developed a versatile FRETbased competitive biomimetic assay using near-infrared (NIR) labeled analyte molecules and luminescent core−shell nanoparticles (NPs) to benefit from the advantages of nanostructured materials in (bio)analytical chemistry.6 To realize the FRET process, we have selected a cyaninelabeled enrofloxacin (NIR-ENR) as acceptor and the Ru(phen)32+ (phen: 1,10-phenantroline) complex as energy donor (Scheme 1). In addition to detection in the NIR region, another advantage of this D−A pair is the long emission lifetime of the Ru(II) dye. This feature allows a facile discrimination of the actual FRET-sensitized cyanine emission at 800 nm, from that arising from direct excitation of the unbound cyanine. Furthermore, the D molecules are encapsulated into silica NPs to bring about signal intensification7,8 and minimize luminescence quenching by dissolved oxygen.9,10 Finally, a thin polymer shell, imprinted with our target analyte, is grown around the silica NP core, providing

olecularly imprinted polymers (MIPs) are regarded as synthetic analogues of antibodies in that they bear cavities which are complementary in size, shape, and chemical functions to a target molecule.1 Such biomimetic materials can be employed as adsorbents for selective binding of the latter, making them useful as stationary phases for smart chromatography-based analysis. However, alternative analytical methods based on fluorescent detection are needed to enable fast, sensitive, user-friendly, affordable sensing devices for health care, food, and environmental testing. The task of coupling an event of binding to the recognition polymer with an optical signal is not obvious, so that relatively few examples of MIPbased (direct) fluorosensors are found in the literature, most of them relying on quantum dots or systems that require UV excitation.2 An elegant possibility of reporting analyte binding with a distinct optical signal change may be Förster resonance energy transfer (FRET). FRET is a photochemical distancedependent process that can reveal the proximity of two species, an energy donor (D)−acceptor (A) pair, by way of their luminescence. An efficient electronic energy transfer from the photoexcited D to ground state A dyes occurs if D and A approach enough (R < 10 nm) and if there is significant spectral overlap between the emission of D and the absorption of A.3 To the best of our knowledge, only one MIP system has been described so far reporting FRET-based detection of the analyte (proteins and peptides).4 Our benchmark target molecule, enrofloxacin, is a broad spectrum antibiotic extensively used in human and veterinary © XXXX American Chemical Society

Received: March 2, 2013 Accepted: May 15, 2013

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Scheme 1. Structures of the FRET Donor (D), Enrofloxacin (ENR), and the Acceptor Fluorophore (A) and Absorption and Emission Spectra of D and A in EtOHa

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The violet region shows the spectral overlap.

selective binding sites for the fluoroquinolone. The resulting core−shell NPs were evaluated in a competitive assay between NIR-ENR and the antibiotic itself. Since the efficiency of the FRET process depends on the distance between D and A, changes on the FRET signal are employed for determining the extent of enrofloxacin binding. Doping of silica NPs has become a popular approach for obtaining brilliant, stable, biocompatible luminescent labels.8 Very often, the Stöber method is followed for their synthesis, and luminophores are added to the reaction mixture for covalent immobilization or physical entrapment. Interestingly, it has been observed that molecular distribution of the dye in the NP can be controlled at will by adjusting the time elapsed before the luminophore is added to the reaction mixture,10 and this setting was crucial to obtain an efficient FRET process from Ru(phen)32+ to the acceptor on the NP surface. Minimizing placement of molecular luminophores in the core would lead to a lower background signal from dyes which do not participate in the FRET process11 and contribute to enhance the signal.12 We also considered that the MIP shell thickness had to be controlled for achieving the adequate FRET to the bound acceptor. The former can be regulated by adjusting the concentration of monomers and cross-linkers in the polymerization mixture.13

Ru(phen)32+ (λemmax = 595 nm, EtOH), as shown in Scheme 1 by their spectral overlap. Calculation of the overlap integral (J in eq 1, Supporting Information) yields the remarkable value of 7.98 × 1015 M−1 cm−1 nm4. Taking into account that the luminescence quantum yield of D, ΦD, is 0.047,15 the obtained Förster distance (D−A distance at which FRET efficiency is 50% or R0) is 4.3 nm. Moreover, the use of a long-lived luminescent donor provides a lower background signal.16

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ENCAPSULATION OF THE FRET DONOR INTO SILICA NPs: EFFECT OF THE DYE ADDITION TIMING The Ru(phen)32+ complex remains entrapped into the negatively charged silica net just by electrostatic interactions.10,17 For the synthesis of luminescent silica NPs (see Supporting Information), TEOS was hydrolyzed in an alkaline (ammonia) ethanolic solution and then the ruthenium complex was added at a certain time after the TEOS hydrolysis started. In all cases, the reaction was left for 10 h (overall time) to ensure complete TEOS consumption. According to the time elapsed before incorporation of Ru(phen)32+, the doped NPs were designated as SiO2Ru-1 (15 min), SiO2Ru-2 (3 h), and SiO2Ru-3 (6 h). The luminescent features of the NPs vary as a function of the dopant addition time as a consequence of the dye distribution within the NPs:10 for SiO2Ru-1, SiO2Ru-2, and SiO2Ru-3, the measured pre-exponentially weighed emission lifetimes τm (in EtOH) were 1.56, 2.64, and 0.42 μs, respectively. The later the Ru(phen)32+ addition, the closer are the luminophore molecules to the NP outermost surface, undergoing a more efficient emission quenching by diffusion of the dissolved O2. Indeed, upon purging with argon, the luminescence intensity of SiO2Ru-3 NPs increased. However, when Ru(phen)32+ is incorporated at the initial stages of nucleation (SiO2Ru-1), the dye molecules accumulate in the NP core to yield aggregates, the emission lifetimes of which (see above) are shorter than those of the isolated luminophores in SiO2Ru-2 NPs due to self-quenching.9

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DESCRIPTION OF THE D−A SYSTEM Taking into account that the FRET acceptor must absorb light in the Ru(phen)32+ emission region (550−700 nm), we chose cyanine IR-797 as the reactive dye for ENR labeling (Schemes 1 and S1, Supporting Information). This dye absorbs and fluoresces in the NIR (maxima at 802 and 823 nm in EtOH, respectively) but, after nucleophilic substitution of its Cl atom with an electron donor N atom, the absorption and emission shift to the blue.14 By reacting IR-797 with ciprofloxacin, the fluorescent derivative of enrofloxacin NIR-ENR is obtained, with ε = 113 000 M−1cm−1 at 705 nm (EtOH). This labeled analyte proved to be an excellent FRET acceptor for B

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Figure 1. Luminescence spectra (uncorrected; λex = 420 nm) for a 0.2 mg/mL−1 suspension of SiO2Ru NPs in MeCN, before (violet) and after (red) addition of 2 μM NIR-ENR: (a) SiO2Ru-1; (b) SiO2Ru-2; (c) SiO2Ru-3. The emission of 2 μM NIR-ENR is shown in black for comparison. (d) Emission decays (λex = 463 nm) of SiO2Ru-3 at 620 nm (black) and 790 nm (red) and of SiO2Ru-3 after addition of NIR-ENR (λem = 790 nm, blue) in air-equilibrated MeCN.

Figure 2. Emission spectra (uncorrected, λex = 420 nm) (cNPs = 0.2 mg mL−1) of (a) SiO2Ru-3c-Ac; (b) SiO2Ru-3c@M1; (c) SiO2Ru-3c@M2; (d) SiO2Ru-3c@N, before (black) and after adding 2 μM NIR-ENR (red) or a mixture of 2 μM NIR-ENR + 20 μM ENR (blue). (e) ENR dose− response curves of SiO2Ru-3c@M2 (red symbols) or SiO2Ru-3c@N (black symbols) NPs in MeCN−HEPES 0.1 M, pH 7.5 (3:7) measured by the area under the emission curve between 525 and 700 nm. Insets: TEM images (scale bar 100 nm) for the corresponding NPs (see magnified picture in Figure S4 in the Supporting Information).

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as EtOH). By adding a 2 μM solution of the NIR-ENR dye to suspensions of the different NPs in acetonitrile (Figure 1), quenching (D) and enhancement (A) of the characteristic emission of the partners could be detected upon excitation at 420 nm, where only D absorbs. As expected, the effect is stronger in the case of SiO2Ru-3 NPs, underlining the

EFFICIENCY OF FRET FOR THE Ru(phen)32+-DOPED NPs In acetonitrile, it was possible to observe the interaction of the positively charged NIR-ENR acceptor dye with the negatively charged surface of the different donor-doped silica NPs (however, no interaction was found in a protic solvent such C

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Information). The elemental analysis shows that the %C increases from 3.5 for SiO2Ru-3c-Ac to 11.6 for SiO2Ru-3c@N and to 4.3 for both, SiO2Ru-3c@M1 and SiO2Ru-3c@M2 NPs, demonstrating the thinner MIP layer compared to the NIP one.

importance of the FRET donor location in the NPs. Therefore, we chose the SiO2Ru-3 NPs for engineering the target core− shell NPs, optimizing the Ru(phen)32+ loading in the first place. In this way, we found that the maximum payload of the NPs without dye leaching was 1.0 × 10−4 mol Ru(phen)32+/mol TEOS (designated SiO2Ru-3c). In order to confirm that the changes observed in the red luminescence of Ru(phen)32+ and in the NIR-ENR emission (Figure 1a−c) arise from the FRET process, emission lifetime measurements were carried out (Figure 1d). A suspension of SiO2Ru-3c NPs in acetonitrile displays a τm of 215 ns at 620 nm (no signal could be observed at 790 nm). When adding NIRENR to the NPs suspension (1 μM final concentration), a biexponential luminescence decay was detected at 790 nm. This decay displays a contribution from the free NIR-ENR emission (0.92 ns in MeCN, Figure S1, Supporting Information) and a slower component (133 ns) which is on the order of magnitude of the SiO2Ru-3c NPs luminescence decay time at 620 nm, where only the donor molecules emit. Therefore, the slow emission component in the NIR can only be attributed to energy transfer from the Ru sensitizer to the cyanine label, the decay kinetics of which is governed by the rate of FRET from the slowly decaying donor.18 Under these conditions, the efficiency of the FRET process is estimated to be an average of 12% (ηet = 1 − τD/τD0),3b a significant value taking into account the distance heterogeneity of the NP-immobilized donor and acceptor pairs.

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COMPETITIVE FRET ASSAYS

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ASSOCIATED CONTENT

Competitive assays were performed in MeCN−HEPES 3:7 (v/ v) mixtures (0.1 M HEPES buffer, pH 7.5),19 by simultaneously adding both NIR-ENR and increasing amounts of the target (ENR) to the NPs suspension. As a control experiment, we also tested NPs which lacked the MIP recognition shell (SiO2Ru-3c and SiO2Ru-3c-Ac). In these cases, a very weak quenching of the Ru(phen)32+ emission was observed upon addition of NIRENR (Figure 2a), compatible with a feeble unspecific interaction with the labeled analyte in rich aqueous medium. However, a pronounced decrease of the emission band at 590 nm was observed with the core−shell engineered NPs (Figure 2b,c). When adding simultaneously NIR-ENR and ENR, the extent of the Ru(phen)32+ donor emission quenching by the FRET acceptor was smaller, showing an effective competition of the antibiotic with its labeled derivative for the MIP binding sites (Figure 2e). The successful molecular recognition of the target by the MIP shell is demonstrated by comparison of its ENR, Norfloxacin, Flumequine, and penicillin G antibiotics binding (Figure S3, Supporting Information) and the absence of competition when nonimprinted NPs are used. Probably, the thicker polymer shell of the latter (Figure 2d) leads unspecifically adsorbed NIR-ENR to quench more strongly the Ru complex emission but no competition is observed by ENR. The measured dose−response curve shows the sensitivity of the NP-based biomimetic FRET assay to the target species, with a limit of detection of 2 μM calculated as the ENR concentration producing an analytical signal that is 3 times the standard deviation of the blank signal.20 It must be stressed that no optimization of the assay was performed and that the FRET signal change occurs immediately after mixing the sample with NIR-ENR, showing the advantage of the NP-based analysis compared to competitive MIP fluoroassays (up to 7 h).21 In conclusion, a new photochemical biomimetic assay for selective target recognition and quantification has been developed by tailoring luminescent core−shell (silica−MIP) NPs. The optical detection is based on the change of the red emission of a core-encapsulated long-lived luminophore undergoing FRET when a NIR-labeled target species binds to the NP shell. The nanoanalytical platform engineered in this way can be tuned to virtually any analyte to benefit from background-free emission intensity and lifetime measurements, the brightness of dyed nanoparticles, the fast kinetics of the assay and, remarkably, the advantage of performing the biomimetic assay in rich aqueous (buffered) media at neutral pH. In this way, the artificial system might become a cheaper more robust alternative to competitive fluorescence immunoassays.

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GROWING A POLYMER SHELL AROUND THE Ru(II)-SILICA CORE On the basis of our experience in enrofloxacin-recognition polymers,4 we selected the following polymerization mixture: ethylene glycol dimethacrylate (EDMA, cross-linker), methacrylic acid (MAA, functional monomer), MeCN (porogen), and 2,2′-azobis(2-methylpropionitrile) (AIBN, radical initiator) (see Supporting Information). Bearing in mind that in our case the calculated Förster distance, R0, is ca. 4 nm, the MIP polymer shell should not significantly exceed that thickness for maximum sensitivity of the assay. To that end, we carefully controlled the monomers concentration after functionalization of the SiO2Ru-3c NPs surface with acryloyl groups (SiO2Ru-3cAc).13 These NPs were added to the polymerization mixture in MeCN, and the radical reaction was thermally initiated in the presence (MIP) or in the absence (nonimprinted polymer, NIP) of the ENR template. In the case of the nonimprinted NPs (SiO2Ru-3c@N), a ratio of 0.08 mmol total monomers per mg of NPs yielded a polymer shell of 7 ± 1 nm (Figure 2). Therefore, we employed the same conditions for growing the MIP layer by adding 1.53 × 10−3 mmol of the template molecules (ENR for SiO2Ru-3c@M1 or tetrabutylammonium ENR for SiO2Ru-3c@M2) per mg of NPs. The presence of enrofloxacin seems to inhibit partially the polymerization, and a very thin layer of MIP was grown, being difficult to observe it by TEM (only a thin layer conglutinating the NPs indicates the presence of polymer). However, further evidence of the silica surface modification was gained from elemental analysis and luminescence spectroscopy (Table S1, Supporting Information, and Figure 2). The average emission lifetime (τm) of the NPembedded Ru complex increases as a result of formation of a polyacrylate layer around the silica core that restricts O2 diffusion to the Ru(phen)32+ sites (for example, from 0.30 μs for SiO2Ru-3c to 2.57 μs for SiO2Ru-3c@N and SiO2Ru-3c@ M1, or 2.28 μs for SiO2Ru-3c@M2, Table S1, Supporting

S Supporting Information *

Experimental details, protocols of chemical synthesis, binding selectivity studies, and calculation of the R0 value. This material is available free of charge via the Internet at http://pubs.acs.org. D

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Letter

(17) Rossi, L. M.; Shi, L.; Quina, F. H.; Rosenzweig, Z. Langmuir 2005, 21, 4277. (18) Augustin, C. M.; Oswald, B.; Wolfbeis, O. S. Anal. Biochem. 2002, 305, 166. (19) This MeCN−HEPES mixture was the most suitable one we found to avoid unspecific NIR-ENR adsorption and aggregation on the NPs surface (see Figure S2, Supporting Information). (20) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 5th ed.; Pearson Education: Essex (UK), 2005. (21) See, for instance: (a) Benito-Peña, E.; Moreno-Bondi, M. C.; Aparicio, S.; Orellana, G.; Cederfur, J.; Kempe, M. Anal. Chem. 2006, 78, 2019. (b) Piletsky, S. A.; Piletska, E. V.; Bossi, A.; Karim, K.; Lowe, P.; Turner, A. P. F. Biosens. Bioelectron. 2001, 16, 701.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.B.D.); orellana@quim. ucm.es (G.O.). Phone: (+34) 91394-4220. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This paper is dedicated to the memory of the late Prof. Nicholas J. Turro. Rest in peace, dear mentor and friend. This work was supported by the Spanish Ministry of Science and Innovation (“Ramón y Cajal” Program and CTQ2009-14565C03) and a FP7Marie-Curie ERG (PERG04-GA-2008239313). The authors thank Dr. A. Auger (UCM) for the enrofloxacin−tetrabutylammonium (TBA) synthesis.

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