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Cavitands Endow All-Dielectric Beads With Selectivity for PlasmonFree Enhanced Raman Detection of Nε‑Methylated Lysine Ivano Alessandri,*,† Elisa Biavardi,‡ Alessandra Gianoncelli,§ Paolo Bergese,§ and Enrico Dalcanale‡ †
INSTM and Chemistry for Technologies Laboratory, University of Brescia, via Branze 38, 25123 Brescia, Italy Department of Chemistry and INSTM Ru, University of Parma, Parco area delle Scienze 17/A, 43124 Parma, Italy § INSTM and Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy ‡
S Supporting Information *
ABSTRACT: SiO2/TiO2 microbeads (T-rex) are promising materials for plasmon-free surface-enhanced Raman scattering (SERS), offering several key advantages in biodiagnostics. In this paper we report the combination of T-rex beads with tetraphosphonate cavitands (Tiiii), which imparts selectivity toward Nε-methylated lysine. SERS experiments demonstrated the efficiency and selectivity of the T-rex-Tiiii assays in detecting methylated lysine hydrochloride (Nε-Me-Lys-Fmoc) from aqueous solutions, even in the presence of the parent Lys-Fmoc hydrochloride as interferent. The negative results obtained in control experiments using TSiiii ruled out any other form of surface recognition or preferential physisorption. MALDI-TOF analyses on the beads exposed to Nε-Me-Lys-Fmoc revealed the presence of the Tiiii•Nε-Me-Lys-Fmoc complex. Raman analyses based on the intensity ratio of Nε-Me-Lys-Fmoc and cavitand-specific modes resulted in a dose−response plot, which allowed for estimating the concentration of Nε-methylated lysine from initial solutions in the 1 × 10−3 to 1 × 10−5 M range. These results can set the basis for the development of new Raman assays for epigenetic diagnostics. KEYWORDS: core/shell colloids, all-dielectric beads, T-rex, plasmon-free SERS, cavitands, Nε-methylated lysine
1. INTRODUCTION All-dielectric nanostructures are intensively investigated for developing analytical platforms based on enhanced vibrational spectroscopy.1,2 Surface-enhanced Raman scattering (SERS) can take full advantage of high-refractive index dielectrics to overcome most of the drawbacks related to the use of plasmonic metals.3 In particular, the strong concentration of the local electromagnetic field, which is at the basis of conventional SERS effect, can severely alter the system under analysis, for example, through the promotion of plasmonassisted reactions4−6 or photothermal degradation of the analytes.7,8 Either denaturation or passivation of specific functional groups are further major issues to be taken into account, primarily in SERS experiments on biological samples.9 As a result, high sensitivity achieved by conventional, metalassisted SERS is often undermined by low reproducibility. However, high-refractive index dielectric materials represent plasmon-free, low-loss alternatives, which can be useful for in situ monitoring of chemical and biochemical processes under real-working conditions.10−14 For example, core/shell SiO2/TiO2 (T-rex) or hollow TiO2 spheres (T-horex) have been recently utilized to detect organic pollutants and small peptides, as well as to monitor their reactions with high spatial control and reproducibility.15−17 These core/shell colloids combine evanescent fields resulting © XXXX American Chemical Society
from total internal reflection and multiple scattering of light at the sphere-to-sphere interface to amplify the optical path length and extract more Raman photons. These spheres can also be assembled to form extended colloidal crystals, adding further benefits coming from analyte preconcentration.17 Moreover, they can be combined with colloidal microlenses to gain extrasensitivity of the Raman signal.18 Another potential advantage is that titania surfaces are easily functionalized using a variety of linkers, offering many possibilities for anchoring different kinds of receptors, which can make T-rex selective toward specific targets.19,20 However, the latter option has not been exploited so far. Here we report the first example of a synergistic combination of T-rex colloids with synthetic receptors for SERS-based (bio)diagnostics. Tetraphosphonate cavitands (Tiiii),21 molecular receptors capable of binding N-methylammonium salts with extremely high selectivity,22,23 were used in a series of proof-of-concept experiments addressed to unambiguously detect methylated ammonium salts in the absence of any Special Issue: Current Trends in Functional Surfaces and Interfaces for Biomedical Applications Received: September 1, 2015 Accepted: November 9, 2015
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DOI: 10.1021/acsami.5b08190 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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the mixture was stirred for 18 h at 45 °C the solvent was removed under reduced pressure. The product was obtained in quantitative yield after sonication of the residue in water followed by vacuum filtration. 1 H NMR (acetone-d6, 300 MHz): δ (ppm) = 7.46 (brs, 4H, ArH); 4.41−4.35 (m, 4H, ArCH); 4.07 (q, 2H, CH2CH2COOCH2CH3, J = 7.08 Hz); 2.57−2.54 (m, 2H, CH2CH2COOEt); 2.27−2.22 (m, 2H + 6H, CH2CH2COOEt + CH2CH2CH3); 2.03 (s, 12H, ArCH3); 1.31− 1.16 (m, 6H + 3H, CH2CH2CH3 + CH2CH2COOCH2CH3); 0.94 (t, 9H, CH2CH2CH3, J = 7.1 Hz). ESI-MS (m/z): 769.41 [M-H]−. Monoester Footed Tetraphosphonate Cavitand 6. To a solution of 5 (153 mg, 0.199 mmol) in freshly distilled pyridine (10 mL), dichlorophenylphosphine (113 μL, 0.83 mmol) was added slowly, at room temperature. After it was stirred for 3 h at 80 °C, the solution was allowed to cool at room temperature, and 900 μL of 55% H2O2 was added. The resulting mixture was stirred for 30 min at room temperature; then, the solvent was removed under reduced pressure, and water was added. The precipitate obtained in this way was collected by vacuum filtration giving the desired product in 69% yield. 1 H NMR (CDCl3, 400 MHz): δ (ppm) = 8.17−8.12 (m, 8H, P(O)ArHO); 7.66−7.58 (m, 4H + 8H, P(O)ArHP + P(O)ArHM); 7.19 (d, 4H, ArH, J = 12 Hz); 4.90−4.82 (m, 4H, ArCH); 4.19 (q, 2H, CH 2 CH 2 COOCH 2 CH 3 , J = 7 Hz); 2.79−2.70 (m, 2H, CH2CH2COOEt); 2.45−2.23 (m, 2H + 6H + 12H, CH2CH2COOEt + CH2CH2CH3 + ArCH3); 1.48−1.43 (m, 6H, CH2CH2CH3); 1.28− 1.22 (m, 3H, CH 2 CH 2 COOCH 2 CH 3 ); 1.13−1.07 (m, 9H, CH2CH2CH3). 31P{1H}NMR (CDCl3, 162 MHz): δ (ppm) = 6.06 (s, P(O)Ph). ESI-MS (m/z): 1281.5 [M + Na]+. Monoacid Footed Tetraphosphonate Cavitand Tiiii-1COOH (Tiiii). Compound 6 (165 mg, 0.133 mmol) was treated with 4 mL of concentrated hydrochloric acid in 15 mL of CH3CN and 3 mL of CHCl3. After 18 h at 110 °C, the solvent was removed under reduced pressure, and the crude was extracted with DCM and water giving compound Tiiii in 92% yield after organic phase evaporation. 1 H NMR (CDCl3, 600 MHz): δ (ppm) = 8.04−7.99 (m, 8H, P(O)ArHO); 7.57−7.54 (m, 4H, P(O)ArHP); 7.50−7.41 (m, 8H, P(O)ArHM); 7.25 (d, 2H, ArH); 4.76−4.69 (m, 4H, ArCH); 2.70− 2.67 (m, 2H, CH2CH2COOH); 2.36−2.22 (m, 8H, CH2CH2COOH + CH2CH2CH3); 2.10 (d, 12H, ArCH3, J = 7.08 Hz); 1.35−1.29 (m, 6H, CH2CH2CH3); 0.98 (t, 3H, CH2CH2CH3, J = 7.3 Hz); 0.94 (t, 6H, CH2CH2CH3, J = 7.3 Hz). 31P{1H}NMR (CDCl3, 162 MHz): δ (ppm) = 11.72 (s, P(O)Ph, complexed by Na). ESI-MS (m/z): 1248.3 [M + NH4]+; 1253.2 [M + Na]+; 1269.1 [M + K]+. Monoester Footed Tetrathiophosphonate Cavitand 7. To a solution of 5 (536 mg, 0.695 mmol) in freshly distilled pyridine (25 mL), dichlorophenylphosphine (396 μL, 2.92 mmol) was added slowly, at room temperature. After it was stirred for 3 h at 80 °C, S8 (178 mg, 5.56 mmol) was added, and the mixture was stirred again for 2 h at 50 °C. The solvent was removed under reduced pressure, and the crude was separated in its components by flash column chromatography (eluent DCM/hexane 8:2 to pure DCM) obtaining 7 in 72% yield. 1 H NMR (CDCl3, 400 MHz): δ (ppm) = 8.23−8.18 (m, 8H, P(O)ArHO); 7.63−7.54 (m, 4H + 8H, P(O)ArHP + P(O)ArHM); 7.27 (d, 4H, ArH, J = 15.5 Hz); 4.78−4.74 (m, 4H, ArCH); 4.20 (q, 2H, CH 2 CH 2 COOCH 2 CH 3 , J = 7.1 Hz); 2.74−2.70 (m, 2H, CH2CH2COOEt); 2.43−2.30 (m, 2H + 6H, CH2CH2COOEt + CH 2 CH 2 CH 3 ); 2.13 (s, 12H, ArCH 3 ); 1.46−1.40 (m, 6H, CH2CH2CH3); 1.28 (t, 3H, CH2CH2COOCH2CH3, J = 7.1 Hz); 1.10−1.08 (m, 9H, CH2CH2CH3). 31P{1H}NMR (CDCl3, 162 MHz): δ (ppm) = 74.93 (s, 4P, P(S)Ph). ESI-MS (m/z): 1345.6 [M + Na]+. Monoacid Footed Tetrathiophosphonate Cavitand TSiiii-1COOH (TSiiii). Compound 7 (420 mg, 0.32 mmol) was treated with 5 mL of concentrated hydrochloric acid in 30 mL of CH3CN and 5 mL of CHCl3. After 18 h at 110 °C, the solvent was removed under pressure, and the crude was extracted with DCM and water giving compound TSiiii in quantitative yield after organic phase evaporation. 1 H NMR (CDCl3, 600 MHz): δ (ppm) = 8.14−8.10 (m, 8H, P(O)ArHO); 7.54−7.50 (m, 4H, P(O)ArHP); 7.48−7.40 (m, 8H, P(O)ArHM); 7.18 (s, 4H, ArH); 4.73−4.64 (m, 4H, ArCH); 2.67−
distinctive spectral fingerprints. This work aims at demonstrating the potential of functionalized T-rex beads as SERS platforms for studying molecular recognition events.
2. EXPERIMENTAL SECTION 2.1. Synthesis of T-rex Beads. T-rex beads were synthesized according to the procedure described in ref 15. Briefly, 2 μm-sized SiO2 spheres were coated by atomic layer deposition (ALD) with a conformal shell of amorphous titania, which is then crystallized into anatase to form the final T-rex bead. The thickness of the titania shell layer can be finely tuned with nanometer-controlled accuracy. For the present experiments, the thickness of the titania shell was set to 100 nm on the basis of our previous works.15,17 2.2. Synthesis of Cavitand Receptors (Tiiii and TSiiii). Res[(3C3H7,1C3H6OH);CH3] and monohydroxy footed silylcavitand 1 were synthesized according to published procedure.24 The scheme of the synthetic sequence is reported in Supporting Information (Figure S1). Monoaldehyde Footed Silylcavitand 2. To a solution of 1 (1.49 g, 1.57 mmol) in 20 mL of dichloromethane (DCM), pyridinium chlorochromate (0.59 g, 2.35 mmol) was added slowly. After it was stirred for 4 h at room temperature, the solvent was removed under reduced pressure, and the crude was purified by flash column chromatography on silica gel by using CH2Cl2 as eluant to give the product in 75% yield. 1 H NMR (CDCl3, 300 MHz): δ (ppm) = 9.85 (s, 1H, CH2CH2CHO); 7.19 (d, 2H, ArH, J = 5.6 Hz); 4.65−4.60 (m, 4H, ArCH); 2.58−2.46 (m, 2H + 2H, CH2CH2CHO); 2.22−2.08 (m, 6H, CH 2 CH 2 CH 3 ); 1.93 (s, 12H, ArCH 3 ); 1.34−1.28 (m, 6H, CH2CH2CH3); 1.03−0.99 (m, 9H, CH2CH2CH3); 0.54 (s, 12H, SiCH3 OUT); −0.67 (s, 12H, SiCH3 IN). ESI-MS (m/z): 973.5 [M + Na]+. Monoacid Footed Silylcavitand 3. To a solution of 2 (1.12 g, 1.17 mmol) in 30 mL of DCM and dimethylformamide (DMF; 1/1, v/v), a 2.4 M aqueous solution of NaH2PO4 (170 mg, 1.22 mmol) was added slowly followed by 56 μL of hydrogen peroxide 50% (1.22 mmol). Subsequently, a 2.4 M aqueous solution of NaClO2 (425 mg, 4.70 mmol) was added using a water bath, and the reaction was stirred for additional 1.5 h at room temperature. The solvent was removed under reduced pressure giving product 3 after extraction in DCM/water in quantitative yield. 1 H NMR (CDCl3, 300 MHz): δ(ppm) = 7.18 (d, 4H, ArH, J = 7.8 Hz); 4.63−4.57 (m, 4H, ArCH); 2.59−2.54 (m, 2H, CH2CH2COOH); 2.38−2.33 (m, 6H, CH2CH2COOH); 2.20−2.13 (m, 6H, CH2CH2CH3); 1.91 (s, 12H, ArCH3); 1.34−1.26 (m, 6H, CH2CH2CH3); 1.00−0.94 (m, 9H, CH2CH2CH3); 0.51 (s, 12H, SiCH3 OUT); −0.69 (s, 12H, SiCH3 IN). ESI-MS (m/z): 989.29 [M + Na]+. Monoester Footed Silylcavitand 4. To a solution of 3 (4.18 g, 4.31 mmol) in 50 mL of DCM, N,N′-dicyclohexylcarbodiimide (980 mg, 4.75 mmol) and 4-dimethylaminopyridine (160 mg, 1.43 mmol) were added. After the mixture was stirred for 10 min, ethanol (377 μL, 6.48 mmol) was added, and the reaction mixture was stirred for 3 h at room temperature. The crude was directly extracted with water, and the organic phase was evaporated under reduced pressure. Purification by silica gel column chromatography with DCM as eluant yielded the desired product 4 (81%). 1 H NMR (CDCl3, 300 MHz): δ (ppm) = 7.18 (d, 4H, ArH, J = 10.1 Hz); 4.59 (t, 4H, ArCH, J = 8.1 Hz); 4.13 (q, 2H, CH 2 CH 2 COOCH 2 CH 3 , J = 7.1 Hz); 2.59−2.52 (m, 2H, CH2CH2COOEt); 2.31−2.25 (m, 2H, CH2CH2COOEt); 2.20−2.13 (m, 6H, CH2CH2CH3); 1.90 (s, 12H, ArCH3); 1.33−1.22 (m, 6H + 3H, CH2CH2CH3 + CH2CH2COOCH2CH3); 1.01−0.93 (m, 9H, CH2CH2CH3); 0.51 (s, 12H, SiCH3 OUT); −0.70 (s, 12H, SiCH3 IN). ESI-MS (m/z): 1012.32 [M + NH4]+; 1017.25 [M + Na]+; 1033.29 [M + K]+. Monoester Footed Resorcinarene 5. To compound 4 (624 mg, 0.63 mmol) dissolved in DMF (17 mL) and CHCl3 (17 mL) in a Teflon flask, 1.2 mL of an aqueous 48% HF solution was added. After B
DOI: 10.1021/acsami.5b08190 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces 2.63 (m, 2H, CH2CH2COOH); 2.42−2.40 (m, 6H, CH2CH2COOH); 2.29−2.20 (m, 6H, CH2CH2CH3); 2.04 (s, 12H, ArCH3); 1.36−1.32 (m, 6H, CH2CH2CH3); 1.00−0.97 (m, 9H, CH2CH2CH3). 31P{1H} NMR (CDCl3, 162 MHz): δ (ppm) = 75.11 (s, 1P, P(S)Ph); 74.96 (s, 2P, P(S)Ph); 74.90 (s, 1P, P(S)Ph). ESI-MS (m/z): 1295.3 [M + H]+; 1312.3 [M + NH4]+; 1317.2 [M + Na]+; 1333.2 [M + K]+. Nε-Me-L-Lys-Fmoc CF3COO− Salt (Nε-Me-Lys-Fmoc). Fluorenylmethoxycarbonyl (Fmoc)-Nε-(Me,Boc)-L-Lys-OH (48 mg, 0.244 mmol), dissolved in 4 mL of DCM, was treated with 800 μL of trifluoroacetic acid (TFA). After 2 h at room temperature, the solvent was removed under reduced pressure, and the crude was dissolved again in DCM and dried three times to completely remove the acid. The product was obtain in quantitative yield. 1 H NMR (MeOD, 300 MHz): δ (ppm) = 7.78 (d, 2H, ArH); 7.71− 7.66 (m, 2H, ArH); 7.43−7.38 (m, 2H, ArH); 7.35−7.29 (m, 2H, ArH); 4.43−4.33 (m, 2H, OCH2/fmoc); 4.25−4.15 (m, 1H + 1H, CH2CH/fmoc + CαHCOOH); 2.98 (t, 2H, CεH2N); 2.69 (s, 3H, NCH3); 2.01−1.87 (m, 1H, CβH); 1.81−1.64 (m, 1H + 2H, CβH′ + CδH2); 1.57−1.46 (m, 2H, CγH2). ESI-MS (m/z): 383.16 [MCF3COO]+. L-Lys-Fmoc Hydrochloride (Lys-Fmoc). Fmoc-L-Lys(Boc)−OH (40 mg, 0.085 mmol), dissolved in 1.5 mL of CH3CN, was treated with 500 μL of concentrated HCl. After 2 h at room temperature, the solvent was removed in vacuo, and the crude was dissolved again in CH3CN and dried three times to completely remove the acid. The product was obtained in quantitative yield without further purification. 1 H NMR (MeOD, 600 MHz): δ (ppm) = 7.70 (d, 2H, ArH); 7.59− 7.56 (m, 2H, ArH); 7.31−7.28 (m, 2H, ArH); 7.23−7.20 (m, 2H, ArH); 4.32−4.30 (m, 1H, OCH/fmoc); 4.26−4.23 (m, 1H, OCH′/ fmoc); 4.13−4.08 (m, 1H + 1H, CH2CH/fmoc + CαHCOOH); 2.82 (m, 2H, CεH2N); 1.80−1.72 (m, 1H, CβH); 1.65−1.58 (m, 1H + 2H, CβH′ + CδH2); 1.40−1.31 (m, 2H, CγH2). ESI-MS (m/z): 369.23 [MCl]+. 2.3. Functionalization of T-rex with Tiiii (T-rex-Tiiii). The Tiiii cavitands were anchored to the T-rex surface by infiltration of a 1 × 10−3 M ethanol solution. When dried, the T-rex substrates were thoroughly washed in Milli-Q water for 1 h, to remove the purely physisorbed materials. The same procedure was performed for functionalizing T-rex with TSiiii cavitands used for control experiments. 2.4. SERS Experiments: Detection of Nε-Methylated and Nonmethylated Fmoc-Lysine. L-Lysine hydrochloride and Nεmethyl-L-lysine hydrochloride were purchased from Sigma-Aldrich and used as received. The Raman spectra were acquired by means of a high-resolution Raman microscope (Labram HR-800, Horiba/Jobin-Yvon), equipped with a He−Ne laser source (λ = 632.8 nm). All the measurements were performed using a 100× optical objective (numerical aperture = 0.9). The lateral resolution was ∼1 μm. Each spectrum resulted from the average of three measurements and is automatically generated by the acquisition software (LabSpec). Ten different homogeneous regions per substrate were analyzed in each experimental run. The selective detection of Nε-methylated lysine was performed by infiltrating T-rex and T-rex-T(S)iiii substrates with a few microliters of the analyte aqueous solution. The surface area of each T-rex section under analysis can be approximated as that of a spherical cap, 0.5 μm in radius (r) and height (h). As a result, the titania surface area probed in each Raman experiment is ∼1.6 × 106 nm2 (2πrh). Assuming that the molecular footprint of each cavitand is 2.5 nm2 and considering the highest occupation of the available sites, about half a million cavitands are probed for each SERS analysis. Nε-methylated (Nε-Me-Lys-Fmoc) and nonmethylated (Lys-Fmoc) lysine aqueous solutions were utilized for SERS detection experiments. All experiments were run at pH 3.5 using hydrochloric acid, to have all guests protonated in solution. Preliminary experiments on both types of lysine solution without Fmoc protective groups (Nε-Me-Lys and Lys) were also performed to prove the importance of the fluorenylmethoxycarbonyl group as Raman reporters (vide infra).
The SERS substrates were kept under the solution droplet for 1 h, to reach the equilibrium. After they dried, the substrates were thoroughly washed in milli-Q water for 1 h, dried, and analyzed. In the second experimental series equimolar 10 μL aliquots of a 1 × 10−3 M of aqueous solution of nonmethylated Fmoc-lysine and of Nεmethylated Fmoc-lysine were mixed. Twenty microliters of a 1 × 10−3 M ethanol solution of Tiiii receptors was added to the 20 μL of the mixed solution and gently stirred at room temperature for 2 h. Afterward, 10 μL of the final solution was infiltrated into the T-rex colloidal crystals. The same procedure was followed to test lysine solutions over a wider concentration range (1 × 10−4 to 1 × 10−6 M). The Raman measurements were performed according to the procedure described above. 2.5. MALDI-TOF/TOF-MS Analyses. All reagents and solvents for chemical analyses, as well as matrix and calibration kits for matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, were purchased from Sigma Italia (Milano, Italy). Samples were suspended in 0.1% TFA and mixed with a solution of α-cyano-4-hydroxycinnamic acid (CHCA; 20 mg/mL) in acetonitrile/ water/TFA (70.0:30.0:0.1, v/v/v). An aliquot (1 μL) of these preparations was deposited on the sample holder and left to dry at room temperature. Experiments were performed on an AB Sciex5800 MALDI-TOF/TOF-MS, equipped with a nitrogen laser (k = 337 nm). Samples were measured in reflector positive mode, for identification of molecular formulas based on precise mass measurements. 1500 laser shots with energy of 3000 (arbitrary unit) were accumulated for each MS spectrum. External mass calibration was based on the monoisotopic values of [2M + H]+ of CHCA and the [M + H]+ of bradykinin [1−7], angiotensin II (human), P14R, and ACTH [18−39] (human) at m/z 757.4000, 1046.5420, 1533.8520, 2465.1990, respectively (ProteoMass Peptide and Protein MALDI-MS Calibration Kit).
3. RESULTS AND DISCUSSION In the present work we tested the analytical capabilities of Trex-Tiiii conjugates through different experiments. We utilized T-rex with micron-sized silica cores (diameter: 2 μm), because they can be directly inspected by the optical microscopes that are usually coupled to commercial Raman spectrophotometers. This choice allows for selecting specific beads or portions of the colloidal crystals and monitoring their Raman response as a function of surface modifications occurring in each experimental run. The titania layer was made of 100 nm thick anatase (see Supporting Information, Figure S2). The importance of endowing T-rex beads with cavitands was assessed through a series of SERS experiments aiming at the unambiguous detection of Nε-methylated lysine salts in aqueous solutions (Figure 1). The Raman analysis of Nε-methylated and nonmethylated lysine from solutions is not trivial, because their low Raman cross-section prevents any direct differentiation based on the presence of the additional methyl group. This can be clearly seen from Raman spectra reported in Figure 2. In general, solutions of amino acids that do not bear any functional groups with large Raman cross sections (e.g., phenyls, pyrrols, thiols) are hardly detectable when their concentration is below 1 × 10−2 M.25,26 T-rex substrates enhance the Raman sensitivity in comparison to the simple microscope glass slides. However, the spectral intensity is still quite low, forcing to increase the acquisition time of each measurement up to quite unpractical values (several minutes). However, most of the amino acids and peptides synthesized for either analytical or biochemical purposes are functionalized by fluorenylmethoxycarbonyl (Fmoc) groups, which serve as protection for amine terminations. The presence of fluorenyl group makes Raman spectra more intense, introducing clear-cut C
DOI: 10.1021/acsami.5b08190 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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detected at ∼1594 cm−1. The presence of Tiiii on the T-rex surface after washing was also confirmed by MALDI analysis (Figure 3c), which showed the distinctive peaks of Tiiii (1231.14 m/z for [Tiiii-H]+), as well as those of its sodium and potassium adducts. Remarkably, Tiiii remains stable in air for months. This can be a key feature in view of fabricating recyclable SERS substrates.30−32 The efficiency of T-rex-Tiiii colloidal crystals in the selective detection of Nε-methylated lysine was tested by means of two different aqueous solutions containing either Nε-methylated or nonmethylated Fmoc-lysine hydrochlorides (1 × 10−3 M). Each solution was infiltrated through distinct colloidal crystal substrates and incubated for 1 h. Afterward, the SERS substrates were thoroughly rinsed with Milli-Q water to remove purely physisorbed guests (Figure 4a). Figure 4b shows the Raman spectra of T-rex-Tiiii substrates before and after infiltration with either Nε-methylated or nonmethylated Fmoc-lysine solutions. On the one hand, the spectra of T-rex-Tiiii substrates infiltrated with nonmethylated probes show no differences in comparison to those of the substrates before infiltration. On the other hand, the substrates infiltrated with Nε-methylated probes exhibit additional peaks, indicated by arrows, which are characteristic of the Fmoc group. MALDI analysis (Figure 4c) shows an intense peak at 1613.6 m/z, which shows the formation of the host−guest complex between Tiiii and Nε-methylated lysine on the T-rex surface. These data indicate that Tiiii receptors do confer remarkable selectivity to T-rex substrates, allowing unambiguous differentiation of Nε-methylated and nonmethylated lysine. The specificity of Tiiii toward its molecular target was also tested in a control experiment involving a closely related tetrathioposphonate (TSiiii) cavitand. TSiiii has the same structure of Tiiii, including the carboxylic termination used for anchoring, except from the upper rim, in which the PO bridging units have been replaced with PS ones (Figure 5a). As demonstrated in previous works, this modification completely shuts off the binding of N-methylated amino acids.33 The Raman spectra of T-rex-TSiiii substrates are analogous to those of the Tiiii counterpart (Figure 5b). Moreover, they do not change upon infiltration with Nεmethylated lysine solutions and washing. MALDI analysis (Figure 5c) confirms the absence of peaks of the host−guest complex, which should appear at 1677.89 m/z. Only the signals from TSiiii and its sodium and potassium adducts are observed. These results demonstrate that Tiiii binds selectively Nε-MeLys-Fmoc at the solid−water interface.
Figure 1. Nε-methylated and nonmethylated lysine used for preliminary Raman detection experiments (see Figure 2). The species labeled with Fmoc protecting groups were also utilized in the following experiments (Figures 3−7).
peaks (see, e.g., the peak at 1608 cm−1 in Figure 2b), which are characteristic of the fluorenyl ring.27,28 However, although the exploitation of Fmoc reporters addresses the intensity issue, it does not help to discriminate between Nε-methylated and nonmethylated lysine. To do so, the Tiiii cavitand is introduced on the surface of T-rex. Its ineffective counterpart (TSiiii) was tested as a control system, to rule out SERS responses due to nonspecific adsorption. In the first experiment series the cavitand receptors were anchored to the T-rex surface through the carboxylic group, purposely introduced as a side-chain linker to the lower rim of the cavitand backbone (Figure 3a). T-rex crystals were infiltrated with a solution of Tiiii in ethanol (pH ≈ 4.5). When anchored, the titania−carboxylate bond remains stable even when the pH is switched to neutral values (vide infra). As already reported in previous works,17 the functionalization can be directly monitored from extinction spectra of T-rex colloidal crystals (see Supporting Information, Figure S3), showing a significant (Δλ = 10 nm) red shift of the Fabry-Pèrot fringes, as a result of the change in the refractive index of the medium upon Tiiii adsorption. A more specific proof is given by the Raman spectra shown in Figure 3b. Strong bands, due to aromatic ring vibrations, dominate the Raman spectrum of Tiiii. In particular, the strongest signal at ∼1000 cm−1 originates from bending vibrations involving the in-phase radial movement of 2,4,6-carbons of the aromatic rings.29 Further distinctive modes of the aromatic quadrant (8a and 8b) are
Figure 2. (a) Raman spectra of 1 × 10−3 M nonmethylated lysine solutions deposited onto microscope glass slides (green) and T-rex (red). Nεmethylated solutions exhibit no significant differences; (b) Raman spectra of 1 × 10−3 M nonmethylated and Nε-methylated lysine solutions protected with Fmoc functional groups. D
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Figure 3. (a) Scheme of T-rex functionalization with Tiiii cavitands; (b) Raman spectra of T-rex-Tiiii beads; (c) MALDI analysis of T-rex-Tiiii (For enlarged images and data analysis see Supporting Information).
Figure 4. (a) Scheme of the molecular recognition experiments; (b) Raman spectra of T-rex-Tiiii beads infiltrated with either Nε-methylated (NεMe-Lys-Fmoc) or nonmethylated (Lys-Fmoc) lysine solutions; (c) MALDI showing the peak at 1613 m/z, which indicates the formation of the Tiiii•Nε-Me-Lys-Fmoc host−guest adduct (For enlarged images and data analysis see Supporting Information).
The final solution, containing cavitands and both types of lysine, was gently stirred at room temperature for 2 h, then used to infiltrate the T-rex colloidal crystals (Figure 6a). When the sample is incubated and washed, the Raman spectra exhibit the
In the last series of experiments, T-rex-Tiiii beads were tested in a competition experiment. Here, the Tiiii cavitands were preliminarily added to an equimolar solution of Nε-methylated and nonmethylated Fmoc-lysine (see Experimental Section). E
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Figure 5. (a) Scheme of the molecular recognition experiments with TSiiii receptor; (b) Raman spectra of T-rex-TSiiii beads before (green) and after (red) infiltration with Nε-methylated lysine (Nε-Me-Lys-Fmoc) solutions; (c) MALDI analysis showing the absence of the peak at 1677.89 m/z, which indicates that TSiiii is unable to bind Nε-Me-Lys-Fmoc (For enlarged images and data analysis see Supporting Information).
signals of both cavitands and Nε-Me-Lys-Fmoc (Figure 6b). MALDI analysis (Figure 6c) confirms that only Nε-methylated species bind Tiiii cavitands at the water−solid interface, whereas physisorbed nonmethylated species are removed upon washing. The same competition experiment was conducted directly on the T-rex-Tiiii beads, obtaining the same results. The outcome of these experiments demonstrates that Tiiii can be used for the selective preconcentration of Nε-methylated lysine from solutions containing analogous nonmethylated targets. To determine the detection limit of T-rex-Tiiii, solutions of Tiiii (1 × 10−3 M) were added to mixed aqueous solutions containing both Nε-methylated and nonmethylated lysine. The concentration of the original lysine solutions was varied from 1 × 10−3 to 1 × 10−6 M, whereas the starting concentration of Tiiii was kept at the fixed value of 1 × 10−3 M. As discussed before, Tiiii receptors ensure the selective capture of Nεmethylated species, forming 1:1 complexes. Thus, in these experimental tests, the molar ratio between Tiiii and Nε-MeLys-Fmoc spans from 1:1 to 1000:1. Upon T-rex infiltration and washing, the Raman spectra were compared through the analysis of the 1500−1700 cm−1 spectral region, characterized by the copresence of the signals of Tiiii and Fmoc rings at 1594 and 1608 cm−1, respectively (Figure 7a). No signals of Nε-Me-Lys-Fmoc were detected for 1 × 10−6 M solutions. Thus, the T-rex-Tiiii system can be used as a Raman assay for sorting and detecting Nε-Me-Lys-Fmoc from solutions in which the concentration of that specific analyte is
1/100 of that of the available binding sites. The analysis of the intensity ratio of those modes as a function of the molar concentration of the initial Nε-Me-Lys-Fmoc solution is reported in a dose−response plot (Figure 7b). These results demonstrate that the analysis based on the intensity ratio can be utilized to roughly estimate the initial concentration of NεMe-Lys-Fmoc in the 1 × 10−3 to 1 × 10−5 M range.
4. CONCLUSIONS Tetraphosphonate cavitands are robust, highly selective receptors, that enable the plasmon-free SERS detection of Nεmethylated lysine hydrochloride, in the presence of the parent lysine hydrochloride. As lysine Nε-methylation is a crucial process in post-translational modifications of proteins that rule gene expressions,34 these results can provide an experimental guideline to design new SERS assays for epigenetic diagnostics. More generally, this work demonstrates that supramolecular receptors35,36 can endow all-dielectric beads, nowadays at the center of many studies on plasmon-free SERS, with high selectivity and specificity. This is a key to achieve reliable Raman data and high sensitivity toward analytes that should be discriminated from other analogous species on the basis of specific functional groups characterized by low Raman cross sections. In this regard, the proof-of-concept experiments discussed above can stimulate future research in the development of dielectric platforms for SERS-based (bio)diagnostics and multiplexed analysis. F
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Figure 6. (a) Scheme of the molecular recognition experiments using Tiiii receptors in aqueous solution containing equimolar (1 × 10−3 M) amounts of both Nε-methylated and nonmethylated lysine; (b) Raman detection of Nε-Me-Lys-Fmoc species upon infiltration and washing; (c) MALDI analysis of T-rex-Tiiii•Nε-Me-Lys-Fmoc beads, showing the peak at 1613 m/z, which indicates the formation of the Tiiii•Nε-Me-Lys-Fmoc host−guest adduct (For enlarged images and data analysis see Supporting Information).
Figure 7. (a) Example of Raman spectra of Nε-Me-Lys-Fmoc solutions that were selectively captured by Tiiii receptors (1 × 10−3 M) from mixed Nεmethylated and nonmethylated aqueous solutions. The values of the original concentration of the Nε-Me-Lys-Fmoc solutions ranged between 1 × 10−3 and 1 × 10−5 M; (b) plot of the intensity ratio of the ∼1608/1594 cm−1 peaks vs the molar concentration of the original Nε-Me-Lys-Fmoc solution. The peaks at 1608 and 1594 cm−1 are attributed to Nε-Me-Lys-Fmoc and Tiiii, respectively. No Nε-Me-Lys-Fmoc signals were observed starting from 1 × 10−6 M solutions. Each experimental point resulted from spectra acquired over 10 different regions of T-rex-Tiiii•Nε-Me-Lys-Fmoc substrates.
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ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author
S Supporting Information *
*E-mail:
[email protected].
The Supporting Information is available free of charge on the
Notes
The authors declare no competing financial interest.
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ACS Publications website at DOI: 10.1021/acs.jpca.5b10015. Scheme of synthesis of receptors; representative SEM image of substrates; functionalization of T-rex monitored by direct observation of optical extinction red shift; MALDI analyses of substrates, substrates infiltrated with Fmoc species, and experiments of selective recognition of Fmoc species from mixed solutions. (PDF)
ACKNOWLEDGMENTS This work was supported by SUPRANANO (INSTM-Regione Lombardia project). Centro Intefacoltà di Misure “G. Casnati” of the Univ. of Parma is acknowledged for the use of NMR facilities. We thank M. Ferroni for support in SEM analysis and N. Bontempi and G. Candiani for fruitful discussions. G
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