Mimicking Horseradish Peroxidase Functions Using Cu2+

Mimicking Horseradish Peroxidase Functions Using Cu2+-Modified Carbon Nitride Nanoparticles or Cu2+-Modified Carbon Dots as Heterogeneous Catalysts Margarita Vázquez-González, Wei-Ching Liao, Rémi Cazelles, Shan Wang, Xu Yu, Vitaly Gutkin, and Itamar Willner* Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: Cu2+-functionalized carbon nitride nanoparticles (Cu2+−g-C3N4 NPs), ∼200 nm, and Cu2+−carbon dots (Cu2+−Cdots), ∼8 nm, act as horseradish peroxidase-mimicking catalysts. The nanoparticles catalyze the generation of chemiluminescence in the presence of luminol/H2O2 and catalyze the oxidation of dopamine by H2O2 to form aminochrome. The Cu2+−g-C3N4-driven generation of chemiluminescence is used to develop a H2O2 sensor and is implemented to develop a glucose detection platform and a sensor for probing glucose oxidase. Also, the Cu2+−C-dots are functionalized with the β-cyclodextrin (β-CD) receptor units. The concentration of dopamine, at the Cu2+−C-dots’ surface, by means of the β-CD receptor sites, leads to a 4-fold enhancement in the oxidation of dopamine by H2O2 to yield aminochrome compared to that of the unmodified C-dots. KEYWORDS: chemiluminescence, dopamine, glucose, cyclodextrin, sensor photoelectrochemical cells,33,34 optical material for sensing,35,36 catalyst for various transformations,37,38 and carrier of heterogeneous catalysts.39 Also, carbon dots (C-dots) attract growing interest as functional material for bioanalytical applications.40−43 The photoluminescent properties of C-dots were used to develop sensing platforms44,45 and were applied for bioimaging.46,47 The surface-modified chemical groups associated with gC3N4 or C-dots (carboxylic acids, amine, hydroxyl) add important functionalities because additional chemical units can be linked to the g-C3N4 or C-dots’ surfaces, thus providing a means for emerging chemical reactivities and potential applications. Here, we report on the synthesis of Cu2+-ionmodified g-C3N4 nanoparticles, Cu2+−g-C3N4 NPs, and Cu2+functionalized C-dots (Cu2+−C-dots) as catalysts mimicking several of the horseradish peroxidase biocatalytic functions. Specifically, we demonstrate that the hybrid heterogeneous catalysts catalyze the generation of chemiluminescence in the presence of luminol/H2O2 and stimulate the catalyzed oxidation of dopamine to aminochrome (for other DNA-

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evelopment of artificial synthetic or biomimetic systems mimicking the catalytic functional horseradish peroxidase (HRP) attracts growing interest. Homogeneous transition metal complex catalysts such as Mn+porphyrin,1−3 heterogeneous catalysts such as iron oxide,4−6 Au,7−9 or Cu10,11 nanoparticles, and heme-modified peptides12−14 (e.g., microperoxidase-11 and hemin/G-quadruplex horseradish peroxidase-mimicking DNAzymes15,16) were reported to act as catalysts mimicking the native enzyme. Different reactions, such as the catalyzed oxidation of the 2,2′azinobis(3-ethylbenzthiazoline-6-sulfonate) dianion (ABTS2−) by H2O2,17−19 the catalyzed generation of chemiluminescence in the presence of H2O2/luminol,20,21 or the catalyzed oxidation of dopamine to aminochrome,22 were driven as HRP-mimicking transformations using the biomimetic catalysts. The interest in developing HRP-mimicking catalysts rests on their application as amplifying labels for sensing events23−25 and their use as catalysts for stimulating different reactions,26,27 such as the H2O2-catalyzed oxidation of NADH28 or the hemin/G-quadruplex-catalyzed oxidation of aniline by H2O2 to form polyaniline.29,30 Carbon nitride (g-C3N4) attracts growing interest as carbon-like material that reveals graphene-like functions.31,32 Various applications of g-C3N4 were recently suggested, including its application as photoactive material in © 2017 American Chemical Society

Received: January 17, 2017 Accepted: February 24, 2017 Published: February 24, 2017 3247

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ACS Nano zyme-mediated48−50 or homogeneous catalyst-mediated51,52 oxidation of dopamine to aminochrome, see references 47−51).

generation of chemiluminescence. Also, unmodified g-C3N4 NPs showed a very low catalytic activity toward the catalyzed generation of chemiluminescence in the presence of luminol/ H2O2 (∼7% of the catalytic activity of the Cu2+−g-C3N4 NPs). Thus, we conclude that the Cu2+ bound to the g-C3N4 NPs reveals unique catalytic properties compared to those of the hydrated homogeneous Cu2+ ions. Figure 1B depicts the derived calibration curve, indicating that H2O2 can be analyzed with a detection limit corresponding to 0.04 mM. The chemiluminescence intensities are controlled by the concentration of H2O2 and by the concentration of the Cu2+−g-C3N4 catalyst (Figure S5). The successful analysis of H2O2 by the Cu2+−g-C3N4 NPs suggests that the NPs could be applied as an optical chemiluminescent probe for analyzing the substrates of H2O2generating oxidases, such as glucose oxidase. Figure 2A depicts schematically the analysis of glucose by the Cu2+−g-C3N4 NPs using chemiluminescence as a readout signal. As the content of H2O2 is controlled by the concentration of glucose, the resulting chemiluminescence relates directly to the concentration of glucose. Figure 2B depicts the chemiluminescence spectra generated by the Cu2+−g-C3N4 NPs in the presence of variable concentrations of glucose. In these experiments, different concentrations of glucose are allowed to react with glucose oxidase (GOx) under aerobic conditions for a fixed time interval of 30 min, and the resulting solution is allowed to react with 5 μg mL−1 Cu2+−g-C3N4 NPs and 0.5 mM luminol. The resulting calibration curve is depicted in Figure 2B, inset. It should be noted that the chemiluminescence intensities generated by the Cu2+−g-C3N4 NPs in the presence of variable concentrations of glucose reveal a linear relationship with the concentration range to 1.0 mM, and at higher concentrations, deviation from linearity with a tendency to saturation was observed. This is due to the saturation of the catalytic sites as the concentration of H2O2 increases. In turn, the sensing of glucose by the chemiluminscence intensities generated by the Cu2+−g-C3N4 NPs at different concentrations of glucose reveals linearity with a broader concentration range of glucose (0−2.5 mM). This is due to the biocatalytic aerobic oxidation of glucose that yields limited nonstoichiometric amounts of H2O2 within the time interval of 30 min, applied to sense glucose. Also the Cu2+−g-C3N4 NPs catalyze the H2O2-driven oxidation of dopamine (1) to aminochrome (2), in analogy to HRP or the hemin/G-quadruplex HRP-mimicking catalyst (Figure 3A). The time-dependent absorbance changes of aminochrome, generated upon oxidation of dopamine, are shown in Figure 3B. Figure 3C depicts the absorption spectra corresponding to aminochrome generated within a fixed time interval of 30 min in the presence of 5 μg mL−1 Cu2+−g-C3N4 NPs and 100 mM H2O2. Evidently, the Cu2+−g-C3N4 NPcatalyzed oxidation of dopamine is controlled by the concentration of dopamine, and as its concentration increases, the rate of the oxidation of dopamine is enhanced. Figure 3D shows the rate of oxidation of dopamine in the presence of variable concentrations of the catalyst. As the concentration of Cu2+−g-C3N4 NPs is higher, the oxidation process is faster. Analyzing the curves using the Michaelis−Menten mode, we derive the value Vmax = 0.24 μM min−1 per 1 mg mL−1 of the Cu2+−g-C3N4 catalyst. Control experiments revealed that Cu2+ ions alone or unmodified g-C3N4 NPs did not lead to the catalyzed oxidation of dopamine to aminochrome (Figure S6), implying that only the Cu2+−g-C3N4 NPs act as effective heterogeneous catalysts for the oxidation of dopamine by H2O2.

RESULTS AND DISCUSSION The g-C3N4 NPs were prepared as reported earlier.53 The particles were reacted with Cu2+ ions to yield the Cu2+-ionmodified g-C3N4 NPs (∼200 nm see transmission electron microscopy (TEM) images, Figure S1). X-ray photoelectron spectroscopy (XPS, Figure S2) indicates that Cu2+ ions are associated with g-C3N4 NPs (atomic concentration of constituents corresponding to Cu 3.12%, O 24.79%, N 39.67%, and C 32.43%). Inductively coupled plasma optical emission spectroscopy (ICP-OES) indicates a coverage of Cu2+ corresponding to 0.046 mg per milligram of Cu2+−g-C3N4 NPs. Because the surface functionalities of the g-C3N4 NPs are unchanged upon binding the Cu2+ ions (see Fourier transform infrared (FTIR) spectra, Figure S3), we suspect that the triazine functionalities associated with the g-C3N4 NPs act as ligands for the association of the Cu2+ ions. The Cu2+-modified g-C3N4 NPs exhibit catalytic properties mimicking HRP functions. For example, the NPs catalyze the generation of chemiluminescence in the presence of luminol and H2O2. Figure 1A depicts the chemiluminescence spectra

Figure 1. (A) Chemiluminescence spectra generated upon the oxidation of luminol by variable concentrations of H2O2 in the presence of 0.5 mM luminol and 5 μg mL−1 Cu2+−g-C3N4 NPs: (i) 0, (ii) 0.05, (iii) 0.1, (iv) 0.25, (v) 0.5, (vi) 1, (vii) 2 mM. (B) Derived calibration curve of chemiluminescence intensities, at λ = 425 nm, generated by the oxidation of luminol with variable concentrations of H2O2. The experiments were conducted in 400 mM phosphate buffer, pH 9.0.

generated in the presence of 0.5 mM luminol and variable concentrations of H2O2. As the concentration of H2O2 increases, the chemiluminescence is intensified. Thus, the Cu2+−g-C3N4 NPs act as a catalytic label for the detection of H2O2. Control experiments (Figure S4) indicate that Cu2+ (in the form of CuCl2) at the same content associated with the gC3N4 NPs does not reveal any catalytic properties toward the 3248

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Figure 2. (A) Analysis of glucose by Cu2+−g-C3N4 NPs using chemiluminescence as a readout signal. (B) Chemiluminescence spectra generated upon the oxidation of luminol with the H2O2 produced by the GOx-catalyzed aerobic oxidation of variable concentrations of glucose in the presence of 0.5 mM luminol and 5 μg mL−1 Cu2+−g-C3N4 NPs: (i) 0, (ii) 0.1, (iii) 0.25, (iv) 0.5, (iv) 0.5, (v) 1, (vi) 2.5 mM. Inset: Derived calibration curve corresponding to the chemiluminescence intensities, at λ = 425 nm, generated by the oxidation of luminol with the H2O2 produced by GOx-catalyzed aerobic oxidation of variable concentrations of glucose. The experiments were conducted in 400 mM phosphate buffer, pH 9.0.

Figure 3. (A) Cu2+−g-C3N4 NP-catalyzed oxidation of dopamine (1) to aminochrome (2) by H2O2. (B) Time-dependent absorbance changes of aminochrome, generated upon the oxidation of variable concentrations of dopamine with 2.5 μg mL−1 Cu2+−g-C3N4 NPs: (i) 0, (ii) 0.05, (iii) 0.1, (iv) 1, (v) 10, (vi) 25 mM. (C) Absorption spectra of aminochrome generated upon oxidation of variable concentrations of dopamine with 5 μg mL−1 Cu2+−g-C3N4 NPs: (i) 0, (ii) 0.05, (iii) 0.5, (iv) 5, (v) 25, (vi) 50 mM. (D) Rates of aminochrome formation as a function of dopamine concentrations in the presence of variable concentrations of Cu2+−g-C3N4 NPs: (i) 1, (ii) 2.5, (iii) 5 μg mL−1. All experiments were conducted in 50 mM MES buffer, including 10 mM KCl, 2 mM MgCl2, pH 5.5, in the presence of 100 mM H2O2.

It should be noted that Cu2+−g-C3N4 NP-catalyzed oxidation of dopamine by H2O2 or the generation of chemiluminescence in the presence of luminol/H2O2 are specific to Cu2+ ions, and g-C3N4 NPs doped with other metal ions such as Ni2+, Co3+, Fe3+, Cd2+, or Zn2+ did not yield any peroxidase-mimicking activities. Also, the classical HRP-catalyzed oxidation of ABTS2− to the colored product ABTS−• does not proceed in

the presence of Cu2+−g-C3N4 NPs. Presumably, the adsorption of the conjugated ABTS2− substrate onto the graphene-like C3N4 NPs blocks the catalytic Cu2+ sites on the surface of the NPs. Similar to the catalytic functions of Cu2+−g-C3N4 NPs, we find that Cu2+-ion-modified carbon dots reveal horseradish peroxidase catalytic functions. The C-dots were prepared by 3249

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Figure 4. (A) Chemiluminescence spectra generated by 3.5 μg mL−1 Cu2+−C-dots, in the presence of 0.5 mM luminol and variable concentrations of H2O2: (i) 0, (ii) 0.25, (iii) 0.5, (iv) 1, (v) 2.5, (vi) 5 mM. Inset: Derived calibration curve corresponding to the chemiluminescence intensities at different H2O2 concentrations. All measurement recorded in 400 mM phosphate buffer, pH 9.0. (B) Schematic oxidation of dopamine (1) to aminochrome (2) by H2O2 in the presence of Cu2+−C-dots. (C) Absorption spectra corresponding to the catalytically generated aminochrome, upon subjecting 50 mM dopamine, in the presence of 20 mM H2O2 and 14 μg mL−1 Cu2+−C-dots for different time intervals. (D) Rates of dopamine oxidation to aminochrome as a function of dopamine concentrations, in the presence of 100 mM H2O2, using variable concentrations of Cu2+−C-dots: (i) 0.87, (ii) 1.75, (iii) 3.5 μg mL−1. All experiments described in (C) and (D) were performed in 50 mM MES buffer, pH = 5.5, that included 10 mM KCl and 2 mM MgCl2.

derived Vmax value for the catalyzed oxidation of dopamine corresponds to 0.7 μM min−1 per 1 mg mL−1 of the Cu2+−Cdot catalyst. Control experiments revealed that no oxidation of dopamine occurred by Cu2+ ions alone or by nonfunctionalized C-dots (Figure S12), implying that the process is catalyzed by the heterogeneous Cu2+−C-dot hybrid. Also, the functionalization of the C-dots with other metal ions, such as Pd2+, Zn2+, Ni2+, Co3+, or Fe3+, did not yield any catalytic C-dots for the oxidation of dopamine. The surface functionalities associated with the heterogeneous nanoparticles allow us to link the nanoparticles beyond the metal ions, other chemical units that could lead to the improved catalytic functions of the system. Toward this goal, we modified the carboxylic acid functionalities associated with the C-dots with amino-β-cyclodextrin (β-CD) to yield β-CDfunctionalized Cu2+−C-dots (Figure 5A). In this system, the binding of dopamine to β-CD receptor sites is anticipated to increase the local concentration of dopamine at the heterogeneous catalyst, thereby enhancing the catalytic process. Figure 5B depicts the rate of dopamine oxidation as a function of dopamine concentrations by the β-CD-functionalized Cu2+− C-dots, curve (i) (average loading of the C-dots corresponds to 0.06 mmol of β-CD per gram of β-CD−Cu2+−C-dots, Figure S13), and by the Cu2+−C-dots, curve (ii). For the application of the β-CD-modified Cu2+−C-dots, in comparison to the Cu2+−C-dots, at lower concentration of the heterogeneous catalyst, see Figure S14. Evidently, the rate of dopamine

microwave-assisted decomposition of urea and citric acid as reported previously.54 Cu2+ ions were linked to the carbon dots to form the Cu2+−C-dot hybrids (∼8 nm; see TEM measurements, Figure S7). XPS measurements (Figure S8) indicate that Cu2+ ions are associated with C-dots (atomic concentration of constituents corresponding to Cu 1.58, O 28.9, N 10.13, and C 59.38%). ICP-OES measurements indicate a coverage of Cu2+ corresponding to 0.14 mg per milligram of Cu2+−C-dots. FTIR spectra suggest that the Cu2+ ions are associated with carboxylate and/or amine functionalities associated with the C-dots due to the appearance of new bands at 1695 and 2924 cm−1, respectively, upon binding of Cu2+ ion to the C-dots (Figure S9). Similar to the Cu2+−g-C3N4 NPs, we find that the Cu2+−Cdots exhibit HRP-like activities. Figure 4A depicts the chemiluminescence spectra generated by the Cu2+−C-dots in the presence of luminol/H2O2. As the concentration of H2O2 increases, the chemiluminescence spectrum is intensified. The resulting calibration curve is shown in Figure 4A, inset. Similarly, the Cu2+−C-dots act as a catalyst for the oxidation of dopamine (1) to aminochrome (2) (Figure 4B). The timedependent absorbance spectra of aminochrome upon the oxidation of 50 mM dopamine (1) by 20 mM H2O2, in the presence of 14 μg mL−1 Cu2+−C-dots, are displayed in Figure 4C. The time-dependent oxidation of variable concentrations of dopamine by 100 mM H2O2, in the presence of variable concentrations of the Cu2+−C-dots, is shown in Figure 4D. The 3250

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activities provide a means to apply these particles for the development of different sensors and as catalysts for other oxidation processes. Also, the results suggest that other metalion-functionalized g-C3N4 NPs or C-dots could act as catalysts for additional chemical transformations. Furthermore, the surface functionalities associated with the g-C3N4 NPs or the C-dots allow further modification with chemical units (e.g., cyclodextrins) that enhance the catalytic function of the heterogeneous nanoparticles.

EXPERIMENTAL SECTION Reagents and Instruments. All materials used in this study were purchased from Sigma and were used without any additional purification. Ultrapure water from a NANOpure Diamond (Barnstead Int., Dubuque, IA) source was used to prepare all buffers. Transmission electron microscopy measurements were carried out on a Tecnai F20 G2 transmission electron microscope at an accelerating voltage of 18 kV. X-ray photoelectron spectroscopy data were obtained with an Axis Ultra photoelectron spectrometer from Kratos Analytical. Ultraviolet−visible spectroscopy measurements were recorded on a Bio-Tek plate reader (monochromator; path length correction applied). Chemiluminescence measurements were carried out using a Varian Cary Eclipse spectrofluorometer with a slit width of 20 nm. Fourier transform infrared spectra were recorded on a Varian 670 spectrometer. Inductively coupled plasma optical emission spectroscopy measurements were carried out on a Perkin Elemer Optima 3000 spectrometer. Synthesis of Cu2+−g-C3N4 Nanoparticles. The bulk g-C3N4 was prepared by pyrolysis of urea following previous procedures with slight modifications. Briefly, urea (11 g) was placed in a crucible and heated at 80 °C for 1 h. Then, the crucible was covered with aluminum foil and heated at 225 °C for 30 min, followed by another 30 min reaction at 350 °C. Finally, the temperature was kept at 540 °C for 4 h. In order to obtain the g-C3N4 nanosheets, 0.2 g of bulk g-C3N4 power was dispersed in 20 mL of 5 M HNO3 and refluxed for 24 h. The white product was centrifuged, washed with water to near-neutral pH, and redispersed in water. The resultant suspension solution was sonicated for 4 h and then centrifuged at 2000 rpm for 10 min to remove the large nanosheets. The product was dried overnight. Cu2+−g-C3N4 NPs were prepared by mixing g-C3N4 nanosheets and CuCl2 under sonication conditions. Briefly, 20 mL of aqueous solution containing 20 mg of g-C3N4 nonosheets was under sonication for 10 min. Subsequently, 20 mL of CuCl2 (0.1 M) was added into the above solution. After sonication treatment for 4 h at 80 °C, the solution was centrifuged and washed with deionized water four times to remove the unbound CuCl2. Finally, the product was dried overnight. Synthesis of Cu2+−C-dots. The C-dots were synthesized according to Wang et al. Citric acid and urea were mixed in water and heated for 4−5 min in a domestic 750 W microwave. The solid was vacuum dried and purified in a centrifuge (30000g for 45 min) to remove large agglomerated particles. The purified C-dots solution was incubated overnight in 0.1 M CuCl2 under N2. The unreacted copper was washed out by three centrifugation steps (30000g for 45 min). Chemiluminescence Studies. In a typical experiment, 105 μL of phosphate buffer solution (400 mM, pH 9), 15 μL of NPs (50 μg mL−1 for Cu2+−g-C3N4 NPs and 35 μg mL−1 for Cu2+−C-dots), 15 μL of 5 mM luminol, 15 μL of variable concentrations of H2O2, with concentrations between 0.5 and 50 mM, were added. The chemiluminescence generated by the system was recorded immediately. For the analysis of glucose, GOx (4 U mL−1 in 80 μL of 10 mM acetate buffer, pH 5) was incubated with variable concentrations of glucose, with concentrations of 1, 2.5, 5, 10, and 25 mM, and the system was allowed to react under oxygen atmosphere at 25 °C for 30 min. Finally, 15 μL of the resulting mixtures were added to 15 μL of Cu2+−g-C3N4 NPs (50 μg mL−1) in 105 μL of phosphate buffer (400

Figure 5. (A) Functionalization of Cu2+−C-dots with β-cyclodextrin. (B) Rates of dopamine oxidation to aminochrome as a function of dopamine concentrations, in the presence of 100 mM H2O2 and (i) 4 μg mL−1 β-CD-functionalized Cu2+−C-dots or (ii) 4 μg mL−1 Cu2+−C-dots. The experiments were performed in 50 mM MES buffer, pH = 5.5, that included 10 mM KCl and 2 mM MgCl2.

oxidation is ∼4-fold enhanced in the presence of the β-CDmodified Cu2+−C-dots.

CONCLUSIONS In conclusion, our study has introduced Cu2+-ion-modified gC3N4 NPs or C-dot hybrids as heterogeneous catalysts mimicking the functions of horseradish peroxidase. Comparison of the catalytic activities of the Cu2+-ion-loaded g-C3N4 NPs and the Cu2+-ion-modified C-dots, normalized to the same content of Cu2+ ions (Figure S15), reveals that the catalytic activity of the Cu2+-ion-modified g-C3N4 NPs is ∼4-fold higher compared to that of the Cu2+-modified C-dots. We have also examined the pH dependence of the peroxidase activities of the Cu2+−g-C3N4 NPs and Cu2+−C-dots, toward the oxidation of dopamine (Figure S16). Interestingly, we find that the Cu2+−gC3N4 NPs reveal the highest activities in the pH region of 7.0 to 8.0, and the Cu2+−C-dots show a high peroxidase activity in the pH range 6.0 to 6.5. That is, the two kinds of Cu2+functionalized NPs broaden the pH range of peroxidase activity compared to that of the native horseradish peroxidase range,55 pH 6.0 to 7.5. It should be noted that many different heterogeneous catalysts were reported to exhibit peroxidasemimicking functions.4−11,56−58 Nonetheless, while most of the heterogeneous catalysts followed the peroxidase-mimicking functions through the colorimetric assays involving the H2O2mediated oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6sulfonate) dianion (ABTS2−) to ABTS−• or of 3,3′,5,5′tetramethylbenzidine (TMB) to 3,3′,5,5′-tetramethylbenzidine diamine (TMBDI), our systems introduce the H2O2-catalyzed oxidation of dopamine to aminochrome and the H2O2catalyzed oxidation of luminol and the accompanying generation of chemiluminescence. The surface functionalities associated with g-C3N4 NPs or C-dots and their catalytic 3251

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ACS Nano mM, pH 9). Then, 15 μL of 5 mM luminol was added, and the chemiluminescence generated by the system was recorded. Dopamine Oxidation Studies. For a typical oxidation reaction, 10 μL of variable concentrations of dopamine, with concentrations between 0.1 and 500 mM, was added over 10 μL of particles (10, 25, or 50 μg mL−1 for Cu2+−g-C3N4 NPs and 8.7, 17.5, or 35 μg mL−1 for Cu2+−C-dots) in 70 μL of MES buffer (50 mM, 10 mM KCl, 2 mM MgCl2, pH 5.5), followed by the addition of 10 μL of 1 M H2O2. The catalytic oxidation of dopamine was followed spectroscopically by measuring the changes in the absorption of the aminochrome product, λmax = 480 nm (ε = 3058 M−1 cm−1). In all measurements, a background signal measured at λ = 800 nm was subtracted. 6-Monodeoxy-6-monoamino-β-cyclodextrin Hydrochloride (β-CD-NH2)-Linked Cu2+−C-dots. To a solution of Cu2+−C-dots (0.3 μg mL−1) were subsequently added equal volumes of EDC and NHS (100 mM in MES buffer 5 mM, pH 7.2) and left to react for 10 min. To the crude NHS-ester were sequentially added 150 μL of 100 mM phosphate buffer, pH 7.2 and 50 μL of the β-CD-NH2 (90 mM in 100 mM phosphate buffer, pH 7.2). The coupling reaction was performed at room temperature for 4 h. The loading of Cu2+−C-dots by β-CD was analyzed by following the decrease in absorbance at 550 nm due to phenolphthalein−CD complex formation54 and using an appropriate calibration curve. βCD−Cu2+−C-dots were isolated from the unreacted β-CD-NH2 by centrifugation (45 min at 30000g and 4 °C), and 2 μL of the resuspended Cu2+−C-dots (1.35 mg mL−1) were added to 18 μL of 0.05 mM Tris-HCl buffer, pH 8.0. To the Cu2+−C-dots solution was added 100 μl of a 5 × 10−5 M phenolphthalein solution that included 125 mM Na2CO3/NaHCO3, pH 10.5. The absorbance changes at λ = 550 nm and the appropriate calibration curve were used to evaluate the average loading of β-CD on the β-CD−Cu2+−C-dots.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00352. TEM, XPS, and FTIR figures; control experiments for dopamine and luminol; chemiluminescence spectra generated by the luminol/H2O2 system in the presence of different concentrations of NPs, β-CD quantification, and rate of dopamine oxidation at lower concentrations of β-CD-functionalized Cu2+−C-dots (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel: 972-2-6585272. Fax: 9722-6527715. ORCID

Itamar Willner: 0000-0001-9710-9077 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study is partially supported by the Israel Ministry of Science and The Minerva Center for Biohybrid Complex Systems. REFERENCES (1) Groves, J. T.; Haushalter, R. C.; Nakamura, M.; Nemo, T. E.; Evans, B. J. High-Valent Iron-Porphyrin Complexes Related to Peroxidase and Cytochrome P-450. J. Am. Chem. Soc. 1981, 103, 2884−2886. (2) Feiters, M. C.; Rowan, A. E.; Nolte, R. J. M. From Simple to Supramolecular Cytochrome P450 Mimics. Chem. Soc. Rev. 2000, 29, 375−384. 3252

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DOI: 10.1021/acsnano.7b00352 ACS Nano 2017, 11, 3247−3253