Ionic Liquid-Functionalized Fluorescent Carbon Nanodots and Their

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Ionic Liquid Functionalized Fluorescent Carbon Nanodots and Their Applications in Electrocatalysis, Biosensing and Cell Imaging Haijuan Li, Limei Chen, Haoxi Wu, Haili He, and Yongdong Jin Langmuir, Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on November 27, 2014

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Ionic Liquid Functionalized Fluorescent Carbon Nanodots and Their Applications in Electrocatalysis, Biosensing and Cell Imaging Haijuan Li,a Limei Chen,a,b Haoxi Wu,a,c Haili Hea,c and Yongdong Jina* a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China b

Department of Cell biology, Basic Medical College, Beihua University, Jilin, 132013, China

c

University of Chinese Academy of Sciences, Beijing 100049, China

*Address correspondence to [email protected]

KEYWORDS: carbon nanodots, electrocatalysis, biosensing, cell imaging, glucose, ionic liquid

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ABSTRACT In this report, ionic liquid functionalized carbon nanodots (IL-CDs) were simply produced by electrochemical exfoliation of graphite rod in the presence of an amino-terminated ionic liquid. And their preliminary applications were exploited. TEM and AFM results showed that these IL-CDs are about 2.6 nm in diameter. The small-sized IL-CDs have strong photoluminescence with a quantum yield of about 11.3% and could be used for cell imaging. Moreover, the IL-CDs exhibit good electron transfer property and nice catalytic activity for O2 and H2O2 reduction reaction. And the as-prepared IL-CDs can be applied as a matrix for immobilizing enzymes (glucose oxidase) to construct biosensors. Due to these favorable properties, the IL-CDs will find more promising practical applications in electrocatalysis, biosensing and bioimaging.


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INTRODUCTION Generally, carbon nanodots (CDs) are quasi-spherical nanoparticles with diameters less than 10 nm.1-3 They are discovered in 2004 and are new members of nanocarbon family. CDs have a series of advantages such as good solubility, desirable biocompatibility, non-poisonous nature, chemical inertness, good conductivity, simplicity in modification, robust photoluminescence to photobleaching, and so on. These properties provide promising potential applications ranging from sensing and bioimaging,4-7 to photocatalysis,8-10 electrocatalysis,11 light emitting diode and solar cells.12,13 The CDs can be obtained by much simpler and faster methods with less expensive instruments than other carbon nanomaterials (carbon nanotubes, fullerenes, nanodiamonds, carbon nanohorns),1,2 which make them promising as alternatives to other carbon nanomaterials.1,2 Nanocarbon materials like graphene, carbon nanotubes and carbon nanohorns have been widely used in the area of electrochemistry and biosensors.14-24 For instance, nanocarbons have been extensively used as electrocatalyst15 and matrix for proteins,16,17 and a variety of biological product like DNA,18 virus,19 antigen,20 disease marker,21 and even whole cell22,23 have been detected. Especially, due to the increase of the diabetes patients in the past years, glucose biosensors using nanocarbons (carbon nanotubes, carbon nanohorns and graphene) and nanocarbon composites with metal nanoparticles have been widely investigated.15-17,25-26 These successful application for carbon nanomaterials can be attributed to their capacity to accelerate electron conductivity during electrochemical process.14 Although CDs have been exploited in a variety of applications, especially in bioimaging, there are seldom reports on using CDs in electrochemical biosensing.23-24 In this study, we exploited CDs for biosensing application and


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using glucose biosensing as a model. The obtained biosensor displayed comparable results with other carbon nanomaterial-based glucose biosensors. However, the synthesis of IL-CDs is much simpler and cheaper than other carbon materials, making it promising for practical applications. Because of their unique properties, such as nonflammable, low vapor pressure, good electrical conductivity, ionic liquids (ILs) have attracted considerable attentions in the past two decades in the area of electrochemistry.25,29-32 In particular, carbon nanoparticles functionalized with ILs have received considerable attentions as the combination of ILs and carbon nanomaterials can enhance the capability of the corresponding composites and the resulting materials have been widely used. For example, Shan et al. constructed a novel graphene/polyethyleniminefunctionalized IL/glucose oxidase (GOx) electrochemical biosensor for glucose detection.25 Guo et al. fabricated an IL functionalized graphene modified electrode for the detection of trinitrotoluene (TNT).29 Lately, Liu and coworkers developed a facile one-step exfoliation method to construct graphene in the electrolyte of IL and water.30 Based on their work, Lu and co-workers found that by adjusting the ratio of water to ILs, a variety of carbon nanomaterials (graphene, photoluminescent carbon nanoparticles and nanoribbons) can be obtained. And through adjusting the ratio of water to ILs and the category of the anion of the ILs, the photoluminescence of the carbon nanoparticles can be adjusted from 2.8% to 5.2%.31 However, they didn’t make further investigation on the applications of these interesting materials. And we supposed that the varieties of the ILs may endow the IL-CDs various properties. Herein an amino-terminated IL, 1aminopropyl-3-methylimidazole tetrafluoroborate ([apmim][BF4]),29 which has good electrical conductivity and exchangeability of the counter-anions, e.g. with enzymes (like GOx), was chosen, and the preliminary application of the obtained IL-CDs was exploited. The obtained IL-


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CDs displayed nice electrocatalytic activity for O2 and H2O2 reduction reaction. Attributed to their good electronic character, the as-prepared IL-CDs-based biosensor accomplished direct electron transmit of GOx and well retained its biological activities, rendering them good platforms for glucose biosensing. The IL-CDs have strong photoluminescence with a quantum yield of about 11.3% and were used in cell imaging. EXPERIMENTAL SECTION Materials. High-purity graphite rods were purchased from China National Medicines Shenyang Co. Ltd.. The IL [apmim][BF4] (>97%) was purchased from Shanghai Cheng Jie Chemical Co. Ltd. Glucose oxidase (EC 1.1.3.4, type X-S, lyophilized powder, 100-250 units/mg, from Aspergillus niger) and D-(+)-glucose (99.5%) were obtained from Sigma. Hydrogen peroxide aqueous solution (30 wt %) was purchased from Beijing Chemical Reagent Co. Other reagents were of analytical grade and used without further purifacation. Aqueous solutions were prepared with double-distilled water from a Millipore system. Electrochemical Synthesis of IL-CDs. Two graphite rods (99%, China National Medicines Shenyang Co. Ltd.) were parallel set into the [apmim][BF4] /water (9/1) solution with a distance of about 3.0 cm. Static potential of 12 V was applied across these electrodes by a direct current power supply. After a short induction period, the electrolyte color became yellow from colorless and afterwards turned into dark brown. After that, the graphite anode became swelled and the outstretched pieces discharged from the anode; and the electrolyte turned into black slurry. The exfoliation process was stopped after 4 h. Then the product was diluted with water and ethanol and centrifuged at a speed of 12000 rpm to separate less-photoluminescent deposit. To acquire


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pure IL-CDs, top of the dispersion was dialyzed with ultrapure water by a dialysis bag with 500 Da MWCO. Preparation of IL-CD-GOx Modified GC Electrodes. IL-CDs (~ 1 mg) was first dispersed in 0.2 ml of 0.5 wt.% Nafion (5% in a mixture of lower aliphatic alcohols and water, SigmaAldrich) solution, then the mixture was agitated to form a uniform Nafion-IL-CDs suspension. Then glassy carbon electrode (GC) was covered by 5 µL of Nafion-IL-CDs suspension and dried at room atmosphere. To prepare glucose biosensor, firstly, GOx dissolved in PBS (0.01 M, pH7.4) was mixed with Nafion-IL-CDs, then, the suspension was dropped on surface of GC. The modified electrode was dried in a refrigerator at 4°C for 24 h. Before use, the modified electrode was washed with PBS for several times. The controll IL-modified electrodes were prepared with 5 µL of 0.5 mg/mL IL aqueous solution by the same procedure, respectively. Electrochemical test. Electrochemical test were carried out in a conventional three-electrode cell. The auxiliary electrode is a platinum wire, the reference electrode is an Ag/AgCl (saturated KCl) and the working electrode is a modified glassy carbon (GC) electrode with 3 mm diameter. Prior to modification, the surface of GC electrodes were carefully polished with alumina slurries to get mirror face. Cyclic voltammetry (CV) measurements were accomplished in a CHI 660 Electrochemical Workstation (CHInstruments, Chenhua Co., Shanghai, China). Cell imaging using IL-CDs. The potential cell imaging capability of the IL-CDs was investigated using HeLa cell as a model. The HeLa cells were incubated in Dulbecco’s minimum essential media plus10% fetal bovine serum, 1% penicillin, and 1% amphotericin B. The HeLa cells were cultured in culture dishes (diameter: 40 mm) at 37 °C in 5% CO2 till approximately 70% confluence is obtained. Then the IL-CDs solution was filtered with a 0.22 µm sterilized filter membrane and introduced into the cells with a final concentration of about 0.2 mg ml-1.


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After incubating for 12 h, the culture media was discharged and the cells were fixed by 90% glycerin. The fluorescence image is taken up by a DMI6000 B inverted microscope (Leica) and a DFC450 digital color camera (Leica), the wavelength of the excitation is about (480 ± 40 nm). Measurement and characterization. Transmission electron microscopy (TEM) micrographs were obtained using a JEOL 2000 transmission electron microscopy operating at 200 kV. Tapping-mode AFM imaging was performed on a Digital Instruments multimode AFM controlled by a Nanoscope IIIa apparatus (Digital Instruments, Santa Barbara, CA) equipped with an E scanner. A standard silicon cantilever tip from Digital Instruments was used. The scan rate was 1-1.5 Hz. X-ray photoelectron spectroscopy (XPS) analysis was carried out on an ESCALAB MK II X-ray photoelectron spectrometer. Fluorescence Spectra were obtained by a Perkin-Elmer LS-55 Luminescence Spectrometer (Perkin-Elmer Instruments U.K.). UV-Vis absorption spectra were recorded by a CARY 500 UV-Vis−NIR Varian40 spectrophotometer.

RESULTS AND DISCUSSION Characterization of IL-functionalized CDs. CDs were prepared mainly based on Lu’s report except for using a new amino-terminated IL, [apmim][BF4] (Scheme 1).26 According to Lu’s report, in pure ILs, the size of the carbon nanoparticles is 2-4 nm; in electrolyte with more water content, the size of the carbon particles will increase to 8-10 nm. When the water content is less than 10%, the particles will be functionalized by IL. In our work, to obtain IL-CDs, a concentrated IL (90%) was used. To avoid the effect of free ILs, the as-prepared IL-CDs were thoroughly dialyzed by ultrapure water with a dialysis bag (500 Da MWCO). The formation of monodisperse IL-CDs can be observed with both TEM and AFM measurements (Figure 1A and 1B). The resultant IL-CDs are well dispersed in water with an average diameter of 2.6 ± 0.6 nm


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(Figure. 1C), consistent with the Lu’s report. HRTEM image inset in Figure 1A displays that the lattice spacing is about 0.27 nm, consistent with the (020) lattice plane of graphite.12

Scheme 1. Illustration of the synthesis of IL-CDs and the combination of ILs to CDs, via covalent or noncovalent interactions. The composition of IL-CDs was further investigated with X-ray photoelectron spectroscopy (XPS). The presence of [apmim] moiety as well as the BF4 counterion is observed in the XPS survey (Figure. 2). The deconvolution of C1s spectrum of CDs indicated the presence of four types of carbon bonds: sp3 C-C (284.6 eV), C-N (286.2 eV), C=O (287.7 eV), and O-C=O (289 eV). The oxidation groups (C=O and O-C=O) imparts water solubility on the CDs. The conspicuous peaks at 399.6 eV and 401.6 eV are attributed to the N 1s of the imidazolium ion and the amino functional group.The pronounced C-N and N 1s peak demonstrates that there are considerable nitrogen-containing functional groups, illustrating the combination of [apmim] with the IL-CDs. The appearance of B 1s peak at 192.2 eV and 194 eV and F 1s at 685.7 eV indicates the existence of the BF4 anion. The presence of these peaks can be ascribed to the combination of ILs to CDs, either by covalent or noncovalent interplay. These XPS results were consistent with the reported exfoliation mechanism31 that involving complicated interaction of water oxidation and ionic liquid insertion on anodic electrode.


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Figure 1. (A) TEM and (B) AFM images of the obtained IL-CDs, (C) the size distribution of CDs determined by TEM, and (D) AFM line scan profile of the IL-CDs. The HRTEM image of single CDs is inserted in (A).


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Figure 2. XPS spectra of the obtained IL-CDs (a) and corresponding high-resolution spectra of C1s (b), O1s (c), N1s (d), F1s (e) and B1s (f) spectra. Photoluminescence of IL-CDs. As shown in the inset of Figure. 3A, the IL-CDs show good solubility, and display relatively strong blue luminescence under 365 nm UV irradiation. Like most luminescent carbon nanoparticles, the IL-CDs also exhibit an excitation-dependent photoluminescence (PL) behavior (Figure 3B). The maximum emission is observed at 485 nm when excited at 400 nm. The quantum yield of the IL-CDs is about 11.3% in contrast with quinine bisulfate (QY 0.54 in 0.1 M H2SO4), which is comparable to QY of CDs synthesized by other methods.33,34

Figure 3. (A) UV-vis absorption and fluorescence spectrum of the as-prepared IL-CDs. Insets are photographs of IL-CDs in aqueous solution (left) and irradiated by 365 nm light (right). The emission spectrum was taken under excitation at 400 nm, and the excitation spectrum was taken


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at the maximum emission wavelength of 480 nm. (B)Emission spectra of IL-CDs at excitation wavelengths varied from 300 nm to 480 nm.

Figure 4. Fluorescence microscopy images of the HeLa cells under 480 ± 40nm excitation with IL-CDs as fluorescent probe. a) bright-field images; b) fluorescent images (scale bar: 20 µm). Cell imaging with IL-CDs. With an attractive set of features including small size, bright luminescence and benign nature, CDs have a great potential in bioimaging applications. Here, using HeLa cell as a model, we investigated he capacity of IL-CDs in biological imaging, After incubated with highly fluorescent IL-CDs for 12 h, the images were collected with an inverted fluorescence microscope by a laser of 480 ± 40 nm (Figure 4). The cells are illuminated brightly with internalized IL-CDs. The IL-CDs are mainly observed in the cell membrane and cytoplasm, while the fluorescence intensity in the nucleus region is very weak, indicating that the IL-CDs did not disrupt genetic information. This observation is in accordance with lateral research on the effect of nanomaterials on living cells, that the nanomaterials did not discompose genetic passage.5, 35-38 These results showed excellent biocompatibility of the IL-CDs, which make them possible to be applied in the area of cell imaging. Electrocatalysis of O2 and H2O2 at the IL-CDs Modified GC Electrode. Compared with conventional fluorescence detection, electrochemical method provides a set of superiorities, like inexpensive and simple equipment, high sensitivity, and good selectivity. In the past two


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decades, electrochemical biosensors based on carbon nanomaterials have been widely used, which is attributed to the excellent electron conduction property of the carbon materials. In this study, we further exploit the application of CDs in electrochemical field. The IL-CDs displayed favorable electrocatalysis toward the reduction of O2 and H2O2. Nafion was used to prevent IL-CDs to desorb from the glassy carbon electrode. A notable O2 reduction peak at ~ - 0.3 V appears at the IL-CDs modified electrode (Figure 5a). The reduction potential is much more positive than that at IL-modified electrodes (Figure 5b). The electrocatalysis of H2O2 at the IL-CDs modified electrode was investigated too (Figure 5c and 5d). Compared with the IL modified electrode, the IL-CDs modified electrode has more positive reduction potential and more conspicuous reduction current, indicating IL-CDs had much better electrocatalytic performance for O2 and H2O2 reduction than control IL-modified electrodes. The nice electrocatalytic performance of IL-CDs for O2 and H2O2 reduction may be attributed to their excellent electron conductivity.

Figure 5. CVs at IL-CDs (a) and IL (b) modified electrodes in 0.1 M PBS solution (pH7.4) saturated with O2 (black line) and degassed with pure N2 (red line). (c) and (d) are IL-CDs and IL


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modified electrodes in 0.1 M PBS solution (pH7.4) saturated with N2 in the absence (red line) and in the presence (black line) of 5 mM H2O2, respectively. Scan rate: 50 mV s-1. Direct electrochemistry and glucose biosensing of GOx immobilized on IL-CDs modified glassy carbon electrode. Figure 6a shows cyclic voltammograms (CV) of the IL-CDGOx modified GC and control IL-GOx modified GC electrodes in N2-saturated PBS solution. The IL-CD-GOx modified electrode showed a pair of well-defined and nearly symmetric redox peak (Figure 6a, red line). The peak current of both the anodic and cathodic increased linearly with the scan rate (correlation coefficient 0.996) (Figure 6b), which indicated that in the investigated potential range the electrode undergoes a surface-controlled process. The control experiments with IL-GOx yielded featureless voltammograms (Figure 6a, black line). This may due to the difficulty of the electron communication from the active redox center of GOx to the electrode. By comparison with the control IL-GOx electrode, we can see that the IL-CD-GOx could improve electron transport between the GOx and electrode substrate, and therefore achieve direct electrochemistry of the immobilized GOx.


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Figure 6. CVs of the IL-CD-GOx (red line) and IL-GOx (black line) modified GC electrodes in 0.1 M PBS solution (pH 7.4) saturated with N2 at a scan rate of 50 mV s-1, respectively. (b) CVs of IL-CD-GOx modified electrode in 0.1 M PBS solution (pH 7.4) at various scan rates from 0.05, 0.10, 0.15, 0.20, 0.3, 0.4 and 0.5 V s-1, respectively. Inset: plot of peak current (ip) vs scan rate. (c) CV measurements at the IL-CD-GOx modified GC electrode in various concentrations of glucose: 0, 0.02, 0.5, 1, 2 mM from outer to inner. The inset is the calibration curve (R = 0.996) corresponding to amperometric responses at - 0.33 V. Scan rate: 50 mV s-1. Figure 6c shows CVs of the IL-CD-GOx modified electrode in PBS solutions with glucose concentrations from 0 to 2 mM. The reduction peak at - 0.33 V, originating from the reduction of O2 to H2O2, decreased linearly with the increase of glucose concentration (Figure 6c), which is


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due to the consumption of O2. The linear range is from 20 µM to 2 mM (R = 0.996), and the limit of detection (LOD) is 7 µM. This result is comparable with other carbon nanomaterial-based GOx biosensors (Table 1). However, the IL-CDs can be obtained more easily and much cheaper than other carbon materials. Table 1: Performance of some carbon based glucose biosensors. Glucose biosensor

Linear Range (mM)

LOD (µM)

Reference

IL-CD-GOx

0.02 to 2

7

This work

Nafionmultiwalled carbon nanotube-GOx

0.025 to 2

4

Tsai et al. 22 (2005)

Nafion-single wall carbon nanohorn-GOx

0 to 6

6

Liu et al. 12 (2008)

graphene/poly ethyleniminefunctionalized IL/GOx

2 to 14

Shan et al. 21 (2009)

Reproducibility test showed that relative standard deviation for 6 successive measurements of 0.5 mM glucose at - 0.33 V was 3.3%. And the biosensor displayed good stability that amperometric response preserved 96% of its initial response in 3 days and 91.8% in 1 week. These results demonstrated that IL-CDs are good matrix to reserve the activity of GOx. This may due to intense electrostatic attraction between positively charged [apmim] moiety and the negatively charged GOx.


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To investigate the application of the biosensor in practical, fingertip blood samples were assayed using the IL-CD-GOx modified electrode. The sample (40 µL) was diluted to 4 mL by 0.1 M PBS (pH 7.4) and the current response at - 0.33 V was recorded. The concentration of glucose was calculated by brought the current response into the linear curve. The glucose concentration of fingertip blood was tested to be 6.4 mM, similar to the value of 6.7 mM obtained by a commercial blood glucose monitor. The recovery value for the assay of added 0.5 and 1 mM glucose was 93.7% and 95.1%. The results of this study revealed that the facilely prepared IL-CDs are very promising nanomaterials to construct stable and reproducible electrochemical biosensor systems. The excellent electron transfer property is convenient to transmit electrochemical signal and the positively charged nature is ideal to immobilize negatively charged enzymes. CONCLUSIONS In this study, ionic liquid functionalized carbon nanodots (IL-CDs) were synthesized by a facile one-step electrochemical exfoliation of graphite, by using an amino-terminated IL ([apmim][BF4]). The facilely prepared IL-CDs exhibit excellent electrocatalytic activities for O2 and H2O2 reduction. Because of their good electronic properties, direct electrochemistry of GOx was achieved on IL-CDs modified GC electrodes, and practical electrochemical glucose sensing with a linear range from 20 µM to 2 mM was obtained. Moreover these IL-CDs exhibit strong photoluminescence with a quantum yield of 11.3 %. Their small size and benign nature make them good fluorescent nanoprobes for biological applications. The facile preparation and high performances of the IL-CDs will find more promising practical applications in electrocatalysis, biosensing and bioimaging.


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AUTHOR INFORMATION Corresponding Author *Tel: +86-431-85262661, Fax: +86-431-85262661, E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by start-up funds from the Changchun Institute of Applied Chemistry of Chinese Academy of Sciences, the Hundred Talents Program of the Chinese Academy of Sciences, and the State Key Laboratory of Electroanalytical Chemistry (No. 110000R387).


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Suggested Figure for the Table of Contents:

One-Step Green Synthesis ofIonic Liquid Functionalized Fluorescent Carbon Nanodots and Their Applications in Electrocatalysis, Biosensing and Cell Imaging

Haijuan Li,a Limei Chen,a,b Haoxi Wu,a,c Haili Hea,c and Yongdong Jina* a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China E-mail:[email protected] b Department of Cell biology, Basic Medical College, Beihua University, Jilin, 132013, China c University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: carbon nanodots, electrocatalysis, biosensing, cell imaging, glucose, ionic liquid

We report the facile synthesis of carbon dots with good electronic properties and strong photoluminescence and their applications in electrocatalysis, biosensing and bioimaging.


Ionic Liquid-Functionalized Fluorescent Carbon Nanodots and Their

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