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Switching Carbon Nanodots from Single Emission to Dual Emission by One-Step Electrochemical Tailoring in Alkaline Alcohols: Implications for Sensing and Bioimaging Lihua Shen, Chenxing Wang, Azhar Abbas, Chunxia Yu, Sichun Zhang, Xinrong Zhang, and Chengxiao Zhang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00278 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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ACS Applied Nano Materials

Switching Carbon Nanodots from Single Emission to Dual Emission by One-Step Electrochemical Tailoring in Alkaline Alcohols: Implications for Sensing and Bioimaging Lihua Shen,*,†,⊥ , Chenxing Wang†, Azhar Abbas∥, #, Chunxia Yu†, Sichun Zhang‡ Xinrong Zhang‡,* and Chengxiao Zhang §* †

College of Chemistry and Chemical Engineering, Xi’an University of Science and

Technology, Xi’an, 710054, China Key Laboratory of Coal Resources Exploration and Comprehensive Utilization, MLR.，

⊥

710021 ‡

Beijing Key Laboratory for Microanalytical Methods, Instrumentation, Department of

Chemistry, Tsinghua University, Beijing 100084, P. R. China § Key

Laboratory of Applied Surface and Colloid Chemistry (Ministry of Education),

School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, China. ∥ Department

of Chemistry, Physical & Theoretical Chemistry Laboratory, University of

Oxford. Oxford OX1 3QZ, United Kingdom. #

Ibn-e-sina block, department of Chemistry, University of Sagodha, Sargodha, 40100,

Pakistan. ABSTRACT Fluorescent carbon nanodots (CNDs) capable of dual emission blue and red light (BR-CNDs) (460 and 610 nm) have been synthesized by a simple and low cost electrochemical method of discharging indole dissolved in ethanol in the presence of alkali. 1

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Presence of alkali played an important role to achieve switching from single blue emission to dual emission (red and blue). The as synthesized CNDs have been thoroughly characterized by different spectroscopic techniques. Moreover, the origin of the red emission has been investigated by analysing the intermediate product of CNDs using gas chromatography-mass spectrometry (GCMS). It was found that, 2-naphthaleneamine, an intermediate product of B-R-CNDs, fetches an extended π-conjugated system and heteroatom functional groups to B-R-CNDs, which are synergistically responsible for emission at longer wavelength. Moreover, alkaline ethanol system played a pivotal role in structural transition from single emission to dual emission, because surface oxidation is caused by hydroxyl radicals produced in the alkaline system. Surface oxidation creates surface defects thereby giving rise to the surface-state-related red emission. The red emission of B-R-CNDs are found highly pH dependent which makes them a potential candidate for application in pH sensing. Moreover, as prepared B-R-CNDs have been used in full color bio-imaging of HepG2 cells, and exhibit an accurate mitochondrial-targeting ability. KEYWORDS: dual emission, carbon nanodots, photoluminescence, bio-imaging, mitochondrial localization 1. INTRODUCTION In recent years, carbon nanodots (CNDs) have attracted vigil eye of researchers because of their unique optical properties, low toxicity, convenience of surface modification, good light stability and biocompatibility.1-8 Moreover, these CNDs have exhibited potential applications in biomedical imaging,9 photo catalysis and multifunctional sensors.10-11 Traditional single emission CNDs, especially blue emission CNDs, limit their application in imaging biological process because they can lead to photodamage to the biological tissue.12-13 Moreover, biological substrates have the 2


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property of blue autofluorescence under UV excitation light.14 Red emission CNDs, on the other hand, have good organ penetration and slight biological tissue photodamage which make them a potential candidate for bio-imaging.15 These single emission CNDs are also susceptible to the environmental effects and fluctuation of the excitation source. So, development of new dual emission CNDs can effectively eliminate shortcomings associated with single emission CNDs and can provide better sensing strategies for biological or biomedical analyses. Up till now, a few methods have been developed for the synthesis of dual emission nanoparticles.16-17 These methods include joining two independent fluorescent chemical compounds18 or two different fluorescent nanomaterials to an inert nanoparticles,19 combining dye molecules with a kind of fluorescent nanoparticles and modification with different ligands20 on the surface of carbon nanoparticles. However, these methods have some drawbacks, such as high cost, complexity of process, long time consumption and incapability of emitting red light. To the date, one-pot synthesis of CNDs having single molecular component and capable of dual emission is a hot topic of research. Liu and coworkers prepared dual-emission CNDs using one step hydrothermal method in alcoholwater binary solution with two emission peaks at 386 nm and 530 nm.21Song et al. developed a new photolunimescent CNDs with a one-pot hydrothermal carbonization method for the ratiometric detection of lysine and pH in cellular systems, with the blue emission peak at 440 nm and red emission at 624 nm.22 However, the origin of the tunable property and red emission are still unclear to date. Here, we have tried to get an insight into the mechanism of formation of these red emission CNDs. CNDs capable of dual emission (blue at 460 nm and red at 610 nm) have been synthesized with a simple and low cost electrochemical method by discharging indole dissolved in ethanol in the presence of alkali. These CNDs are thoroughly characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectroscopy (UV), photoluminescence spectroscopy (PL), high resolution transmission electron microscopy (HRTEM) and GCMS. This detailed spectroscopic and optical characterization of B-R-CNDs revealed that adding alkali in the discharging process played a pivotal role for synthesis of red emission CNDs, and 23



naphthaleneamine was an important intermediate in the formation of B-R-CNDs. UV and PL properties of CNDs synthesized by discharging 2-naphthaleneamine and naphthalene dissolved in ethanol in the presence and absence of alkali were compared favorably. Two factors were synergeticly responsible for the red emission: i) hydroxyl radicals produced in an alkali-ethanol system lead to surface oxidation of CNDs, which creates surface defects thereby giving rise to the surface-state-related red emission PL. ii) Precursor containing an extended π-conjugated system with a hetero atom (N and O) as a part, can bring heteroatoms containing groups to CNDs. Moreover, as prepared B-R-CNDs have been successfully used for full color bio-imaging of HepG2 cells, and exhibit an accurate mitochondrial-targeting ability. 2. EXPERIMENTAL SECTION 2.1. Materials. Platinum wire electrodes (Pt > 99.9%), ethanol (99.97%), sodium hydroxide (electronic grade) were purchased from Sigma-Aldrich (USA). Indole (analytical grade) was obtained from Aladdin (Shanghai, China). A dialysis membrane of 3500 Da Molecular Weight Cut Off (MWCO) was purchased from the Beijing Ruida Henghui Science & Technology Development Co. Ltd. Dimethyl sulfoxide (DMSO) was obtained from Dengfeng Co. Ltd (Tianjin, China). MitoTracker Deep Red FM (M22426) was supplied by Life Technologies Corporation. Ultrapure water was used throughout the experiments. 2.2. Instruments. The electrodes were ignited and maintained liquid ionization microplasma using a 35 kHz high-frequency discharge, which was generated from an HT-106B neon power supply (TDGC2-0.5KVA, Guangzhou Xinxing Neon Light Supply, China) at input voltages of 45-65 V of alternating current (AC), as shown in Figure S1. The two platinum wire electrodes were kept 2 cm apart in a beaker for all the experiments. The surface morphology and structural properties of CNDs were investigated using HRTEM (FEI, US, Tecnai G2). PL spectra were recorded by a fluorescence spectrophotometer (LS 55, Perkin 4


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Elmer, USA). UV spectra were recorded by UV-vis spectrophotometer (TU-1901, Persee, Beijing, China). FTIR spectra measurements were performed on a GX FTIR spectrometer (Perkin Elmer Co. USA). XPS was recorded by K-Alpha photoelectric spectrometer (Thermos-electric company, US). GCMS were performed on a QP2020 spectrometer. HepG2 cell images were observed with confocal laser scanning microscopy (Nikon A1). 2.3 Synthesis of carbon nanodots 2.3.1 Synthesis Scheme Scheme 1 illustrates the procedure of synthesis CNDs with single emission blue light (B-CNDs) and B-R-CNDs by a one-pot simple, cost effective and novel electrochemical method of discharging indole dissolved in ethanol in the absence and presence of alkali, respectively. Unlike traditional electrochemical methods in which graphite based electrodes are used as a carbon source to prepare CNDs, here, two platinum (Pt) wires were used as electrodes. Ethanol was used as solvent and indole was used as a carbon source. Scheme 1. Schematic illustration for the synthesis of B-CNDs and B-R-CNDs with electrochemical method.

Single channel Discharge by AC power In the absence of NaOH Dissolved in ethanol

λem=425 nm Double channel

NH

λem=460 nm In the presence of NaOH

λem=610 nm

i) Synthesis of B-CNDs. Indole (1320 mg) dissolved in ethanol (50 mL) was discharged by two Pt wires electrodes using an AC power supply for 2 h. The constant voltage mode was used 2200VAC. The change in color of the mixture from colorless to light yellow indicates the formation of B-CNDs. These B-CNDs emit blue light under UV 5



irradiation at 365 nm. These B-CNDs were dialyzed in ethanol with a molecular weight cutoff of 3500 Da in a dialysis bag for 48 h to remove redundant reagent and small molecules. ii) Synthesis of B-R-CNDs. A mixture of indole (1320 mg) and NaOH (450 mg) dissolved in ethanol (50 mL) was discharged by two Pt wires electrodes using an AC power supply for 3.5 h. The constant voltage mode was used 2200 VAC. The change in color of the mixture from colorless to orange indicates the formation of B-R-CNDs. These B-R-CNDs show dual emissions at 460 and 610 nm under UV irradiation at 365 nm and 532 nm, respectively. These B-R-CNDs were purified by dialysis with the same procedure of B-CNDs. 2.4 Cytotoxicity Assays and Cellular imaging The cytotoxicity of B-R-CNDs was assessed through MTT experiment. Typically, 100 µL of cells were seeded in a 96-well plate with a density of 1×105 cells per mL and allowed to adhere overnight. Then, the culture medium was discarded and then cells were treated with Dulbecco's Modified Eagle's Medium (DMEM), adding various concentrations of B-R-CNDs (2.5-50 µg/mL) for another 24 h. At the end of the incubation, the culture medium was removed, and 10 µL of MTT (5.0 mg/mL in PBS) was added into each well. After additional 3 h incubation, the DMEM was removed and 100 µL of DMSO was added into each well to dissolve MTT. Finally, the optical density of each sample was recorded using a microplate reader (Varioskan Flash) at a wavelength of 450 nm. For living cell imaging, HepG2 cells were seed on 18×18 mm glass coverslip mounted in custom-designed chambers at 37 oC. The as prepared B-R-CNDs were dispersed in DMSO with a concentration of 5 mg/mL and then was centrifuged to remove additional sediment. HepG2 cells were incubated with the dispersions of B-R-CNDs of 50 µg/mL for 16 h at 37 oC, and then washed twice with phosphate buffered solution (PBS) for removing the free B-R-CNDs. The HepG2 live cells were tracked by confocal laser scanning microscopy with laser excitation at 400 nm, 440 nm and 560 nm. The B-R-CNDs has also been used to monitor mitochondria. HepG2 live cells were incubated with 10 µL of 5 mg/mL B-R-CNDs for 12 h followed by washing with PBS. And then were incubated with 100 nM Mito Tracker Deep Red FM (a mitochondrial 6


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indicator) at 37 oC for 20 min. The cells were rinsed with PBS, and then imaged immediately by confocal laser scanning microscopy. 3. RESULTS AND DISCUSSION 3.1 Characterization of B-CNDs and B-R-CNDs The surface functional groups of as synthesized B-CNDs and B-R-CNDs were identified by FTIR spectroscopy, as shown in Figure 1a. Both B-CNDs and B-R-CNDs show similar characteristic absorption bands. The broad bands at 3360 cm-1 are attributed to stretching vibrations due to O-H.23 The bands at 2850-2980 cm-1, 1660 cm-1 , 13801060 cm-1 are due to stretching vibration of -CH2-, C=O and C-N/C-O, respectively.24-27 While the band at 669-882 cm-1 is due to bending vibration of C-H.28 Some other additional new peaks were observed in B-R-CNDs at 2530 cm-1, 2260 cm-1, 2140 cm-1 and 1920 cm-1 which can be attributed to the stretching vibration of -OH, -N=C=O, N=C=N and asymmetric stretching of C=O, respectively. These observations suggest that B-R-CNDs contain many C=O bonds, nitrogenous and oxygenous functional groups such as -N=C=O, N=C=N29 and electrochemical carbonization in alkaline medium can successfully bring oxygenous and nitrogenous functional groups to B-R-CNDs. The valence states of the elements within the B-CNDs and B-R-CNDs were analyzed by XPS, as shown in Figure 1 (b-h). Three peaks for both of CNDs appeared at approximately 284.08 eV, 397.08 eV and 532.08 eV, which were attributed to C 1s, N 1s and O 1s, respectively. It demonstrated that the two CNDs have the same elemental composition (i.e., C, N and O). The high resolution C 1s spectrum of B-CNDs (Figure 1c) could be deconvoluted into three peaks at 284.5 eV (C=C), 285.6 eV (C-N/C-O) and 288.1 eV (O-C=O).30 B-R-CNDs (Figure 1d) also have three similar peaks at 284.6 eV (C=C), 292.7 eV (O-C=O), and 287.7eV (C=O/C=N) (Figure 1d). The N 1s band of B-CNDs contain two peaks at 399.3 eV for pyridinic N and at 400.4 eV for pyrrolic N (Figure 1e), while N 1s band of B-R-CNDs (Figure 1f) reveals the presence of pyridine-like N (397.5 eV) and pyridinic N (399.5 eV).31-34 The O 1s spectrum of B-CNDs (Figure 1g) shows two peaks at 532.1 eV and 534.0 eV, which can be assigned to C=O and C-OH/C-O-C, 7



respectively. B-R-CNDs also have two peaks at 531.5 eV (C=O) and 532.9 eV (C-OH/CO-C), similar to O 1s spectrum of B-CNDs.35-36 Moreover, EDS element mapping was carried out for B-R-CND (Figure S2). This EDS element mapping clearly showed that C, N, O were involved in the B-R-CNDs. From the above results, it is obvious that nitrogen has been successfully doped into the two kinds of CNDs. The only difference is that nitrogen element is in a state of pyridine-like N in B-R-CNDs instead of pyrrolic N in BCNDs. Moreover, C-N/C-O bonds are present in B-CNDs while C=O/C=N in B-R-CNDs. The analysis also confirmed that CNDs contained large π-conjugated system in their structure and a large number of nitrogenous and oxygenous groups. UV-vis absorption and PL spectra of B-CNDs and B-R-CNDs were recorded to study the optical properties of the as prepared CNDs ( Figure 2a and 2d). The B-CNDs and BR-CNDs both show three absorption bands at the same position (217, 268 and 288 nm). But B-R-CNDs show two additional absorption bands at 522 and 575 nm. The first three bands in both B-CNDs and B-R-CNDs are related to the π-π* transition from C=C bond, C=N and n-π* from C=N, C=O and C-O.37-38 The appearance of the two new absorption bands (522 and 575 nm) in B-R-CNDs are indicative of π-π* transition in an extended conjugation system and a low energy n-π* transition due to nitrogen part of this conjugated system, respectively.39 Moreover, it is found that the absorption bands at 575 nm is perfectly consistent with the maximum excitation wavelength (λex) of B-R-CNDs at the emission wavelength (λem) of 610 nm, which suggests that the absorption band at 575 nm contribute to the red PL emission at 610 nm.

8


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(a)

C=O

O-H O-H

CH2

(b)

282

N=C=O N=C=N

C=C (284.5 eV) C-O/C-N (285.6 eV) C=O (288.1 eV)

284 286 288 Binding Energy (eV)

N1s

(d)

200 400 600 800 1000 1200 Binding Energy (eV)

290

C=C (284.6 eV) C=O/C=N (287.7 eV) O-C=O (292.7 eV)

B-R-CNDs C1s

282 284 286 288 290 292 294 296 Binding Energy (eV)

pyridine N (399.3 eV) pyrrolic N (400.4 eV)

(f)

B-R-CNDs N1s

pyridine-like N(397.5 eV) pyridinic N (399.5 eV)


B-CNDs O1s

528

404

C=O (532.1 eV) C-OH/C-O-C (534.0 eV)

530 532 534 536 538 Binding Energy (eV)

(h)

396

397 398 399 400 401 Binding Energy (eV)

402

B-R-CNDs

C=O (531.5 eV)

O1s

C-OH/C-O-C (532.9 eV)

Intensity (a.u.)

396 (g)

0

Intensity (a.u.)

B-CNDs N1s

O1s

C-N/C-O

Intensity (a.u.)

(e)

C1s

Intensity (a.u.)

Intensity (a.u.)

B-CNDs C1s

N1s

C-H

3200 2400 1600 800 Wavenumber (cm-1) (c)

C1s

C=O C=O

B-R-CNDs B-CNDs

O1s

Intensity (a.u.)

Transmittance

B-R-CNDs B-CNDs

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


540

528


540

Figure 1. The FTIR spectra (a), XPS spectra (b) and high resolution XPS spectra of C 1s (c, d), N 1s (e, f) and O 1s (g, h) of B-CNDs and B-R-CNDs. 9



The PL spectra of B-CNDs and B-R-CNDs are shown in Figure 2b and 2e. B-CNDs exhibited an excitation-independent emission peak centered at 425 nm. The emission peaks keep their position unchanged and its highest emission intensity is obtained when the λex = 380 nm. This B-CNDs sample was light yellow in daylight and emitted blue light under UV irradiation of 365 nm (insets of Figure 2b). The B-R-CNDs was orange in daylight and is capable of emitting blue light under UV irradiation at 365 nm, and emitted red light under visible light at 532 nm (insets of Figure 2e). When the λex varied from 340 to 400 nm, there were two emission peaks, one fixed at 460 nm, and the second centered at 610 nm. When the λex varied from 400 to 480 nm, the former λem varied with λex and shifted to the longer wavelength, while the later λem remained fixed at 610 nm with gradual decrease in PL intensity. When the λex varied from 500 to 580 nm, only one strong emission peak appeared at 610 nm with gradual increase in PL intensity. The B-R-CNDs had a satisfactory relative quantum yield of 13.75% (Figure S3) and absolute quantum yield of 22.61% (Figure S3) at the maximum λem at 610 nm. The influence of the discharge time on the PL intensity of B-CNDs and B-R-CNDs have also been investigated ( Figure S4 and Figure S5). Figure S4 revealed that the highest PL intensity has been obtained when the discharge time is up to 2 h for B-CNDs. While for B-R-CNDs (Figure S5), the PL intensity of red emission (610 nm) increase with discharge time, and reached a maximum at the discharge time of 3.5 h. So, 3.5 h sample was an optimized sample and used during the whole subsequent studies. The morphology and size of the as-synthesized B-CNDs and B-R-CNDs were investigated by HRTEM. These HRTEM images clearly revealed that B-R-CNDs were spherical and well monodispersed particles with an average size of about 2.32 nm (Figure 2f), while in case of B-CNDs the particles were of spheroid shape with bigger size of 17.30 nm (Figure 2c). It was also revealed that B-R-CNDs had a well-resolved lattice spacing of 0.21 nm (Figure S6f), and was attributed to the (100) lattice plane of graphite, while, BCNDs had amorphous structure without lattice (Figure S6c). It can be clearly seen that larger B-CNDs aggregate (~17.30 nm ) were composed of a few single smaller CNDs (~2 nm) (Figure S6g, h). Regarding PL properties of CNDs, the λem band of B-CNDs centred at 425 nm, and λem band of B-R-CNDs centred at 460 nm and 610 nm. Combining PL 10


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properties with HRTEM, we can infer if PL of CNDs would have originated from quantum size effects, the λem of B-R-CNDs would have shown a blue shift as compared with 425 nm because B-R-CNDs has a smaller size. However, B-R-CNDs have a λem band at 460 nm, i.e., a red shift as compared with 425 nm. So, we suppose that the PL emission from

217nm

1.0 0.8 0.6 0.4

268 nm

0.2

288nm

0.0 200

a) 3.0

Absorbance Ex

1.4

522 nm

1.2

578 nm

240

575 nm

1.0

120

0.8 0.6

1.0

480

560

0

640

Wavelength (nm)

0.5

d)

350

PL Intensity (a.u.)

Absorbance

217 nm

1.5

0.0

300

Wavelength (nm)

2.5 2.0

250

268 nm 288 nm

200

250

300

350

400

450

Wavelength (nm)

150

17.30  4.04 nm

20 15 10 5 0

9 12 15 18 21 24 27 30 Size (nm)

50 0

350

400

450

500

550

600

Wavelength (nm)

600 450

c)

200 nm

340nm 360nm 380nm 400nm 480nn 500nm 520nm 540nm 560nm 580nm

750

16

2.32  0.54 nm

12 8 4 0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Size (nm)

300 150 0

e)

25

100

b)

500

300nm 320nm 340nm 380nm 400nm

200

Precentage (%)

Absorbance

1.2

PL Intensity (a.u.)

1.4

Precentage (%)

PL Intensity (a.u.)

B-R-CNDs does not originate from the quantum confinement effects.

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


400 450 500 550 600 650 700

50 nm

f)

Wavelength (nm) Figure 2. UV-vis absorption spectra, PL emission spectra and HRTEM images of B-CNDs

(a, b, c) and B-R-CNDs (d, e, f). Inset in ‘b’ shows photographs of B-CNDs in daylight (left) and irradiated by UV light at 365nm (right). Inset in ‘c’ shows size distribution of BCNDs. Inset in ‘e’ shows photographs of B-R-CNDs in daylight (left), irradiated by UV light at 365 nm (middle) and irradiated by visible light at 532 nm (right). Inset in ‘d’ shows the absorption spectrum and the excitation spectrum of the B-R-CNDs at the emission wavelength of 610 nm. Inset in ‘f’ shows size distribution of B-R-CNDs. 3.2 Origin of red emission Possible mid-product or intermediate

In order to have an insight into the origin

of red emission, GCMS studies were carried out (Section II in Supporting information). Figure S7 shows GCMS of mid-products of the reaction I from which B-CNDs were 11



obtained. According to the position of the peak, retention time of 6.690 min, 10.800 min and 12.085 min were assigned to 2-ethyl-1-hexanol, indole and 3-methylindole, respectively ( Table S1). Figure S8 shows GCMS of the mid-products of the reaction II from which B-R-CNDs were obtained. The retention time at 10.810 min, 12.080 min and 14.345 min were attributed to the indole, 3-methylindole and 2-naphthalenamine, respectively ( Table S2). Comparison of the mid-products obtained in these two reactions evidenced that 2-naphthalenamine is the mid product which only appears in reaction II. Based on these results, we can hypothesize that 2-naphthalenamine (an intermediate in reaction II) can give rise to final products capable of red emission. Optical properties of the CNDs obtained from 2-naphthalenamine and naphthalene

In order to corroborate our hypothesis, 2-naphthalenamine was used as

precursor to obtain CNDs. When 2-naphthalenamine was discharged in ethanol without NaOH, the obtained CNDs only have an absorption band at 223, 288, and 348 nm (Figure 3a). However, when 2-naphthalenamine was discharged in ethanol with NaOH, the obtained CNDs have absorption band not only in the ultraviolet light range (237, 275, 340 nm) but also in the visible light range (475 nm) (Figure 3b). The new emerging absorption bands were mostly contributed by the extended π-conjugated system.39 When 2naphthalenamine was discharged in ethanol without alkali, coincidentally, the obtained CNDs showed dual emission properties (425 nm and 460 nm) and exhibited excitationdependence PL. More importantly, addition of alkali in discharge process also caused a red shift in the second emission band (from 462 nm to 591 nm). The second emission band at 591 nm is near 610 nm which was appeared in B-R-CNDs. So, we can infer that the red emission of B-R-CNDs might be associated with nitrogen containing amino group of 2naphthalenamine as an intermediate of B-R-CNDs.

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0.2

348 nm

2.0 1.5

1.2 0.8 0.4

1.0 0.5

340 nm

0.0 300

275 nm

475 nm

400 500 600 Wavelength (nm)

720 425 nm

340 nm

600 480 360 240 120

0.0 0.0 200 250 300 350 400 450 500 200 250 300 350 400 450 500 Wavelength (nm) Wavelength (nm)

239 nm

0.1

308 nm

1.5 1.0 0.5

0.0 300 400 Wavelength (nm)

500

385 nm

450 300 150

275 nm 200


600

220 nm PL Intensity (a.u.)

0.2

2.0 Absorbance

0.3

208 nm 216 nm

0 300

d) in the presence of NaOH

280 nm 300 nm 320 nm 340 nm 360 nm 380 nm 400 nm 420 nm 440 nm 460 nm 480 nm

0.0 200 250 300 350 400 450 500 Wavelength (nm)

700

350


320 nm 340 nm 360 nm 380 nm 400 nm 440 nm 460 nm 480 nm

428 nm

120 80 40

591 nm 400 450 500 550 600 650 700 Wavelength (nm)

h) in the presence of NaOH

240 nm 250 nm 260 nm 270 nm 280 nm 290 nm 300 nm 310 nm 320 nm 330 nm

500

0 300

160

0

g) in the absence of NaOH

f) in the presence of NaOH

e) in the absence of NaOH

460 nm

PL Intensity (a.u.)

288 nm

Absorbance

Absorbance

0.6

237 nm

PL Intensity (a.u.)

2.5

Absorbance

223 nm

0.4

c) in the absence of NaOH

b) in the presence of NaOH

a) in the absence of NaOH

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PL Intensity (a.u.)

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550

393 nm 414 nm

400 300 200

R

100 0

260 nm 280 nm 300 nm 320 nm 340 nm 360 nm 380 nm 400 nm 420 nm

350 400 450 500 550 600 650 700 Wavelength (nm)

Figure 3. UV-vis absorption spectra and PL emission spectra of the CNDs obtained from discharging of 2-naphthalenamine (a, b, c, d) and naphthalene (e, f, g, h) in ethanol in the absence and presence of alkali. Inset in ‘a’, ‘e’ and ‘h’ show photographs of CNDs in daylight (left) and irradiated by UV light at 365 nm (right). Inset in ‘d’ show photographs of CNDs in daylight (left), irradiated by UV light at 365 nm (middle) and irradiated by visible light at 532 nm (right). To verify the role of amino group of 2-naphthaleneamine in the formation of B-RCNDs, CNDs using naphthalene as precursor were also synthesized. When naphthalene was discharged in ethanol without alkali, the absorption spectrum of the synthesized CNDs (Figure 3e) had three absorption peaks at 208 nm, 216 nm and 239 nm which were attributed to π-π* transition of C=C.37-38 A shoulder peak appeared at 308 nm which belonged to n-π* transition of C=O. These CNDs were light yellow under daylight and emitted blue light under UV irradiation at 365 nm (inset of Figure 3e). These CNDs exhibited excitation-independent PL properties with a maximum λem at 385 nm (Figure 3g), which was a blue emission compared with that of CNDs obtained from 2naphthalenamine. Moreover, these CNDs obtained from naphthalene did not exhibit dual emission properties. However, discharging of naphthalene in the presence of alkali resulted in CNDs which showed absorption peaks at 220 nm and 275 nm ( Figure 3f). The 13



PL properties of these CNDs are also different from the aforementioned CNDs obtained without alkali (Figure 3h). The maximum λem has shifted slightly from 385 nm to 393 nm, and a new shoulder emission band has also appeared at 414 nm. Moreover, its PL exhibited excitation-dependent properties. It can be seen that the presence of alkali during the discharge process gave rise to an obvious change in the optical properties of the CNDs, suggesting the change of the structure or surface state of the CNDs. Literature also reveals the potential of NaOH/EtOH system for generating hydroxyl radical during the discharging process which has high oxidation ability.40 Generation of hydroxyl radical from NaOH/EtOH system (during the discharge process) can be evidenced by the fact that the color of the methylene blue fades in presence of this alkaline ethanol system ( Figure S9).41 Thus, it can be concluded that NaOH/EtOH system can lead to surface oxidation of the CNDs, and surface defects created by surface oxidation serve as capturing centers of excitons, thereby giving rise to the surface-state-related PL.42-43 Oxidation by NaOH/EtOH discussed above was very similar to HNO3-oxidation43or electrochemical oxidation previously used to prepare CNDs.44 These experiments showed that the higher the surface oxidation of the CNDs, the more surface defects were present on the CNDs. The more the surface defects, the narrower was the energy gap of the surface state, resulting in a red shift in emission wavelength. From the comparison of the optical properties of CNDs obtained from 2-naphthalenamine and naphthalene, we can infer that addition of alkali during the discharge process might lead to a significant change in optical properties, especially a red-shift in PL, no mater what the carbon source is. However, only the presence of alkali during discharging of carbon source is not enough to produce red emission CNDs. Presence of amino group in the carbon source and then discharging in alkali/EtOH is necessary for producing red emission CNDs. That’s why discharging of naphthalene, which has the same benzene ring structure did not give rise to red emission CNDs but that of 2-naphthalenemine resulted in CNDs capable of red emission. That means amino group played a crucial role in the formation of dual emission CNDs. We speculate that amino group containing structure help to bring an extended conjugated π-electron into the system45, and this extended π-electron system can strongly couple with surface electronic states created by the surface oxidation, consequently 14


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altering the overall electronic structure of the CNDs.

The changes in the surface

chemistry and π-electron system of the CNDs both have significant influence on the energy gaps, leading to narrowing of the energy levels. Such narrowing of energy gaps consequently causes the red-shifted PL emission of the CNDs.21 In short, we can infer that two factors were synergeticly responsible for the red emission: i) hydroxyl radicals produced in an alkali/EtOH system lead to surface oxidation of CNDs, which creates surface defects thereby giving rise to the surface-state-related red emission PL and ii) precursor containing an extended π-conjugated system with a hetero atom (N and O) as a part, can bring heteroatoms containing groups to CNDs. 3.3 pH sensing The B-R-CNDs were found sensitive to the pH of the medium. Figure 4 shows photographs of color change of B-R-CNDs before and after adding HCl and NaOH under λex = 532 nm. In strong alkaline medium, B-R-CNDs emitted red light. But when HCl (strong acidic conditions) was added to them, the emission of red light disappeared. When NaOH (strong alkali) was added again, these B-R-CNDs again emitted red light. +HCl

+NaOH

(a)

(b)

(c)

Figure 4. Photographs of color change of B-R-CNDs before (a) and after adding HCl (b), and NaOH (c) under λex = 532 nm visible light irradiation.

15



a)

b)

250 200 150 100 50

700 600 500 400 300 200 100

800

PL Intensity (a.u.)

13.9 13.1 12.0 11.0 10.0 9.0 8.0

PL Intensity (a.u.)

300 PL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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600 400 200 0 10

11

12 pH

13

14

13.9 13.1 12.0 11.0 10.0 9.0

0

0 450 500 550 600 650 600 620 640 660 680 700 720 Wavelength (nm) Wavelength (nm) Figure 5. PL spectra of B-R-CNDs at different pH value when λex = 400 nm (a) and λex = 560 nm (b). Figure 5 shows the dependence of PL of B-R-CNDs on pH by adding NaOH or HCl. Under strong acidic conditions, the intensity of red emission (λem = 610 nm) was very weak. The more alkaline the medium, the higher the PL intensity of red emission (λem = 610 nm). When the pH was increased from 10 to 13.9, the PL intensity increased linearly. However, the PL emission of blue light was less affected by pH values from 8 to 13.9. That means the alkaline medium was responsible for red emission. This pH-dependent emission properties may be associated with the protonation and deprotonation of surface functional groups of CNDs.21 This pH dependent emission properties makes them a potential candidate for application in pH sensing, especially for extreme alkali condition, eg. Bacillus subtilis.46 The reason for the red emission in B-R-CNDs can be attributed to the presence of the extended conjugation system in the structure of B-R-CNDs. The equilibrium in equation 1 shifts to right at lower pH. The lone pair of electrons of hetro atom (nitrogen or oxygen) in the B-R-CNDs structures (see Figure 6) does not show any conjugation with π-electron system at acidic pH because of protonation of surface groups at lower pH. Hence conjugation is somewhat decreased showing no red emission at low pH. But at the high pH, the equilibrium shifts to the left. Nitrogen or oxygen of surface functional groups are no more protonated and lone pair of electrons is involved in conjugation with π-electron 16


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system, shifting the emission wavelength to the red. ∅ + H2O

∅H + + OH-

(1)

Where ‘Ø’ is extended π conjugation system containing nitrogen and oxygen fluorophores and surface functional group, as shown in Figure 6.

Figure 6. Schematic diagram of extended π conjugation system containing nitrogen and oxygen fluorophores and surface functional group. 3.4 Cell Imaging and mitochondria position targeting The cytotoxicity of the B-R- CNDs was evaluated by using a standard MTT assay46 with HepG2 cells. Figure S10 shows the cytotoxicity studies of B-R-CNDs. Over 85% cell viability was observed after incubation of HepG2 with B-R-CNDs at concentrations ranging from 2.5 to 50 µg/mL for 24 hours, thus confirming the low cytotoxicity of B-RCNDs. High brightness, good stability and low toxic behavior of CNDs make them a promising material for biological applications. To assess the prospects of B-R-CNDs as bioimaging materials, practical imaging application of B-R-CNDs in living cells was tested by using confocal laser scanning microscopy. HepG2 is a cell line derived from the liver tissue of a patient with hepatocellular carcinoma, and here these were used to explore the potential of B-R-CNDs as a bioimaging agent. The specific labeling of live cell 17



membranes (HepG2 human liver cancer cells) indicated the successful bioconjugation of B-R-CNDs with a bio-anchored membrane. The cellular uptake of B-R-CNDs by HepG2 live cells was tracked by confocal microscopy with laser excitation at 400, 440 and 560 nm, as shown in Figure 7. After incubation with B-R-CNDs, the cells showed an obvious bright blue, green and red

emission at the test excitation conditions (Figure 7a, b, c).

These results clearly confirmed that the B-R-CNDs could readily penetrate into the cell membrane and provided full-color (blue, green, red) emission characteristic in a cellular environment.

Figure 7. Confocal laser scanning microscopy (CLSM) images of HepG2 cells cultured with B-R-CNDs at 37 °C for 24 h at λex = 400, 440 and 560 nm, respectively (a, b, c) and overlay of the blue, green and red channels (d). scale bar：10 µm. Furthermore, in order to confirm whether B-R-CNDs could be used to image mitochondria in the cytoplasm region, a commercial probe for mitochondria, Mito-Tracker deep red (100 nM), was used as indicators and compare to evaluate the sensitivity of B-RCNDs. The purple PL images (Figure 8c) stained by Mito-Tracker deep red matched well with the blue photoluminescence images stained by B-R-CNDs (Figure 8b),and the 18


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colocalization coefficient was assessed by Pearson’s correlation factor (Rr) with Rr = 0.962 (Figure 8e). The merged images and the correlation mapping of the PL intensities showed a good colocalization of B-CNDs and Mito Tracker, indicating that B-R-CNDs was predominantly accumulated in mitochondria.

Figure 8. PL images of B-R-CNDs in HepG2 cells costained with Mito-Tracker deep red (100 nM): (a) bright field image, (b) blue emission from B-R-CNDs in DMSO at λex = 400 nm , (c) purple emission from Mito-Tracker red at λex = 640 nm , (d) overlay of the blue and purple channels, (e) colocalization analysis, (f) intensity profile of region cross costain image. scale bar:10 µm. CONCLUSION A simple and effective electrochemical strategy was used to prepare dual emission CNDs based on the precursor indole dissolved in ethanol. Here, alkali-ethanol system played a crucial role in structural transition from single emission to dual emission. And hydroxyl radical generated by alkali-ethanol system lead to surface oxidation, which creates surface defects thereby giving rise to the surface-state-related red emission. The carbon sources that can bring an extended π-conjugated system and heteroatom functional group (C=O, C=N, -OH and pyridine-like N) to the CNDs are synergistically responsible 19



for emission at longer wavelength. The facile preparation and unique optical features make the B-R-CNDs potentially useful in numerous applications such as optoelectronic applications and multiplexed PL bioimaging. Importantly, the B-R-CNDs exhibit ensemble emissions as full-color cell imaging in HepG2 living cells and are successfully applied for locating mitochondria in HepG2 living cells. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures and analytical data (PDF) AUTHOR INFORMATION Corresponding Author *[email protected]; *[email protected]; *[email protected] ORCID Lihua Shen: 0000-0001-8882-8985 Chengxiao Zhang: 0000-0003-2829-5122 Chenxing Wang：0000-0001-8914-7217 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported financially by the National Natural Science Foundation of China (No. 21505102, 21621003, 21475082, 21390410). Thanks to Mr. Zhang Hu from Xi'an University of Technology for provision of TEM analyses. REFERENCES (1) Zhao, A.; Chen, Z.; Zhao, C.; Gao, N.; Ren, J.; Qu, X. Recent Advances in Bioapplications of C-dots. Carbon 2015, 85, 309−327. (2) Miao, P.; Han, K.; Tang, Y.; Wang, B.; Lin, T.; Cheng, W. Recent Advances in Carbon 20


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TOC graphic

＋Ethanol NH

Single channel

Bio-imaging

Discharge λem=425 nm Double channel

Mitochondrial localization

λem=460 nm ＋NaOH

λem=610 nm

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Switching Carbon Nanodots from Single Emission ... - ACS Publications

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