Carbon dots isolated from chromatographic fractions for sensing applications

Lizhen Liu ab, Feng Feng*ab, Man Chin Paauc, Qin Huc, Yang Liuc, Zezhong Chenb and Martin M. F. Choi§ *c
aSchool of Chemistry and Materials Science, Shanxi Normal University, Linfen 041004, China. E-mail: feng-feng64@263.net; Fax: +86-352-6100028; Tel: +86-352-7157968
bCollege of Chemistry and Chemical Engineering, Shanxi Datong University, Datong 037009, China
cPartner State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, China. E-mail: mmfchoi@gmail.com; Fax: +852-34117348

Received 16th October 2015 , Accepted 7th December 2015

First published on 9th December 2015


Abstract

A fast and easy approach to synthesise carbon dots (C-dots) by simply mixing acetic acid, N-acetyl-L-cysteine (NAC), water, and diphosphorus pentoxide has been developed. The synthesised C-dots sample has been found to be a relatively complex mixture, and its complexity can be reduced significantly by high-performance liquid chromatography (HPLC). The separated C-dots fractions are collected and characterised by UV-vis absorption spectroscopy, photoluminescence (PL) spectroscopy, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy. The C-dots fractions display unique absorption bands and specific emission wavelengths which are fully anatomised by MALDI-TOF MS, displaying their fragmentation mass ion features. The core sizes of some selected C-dots are 2.51, 2.83, 2.90, 3.17, 3.39, and 3.44 nm, consistent with their HPLC elution order. The fractionated C-dots show profound differences in emission quantum yield, allowing the brighter C-dots to be isolated from an apparent low quantum yield mixture. These brighter C-dots fractions can be used as fluorescent probes for sensitive detection of Fe3+ and Hg2+.


Introduction

In recent years, carbon dots (C-dots), which are small carbon nanoparticles (C-NPs) less than 10 nm in size, have attracted broad attention from both academia and industry because of their outstanding advantages such as stable photoluminescence (PL) properties, robust chemical inertness, biocompatibility, versatile surface chemistry and low toxicity.1–4 Until now, various C-dots have been prepared from graphite,5 multiwalled carbon nanotubes,6 activated carbon,7 graphene,8 carbon soot,9 natural plants,10 and molecular precursors11–16 via a variety of methods including laser ablation,5,17,18 electrochemical oxidation,6,19,20 pyrolysis,21,22 microwave treatment,23–25 and self-catalysis.26 The application potential of C-dots is huge, for example, in bioimaging,18,27,28 sensing,29,30 photocatalysis,31,32 optoelectronics,12,33 and drug delivery.34–37 Compared to highly toxic organic dyes and heavy metal-based quantum dots, C-dots are a promising alternative in biomedical and other applications. While the typical PL properties of C-dots play a dominant role in rendering their viability in a number of applications, the origins of C-dots luminescence are still under investigation. The role of surface functionality on C-dots appears to have a strong influence on their PL properties and their emissions are believed to be due to fluorescence resonance energy transfer,26 and surface energy trap states or bright edge states.1,38

Because of the intense interest in the synthesis, applications, and fundamental studies of C-dots, there is a need for analytical methodology that can fractionate these materials. This can enhance the applications of the fractionated C-dots, promote further studies and shed light on their unexplained fundamental properties. Typically, other constituents and inhomogeneity of the surface-attached functionalities and sizes may be present in an as-synthesised product. We have been interested in developing fractionation methods to investigate C-dots synthesised with a typical bottom-up and wet-chemical approach, facilitating more precise studies of the individual C-dots species in an as-synthesised C-dots product. Herein, a self-promoted and self-controlled method to synthesise C-dots with NAC as N and S dopant,39 HAc as carbon source and diphosphorus pentoxide as dehydrating agent26,40 is adopted since it allows for straightforward production of a mixture of C-dots containing doped C-dots or undoped C-dots and small-sized nanodomains of graphitic carbon. The aim of this work is to separate and collect various C-dots fractions from a complex mixture of C-dots. The collected C-dots fractions are further characterised and applied to fluorescence detection.

Gel electrophoresis,41 capillary electrophoresis (CE),42–44 and anion-exchange high-performance liquid chromatography (AE-HPLC)45,46 have been applied to separate and fractionate C-NPs. Gel electrophoresis benefits in identifying the relationship between the mobility and colour of the fluorescent C-NPs but the separation efficiency is low. CE achieves the separation of C-NPs with satisfactory resolution; unfortunately, it is difficult to collect the sample due to the low sample injection quantity. AE-HPLC can achieve high peak resolution and its preparative scale property allows for multiple-collection of individual fractions of C-NPs; however, it requires relatively expensive ion-exchange column and ammonium acetate or carbonate as the eluent. The separation largely depends on the pH of eluent and the search of the optimal separating condition is time-consuming. In addition, AE-HPLC only separates charged components but not the neutral C-NPs entities. Reversed-phase (RP)-HPLC is a better analytical separation technique in dealing with a complex mixture of neutral and charged C-NPs. Compared with AE-HPLC, RP-HPLC column possesses higher separation efficiency and is less expensive. Thus, it has rapidly emerged as one of the most powerful approaches for high-efficient separation of C-NPs.39,40,47,48 However, to our knowledge, there is not much work on applying this technique to select and harvest the brighter C-dots fractions for sensing applications.

In this article, we for the first time report the application of RP-HPLC for efficient separation and isolation of C-dots fractions for further characterisations and sensing applications. The separated C-dots fractions provide valuable insight into the complexity and contents of a given C-dots batch. Coupling with other appropriate spectroscopic techniques, one can assess the photophysical properties of the individual fractionated C-dots species with respect to their size and morphology. More importantly, the absorption and PL characteristics and emission quantum yield (ΦS) of each C-dots species can be more precisely and accurately acquired. The high ΦS C-dots fractions were collected and used as fluorescent probes for sensitive detection of Fe3+ and Hg2+. Our proposed HPLC technology could open up new initiatives on extensive studies of individual C-dots species in the biomedical, catalysis, electronic and optical device, energy storage, material and sensing fields. It can also provide a methodology to select and harvest the most fluorescent C-dots fractions for applications in sensors, bioimaging and optoelectronics.

Experimental

Materials

Diphosphorus pentoxide (P2O5) and 2,5-dihydroxybenzoic acid (DHB, 98%) were obtained from Sigma (St. Louis, MO, USA). Glacial acetic acid (HAc) was from Fisher Chemicals (Fair Lawn, NJ, USA). N-Acetyl-L-cysteine (NAC, 99%) was obtained from International Laboratory (San Bruno, CA, USA). Potassium bromide (KBr) was purchased from Aldrich (Milwaukee, WI, USA). Methanol (MeOH) was from Labscan (Bangkok, Thailand). Fe(NO3)3, Ca(NO3)2, MnCl2, Al(NO3)3, Ba(NO3)2, AgNO3, Pb(NO3)2, HgCl2, Co(NO3)2, Cu(NO3)2, Zn(NO3)2, Ni(NO3)2, Cr(NO3)2, Cd(NO3)2, KNO3, NaNO3, and Mg(NO3)2 were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Distilled deionised (DDI) water was obtained from a Millipore Milli-Q-RO4 water purification system with a resistivity higher than 18 MΩ cm−1 (Bedford, MA, USA). All reagents of analytical reagent grade or above were used as received without further purification.

Synthesis of C-dots sample

The synthesis of C-dots is self-promoted and self-controlled by the chemical and physical properties of the reactants without any external treatment. Various amounts of NAC (0.00–0.080 g) were dissolved in a solution of 1.0 mL HAc and 80 μL DDI water and sonicated in a water bath for 30 min. The obtained homogeneous mixture was quickly added to 2.5 g P2O5 in a 25 mL beaker without stirring. The synthesis was carried out in a fumehood to prevent inhalation of acetic acid vapour. After the reaction, the beaker was cooled down within 10 min to obtain the dark brown solid. The crude product was then dispersed in water and purified by dialysis with a Spectrum Laboratories cellulose ester dialysis membrane tube (molecular weight cut-off 1000 Da) (Rancho Dominguez, CA, USA) in DDI water with stirring and recharging with fresh DDI water every 24 h for 7 days. Finally, the product from the dialysis membrane tube was centrifuged at 8000 rpm for 10 min to remove the supernatant. The dark brown deposits were collected and freeze-dried to obtain the C-dots product. In our preliminary work, the use of higher amounts of NAC (>80 mg) for synthesis of C-dots is not viable as NAC does not completely dissolve in a solution of 1.0 mL HAc and 80 μL DDI water. As such, C-dots samples synthesised with NAC > 0.08 g are not investigated in this study.

Characterisation

The UV-vis absorption spectra of the as-prepared C-dots product and the collected HPLC C-dots fractions were acquired with a Varian Cary 300 scan UV-vis absorption spectrophotometer (Palo Alto, CA, USA) at 200–700 nm. The PL spectra of the as-prepared C-dots product and the collected HPLC C-dots fractions were determined on a Photon Technology International QM4 spectrofluorometer equipped with a photomultiplier tube detector (Birmingham, NJ, USA).

The Fourier transform infrared (FTIR) spectrum of the as-prepared C-dots product was obtained from a Nicolet Magna 550 FTIR spectrometer (Thermo Scientific, West Palm Beach, FL, USA) with the KBr pellet technique ranging 500–4000 cm−1. The C-dots/KBr disk was obtained by mixing few milligrams of C-dots with ca. 100 mg KBr powder, grinding to approximately 2 μm in an agate mortar, and subjecting to 29.7 MPa pressure by a pressing machine.

The X-ray photoelectron spectra (XPS) of the as-prepared C-dots product were acquired on a Leybold Heraeus SKL-12 X-ray photoelectron spectrometer (Shenyang, China). Spectra were processed by the Casa XPS v.2.3.12 software using a peak-fitting routine with symmetrical Gaussian–Lorentzian functions.

Elemental analyses (C, H, O, N and S) were carried out on an Elementar Analysensysteme vario EL cube elemental analyser (Hanau, Germany), and phosphorus (P) was determined by a Thermo Scientific iCAP 6300 inductively coupled plasma-optical emission spectrometer (West Palm Beach, FL, USA). Analyses were performed in triplicate, and the average values were obtained.

Reversed-phase high-performance liquid chromatography

Chromatographic separation was conducted on a Waters (Milford, MA, USA) instrument comprising a 2695 separations module capable of gradient elution and a 2475 multi-wavelength fluorescence detector (FD). Fluorescence chromatogram was obtained at excitation/emission wavelengths (λex/λem) of 300/450 nm. An Agilent TC-C8 chromatographic column (4.6 mm i.d. × 150 mm) packed with 5 μm octyl-bonded silica (C8) particles (130 Å pore size) (Santa Clara, CA, USA) was used for separation of C-dots. 10 mg C-dots were dissolved in 10 mL methanol and the C-dots solution was pre-filtered through an Alltech 0.2 μm cellulose acetate membrane syringe filter (Deerfield, IL, USA) before injections.

The mobile phase containing MeOH and Milli-Q water was filtered through 0.45 μm cellulose acetate membrane filters (Alltech) prior to use. The injection volume was 10 μL and the column temperature was maintained at 25 °C. A gradient elution programme was applied at a flow rate of 0.80 mL min−1 as follows: 45% v/v MeOH from 0.0 to 10 min, linearly increased to 50% v/v MeOH from 10 to 20 min, then linearly increased to 80% v/v MeOH from 20 to 50 min and finally linearly increased to 100% v/v MeOH from 50 to 60 min. The selected HPLC fractions were collected manually based on the appropriate fluorescence signal threshold. The collected HPLC C-dots fractions were purged and pre-concentrated by a stream of nitrogen (N2) at room temperature prior to UV-vis absorption, PL, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometric (MALDI-TOF MS), transmission electron microscopic (TEM) measurements and sensing applications.

Mass spectrometry

The concentrated HPLC C-dots fractions were analysed by a Bruker Autoflex MALDI-TOF mass spectrometer (Bremen, Germany). Each fraction was mixed with a 1.0 M solution of DHB in MeOH/water (1[thin space (1/6-em)]:[thin space (1/6-em)]1 v/v), respectively. Then 4.0 μL of this mixture was deposited on a MALDI target plate and air-dried. The sample was irradiated by a pulsed N2 laser working at 337 nm (3 ns pulse, 3 Hz). In general, 30 laser shots were averaged for each spectrum.

Transmission electron microscopy

The TEM images of the selected concentrated HPLC fractions were obtained with a JEOL JEM-1011 transmission electron microscope (Tokyo, Japan) operating at 200 kV. Sample was prepared by casting and evaporating a droplet of solution of HPLC fraction onto Agar Scientific 400 mesh copper grids (Stansted, Essex, UK). Particle size was determined by the ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA).

Fluorescent sensors for Fe3+ and Hg2+ detection

Briefly, 10 μL of HPLC C-dots fraction solution or as-synthesised C-dots solution was added to 2.0 mL DDI water, followed by the addition of different amounts of Fe3+/Hg2+ ions. The mixture was shaken thoroughly at room temperature prior to fluorescence measurement. The selectivity for Fe3+/Hg2+ was confirmed by adding other metal ions solutions instead of Fe3+/Hg2+ ions in a similar way. All fluorescence measurements were performed at room temperature under ambient conditions. All the fluorescence intensities were an average of three independent measurements.

Results and discussion

Characterisation of the as-synthesised C-dots

Six types of C-dots were synthesised using various NAC/HAC: 0.00, 0.005, 0.007, 0.010, 0.015 and 0.025. Among them, the C-dots synthesised with NAC/HAC of 0.025 produces the strongest fluorescence. As such, it was chosen for further characterisation and studies.

In quest of exploring the optical properties of the as-prepared C-dots, the UV-vis absorption and PL spectra are acquired. Fig. S1 displays the UV-vis absorption spectrum of the as-prepared C-dots. It comprises an absorption peak at 210 nm, a shoulder peak at 250 nm and a small shoulder peak at 300 nm corresponding to the π → π* transition of the aromatic sp2 domain,49,50 the multiple polyaromatic chromophores51–54 and the n → π* transitions of C[double bond, length as m-dash]O,49,52,54 respectively. The C-dots sample depicts a small λem peak at 330 nm and a strong λem peak at 480 upon λex at 300 nm. The inset in Fig. S1 depicts the photographic images of the C-dots solution under daylight (right) and UV irradiation (365 nm, left). The C-dots is a clear transparent yellow solution, exhibiting a strong yellowish green colour under a hand-held UV lamp. The ΦS of the as-prepared C-dots is 4.65% (Fig. S2 and ESI). Fig. S3 displays the PL spectra of C-dots under various λex. It is observed that C-dots show both λex-independent and λex-dependent emission behaviour. The λem is 480 nm at λex 250–320 nm. The λem is red-shifted from 457 to 550 nm when the λex moves from 340 to 500 nm. The λex-dependent PL behaviour is common with other C-dots.55–62 This behaviour is possibly attributed to the optical selection of different-size nanoparticles (quantum effect) and different emissive energy trap sites on the surface of C-dots.63,64 The dual emission bands are found at λex 250–330 nm. The formation of dual emission bands is ascribed to the zigzag effect (π → π* transition at the shorter wavelength region) and interstate to band transition (n → π at the longer wavelength region).65

IR spectroscopy is a useful technique to characterise the surface functionality of C-dots. Fig. S4 depicts the IR spectrum of the as-prepared C-dots, showing a broad absorption band around 3432 cm−1 corresponding to the O–H stretching of the surface-attached carboxylic acid group. The peaks at 2931 and 2846 cm−1 correspond to the C–H stretching vibrations. The C-dots exhibits other characteristic absorption bands at 1716, 1656 and 1400 cm−1, corresponding to the C[double bond, length as m-dash]O, C[double bond, length as m-dash]C stretching vibrations, and –CH3 umbrella bend, respectively.26 In addition, the peak at 1160, 1085 and 960 cm−1 are related to the vibration of P[double bond, length as m-dash]O, P–O–C and P–O–H, respectively,66 indicating that C-dots contains a surface attached phosphate moiety. In summary, the as-prepared C-dots comprises sp3 (C–C), sp2 (C[double bond, length as m-dash]C), sp2 (C[double bond, length as m-dash]O) carbon atoms and phosphocarbonaceous moieties.

To gain further insight into the surface functional groups and element states of C-dots, XPS of C-dots were acquired. Fig. S5A depicts the survey scan of the as-synthesised C-dots. Six peaks centred at 534, 401, 287, 230, 166, and 135 eV, associated with O1s, N1s, C1s, S2s, S2p, and P2p are observed, inferring the presence of C, O, P, N, and S in the as-synthesised C-dots. These results further confirm the incorporation of heteroatoms N and S from NAC into C-dots. But the intensity of peak associated with N1s, S2s and S2p are much weaker, confirming that the C-dots are slightly doped by N and S. Fig. S5B and C display the C1s and O1s XPS spectra of C-dots. The C1s XPS spectrum can be deconvoluted into five peaks at 284.2, 286.1, 286.8, 288.4, and 290.8 eV corresponding to C[double bond, length as m-dash]C, C–O, C–OH, C–O–C, and O[double bond, length as m-dash]C–OH, respectively.5,67,68 The O1s XPS spectrum is fitted into three peaks at 532.1, 533.7 and 535.3 eV associated with C[double bond, length as m-dash]O, C–O–C and O[double bond, length as m-dash]C–OH, respectively.67,69,70 Fig. S5D and E display the N1s and S2p XPS spectra of C-dots. The N1s XPS spectrum can be deconvoluted into three peaks at 399.7, 401.5 and 403.4 eV corresponding to C–N–C, N–H and N–O, respectively,67,71,72 The S2p XPS spectrum is fitted into two peaks at 165.6 and 169.3 eV associated with C–S and S–O, respectively.67,73,74 Fig. S5F reveals the P2p XPS spectra of C-dots. A deconvoluted P2p XPS spectrum shows a peak at 135.6 eV for PO43−. In summary, the XPS data show the presence of C[double bond, length as m-dash]C, C–O, C[double bond, length as m-dash]O, C–OH, COOH and PO43− surface-functionalities on the C-dots which concur with the IR data. In addition, the XPS analysis confirms the presence of trace N and S in the C-dots.

RP-HPLC separation of the as-synthesised C-dots

It has been reported that the surface-doped N and S atoms on C-dots are the main factors for enhancing the PL of C-dots.75–77 As such, a series of C-dots samples were synthesised with different mole ratios of NAC/HAc as the initial reagents. These C-dots samples were applied to HPLC-FD. Fig. 1A depicts the fluorescence chromatograms of C-dots synthesised with different mole ratios of NAC/HAc. Numerous well separated peaks are observed, inferring that the as-synthesised C-dots are composed of different C-dots species. Our developed HPLC-FD can be successfully applied to separate complex C-dots samples. Peaks labelled with asterisks are nitrogen (N), sulfur (S) and phosphorous (P) co-doped C-dots (N,S,P-C-dots) since these peaks are not found when NAC is absent. When the mole ratio of NAC/HAc increases from 0.0050 to 0.025, the intensity of the N,S,P-C-dots peak increase progressively, indicating that more N,S,P-C-dots are formed. Other elution peaks do not change much. The elemental analysis also confirms the observations obtained from HPLC as depicted in Table S1. N and S are found in the C-dots samples using NAC as the precursor, indicating that N and S originated from NAC could co-dope into C-dots. Higher N and S contents are found with the increase in the mole ratio of NAC/HAc. These results are consistent with the HPLC analysis. Among these C-dots products, NAC/HAc = 0.025 could produce more N,S,P-C-dots with stronger fluorescence emissions. As such, C-dots sample synthesised with NAC/HAc = 0.025 was chosen for most studies. In order to achieve a better understanding of the properties of the as-synthesised C-dots, sixteen separated fractions labelled in Fig. 1B of the C-dots (NAC/HAc = 0.025) were collected for further characterisation by UV-vis absorbance and PL spectroscopy, TEM and MS.
image file: c5ra21137a-f1.tif
Fig. 1 (A) Fluorescence chromatograms of methanol solutions of C-dots (1.0 mg mL−1) synthesised with different mole ratios of NAC/HAc (0.00–0.025) in the initial reagents. (B) The expanded fluorescence chromatogram of C-dots synthesised with a mole ratio of NAC/HAc at 0.025. The chromatograms are acquired by monitoring the fluorescence detector at excitation/emission wavelengths of 300/450 nm and are offset for clarity and ease of comparison.

Absorption and photoluminescence of HPLC fractions of C-dots

To further understand the optical properties of the C-dots species in the as-synthesised C-dots products, the UV-vis absorption and PL spectra of the HPLC fractions of the C-dots are displayed in Fig. S6. All fractions of C-dots display a strong absorption peak at 202–208 nm ascribed to the π → π* transition of the aromatic sp2 domain and a broad shoulder absorption peak between 250 and 300 nm corresponding to the n → π* transitions of C[double bond, length as m-dash]O bond. For fraction 5, an additional absorption peak at 229 nm is found, probably attributing to the N,S doping to the C-dots. All fractions depict strong λem peaks at the short wavelength regions upon λex at 250–300 nm. For fractions 1–3, 7–13 and 16, the main broad emission bands are bathochromically shifted with an increase in λex at 310–500 nm, indicating that the PL band can be tuned by adjusting the λex. Fractions 4–6, 14 and 15 show both λex-independent and λex-dependent emission behaviour at λex 310–500 nm. For fraction 4, the main broad λem is 412 nm at λex 310–340 nm and it is red-shifted with an increase in λex 350–500 nm. For fraction 5, the main broad λem is 420 nm at λex 310–370 nm and it is red-shifted with an increase in λex 380–500 nm. For fraction 6, the main broad λem is 449 nm at λex 310–370 nm and it is red-shifted with an increase in λex 380–500 nm. For fraction 14, the main broad λem is 445 nm at λex 310–380 nm and it is red-shifted with an increase in λex 390–500 nm. For fraction 15, the main broad λem is 446 nm at λex 320–380 nm and it is red-shifted with an increase in λex 390–500 nm. The PL spectra for fractions 1–16 are different attributing to their differences in chemical composition, particle size, lattices and graphitic carbons. Table 1 summarises the ΦS of fractions 1–16. Each fraction has its own ΦS ranging 0.82–8.12%. The variation of ΦS is attributed to the difference in the degree of graphitisation and surface-functionality of the C-dots fractions. In essence, we can conclude that the fractions in the C-dots mixture do indeed possess unique absorption features which lend themselves to unique emission characteristics. These findings support our earlier idea that an as-synthesised C-dots product is a complex mixture containing various C-dots species. Each fraction has its own spectral characteristics and is free from each other's interference.
Table 1 The quantum yields (ΦS) of fraction 1–16 labelled in the HPLC chromatogram of the as-synthesised C-dots
Fraction 1 2 3 4 5 6 7 8
ΦS (%) 8.12 1.05 0.82 1.34 6.58 3.21 2.05 1.16
Fraction 9 10 11 12 13 14 15 16
ΦS (%) 2.09 2.13 2.04 1.81 1.38 1.97 2.36 1.37


Mass spectrometric analysis of C-dots fractions

Mass spectrometry has been an indispensable tool in chemistry and biology to determine the mass of C-dots and so was used to characterise C-dots fractions 1–16 from the HPLC separation. Fig. S7 display the MALDI-TOF MS of fractions 1–16. The possible largest mass C-dots species are labelled on all the MS with red asterisks and all the mass-to-charge ratio (m/z) peaks are below 4200 Da. The largest mass ions on the MS approximately indicate the relative molecular mass of the C-dots species, ranging 2757–4144 Da for fractions 1–16. The HPLC elution order basically follows the molecular mass of the separated C-dots species with some exceptions. It is well known that C-dots are composed of sp2-hybridised carbons cores.78,79 The increase in core size also indicates the increase in the number of carbons of the C-dots. In addition, it has been reported that particle size is proportional to the cube root of the mass of NPs.80 As such, stronger interaction between C-dots and C8 stationary phase takes place when the molecular mass (or size) of C-dots increases under the HPLC condition. We postulate that the separation is not only governed by molecular mass or size but also the shape and surface-functionality of the C-dots.

Each fraction displays regular mass spacing derived from carbonisation of acetic acid and P2O5. Fig. 2 depicts the expanded MS of fractions 1, 5, 6, and 14 in the mass range 1000–1400 Da, respectively. All the expanded MS show major mass spacing in the alternating mass units of 284 which is derived from the C4H14P2O10 moiety. The insets in Fig. 2 display its chemical structure. In addition, a serious of minor mass spacing in the mass units of 45, 43, 32, 31, 17, 15, and 12 corresponding to the loss of a –COOH, –CH3CO, –CH3OH, P, –OH, –CH3, and C moieties are commonly found for all fractions. More importantly, minor mass spacing of 58, 33, 15, and 14 corresponding to –CH3CONH, –SH, –NH, and N moieties are found for fraction 5 which is not observed in the MS of other fractions. These observations further confirm that fraction 5 is N,S,P-C-dots and their surfaces are composed of amide and alkyl sulfide functionalities. In summary, the MS data help us comprehend the chemical composition of the as-synthesised C-dots, concurring with the IR and XPS data.


image file: c5ra21137a-f2.tif
Fig. 2 Expanded mass spectra in the mass range 1000–1400 Da of fractions 1, 5, 6, and 14.

Transmission electron microscopy of C-dots fractions

TEM analysis was also implemented to determine the particle size of the isolated C-dots fractions. Since fractions 1, 5, 6, 10, 14, and 15 possess some intense fluorescence signals that the concentration of C-dots species in these fractions are relatively higher, it is easier to capture their TEM images. Fig. 3 displays their TEM images. The average C-dots size obtained by counting randomly 100 particles from various spots of the fractions during TEM imaging increases in the order: fraction 1 (2.51 nm) < fraction 5 (2.83 nm) < fraction 6 (2.90 nm) < fraction 10 (3.17 nm) < fraction 14 (3.39 nm) < fraction 15 (3.44 nm). The TEM results show that an as-synthesised C-dots product indeed comprises C-dots of various core sizes. The smaller C-dots species are eluted first and followed by the larger ones in HPLC. The size-dependent elution behaviour is similar to other types of NPs.81
image file: c5ra21137a-f3.tif
Fig. 3 TEM images and the particles size distribution of fractions 1, 5, 6, 10, 14, and 15.

Sensing applications

RP-HPLC is a useful technique to separate different C-dots species for anatomising their properties. The fractionation allows us to isolate highly PL nanomaterials from the as-synthesised product. Among the HPLC fraction, fractions 1 and 5 display the highest ΦS of 8.12 and 6.58% respectively and good water-solubility whereas the unfractionated C-dots mixture has ΦS of only 4.65% and lower water-solubility. In addition, fractions 1 and 5 possess different chemical compositions. Fraction 1 is undoped C-dots while fraction 5 is N,S,P-C-dots. Since fractions 1 and 5 possess high ΦS, good water-solubility and differ in surface activity groups, it will be of great advantages to leverage their unique properties for a useful end-application. In view of this, we further explored the feasibility of using fractions 1 and 5 for the detection of heavy metal ions. The dramatic PL quenching behaviour of fraction 1 can be observed when Fe3+ is added into the solution as depicted in Fig. 4A. It is obvious that the PL intensity decreases as the concentration of Fe3+ increases. The fluorescence quenching data follow the Stern–Volmer equation:82,83
 
Fo/F = KSV [Fe3+] + 1 (1)
where Fo and F are the PL intensities of fraction 1 at λex/λem of 350/436 nm in the absence and presence of Fe3+, respectively and KSV is the Stern–Volmer constant. The plot in the inset of Fig. 4B fits the linear concentration range 0.10–10 μM. The correlation coefficient (r2) and KSV are 0.9977 and 0.2173, respectively. The detection limit is estimated to be 31.5 nM at a signal to noise ratio (S/N) of 3.

image file: c5ra21137a-f4.tif
Fig. 4 (A) Fluorescence spectra of fraction 1 with different Fe3+ concentrations (top to bottom: 0.0, 0.10, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 15.0, 20.0, 30.0, 40.0, 60.0, 80.0, and 100 μM). (B) The plot of Fo/F against Fe3+ concentration, where Fo and F are the luminescence intensities of fraction 1 in the absence and presence of Fe3+. Each data point is the average of three measurements. The error bars indicate the standard deviation of the measurements. The inset displays the linear relationship between Fo/F and Fe3+ concentration at the lower range.

We also explored the feasibility of using fraction 5 for heavy metal ions detection. The dramatic quenching behaviour in the PL intensity of fraction 5 was observed when it was exposed to Fe3+ and Hg2+ as shown in Fig. 5A and C, respectively. It is obvious that the PL intensity at λex/λem of 350/420 nm decreases with the increase in Fe3+ and Hg2+ concentrations. For Fe3+, the linear plot in the inset of Fig. 5B fits the range 0.10–6.0 μM. The r2 and KSV are 0.9973 and 0.1869, respectively. The detection limit is estimated to be 49.6 nM at S/N of 3. For Hg2+, the linear plot in the inset of Fig. 5D fits the range 0.010–2.0 μM. The r2 and KSV are 0.9970 and 0.6041, respectively. The detection limit is estimated to be 15.3 nM at S/N of 3. These results demonstrate that fractions 1 and 5 can serve as probes for the detection of Fe3+ and Hg2+.


image file: c5ra21137a-f5.tif
Fig. 5 (A) Fluorescence spectra of fraction 5 with different Fe3+ concentrations (top to bottom: 0.0, 0.10, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 30, 40, 60, 80, and 100 μM). (B) The plot of Fo/F against Fe3+ concentration. (C) Fluorescence spectra of fraction 5 with different Hg2+ concentrations (top to bottom: 0.0, 0.010, 0.050, 0.10, 0.20, 0.40, 0.60, 0.80, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.5, 3.0, 4.0, 6.0, 8.0, 10, 15, 20, 30, 40, and 50 μM). (D) The plot of Fo/F against Hg2+ concentration. Each data point is the average of three measurements. The error bars indicate the standard deviation of the measurements. The insets display the linear relationship at the lower concentration ranges.

In addition, to demonstrate that fractions 1 and 5 are superior to as-synthesised C-dots mixture as the fluorescence probes for detection of Fe3+ and Hg2+, we further examined the PL intensity changes of the as-synthesised C-dots product and fractions 1 and 5 in the presence of Fe3+ and Hg2+ under the same conditions, respectively. Fig. S8 depicts the PL intensity changes of C-dots products and fractions 1 and 5 in the presence of low concentration of Fe3+ (4.0 μM) and Hg2+ (1.0 μM) and high concentration of Fe3+ (60 μM) and Hg2+ (20 μM). In both cases the PL intensity changes of fractions 1 and 5 are larger than the as-synthesised C-dots product in the presence of Fe3+. The fluorescence intensities of fraction 5 greatly decrease in the presence of Hg2+. By contrast, the fluorescence intensity of the as-synthesised C-dots product is almost kept unchanged in the presence of Hg2+. These results further demonstrate that fractions 1 and 5 are indeed superior to the as-synthesised C-dots product for Fe3+ and Hg2+ detection.

To evaluate the selectivity of fractions 1 and 5 as fluorescent probes, we examined the PL intensity changes in the presence of representative metal ions under the same conditions such as Ni2+, Co2+, Cr2+, Ba2+, Cd2+, Mn2+, K+, Ca2+, Na+, Ag+, Cu2+, Mg2+, Al3+, Zn2+, and Pb2+ as shown in Fig. 6. It is clear that Fe3+ can strongly quench the intrinsic fluorescence of fraction 1. The fluorescence intensities of fraction 5 greatly decrease in the presence of Fe3+ and Hg2+. By contrast, the other ions display weak or even negligible effects on the fluorescence intensities of fractions 1 and 5. These results demonstrate that fractions 1 and 5 are highly selective to Fe3+ and Hg2+ over the other metal ions. The high selectivity of fractions 1 and 5 for Fe3+ is ascribed to the fact that Fe3+ has stronger binding affinity with hydroxyl and carboxylic groups on the surfaces of C-dots, which has been widely used for the detection of Fe3+ ions.84–86 Fluorescence quenching may contribute to nonradiative electron-transfer that involves partial transfer of an electron in the excited state to the d orbital of Fe3+.85 In addition, fraction 5 shows high selectivity to Hg2+, attributing to the stronger affinity between Hg2+ and thiol groups of N,S,P-C-dots and facilitating the nonradiative recombination of excitons via an effective electron transfer process.87


image file: c5ra21137a-f6.tif
Fig. 6 (A) Fluorescence responses of fraction 1 in the presence of different interfering metal ions. 100 μM for Ni2+, Co2+, Cr2+, Ba2+, Cd2+, Mn2+, K+, Ca2+, Na+, Ag+, Cu2+, Mg2+, Al3+, Zn2+, Pb2+, Fe3+, and Hg2+. (B) Fluorescence responses of fraction 5 in the presence of different interfering metal ions. 50 μM for Ni2+, Co2+, Cr2+, Ba2+, Cd2+, Mn2+, K+, Ca2+, Na+, Ag+, Cu2+, Mg2+, Al3+, Zn2+, Pb2+, Fe3+, and Hg2+. Each data point is the average of three measurements. The error bars indicate the standard deviation of the measurements.

Conclusion

We have shown that as-synthesised C-dots derived from acetic acid, NAC and P2O5 exists as a complex mixture, and its complexity can be significant reduced by RP-HPLC fractionation. HPLC fractionation can reveal the unique absorption and emission characteristics, and ΦS of each C-dots species, which would be otherwise misled by only studying the unfractionated C-dots mixture. By fractionation into a single entity, as revealed by MALDI-TOF MS and TEM method, one can better understand the morphology and chemical composition of C-dots. The selected C-dots fractions display high ΦS and good water-solubility. They possess various surface functional groups and have potential for serving as fluorescence probes.

This work demonstrates the significance and importance of fractionating various C-dots species in an as-synthesised sample by HPLC. Their fundamental properties can be truly revealed, and their applications in different fields can be established. The selected C-dots fractions were successfully applied to detect Fe3+ and Hg2+ with high sensitivity and selectivity. The high ΦS, good water-solubility and different surface functionalities of these selected C-dots fractions give these nanoprobes advantages over the widely used unfractionated C-dots mixture. This work also emphasise the importance of the fractionation process when studying nanomaterials, whether luminescent or otherwise, to properly evaluate the material's characteristics. Isolation from what can be a rather complicated mixture is critical to properly assess fundamental properties of nanomaterials before establishing their applicability.

Acknowledgements

Financial supports from the National Nature Science Foundation of China (21175085 and 21375083), Hundred Talent Programmer of Shanxi Province, and HKBU Faculty Research Grant (FRG1/13-14/039) are gratefully acknowledged. We would express our sincere thanks to Ms Winnie Y. K. Wu of the Institute of Advanced Materials for taking the TEM image and Ms Silva T. Mo of the Department of Chemistry, Hong Kong Baptist University for acquiring the MALDI-TOF MS. The TEM was supported by the Special Equipment Grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Grant SEG_HKBU06).

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Footnotes

Electronic supplementary information (ESI) available: UV-vis absorption and PL spectra of the C-dots in methanol solution, plots of integrated PL intensity against absorbance of C-dots and quinine sulfate, PL spectra of C-dots at different λex 250–500 nm, IR spectrum and XPS spectra of C-dots, elemental analysis of the C-dots samples, UV-vis absorption and PL spectra at different λex of fractions 1–16, MALDI-TOF mass spectra of fractions 1–16, and PL intensity changes of the as-synthesised C-dots products and fractions 1 and 5 in the presence of Fe3+ and Hg2+. See DOI: 10.1039/c5ra21137a
Exchange student on visit to Hong Kong Baptist University.
§ Present address: Acadia Divinity College, Acadia University, 15 University Avenue, Wolfville, Nova Scotia, B4P 2R6, Canada.

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