From the traditional way of pyrolysis to tunable photoluminescent water soluble carbon nano-onions for cell imaging and selective sensing of glucose

Abstract

The traditional pyrolysis of vegetable ghee leads to the fabrication of graphitic photoluminescent, water soluble carbon nano-onions (wsCNO) with tunable photoluminescence without using any metal catalyst. Simple oxidative treatment by nitric acid fabricated a high density “self-passivated” water soluble version. As-synthesized wsCNO possessed tunable photoluminescence behavior from the visible-to-near infrared region. Further small sized wsCNO separated from the bulk as-synthesized wsCNO via gel filtration achieved a highly fluorescent colored fraction, used for cell imaging (Escherichia coli and Pseudomonas putida) and selective, immediate sensing of glucose molecules based upon a simple fluorescence “turn-off”/“turn-on” technique.

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1. Introduction

Nano-sized fluorescent nanocrystals, generally referred as quantum dots (QDs), have attracted a lot of interest for imaging, especially in the biomedical sciences.^1,2 However, being metallic in nature their toxicity significantly challenges the excellent optical advantageous performances.^3–5 Tremendous efforts have been made to find a non-toxic alternative of QD-type photoluminescent probe with comparable optical properties. The outcome of these significant efforts has resulted in the discovery of a new class of fluorescent nano-carbons (FNCs),^6–12 referred as a non-toxic fluorescent probe, having optical properties comparable with QDs. FNCs predominantly consist of carbon dots (CD),^6,13,14 carbon nano-diamonds,⁷ graphene quantum dots,^8,9,15 carbon nano rods^16,17 and carbon nano-onions (CNO).^10–12,18 Among all of these, the CD have been studied the most^6,14,19–24 as a fluorescent probe for non-toxic biological imaging purposes, whereas CNO have been investigated least.^{10,11,25–27} Because of the presence of a well-defined band gap, QDs shows quantum confinement effects as a result of which, its size dependent electronic and optical properties can easily be tuned. However, FNCs do not have any theoretically defined band gap based fluorescence color related size distributions of nanoparticles. Because of this, these are different from QDs and have the advantageous, significant properties of tunable photoluminescence from the single particle^10,11,28,29 together with high solubility, stability, and non-toxicity. Among the FNCs, CNO is an emerging class of quasi-spherical nano carbon, which typically consists of successive layer of graphene around a filled or hollow core and they offer a high aspect ratio and conductivity.³⁰ Morphologically, graphitic structures of CNO are in between fullerene and graphitic nanotubes having closed graphitic shells which are placed on one on another.^11,31,32 So far, less attention have been given to CNO even though the prospective applications have already discussed. CNO exhibited the characteristic, onion-like morphology and small domains of graphitic sp² carbons with localization of the π electrons and dangling bond defects in the periphery.³⁰ As a consequence, it could be envisioned as an enclosed graphitic shell with excellent optical and electrochemical properties that render them a potential nanomaterial for industrial, biomedical and electronic applications.^33–36 From the time of their discovery¹² until the present date, only a limited number of synthetic methods for CNO have been available and it can be synthesized mainly by using high energy techniques. Such as the arc-discharge method, in which two graphite electrodes are arced under water for the synthesis of CNO on the water surface.³² Annealing of nano-diamond for 1.5 h at a temperature range of 500–1400 °C under vacuum³⁷ and electron transfer in a nano-diamond at 200 keV has also been used.³⁸ Another method is the thermal reduction of glycerin and magnesium at 650 °C for 12 h in an autoclave.³⁹ Chemical vapor deposition has also been used and involves methane and depositing CNO on a silicon surface.⁴⁰ Hou et al. used the catalytic synthetic method by using nickel as a catalyst in the counter flow diffusion of flames of ethylene, methane and nitrogen.⁴¹ Han et al. reported the thermal treatment of copper(II) chloride dihydrate and calcium carbide in an autoclave at 600 °C for 10 h for synthesis of CNO⁴² and via the flame pyrolysis of naphthalene.⁴³ Besides this, a very easy and cost effective method was also reported using the pyrolysis technique over waste wood known as “wood wool”.^10,35 Among all the reported methods, annealing of nano-diamond and arc discharge have been the most commonly used methods until now. So far CNO have shown significant potential in the field of lithium ion batteries,⁴² as supercapacitor electrodes,^31,44 capacitors,⁴⁵ optical limiters,⁴⁶ catalysis,^47,48 sensing,⁴⁹ tribology,⁵⁰ fuel cells,⁵¹ and terahertz shielding.⁵²

Until now, only a few reports have been available on the investigation of biological applications of CNO and it is still necessary to understand in detail their non-toxic nature,^{10,11,27,30,35,53} and tunable photoluminescence properties.^10,11,25,26 Like other forms of nano-carbons, their insolubility restricted many of their potential applications. To make these soluble in organic solvents various adducts of CNO were formed based upon simple organic chemistry.^54,55 Ghosh et al. used a simple one-step oxidative approach for the formation of multicolored fluorescent, water soluble carbon nano-onions (wsCNO) and these were used for the full, life cycle imaging of Drosophila melanogastor.¹⁰ In another study, Sonkar et al. showed the non-toxicity of wsCNO in the food chain of two model organisms: prokaryotic Escherichia coli cells and eukaryotic Caenorhabditis elegans.¹¹ Recently Giordani et al. investigated various routes for the modification of CNO surface using diverse functional groups for potential applications in biomedical imaging.^25,26,56 Low toxicity with biocompatibility, and ease in cellular uptake with good penetration capability are the important aspects for bioimaging.^10,11,34,35 Readily achievable tunable photoluminescence in the green-red region with a slight extension in the near infrared (NIR) region have added an additional advantage,^25,36 in contrast to the blue-green emissions of other fluorescent nanocarbons. Imaging in the NIR region is of huge significance as it offers high biological transparency,⁵⁷ because of low light scattering, deeper penetration, and low background signal.⁵⁸ Sensing of blood glucose has gained extensive attention worldwide because diabetes mellitus is one of the major health problems.⁵⁹ It has become a global issue and according to the World Health Organization (WHO), approximately 300 million people suffering from diabetes and this figure will increase up to 600 million by 2030.⁶⁰ So glucose detection is highly significant in the clinical diagnosis and biochemical study of this illness. Most of the sensors used for glucose detection are either, based upon an electrochemical method, enzyme-based techniques (glucose oxidase) or microdialysis probes.^60,61 However, many of these methods suffer from instability, environmental sensitivity, cytotoxic effect of the probe and require sophisticated, expensive instrumentation, chemicals (enzymes), and the complex synthetic procedures are time consuming.

In the research reported in this paper, a traditional pyrolysis route was adopted for the synthesis of CNO using vegetable ghee as low cost available carbon source. To separate the most fluorescent fraction of wsCNO from the bulk as-produced wsCNO, gel filtration was done and this fraction was used for the imaging of E. coli and P. Putida cells. The wsCNO possess a high density of carboxylic and hydroxyl groups on their surface making them a stable species in aqueous solution, and thus, they can readily diffuse into the cell without causing any cell damage^10,11 and ultimately excreted from the body.¹⁰ Furthermore, this most fluorescent fraction can be utilized as the non-enzymic sensor for selective and immediate sensing of “glucose” via a fluorescent “turn-off”/“turn-on”⁶² mechanism.

2. Experimental section

2.1. Materials

All the experiments were performed in deionised water. Vegetable ghee (Dalda), was procured from a local market in Jaipur, India. All the chemicals and solvents were of analytical grade and used as-obtained without any further purification. Methylene blue (MB), glucose, dopamine and amino acids were purchased from Sigma-Aldrich (India).

2.2. Measurements

Solid state Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vector 22 FTIR spectrometer. Raman spectra were recorded on a WITec Raman spectrometer with 532 nm laser excitation. Thermogravimetric analysis (TGA) was conducted under a nitrogen atmosphere with a Mettler thermal analyzer at a heating rate of 10 °C min⁻¹. Weight loss versus temperature were recorded starting from room temperature up to 900 °C. X-ray photoelectron spectroscopy (XPS) was performed on a Scienta Omicron ESCA⁺ morphology and structure analysis of the wsCNO were performed using high-resolution transmission electron microscopy (TEM/HRTEM) on a FEI Tecnai G2 20 at 200 kV and high-resolution field-emission scanning electron microscopy (FESEM) was performed using a Zeiss SUPRA 4A and a FEI Tecnai G2 20 at 200 kV. The samples for FESEM and TEM analysis were prepared by dropping an aqueous solution of wsCNO onto the surface of a brass stub and a carbon coated copper grid, respectively, followed by the evaporation of water under a 100 W table lamp. Fluorescence spectra in an aqueous medium were recorded on a PerkinElmer LS-55 fluorescence spectrometer. Quantum yield values of wsCNO were measured using the same procedure described in an earlier paper by Tripathi et al.¹⁶ E. coli and P. putida and wsCNO imaging, were performed using a Leica DM 2500 inverted microscope (Leica, Heerbrugg, Switzerland) under 488 nm and 562 nm band pass filters. The sample for fluorescence microscopic imaging was prepared by dropping the diluted aqueous solution of wsCNO on a glass slide and forming a smear for the analysis. The ultraviolet-visible (UV-Vis) absorption spectra of wsCNO were recorded on a PerkinElmer Lamda 35 spectrometer at room temperature. Surfacial charge analysis for wsCNO in aqueous medium was done with zeta potential measurement on a Beckman Coulter Delsa™Nano.

2.3. Synthesis of wsCNO

The CNO were synthesized using simple and traditional pyrolysis of vegetable ghee as a low cost source of carbon with a cotton wick. The soot was collected in an inverted borosilicate glass beaker to avoid any chance of metal impurities. The as-collected soot was purified using a Soxhlet extraction technique^34,63 in which soot was washed with different boiling solvents (acetone, methanol, acetonitrile and petroleum ether) to remove any unburnt hydrocarbons and impurities. The water solubility for the Soxhlet purified soot was achieved by refluxing it with 60% nitric acid (HNO₃) solution for 12 h, and the excess nitrate was removed by repeatedly washing with deionized water in a water bath according to a previously reported method.^64–66 The yield of the water soluble product (as wsCNO) was ∼87% (based on the CNO used for oxidation). To isolate the most fluorescent fraction of wsCNO with a narrow size distribution, high-speed centrifugation was performed followed by a gel filtration separation method using a Sephadex G-100 column as described earlier.^67,68 The overall yield of the light yellow colored fraction of wsCNO was ∼17% (based on the amount of CNO used for oxidation) having the quantum yield value of ∼1.9% and this was used further for cell imaging and selective detection of glucose molecules.

2.4. Cell staining

Cells of the DH5α strain of E. coli and the MTCC 2445 strain of P. putida were incubated with the wsCNO (100 ppm) in 20 mL of freshly prepared food medium (Luria-Bertani media for E. coli and minimal media for P. putida cells), as described earlier.^10,34,64,69 Then, the cells with the remaining broth containing wsCNO, were washed thoroughly, three times, with phosphate buffered saline. The sample for cellular imaging was prepared by dropping a droplet of bacterial suspension (8–10 μL) on a glass slide.

2.5. Fluorescence detection of glucose

Glucose sensing was performed at room temperature in aqueous solution. Initially 1 mL of MB (6.2 × 10⁻⁴ M) was added to an aqueous solution of wsCNO (1.5 × 10⁻⁴ g mL⁻¹) by continuous addition of 0.1 mL MB solution in 10 steps. After adding MB, the fluorescence emission spectra were recorded immediately. Deionized water was added to make the final volume up to 3 mL in each step. Glucose solution (0.1 mL; 1.8 × 10⁻² M) was added to the MB–wsCNO solution to restore the fluorescence intensity. Interferences which originated from other protein, or amino acids were investigated individually. All the fluorescence spectral measurements were done at ambient conditions at the same excitation wavelength of 460 nm.

3. Results and discussion

The strong oxidizing nature of HNO₃ assists the incorporation of hydrophilic carboxylic (–COOH) and hydroxyl (–OH) groups on to the surface of CNO. These groups impart its solubility in aqueous media with multi-colored emissive tunable photoluminescent properties, which represented the characteristic features of FNCs.^21,70 Furthermore, the size selective separation was achieved using high-speed centrifugation followed by gel filtration to achieve the most fluorescent fraction of wsCNO having the quantum yield value of ∼1.9%. Most importantly, the high-density surface of carboxylated wsCNO exhibited a broad range of tunable photoluminescence emissions across a wide range of the visible spectrum with a very slight extension in the NIR region.

3.1. FTIR/Raman/XPS/powder XRD/TGA analysis

The FTIR spectra of CNO and wsCNO are shown in Fig. 1(a). The FTIR spectrum of CNO (Fig. 1(a)-solid line) shows a sharp peak around ∼2930 cm⁻¹ which is because of the presence of sp³ (C–H) stretching vibrations, and a strong peak at around ∼1654 cm⁻¹ shows the presence of (C=C) stretching vibrations. The FTIR spectrum of wsCNO (dotted line) as displayed in Fig. 1(a) shows the characteristic peaks of –OH (broad) at 3402 cm⁻¹, –C=C– at 1578 cm⁻¹ and –C=O at 1716 cm⁻¹ (a sharp peak), which confirms the presence of surfacial carboxylic and hydroxyl type hydrophilic groups.^16,34 It also shows the presence of sp³ carbon because of the existence of a characteristic C–H peak at 2915 cm⁻¹. A peak at 1214 cm⁻¹ showed the presence of a –C–O group in the wsCNO. To investigate the negative surface functionalities, zeta potential analysis was performed, which showed a high negative value of −40.04 mV, confirming the presence of high density negatively charged surface groups.⁷¹ To quantify the sum of negative surface carboxyl groups present on the surface of wsCNO, a very simple acid–base titration was performed to calculate its weight percentage and was found to be in the range of 15–16.7% (varied batch-to-batch).^16,64,72 This high density carboxylation leads to high solubility and stability of wsCNO in aqueous solution. As well as the solubility, high density “self-passivated” wsCNO generated by surface carboxylation will be useful for performing a huge range of functionalization chemistry of wsCNO with^30,73,74 or without bio-molecules (such as oligomeric^67,73,74 and monomeric amines)⁷⁰ based upon the simple amide linkage chemistry.

Fig. 1 (a) FTIR spectra of CNO (solid line) and wsCNO (dotted line); (b) Raman spectra showing D and G band of Soxhlet purified soot (before derivatization solid line) and wsCNO (after derivatization, dotted line); (c) XPS spectrum showing elemental C and O in the wsCNO; (d and e) short scan high-resolution XPS for carbon and oxygen; (f) TGA graphs of Soxhlet purified (solid line) and wsCNO (dotted line); (g) powder XRD showing two prominent peaks corresponding to the graphitic carbon in wsCNO.

Raman spectra were used to understand the extent of derivatization concerning the ratio of intensities of the G and D bands (I_G/I_D) and for structural elucidation of graphitic materials regarding band positions.⁴³ Fig. 1(b) shows the Raman graphs of Soxhlet purified CNO (solid line) and its water soluble version (dotted line). Both Soxhlet purified soot and wsCNO exhibited two prominent characteristic bands for carbon atoms present in the graphitic framework. The graphitic (G-band) at ∼1600 cm⁻¹ and disordered (D-band) at ∼1349 cm⁻¹ corresponds to the E_2g mode of graphitic carbon in a two-dimensional hexagonal lattice and a dangling bond in the disordered graphitic shells present in CNO, respectively.⁷⁵ Both D and G bands show the downward shift at ∼1330 cm⁻¹ (from 1349 cm⁻¹) and ∼1582 cm⁻¹ (from 1600 cm⁻¹), which corroborate the weakening of the graphitic structure in the form of bending and destructions of bonds influenced by the oxidative treatment,^{10,11,34,64,76} inferring the introduction of a high-density surface defect.^10,11,34 Incorporation of high-density defects in the outermost shell of the nano-onions containing higher number of shells resulted in a disordered like spectrum as reported by Choucair and Stride.⁴³ Incorporation of high-density surface functionalization is calculated using the I_G/I_D ratio. The wsCNO I_G/I_D ratio (∼0.18) was found to be much smaller in comparison with the Soxhlet purified CNO I_G/I_D ratio (∼0.43). The decrease in the values of I_G/I_D ratio for wsCNO strongly supports the formation of high degree of surface defects which arise from the transformation of sp² clusters of graphitic carbons in disordered functionalized sp³ hybridized carbon atoms.^23,64

The presence of negative hydrophilic groups as surface elements and chemical composition was determined using XPS measurements. The XPS spectrum of wsCNO shown in Fig. 1(c) clearly shows that carbon (C 1s) and oxygen (O 1s) are present on the surface of wsCNO at 286 eV and 535 eV, respectively. Fig. 1(d) and (e) describes the high-resolution short scanned XPS spectra of C 1s and O 1s, respectively. Short scanned carbon, C 1s analysis as displayed in Fig. 1(d) shows the presence of three different peaks located at 285.8 eV, 286.7 eV and 287.6 eV, which corresponds to the presence of C=C, C–O and C=O respectively, and indicates the presence of carboxylic acid functionalities.⁷⁷ The short scan high-resolution XPS survey for the O 1s region shows the presence of three major peaks at 533.7 eV, 535.1 eV and 536.2 eV corresponding to the presence of C–O, C=O, and COO⁻, respectively.⁷⁸ The results of surface functionality analyses using XPS are in agreement with the results from FTIR analysis.⁷⁹ TGA analysis was carried out to understand the thermal stability of wsCNO.³⁴ The comparative TGA analysis for both the samples, Soxhlet purified CNO (solid line), and wsCNO (dotted line) are displayed in Fig. 1(f). Both the samples show a gradual degradation in weight starting from 35 °C. TGA analysis shows that wsCNO are thermally less stable in comparison with Soxhlet purified soot because of the presence of a high degree of surfacial oxygen species (–COOH/–OH) generated from the destruction of surfacial graphitic framework of wsCNO during its oxidative process.⁶⁴ A weight loss of 24.1% was observed for wsCNO in comparison with a loss of ∼8.1% for Soxhlet purified CNO under the same temperature conditions. Fig. 1(g) shows the powder X-ray diffraction (XRD) spectra of wsCNO having the characteristic peaks of graphitic carbon with an inter-planar distance of 0.32 nm.⁸⁰

3.2. Microscopic studies

The physical appearance and internal detailed microstructure characterization of soluble nano-onions were carried out using FESEM, TEM, and HRTEM. Fig. 2(a) shows a high magnification FESEM image, without presence of any amorphous type carbon. Fig. 2(b) illustrates the well dispersed and spherical nature of wsCNO showing some opacity because of the overlapping of the nanoparticles. Based on Fig. 2(b), the average size distribution of wsCNO was calculated statistically and the size distribution of wsCNOs (diameter ranging from 10–50 nm) in the form of a histogram is shown in Fig. 2(c). The size range of the majority of the wsCNO were 20–40 nm. HRTEM images of nano-onions as shown in Fig. 2(d) and (e) illustrate the presence of a closed quasi-spherical shape having graphitic inter-planer fringes with a lot of defects. HRTEM images show the presence of well-resolved lattice fringes with 0.32 nm lattice spacing, which is indicative of their graphitic nature.⁸¹ HRTEM images clearly reveal the closed cage structure of well defined concentric shells and their crystallized structure. Surfacial defects as marked by black arrows are in good agreement with the Raman analysis regarding I_G/I_D ratio.

Fig. 2 (a) High-resolution FESEM image of wsCNO; (b) low magnification TEM image showing the nearly monodispersed wsCNO; (c) size (diameter) distribution histogram; (d and e) high-resolution TEM image of wsCNO; (e) crystalline lattice planes of wsCNO with a lot of surfacial defects (marked by black arrows).

3.3. Absorption, photoluminescence emission–excitation study and photostability test under high ionic conditions

The wsCNO are highly soluble in water and remain soluble for several months. The absorption spectrum as demonstrated in Fig. 3(a) exhibits a characteristic absorption band which is attributed to the typical absorption of conjugated π-plasmons of sp² domains.^34,45,64 The excitation dependent tunable photoluminescence was studied at room temperature in an aqueous solution. wsCNO emits in a broader region of the visible spectrum with its extension to NIR (from 550 nm to 800 nm), as displayed in Fig. 3(b), when excited from 400 to 660 nm with a progressive increment of 20 nm. The emission maximum is centered in the greener region of spectrum at about ∼550 nm (excitation at 400 nm) with the quantum yield value of ∼1.9%.⁶¹ Fig. 3(c) shows the magnified image of Fig. 3(b), revealing the tunable shifting of emissions towards the redder region of the spectrum with its NIR extension. The characteristics of the multi-colored tunable excitation dependent emission profiles of wsCNO continuously shifted towards the redder region of the spectrum which is the characteristic features of FNCs.^28,46 The possible mechanism of a multi-colored tunable photoluminescence emissive profile from the single particle of wsCNO involves the radiative recombination of quantum confinement effects of excitations,^{6,10,11,67,70,82} emissive surface traps, dipole emitted centers, and the coupling of electron–phonons present over the defective surfaces of wsCNO.^83,84 Full width at half maximum measurements have the value of ∼65.23 nm, showing the small size distribution of wsCNO. Fig. 3(d) shows the excitation (photoluminescence) spectrum at 460 nm emission wavelengths for wsCNO with four absorptions centered at 220 nm (5.63 eV), 239 nm (5.18 eV) and 285 nm (4.35 eV) and 307 nm (4.0 eV) which confirms the presence of various types of light emitting centers.^82,85 Not only is the tunable photoluminescence important, but photostability is also another important issue related to its various uses in biological applications, because of the very different ionic environmental conditions of biological cells.

Fig. 3 Multi-colored emissive tunable emissions profile of wsCNO; (a) absorbance spectra of wsCNO in aqueous solution; (b) excitation-dependent emission from 400–660 nm with progressive increments of 20 nm; (c) magnified image of (b) showing NIR emission profiles; (d) photoluminescent excitation spectrum of wsCNO at 640 nm excitation wavelength; (e) time dependence photostability test of wsCNO with continuous excitation at 400 nm wavelength for 5 h; (f and g) photostability test in the presence of a high ionic strength of sodium nitrate and barium chloride; (h) and (i) fluorescence microscopic images of wsCNO under 488 nm (green) and 562 (red) nm bandpass filter, respectively. Scale bar: 10 μm.

To understand better the photostability, two different sets of experiment were performed: continuous photo-excitation and testing its stability in the presence of high ionic strength solutions. wsCNO exhibits excellent photostability, and no sign of reduction in photoluminescence intensity was observed after five hours of continuous excitation with 400 nm wavelength as displayed in Fig. 3(e). Additionally, the photoluminescence emission remains unaffected in the presence of high concentrated solutions of Na⁺ and Ba²⁺ ions (1 × 10⁻⁵ to 0.1 M) (Fig. 3(f) and (g)), respectively. The fluorescence microscopic images of wsCNO under excitation at 488 nm and 562 nm are demonstrated in Fig. 3(h) and (i), respectively. High photostability towards long-term continuous excitation and in a high ionic strength solution indicates its significant potential for fluorescent cell imaging and it could also be used for sensing purposes.

3.4. Fluorescence imaging of E. coli and P. putida cells

E. coli and P. putida cells were used for the imaging purposes. A sample of wsCNO with a concentration of 100 ppm was mixed with 20 mL of the specific food media of E. coli and P. putida, in comparison with the control media (media without wsCNO). Cell imaging experiments were performed with three replicates each time, with and without the presence of wsCNO. Fig. 4 shows the fluorescent images of wsCNO labelled E. coli (a and b), and P. putida (c and d) under the green and red channel of fluorescence microscope, respectively. This simple and easy labelling of wsCNO via oral ingestion (mixing with food only) will be researched further for specified imaging of different parts of cell based upon the attachment of different surface functionalities which can selectively attach inside the different regions of cells.

Fig. 4 Fluorescence microscopic images of E. coli (a) and (b) and P. putida (c) and (d) under 488 nm (green) and 562 nm (red) band pass filters, respectively.

3.5. Glucose sensing

The most fluorescent fraction of “self-passivated” wsCNO, was used for the development of selective sensing of glucose based upon the simple fluorescence “turn-off/turn-on” mechanism.⁶⁴ Fluorescence intensity of wsCNO was efficiently quenched by MB to its maximum (fluorescent turn-off) and would be “turned-on” after the addition of glucose. The mechanistic illustration for the development of wsCNO (fluorescence “turn-off/turn-on”) based glucose biosensor is shown in Fig. 5(a). When wsCNO interacted with MB, the fluorescent emission profiles of wsCNO were quenched to its maximum by the stepwise addition of MB. Because of the efficiency of high density, negatively charged wsCNO interacted with MB molecules via surfacial charge-transfer and hydrophilic interactions⁶⁴ that resulted in the fluorescence “turn off” for wsCNO as illustrated in Fig. 5(b). The intensity of the ∼623 nm centred emission peak of wsCNO (1.5 × 10⁻⁴ g mL⁻¹) was completely quenched by MB within 20 minutes by the gradual addition of 1 mL MB (6.2 × 10⁻⁴ M) in 10 steps (0.1 mL in each step) (Fig. 5(b)). The maximally quenched fluorescence intensity of wsCNO (Fig. 5(c) blue trace) was recovered by the addition of glucose (0.1 mL, 1.8 × 10⁻² M). The photoluminescent emission in wsCNO occurred because of the recombination of the electron–hole pair via photogeneration on the surface.^{6,10,11,16,23,34} After the adsorption of MB on the wsCNO surface, electron transfer from wsCNO to MB diminished the recombination of electron–hole pair and resulted in fluorescence quenching. The interaction of surfacial hydrophilic groups with MB and better availability of the CNO surface for adsorption of MB act as the driving force for the fluorescence “turn off”. The possible mechanism for the fluorescence “turn-on” could be because of H-bonding interaction between primary alcoholic group of glucose and nitrogen of the centre ring of surface adsorbed MB. This causes the replacement of surface adsorbed MB molecules by glucose from the wsCNO, and ultimately leads to its fluorescence restoration, termed as fluorescence “turn-on” as shown in Fig. 5(c) (red trace).^86–90 Glucose has two types of –OH groups, four similar types of secondary alcoholic groups and a single primary alcoholic group which is a bit more acidic in comparison with other four.⁸⁷ This primary alcoholic group has the tendency to bind with MB via H-bonding,^86–90 and ultimately causes the reduction of MB. This simple reduction chemistry of MB by the protonation from glucose is very well known and is described in the literature.^86–90 The emission intensity continuously decreases with MB addition until a maximum is reached as shown by the Stern–Volmer plots in Fig. 5(d). Also, wsCNO have a unique morphology, and a significant number of defects in the forms of dangling bonds,³³ which can also lower the activation energy of desorption of MB and selective sensing of glucose by fluorescence “turn-on”.

Fig. 5 (a) A schematic presentation showing the mechanism based on a fluorescence “turn-off” and “turn-on” method via the adsorption of MB and glucose molecules on wsCNO, respectively, for sensing glucose; (b) photoluminescence emission spectra from stepwise addition of 0.1 mL aqueous solutions of MB showing a maximal quenched solution; (c) fluorescence “turn on” after the replacement of MB molecules with glucose from wsCNO surface; (d) relationship between the photoluminescence intensity of wsCNO and MB concentration; (e) selectivity of a wsCNO-based fluorescence sensor for glucose.

In addition to sensing, it is known that selectivity is a prime parameter for developing a sensor for biological applications in order to utilize its maximum potential inside very complicated cellular environments.⁹¹ A non-enzymic, wsCNO-based sensor was developed for the selective detection of glucose molecules. The selectivity of glucose, was explored with some common interferants, such as dopamine, uric acid and amino acids (cysteine, methionine, aspartic acid, tryptophan, and valine; 2.4 × 10⁻² M) at identical experimental conditions. Fig. 5(e), shows the selective detection of glucose by comparison with other methods based on the fluorescence “turn-off”/”turn-on” mechanism of wsCNO. The comparison between wsCNO and various fluorescence sensors from the literature review for the detection of glucose are presented in Table S1 (ESI†). Although, a few materials have a lower detection limit than wsCNO (1.3 × 10⁻² M) but the cost effective nature and simplicity of the detection process with wsCNO used here are advantageous in terms of immediate response time (restoration of maximally quenched fluorescence intensity) within a few seconds.^60,92–95

4. Conclusions

Large-scale fabrication of pure wsCNO via a simple and convenient route is described in the present work. The highly defective outer surfaces impart tunable photoluminescence properties to “self-passivated” wsCNO and make them a promising candidate for use in biological imaging and sensing applications. The most fluorescent fraction was separated using gel filtration and applied as the fluorescent probe for the imaging of E. coli and P. putida cells. Furthermore, the same fraction of wsCNO was used in this research for the selective and immediate sensing of glucose molecules, based upon the fluorescence “turn-off”/“turn-on” technique (detection limit = 1.3 × 10⁻² M). The ease of synthesis of wsCNO by using vegetable ghee as a carbon precursor could lead the way for the fabrication of large scale, pure and NIR-emissive wsCNO for multi-functional applications.

Acknowledgements

KMT thanks CSIR, New Delhi for a Senior Research Fellowship, AB and AS thank MNIT Jaipur for doctoral assistantships, NRG thanks DST, New Delhi for funding under the DISHA scheme [DST/DISHA/SoRF-PM/024/2013], SV thanks CSIR, New Delhi for a doctoral assistantship and SS thanks DST for a Ramanna Fellowship. SKS thanks DST New Delhi for funding [SB/EMEQ-383/2014] and the Material Research Centre, MNIT Jaipur for HRTEM and XPS analysis. SKS thanks Dr Vivek Verma of the Materials Science and Engineering Department, IIT Kanpur for helping in the cell imaging experiment.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04030f

‡ Both the authors contributed equally.

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