Journal of Materials Chemistry B - Indian Institute of ...iiti.ac.in/people/~xray/C7TB02484C.pdf · Nature oﬀers a wealth of ... degrees in Nanotechnology from University of Rajasthan,

8904 | J. Mater. Chem. B, 2017, 5, 8904--8924 This journal is©The Royal Society of Chemistry 2017

Cite this: J.Mater. Chem. B, 2017,

5, 8904

Sustainable carbon-dots: recent advances in greencarbon dots for sensing and bioimaging

Vinay Sharma, a Pranav Tiwarib and Shaikh M. Mobin *abc

The past decade has witnessed the emergence of carbon dots (c-dots), outshining other members of

the carbon family because of their outstanding properties in fluorescence, cytocompatibility,

photostability, electronic, mechanical and other chemical properties. This has resulted in an increasing

number of applications in bioimaging, sensing, photovoltaic and medicine. Nature offers a wealth of

exciting precursors that motivate constant persuasion of benign synthetic routes. Consequently, the past

5 years has seen a tremendous rise in green synthetic approaches of c-dots. This study reviews the

journey of green c-dots by means of green sources of synthesis and their applications, with the major

focus on various sensors and bioimaging probes.

1. Introduction

Fluorescent carbon nanoparticles were accidentally discoveredby Xu et al.1 in 2004 during electrophoretic purification ofsingle-walled carbon nanotubes (SWCNTs). In 2006, Sunet al.2 reported synthesis of fluorescent carbon particles of sizeless than 10 nm, which they called ‘carbon dots’. There fol-lowed intensive research exploring this fascinating materialwith extraordinary potential. Carbon-dots have unprecedentedand remarkable properties, including multicolour wavelengthtuned emission, up-conversion photoluminescence, high

quantum yield, aqueous dispersibility and high biocompatibility.In recent years, various chemical precursors have been identifiedfor synthesis of c-dots, including citric acid,3–5 ammoniumcitrate,6 ethylene glycol,7 benzene,8 phenylenediamine,9 phyticacid,10 EDTA11,12 and thiourea13 etc. Various synthetic routes havebeen used to convert these precursors into fluorescent carbondots, including hydrothermal,14–18 solvothermal,19–21 electro-chemical,22–24 microwave assisted pyrolysis,10,25–27 ultra-sonication,28,29 simple heating,30,31 arc discharge, laser ablation2,32

and chemical oxidation33 etc.Several reviews have described methods for synthesis and

applications of carbon dots,34,35 surface engineering,36 photo-luminescence, hetero-atom doping,37 electroanalysis,38 electro-chemical biosensors39 and fluorescence-based sensing andbioimaging.40 However, to date there has not been a reviewexplicitly describing green sources of synthesis, properties andapplication. This article attempts to fill the gap. Green chemistry

a Center for Biosciences and Bio-Medical Engineering, Simrol, Khandwa Road,

Indore 453552, Indiab Discipline of Metallurgy Engineering and Materials Science, Simrol,

Khandwa Road, Indore 453552, Indiac Discipline of Chemistry, Indian Institute of Technology Indore, Simrol,

Khandwa Road, Indore 453552, India. E-mail: [email protected]

Vinay Sharma

Vinay Sharma was born in 1989 inRajasthan province of India. He iscurrently working as a researchscholar at Indian Institute ofTechnology Indore. He obtainedhis BTech (Hons.) and MTech(Hons.) degrees in Nanotechnologyfrom University of Rajasthan, Indiain 2012. His research work isdevoted to the development of bio-medical sensors based on nano-materials and their intracellularapplicability. Pranav Tiwari

Pranav Tiwari is a research scholarat Discipline of Metallurgy Engineer-ing and Materials Science, IITIndore. He obtained BTech andMTech in Nanotechnology fromCentral University of Jharkhand,India. He worked as projectassistant at Indian Institute ofScience (IISc) prior to joining IITIndore. His research work isrelated to graphitic nanomaterials.

Received 15th September 2017,Accepted 24th October 2017

DOI: 10.1039/c7tb02484c

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has advantages of reducing chemical exposure, reducing waste,producing cheap, renewable and abundant biomass, and havingpotential for scale-up. Liu et al.41 caused a paradigm shift withuse of grass as a natural carbon source, beginning a trend for useof biomass for synthesis of carbon dots.

2. Carbon dots derived from greenprecursors (green c-dots)

Initially, synthesis of c-dots was limited to use of carbonaceousmaterials, which resulted in c-dots with low quantum yield (QY)and limited solubility. Further developments resulted in surfacepassivated c-dots with better solubility and QY; however, thetrend for green synthetic approaches led to ever-increasingattention on green synthesis of c-dots. ‘Green’ c-dots can bedefined as carbon quantum dots synthesized using ‘greenprecursors’ as the source of carbon; the term ‘green precursors’refers to substances which are either naturally occurring or arederivatives of renewable natural products or processes.

Various green carbon precursors have been investigated inattempts to achieve a simple, cost-effective, environmentallyfriendly method with exciting properties. The c-dots have beengrouped into different classes on the basis of the chosen greensource (Fig. 1). Most of these green methods were optimizedconcerning the QY achieved; hence this section also discussesthe QY obtained with respect to different green sources.

2.1 Pioneer work

Hsu et al.42 explored use of coffee grounds for synthesis ofgreen c-dots with diameter 5 � 2 nm and a QY of 3.8%. Theyproposed that use of hydrophilic precursors results in high QYcompared with use of hydrophobic precursors. Liu et al.41

reported synthesis of c-dots using hydrothermal treatment ofgrass at 180 1C, the resultant c-dots were nitrogen-doped and

were soluble in water. Elemental analysis revealed the presenceof 41.54 wt% carbon, 4.23 wt% nitrogen, 4.18 wt% hydrogenand 50.05 wt% oxygen and the QY was obtained to be 6.2%. Theauthors observed that higher reaction temperatures led to lowerparticle size and higher QY of c-dots. These pioneering greensynthesis works started a trend which resulted in massiveattention towards green sources for synthesis of c-dots.

2.2 Fruits, fruit juices and fruit peels as carbon sources

Fruit and fruit derivatives encompass an essential and signifi-cant portion of green sources used for c-dot synthesis. Follow-ing from use of grass, fruit sources such as pomelo peel wereused as precursors for hydrothermal treatment at 200 1C, withthe resultant c-dots having an average diameter of 2–4 nm withQY of 6.9%, higher than values for c-dots from grass and coffeeground sources.43 Further improvement in QY to 26% wasachieved by Sahu et al.44 using orange juice as a carbon source.The authors isolated highly fluorescent small c-dots and lessfluorescent big coarse particles simply by controlling thecentrifugation speed (Fig. 2). The synthesis was performed atrelatively low temperature, i.e. 120 1C, and the average particlediameter obtained was 1.5–4.5 nm.

Carbonization of watermelon peel was next explored forc-dot synthesis at 220 1C. The resultant particles were of sizeB2 nm, with QY of 7.1%, along with a fluorescence lifetime of5.72 � 0.05 ns. The elemental analysis revealed 64.65 wt%carbon, 7.67 wt% hydrogen, 1.13 wt% nitrogen, and oxygen wascalculated to be 26.55 wt%.45

In addition to attempts to improve QY, it was also seen as achallenge to increase synthesis yield. Karak et al.31 used bananajuice (Musa acuminate) as a green carbon precursor to achieve ahigh yield of c-dots and claimed to have obtained 58% synthesisyield and a QY of 8.95%. In this process, c-dots were prepared bysimply heating using an oven at 150 1C for 4 h, with the resultantaverage particle size estimated to be 3 nm using a transmissionelectron microscope (TEM). Karak et al. also proposed a possiblemechanism of synthesis which includes hydrolysis, dehydrationand decomposition of various carbohydrates to yield solubleketones, furfural aldehydes and different organic acids. Further,polymerization, aromatization and carbonization followed by anuclear burst are believed to be a route of synthesis. Hydro-thermal carbonization of apple at 150 1C, was reported tosynthesize c-dots with surface functional groups such as hydroxyl,amino, keto and carboxylic acid with a QY of 4.27%.46

Nitrogen-doping in c-dots has gained significant attentionas a potential method to obtain high QY and improve theintrinsic low emission efficiency. The fruit extract of Prunusavium was studied for N-doped c-dots, with aqueous ammoniaused as the nitrogen dopant, to achieve a QY of 13%.47

Other green sources used in this class were papaya48 andpeach,49 which were also subjected to hydrothermal treatmentfor c-dot synthesis. Doping of nitrogen using ethylene diaminewas reported to enhance the QY of peach-derived c-dots from5.31% to 28.46% by passivizing the surface of c-dots. Thehydrothermal treatment of limeade50 and lemon peel51 resultedin c-dots with QY of 53.6% and B14%, respectively.

Shaikh M. Mobin

Dr Shaikh accomplished hisBachelor’s and Master’s fromWilson College, University ofMumbai with major in Chemistryand PhD from Mumbai Universityin Chemistry. In 2012, he joinedIIT Indore and now working as anAssociate Professor in Discipline ofChemistry. He had develop hisresearch group working in widearea of research including Single-Crystal-to-Single-Crystal (SCSC)Transformation, optical and electro-chemical sensing, metal nano-oxide

materials derived by employing metal complexes as single-sourcemolecular precursors as catalyst in organic transformation andgreener c-dots. Moreover, the research group designs and synthesizessmall molecules as cellular organelles target, also cell imaging anddocking.

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Recently, mangosteen pulp52 and pulp-free lemon juice53

have been carbonized to obtain nitrogen-doped c-dots withnitrogen content of 3.16% and up to 15%, respectively. Thehigh nitrogen content also resulted in high QY of 28% in thecase of lemon juice as a precursor. Here, no separate nitrogen

source was used for surface passivation with the intrinsicnitrogen of the biomass being used. The fruit extract ofHylocereus undatus was supplied with ammonia as a nitrogensource by Arul et al.,54 and a nitrogen content of 5.82% wasobtained.

The degree of surface functionalization depends on thegreen precursor used, which in turn reflects variations inoptical behaviour. This highlights the importance of exploringdifferent natural precursors for green c-dot synthesis.

2.3 Beverages as carbon source

Beverages are an important class of edible green sources whichcan be exploited for green c-dot synthesis. Zhu et al.55 used soymilk as a green source and synthesized c-dots using hydro-thermal treatment at 180 1C, where a simultaneous process ofcarbonization, surface functionalization and doping resulted inbifunctional c-dots with a QY of 2.6%.

Instant Nescafe coffee powder was the first beverageexplored for natural presence of c-dots. Jiang et al.56 extractednatural c-dots by simply mixing the coffee powder in hot water

Fig. 1 Some of the green sources used for development of green carbon dots.

Fig. 2 Hydrothermal preparation of c-dots using orange juice. (Repro-duced from Sahu et al.44 with permission from The Royal Society ofChemistry).

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followed by centrifugation and filtration. A relatively highnitrogen content of 7.8% was found in c-dots prepared in thisway, and the QY was 5.5%, higher than that for soy milk-derivedc-dots. The presence of c-dots in such highly consumed day-to-day food items provides clear evidence of non-toxicity andsafe use.

Wang et al.57 envisioned use of milk as an inexpensive andrenewable carbon source. Hydrothermal treatment of milk at180 1C resulted in self-passivated nitrogen-doped c-dots withQY of 12%. The hydrothermal treatment of milk at 180 1C for aperiod of 8 h is reported to synthesize c-dots with averagediameter 5 � 0.27 nm. Sulfur- and nitrogen-doping in milk-derived c-dots using L-cysteine and urea, respectively, causesfurther reductions in average particle diameter to 4 � 0.07 and3 � 0.07 nm. Examination of effect of heating time on QYrevealed that the QY reaches its maximum in 8 h. Under similarconditions, milk c-dots have a QY of 7.55% whereas c-dotsderived from carrot have a QY of 5.55%.58

The presence of c-dots in commercial beer was investigatedby Wang et al.59 In this process, simple condensation using arotary evaporator, chromatographic fractionation by SephadexG-25 gel filtration using water as an eluent, and lyophilizationwas performed to obtain c-dots with QY 7.39%. The method didnot require any hydrothermal or microwave treatment (Fig. 3).

Wei et al.60 observed the presence of natural c-dots in a cupof tea. Even after extraction of natural c-dots from the tea, thescope of carbonization of tea for higher yield remained. Con-sequently, the trend of employing beverages as precursorcontinued by Song et al.,61 who used black tea for hydrothermalcarbonization and nitrogen doped c-dots were obtained.

2.4 Animals and animal derivatives as carbon source

Animals and their derivatives have been used for humanconsumption and hence their possible use as green carbonsources could not be ignored.

Wang et al.62 used chicken egg as a precursor for amphi-philic c-dots considering affordability and reduced toxicity. Thesynthesis was governed by plasma treatment and the synthesis

yield was 5.96%. The presence of nitrogen in proteins allows forthe possibility of self-passivation, doping and high QY. Wuet al.63 reported synthesis of amphoteric nitrogen doped c-dotsusing a natural protein Bombyx mori silk, which has an intrin-sically high nitrogen content of B18%. The synthesis wasperformed using hydrothermal treatment at 180 1C. The struc-tural characterization of the as-prepared c-dots was performedby TEM, XPS and Raman spectroscopy. Elemental analysisrevealed a nitrogen content of 10.45%, higher than that ofc-dots prepared using soy milk, banana juice or grass.

Following the trend of using edible animal derivatives ascarbon sources, Wang et al.64 reported that overcooked beefmeat was a source of carbon for synthesis of c-dots. The authorsstated that the as-synthesized c-dots incorporated a mixtureof various fractions having different surface passivation anddifferent QY, which were separated using Sephadex G-100 gelcolumn to obtain a QY up to 40%. This study corroborates thatthe core structure and composition of c-dots synthesized fromdiverse precursors are intrinsically versatile.

Further, Yang et al.65 conceived the idea of using honey as acarbon precursor and obtained N-doped c-dots with a high QYof 19.8% by low-temperature synthesis at 100 1C. Also, naturaloccurrence of c-dots in raw honey has been reported recently.66

It is clear that use of precursors with multiple elements canlead to self-passivation and heteroatom doping. A major wasteproduct of the poultry industry is feathers, which are rich incarbon, nitrogen, oxygen and sulfur. Use of feathers in synthesisof heteroatom-doped c-dots was envisioned by Liu et al.,67 usinga microwave-assisted method. The elemental composition ofas-synthesized c-dots revealed the presence of 48.4% C, 16.3%N, 1.90% S and 33.3% oxygen. The QY was found to be 17.1%.The high content of oxygen and nitrogen suggests that the c-dotswere rich in surface carbonyl, amide, hydroxyl and carboxylfunctional groups.

The silkworm chrysalis is rich in protein and chitosan, andtherefore has potential as a natural carbon source for synthesisof self-passivation and doped c-dots. This was explored by Fenget al.,68 using microwave-assisted synthesis. The average dia-meter of obtained c-dots was relatively high, i.e. 19 nm with asize distribution of 13–26 nm, and intrinsic nitrogen-doping of5.72% was achieved. The QY was fairly high, i.e. 46%. Themicrowave treatment was chosen because of its reduced timeconsumption, and precise control of pressure and temperatureresulting in uniform c-dots with favourable reproducibility(Fig. 4).

A further improvement in QY was achieved by D’souzaet al.69 using Indian prawn (dried shrimp) for synthesis ofc-dots using hydrothermal treatment. In this process, EtOH :water (1 : 1) was used as a solvent and a hydrothermal treatmentwas performed at 170 1C for 12 h. A very high fluorescencequantum yield of 54% was obtained, with an average particlediameter of B6 nm.

A range of other animal-derived carbon sources such aspigskin (QY 24.1%),70 prawn shell (QY 9%)71 and wool derivedfrom domestic sheep (QY 22.5%)72 have been used for c-dotsynthesis, but, although respectable QY was reported in all

Fig. 3 Isolation of c-dots from commercial beer. (Reproduced fromWang et al.59 with permission from The Royal Society of Chemistry).


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cases, there were no increases in QY beyond those alreadyreported.

Enhancement of or addition to the properties of green c-dotsbecame the next attraction. The crab shell is a source of chitin,which is a linear polysaccharide widely used in biomedicalapplications. Yao et al.73 used crab shell as a carbon source formicrowave-assisted pyrolysis. To provide magnetic propertiesto these c-dots, metal ions such as Gd3+, Eu3+ and Mn2+ werechosen as dopants having seven, six and five unpaired elec-trons, respectively. Dried crab shells were ground and dissolvedin 1% acetic acid solution with stirring at room temperature toremove calcium carbonate from the crab shell. The requisitedopant was added to the supernatant solution which wassubjected to microwave pyrolysis at 220 1C for 10 minutes.The purified products were termed as Gd@CQDs, Mn@CQDs,and Eu@CQDs, respectively (Fig. 5). The maximum QY wasfound in the case of Gd3+ doping (19.84%), followed by Eu3+

(14.97%) and Mn2+ (12.86%).Recently, Lin et al.74 subjected lactic acid bacteria Lactobacillus

plantarum to hydrothermal carbonization. The as-synthesized

c-dots, termed CDs-605, were negatively charged, self-passivatedand composed of C, N, O, H, S and P, eliminating the need forany additional effort for hetero-atom doping, which is known tohave a positive impact on the properties of c-dots. A culture ofL. plantarum grown overnight was heat-treated at 200 1C for 24 hto achieve CDs-605, with QY of 16%.

2.5 Flour and bakery products as carbon sources

Chattopadhyay et al.75 explored the presence of carbon nano-particles in food caramels, namely jaggery, bread and sugarcaramel, in an attempt to alleviate any nanoparticle-associatedtoxicity, with the derived c-dots showing relatively lower QY of0.55%. 1.2% and 0.63%, respectively. The particle size wasfound to be 4–30 nm. Qin et al.76 used flour as a carbon sourcefor microwave-assisted synthesis of c-dots with diameter in therange of 1–4 nm. They explored the effects of temperature andconcentration on particle size, and revealed that decrease inreactant concentration and increase in temperature leadto a smaller size of c-dots. In this procedure, concentrationsranging from 20 mg mL�1 to 120 mg mL�1 were used againsttemperature ranging from 160 1C to 200 1C, with time keptconstant at 20 min. Four kinds of c-dots were produced withsize range 1–30 nm. The maximum QY obtained was 5.4%.

Oatmeal is known to have a high carbohydrate and proteincontent, suggesting its potential in synthesis of self-passivatedc-dots. Yu et al.77 succeeded in obtaining a high QY of 37.40%by hydrothermal treatment of oatmeal at 200 1C, resulting inN-doped c-dots with 2.92% N content.

2.6 Human derivatives as carbon source

The attractive potential for tuning properties of c-dots by self-passivation and heteroatom-doping led researchers to attemptco-doping of multiple elements in c-dots. In the first such useof human derivatives, Sun et al.78 developed nitrogen andsulfur co-doped c-dots (S–N–C dots) from hair fibers, to give apromising low-cost green source of carbon. However, the use ofsulfuric acid as a sulfur source is controversial in terms of greenmethods. The particle size was found to be smaller at a highertemperature, which is in agreement with Qin et al.76 The hightemperature also resulted in a higher S content and highemission wavelength. The effect of temperature was also seenin the c-dot yield, where 40 1C, 100 1C and 140 1C temperatureyielded 9%, 16% and 29% of mass yields, respectively. TheS–N–C-140 dots showed maximum S and N contents of 5.08%and 5.64%, respectively. The maximum QY was found to be11.1% in the case of S–N–C-40.

In the next development, Liu et al.,79 demonstrated solvent-free synthesis of c-dots by thermal decomposition of humanhairs under nitrogen atmosphere at 300 1C, resulting in highlyfluorescent and polymer compatible c-dots without any surfacepassivation. Keratin, the main component of hair, is rich in C,N and O contents, hence the derived c-dots were N-doped andwere shown to have a QY of 17.3% as multidimensionalfluorescent hybrids (Fig. 6).

In another solid-state synthesis, Guo et al.80 carbonizedhuman hair at 200 1C and claimed to achieve a synthesis yield

Fig. 4 Microwave-assisted synthesis of c-dots from live silkwormchrysalis having blue photoluminescence. A and B contained different con-centrations of c-dots, i.e., 5 and 0.83 mg mL�1, respectively. (Reproduced fromFeng et al.68 with permission from The Royal Society of Chemistry).

Fig. 5 Preparation of magnetofluorescent c-dots using crab shell as acarbon source. (Reproduced from Yao et al.73 with permission from TheAmerican Chemical Society).


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of 95%, the highest to date. However, the QY obtained was10.75%, which is lower than from hydrothermally hair-derivedc-dots.81

In an interesting piece of work by Essner et al.,82 thermalupcycling of human urine was executed to obtain c-dots,termed ‘‘pee-dots’’. The remediation of waste material suchas sewage, along with the worldwide issue of polluted water, isan important environmental concern and upcycling not onlyremoves pollutants but also generates cleaner end-products.Interestingly, ‘‘dietary doping’’ has been attempted in theurine sources to alter the optical properties of these c-dots.An unmodified diet, an asparagus-rich diet and vitaminC-supplemented dietary intake resulted in different constituentsin the obtained urine, which was heated at 200 1C for 12 hours toachieve carbonization.

2.7 Vegetables and spices as carbon sources

The impressive results from use of fruits as carbon sources forc-dot synthesis led to exploitation of other plant-derivedsources including vegetables and spices for this purpose. Yinet al. obtained a high QY of 19.3% from hydrothermal treatmentof sweet red pepper at 180 1C.83 In this case the QY increasedwith increasing reaction temperature up to 180 1C. Li et al.84

synthesized intrinsic nitrogen-doped c-dots by hydrothermaltreatment of ginger.

A high yield self-passivated c-dot synthesis methodology wasintroduced by Alam et al.,85 using cabbage as the carbonsource. In the environmentally friendly method, a domesticfruit juicer was used to smash the cabbage for hydrothermaltreatment, with the crushed cabbage then carbonized at 140 1Cfor 5 h. The product was further centrifuged and dialysed asshown in Fig. 7. Carbon, nitrogen and oxygen were present inthe atomic rations of 66.5%, 4.61% and 28.73%, respectively,and the QY was found to be 16.5%. The synthesis yield was7.076%, which was higher than some other reports and it wasclaimed that the methodology could be used for large-scalesynthesis.

Li et al.86 reported synthesis of nitrogen-doped c-dots witha QY of 9.3% by carbonization of Chinese yam at 200 1C.

Another example of self-passivated c-dots was given by Zhaoet al.,87 with garlic as the sole source of carbon, nitrogen andsulfur for synthesis of N,S co-doped c-dots, with 6.9% N and1% S in the resultant c-dots having QY of 17.5%.

Onion waste is rich in dietary fibres and non-structuralcarbohydrates, and thus was used to make N-doped c-dotsusing ethylenediamine as a nitrogen source. This hydrothermalmethod provided a production yield of 6.06%, with a highnitrogen content of 9.34%.88

Liu et al.89 reported use of rose-heart radish for synthesis ofc-dots, with ammonia used as the nitrogen source for achievingsurface passivation, obtaining a QY of 13.6%.

Direct hydrothermal carbonization of carrot juice was per-formed by Jin et al.,90 who also conjugated the c-dots with Nileblue and polyethyleneimine to obtain CD/PEI/NB composite.

Hu et al.,91 for the first time, used two natural biomasses toobtain heteroatom-doped c-dots. Water chestnut and onionwere heated together in an autoclave at 180 1C to obtain Sand N co-doped c-dots with a QY of 12%. To obtain a longemission wavelength and high QY, the ratio of water chestnutto onion was optimized to be 2 : 3. The authors stated that highreaction temperature and a short reaction time were favourableto obtain small sized c-dots.

2.8 Waste material as a source of carbon

Wei et al. synthesized c-dots using paper ash92 and wastepaper93 by simple carbonization and hydrothermal treatment,respectively. The paper ash method presents uncontrollablereaction conditions and open flames, whereas the hydrothermalmethod offers more control of the reactant and reaction condi-tions. Both methods resulted in satisfactory QY of 9.3% and10.8%, respectively. Interestingly, the hydrothermally synthesizedc-dots were nitrogen-doped with a marginal N content, the atomicC : N : O was 74.1 : 1.8 : 24.1, while the paper ash driven c-dotscontained 68.7 wt% C, 7.2 wt% H and 24.1 wt% O (Fig. 8).

Kitchen waste and waste frying oil have been explored aspotential carbon sources, but these yielded very low QY of 3.1%

Fig. 6 Human hair derived c-dots as multidimensional fluorescenthybrids. (Reproduced from Liu et al.79 with permission from The RoyalSociety of Chemistry).

Fig. 7 Cabbage-derived hydrothermally synthesized c-dots. (Repro-duced from Alam et al.85 with permission from The Royal Society ofChemistry).


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and 3.66%, respectively.94,95 Park et al.96 and Purkayasthaet al.97 processed food waste and agricultural waste, respec-tively, into c-dots. In a room-temperature ultrasonic-assistedmethod, 100 kg of food waste was converted to 120 g of c-dots.Oilseed press cake/spent meal was used for c-dot synthesis byhydrothermal carbonization at 180 1C under reflux conditions,with QY found to be 9.2%.

Vegetable waste such as onion peel,98 cornstalk99 andpseudo banana stem100 have been explored recently for c-dotsynthesis. Microwave treatment of onion peel in an ethanolicalkaline solvent results in N–S–P co-doped c-dots because of thepresence of polyphenols and minerals such as phosphorus,sulfur and nitrogen. However, quantification of the differenthetero-atoms was not provided in the studies.

2.9 Plant leaves and derivatives as a source of carbon

Successful use of edible plant derivatives such as fruits andvegetables indicated the possibilities for use of non-edibleplant parts for c-dot synthesis.

Liu et al.101 synthesized branched polyethylenimine cappedc-dots (BPEI-CQDs) using leaves of bamboo. In this procedure,the leaves were embrittled with liquid nitrogen and the resultantpowder was subjected to hydrothermal treatment at 200 1C for6 h. The cationic BPEI was physically absorbed on CQD usingvigorous stirring, resulting in surface-passivized CQD withQY 4 7.1% and average particle diameter of 3.6 nm.

After developing amphiphilic c-dots using eggs, Wang et al.reported c-dots using s mixture of plant leaves102 and humanhair79 using simple pyrolysis. A series of plant leaves includinglotus, camphor, ginkgo pine, palm, osmanthus, bamboo, orien-tal plane and maple were pyrolyzed in a temperature range250–400 1C under a nitrogen atmosphere, without any solvent.

The optimum temperature was found to be 350 1C; it is believedthat carbonization is incomplete at lower temperatures andthat higher temperatures lead to over oxidation of c-dotsreducing the PL intensity. The optimum QY was 16.4%,15.3% and 11.8% for oriental plane, lotus and pine leaves,respectively. Interestingly, plasma treatment and microwaveirradiation of the pyrolyzed leaves resulted in enhancement ofPL intensity up to 25% and 100%, respectively. The authorsstated that the radiation treatment contributed to monodispersionand uniform size particles with additional functionalization, lead-ing to higher PL intensity (Fig. 9). Gao et al.103 reported use ofwillow leaves as a natural carbon source for c-dot synthesis.

Microwave irradiation of rose petals104 is another potentialgreen method. Gopinath et al.105 reported in situ self-passivation of c-dots synthesized using coriander leaves underhydrothermal carbonization at 240 1C. Xu et al.106 used aloe as agreen carbon source for hydrothermal treatment and preparedc-dots with QY of 10.37%. Thambiraj et al.107 used sugarcanebagasse pulp in a low-temperature synthesis methodology usingcombustion at 60 1C followed by extraction and ultrasonication.

Ramanan et al.108 used harmful eutrophic algal blooms asa carbon source and used a domestic microwave oven forsynthesis of c-dots. Conversion of such a harmful waterpollutant into c-dots is of great interest. Shiitake mushroomis an edible fungus, which is high in nutrition and rich incarbohydrate, protein, lipid and amino acids. Exploiting thepresence of high carbon and nitrogen with carboxyl and aminegroup, Wang et al.109 synthesized self-passivated c-dots byhydrothermal treatment of Shiitake mushroom with 5.5% QYand 6% nitrogen content.

Another non-edible plant source was Ocimum sanctum, theleaves were hydrothermally treated to obtain c-dots with QY9.3%.110 Amjadi et al.111 synthesized nitrogen-doped c-dots usinghydrothermal treatment of brown lentil and obtained a QY of 10%.

3. Properties

C-Dots primarily consist of C, H, N and O elements, which arepresent in the form of various functional groups, and provide

Fig. 8 Synthesis of paper ash-driven c-dots. (Reproduced from Weiet al.92 with permission from The Royal Society of Chemistry).

Fig. 9 Plant leaf-driven c-dots with plasma and microwave irradiation-enhanced fluorescence. (Reproduced from Wang et al.102 with permissionfrom The Royal Society of Chemistry).


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good water solubility with scope for further functionalization.The advantage of green sources over chemical entities is thatmost of these methods do not require separate reactants fordoping, post-modifications or surface passivation becauseof the presence of various carbohydrates, protein and otherbiomolecules which provide self-passivation to green c-dots.This section briefly describes some of the most importantproperties of green c-dots.

3.1 Structural properties

Powder X-ray diffraction (XRD) of c-dots usually shows anamorphous nature because of disordered carbon atoms. ThePXRD spectra of green c-dots typically show a broad diffractionpeak in the 2y range 201–251 and interlayer spacing (d) between0.31 and 0.38 nm.68,82,98,109 The deviation in d-spacing value ofthe (002) plane from the normal graphene sheet represents theslight graphitic nature of c-dots with increased amorphousnature. The average particle size/diameter in most greenc-dots is less than 10 nm with spherical shape; however, excep-tions do exist, for example, silkworm chrysalis-made c-dots werefound have size of 19 nm.68 As shown in Fig. 10, lemon juice-derived c-dots (R-CD)53 exhibit a broad diffraction peak at 251 withd-spacing of 0.32 nm and an average particle size of 4.6 nm. AFMshowed thickness of about 1.5 nm corresponding to 3–5 graphene

sheets, with a high degree of graphitization as determined by theRaman spectrum.

The elemental composition and surface functional groups ofc-dots can be analysed by XPS and FTIR. A classic example ofself-passivated c-dots is the use of onion peels as a carbonsource. The XPS spectrum (Fig. 11) shows presence of nitrogen,phosphorus and sulfur in c-dots without any additionalreagent.98 The FTIR spectrum of c-dots was employed forsurface functional group determination of garlic-based c-dots.87

Typical peaks at 1720 and 1620 cm�1 were assigned to CQOand CQC, and stretching vibration of O–H was observed atB3450 cm�1. The presence of C–S could be affirmed with a peakat 680 cm�1 and presence of C–N exhibited a peak at 1400 cm�1.

3.2 Optical properties

3.2.1 Absorbance. The UV-visible absorption spectra ofgreen c-dots are typically shown to have a peak in the UV regionwith a tail extending in the visible region. The absorptionlocated in 230–270 nm is generally attributed to p–p* transitionof CQC and the peak/shoulder located around 300–330 nm isattributed to n–p* transition of CQO.61,80,108,109 Hu et al.91

synthesized dual biomass-derived S/N co-doped c-dots andassigned the peak at 333 nm to the trapping of excited state

Fig. 10 (a) TEM and HR-TEM, (b) AFM (c) PXRD (d) Raman spectra of R-CD derived from lemon juice. (Reproduced from Ding et al.53 with permissionfrom The Royal Society of Chemistry).


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energy. Yao et al.73 also obtained a minor peak at 350 nmbecause of trapping of excited state energy of surface groups.

3.2.2 Fluorescence and wavelength tuned emission.Fluorescence is the most exciting property of green c-dots. Asize-dependent absorption or emission is a salient feature ofquantum confinement. Wavelength tuned emission is a widelyobserved phenomenon in c-dots. There are a number of possibleexplanations for this, including size dependence, surface defects,surface states, the degree of oxidation etc. Wang et al.48 ascribedthe l tuned emission in papaya-based c-dots to optical selection of

surface defect states near the Fermi level. Wei et al.60 exploredthe optical properties of tea-derived c-dots (CND), which showa single absorption peak at 273 nm corresponding to p–p*transition (Fig. 12(a)). Interestingly, the fluorescence behaviorwas unusual, with two peaks seen in PL spectra when the CNDwere excited at l – 320–400 nm. The first peak showed anexcitation-dependent emission and shifted from 447 nm to464 nm because of particle size distribution and surfaceemissive traps. The second peak located at 516 nm wasattributed to the presence of Mn(II) in CND, and showed

Fig. 11 (a) XPS survey scan (b) C 1s, (c) O 1s, (d) N 1s, (e) S 2p and (f) P 2p spectra of onion-derived carbon nanodots. (Reproduced from Bankoti et al.98

with permission from The Royal Society of Chemistry).

Fig. 12 (a) Absorption and PL spectra (lex. – 360 nm) (b) excitation-dependent emission, in tea-derived carbon nanodots. (Reproduced from Wei et al.60

with permission from The Royal Society of Chemistry).


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enhanced fluorescence with red shift in excitation wavelength(Fig. 12(b)).

The wavelength tuned emission made it possible to obtainblue, green, yellow and red emission from c-dots. However, theQY obtained for yellow and red colors was relatively very low.112

In pursuit of this, efforts were made towards excitation-independent long wavelength emission in c-dots. Long wave-length emission in chemically synthesized c-dots has beenadequately reported, and individual colored emissions such asyellow,113–116 yellow-green,117 orange,118,119 and red120,121 wereobtained. However, only a handful of reports described longwavelength emission in green c-dots. Ding et al.53 used lemonjuice for synthesis of red emitting c-dots (R-CDs) using ahydrothermal-assisted method. The hydrothermally synthe-sized c-dots, which were a mixture of c-dots with different PLcolors, were purified using silica column chromatography toobtain R-CDs. The R-CDs exhibited a high QY of 28% with anexcitation-independent emission maximum at 631 nm. Toexplore the role of surface states in red emission, the R-CDswere reduced with NaBH4, resulting in orange emissive reducedR-CDs with the emission maxima blue-shifted to 589 nm(Fig. 13). The long wavelength emission offers deep tissueimaging, low background signal and reduced destruction ofbiological samples.

The optical properties of c-dots remain controversialbecause of lack of exact mechanism. Various explanations havebeen given, which hints towards co-existence of multiple phe-nomena like quantum confinement, emissive traps, surfacedefects, aromatic structure etc.

3.2.3 Up-conversion photoluminescence. Another interest-ing optical behavior observed in green c-dots is the presence ofup-conversion fluorescence, where the emission wavelengthis shorter than the excitation wavelength. Sun et al.78 reportedup-conversion in hair-derived, S and N co-doped c-dots. In thiscase, the emission wavelength shows a red shift with increasingexcitation wavelength from 600 to 900 nm. Multiphoton activeprocess and anti-stokes PL form the most commonly used mecha-nism responsible for up-conversion PL. Up-conversion fluorescencebehavior was observed in many other green c-dots.46,56,58,63,65,83,85

4. Sensors based on green carbon dots

The very small size, high surface to volume ratios and highsurface functionalization makes carbon dots very reactive andthey can easily interact with any chemical species, whichultimately results in changes to their properties, in particular,

optical properties. The tunability in the emission spectrum,narrow spectral bands and easy functionalization makes c-dotsan excellent candidate for fluorescence-based specific sensingapplications.122

4.1 Metal ion sensors

Metal ions interact with the carbon dots via surface bondingwith high selectivity and sensitivity, which results in formationof new electron–hole recombination via an energy transferprocess and results in change in fluorescence intensity ofc-dots and acts as a measurable response signal.41 A range ofvarious metal ion sensors developed using green c-dots isdescribed in this section.

4.1.1 Mercury (Hg2+) sensor. The heavy metal ion mercuryposes a serious environmental threat because of its hightoxicity and bioaccumulation. Green c-dots have beenemployed for selective and sensitive Hg2+ detection at variousoccasions. Sun et al.43 synthesized green c-dots which canselectively sense Hg2+ with a detection limit of 0.23 nM. In thisstrategy, Hg2+ interacted with greater affinity with carboxylgroups present on the surface of c-dots, resulting in creationof new non-radiative electron–hole pairs and leading to fluores-cence quenching of carbon dots. Apart from this, the synthe-sized c-dots exhibited an increase in fluorescence intensity withincrease in pH (from pH 1 to pH 7) and then a gradual decreasein the range pH 8 to pH 13. The authors also employed thesensor for real samples. The same group employed flour-derived c-dots for Hg2+ detection with a detection limit of0.5 nM and linear range 0.0005–0.01 mM.76 This system wasalso pH-dependent and was more effective in basic pH. Chenet al.123 used vitamin source folic acid as a carbon source forsynthesizing nitrogen-doped c-dots (N-CQDs) for Hg2+ detec-tion. The folic acids have –NH2 and –COOH functional groupspresent in the structure, which results in various oxygenatedand nitrous groups on the c-dot surface. The N-CQDs interactswith Hg2+ and causes a non-radiative electron transfer. Also, theHg2+ induces conversion of the –CONH– functional group toopen ring amide from bridged cyclic amide and contributes tofluorescence quenching.124 Further, it was observed that thec-dots-Hg2+ system is sensitive to iodide and results in removalof Hg2+ from carbon dot surface and recovery of fluorescence ofcarbon dot.

Feng et al.125 used strawberry as the carbon source forsynthesis of fluorescent nitrogen-doped carbon nanoparticles(FNCPs) for selective Hg2+ sensing in the linear range 10 nM to50 mM and detection limit of 3 nM. Yeng’s group achieved adetection limit of 1 nM using tea-derived c-dots for Hg2+ withreal sample applicability.60

Human derivatives such as pee and hairs have also beenused successfully as Hg2+ sensors.80,82 Hair-derived c-dots(CQD) showed high selectivity and sensitivity towards Hg2+

with a limit of detection of 10 nM and linear response in therange of 0 to 75 mM (Fig. 14). The CQD showed dynamicquenching with a rise in fluorescence lifetime from 5.15 ns to6.22 ns as a function of Hg2+ concentration. On the other hand,the UV visible absorption peaks of CQD disappeared after

Fig. 13 Synthesis of red-emitting lemon juice-derived c-dots andorange-emitting NaBH4 reduced c-dots. (Adapted from Ding et al.53).


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addition of Hg2+, indicating a static quenching mechanism.Hence, it was concluded that both static and dynamic quench-ing coexist in this case.

Chen et al.126 made carbon dots using Jinhua bergamot,which acts as a dual sensor for Hg2+ and Fe3+ ions. Hg2+ atomsquench the fluorescence intensity of carbon dots via staticquenching. The detection limit was 5.5 nM for Hg2+ and thelinear range was between 0.01 and 100 mM, whereas for Fe3+ thedetection limit was 0.075 mM and the linear range 0.025–100 mM.

4.1.2 Lead (Pb2+) sensor. Another toxic heavy metal, lead(Pb2+), has gained increasing attention for its drastic effects onthe environment, humans and animals. Sahu et al.110 used anOcimum sanctum-derived green c-dot sensor for Pb2+ with detec-tion limit 0.59 nM and linear detection range of 0.01–1.0 mM.The selective nature of this sensing system results from efficientbinding affinity between the vacant d-orbitals of Pb2+ ions andamine groups present on the c-dot surface. The nitrogen atomspresent on the surface of c-dots donate electron pairs to vacantd-orbitals of Pb2+ ions and cause non-radiative electron transfer.This leads to quenching of c-dots in the presence of Pb2+.

4.1.3 Iron (Fe3+) sensor. Fe3+ ions play a vital role inbiological functions and any abnormalities can cause severedisease such as heart failure, Alzheimer, Parkinson etc., hencedetection in the biological system is imperative. The first workon Fe3+ detection using green c-dots was reported by Chenet al.102 using leaf-derived c-dots. The c-dots synthesized usingoriental plant leaves detected Fe3+ by a fluorescence quenchingprocess. The linear response was found to be in the range of0–100 mM. Dou et al.65 used honey-derived c-dots for Fe3+

sensing, which was governed by the interaction of Fe3+ ionswith various functional groups such as –COOH, –OH and –NH2

present on the surface of c-dots. These interactions result inaggregation of c-dots and lead to fluorescence quenching with a

detection limit of 1.7 � 10�9 mol L�1. This sensor was alsoemployed for Fe3+ detection in human blood samples, withsatisfactory recovery. Animal derivative wool-derived nitrogen-doped c-dots were used by Shuang et al.72 for Fe3+ detectionwith LOD 10 nM.

In recent years, Fe3+ has been one of the most exploredmetal ions detected using green c-dots derived from corianderleaves105 (LOD – 0.4 mM), papaya48 (LOD – 0.29 mmol L�1), roseheart radish89 (LOD – 0.13 mM), Prunus avium47 (LOD – 0.96 mM),and banana100 (LOD – 6.5 � 10�9 M) etc.

Onion waste-based c-dots88 could detect Fe3+ in linear range0–20 mM and LOD 0.31 mM. The TCSPC data revealed a decay influorescence life-time from 5.71 ns to 4.89 ns as a function ofFe3+ concentration, exhibiting dynamic quenching. This sensoralso worked well with tap and lake water samples.

4.1.4 Copper (Cu2+) sensor. The pioneer green c-dots(nitrogen-doped carbon-rich photoluminescent nanodots,PPNDs) synthesized by Sun et al.41 using grass can be used tosense selectively the presence of Cu2+ ions via fluorescencequenching, with LOD as low as 1 nM. The PL quenching arisesfrom both static and dynamic modes. The presence of nitrogenand oxygen atoms in PPNDs makes the sensing system highlyselective because of higher thermodynamic affinity and fasterchelating with Cu2+. It has been reported that Fe3+ interfereswith Cu2+ detection, but this effect can be circumvented usingsodium hexametaphosphate as a chelating agent for the Fe3+

ions. Bamboo leaf-derived c-dots101 were capped with BPEI forselective detection of Cu2+ ions, with a limit of detection of115 nM. The increasing Cu2+ concentration caused PL quench-ing along with a blue shift in emission resulting from adsorp-tion of Cu2+ and desorption of BPEI from the c-dot surface.

4.1.5 Cobalt (Co2+) sensor. Shuang and coworkers70 usedpigskin-derived nitrogen-doped c-dots (N-CDs) for detection of

Fig. 14 Human hair-derived CQD for selective sensing of Hg2+. (Reproduced from Guo et al.80 with permission from nature publishing group).


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Co2+ ions. The detection limit of the sensing system was 6.8 �10�7 M. The sensing capacity was in the range of 1 � 10�6 M to3 � 10�4 M. The excitation and emission spectra of N-CDs wereshown to have precise overlapping with absorption spectra ofCo2+, leading to the fluorescence quenching of NCDs by aninner filter effect. Both dynamic and static quenching mechanismswere stated to be occurring in this sensing system.

4.1.6 Gold (Au3+) sensor. Zhou et al.49 used nitrogen-dopedcarbon dots derived from peach gum as a gold ion sensor withLOD 6.4 � 10�8 M. The sensing response time was less than aminute. The selective sensing behavior resulted from a synergisticeffect of electron transfer and fluorescence resonance energytransfer between nitrogen-doped c-dots and Au nanoparticles.The Au3+ can cause electron transfer from N-CDs to Au3+, andFRET can happen as Au particles act as an acceptor to absorb lightemitted by nitrogen-doped c-dots.

4.1.7 Chromium (Cr6+) sensor. High-valent chromium ionCr6+ is carcinogenic and highly toxic because of its highoxidation state and high mobility. It is also associated withgeneration of reactive oxygen species and hence has toxicimplications.127 Tyagi et al.51 used lemon peel-derived c-dotsfor sensing of Cr6+ in linear range 2.5–50 mM with LOD of73 nM. The presence of a vacant d-orbital in Cr6+ and low lyingd–d transition state assist non-radiative electron–hole pairrecombination and lead to fluorescence quenching of c-dotsin the presence of Cr6+.

4.2 Anion sensors

4.2.1 Hypochlorite (ClO�) sensor. Hypochlorite is animportant reactive oxygen species (ROS) which acts as a

microbicidal mediator in the immune system and is also usedfor treating water or food surfaces. The high level of hypochloritecan be a health hazard. Yao et al.83 explored sweet pepper-deriveddown and upconversion fluorescent c-dots as a dual readout assayprobe for detecting hypochlorite (ClO�). It was reported thatvarious hydroxyl groups present on the c-dot surface act as reducinggroups and are oxidized in the presence of ClO� group. Thischange in the surface state leads to PL quenching. The quenchingbehavior is observed during both down and up-conversion withsimilar detection limits of 0.05 and 0.06 mg mL�1, respectively.Zhan et al.128 used fish scale-derived nitrogen-doped c-dots fordetection of hypochlorite in the range 0–10 mM via fluorescencequenching. The hypochlorite sensing was attributed to selectivephotoinduced electron transfer from N atoms to ClO�.

4.2.2 Sulfide (S2�) sensor. A ratiometric two-photon turn-on probe for sulfide (S2�) detection was developed by Sunet al.,90 using carrot-derived green c-dots. These c-dots wereconjugated with polyethyleneimine (PEI) and Nile blue (NB)chloride to develop CDs/PEI/NB nanocomposite. The fluores-cence of CDs/PEI/NB was quenched by Cu2+ ions via an innerfilter effect. The addition of S2� leads to recovery of quenchedfluorescence by combining with Cu2+ ions to form stablespecies and separates Cu2+ from c-dots. The sensing systemwas highly selective for S2� ions in the concentration range0.1–8 mM and a LOD of 0.06 mM was obtained (Fig. 15).

4.2.3 Thiosulfate (S2O32�) sensor. The pseudostem of

banana plant-derived c-dots was employed as a turn-on sensingprobe for S2O3

2� detection.100 The c-dots showed quenching inthe presence of Fe3+, which could be recovered using S2O3

2�.The LOD of the CD/Fe3+ system for S2O3

2� was 8.47 � 10�7 M.

Fig. 15 Carrot juice-derived c-dots composited with PEI and NB for ratiometric turn-on sensing of S2�. (Reproduced from Sun et al.90 with permissionfrom Elsevier).


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The ‘‘on–off’’ behavior of the c-dots was attributed to thecarboxyl groups on the surface and the affinity of Fe3+ towardsoxygen. However, a strong interaction between S2O3

2� and Fe3+

derived recovery of fluorescence by means of dissociation ofFe3+ from the surface of c-dots.

4.3 pH sensors

pH homeostasis has important physiological significance andhence development of pH sensors is an area of keen research.The waste frying oil-derived sulfur-doped c-dots95 exhibitpH-sensitive behavior and are reported to act as a pH sensorin the range 3 to 9 (Fig. 16). The fluorescence intensity shows alinear increase in pH range 3 to 9 and also shows reversiblebehavior. The pH-responsive behavior resulted from increas-ing degree of deprotonation with increasing pH leading to ahigher concentration of carboxyl groups on the c-dot surface,which act as a fluorophore species, hence the intensity isincreased. However, in pH range 9–12 the carboxyl groupattains maximum concentration and further increase in intensityslows down.

4.4 Chemical sensor

Antibiotics are used in therapy against infectious diseasecaused by bacteria. Tetracycline is a commonly used antibiotic;however, its overdose can cause side effects such as abnorm-ality in growth and formation of teeth. Yang and colleagues104

used rose flower-derived c-dots as a sensor for tetracycline bymeasuring variation in fluorescence intensity. The LOD was3.3 � 10�9 mol L�1 and the linear range was 1 � 10�8 mol L�1

to 1 � 10�4 mol L�1.Tartrazine is a yellow-colored synthetic compound used as

a colorant in the food and beverages industry. However, it hastoxic effects such as neurobehavioural toxicity, and toxicity tothe reproductive system. Also, it causes allergic and intoler-ance reactions among asthmatics patients, and aspirin toler-ance. Liao and group106 used aloe-based carbon dots toselectively sense tartrazine through fluorescence quenchingin c-dots with a detection limit 73 nM and linear range of0.25–30.25 mM. The quenching of fluorescence arises fromground complex formation between tartrazine and the c-dots.The sensor worked satisfactorily in real samples.

4.5 Temperature sensors

A green strategy for development of thermosensitive c-dots wasreported by Wu et al.129 using egg white as a carbon source. Theas-synthesized c-dots were used for making microgel, whichresponds in the temperature range 20–54 1C with good rever-sibility under a continuous heating–cooling cycle.

4.6 Bacterial sensor

The upsurge in multi-drug resistant bacteria means that it isimperative to develop sensors for detection of bacterial specieswith high sensitivity and selectivity. Papaya-derived c-dots wereused as a sensor for pathogenic bacterial species E. coli.O157:H7 with a detection limit of 9.5 � 104 cfu mL�1. Thesensor responded linearly in the range of 105–108 cfu mL�1.The presence of E. coli O157:H7 resulted in a significantincrease in the fluorescence intensity of c-dots. The enhance-ment in fluorescence was attributed to interaction of mannosepresent in c-dots with the FimH proteins on the tip of thefimbriae of E. coli O157:H7, providing the basis for quantifica-tion of bacterial species. However, the response of this sensortowards other bacterial species was not discussed.

5. Green carbon dots as bioimagingprobes

The inherent fluorescence, higher resistance to photobleach-ing, high aqueous solubility, improved biocompatibility andlow cytotoxicity of c-dots make them ideal as candidates forbioimaging candidates. These excellent optical and biologicalproperties have resulted in extensive studies on c-dots forbioimaging. This section summarizes some of the greenc-dots applied for cellular and animal imaging.

5.1 Mammalian cell imaging

The extremely small size of c-dots means they can be taken upeasily by cells which can be imaged for intracellular fluores-cence. Chang et al.,42 used coffee grounds-derived c-dots forcellular imaging of LLC-PK1 cells. The c-dots were internalizedby endocytosis and localized in the cell membrane andcytoplasm. Sahu et al.44 used orange juice-derived c-dotsfor imaging of human osteosarcoma (MG-63) cell lines.

Fig. 16 Waste frying oil-derived S-doped c-dots for pH sensing. (Reproduced with permission from Hu et al.95 with permission from Elsevier).


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The excitation-dependent emission was also seen in an intra-cellular environment where lex – 488 nm resulted in greenintracellular fluorescence while at lex – 405 nm, a blue signalwas observed. These c-dots were localized in the cytoplasm andleft the nuclei zone clear. Green c-dots derived from grape juice

and human hair have also been used for imaging cytoplasm ofHeLa cells.78,130 The human lung cancer cell line (A549) wastreated with paper ash-derived c-dots92 and Bombyx morisilk-derived c-dots.63 It was observed that c-dots were able topenetrate the cell nucleus through nuclear pores along with thecell membrane and cytoplasm. Similar whole cell imaging withclear nuclear penetration was observed by Shuang et al.,131

using petals-based c-dots in A193 cells. Alam et al.85 exploitedthe fluorescent nature of c-dots prepared from cabbage forimaging HaCaT cells. Clear wavelength tuned multicolouremission was observed from the cytoplasm and cell membrane.

Gopinath et al.105 used coriander leaf-derived c-dots forimaging of A549 and L-132 cells. The cells were counterstainedwith Hoechst 33342 dye to investigate the nuclear localizationof c-dots. No clear overlap of green fluorescence coming fromc-dots with a blue signal of Hoechst revealed that the c-dotscould not penetrate the nuclear membrane and were distributedin the cytoplasm. Algal bloom-derived c-dots108 exhibit a similarcell membrane and cytoplasmic fluorescence in MCF-7 cells.Waste onion peel-synthesized nitrogen, sulfur and phosphorousco-doped c-dots were also shown to have localization in thecytoplasmic area of MG63 and HFFs cells.98 Cow manure is awaste biomass which was used by Neto et al.132 for bioimaging inMCF-7 cell lines. Interestingly, it was observed that unmodifiedc-dots derived from cow manure could stain the cytoplasm ofcells, whereas the c-dots modified through acid activation couldspecifically stain the nucleoli region of cells. The effect ofcell fixation on subcellular localization was also analyzed; theunmodified c-dots with cell fixation showed dispersed fluores-cence in the cytoplasm region because of an improved associa-tion of c-dots with the cytoplasm target. The higher fluorescencesignal in fixed cells treated with modified c-dots was attributed

Fig. 17 Cellular bioimaging experiments for the modified c-dots with theMCF-7 cell line. (A) and (B) show the modified c-dots staining pattern(green) for the fixed cells, whereas (C) and (D) show staining for live cells.(B) and (D) also show the nucleus staining with commercially availableDAPI (blue). (Reproduced from Neto et al.132 with permission from JohnWiley and Sons Ltd).

Fig. 18 Fluorescence images of (a–d) HeLa and (e–h) HepG2 cells incubated with FA-Gd@CQDs, fixed and stained with NucView agent. (a and e)Bright-field transmission images; (b and f) NucView fluorescence images; (c and g) FA-Gd@CQD fluorescence images; and (d and h) overlay offluorescence images. Scale bars = 40 mm. (Reproduced from Yao et al.73 with permission from American Chemical Society).


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to structural changes in biomolecules because of fixation.A counterstaining study with DAPI was also conducted, andprovided strong evidence of nucleoli staining (Fig. 17). A similarstaining trend has been observed in other cell lines such asMDA-MB-231, Caco-2 and DU145.

In a strategic cell targeting study, waste crab shell-basedGd3+-doped magneto-fluorescent carbon dots73 (Gd@CQDs)were conjugated with folic acid (FA-Gd@CQDs) for specifictargeting of folate receptor-positive HeLa cells and folatereceptor-negative HePG2 cells. It was observed that the bluefluorescence signal was much more intense in receptor-positiveHeLa cells treated with FA-Gd@CQDs compared with receptor-deficient HepG2 cells. The addition of free folic acid to theculture medium led to cellular binding with free folic acid andweak intracellular fluorescence for FA-Gd@CQDs-treated HeLacells. Also, control Gd@CQDs-treated HeLa cells gave very weakfluorescence. These results verify the role of folic acid in specific

HeLa cell targeting as cellular uptake of FA-Gd@CQDs occurs viafolate receptor-mediated endocytosis (Fig. 18). This work givesnew insights into strategic developments in specific cell target-ing using green c-dots.

5.2 Bacterial and fungal cell imaging

Green c-dots are being explored in attempts to achieve real-time, long-term and multiplex imaging of bacteria and fungi.Apple juice-derived c-dots have been investigated for imaging ofbacteria (Mycobacterium tuberculosis and Pseudomonas aeruginosa)and fungal cells (Magnaporthe oryzae), and showed intra-cellular wavelength tuned emission. The c-dots were taken upby the cells through entrapping endosomes, multivesicularbodies, and lysosomes via endocytosis. The hydrophilic natureof c-dots was helpful in internalization in the cytosol andnuclear region.46 The same group further explored pomegranate-derived c-dots for imaging Fusarium avenaceum and

Fig. 19 In vivo bio-distribution of FCP-B, FCP-G and FCP-Y in balb/c nude mice after tail vein injection of 5 mg kg�1 of body weight. The in vivo bio-distribution and corresponding intensities of FCP-B (a and b), FCP-G (c and d) and FCP-Y (e and f) respectively; the normalized intensity from dissectedorgans (n = 3). (Reproduced from Park et al.136 with permission from The Royal Society of Chemistry).


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Table 1 Overview of green carbon dots: synthesis conditions and their applications

Precursor Synthesis methodSyn.temp. (1C)

Heatingtime (h)

Size(nm) QY (%) Sensing

Bio-imaging

Intracellularsensing Ref.

Apple juice Hydrothermal 150 12 4.5 4.27 ‘ | ‘ 46Bagasse Hydrothermal 180 3 1.8 12.3 ‘ | ‘ 141Banana juice Heating 150 4 3 8.95 ‘ ‘ ‘ 31Bee pollens Hydrothermal 180 24 1.1–2.1 6.1–12.8 ‘ | ‘ 142Bombyx mori silk Hydrothermal 190 3 5 13.9 ‘ | ‘ 63Bread Acid oxidation 60 4 2–10 4.5 ‘ | ‘ 143Cabbage Hydrothermal 140 5 2–6 16.5 ‘ | ‘ 85Carica papaya juice Hydrothermal 125–170 12 3 7 ‘ | ‘ 144Chicken egg Plasma 2.2–3.4 6–8 ‘ ‘ ‘ 62Cocoon silk Hydrothermal 200 6–48 70 38 Hg2+ and Fe3+ | ‘ 145Coffee grounds Heating 300 2 5 � 2 3.8 ‘ | ‘ 42Cow manure Chemical oxidation 120–300 72 4.8 65 ‘ | ‘ 132Dried shrimp Hydrothermal 170 12 6 54 ‘ | ‘ 69Enteromorpha prolifera Hydrothermal 180 3–10 2.75 � 0.12 8 Fe3+ | ‘ 146Eggshell membrane Combustion/microwave 400 2 5 14 Glutathione ‘ ‘ 147Flour Microwave 180 20 1–4 5.4 Hg2+

‘ ‘ 76Garlic Hydrothermal 200 3 11 17.5 ‘ | ‘ 87Garlic Microwave 700 Wa 2 minb 5 5 ‘ | ‘ 148Ginger Hydrothermal 300 2 4.3 13.4 ‘ | ‘ 84Grape peel Hydrothermal 180 6 1.5–3.0 3.1 Fe3+ | ‘ 94Grape Juice Hydrothermal 180 12 2.7 � 0.5 13.5 ‘ | ‘ 130Grass Hydrothermal 150–200 3 3–5 2.5–6.2 Cu2+ | ‘ 41Hair fibre Acid treatment 40–140 24 2–10 11.1 ‘ | ‘ 78Hair Thermal treatment 200 24 2–8 10.75 Hg2+

‘ ‘ 80Hair Thermal decomposition 300 2 2.3 17 ‘ ‘ ‘ 79Hair Hydrothermal 200 6 29–80 24.8 Hg2+

‘ ‘ 81Honey Hydrothermal 100 2 2 19.8 Fe3+ | ‘ 65Jinhua bergamot Hydrothermal 200 5 10 50.78 Hg2+ and Fe3+

‘ ‘ 126Konjac flour Pyrolysis 470 1.5 3.37 13/22 Fe3+and L-lysine | ‘ 149Lemon juice Hydrothermal 190 10 4.6 28 ‘ | ‘ 53Lemon Peel Hydrothermal 200 12 1–3 14 Cr6+

‘ ‘ 51Lychee seed Carbonization 300 2 1.12 10.6 Methylene blue | ‘ 150Mango Carbonization 80–100 0.3–1 5–15 0.48–3.92 ‘ | ‘ 136Milk Hydrothermal 180 2 3 12 ‘ | ‘ 57Milk Hydrothermal 180 2–8 3–5 5.86–7.55 ‘ | ‘ 58Neem gum Biogenic RT 3 5–8 ‘ ‘ | ‘ 151Nescafe Heating 90 0.25 4.4 5.5 ‘ | ‘ 56Orange juice Hydrothermal 120 2.5 1.5–4.5 26 ‘ | ‘ 44Orange waste peel Hydrothermal 180 12 2–7 36 ‘ ‘ ‘ 152Onion waste Hydrothermal 120 2 7–25 28 Fe3+ | | 88Onion peel Microwave 1000 Wa 1–3 minb 2–4 ‘ ‘ | ‘ 98Overcooked BBQ Heating ‘ 5 ‘ 40 ‘ ‘ ‘ 64Papaya Hydrothermal 200 5 2–6/8–18 18.39–18.98 Fe3+ | ‘ 48Peanut shell Carbonization 250 2 0.4–2.4 9.91 ‘ | ‘ 153Pigskin Hydrothermal 250 2 3.5–7.0 24.1 Co2+ | | 70Pipe tobacco Hydrothermal 180 8 5 3.2 Cu2+

‘ ‘ 154Plant leaf Pyrolysis 250–400 2 3.7 11.8–16.4 Fe3+

‘ ‘ 102Plant soot Reflux with acid 110 20 2–4.3 0.72–4.28 ‘ | ‘ 137Pomelo peels Hydrothermal 200 3 2–4 6.9 Hg2+

‘ ‘ 43Potato Hydrothermal 170 12 0.2–2.2 6.14 ‘ | ‘ 155Soy milk Hydrothermal 180 3 13–40 2.6 ‘ ‘ ‘ 55Soya bean grounds Heating 260 0.5 13 3 ‘ | ‘ 156Spider silk Hydrothermal 200 72 178 17.6 ‘ | ‘ 157Strawberry juice Hydrothermal 180 12 5.2 6.3 Hg2+

‘ ‘ 125Sugar cane juice Hydrothermal 120 3 2.71 5.76 ‘ | ‘ 135Sweet potato Hydrothermal 180 18 2.5–5.5 8.64 Fe3+ | | 138Sweet potato Hydrothermal 180 3 1–3 2.8 Hg2+

‘ ‘ 158Traditional Chinesemedicine

Hydrothermal 200 3 35 11.5 Hg2+‘ ‘ 159

Trapa bispinosa peel Thermal oxidation 90 2 5–10 1.2 ‘ | ‘ 160Vitamin B1 Carbonization 90–130 2 1–6 76 ‘ | ‘ 161Waste frying oil Heating with acid 100 5 minb 1–4 3.66 pH sensing | ‘ 95Watermelon peels Carbonization 220 2 2 7.1 ‘ | ‘ 45Willow bark Hydrothermal 200 3 1–4 6 ‘ ‘ ‘ 162Willow leaves Hydrothermal 180 24 2–4 ‘ ‘ ‘ ‘ 103

a Microwave power is denoted instead of temperature. b Time in minutes.


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Pseudomonas aeruginosa cells.133 A similar multicolor cytoplasmicfluorescence was obtained.

Das et al.134 applied luminescent c-dots derived from gramfor imaging of E. coli. A clear multicolor emission was obtainedfrom the cell surface, which was attributed to the affinity ofcarboxylic groups of c-dots with the cell surface. On the otherhand, Kailasa et al.135 achieved E. coli. membrane permeationby Saccharum officinarum derived c-dots and an intracellularemission was obtained. A similar trend was observed in thecase of yeast cells.

5.3 In vivo and ex vivo imaging

The in vivo bio-distribution of c-dots derived from mango fruitwas investigated by Park et al.136 The controlled carbonizationof mango fruit yielded different colors of fluorescent carbonparticles termed FCP-B, FCP-G and FCP-Y for blue, green andyellow emitters, respectively. Athymic nude mice were treatedwith FCP for 24 h by tail vein injection, and it was concludedthat bio-distribution is size-dependent and small particles arerapidly cleared from the body. The relatively large-sized FCP-Ywas accumulated in the liver, while FCP-B and FCP-G werefound in the urinary bladder (Fig. 19).

Tan et al.137 performed ex vivo imaging of guppy fishincubated in water treated with plant soot-derived c-dots. Similarsmall animal imaging was exhibited in Nescafe-derived c-dot-treated guppy fish.56

6. Intracellular sensing using greencarbon dots

The elaborate sensing and bioimaging studies of green c-dotsled to efforts to combine the two and sense analytes in anintracellular environment through bioimaging.

The intracellular sensing of pH and Cu2+ was investigated byShuang et al.14 using leek-derived blue fluorescence c-dots(B-CDs). The B-CDs showed turn-on fluorescence in the pHrange 3–11. The intracellular strong blue fluorescence in HeLacells treated with B-CDs at pH = 10 faded as the pH decreased.Similarly, the Cu2+ responsive fluorescence behavior of B-CDswas sustained in live HeLa cells and the fluorescencedisappeared in 60 seconds in the presence of 100 mM Cu2+.

Another important study for intracellular pH sensing camefrom the c-dots (MCDs) prepared by Wang et al.109 usingshiitake mushroom as a green source. The MCDs were respon-sive in pH range 4–8 with high fluorescence at lower pH. In thiscase, the pH sensing was demonstrated by a lysosome localiza-tion study owing to the lysosomal pH in the range of 4–5.5. Thehigher emission from lysosome compared with cytosol wasevident from a lysotracker red overlapping study, which demon-strated the applicability of MCD as a pH sensor and lysosomeimaging agent in living cells.

Shau et al.110 used Ocimum sanctum-derived c-dots for Pb2+

sensing in MDA-MB-468 cells via a fluorescence quenchingphenomenon. Guttena et al.21 successfully detected Fe3+ ions

in HeLa cells by fluorescence quenching of onion-derivedc-dots.88

Hu et al.91 used dual biomass, water chestnut and onion-derived sulfur and nitrogen co-doped carbon dots (S,N/CDs) forquantification and imaging of coenzyme A (coA). The S,N/CDsshowed a turn-off in the presence of Cu2+ by making a S,N/CDs-Cu(II) complex, which acts as a turn-on probe for detection ofcoA. The T24 cells pre-treated with 15 mM N-ethylmaleimideand incubated with S,N/CDs-Cu(II) showed no fluorescence butcells pre-treated with 150 mM coenzyme A and incubated withS,N/CDs-Cu(II) showed stronger fluorescence which confirmsselective imaging of coenzymeA in T24 cells.

Cai et al.138 employed sweet potato-derived c-dots forsensing of Fe3+ in HeLa and HepG2 cell lines, while Lonshakovet al.52 recently used mangosteen pulp-derived c-dots forsensing Fe3+ in yeast cells using a fluorescence quenchingmechanism.

Unfortunately, none of the intracellular sensing work pro-vides a methodology for intracellular quantification of analytesand further work is required to understand the mechanisticviewpoint.

Table 1 summarizes some of the green c-dots with theirfeatures and application in bioimaging or sensing. The greenc-dots offer the following advantages over chemically synthesizedc-dots, making them a promising next-generation material:� The green c-dots are often self-passivated, eliminating the

need for separate passivation agent and any processing forthe same. For instance, the nitrogen and sulfur doping inchemically synthesized c-dots is achieved by strong acids suchas HNO3 and H2SO4.139,140 On the contrary, N, P and S richbiomass such as onion, ginger, garlic etc. itself provides theheteroatom dopant without any hazardous acid treatment.� The synthesis of green c-dots is relatively cheap compared

with expensive chemically synthesized c-dots. The use of wastematerial or abundant biomass such as grass etc. reduces theprecursor cost to almost nil compared with any chemicalprecursors. The low precursor cost provides the capability forlarge-scale synthesis and easy scale-up.� The green c-dots use renewable natural resources which,

in turn, provide sustainability.� The synthesis of green c-dots offers an environmentally

benign approach by minimizing use of hazardous chemicalreactants, providing accident prevention along with obviousenvironmental preservation.� The use of waste material for c-dot synthesis reduces

pollutants and prevents pollution in a cost-effective mannerand offers new avenues for waste management.� The green precursors have an edge over chemical precursors

in terms of availability and affordability.

7. Conclusion and outlook

Carbon dots have proven benefits over conventional semicon-ducting quantum dots and organic fluorophores for bioimagingand sensing applications resulting from their easy synthesis, low


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cytotoxicity and superior optical properties. The obviousadvantages offered by green chemistry have inspired greatefforts in synthesis and applications of green c-dots. Theapparent progress in development of various green c-dots fora variety of applications truly make these a next-generationmaterial, distinguished from other carbon structures. The bio-inspired green precursors could eliminated the requirementfor chemical passivation of c-dots. Most green c-dots are self-passivated because of the presence of various functionalgroups by virtue of carbohydrates and proteins in the greenprecursor itself. The hetero-atom doping achieved by self-passivation has a beneficial impact on the optical propertiesleading to well-versed applications in the bio-medical field.However, the complex precursor composition could lead toheterogeneity in green c-dots and deeper investigation isrequired into separation, purification and homogenization.Further exploration of green sources is still desired.

The synthesis mechanism is still not fully understood andprecise control of size, surface functionality and properties isstill far away. Importantly, diversity in composition of samplesof different origin from the same green source can be a bottle-neck for reproducibility of green c-dots. For instance, thecomposition of human hair, widely explored as a green carbonsource, is affected by aging and genetics. Similarly, the compo-sition of human urine can vary according to different physio-logical and dietary conditions. Hence, the otherness in naturalconditions could be a setback for green c-dots and must beaddressed to enhance the plausibility of green c-dots. One wayto achieve this would be thorough characterization of startingmaterial, ensuring uniformity of green precursors and repro-ducibility of green c-dots.

Green c-dots have shown an excellent response for sensingof metal ions and anions with high sensitivity and selectivity,having applicability in real-life samples. In spite of great atten-tion to metal ion detection using green c-dots, studies for someimportant toxic metals such as As3+, Po3+, Mn2+ and Cd2+ aresparse. To realize the full potential of green c-dots, furtherresearch efforts should be directed towards these metal ions.Also, the readily detected metals such as Hg2+, Fe3+ should betargeted for simultaneous multiple metal detection with appre-ciable selectivity and sensitivity, which may lead to fabricationof devices for real-time detection.

Another exciting area for further development is specifictargeting of cellular organelles with green c-dots. The function-alization of green c-dots with specific receptors for targetingorganelles such as endoplasmic reticulum, golgi body, mito-chondria, lysosome and nucleolus etc. is expected to become areality in the near future. The c-dot based bioimaging probescould be tuned for high intracellular photostability for long-term imaging. The desire of high wavelength emission for deeptissue imaging must be realized for full use of the excellentoptical tuning of green c-dots.

The field of energy and storage is yet to be explored usinggreen c-dots and the enormous potential shown by these nono-lights advocates hopes of achieving green energy systems orgreen electronics using c-dots.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

V. S. gratefully acknowledges UGC, New Delhi for researchfellowship. P. T. thanks MHRD for research fellowship. SMMthanks SERB-DST (Project no. EMR/2016/001113), Govt. of Indiafor a research grant.

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