nanomaterials Article

Cultivating Fluorescent Flowers with Highly Luminescent Carbon Dots Fabricated by a Double Passivation Method Shuai Han 1,2 , Tao Chang 1 , Haiping Zhao 2 , Huanhuan Du 1 , Shan Liu 1 , Baoshuang Wu 1 and Shenjun Qin 2, * 1

2

*

College of Materials Science and Engineering, Hebei University of Engineering, Handan 056038, China; [email protected] (S.H.); [email protected] (T.C.); [email protected] (H.D.); [email protected] (S.L.); [email protected] (B.W.) Key Laboratory of Resource Exploration Research of Hebei Province, Hebei University of Engineering, Handan 056038, China; [email protected] Correspondence: [email protected]; Tel.: +86-0310-857-7902

Academic Editor: Shirley Chiang Received: 22 April 2017; Accepted: 28 June 2017; Published: 7 July 2017

Abstract: In this work, we present the fabrication of highly luminescent carbon dots (CDs) by a double passivation method with the assistance of Ca(OH)2 . In the reaction process, Ca2+ protects the active functional groups from overconsumption during dehydration and carbonization, and the electron-withdrawing groups on the CD surface are converted to electron-donating groups by the hydroxyl ions. As a result, the fluorescence quantum yield of the CDs was found to increase with increasing Ca(OH)2 content in the reaction process. A blue-shift optical spectrum of the CDs was also found with increasing Ca(OH)2 content, which could be attributed to the increasing of the energy gaps for the CDs. The highly photoluminescent CDs obtained (quantum yield: 86%) were used to cultivate fluorescent carnations by a water culture method, while the results of fluorescence microscopy analysis indicated that the CDs had entered the plant tissue structure. Keywords: carbon dots; surface passivation; luminescence; Ca(OH)2 ; flowers

1. Introduction As an emerging class of photoluminescent nanomaterials, carbon dots (CDs) have attracted considerable attention owing to their outstanding photostability, high biocompatibility, and tunable photoluminescence (PL) [1–5]. As this research field evolves, it is now widely recognized that the surface state of CDs significantly affects their fluorescence properties, and consequently their applicability in bioimaging, fluorescent inks, and so on [6–10]. Accordingly, it is especially important to enhance the PL by adjusting the surface chemistry of CDs [11,12]. Moreover, it is important to expand the applications of these types of materials, for example, to novel applications in biological technologies [13,14]. Citric acid, a surface passivation agent containing amino groups, has reportedly been used to prepare highly photoluminescent CDs by a hydrothermal method [15]. However, the active functional groups, such as amino and carboxyl groups, could be overconsumed during the reaction process, which might limit the PL quantum yield (QY) of the product [16]. On the other hand, the radiative recombination luminescence of the excitons could be absorbed by electron-withdrawing groups on the surface of the obtained CDs, such as carboxy and nitro groups, which might further limit the PL efficiency of the CDs [17]. Thus, it would be feasible to enhance the PL QY by choosing an appropriate agent that not only protects the active functional groups from overconsumption during the reaction process, but also reduces the number of electron-withdrawing groups on the CD surface. Nanomaterials 2017, 7, 176; doi:10.3390/nano7070176

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Nanomaterials 7, 176 process, but also reduces the number of electron-withdrawing groups on 2 of 10 during the 2017, reaction the CD surface. With the rapid rapid development development of of nanotechnology nanotechnology and and biology, biology, the the applications applications of of biocompatible biocompatible With the nanomaterials become more and more attractive [18–22]. As a cross study of nanotechnology and nanomaterials become more and more attractive [18–22]. As a cross study of nanotechnology and phytobiology,the thecultivation cultivation of function-specific plants nanomaterials has become a hot phytobiology, of function-specific plants usingusing nanomaterials has become a hot research research topic; for example, modified single-walled carbon nanotubes have been transported into topic; for example, modified single-walled carbon nanotubes have been transported into plant leaves plant leaves to monitor aromatic nitro compounds [23]. As a type of function-specific plant to monitor aromatic nitro compounds [23]. As a type of function-specific plant materials, fluorescent materials, fluorescent flowers, which fluorescence under (UV) light,but arecould not only flowers, which emit fluorescence underemit ultraviolet (UV) light, areultraviolet not only ornamental, also ornamental, but could also facilitate research in plant cell biology, environmental botany, and on; facilitate research in plant cell biology, environmental botany, and so on; consequently, they havesobeen consequently, they have been attracting increasing research interest [24]. To date, fluorescent attracting increasing research interest [24]. To date, fluorescent flowers have been obtained mainly by flowers have been obtained mainly by transgenic technology [10]. However, this technology has transgenic technology [10]. However, this technology has disadvantages; for example, it is not cost disadvantages; for example, it is not cost effective and requires complex operations [25]. effective and requires complex operations [25]. In waschosen chosen as as aa representative representative passivating passivating agent agent to In this this work, work, calcium calcium hydroxide hydroxide [Ca(OH) Ca(OH)22]was to 2+ could protect the carboxyl groups produce CDs with much higher PL. In the reaction process, Ca 2+ produce CDs with much higher PL. In the reaction process, Ca could protect the carboxyl groups of of acidoverconsumption from overconsumption during and dehydration and and the citriccitric acid from during dehydration carbonization, andcarbonization, the electron-withdrawing electron-withdrawing groups could be converted to (–OH) electron-donating groups by the. groups could be converted to electron-donating groups by the hydroxyl ions (–OH) from Ca(OH) 2 hydroxyl ions from Ca(OH) 2. Thus, Ca2+ chelation in combination with hydroxyl group passivation, 2+ Thus, Ca chelation in combination with hydroxyl group passivation, which we refer to as a double which refer to as a double passivation strategy, attempted. Then, highly surfacewe passivation strategy, wassurface attempted. Then, the highlywas photoluminescent CDsthe obtained photoluminescent CDs obtained were used to cultivate fluorescent method. carnations To by the a water-culture were used to cultivate fluorescent carnations by a water-culture best of our method. To the best of our knowledge, this is the first study to cultivate fluorescent using knowledge, this is the first study to cultivate fluorescent flowers using nanotechnology.flowers Furthermore, nanotechnology. the test results of anthat insect test showed theattractive fluorescent the results of an Furthermore, insect attraction showed theattraction fluorescent flowers arethat more to flowers are more attractive to insects than nonfluorescent ones in an outdoor environment, insects than nonfluorescent ones in an outdoor environment, benefiting from the UV irradiation from benefiting from the UV irradiation from solar spectrum. solar spectrum.

2. 2. Results Results 2.1. Physicochemical Characterization of CDs Transmission electron (TEM) was used to characterize the the micromorphology of the Transmission electronmicroscope microscope (TEM) was used to characterize micromorphology of threethree obtained CD samples. A TEM 1) of Ca-2-CD indicates indicates a uniformasize (diameter: the obtained CD samples. A image TEM (Figure image (Figure 1) of Ca-2-CD uniform size 2–5 nm) without apparent aggregation. A high-resolution TEM (HRTEM) reveals that most of (diameter: 2–5 nm) without apparent aggregation. A high-resolution TEMimage (HRTEM) image reveals the Ca-2-CD are amorphous without any lattices [26,27]. TEM images (Figure S1) that most of particles the Ca-2-CD particles are amorphous without anyAdditional lattices [26,27]. Additional TEM show that the three samples (ECD, Ca-1-CD, and Ca-2-CD) had similar narrow size distributions images (Figure S1) CD show that the three CD samples (ECD, Ca-1-CD, and Ca-2-CD) had similar and morphologies (average 3.3, 3.3, and 3.1 nm, respectively), that adding narrow size distributions anddiameters: morphologies (average diameters: 3.3, 3.3, andindicating 3.1 nm, respectively), Ca(OH)2 had significant effect on no thesignificant morphology of the CDs. indicating thatno adding Ca(OH) 2 had effect on the morphology of the CDs.

Figure Figure 1. 1. TEM TEM image image of of Ca-2-CD Ca-2-CD (inset: (inset: HRTEM HRTEM image). image).

The FTIR results (Figure 2) revealed that all three samples showed O–H and N–H stretching at The FTIR results (Figure 2) revealed that all three samples showed O–H and N–H stretching at bending at 1570 cm−1, and stretching at 1635 3200–3700 cm−1−,1C=C stretching at 1420 cm−1, −N–H 1 , N–H −1 , C=O 3200–3700 cm , C=C stretching at 1420 cm bending at 1570 cm and C=O stretching −1 at −1 cm [28]. However, both Ca-1-CD and Ca-2-CD exhibited a similar prominent peak at 1400 cm−1, − 1 1635 cm [28]. However, both Ca-1-CD and Ca-2-CD exhibited a similar prominent peak at 1400 cm , which is ascribed to the carboxylate anion (–COO−) and indicates successful anchoring of Ca2+ [29]. which is ascribed to the carboxylate anion (–COO− ) and indicates successful anchoring of Ca2+ [29].

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−1−1 Moreover,itititisisis worth noting that the intensity intensity ofstretching O–H stretching stretching (3200–3700 cm and C=O C=O Moreover, worth noting thatthat the intensity of O–H (3200–3700 cm−1 ) andcm C=O stretching Moreover, worth noting the of O–H (3200–3700 )) and −1 − 1 −1 stretching (1635 cm ) gradually decrease as the amount of Ca(OH) 2 increases, indicating conversion (1635 cm ) gradually decrease as the amount of Ca(OH) increases, indicating conversion of the stretching (1635 cm ) gradually decrease as the amount of Ca(OH) 2 increases, indicating conversion 2 of the carboxyl groups to hydroxyl groups. XPS survey spectra provide a consistent result: the carboxyl groupsgroups to hydroxyl groups. groups. XPS survey consistent result: the result: numberthe of of the carboxyl to hydroxyl XPSspectra surveyprovide spectra aprovide a consistent numberheteroatoms ofsurface surfaceheteroatoms heteroatoms (Caand andO) O) increased asthe the amount of Ca(OH) 2increased increased (Figure surface (Ca and O) (Ca increased asincreased the amount of Ca(OH) increased (Figure 3a–c) [30,31]. number of as amount of Ca(OH) 2 (Figure 2 3a–c)[30,31]. [30,31]. Therefore, possiblefor mechanism forthe theofformation formation ofCDs CDspassivated passivated with Ca(OH) is Therefore, a Therefore, possible mechanism the formation CDs passivated with Ca(OH) proposed: 3a–c) aapossible mechanism for of with 2 2is 2 isCa(OH) proposed: asthe the amountof Ca(OH)2 2increased, increased, moreCa Cacations cations wereon anchored onthe theCD CD surface, as the amount ofamount Ca(OH) increased, more Ca cations were anchored the CD on surface, which also proposed: as Ca(OH) more were anchored surface, 2of whichalso alsothe converted the carboxyl groupsto to hydroxyl groups onthe theCD CDsurface. surface. converted carboxyl groups to hydroxyl groups on the CD surface. which converted the carboxyl groups hydroxyl groups on

Figure2.2. 2.FTIR FTIRspectrum spectrumof ofthe thethree threeCD CDsamples. samples. Figure Figure FTIR spectrum of the three CD samples.

(a) (a)

(b) (b)

(c) (c)

Figure 3. XPSspectra spectra ofthe the threeCD CD samples.(a) (a) ECD;(b) (b) Ca-1-CD;(c) (c) Ca-2-CD. Figure Figure3.3.XPS XPS spectraof of thethree three CDsamples. samples. (a)ECD; ECD; (b)Ca-1-CD; Ca-1-CD; (c)Ca-2-CD. Ca-2-CD.

2.2.Photoluminescence PhotoluminescenceProperties Properties 2.2. 2.2. Photoluminescence Properties TheUV–Vis UV–Visspectra spectraof ofall allthree threesamples samplesshowed showedobvious obvioussimilar similarabsorption absorptionpeaks peaksin inboth boththe the The The UV–Vis spectra of all three samples showed obvious similar absorption peaks in both the far-UV region (225–250 nm) and near-UV region (300–350 nm), which could be ascribed to the π–π* far-UV region (225–250 nm) and near-UV region (300–350 nm), which could be ascribed to the π–π* far-UV region (225–250 nm) and near-UV region (300–350 nm), which could be ascribed to the π–π* and andn–π* n–π*transitions, transitions,and andindicated indicatedthe theexistence existenceof ofaromatic aromaticstructures structuresin inthe theCDs CDs(Figure (Figure4)4)[32]. [32]. and n–π* transitions, and indicated the existence of aromatic structures in the CDs (Figure 4) [32]. However, 2 , and However, the absorbance in the two regions decreased with an increasing amount of Ca(OH) However, the absorbance in the two regions decreased with an increasing amount of Ca(OH)2, and the absorbance in the two regions decreased with an increasing amount of Ca(OH)2 , and Ca-2-CD Ca-2-CD exhibited a slight blue shift (centered at 237 nm and 345 nm for the π–π* and n–π* Ca-2-CD exhibited a slight blue shift (centered at 237 nm and 345 nm for the π–π* and n–π* exhibited a slight blue shift (centered at 237 nm and 345 nm for the π–π* and n–π* transitions) transitions)compared comparedwith withECD ECD(centered (centeredatat240 240nm nmand and353 353nm nmaccordingly). accordingly).We Wehypothesize hypothesizethat that transitions) compared with ECD (centered at 240 nm and 353 nm accordingly). We hypothesize that the carboxyl the carboxyl groups are auxochromes that can donate their lone-pair electrons to the antibonding the carboxyl groups are auxochromes that can donate their lone-pair electrons to the antibonding groups are auxochromes that can donate their lone-pair electrons to the antibonding orbitals of the orbitals ofthe thesp sp2 2carbon carbonclusters, clusters,increasing increasingthe theliquidity liquidityof ofthe theelectron electroncloud cloudin inthe theconjugation conjugation orbitals of 2 sp carbon clusters, increasing the liquidity of the electron cloud in the conjugation system and thus system and thus decreasing their energy gaps and increasing the absorbance [33]. Consequently, as system and thus decreasing their energy gaps and increasing the absorbance [33]. Consequently, as decreasing their energy gaps and increasing the absorbance [33]. Consequently, as the number of the number of carboxyl groups on the CD surface decreased, the absorbance in the optical spectrum the number of carboxyl groups on the CD surface decreased, the absorbance in the optical spectrum carboxyl groups on the CD surface decreased, the absorbance in the optical spectrum decreased and decreasedand andaablue blueshift shiftoccurred. occurred.Furthermore, Furthermore,the theenergy energygaps gapsof ofthe thethree threeCD CDsamples sampleswere were decreased a blue shift occurred. Furthermore, the energy gaps of the three CD samples were measured using measured using cyclic voltammetry, which indeed showed a gradual increase with the increasing measured using cyclic voltammetry, which indeed showed a gradual increase with the increasing cyclic voltammetry, which indeed showed a gradual increase with the increasing Ca(OH)2 content, Ca(OH)2 2content, content,and andwas wasconsistent consistentwith withour ourhypothesis hypothesis(Figure (FigureS3). S3). Ca(OH) and was consistent with our hypothesis (Figure S3).

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−1).−(a) 1 Figure 4. 4. UV-Vis adsorption spectrum of the three CD samples ininaqueous solution (0.1 μg·mL Figure spectrumof ofthe thethree threeCD CDsamples samplesin aqueous solution (0.1 µg·mL −1). Figure 4. UV-Vis adsorption spectrum aqueous solution (0.1 μg·mL (a)). ECD; (b) Ca-1-CD; (c) Ca-2-CD. (a) ECD; Ca-1-CD; (c) Ca-2-CD. ECD; (b) (b) Ca-1-CD; (c) Ca-2-CD.

The smaller smaller number of carboxyl groups on the CD surface also affected the PL PL behavior. As numberof ofcarboxyl carboxylgroups groupson onthe the surface affected behavior. The smaller number CDCD surface alsoalso affected the the PL behavior. As shown in Figure 5, the emission maximum is blue-shifted from 438 nm (ECD) to 433 nm (Ca-2-CD). As shown in Figure 5, the emission maximum blue-shiftedfrom from438 438nm nm(ECD) (ECD)toto433 433nm nm(Ca-2-CD). (Ca-2-CD). shown in Figure 5, the emission maximum is is blue-shifted The excitation excitation maximum also exhibits exhibits corresponding corresponding blue blue shifts shifts from from 362 362 to 357 nm (monitored at The maximum also exhibits corresponding blue shifts from 362 to 357 nm (monitored at the correspondingemission emission maximum). Clearly, new emission centers were generated as the Clearly, newnew emission centers were generated as the amount the corresponding corresponding emissionmaximum). maximum). Clearly, emission centers were generated as the amount of Ca(OH) 2 increased. The QY, which is one of the most important function indicators, was of Ca(OH) increased. The QY, which is one is ofone the of most function indicators, was was also amount of 2Ca(OH) 2 increased. The QY, which the important most important function indicators, also measured. Interestingly, the QY of the CDs was found to increase with increasing Ca(OH) measured. Interestingly, the QY the of CDs found increase with increasing Ca(OH)2Ca(OH) content22 also measured. Interestingly, theofQY thewas CDs was to found to increase with increasing content (Table S1). The QY of ECD73.1%, reached 73.1%, whereas that of Ca-2-CD was further to enhanced to (Table S1). The S1). QY of ECD whereas of Ca-2-CD further enhanced as high to as content (Table The QYreached of ECD reached 73.1%, that whereas that of was Ca-2-CD was further enhanced as high as 86.0%, which is almost equal to that of organic dyes or semiconductor quantum dots [34]. 86.0%, is almost to that of organic or semiconductor quantum dots [34]. Because of as highwhich as 86.0%, whichequal is almost equal to that dyes of organic dyes or semiconductor quantum dots [34]. Because of thehigh extremely high PL, blue fluorescent emission from the aqueous solution of be Ca-2-CD the extremely PL, blue fluorescent fromemission the aqueous of Ca-2-CD could easily Because of the extremely high PL, blueemission fluorescent fromsolution the aqueous solution of Ca-2-CD could be easily observed under natural light (Figure S2). observed underobserved natural light (Figure S2). could be easily under natural light (Figure S2).

(a) (a)

(b) (b)

(c) (c)

Figure 5. Excitation-dependent PL of the CD samples in aqueous solution. (a) ECD; (b) Ca-1-CD; (c) Figure inin aqueous solution. (a) (a) ECD; (b) (b) Ca-1-CD; (c) Figure 5. 5. Excitation-dependent Excitation-dependentPL PLofofthe theCD CDsamples samples aqueous solution. ECD; Ca-1-CD; Ca-2-CD and the excitation spectrum monitored at the maximum emission peak(black line), (a) λem = em = Ca-2-CD and the excitation spectrum monitored at the maximum emission peak(black line), (a) λ (c) Ca-2-CD and the excitation spectrum monitored at the maximum emission peak (black line), 438 nm, λem = 434 nm, λem = 433 nm. 438 λem = 434 nm, λem = 433 nm. (a) λnm, em = 438 nm, λem = 434 nm, λem = 433 nm.

The chelate rings of metallic chelates are known to possess excellent thermal stability, and it has The chelate rings of metallic chelates are known to possess excellent thermal stability, and it has chelate of metallic chelates are known possess by excellent thermaleffect stability, and it has beenThe proven thatrings the active functional groups can be to protected the chelating in the reaction been proven that the active functional groups can be protected by the chelating effect in the reaction 2+ been proven that the active functional groups can be protected by the chelating effect in the reaction process [16,35]. Thus, chelation of citric acid by Ca2+ can protect the carboxyl groups of citric acid process [16,35]. Thus, chelation of citric acid by Ca2+ can protect the carboxyl groups of citric acid process [16,35]. Thus, chelation citric acid by can protect On the carboxyl of citric acid from overconsumption during of dehydration andCacarbonization. the othergroups hand, the radiative from overconsumption during dehydration and carbonization. On the other hand, the radiative from overconsumption during dehydration and carbonization. On the other hand, the radiative recombination luminescence of the excitons could be absorbed by the electron-withdrawing groups, recombination luminescence of the excitons could be absorbed by the electron-withdrawing groups, recombination luminescence of the excitons could beofabsorbed by theCDs, electron-withdrawing such as carboxy and nitro groups, on the surface the obtained which might limitgroups, the PL such as carboxy and nitro groups, on the surface of the obtained CDs, which might limit the PL such as carboxy groups,electron-donating on the surface ofgroups, the obtained CDs, which and might limit groups, the PL efficiency of the and CDs.nitro However, such as hydroxyl amino efficiency of the CDs. However, electron-donating groups, such as hydroxyl and amino groups, efficiency of theaCDs. However, electron-donating such of as the hydroxyl and amino could produce conjugation effect with the surfacegroups, sp2 clusters CDs, which could groups, further could produce a conjugation effect with the surface sp22 clusters of the CDs, which could further could produce a conjugation effectbetween with thethe surface clusters of the CDs, could further increase the transition probability lowestspexcited singlet state andwhich the ground state of increase the transition probability between the lowest excited singlet state and the ground state of 2 clusters, increase the sp transition probability between excited singlet the ground of the surface thus enhancing thethe PLlowest QY [36,37]. Thus, thestate QY and of Ca-2-CD was state further the surface sp22 clusters, thus enhancing the PL QY [36,37]. Thus, the QY of Ca-2-CD was further the surface sp clusters, thus enhancing the PL QY [36,37]. Thus, the QY of Ca-2-CD was further enhanced owing to the conversion of electron-withdrawing groups to electron-donating groups enhanced owing to the conversion of electron-withdrawing groups to electron-donating groups enhanced to the conversion of electron-withdrawing groups todouble electron-donating (–OH) (–OH) by owing hydroxyl ions released from Ca(OH)2. Consequently, the passivationgroups strategy was (–OH) by hydroxyl ions released from Ca(OH)2. Consequently, the double passivation strategy was found to produce the highly luminescent sample Ca-2-CD, as shown in Figure 6. found to produce the highly luminescent sample Ca-2-CD, as shown in Figure 6.

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by hydroxyl ions released from Ca(OH)2 . Consequently, the double passivation strategy was found to Nanomaterials 7, 55 of produce the2017, highly luminescent sample Ca-2-CD, as shown in Figure 6. Nanomaterials 2017, 7, 176 176 of 10 10

“double passivating strategy”. Figure 6. 6. Schematic representation representation of of the the “double “double passivating passivating strategy”. strategy”.

Time-resolved PL spectra of three CD samples were recorded (λex = 360 nm) and fitted as twoTime-resolved PL spectra of three CD samples were recorded ex(λex = 360 nm) and fitted as component exponential decay functions, which all contain one fast and one slow components. The two-component exponential decay functions, which all contain one fast and one slow components. average of these components can be correlated to the energy transfer process among the sp22 clusters The average of these components can be correlated to the energy transfer process among the sp2 with different energy gaps. As shown in Figure S4 and Table S2, the average lifetimes of the three clusters with different energy gaps. As shown in Figure S4 and Table S2, the average lifetimes samples decreased with increasing Ca(OH)22 content in the reaction process. For the CD samples, of the three samples decreased with increasing Ca(OH)2 content in the reaction process. For the fewer and fewer electron-withdrawing groups were attached on the CD surface with increasing CD samples, fewer and fewer electron-withdrawing groups were attached on the CD surface with Ca(OH)22 content, which made the energy gaps of the ECD be more and more simple, and increasing Ca(OH)2 content, which made the energy gaps of the ECD be more and more simple, subsequently, the frequency of the energy transfer process decreased. As the result, the average and subsequently, the frequency of the energy transfer process decreased. As the result, the average lifetime of the three samples decreased with increasing Ca(OH)2 content in the reaction process. lifetime of the three samples decreased with increasing Ca(OH)22 content in the reaction process. 2.3. Biological Biological Characteristics Characteristics 2.3. The MTT MTT assay assay method method was was used used to to evaluate evaluate the the cytotoxicity cytotoxicity of of the the as-prepared Ca-2-CD before before The as-prepared Ca-2-CD application (Figure (Figure 7). 7). It It is is noteworthy noteworthy that that the the obtained obtained CDs CDs exhibit exhibit very very little little toxicity toxicity toward toward HeLa HeLa application −1 −11) and after long incubation times (36 h); hence, they are safe and − cells even at high doses (0.4 mg·mL cells even at high doses (0.4 mg·mL ) and after long incubation times (36 h); hence, they are safe and highly suitable suitable for for use use in in biological biological applications applications [38]. [38]. highly

−1 ) −1 −1) Figure MTT assay assay for for cytotoxicity cytotoxicityvalues valuesversus versusincubation incubationconcentrations concentrations(0–400 (0–400μg·mL µg·mL Figure 7. Figure 7. 7. MTT MTT assay for cytotoxicity values versus incubation concentrations (0–400 μg·mL ) of of of Ca-2-CD. Ca-2-CD. Ca-2-CD.

Because of the high QY and low cytotoxicity, an exploratory experiment was performed to assess the potential application of Ca-2-CD for cultivating fluorescent flowers. As shown in Figure 8, cut carnations cultivated using DI water were observed to be nonfluorescent upon UV light irradiation; however, bright blue-light emission was observed under UV light irradiation from carnations cultivated using Ca-2-CD solution, which could be attributed to the CDs that had entered

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Because of the high QY and low cytotoxicity, an exploratory experiment was performed to assess the potential application of Ca-2-CD for cultivating fluorescent flowers. As shown in Figure 8, cut carnations cultivated using DI water were observed to be nonfluorescent upon UV light irradiation; however, bright blue-light emission was observed under UV light irradiation from carnations cultivated Nanomaterials 2017, 7, 176 6 of 10 using Ca-2-CD solution, which could be attributed to the CDs that had entered the plant. Interestingly, Nanomaterials 2017, 7, 176 6 of 10 the plant. Interestingly, margin of the petals was found fluorescence. to produce more fluorescence. It are the margin of the petals wasthe found to produce more intense It isintense thought that the CDs isenough thought that CDs the are margin small enough to be transported bybody the channels in the body when smallthe to bethe transported by the in the plant when they areplant dispersed in Itwater, plant. Interestingly, of channels the petals was found to produce more intense fluorescence. they are dispersed inreleased water, and the water could beand released through their and petals by is thought that thebeCDs are small enough to be transported by the channels in leaves the plant body when and the water could through their leaves petals by transpiration [39]. The margin is transpiration [39]. The margin is much thinner than other parts of the petals, making its air contact they are dispersed in water, and the water could be released through their leaves and petals by much thinner than other parts of the petals, making its air contact area much larger. Thus, transpiration area much larger. Thus, transpiration would be much more intense the margin, so transpiration [39]. intense The margin is much thinner than other parts ofaccumulate the at petals, making itsthe air margin contact would be much more at the margin, so the margin would much larger amounts of would accumulate much larger amounts of Ca-2-CD, ultimately producing stronger fluorescence. area much larger. Thus, transpiration would be much more intense at the margin, so the margin Ca-2-CD, ultimately producing stronger fluorescence. Furthermore, the longevity of the fluorescent Furthermore, the longevity of theamounts fluorescent cut flowers was observed comparing with that of the would accumulate much larger of Ca-2-CD, ultimately producing stronger fluorescence. cut flowers was observed comparing with that of the ordinary ones, which showed that there ordinary ones,the which showed thatfluorescent there is no cut obvious influence of CDs-doping on the of is no Furthermore, longevity of the flowers was observed comparing withlongevity that of the obvious influence of CDs-doping on the longevity of the cut flowers (Figure S5). the cut flowers (Figureshowed S5). ordinary ones, which that there is no obvious influence of CDs-doping on the longevity of the cut flowers (Figure S5).

Figure 8. Photographs of carnation cultivated with D-I water(a,b) (a,b)and andCa-2-CD Ca-2-CD solution solution (c,d); Figure 8. Photographs of carnation cultivated with D-I water (c,d); before before (a,c) (a,c) and after (b,d) UV irradiation. Figure 8. Photographs of carnation cultivated with D-I water (a,b) and Ca-2-CD solution (c,d); before and after (b,d) UV irradiation. (a,c) and after (b,d) UV irradiation.

Fluorescence microscopy was used to investigate the distribution of Ca-2-CD in the plant body

Fluorescence microscopy was to investigate thedistribution distribution Ca-2-CD in plant theshowed plant at theFluorescence micro level. As illustrated inused Figure petal tissue from the nonfluorescent microscopy was used to9c,d, investigate the of of Ca-2-CD inflowers the bodybody no fluorescence. However, an overlay of fluorescence and bright-field images of petal tissue from at theat micro level. As illustrated in Figure 9c,d, petal tissue from the nonfluorescent flowers showed the micro level. As illustrated in Figure 9c,d, petal tissue from the nonfluorescent flowers showed fluorescent carnations (Figure 9a,b) demonstrated that fluorescence could be observed in the tissue no fluorescence. However, ananoverlay andbright-field bright-field images of petal tissue no fluorescence. However, overlayof of fluorescence fluorescence and images of petal tissue from from fluid and cells, indicating that the CDs had enteredthat the plant tissue structure. fluorescent carnations (Figure 9a,b) demonstrated fluorescence could be be observed in the fluorescent carnations (Figure 9a,b) demonstrated that fluorescence could observed intissue the tissue and cells, indicating that CDshad hadentered entered the the plant fluid fluid and cells, indicating that thethe CDs planttissue tissuestructure. structure.

Figure 9. Fluorescence images of the petal tissue of fluorescent carnation at a micro level (b), Bright field transmission image (a); non-fluorescent carnation at a carnation micro level. field transmission Figure 9. Fluorescence images of the petal tissue of fluorescent atatBright aamicro level (b), Figure 9. Fluorescence images of the petal tissue of fluorescent carnation micro level (b), Bright field image (c), Under UV light (d). field transmission (a); non-fluorescent carnation at alevel. microBright level. Bright field transmission transmission image (a);image non-fluorescent carnation at a micro field transmission image (c), image (c), Under UV light (d). Under UV light (d).

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3. Methods 3.1. Materials Citric acid and ethylenediamine were obtained from Aladin Ltd. (Shanghai, China). Carnations were purchased from Aimei Flowers Ltd. (Kunming, China). Deionized (DI) water was used for the experiments. 3.2. Measurements Transmission electron microscopy (TEM) was performed on a JEOL-2010 instrument (JEOL, Tokyo, Japan) at 200 kV. Fourier transform infrared (FTIR) spectra were collected in the wavenumber range of 4000–400 cm−1 using a Nicolet 360 FTIR spectrometer (Nicolet, Madison, WI, USA). X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer (VG Scientific, London, England) with monochromatic Al Kα radiation (hν = 1486.6 eV), and the binding energy calibration was based on C 1s (284.6 eV). UV–vis absorption spectra were recorded on a PerkinElmer Lambda 950 spectrophotometer (PerkinElmer, Waltham, MA, USA). Excitation and emission spectra were collected using a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The QY of the CDs was measured at an excitation wavelength of 360 nm using quinine sulfate as a standard (QY = 54%) [6]. Confocal microscopy analysis was performed using an Olympus FluoView 500 laser scanning confocal microscope (Olympus, Tokyo, Japan), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of the obtained CDs was used to quantify the viability of HeLa cells [40]. 3.3. Preparation of Carbon Dots Citric acid (10 g), ethylenediamine (5 mL), and Ca(OH)2 (5.2 g) were dispersed in DI water (10 mL); the mixture was then transferred to an autoclave (100 mL) lined with poly(tetrafluoroethylene) (Teflon) and heated at 200 ◦ C for 3 h. After the reaction, the reactors were cooled to room temperature naturally. The product, which was brown and transparent, was subjected to dialysis to obtain the CDs, denoted as Ca-2-CD. Simultaneously, to better understand the role of Ca(OH)2 in the formation of highly fluorescent CDs, parallel experiments were conducted with either 0 or 2.6 g of Ca(OH)2 . The corresponding products are denoted as ECD and Ca-1-CD, respectively. 3.4. MTT Assay of Cell Viability The cytotoxicity of Ca-2-CD was assessed by MTT assay. Briefly, approximately 1 × 104 HeLa cells per well were seeded into a 96-well plate. After overnight culturing, the obtained Ca-2-CD was added to each well at concentrations ranging from 0 to 400 µg·mL−1 in Dulbecco’s modified Eagle’s medium. After incubation for 12 h, the metabolically active cells were then detected by adding MTT (5 mg·mL−1 in phosphate-buffered saline) for another 4 h, and the plates were read at 570 nm with a 630 nm reference. Then, the mean and standard deviation for triplicate wells were recorded. 3.5. Preparation of Fluorescent Flowers Ca-2-CD solution was prepared with a concentration of 0.5 g·L−1 . Cut carnations (flower diameter, 4–5 cm; stem length, 18–20 cm; cutting angle, 45◦ ) were cultivated with the Ca-2-CD solution for 6 h at 25 ◦ C. As a comparative test, other cut flowers were cultivated using DI water. To observe the fluorescence properties, the flowers were exposed to UV light with a wavelength of 365 nm below a 6 W UV lamp (10–20 cm apart) in a dark room. 3.6. The Longevity-Observing Tests of the Cut Flowers Six carnations were put together as one bunch, then, the bunch of carnations was inserted in the Ca-2-CD solution with a concentration of 0.5 g·L−1 . During the cultivation, the solution

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was freshly changed every 24 h. The longevity of the fluorescent cut flowers was observed and recorded. As a comparative test, the other bunch of cut flowers was cultivated using DI water. The longevity-observing tests were arranged in four groups to make analysis. 3.7. Confocal Microscopy Confocal microscopy analysis was performed using an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). Briefly, a small piece of skin was torn from the petal of a fluorescent carnation using tweezers and quickly fixed on a slide for inspection and bioimaging using the fluorescence microscope. 4. Conclusions In summary, a double passivation strategy was developed to fabricate highly luminescent CDs with the assistance of Ca(OH)2 . In the reaction process, Ca2+ could protect the active functional groups from overconsumption during dehydration and carbonization, and electron-withdrawing groups could be converted to electron-donating groups (–OH) on the CD surface by hydroxyl ions. Then, the highly photoluminescent CDs obtained were used to cultivate fluorescent carnations by a water-culture method. Further, the results of an insect attraction test showed that the fluorescent flowers are more attractive to insects than non-fluorescent ones in an outdoor environment. Our work provides a new method of preparing highly luminescent CDs and is expected to afford a new way to obtain function-specific plants. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/7/7/176/s1. Figure S1: TEM images of ECD (a), Ca-1-CD (b), Ca-2-CD (c), Figure S2: The Ca-2-CD aqueous solution under natural light irradiation (0.1 µg·mg−1 ), Figure S3: The cyclic voltammogram of the ECD (a), Ca-1-CD (b), Ca-2-CD (c) and 0.1 mol/L KCl aqueous solution, Figure S4: Luminescence decay curve of the three CD samples recorded at room temperature in aqueous solution (a. ECD, b. Ca-1-CD, c. Ca-2-CD), Figure S5: The results of the longevity-observing tests (left, the longevity of ordinary carnations; right, the longevity of fluorescent carnations), Table S1: The QY results of the three CD samples in aqueous solution, Table S2: The lifetimes (τ) and the average lifetimes () of the three CD samples. Acknowledgments: This work was supported by the Science Foundation of Hebei Province (No. D2014402046), and the Program for One Hundred Innovative Talents in Universities of the Hebei Province (No. BR2-204). The authors are indebted to Lianwei Kang and Jianjun Liu for the use of lyophilizer, Juan Feng (Northwest University, Xi’an) for carrying out the TEM measurements. Author Contributions: Shuai Han and Shenjun Qin fabricated all the nanostructures and drafted the manuscript. Tao Chang and Haiping Zhao performed the fluorescence microscope tests on the composite nanostructures. Huanhuan Du and Shan Liu planned the whole work, and revised the manuscript. Baoshuang Wu performed the biological test. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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