Lu ChenLei ZhangShen-long JiangQun Zhang

Department of Chemical Physics,Hefei National Laboratory for Physical Sciences at the Microscale,Synergetic Innovation Center of Quantum Information and Quantum Physics,University of Science and Technology of China,Hefei 230026,China

I.INTRODUCTION

Owing to its unique optical,electrical,and physical/chemical properties[1–6],graphene,a well-known two-dimensional atomic crystal,has been subjected to extensive research in recent years.With its large-scale production being possible during the last decade[7],graphene has been widely used in such applications as solar cells,lithium atomic batteries,supercapacitors,sensors,and detectors[8–11].Meanwhile,the field has also witnessed a boom in the research of graphene oxide(GO).GO has been not only used as an efficient intermediate for the synthesis of chemically modi fied graphene[12],but also found to hold promise in a variety of other applications.For instance,GO is known as an environment-friendly sensor for the detection of such biomolecules as DNA and ATP due to its capability in quenching the fluorescence of dye molecules[13–15].Certainly,it is important to gain insights into the mechanisms underlying the fluorescence quenching of molecules by GO,which would help improve the accuracy of the relevant quantitative measurements.

Recently,there emerged researches related to the topic of fluorescence quenching of molecules by GO[16–21],among which the mechanistic information still remains limited[19–21].The current work is devoted to deepening the understanding about the involved fluorescence quenching mechanisms from a perspective pertaining to the functional groups on the GO nanosheet as well as the quenching regimes(i.e.,dynamical and/or static).Herein,the dye molecule of Rhodamine 6G(R6G)was taken as a representative fluorophore,whose fluorescence quenching behavior and mechanisms were examined by analyzing the fluorescence emission spectra recorded on a set of controlled GO samples with varied C/O ratios.It was found that the carbonyl groups on the GO nanosheet play a dominant role in quenching the R6G fluorescence.Moreover,the “sphere of action”model turned out to nicely accommodate the data responsible for the static part of fluorescence quenching,and such a static quenching was further identified,with the help of the input from the absorption spectral evolution,to feature the formation of ground-state complex between R6G and GO.

II.EXPERIMENTS

The commercially available GO powder(platelet diameter～5 μm,thickness 0.8?1.2 nm,Nanjing XFNANO Materials Tech.Co.Ltd.)was used as the precursor sample,whose C/O ratio was determined to be～2.1 by the X-ray photoelectron spectroscopy(XPS)analysis[20,22].This precursor GO sample is denoted as GO-2.1 hereafter.Following a documented method[23],we also prepared two other GO samples with larger C/O ratios.The relevant details are briefed as follows.Firstly,the concentrated GO-2.1 aqueous solution was prepared by dispersing 200 mg of GO-2.1 powder into 80 mL of deionized water,followed by a 5-h sonication.Secondly,533μL of ammonium hydroxide(AR grade)and 30μL of hydrazine hydrate(85%,AR grade)were added into 15 mL of the concentrated GO-2.1 aqueous solution,after which the mixture was allowed to react at 95°C for a controlled period of time,i.e.,0,5,or 10 min.Finally,each post-reaction mixture was diluted to yield the investigated precursor solution with a GO concentration of 500μg/mL.The C/O ratios of the treated samples with 5 and 10 min were determined to be～2.9 and～3.5 by the XPS analysis,and hence denoted as GO-2.9 and GO-3.5,respectively.The precursor R6G solution was prepared by dissolving the R6G dye powder in deionized water,forming a concentration of 88.7μmol/L.In the following fluorescence and absorption spectroscopic measurements,the R6G concentration,[R6G],in all of the R6G/GO mixed solutions was kept constant at 35.5μmol/L,and the concentrations of different GO quenchers,[GO-2.1],[GO-2.9],and[GO-3.5],were varied in the mass concentration ranges of 0?200,0?150,and 0?40 μg/mL,respectively.Note here that the XPS analysis for determining the C/O ratios of the GO samples was conducted after a vacuum drying treatment on their solutions at 50°C for 5 h.

The XPS spectra were recorded on an ES-CALAB 250 system(Thermo-VG Scientific).The steady-state absorption spectra were registered on an ultravioletvisible(UV-Vis)spectrophotometer(Persee).The steady-state and time-resolved fluorescence measurements were performed on an FLS920 fluorescence spectrometer(Edinburgh).All of the steady-state fluorescence spectra(520?700 nm)were calibrated against the sensitivity of the photomultiplier tube equipped with the fluorescence spectrometer.The peak intensity of the R6G fluorescence emission at 500 nm was calibrated against the R6G absorption intensity with the absorption contribution from GO at the same wavelength being excluded.For the fluorescence lifetime measurements,the excitation at 500 nm was provided by an SC400-2 supercontinuum laser source(Fianium)with a 6-ps pulse width and the emission was monitored at 560 nm by means of time-correlated single photon counting.The fluorescence lifetimes were obtained by a least-squares fit of the experimental data with the instrument response function deconvoluted.All the above measurements were carried out under ambient conditions.

III.RESULTS AND DISCUSSION

Shown in FIG.1(a),(b),and(c)are the C1s XPS spectra for GO-2.1,GO-2.9,and GO-3.5 samples,respectively.As for GO-2.1(FIG.1(a)),its C1s XPS pro file can be decomposed to four bands peaking at about 284.8,286.6,287.0,and 288.5 eV,attributable to the sp2/sp3carbon,epoxy/hydroxyl,carbonyl,and carboxyl groups,respectively[22].With increasing the C/O ratios,both the carbonyl and carboxyl bands get suppressed,as is seen from the comparison between FIG.1(b,c)and FIG.1(a).Particularly,the carbonyl band vanishes as the C/O ratio is elevated from 2.1 to 3.5.Once the GO samples are reduced from GO-2.1 to GO-3.5,the carbon proportions(in percentage)for the sp2and sp3carbon and epoxy and hydroxyl bands increase while those for the carbonyl and carboxyl bands decrease,as listed in Table I.

FIG.1The C1s XPS spectra recorded on(a)GO-2.1,(b)GO-2.9,and(c)GO-3.5 samples.

With the three GO samples serving as fluorescence quencher,the corresponding quenching effect on the R6G fluorescence emissions(in the wavelength range of 520?700 nm,peak at～560 nm)turns out to become more pronounced with increasing the GO concentration,as shown in FIG.2.Clearly,the addition of smaller amounts of reduced GO samples(i.e.,GO-2.9 and GO-3.5)brings about similar or more significant quenching effect as compared to GO-2.1.These observations,in conjunction with the above XPS results(Table I),suggest that the substantial enhancement of the quenching effect by GO-2.9 and GO-3.5 is dominantly linked to the carbonyl groups,as the carbon proportion of the carbonyl band experiences a drastic decrease from 31.7%(GO-2.1)to 7.8%(GO-2.9)and even to 0%(GO-3.5).The fact that the amount reduction of the carbonyl groups on the GO nanosheet greatly promotes the R6G fluorescence quenching can be understood asfollows.As demonstrated in the R6G/GO system,the photoinduced electron transfer from R6G to GO constitutes one of the major mechanisms responsible for fluorescence quenching[20].In terms of electronegativity,the carbonyl group stands out among others(e.g.,the hydroxyl,carbonyl,and epoxy groups).The existence of carbonyl groups with high electronegativity hampers the process of photoinduced electron transfer from the excited R6G fluorophore to the GO nanosheet,and hence their removal from the GO nanosheet plays a dominant role in enhancing the observed fluorescence quenching effect.

TABLE I The carbon proportions for the four XPS bands.

FIG.2 The fluorescence emission spectra of R6G-GO systems with varied concentrations of GO:(a)[GO-2.1]=0,10,20,40,60,80,100,125,150,175,and 200 μg/mL;(b)[GO-2.9]=0,5,10,20,30,40,60,80,100,125,and 150 μg/mL;(c)[GO-3.5]=0,2,4,8,12,16,20,25,30,35,and 40μg/mL.For all of the R6G-GO systems,[R6G]was kept constant at 35.5μmol/L.

Fluorescence quenching is a result of a variety of molecular interactions,including but not limited to electron/energy transfer,excited-state reactions,ground-state complex formation,molecular rearrangements,and collisional quenching[24].From a regime perspective,the quenching mechanisms can be generally categorized into the dynamic and static regimes.Under each pure regime,there exists a linear relationship between theF0/Fvalue and the quencher concentration,whereF0(F)is the fluorescence intensity in the absence(presence)of the quencher.As shown in FIG.3(a1),(b1),and(c1)(for GO-2.1,GO-2.9,and GO-3.5,respectively),none of theF0/Fversus[GO]plots exhibit such a linear relationship,indicating that the R6G fluorescence quenching by GO should be a combination of dynamic and static mechanisms.Of particular interest is the static quenching as it contains molecular interaction information.Given that the dynamic contribution is proportionally correlated toτ0/τ,whereτ0(τ)is the fluorescence lifetime in the absence(presence)of the quencher,the static contribution can be routinely retrieved by dividing the total value ofF0/Fby the dynamic part ofτ0/τ,i.e.,(F0τ)/(Fτ0)[24].For the current case,the corresponding contributions are plotted in FIG.3(a2),(b2),and(c2)(dynamic)and FIG.3(a3),(b3),and(c3)(static)for GO-2.1,GO-2.9,and GO-3.5,respectively.In our previous work[20]on the R6G fluorescence quenched by a larger GO sample with a platelet diameter of～45μm(i.e.,roughly one order of magnitude larger than the current GO sample whose diameter is～5μm),the static quenching contribution was found to be in the form of

whereandare the static Stern-Volmer constants,under the assumption that a stable non fluorescent ground-state complex is formed between the R6G fluorophore and the GO quencher.Physically,is the equilibrium constant for the complex formation between the R6G fluorophore monomer(dimer)and the GO quencher[20].In the current case,however,the static quenching data of GO-2.9 and GO-3.5 cannot be fitted well by such a binomial function(the right-hand side of Eq.(1)).The relevant fitting turned out to yield unsatisfactoryR2values that are smaller than 0.95,as given in FIG.3(b3)and(c3).As for GO-2.1(FIG.3(a3)),although such a fitting turned to yield a reasonableR-square value(i.e.,0.973),the resultingandconstants were both found to be negatively valued,which obviously lacks physical meaning.The failure of binomial fitting suggests that for the current R6G/GO system with a much smaller size of GO platelet,the model that solely takes into account the formation of ground-state complex(Eq.(1))cannot be adopted to describe the static quenching effect observed in the current R6G/GO system.

FIG.3 Fluorescence quenching of R6G by GO.The F0/F values are plotted as a function of(a1)[GO-2.1],(b1)[GO-2.9],and(c1)[GO-3.5].The τ0/τ values are plotted as a function of(a2)[GO-2.1],(b2)[GO-2.9],and(c2)[GO-3.5].The values(F0τ)/(Fτ0)are plotted as a function of(a3)[GO-2.1],(b3)[GO-2.9],and(c3)[GO-3.5],with a binomial fitting being incorporated.The(F0τ)/(Fτ0)values are plotted as a function of(a4)[GO-2.1],(b4)[GO-2.9],and(c4)[GO-3.5],with an exponential fitting being incorporated.The ln[(F0τ)/(Fτ0)]values are plotted as a function of(a5)[GO-2.1],(b5)[GO-2.9],and(c5)[GO-3.5],with a linear fitting being incorporated.Note that F0(F)and τ0(τ)stand for the R6G fluorescence intensity and lifetime,respectively,in the absence(presence)of GO.

Alternatively,we resorted to a general model of quenching “sphere of action” [24],which reads

FIG.4 Steady-state absorption spectra(425?625 nm)of R6G recorded on the three R6G/GO systems with varied GO concentrations(annotated in each subgraph):(a)GO-2.1,(b)GO-2.9,and(c)GO-3.5.These spectra were all calibrated against the absorbance contribution from GO in each R6G/GO system.

whereVis the volume of the action sphere andNAis Avogadro constant.It turned out that the static quenching data of GO-2.1,GO-2.9,and GO-3.5 can be fitted nicely by such an exponential function(the right-hand side of Eq.(2))with satisfactoryR2values that are all larger than 0.98,as given in FIG.3(a4),(b4),and(c4),respectively.From the excellent linear fit ofversus[GO],as shown in FIG.3(a5),(b5),and(c5),one can derive theV NAvalues:0.0152,0.0358,and 0.1243(μg/mL)?1for GO-2.1,GO-2.9,and GO-3.5,respectively.In order to evaluate theVvalue in the unit of(μm)3,one need to obtain the molar concentration of GO.Given that the diameter of the GO platelet is～5μm and the length of carbon?carbon bonds on the graphene nanosheet is approximately 1.42?A[25],a single GO-2.1 platelet contains～7.5×108carbon atoms and～3.6×108oxygen atoms,and hence its molar mass can be estimated to be～1.48×1010g/mol.As such,for all of the GO samples used in the experiment the mass concentration of 1μg/mL corresponds to the molar concentration of～6.8×10?14mol/L,which is much lower than the molar concentration of R6G(～35.5 μmol/L)that was kept constant in the R6G/GO solutions.Therefore,theVvalues can be determined to be 371.2,874.3,and 3035.4(μm)3,corresponding to the effective “sphere of action”radii of 4.46,5.93,and 8.98 μm for the R6G/GO-2.1,R6G/GO-2.9,and R6G/GO-3.5 systems,respectively.Notably,the effective radii of action sphere derived here(roughly in the range of 4?9μm)are larger than the sum of the radii of R6G fluorophore and GO quencher(～2.5 μm).

Last but not least,we have also examined the steadystate absorption spectra(425?625 nm)of R6G recorded on the three R6G/GO systems,as shown in FIG.4.From the spectral evolution with varied GO concentrations,one can clearly see a red-shift of the predominant 525-nm peak.As is well known,this 525-nm peak is associated with the R6G monomer,while its adjacent shoulder peak at～500 nm is responsible for the R6G dimer[26].Accompanied by this red-shift of the 525-nm peak,there emerges a new peak at～550 nm.This 550-nm peak is most likely linked to the formation of ground-state complex between the R6G fluorophore and the GO quencher.With increasing the concentrations of GO in both R6G/GO-2.9 and R6G/GO-3.5 systems,absorption from such a ground-state complex gets more pronounced.Markedly,this ground-state complex absorption centered at～550 nm possesses a severe overlap with the pro file of the R6G fluorescence emissions centered at～560 nm(see FIG.2).This high-degree,spectral overlap suggests that the fluorescence emissions of R6G(520?700 nm)are quenched,in a static fashion,via substantial absorption by the ground-state complex formed between R6G and GO.

IV.CONCLUSION

In summary,we have investigated the fluorescence quenching of Rhodamine 6G(R6G)by a set of controlled graphene oxide(GO)samples with varied C/O ratios.We found that the carbonyl groups on the GO nanosheet play a dominant role in quenching the R6G fluorescence and that the static quenching can be described by the “sphere of action” model.With the assistance of the absorption spectra,we also identi fied that signi ficant absorption of the R6G fluorescence by the ground-state complex formed between R6G and GO accounts for the static quenching.This work provides useful information on the fluorescence quenching mechanisms in the R6G/GO system.

V.ACKNOWLEDGEMENTS

This work was supported by the Ministry of Science and Technology of China(No.2016YFA0200602),the National Natural Science Foundation of China(No.21573211andNo.21633007),andtheFundamental Research Funds for the Central Universities(No.WK2340000063).

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