一鍋煮串聯合成方法在熒光碳點識別基團調控中的研究

王梅梅李 莉陳琦棟王歡歡

(新疆農業大學化學工程學院，新疆烏魯木齊 830052)

1 Introduction

Carbon dots(CDs)with features of low toxicity， tunable excitation and emission spectra， and high photo stability have been proposed in application for bioimaging and theranostics， sensor， antibacterial and antioxidant property， photo catalysis，etc.[1-5].Fabricating novel CDs with desired physical and chemical properties is concerned with the precursors and the synthesis conditions involved.Thereby，various carbon sources such as bulk carbon， natural bio-resources， polymer even small molecules have been employed as precursors[6-7].Under the ultrasonic， microwave， light or thermal conditions，the precursors engage in decomposition/fusion/carbonization reactions in a random manner[8-10].Consequently the extent of π-conjugate domain generated in the reactions determines the luminescence property of the obtained CDs[11].However the“conundrum”reactions weaken the rational construction of CDs[12].Therefore，the value of structural-design ability has been stressed.The controllable synthesis of such materials points to the principle and guide for the size，element and functional group content.In terms of element composition，precursors constituted of versatile elements were adopted to contribute to hetero-doped CDs with tunable luminescence property due to the inheritance of element from precursors to the target materials.Boron[13-14]，nitrogen[15]， phosphorus[16]， sulfur[17]， fluorine[18-19]even metallic element[20-22]doping and/or co-doping[23-24]carbon dots exhibit varied luminescence property and target affinity.However，the hetero-doping strategy presents the limitation on resolution of functional groups content.Hence post-modification has been proposed to create a tailorable structure in specific synthesis protocol[25].By taking the advantage of the reactivity of the residue amine or carboxyl groups in the edge，various functional groups are attached onto the surface of the materials[26-29].On minus side，the fluctuation of grafting yield has much impact on the quantitative analysis.

Other than element doping and post modification，the question of tentative access to create a defined CDs structure has been addressed by chemists.Despite the“conundrum”reactions involve ambiguous bond rupture and formation，the plausible reactions originated from small molecules are confined into the scope of organic reactions.And plenty of CDs derived from versatile small molecules are readily for establishment of quasi-Scifinder for CDs synthesis[30].Furthermore the synthesis of carbonaceous materials could adopt the principles and strategies of molecules synthesis such as retrosynthesis[31].The scope of the organic synthetic strategies has been expanded to preparation of inorganic material and supramolecularcrystalengineering， in which the premise is that the synthons are chemoselective[32-34].If the synthons for CDs behave chemoselective，the organic synthetic strategies could likely be alternative rational design access.In other words，a regiospecific reaction restricts the tendency of other plausible reaction to yield the favored CDs structure especially in a multi-steps scheme.However，the outcome of rational design is rather limited due to the harsh hydrothermal treatment in the preparation of carbon dots[35].Hence evaluating regionselectivity of precursors in hydrothermal reaction is critical to render a successful controllable synthesis of CDs.To a certain extent，functional group content and structural instability of the precursors in the harsh environment are responsible for the orientation of reaction.A survey of the literatures illuminates that aromatic amine in the instable compound like dopamine[36]， phenylenediamine[37-38]can easily form stable luminescence dot via self-polymerization into nitrogen doped conjugate structure.

Inspired by the investigation of aromatic amine，we have chosen diaminouracil as a potential precursor to implement rational preparation of carbon dots[39].Thanks to the amine group，diaminouracil is an antioxidant that can capture reactive oxygen radical species[40-42].Moreover，it is basic skeleton to prepare fused uracils[43]and a good chelator for metalions[44]. From perspective offunctional groups，amino group is sensitive and active in oxidation reaction.The nature of antioxidant enables amino group undergoing oxidate-induced cyclization to build pyrazine ring，while the uracil moiety is sensitive in alkaline amended solution.Thus，it undergoes alkaline hydrolysis to transform to amine and carboxyl group.In this regard，metabolism of diaminouracil is regioselective.We are prone to practically verify the rational design based on the precursor's regioselectivity through a two-step linear sequence hydrothermal reaction.In the first step，a common hydrothermal condition is utilized to make the amino groups， the predominant reactive sites， to form nitrogen rich conjugate structure as chromophore[45].The residual uracil receptor would conduct coordination behavior with mercury and silver ion.Further addition of alkaline base induces a sequential hydrolysis reaction.The uracil receptor would convert to amine and carboxyl group in strongly basic medium.The reaction makes it possible to get a zwitterion symbol carbon dots.In comparison with the carbon dot with uracil ligand，the zwitterion carbon dot will display less affinity for silver and mercury ions.With the two distinct function and structure characteristic in hands，controllable preparation of diaminouracil derived carbon dots in a sequential reaction would be feasible.

2 Experiments

2.1 Materials

Diaminouracil were prepared according to the literature[46].All other chemicals and solvents of analytical grades were purchased from Aladdin Reagent Co.Ltd.(Shanghai，China).Stock solutions of cations were prepared from their nitrate salts(analytical grade)，except for HgCl2.

2.2 Synthesis of CDHBand CDZW

The schematic diagram of all synthesis procedures was shown in Fig.1.CDHBwere firstly fabricated via a one-step hydrothermal method from diaminouracil.The suspension(101.1 mg diaminouracil＠11 mL H2O)was subsequently transferred to a 25 mL Teflon-lined stainless steel autoclave and incubated for 4 h at 160℃until the reaction was completed.After cooled to 50℃，the synthesized darkbrown stock solution was filtered with a 0.22 μm filters(Millipore Corp.， Bedford， MA， USA) to remove large particles.With the temperature down to room temperature(r.t.)，the solution turned into turbidity.Then the suspension was centrifuged(11 000 r/min，30 min)to get dark-brown aggregates.

Without further treatment，crude CDHBsolution in the autoclave was added 5 mol/L sodium hydroxide(5 mL).Then the autoclave was resealed and further incubated for 4 h at 180℃until the reaction was completed.The synthesized CDZWdark-brown stock solution was centrifuged(11 000 r/min，30 min)，and the supernatant was filtered with a 0.22 μm filters(Millipore Corp.， Bedford， MA， USA)to remove large particles and aggregates.The result solution was then neutralized with 7 N HCl to pH 7-8.

2.3 Samples Preparation

All the dots were freeze dried and redispersed in the aqueous solution for TEM and emission characterizations.

2.4 Characterizations

The PL spectra were measured using a Cary Eclipse fluorescence spectrophotometer.The excitation and emission slits were set at 5 nm.The absolute quantum yields were measured on Hamamatsu C9920-02G.High Resolution Transmission Electron Microscope(HRTEM) images and selected-area electron-diffraction were obtained using a FEI Tecnai F30 microscope.X-ray photoelectron spectroscopy(XPS)was performed using a Thermo escalab 250Xi instrument.The Infrared Spectroscopy(IR)spectra were obtained using a Thermo Fisher iS50 spectrometer.

3 Results and Discussion

As shown in Fig.1，a two-step sequential reaction with the diaminouracil as precursors was designed.In the first step，diaminouracil undergoes oxygen-condensation of quinone-imine or the quinone with the amino group subsequent third-ring closure to yield chromophore containing pyrimidopteridine moiety[39].Under hydrothermal conditions，the condensation reaction generates a large conjugated sp2domain，and the uracil skeleton retains as the receptor.Subsequently without any further treatment，the second step was carried out under hydrolysis condition.The addition of sodium hydroxide transforms the imine group in the uracil receptor to amine and carboxyl groups.

Fig.1 Schematic diagram of all synthesis procedures

Firstly，the morphologies of the CDHBgenerated in the first step and the CDZWin the second step were characterized by transmission electron microscopy(TEM).Fig.2 shows TEM images of the CDHBand CDZW.CDHBhave a uniform dispersion and an average diameter of c.a.3 nm like typical dots，while diameter of CDZWis relative larger(c.a.5 nm).The lattice fringes of the two dots，with an interlayer distance of 0.21 nm，may correspond to the facet of graphitic carbon(100)[47].Thus，the two dots consisted of a nanocrystalline sp2carbon core.And a medium-intensity ring in the selected-area electron-diffraction pattern (Fig.2(a)) also revealed the existence of crystalline structure in CDHB.

Fig.2 HR-TEM images and FT-IR spectra of the CDs.(a)-(b)HR-TEM images of CDHBand CDZWnanoparticles(insets： lattice fringe images and SAED pattern for CDHB).(c)-(d)FT-IR spectra of CDHBand CDZW，respectively.

To demonstrate the site-specific changes on the dots'ligands in the line sequence，the investigations of FT-IR and XPS spectra of the two materials were conducted.It turns out that the structures'distinctions between CDHBand CDZWproduce significantly different vibration frequency and binding energy.The Fourier transform infrared(FT-IR)spectra of CDHB(Fig.2)show stretching vibrations for N—H(3 178 cm-1)， imideⅠ(1 714 cm-1)， imideⅡ(1 400 cm-1)，imideⅢ(1 096 cm-1)bonds and imideⅣ(755 cm-1)，manifesting the presence of imide group[48].Whereas the Fourier transform infrared(FT-IR)spectra of CDZW(Fig.2(d))show stretching vibrations for O—H(3 340 cm-1)，N—H(3 152 cm-1)，C==O(1 640 cm-1).The stretching vibrations of C—O at 1 117 cm-1and 906 cm-1also implicate the presence of—COOH group.In coincidence with the signals of FT-IR，the XPS peak has demonstrated the evident distinction on the element composition and the electronic property of the functional groups.According to peak analysis in XPS(Fig.3)，CDHBis composed of C 1s(45.98%)，N 1s(28.51%)and O 1s(25.52%).Whereas CDZWis composed of C 1s(52.69%)，N 1s(12.05%)and O 1s(35.26%).The distinctions of oxygen content and nitrogen content likely produce two types of structure defects and ligands on the surfaces.CDHBcould be ascribed to N-defect dots and CDZWcould be ascribed to O-defect dots[49].As shown in Fig.3(c)，deconvolution of C 1s peak in CDHBreveals abundant component of diverse carbonaceous groups.It partially displays similar characteristic peaks with uracil[50].The peak at 288.9， 287.7， 286.2， 285.1，284.6 eV can be assigned to the binding energy of carbon at the groups of(NH)2—C==O， O==C—NH， C==N， C—O， C—C/C==C respectively[50-51].The binding energy of 290.4 is correspondence with the π-π*[52].The component of C 1s in CDZWis relative simple.It could be deconvoluted into four peaks at 288.5， 286.8， 285.1， 284.6 eV，which could be attributed to the group of O==C—O，C==N， C—O， C—C/C==C respectively，shown in Fig.3(f).The distinction of the two deconvolution confirms the receptor changes.The element ratio of the two dots is distinctive.The CDHBincludes more nitrogenous ingredient，which ascribed to the uracil receptor，while CDZWexhibits lower nitrogenous constitution.The high resolution N 1s spectra(Fig.3(b))were resolved into three characteristic peaks at 401.4，400.0，399.2 eV corresponding to the nitrogen at graphite N，imide/amide，C==N，respectively[50].As for the deconvolution of CDZW(Fig.3(e))，there are three peaks of 401.4，400.2，399.2 eV[53].The shift of nitrogen binding energy from 400.0 to 400.2 eV indicates the ring-opening of uracil receptors.These data imply that the amino groups in diaminouracil are the predominant reactive sites in the first step，subsequently the imide group in the obtained uracil is the main reactive sites in the second step.

Fig.3 XPS spectra of CDHB(a)and CDZW(d)，high resolution N 1s spectra of CDHB(b)and CDZW(e)，and high resolution C 1s spectra of CDHB(c)and CDZW(f).

Fig.4 Normalized emission spectra of various amount of CDHBand CDZW

The concentration dependent emission spectra in Fig.4 showed that the CDHBaggregates(8.16 mg/mL)initially exhibited the spectroscopic features with a weak fluorescence emission at 527 nm.After being diluted，the mixture exhibits a blue shift accompanied with a distinct intensity increase in the fluorescence spectrum.In a final state，a diluted aqueous solution of CDHB(7.97 μg/mL)was tested with a blue shift of 84 nm since it is believed as a reliable solution to get a good dispersion of dots in the forms of individual monomers.And the absolute quantum yield of the fluorescence of the diluted solutions is 18.5%.Moreover，the dots in diluted solution also show remarkable excitation dependent emission spectra.Therefore，it illustrates that the emission wavelength of CDHBis closely controlled by the concentration.Moreover，the high concentration induces the self-quench of dots aggregates.Although high concentration enhances the extent of aggregation of nanomaterials in solutions， π-π stacking， hydrophobic interaction and the hydrogen bonding interaction among the dots are responsible for the concentration dependent bathochromic characteristic[54].At high concentration，the surface resonance energy is easier to transfer between dots aggregates，hence resulted in green-yellow emission[55].Besides，in the process of concentration increasing，a major energytransfer processes responsible for self-quenching because of the π-π interaction[56].As evidenced by the characterization above，CDHBbear uracil receptors which have been proven to be good hydrogen bonding substrates.With typical hydrogen bonding donor—NH and acceptor—C==O on the dots，aggregates of CDHBfromed with a specific head-to-tail dots arrangement[57].Generally，the luminescence of CDs is relative with core region and surface state.Emission from surface states is always red shifted compared to those originating from the core[58].The hybridization of carbon backbone resulting from aggregate behavior strengths the emission from surface.Thus CDHBexhibits concentration-dependent redshift emission.

In contrast to CDHB，zwitterated CDZWshowed almost no appearances of aggregates for hours.When the transparent solution temperatures drop without any disturbance after transferring from calve，no evident precipitates get produced.According to the study of the emission spectra at various concentration from 58.4 mg/mL to 3.65 mg/mL，CDZWdisplay no evident self-assembly behavior.The test of emission is non concentration-dependent.And the absolute quantum yield of the fluorescence of the solutions is 7.2%.As mentioned in characterizations above，the receptor of uracil has been transformed to carboxyl groups.These oxygen-related groups are regarded as electron traps，causing suppression of fluorescence due to non-radiative relaxation[59].Meanwhile，introduction of hydrophilic oxygen-related groups(—COOH)after alkaline treatment enhances the emission from surface states[60].And a relative larger core size of CDZWmakes it displaying in a fashion of red-shift in comparison of CDHBin diluted solution from the perspective of emission peak.Similar to CDHB， the π-π interaction drives CDZWfluorescence quenching with concentration increasing[61].The distinctions on spectra of the dots imply that the receptor domain transformation also influences the photoluminscence behavior.The phenomena are coincidence with the characterizations in FTIR and XPS spectra.

To further make clear how structure changes shape the binding affinity property，emission spectra were recorded at various metal ions.It is well known that the uracil shows high affinity towards silver and mercury.While bond rupture at the uracil moiety would shift the binding affinity.The results also strengthen the evidence obtained in the structure characterizations.As shown in Fig.5，when excited at 410 nm，the addition of silver and mercury triggered remarkable fluorescence emission quench of CDHB.The coordination process was completed after the addition of silver and mercury，implying that surface of CDHBbears the silver receptors.And such receptors could greatly be uracil.The above findings suggest that CDHBmay be promising as an efficient sensing platform for silver ions.

Fig.5 Emission profiles and quench mechanism of CDHB(128 μg/mL)upon addition with silver(0.08 μmol)(a)and CDZW(3.65 mg/mL)in the presence of nickel(0.36 μmol)(b)in an aqueous solution respectively(λex=410 nm).The intensity quenched index(I0/I-1)in the presence of various metal ions for CDHB(c)and CDZW(d).

The original fluorescence emission decreased(λex=410 nm)with addition of 0.08 μmol silver ions.It was noted that the index(I0-I)/Iat 463 nm gradually increased to 3.56 fold as the concentration of silver ions increased.These changes in the optical properties resulted from the coordination of CDHBwith silver ions at a molecular level.To evaluate their selectivity，the responses of CDHBtoward other competitive heavy metal ions were also tested under identical conditions.The same amount of other metal ions， namely Fe3+， Cu2+， Zn2+， Ni2+， only caused negligible or slight changes in the spectra.

In contrast to CDHB，zwitterated CDZWshowed almost no response towards silver and mercury.The emission intensity change are negligible upon addition of the two cations.Whereas nickel exhibits remarkable fluorescence quench.The test of emission towards other heavy metal ions such as Fe3+，Cu2+，Zn2+manifests the affinity selectivity for nickel.As mentioned above，the receptor transformation from uracil to amine and carboxyl groups induced the shift of binding affinity.

The results of the above competition experiments can be explained based on different stabilities of the metal complexes，which originate from the different coordination modes of receptors with the tested cations as confirmed in the literatures[21-22].All the spectra responses are originated from the coordination mode at a molecular level.

4 Conclusion

In summary，a linear sequence of stepwise hydrothermal reactions was utilized to construct two types of carbon dots skeletons with distinct receptor moieties.The uracil receptor makes the CDHBprone to form aggregates and exhibit high affinity towards silver，mercury ions in neutral solution.Opposite to CDHB， with amine and carboxyl moieties， CDZWbear the feature of zwitterion which performs improved solubility， stability in neutral aqueous solution， and displays high affinity for nickel ions.Via characterizations and properties investigations，principles of the organic synthesis serve the goal of rational preparation of carbon dots.With the consideration of the lack of accurate characterization methods for carbon dots，a rational synthesis scheme helps to bridge the relationships between the structures and the stimulus-responsive properties.Meanwhile，discovery of novel organic molecule and its orientation of chemical reaction will give us flexibility in a fashion that perfect the carbon dots skeletons with desired functionality.