PREPARATION AND CHARACTERIZATION OF WATERSOLUBLE NEAR-INFRARED EMITTING PbSQUANTUM DOTS

Cao Jie,Li Shan,Deng Dawei,Gu Yueqing

(School of Life Science and Technology,China Pharmaceutical University,Nanjing,210009,P.R.China)

INTRODUCTION

Semiconductor quantum dots(QDs),with tunable size and good optical properties,have attracted great research interest in the past two decades[1-4]. Among th em, the near-infrared(NIR)-emitting QDs with emission wavelengths between 700 nm and 1 000 nm areof particular interest to researchers because of the capability of penetrating living tissues several centimeters due to the low absorbance of tissue intrinsic chromophores[5-6].

To date various kinds of NIR-emitting QDs have been synthesized by organometallic routes or aqueous methods,including Cd Te[7],Cd Te/Cd S[8], Cd Te/Cd Se[9], Cd HgTe,Cd HgTe/Cd S[10],etc.To thebest of our knowledge,these Cd-,Hg-,Te-and Se-based QDs all are highly toxic.And the synthetic conditions are also complicated,thus needing high reaction temperature(≥ 100℃ ),long refluxing time and the protection of inert atmosphere.These shortcomings will deter their applications in biomedical fields. Compared with these materials,Pb S is lowly toxic and suitable for the synthesis of NIR-emitting QDs.However,in previous studies[11],the stabilizing agents(thioglycerol(TGL)and dithioglycerol(DTG)),commonly used for synthesizing PbSQDs,areall toxic carcinogenic substances with an awful odor.Besides this,the resulting water-soluble PbS QDs exhibit the emission in the range of 1 000 nm to 1 400 nm,leaving the facile and environmentally friendly aqueous synthesis of small PbS QDs with PL emission in the range of 700 nm to 1 000 nm still challenging.

In this paper,a new facile way is explored to prepare water-soluble Pb S QDs,using N-acetyl-L-cysteine,which is a derivate of L-cysteine as a stabilizing agent.Compared with the previous aqueous synthetic approach of PbS QDs(the stabilizers:TGL and DTG,and PL emission:from 1 000 nm to 1 400 nm),our strategy is more facile,and the resulting NIR-emitting PbS QDs are more promising in the biological application,especially in the in vivo imaging(the stabilizer N-acetyl-L-cysteine(NAC)is non-toxic and contains a—COOH terminus,and the PL emission of the resulting QDs is in the range of 895—970 nm).

1 EXPERIMENT

1.1 Chemicals

L-cysteine(99+%),NAC(99+%),glutathione(98+%),lead(II)acetate trihy-drate(99+%),Na2 S?9H2O(98+%),and sodium hydroxide(96+%)are of analytical grade and used as received.The water used in all experiments is deionized to a resistivity of 18.2 MΩ? cm.

1.2 One-pot facile synthesis of PbSusing dif ferent capping agents

Similar to the previous strategy[12],the typical route to synthesize water-soluble NAC-capped Pb SQDs using NACis as follows:Firstly,under ambient atmosphere, at room temperature,deionized water(2 mL)is added dropwise to NAC(49 mg)under magnetic stirring.Secondly,the aqueous solution of NAC(0.3 mmol)is adjusted to p H=8.0 by adding dropwise 0.2 mmol/L NaOH.Then,1 mL 0.1 mmol/L lead(Ⅱ)acetate solution is introduced into the solution of NAC on strong magnetic stirring.Finally,a 2 m L 0.025 mmol/L solution of Na2 S is slowly added dropwise to the system.The solution instantly turns from buff to dark-brown,indicating the formation of Pb S QDs.Stirring lasts for 5 min.Here,the total volume of the original solution is 50 mL,and the initial molar ratio of Pb/S/NAC is set to 1∶ 0.5∶ 3 unless otherwise specified.Water-soluble PbS QDs using glutathione(GSH)or L-cysteine as its capping agent is synthesized as the same as above-mentioned.

1.3 Characterization of PbSQDs in vitro

S2000 eight-channel optical fiber spectrographotometer (Ocean Optics Corporation,America),and a NL-FC-2.0-763 semiconductor laser(λ=765.9 nm,Enlight,China)light is utilized for fluorescence spectra detection.754-PC UV-Vis spectrophotometer(Jinghua Technological Instrument Corporation,Shanghai,China)is used for UV-Vis spectra measurement.All optical measurements are performed at room temperature.PL quantum yields(QYs)of PbS QDs in water are calculated by comparing their integrated emission to that of 20%aq DMSO solution of cypate(theabsorption and PL emission peaks of cypate are at 790 nm and 810 nm,respectively;the PL QY is 12%)supplied by Samuel Achilefu(Department of Radiology,Washington University at St.Louis).PH meter(PHS-25)is purchased from Shanghai Scientific Instrument Corporation,Shanghai,China.JEM-2100 transmittance electron microscope(JEOL,Japan)is used to evaluate the morphology and crystal structure of QDs.Powder XRD measurement is carried out using a Philips X′Pert PRO X-ray diffractometer(λ=1.541 78× 10-10).

1.4 Non-invasive real-time in vivo optical imaging of PbSQDs in mouse model

To capture the real-time surface maps of the fluorescence signals in living animals,a NIR imaging system is firstly constructed[13]. The schematic diagram of the imaging system is shown in Fig.1.A NL-FC-2.0 laser light(λmax=765.9 nm)is coupled into NIR optical fiber and defocused to provide a broad spot with even optical density shining on the surface of themouse.A high sensitive NIR CCD camera(Princeton,America)is positioned at 20 cm above the subjects.An 800 nm long pass filter(Chroma,Rockingham,V T)is put ahead of the CCD to block the excitation and ambient light,thus capturing the emitted NIRfluorescence(PL peak＞800 nm)from the molecular probes.The fluorescence imageis acquired and disposed by the matched software.

Fig.1 NIRimaging system

Prior to the NIR imaging experiment,Kunming(KM)mouse needs to be denuded through an artificial method(pruning).The denuded mice are put back to theanimal house and ready for experiment after 24 h.In a typical imaging experiment,the denuded mouseis immobilized in a special jig,and then Pb S QD aqueous solution(9μg/g)is injected through the right leg into the mouse.NIRimaging is performed in a dark room by using the NIR imaging system,where the light intensity of laser light from the fiber bundle is adjusted to 36×10-3W.For each mouse,a background image(prior to injection)is firstly acquired.The fluorescence image is then collected after the injection.

2 RESULTSAND DISCUSSION

2.1 Synthesis of NIR-emitting NAC-capped PbS QDs

When the solution of Na2Sis slowly added dropwise to the system containing 0.1 mmol Pb2+ions and 0.3 mmol NACat p H 8.0,the initial solution turns from pale yellow to darkbrown instantly,which indicates the formation of PbS QDs.In this study,in order to explore the influence of ligand on the synthesis of watersoluble Pb S QDs,three different ligands L-cysteine and its derivates(NAC and GSH)are selected.Fig.2 shows the absorption and PL spectra of the as-prepared PbS QDs when using L-cysteine,NAC and GSH as its capping agent,respectively.As shown in Fig.2(a),the fluorescenceintensity of NAC-stabilized PbSQDs is the highest,compared with that of L-cysteine-or GSH-stabilized Pb SQDs.The absorption shoulder(905 nm)and PL peak(950 nm)of NAC-stabilized PbSQDs are all located in the near-infrared spectral region. These experimental results demonstrate that NACis an effective ligand for the synthesis of water-soluble PbS QDs.That is to say,water-soluble NIR-emitting PbS QDs can be prepared by using NAC as a capping agent,besides the mixture of TGL and DTG reported previously[11,14].For the low toxicity and—COOH terminus,the as-prepared PbS QDs have more perspective in biological application,especially in the non-invasive in vivo biomedical imaging.

Fig.2 PL and absorption spectra of as-prepared Pb S QDs

2.2 Optimization of synthesis conditions

The influence of the precursor Pb/S molar ratio,the Pb/NAC molar ratio,and the p H of original solution on optical properties of NAC-stabilized Pb S QDs is further explored.Figs.3(a,b)show the PL and the absorption spectra by changing theprecursor Pb/Smolar ratio,respectively,where the concentrations of NAC and Pb2+ions are set to 3 mmol/L and 2 mmol/L,respectively,and only varies the added molar amount of Na2 S(the sulfur source of Pb SQDs).With decreasing the Pb/Smolar ratio from 1∶ 0.2 to 1∶ 0.8,the emission peak red shifts from 897 nm to 922 nm,which points to the increasein size of QDs.The PLintensity will reach a maximum at the Pb/S molar ratio of 1∶0.4.Similar to PL spectra,Fig.3(b)shows the absorption spectra also red shifts gradually with the decrease of the Pb/Sfeed ratio.These experimental results suggest that a part of the newly formed Pb S by the reaction between the successively introduced S2-anions and the Pb2+ions in the present synthetic system will deposit on the surface of the initially formed small PbS"core"QDs,resulting in the symmetrical increase in size of QDs.

Fig.3 PL and absorption spectra of NAC-stabilized PbSQDs

Fig.4 PL and absorption spectra of NAC-stabilized Pb SQDs

Fig.4 shows the PL(a)and absorption(b)spectra of PbS QDs obtained by changing the precursor NAC/Pb molar ratio.When tuning the NAC/Pb feed ratio from 1.5∶ 1 to 4∶ 1,PL peak position of as-synthesized PbS QDs red shifts correspondingly from 917 nm to 968 nm,which also indicates the increase in sizes of QDs.This experimental result is similar to the previous report[12].Furthermore,when the NAC/Pb feed ratio is 1.5∶1,it is optimal for the preparation of water-soluble NIR-emitting Pb SQDs.At this time,PL quantum yield is above5%.In the process of QD preparation,we attend that when the NAC/Pb molar ratio is less than 1.5∶1,the resulting PbSQDs are not stable,and will precipitate at room temperature.

In the study,the effect of the p H of the solution on the optical properties of NAC-capped PbS QDs is also investigated,and the result is shown in Fig.5.The p H of the solution of PbS QDs is adjusted to 7.2,8.0,10.0 and 11.2 and 12.0 with 0.2 mol/L NaOH solution,and PL spectra are measured under the same conditions.Fig.5(a)shows that the QDs formed at p H 8.0 show the strongest PL emission.When the p H adjusts to 7.2 below or 12.0 above,the PL intensity decreases apparently.As we known,in basic aqueous solutions,the sulfur atoms and the carboxylic acid group of NAC will be deprotonated.Thus,NAC can react readily with Pb2+ions to form the Pb-NAC complex,and the negative charges of carboxylate groups locate on the surface of the QDs repel each other,which are beneficial in preventing aggregation and stabilizing QDs,and result in higher PL efficiency.However,under neutral or weak acidic conditions,the thiol molecules will be protonated,leading to the detachment of the capping agent from QDs.

Fig.5 PL and absorption spectra of NAC-stabilized PbS QDs obtained by changing solution p H(precursor molar ratio of Pb/S/NAC=1∶ 0.5∶ 3, concentration of Pb2+ ions=2 mmol/L)

As such, the QD-ligand complexes are destroyed,and consequently decrease their PL emissionintensity.This experimental result confirms that at normal physiological p H(7.4),the produced NAC-capped PbS QDs are stable and still show a strong NIR fluorescence emission,which also indicates that it is possible to use these NIR-emitting QDs for noninvasive in vivo biomedical imaging.

2.3 Morphological and structural analyses of as-synthesized water-soluble NIR-emitting NAC-stabilized PbSQDs

As described above,using NAC as a stabilizer,water-soluble PbS QDs with strong NIR fluorescence are synthesized,and its reaction conditions are optimized systematically.Here,the morphology and crystal structure of these NAC-capped PbSQDs are characterized further.

The typical TEM image of NIR-emitting PbS QDs(λmax=900 nm)synthesized under optimized conditions is shown in Fig.6.Theinset shows a HRTEM image of the same sample.It is quite evident that these QDs areclose to spherical with well monodispersity,and remain well separated.

Fig.6 Typical TEM image of as-prepared NAC-capped Pb S QDs(λmax=900 nm)

The corresponding average size is less than 3 nm.Fig.6 displays the typical high-resolution TEM image of the NIR-emitting PbS QDs.The lattice spacing is 0.29 nm,corresponding to the(200)plane of the cubic structure of PbS.This experimental observation reveals that these QDs are cubic PbS.At the same time,the lattice fringes in the HRTEM image are not so clear as that appeared in our previous studies[14]because the sizes of as-prepared Pb S QDs are extremely small.

Fig.7 shows typical X-ray diffraction pattern of the powdered Pb S QDs stabilized by NAC.All reflections match well the reference powder diffraction pattern of PbS.The positions of these diffraction peaks show that the resulting NAC-stabilized Pb S QDs have a cubic crystal structure.Because the sizes of as-prepared PbS QDs are so small,the half band width is broader than the previous studies[8].Thus,the experimental result from XRD measurement is well in agreement with HRTEM imaging,confirming the structure of the produced PbS QDs to be face-centered cubic.

Fig.7 Typical X RD pattern of NAC-stabilized Pb SQDs(λmax=900 nm)

2.4 NIR fluorescence imaging in living animals with NAC-capped PbSQDs

As described above,the KM mouse is denuded and immobilized in the special jig after 24 h of further feeding,and then ready for the noninvasiveinvivo imaging.Thebackground images(prior to injection)of the mouse are firstly collected,as shown in Fig.8(a),which indicates that under the radiation of NIRlaser light(λmax=765.9 nm),endogenously generated fluorescence(i.e.,intrinsic fluorescence or autofluorescence)is weak.Next,a 3 mmol/L aqueous solution of Pb S QDs is injected into the right legs of the mouse.The mouse is imaged further.As shown in Fig.8(b),the right leg(pseudo-color)injected with QDs becomes much brighter than the other parts of the body.These experimental results confirm that the NIR fluorescence emission of PbSQDs from the excitation of laser light can penetrate living tissue of the mouse,and finally is detected by NIRin the vivo imaging system.

Fig.8 Background fluorescent image recorded by 765.9 nm excitation

3 CONCLUSION

In summary,by using NACas the stabilizer,high-quality water-soluble PbS QDs with strong NIR fluorescence emission are successfully synthesized through a one-pot facile,environmental friendly room temperature approach.The size and the PL emission peaks of theas-prepared PbSQDs can be tuned from 895 nm to 970 nm by simply changing the feed ratios of the precursors.Under normal physiological conditions,the produced NIR-emitting QDs are highly fluorescent.By means of our noninvasive in vivo NIR imaging system,the NIRfluorescenceof the PbS QDs in living tissues generated from the excitation with semiconductor laser can be detected easily.As a result,the high-quality water-soluble NIR-emitting PbS QDs show great potential in labeling biological molecules and cells for various noninvasivein vivo NIRfluorescence imaging applications.

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