Facile synthesis of N-acetyl-l -cysteine capped CdHgSe quantum dots and selective determination of hemoglobin

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 198–203

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Facile synthesis of N-acetyl-L-cysteine capped CdHgSe quantum dots and selective determination of hemoglobin Qingqing Wang, Guoqing Zhan ⇑, Chunya Li ⇑ Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 N-acetyl-L-cysteine (NAC) capped

CdHgSe quantum dots (QDs) were facilely synthesized.  The NAC capped CdHgSe QDs were characterized thoroughly.  Selective determination of Hb was demonstrated based on the NAC capped CdHgSe QDs.

a r t i c l e

i n f o

Article history: Received 14 February 2013 Received in revised form 21 July 2013 Accepted 2 August 2013 Available online 9 August 2013 Keywords: N-acetyl-L-cysteine CdHgSe quantum dots Fluorescence Hemoglobin

a b s t r a c t Using N-acetyl-L-cysteine (NAC) as a stabilizer, well water-dispersed, high-quality and stable CdHgSe quantum dots were facilely synthesized via a simple aqueous phase method. The as-prepared NAC capped CdHgSe quantum dots were thoroughly characterized by fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. A novel method for the selective determination of hemoglobin (Hb) was developed based on fluorescence quenching of the NAC capped CdHgSe quantum dots. A number of key factors including pH value of phosphate buffer solution, quantum dots concentration, the adding sequence of reagents and reaction time that influence the analytical performance of the NAC capped CdHgSe quantum dots in Hb determination were investigated. Under the optimal experimental conditions, the change of fluorescence intensity (DI) was linearly proportional to the concentration of Hb in the range of 4.0  109–4.4  107 mol L1 with a detection limit of 2.0  109 mol L1. The developed method has been successfully employed to determine Hb in human urine samples. Ó 2013 Published by Elsevier B.V.

Introduction Semiconductor nanocrystals (or quantum dots, QDs) have attracted considerable interest in the past two decades because of their excellent properties such as broad excitation band, narrow emission spectra, and higher resistance to photobleaching than their organic counterparts [1–5]. Since could be applied as optical ⇑ Corresponding authors. Tel.: +86 2767842752. E-mail addresses: [email protected] (G. Zhan), [email protected] (C. Li). 1386-1425/$ - see front matter Ó 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.saa.2013.08.001

amplifier media for telecommunications systems, ternary cadmium–mercury chalcogenides are of especial interest in electroluminescence devices and biological field [6]. Ternary QDs containing Hg are considered as potential candidates for infrared spectroscopic determination and solar cell applications due to the manipulability of bandgap, higher absorption coefficient and wider spectral sensitivity [7,8]. The presence of Hg is an important factor which influences size, shape and others of the fabricated QDs thus leading to the change of their fluorescence qualities. According to Hankare’s report, the control of size and shape of the QDs

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could be fulfilled by using different ratio of [Hg]/[Cd] [8]. Furthermore, the substitution of Cd by Hg in CdSe nanocrystals may lead to the red shift of fluorescence wavelength. Thus, the fluorescence interference from background, especially from biomolecules, can be reduced or even be eliminated. Many methods, ranging from high temperature hot injection method to aqueous precipitation route, have been developed for the preparation of mono-dispersed QDs. Although these routes are successful, there are still some inherent limitations for their fabrication including high-cost, environmentally unfriendly and rigorous experimental conditions. Moreover, in order to be prevented from aggregation, QDs are commonly capped with organic ligands such as mercaptopropionic acid (MPA) and thioglycolic acid (TGA). Some of these organic ligands have obnoxious odor and are carcinogenic. Therefore, stringent safety conditions are often required for their use [9,10]. Thus, preparation of QDs with high photostability and high emission quantum yield in an inexpensive and facile method is still of great interest to researchers. To meet this challenge, we use N-acetyl-L-cysteine as an effective stabilizer to synthesis QDs with a one-pot method. N-acetyl-L-cysteine (NAC) is known as an antioxidant and can protect cells against oxidative stress and QDs-induced cytotoxicity [11,12]. In addition, as a derivative of L-cysteine, NAC possesses excellent biocompatibility and good water solubility. It is also low cost, stable, nonvolatile, and odorless [11–16]. To our best known, NAC has not been used as a stabilizer in the synthesis of CdHgSe QDs in aqueous solution. In this work, NAC capped CdHgSe QDs was synthesized with an inexpensive and facile method. The as-prepared NAC capped CdHgSe QDs possess excellent water solubility and stability. Quantum yield (QY) of NAC capped CdHgSe QDs is estimated to be 40%, and is higher than CdHgTe (12%) and CdHgTe/ CdS (15%) quantum dots [17]. In addition, the doping of Hg into CdSe QDs resulted in significant increase of fluorescence intensity and red shift of emission wavelength, thus improving the quality of the quantum dots itself and reducing the fluorescence interferences from background. Meanwhile, the carboxyl groups existed in NAC is expected to form complex with metal ions, to aid solubilization in water and to provide active sites for further functionalization of the synthesized QDs. The above properties of the asprepared NAC capped CdHgSe QDs enable its application in biological analysis. Hemoglobin is the main component of red blood cells, and plays an important role in transporting oxygen from the lungs to the different organs for their proper function. The structure of hemoglobin and its sophisticated function for oxygen delivery and mechanism for oxygen binding have been fully investigated [18– 22]. Hemoglobin concentration in urine samples has been used as an important index for diagnosis of hemolytic disease and nephritis, damages of the red cells from mechanochemistry, immune process or radiation. Various methods including UV spectrophotometric method [23], high performance liquid chromatography [24], electrochemical measurement [25,26] have been developed for the determination of Hb in complex matrix. Unfortunately, some of them suffered from either expensive, complex or the necessity to work in organic solvents. Compared to electrochemical measurements, limited investigation has been spend on the determination of Hb using QDs with fluorescence spectroscopy, especially fluorescence quenching method. Herein, a facile method for the fabrication of water-dispersed NAC capped CdHgSe QDs was present. Influences of pH values, the ratio of [Hg]/[Cd], reaction temperature and time on the fluorescence properties of NAC capped CdHgSe QDs were investigated thoroughly. Based on fluorescence quenching method, selective determination of trace amount of Hb using the NAC ca-

199

pped CdHgSe QDs as fluorescent probes were successfully performed. The practical application of the developed method was demonstrated by determining Hb in human urine samples. Experimental Reagents NAC was purchased from Shanghai Aladdin Reagent Inc. (Shanghai, China). Powdered Hb was bought from Shanghai Bio Science & Technology. Co., Ltd. (Shanghai, China), and was dissolved into phosphate buffer solution to obtain 1.0  106 mol L1 Hb standard solution. Selenium powder and NaBH4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). CdCl22.5H2O was purchased from Chengdu Chemical Reagent Plant (Chengdu, China). All water used in the experiment was ultrapure water. Apparatus All fluorescence spectra of the NAC capped CdHgSe QDs were measured using a Perkin Elmer LS-55 luminescence spectrometer. The excitation wavelength (kex) at 390 nm was selected for all experiments. All pH values were measured with pHS-3 meter (Shanghai, China). A Tecnai G2 20-TEM operating at 200 kV was used for morphologic characterization. FT-IR spectra were recorded on a Nexus 470 spectrophotometer using KBr pellet. Energy-dispersive X-ray spectroscopy (EDS) was captured using an FEI Sirion 200 scanning electron microscope. A VG MultiLab 2000 photoelectron spectrometer was used to obtain X-ray photoelectron spectrum (XPS). The QY of CdHgSe QDs was measured according to the literature [27]. Rhodamine B in ethanol was chosen as the reference standard [28]. Preparation of NAC capped CdHgSe QDs The typical procedure for the synthesis of NAC capped CdHgSe QDs was similar to previously reported method with some modifications [29]. NaBH4 was used to react with selenium powder at a molar ratio of 2:1 in ultrapure water to produce NaHSe solution. 20.00 mL of CdCl2 solution (0.002 mol L1), NAC (0.0157 g) and 10 mL of ultrapure water were loaded into a 100 mL three-neck flask under protection of N2. The pH value of the reaction mixture was adjusted to 11.5 by adding sodium hydroxide solution dropwise. Then 0.01 mol L1 HgCl2 solution was added dropwise. After homogeneous mixing, NaHSe solution (84 lL) was injected into the reaction mixture using a syringe, and vigorously stirred for 4 h at 80°C. The concentration of NAC capped CdHgSe QDs estimated from NaHSe added was 5.90  104 mol L1. The obtained NAC capped CdHgSe QDs solution was stored in a refrigerator and found to be stable at least for 4 months. Measurements for Hb determination NAC capped CdHgSe QDs solution and various amounts of freshly prepared Hb standard solution were mixed in phosphate buffer solution, and equilibrated for 15 min before the spectral measurements. Fluorescence spectra were recorded at the excitation wavelength of 390 nm and the emission wavelength of 598 nm. The slit widths for excitation and emission were 7 nm and 15 nm, respectively. The change of fluorescence intensity (DI) between the NAC capped CdHgSe QDs solution and the mixed solution was used for Hb determination.

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Results and discussion Characterization of NAC capped CdHgSe QDs Transmission electron microscopy was employed to characterize the morphology of the as-prepared NAC capped CdHgSe QDs. As shown in Fig. S1, the TEM image reveals that the shape of the NAC capped CdHgSe QDs was spherical and well proportioned. The average size of QDs is estimated to be 1–2 nm. FTIR spectra of NAC (a) and the NAC capped CdHgSe QDs (b) were displayed in Fig. 1. In Fig. 1a, the characteristic peak at 2548 cm1 is corresponding to S–H stretching vibration. The peak located at 1718 cm1 is attributed to the asymmetric stretching of carboxyl group. The peak located at 1585 cm1 is ascribed to the flexural vibrations of amide linkage. Furthermore, peaks located at 2900 cm1 and 2808 cm1 are due to C–H stretching vibrations. However, in the case of NAC-capped CdHgSe QDs (curve b), the characteristic peak of S–H disappeared, indicating the S–H bond was cleaved and formed new bond between NAC and CdHgSe QDs. In addition, the characteristic absorptions of methyl and methylene are still observed obviously in curve b. The above results indicate that the NAC has been successfully modified onto the CdHgSe QDs surface. XPS was used to search for the presence of particular elements in the NAC capped CdHgSe QDs surface. From Fig. 2, it was found that C1s levels at 284.6 eV, O1s level at 530.9 eV, N1s level at 399.2 eV and S2p level at 159.9 eV were existed in the NAC capped CdHgSe QDs. In addition, Cd3d core levels at 404.6 eV, Hg4f level at 100.1 eV and Se3d level at 53.6 eV are assigned to be the CdHgSe QDs. These results suggested that the NAC capped CdHgSe QDs have been successfully synthesized with a facile method. In addition, the EDS technique was employed to analysis the composition of the NAC capped CdHgSe QDs. Seen from Fig. 3, Cd, Hg, Se and S elements are found to exist in the NAC capped CdHgSe QDs. The existence of S element indicates the successful modification of NAC onto the CdHgSe QDs surface. In the further analysis, we can see that the actual constituent ratio of Cd to Hg is estimated to be 4:1, which is higher than the feed ratio (1:1). There would be two possible reasons for this phenomenon. The first reason might be the loss of the volatile Hg. The embedding of Hg element into the interior of the NAC capped CdHgSe QDs during the growth process would be the second reason. EDS is only a micro-area technique for surface analysis. Hg element in the interior part of NAC capped CdHgSe QDs would be difficult to be detected quantitatively, thus resulting in the high ratio of Cd to Hg.

The fluorescence intensity of the NAC capped CdHgSe QDs was seriously influenced by pH value during its preparation. As shown in Fig. 4, in the pH value range from 10.94 to 11.50, the fluorescence intensity of the NAC capped CdHgSe QDs increased dramatically. With further increasing pH values from 11.50 to 11.94, the fluorescence intensity of the NAC capped CdHgSe QDs decreased reversely. The participation of OH in the formation of the NAC capped CdHgSe QDs may be responsible for this result. The added amount of HgCl2 in the fabrication of the NAC capped CdHgSe QDs was controlled by adjusting the volume of

Fig. 1. FTIR spectra of NAC (a) and the NAC capped CdHgSe QDs (b).

Fig. 4. Influence of pH values on the fluorescence intensity of the NAC capped CdHgSe QDs. (From a to f is the fluorescence spectra of QDs prepared at pH values of 10.94, 11.40, 11.50, 11.60, 11.78, 11.94.)

Fig. 2. XPS spectrum of the NAC capped CdHgSe QDs.

Fig. 3. EDS spectrum of the NAC capped CdHgSe QDs.

Optimization of preparation conditions

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0.01 mol L1 HgCl2 solution. It was found that the added amount of HgCl2 not only affect the fluorescence intensity of the NAC capped CdHgSe QDs but also the emission wavelength. From Fig. 5, we can see that the fluorescence intensity of QDs enhanced greatly with the volume of HgCl2 solution increasing from 0 mL to 4 mL. In addition, the emission wavelength shifted from 475 nm to 578 nm. The red shift of emission wavelength will profoundly benefit the biologic analysis due to the avoidance of the interference from the fluorescence background of biomolecules. However, when the added amount of HgCl2 solution was more than 4 mL, precipitation reaction occurred due to the formation of Hg(OH)2, thus leading to fluorescence quenching of the NAC capped CdHgSe QDs. Furthermore, the reaction temperature and time were also optimized. The reaction had better to be conducted at 80 °C for 4 h. Interaction between NAC capped CdHgSe QDs and Hb The fluorescence intensity of the NAC capped CdHgSe QDs decreased with the addition of Hb, indicating a fluorescence quenching interaction between the QDs and Hb. The mechanism of the fluorescence quenching process was investigated and assumed to be a dynamic quenching process using Stern–Volmer equation [30]:

F 0 =F ¼ 1 þ K q s0 ½Q  ¼ 1 þ K SV ½Q 

found to decrease with Hb concentration increasing, indicating Hb possesses quenching effect on the fluorescence intensity of the NAC capped CdHgSe QDs. Two possible reasons lead to the fluorescence quenching of the NAC capped CdHgSe QDs. Firstly, from the view of chemical structure, surface state change may result in the change of the fluorescence performance of the NAC capped CdHgSe QDs. The isoelectric point of Hb is less than pH value of the solution, therefore, Hb molecules would be charged with positive charge. The NAC capped CdHgSe QDs which loaded with negative charge will be encouraged to react with Hb through electrostatic attraction, even to form ionic bonds. In addition, thermodynamic parameters including enthalpy change (DH), free energy (DG) and entropy change (DS) were examined to confirm the interaction forces between the NAC capped CdHgSe QDs and Hb. If the temperature does not vary significantly, the enthalpy change can be regarded as a constant. The thermodynamic parameters can be evaluated from the van’t Hoff equation:

DH DS þ RT R DG ¼ DH  T DS ¼ RT ln K ln Ka ¼ 

ð3Þ

where R is the gas constant, T is the experimental temperature and K is the binding constant at corresponding T. K can be calculated from the binding constant formula [32]:

ð1Þ log

Where F and F0 are the fluorescence intensity at 590 nm in the presence and absence of Hb, respectively; KSV is the Stern–Volmer quenching constant; s0 is the average life-time of biomolecules without quencher (s0 = 108 s [31]) and [Q] is the quencher concentration ([Hb]). The Stern–Volmer plots of the fluorescence quenching of the NAC capped CdHgSe QDs by Hb at different temperatures were displayed as Fig. S2. The corresponding Ksv for the interaction between Hb and QDs were calculated to be 1.47  106 L mol1 (R = 0.9908) at 290 K and 2.521  106 L mol1 (R = 0.9925) at 310 K. The results show that the Stern–Volmer quenching constant KSV is positively correlated with temperature, proving the quenching mechanism is dynamic quenching process. No absorption wavelength change of QDs in the presence and absence of Hb, which indicated that the quenching process occurred in excited state. Hence, the probable quenching mechanism of the NAC capped CdHgSe QDs by Hb was confirmed. The binding reaction between them was initiated by dynamic collision. Fluorescence spectra of the NAC capped CdHgSe QDs in the presence of Hb with varying concentrations were shown in Fig. 6. The fluorescence intensity of the NAC capped CdHgSe QDs was

ð2Þ

  F0  F ¼ log K þ n log½Q  F

ð4Þ

Fig. 6. Fluorescence spectra of the NAC capped CdHgSe QDs interacted with different concentration of Hb. (From a to f, the Hb concentration is 0, 4.0  109, 8.0  108, 1.4  107, 2.0  107, 4.4  107 mol L1.)

200

160

I

120

80

40

0 0 Fig. 5. Fluorescence spectra of the NAC capped CdHgSe QDs fabricated using the different volume of 0.01 mol L1 HgCl2 solution. (From a to f, the volume of HgCl2 solution is 0, 1.5, 2.5, 3.0, 3.5 and 4.0 mL, respectively.)

100

200

300

400

Concentration of Hb (n mol.L-1) Fig. 7. Calibration curve for the determination of Hb.

500

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Table 1 Interferences of coexisting substances for the determination of Hb. Substances

Change of fluorescence intensity (%)

Substances

2+

4.8

Ca

Fe3+

1.7

Mg2+

Pb2+

Fe

2+

Change of fluorescence intensity (%)

Substances

2.2

L-proline

0.04

L-phenylalanine

1.8

L-histidine

4.8

5.9

1.6

Al3+

K+

2.9

Zn2+

Ag+

4.8

L-methionine

1.0

L-arginine

Co2+

0.71

L-valine

4.4

BSA

12 0.35

where n is the number of binding sites. DH was calculated to be 73.16 kJ mol1, DG was 34.29 kJ mol1 at 290 K and 31.61 kJ mol1 at 310 K, DS was 134 J mol1 K1. The negative value for DG means the interaction process is spontaneous at the standard state. The negative values of DH and DS indicate that the hydrogen bonds played an important role in the binding of QDs to Hb [33]. Based on the fluorescence quenching of the NAC capped CdHgSe QDs, a sensitive method for the determination of Hb was developed.

Optimization of experimental conditions The influence of pH values on the fluorescence intensity of 5.9  105 mol L1 NAC capped CdHgSe QDs interacted with 3.0  107 mol L1 Hb solution. The plot between the change of fluorescence intensity (DI = F0F) and the pH values of solution was shown in Fig. S3. When pH values varied from 5.0 to 6.0, DI increased gradually. With further increasing of pH value, DI decreased reversely. The maximal DI value was obtained at about pH 6.0, and this pH value was selected for the further determination of Hb. Effect of the NAC capped CdHgSe QDs CdHgSe concentration on the determination of Hb was shown in Fig. S4. At lower concentration, DI increased with the NAC capped CdHgSe QDs concentration increasing, possibly due to the incomplete interaction of the NAC capped CdHgSe QDs with Hb. However, DI decreased reversely when the NAC capped CdHgSe QDs concentration was higher than 5.9  105 mol L1. The overloading of the NAC capped CdHgSe QDs leads to the excessive interaction sites than the Hb supplied, thus resulting in the decrease of DI. Consequently, the sensitivity for the determination of Hb decreased correspondingly, and the appropriate NAC capped CdHgSe QDs concentration for Hb determination was 5.9  105 mol L1. Effects of the mixing sequence and reaction time for Hb determination were also investigated. The optimized mixing sequence was 0.05 mol L1 phosphate buffer solution at pH 6.0, Hb solution and followed by the drop addition of the NAC capped CdHgSe QDs solution. The suitable reaction time, which was evaluated by the values of DI at the same concentration of Hb and the NAC capped CdHgSe QDs, was 15 min. The fluorescence intensity of the obtained mixture can be maintained stably at least 2 h at room temperature.

Analytical characteristics The change of fluorescence intensity (DI) of the NAC capped CdHgSe QDs was employed to evaluate the analytical performance of the proposed method for the determination of Hb. As shown in Fig. 7, under the optimal experimental conditions, DI was found to be proportional to the concentration of Hb (c) in the range from 4.0  109 mol L1 to 4.4  107 mol L1 with an equation of DI = 0.434 c (nmol L1) + 5.43 (R = 0.998). The detection limit was estimated to be 2.0  109 mol L1 (S/N = 3).

L-lysine

Change of fluorescence intensity (%)

3.4 1.7 5.5

The specificity of the proposed method was also investigated. The influences of various metal ions, amino acids and bull serum albumin (BSA) on the determination of 1.0  107 mol L1 Hb were examined. The concentration of these interferences, 2.0  106 mol L1, was 20 times of Hb concentration. As shown in Table 1, the relative deviation for the fluorescence intensity was very slight (<5.0%). These results demonstrated that most metal ions, amino acids and bull serum albumin have noninterference on the determination of Hb, showing good selectivity of the method. However, as shown in Fig. S5, cytochrome C, protoporphyrin IX, myoglobin and heme gave positive interferences. We can draw a conclusion reasonably that the NAC capped CdHgSe QDs might be suitable for the determination of these type of compounds with different sensitivity. To evaluate its practical utility, the proposed method was employed to determine trace amount of Hb in human urine samples with standard addition method. Prior to being determined, human urine samples were centrifuged, and were then diluted with 100fold ultrapure water. The calculated average concentration for Hb in the human urine samples was 3.14  108 mol L1 (n = 5, RSD = 2.6%). The recoveries for the addition of Hb into human urine samples were in the range of 94.27–102.1%. The concentration of Hb in the tested human urine samples was also confirmed with the reported method [34], and the obtained average value is 3.01  108 mol L1 (n = 5, RSD = 4.3%). The relative deviation is 2.1%, indicating that the NAC capped CdHgSe QDs is a promising and reliable tool for the determination of Hb, and possesses potential applications in the analysis of the relevant real samples.

Conclusions A facile method for the fabrication of the NAC capped CdHgSe QDs was developed. The as-prepared NAC capped CdHgSe QDs have been demonstrated to possess superior stability, good water solubility and high quantum yield. The fluorescence intensity of the NAC capped CdHgSe QDs was found to be strongly dependent on pH value, temperature, time, and the amount of HgCl2. The dramatic decrease of the fluorescence intensity of the NAC capped CdHgSe QDs upon interacting with Hb enable to develop a sensitive and selective method for Hb determination. The proposed method was proved to be low cost, reproducible and reliable for the analysis of Hb in human urine samples. Under further way, the NAC capped CdHgSe QDs would be a promising probe for the diagnosing of nephritis and other related diseases.

Acknowledgements The authors gratefully acknowledge the financial supports from Natural Science Foundation of China (No. 21275166), Wuhan Science and Technology Bureau (No. 201160723224), South-Central University for Nationalities (No. XTZ09005).

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