Mn-doped ZnS quantum dots with a 3-mercaptopropionic acid assembly as a ratiometric fluorescence probe for the determination of curcumin

Abstract

Mn-doped ZnS quantum dots capped with 3-mercaptopropionic acid (MPA) were synthesized by a facile method in aqueous solution as a ratiometric fluorescent (I_{590 nm}/I_{458 nm}) probe for curcumin. Curcumin caused defect-related fluorescence quenching of the probe at 458 nm, whereas the orange transition emission of the probe at 590 nm changed only negligibly with variations in curcumin concentration. The MPA-assembled Mn-doped ZnS quantum dots were used for the simple, sensitive and non-toxic determination of curcumin. Under the optimized conditions, the response to curcumin was linear in the range 0.16–16.9 μM with a detection limit of 37 nM. The method was successfully applied to the determination of trace amounts of curcumin in urine samples with recoveries of 97.73–109.73%.

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1 Introduction

Curcumin (Fig. 1) is found in turmeric powder, turmeric root tubers and zedoary (white turmeric), commonly used in cosmetics and spices.¹ Curcumin also has beneficial pharmacological effects, including anti-inflammatory,² antioxidant,³ anti-cancer,^4,5 anti-HIV⁶ and antimicrobial⁷ properties. The quantification of curcumin has therefore attracted interest in both clinical medicine and pharmacology.^8,9 Various methods have been developed for the determination of curcumin, including high-performance liquid chromatography,^10–12 high-performance thin-layer chromatography,¹³ dispersive liquid–liquid microextraction,^14,15 electrochemical methods,¹⁶ fluorimetry^17,18 and UV-visible spectrophotometry.¹⁹ Compared with other methods, fluorescence spectrometry is of interest because it is facile, rapid, sensitive, selective and cost-effective.²⁰ Wang et al.²¹ developed a fluorimetric method for the determination of curcumin based on the sensitizing action of a mixed micelle. Chen et al.²² used a resonance light scattering technique to detect the complex formed between Cu(II) ions and curcumin with a detection limit of 0.19 μM. Rahimi et al.²³ described the use of high-performance liquid chromatography for the determination of curcumin in human urine samples. High sensitivity and selectivity are highly desirable in the determination of curcumin.

Fig. 1 Chemical structure of curcumin.

Semiconductor quantum dots (QDs) are robust inorganic chromophores that combine an efficient broadband absorption with a narrowband fluorescence spectrum. Doping QDs with trace impurities enables the number of emission centres to be modified or increased.^24,25 QDs have therefore become very attractive nanomaterials for bioimaging and drug detection. It has been reported that the replacement of cadmium in cadmium chalcogenide QDs with zinc as the absorption zone resolved toxicity problems while retaining the original advantages of this method.²⁶ Intentionally importing transition metal ions (Mn, Cu, Ag and Eu) as the emissive centre in bulk semiconductors has been shown to improve their optical properties.²⁶ Manganese (Mn²⁺)-doped QDs, particularly using ZnSe or ZnS as the host, are a novel category of luminescent materials that not only minimize self-absorption, but also enhance the quantum efficiency. The defect-related emission of Mn-doped ZnS QDs leads to fluorescence at 458 nm resulting from the band gap transition of ZnS, whereas an orange emission at 590 nm originates from the ⁴T₁ → ⁶A₁ d–d transition of the Mn²⁺ on the Zn²⁺ sites, where Mn²⁺ is tetrahedrally coordinated by S²⁻.²⁷ Mn-doped ZnS QDs have been used as a fluorescent platform for the determination of L-tryptophan and proteins,^28,29 4-nitrophenol³⁰ and TNT.³¹ We therefore investigated the use of Mn-doped ZnS QDs to construct a detection system for curcumin.

We synthesized Mn-doped ZnS QDs in aqueous solution using 3-mercaptopropionic acid (MPA) as the stabilizer. The as-prepared MPA-capped Mn-doped ZnS QDs were characterized by TEM, IR spectra, XRD, UV-visible spectrophotometry, fluorimetry, size distribution and zeta potential measurements. Using changes in the ratiometric emission response of Mn-doped ZnS QDs in the presence of curcumin, a highly sensitive and selective method was established and applied to the determination of curcumin in human urine samples. The as-synthesized Mn-doped ZnS QDs were shown to be good candidates for the ratiometric fluorescence determination of curcumin in the fields of biology, chemistry, medicine and clinical analysis.

2 Experimental

2.1 Reagents

3-Mercaptopropionic acid (MPA), sodium sulfide nonahydrate (Na₂S·9H₂O), manganese chloride (MnCl₂) and heptahydrate zinc sulphate (ZnSO₄·7H₂O) were purchased from Aladdin. β-Glucuronidase (type IX-A from Escherichia coli) was purchased from Sigma. All chemicals and solvents were of analytical-reagent grade and were used without further purification. The solutions of metal cations were prepared from their chlorine salts. A phosphate buffer solution (0.1 M, pH 7.4) was prepared by mixing solutions of sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄) and sodium hydroxide (NaOH). Doubly distilled water (18.2 M cm⁻¹) from a Millipore Milli-Q system was used to prepare all the aqueous solutions.

2.2 Apparatus

A Mettler Toledo FE20 pH meter was used to determine the pH. The fluorescence spectra were measured on a Hitachi F-4500 fluorescence spectrophotometer. UV-visible absorption spectra were collected using a Hitachi TU-1901 double-beam UV-visible spectrophotometer. Infrared spectrometry was carried out using an FTIR-8400S spectrometer. The appearance and size of the nanoparticles were examined by a JEOL JEM-2100 high-resolution transmission electron microscope (Tokyo, Japan). Fluorescence lifetime assays were completed with an FLS-920 Edinburgh fluorescence spectrophotometer. The XRD spectra were obtained on a D8 Advance Bruker X-ray diffractometer (Cu Kα radiation). The size distribution and zeta potential measurements were performed with a Malvern Instruments Nano-ZS90 Zetasizer. LC-MS spectra were recorded on a Shimadzu Instruments LCMS-8030 instrument.

2.3 Procedure

2.3.1 Synthesis of the Mn-doped ZnS QDs. MPA-capped Mn-doped ZnS QDs were synthesized via an aqueous route according to the previously reported reference method.³² For 3% Mn doping, the synthetic procedure was as follows: 5 mL of 0.1 M ZnSO₄, 1.5 mL of 0.01 M MnCl₂ and 20 mL of 0.1 M MPA were added to a three-necked flask and then diluted to 45 mL with doubly distilled water. The pH was adjusted to 11 with 1 M NaOH and stirred under nitrogen at room temperature for 30 min. A 5 mL volume of 0.1 M Na₂S was then quickly injected into the solution. Stirring was continued for 30 min until Mn-doped ZnS nanocrystals with 3 at% Mn were obtained. The solution was then heated at 50 °C in air for 2 h to form MPA-capped Mn-doped ZnS QDs. The MPA-capped Mn-doped ZnS QDs were precipitated by anhydrous ethanol and the precipitate was centrifuged and washed with ethanol, then dried under vacuum. The as-prepared QD powder was highly soluble in water.

2.3.2 Measurement procedures. A 2 mL volume of 5 mg mL⁻¹ Mn-doped ZnS QDs and 3 mL of 0.1 M PBS (pH 7.4) were mixed in a 10 mL calibrated test-tube, then different volumes of curcumin were added stepwise to the mixture, followed by thorough shaking for 5 min. Using an excitation wavelength of 315 nm, the fluorescence spectrum of the mixture showed two emission peaks at 458 and 590 nm; the ratiometric fluorescence emission was used for quantitative analysis. The excitation and emission slit widths were both 10 nm.

3 Results and discussion

3.1 Characterization of the MPA-capped Mn-doped ZnS QDs

The as-prepared MPA-capped Mn-doped ZnS QDs were characterized by TEM and XRD, and FTIR, UV-visible and fluorescence spectrometry. The TEM images (Fig. 2) showed that the Mn-doped ZnS QDs had a spherical shape and were almost uniformly dispersed with diameters of about 2.8 ± 0.1 nm. The XRD patterns of the Mn-doped ZnS QDs showed a cubic (or zinc blende) structure with peaks corresponding to the (111), (220) and (311) planes (Fig. 3), consistent with the standard cubic bulk ZnS peak position in JCPDS file no. 77-2100. Fig. 4 shows the IR spectra of the MPA and MPA-capped Mn-doped ZnS QDs. The absence of the stretching bond of the sulfhydryl group between 2682 and 2561 cm⁻¹ showed the attachment of the MPA ligand through covalent bonds between thiols and the surface Zn atoms of the Mn-doped ZnS QDs. As a result, the solubility of the QDs in water was improved. Fig. 5 shows the UV absorption spectra of the Mn-doped ZnS QDs and curcumin; the Mn-doped ZnS QDs had an obvious ultraviolet absorption peak around 315 nm, which showed a quantum confinement effect.³³ Fig. 6(1, 1′) shows that the Mn-doped ZnS QDs emitted a strong fluorescence at about 458 and 590 nm when excited at 315 nm. The fluorescence emission at 458 nm resulted from the defect-related emission of ZnS. With Mn²⁺ as the dopant, the orange emission at about 590 nm originated from the ⁴T₁–⁶A₁ transition of the Mn²⁺ ion on the Zn²⁺ sites, which indicated that Mn had entered into the ZnS lattice to form the Mn-doped ZnS QDs. It was noted that curcumin had a significant fluorescence emission around 520 in the polar solvent ethanol (2, 2′) and a very weak fluorescence emission in the PBS used here (3, 3′).³⁴ The fluorescence of curcumin interfered negligibly with our assay. The size distribution of the Mn-doped ZnS QD particles was also examined by DLS (Fig. S1, ESI†).

Fig. 2 TEM image of Mn-doped ZnS QDs.

Fig. 3 XRD spectrum for Mn-doped ZnS QDs.

Fig. 4 FTIR spectra of MPA and MPA-capped Mn-doped ZnS QDs.

Fig. 5 UV-visible absorption spectra of Mn-doped ZnS QDs and curcumin.

Fig. 6 Fluorescence spectra of 16.9 μM curcumin and 1 mg mL⁻¹ Mn-doped ZnS QDs. (1, 1′) Mn-doped ZnS QDs in PBS with pH 7.4; (2, 2′) curcumin in ethanol; and (3, 3′) curcumin in pH 7.4 PBS.

3.2 Optimization of factors influencing determination of curcumin

3.2.1 Effect of pH. The ratiometric change in emission response (I_{590 nm}/I_{458 nm}) of the MPA-capped Mn-doped ZnS QDs was dependent on pH (Fig. 7). The fluorescence ratiometric emission of the MPA-capped Mn-doped ZnS QDs increased slightly in the absence and presence of curcumin at pH 5–7 as a result of the dissociation of the QDs and the capping agent based on protonation of the surface-binding sulfhydryl compounds (Fig. 8). Between pH 7.0 and 8.0, the fluorescence ratiometric emission of both the MPA-capped Mn-doped ZnS QDs and curcumin increased dramatically because the covalent bond between the QDs and the capping molecules was enhanced; it is possible that the carboxyl group lost a proton. However, the ratiometric emission began to decrease with increasing pH, which might be caused by the disintegration of MPA in extremely basic media and the formation of metal hydroxide precipitates.^35,36 This phenomenon could also be attributed to electrostatic repulsion between negatively charged Mn-doped ZnS QDs (Fig. S2, ESI†) and the curcumin anion (Fig. 9).³⁷ Based on these factors, the neutral form of curcumin was chosen as suitable for our study system. pH 7.4 was chosen as the optimum condition for the determination of curcumin in subsequent studies.

Fig. 7 Effect of pH on the changes in the ratiometric emission response (I_{590 nm}/I_{458 nm}) at 458 and 590 nm of the Mn-doped ZnS QDs in the absence and presence of 1 μM curcumin.

Fig. 8 Protonation action of the Mn-doped ZnS QDs and the MPA capping agent.

Fig. 9 Prototropic equilibria of curcumin.

3.2.2 Effect of reaction temperature. The effect of temperature on the fluorescence emission of curcumin was examined. Fig. 10 shows that the fluorescence ratiometric emission decreased gradually with increasing reaction temperature from 20 to 80 °C. At temperatures >25 °C, the kinetic energy of the QDs increased, which caused an increase in the dissociation rate of the MPA ligand stabilizing the QDs; thermal quenching might also decrease the fluorescence intensity as the temperature increases.³⁸ It was obvious that a lower temperature of 15 °C could give rise to a higher fluorescence than a temperature of 20 °C. A lower temperature would therefore be better from the point of view of an assay. However, we chose room temperature (20 ± 2 °C) as the measurement temperature for convenience.

Fig. 10 Effect of reaction temperature on the fluorescence intensity of the Mn-doped ZnS QDs in the absence and presence of 1 μM curcumin.

3.3 Determination of curcumin using MPA-capped Mn-doped ZnS QDs

We investigated the fluorescence sensing of curcumin using the MPA-capped Mn-doped ZnS QDs under optimum conditions and found that these QDs were sensitive to curcumin. Fig. 11 shows that the fluorescence intensity of the Mn-doped ZnS QDs at about 458 nm decreased as curcumin was added, whereas the fluorescence intensity at 590 nm changed only negligibly. We compared the linearity of the ratiometric response of I₅₉₀/I₄₅₈ with that of I₄₅₈/I₅₉₀ (Fig. 12). The I₅₉₀/I₄₅₈ response showed good linearity, whereas I₄₅₈/I₅₉₀ showed a poor linearity; I₅₉₀/I₄₅₈ was therefore chosen for the calibration graph. The fluorescence emission at 458 nm results from the band gap transition of ZnS, whereas that at 590 nm is attributed to the ⁴T₁ → ⁶A₁ d–d transition of Mn²⁺ on the Zn²⁺ sites.²⁷ Fig. 12 shows that the presence of curcumin only affected the band gap transition of ZnS and had no effect on the ⁴T₁ → ⁶A₁ d–d transition of Mn²⁺ on the Zn²⁺ sites. The quenching of fluorescence emission at 458 nm might be due to ultrafast electron transfer from the QDs to curcumin during the band gap transition of ZnS³⁹ (Fig. S3, ESI†). Thus the ratio I₅₉₀/I₄₅₈ for the Mn-doped ZnS QDs gradually increased with increasing curcumin concentration to 16.9 μM. When the surface of the Mn-doped ZnS was fully saturated with curcumin, I₅₉₀/I₄₅₈ was at a maximum. The ratiometric emission I₅₉₀/I₄₅₈ of the Mn-doped ZnS QDs had a linear correlation with the concentration of curcumin in the range 0.16–16.9 μM, with a linear regression equation of I₅₉₀/I₄₅₈ = 0.5181 + 0.0344C, where C is the concentration of curcumin in μM (r = 0.9993). The limit of detection and the relative standard deviation were 37 nM and 3.19% (n = 11), respectively.

Fig. 11 Fluorescence spectra of MPA-capped Mn-doped ZnS QDs at various concentrations of curcumin.

Fig. 12 Changes in the ratiometric emission response of Mn-doped ZnS QDs with different concentrations of curcumin: (A) I₅₉₀/I₄₅₈ and (B) I₄₅₈/I₅₉₀. Inset: linearity in the range 0.16–16.9 μM.

3.4 Interaction between Mn-doped ZnS QDs and curcumin

We used time-resolved fluorescence spectrometry to determine the nature of the fluorescence quenching by curcumin of the defect-related emission at about 458 nm. Fig. 13 shows the time decay curves; the average lifetime values decreased from 14.13 to 10.34 ns using the expression τ_av = ∑a_iτ_i²/∑a_iτ_i.⁴⁰ The fluorescence lifetime of the MPA-capped Mn-doped ZnS QDs decreased in the presence of curcumin, which might result from ultrafast electron transfer from the QDs to curcumin.³⁹ The quenching of the fluorescence intensity at about 458 nm appeared to be a result of spectroscopic photo-induced electron transfer.^41,42 Fig. 14 shows that the electrons in the holes were excited from the valence band to the conduction band and transited to the initial condition following the solid arrow to generate the two emissions. The UV-visible absorption of curcumin was around 260 and 425 nm (Fig. 5), which was close to the band gap of the Mn-doped ZnS QDs. Therefore the electrons in the conductive band of the Mn-doped ZnS QDs could transfer directly to the lowest unoccupied molecular orbital of the UV and the visible band of the curcumin following the paths shown as dashed arrows. The excited electrons tended to go back by the dashed paths and quenching was generated. The fluorescence resonance energy transfer also possibly contributed to the quenching as a result of the existing overlap bands between the absorption band of curcumin and the defect-related emission band of Mn-doped ZnS QDs.⁴³

Fig. 13 Fluorescence decay of Mn-doped ZnS QDs in the absence and presence of curcumin.

Fig. 14 Schematic illustration of the electronic transitions in the luminescence from Mn-doped ZnS QDs and the fluorescence quenching action of electrons transferring from the QDs to curcumin.

3.5 Selectivity of MPA-capped Mn-doped ZnS QDs for the determination of curcumin

We investigated the selectivity of the Mn-doped ZnS QDs towards curcumin. The interferents investigated included metal ions commonly found in biological fluids (NH⁴⁺, K⁺, Na⁺, Li⁺, Fe²⁺, Zn²⁺, Ca²⁺, Mg²⁺, Fe³⁺), small organic molecules (sodium citrate, glucose, lactose) and some amino acids (D,L-phenylalanine, L-methionine, L-tyrosine, L-proline, L-leucine, D,L-histidine, L-valine, L-threonine, D,L-aspartic acid, L-serine). Fig. 15 shows that the quenching of the ratiometric emission of the QDs resulting from the addition of 1.0 μM curcumin was unaffected by 100-fold excesses of NH⁴⁺, K⁺, Na⁺, Li⁺, small organic molecules and some amino acids and also unaffected by a 30-fold excess of Fe²⁺, Zn²⁺, Ca²⁺, Mg²⁺ and Fe³⁺. The Mn-doped ZnS QDs offered excellent selectivity for the determination of curcumin.

Fig. 15 Ratiometric emission response (I₅₉₀/I₄₅₈) of Mn-doped ZnS QDs for various potential interferents.

3.6 Determination of curcumin in urine samples

To demonstrate the practicality of the ratiometric fluorescence probe for the determination of curcumin, the detection performance was evaluated in human urine samples obtained from volunteers. All the experiments were performed according to the relevant laws and institutional guidelines and informed consent was obtained from the volunteers. The volunteers took trace amounts of curcumin orally in the morning and urine samples were collected in dark glass bottles after 3 h. The results obtained by this method showed that the assay could quantitatively detect the curcumin added exogenously. To validate the selectivity of the assay, we further determined the concentration of endogenous curcumin using the standard LC-MS method^43,44 and compared these results with our proposed method (Table 1). The concentration of endogenous curcumin determined by the two methods was comparable and consistent. The recoveries were in range 97.73–109.73%. These results illustrate that this method could be used in clinical applications.

Table 1 Determination of curcumin in urine samples by the proposed method and LC-MS

Urine sample	LC-MS (nM) (n = 3)	This method (nM) (n = 3)	Concentration of curcumin (μM)		Recovery (%)	RSD (%)
			Added	Found
1	553.13 ± 5.32	561.00 ± 7.02	1.50	2.05	99.33	1.41
			2.20	2.88	105.45	2.33
2	621.24 ± 3.98	617.00 ± 6.49	1.50	2.16	103.33	1.92
			2.20	2.76	97.73	2.60
3	593.76 ± 8.51	584.00 ± 8.27	1.50	2.23	109.73	3.91
			2.20	2.69	105.50	2.74

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4 Conclusions

We have developed a sensitive and selective probe to determine curcumin based on a ratiometric emission platform. The ratiometric fluorescence probe was constructed in a facile manner using the change in fluorescence intensity ratio of two different emission wavelengths with the variation in curcumin concentration. The probe was successfully applied to the determination of curcumin in human urine. The MPA-capped Mn-doped ZnS QDs ratiometric probe was highly selective, sensitive, non-toxic, simple, fast and low cost. This work is a useful reference for further exploration and practical applications in this area of analysis and chemical sensing.

Acknowledgements

Financial support was received from the National Natural Science Foundation of China (nos 21175086 and 21175087), the Shanxi Province Personnel Returning from Overseas Fund and the Shanxi Province Hundred Talents Project.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra01412c

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