Carbon dots as fluorescent off–on nanosensors for ascorbic acid detection

Abstract

An “off–on” approach for the detection of ascorbic acid using carbon dots as a fluorescent probe is presented, which is based on the fluorescence recovery of the quenched C-dots/Fe³⁺ complex when ascorbic acid is introduced. This sensor was also employed to determine the encapsulation efficiency of ascorbic acid in a liposome formulation.

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Ascorbic acid (AA, also known as vitamin C), is a strong antioxidant and a cofactor in at least eight essential enzymatic reactions. Because AA can efficiently scavenge toxic free radicals and other reactive oxygen species (ROS) that are associated with several forms of tissue damage, disease and the process of aging formed in cell metabolism, it has been used for the prevention and treatment of scurvy, cardiovascular disease, cancer, age-related macular degeneration, gout, heavy metal toxicity, diabetes and AIDS.¹ In addition, AA is a vital vitamin in the human diet. However, AA is sensitive to various environmental factors, such as temperature, oxygen concentration, metallic ions and UV exposure. This vulnerability causes AA to suffer from low efficiency of absorption or utilization in food products that are fortified with AA in order to supply the recommended daily allowance. In order to reduce degradation and improve the absorbance of AA, some nanocarriers are used to protect AA from unwanted interactions, such as microemulsions,² micelles³ and liposomes.^4,5 Liposomes are spherical vesicles made of phosphatidylcholine-enriched phospholipids for the delivery of active cosmetic/drug materials, and they have been used in the food industry to encapsulate AA. To evaluate the concentration of AA in liposomes, an assay is needed to determine the entrapment efficiency of AA in the liposome.

In the past ten years, various approaches have been developed for the quantitative determination of AA, such as titration,⁶ colorimetry,⁷ electrochemical methods⁸ and fluorescence assays.^9–12 Among the above methods, the strategy based on changes in fluorescence intensity or anisotropy¹³ possesses the advantages of operational simplicity, high sensitivity and real-time detection. In fluorescence assays, fluorescent nanomaterials, such as semiconductor quantum dots,¹⁰ novel metal nanoparticles¹² and rare earth doped luminescent nanomaterials¹¹ have been employed for AA detection. However, these assays suffer from the usage of hazardous^6,9,10 or precious materials,¹² complicated processes,⁷ complex modified electrodes⁸ and limited sensor range.^9,11,12 Therefore, superior methods for the detection of AA are still greatly needed.

As a new member of the nanomaterials family, fluorescent carbon dots (C-dots) are small carbon nanoparticles with sizes below 10 nm, and have many intriguing merits.^14,15 As an alternative to semiconductor quantum dots, C-dots have shown versatile applications in bio-imaging, sensors, photo-catalysis and optronics based on their exciting fluorescent, nontoxic, biocompatible and electronic properties.^16,17 Functionalized C-dots have great potential in analytical applications, and have been used to sense pH, metal ions and molecules.^18–20 Among C-dots based sensors, “off–on” sensors have appeared as a novel type of sensor with a flexible functional platform.²¹ In this sensing system, the PL signal of the carbon dots is initially quenched by one substance, and then recovered by the analyte. An “off–on” fluorescent sensor has been reported based on a C-dots/Cr(VI) system for AA determination; however, Cr(VI) is hazardous, and the sensor range was limited.⁹ Therefore, it is still highly desirable to explore improved off–on sensors for AA determination.

In this communication, a novel fluorescent nanosensor based on C-dots for AA detection, with the merit of a wide detection range, low detection limit, and high selectivity, was developed. In this sensing method, the PL of the C-dots sensor is quenched by Fe³⁺ and then recovered after the addition of AA, as shown in Scheme 1. The detection range, limit of detection and the selectively of this off–on sensor were studied, and the PL recovery mechanism was explored. This off–on method was employed to determine the encapsulation efficiency of AA in a liposome formulation.

Scheme 1 Schematic illustration of the AA sensor based on the off–on fluorescent probe of C-dots adjusted with Fe³⁺.

C-dots were prepared by a M–H method using AA as the carbon source.²² The details of the synthetic process are shown in the ESI.† The morphology analysis, XRD and FTIR spectrum of the C-dots are presented in Fig. S1.† The diameters of the C-dots, obtained from TEM and dynamic light scattering (DLS), were about 3 nm and 6 nm (Fig. S1(b) and (d)†), respectively. The hydrodynamic diameter of 6 nm (DLS) was larger than the diameter of 3 nm (TEM) because the hydrodynamic diameter includes all solvent molecules attracted to the surface of the C-dots. It can be seen that the non-crystalline hydrophilic carbon dots are spherical and well dispersed. The C-dots showed highly luminescent fluorescence and excellent optical stability, and the intensity was independent of the pH value in aqueous solution, as shown in Fig. S2.† AA detection can proceed in aqueous solution without buffer. The superior fluorescence properties of C-dots favour their application as a sensor.

In the “off” step of the “off–on” process, ferric chloride (FeCl₃) aqueous solution was employed to quench the PL intensity of the C-dots. In order to optimize the concentration of C-dots for AA detection, the concentration dependent PL behaviour of the C-dots in aqueous solution was studied. As shown in Fig. 1(a), the PL intensity of the C-dots increased with concentration when the concentration was below 0.05 mg mL⁻¹, but it decreased with concentration when the concentration was greater than 0.05 mg mL⁻¹. The decrease of PL intensity at high concentrations was ascribed to self-absorption quenching.²³ As a result, the concentration of C-dots used in the following experiments was chosen to be 0.05 mg mL⁻¹. The effect of incubation time on the PL intensity of C-dots/Fe³⁺ was investigated to assure the accuracy of this analytical procedure (Fig. 1(b)). C-dots (0.05 mg mL⁻¹) and Fe³⁺ solution (1 mM) were mixed, and the PL intensity of the mixture stabilized within 3 min. Therefore, the optimal incubation time was confirmed as 3 min. The effect of Fe³⁺ on the PL spectra of the C-dots was studied to optimize the concentration of Fe³⁺ for further AA detection. The PL intensity of the C-dots decreased sharply with increasing Fe³⁺ concentration when the concentration of Fe³⁺ was below 0.6 mM, and the decreased slope flattened when the concentration of Fe³⁺ approached 1 mM, which indicated a quenching saturation at 1 mM. Therefore, the optimal Fe³⁺ concentration was chosen as 1 mM for quenching the PL of the C-dots. This fluorescence quenching may be attributed to non-radiative electron transfer from the excited state of the C-dots to the d orbital of the Fe³⁺ ions.²⁰

Fig. 1 (a) Concentration dependence of the PL behaviour of C-dots in aqueous solution. (b) Kinetic behaviour of the PL intensity of the C-dots/Fe³⁺ system. (c) PL spectra of the C-dots in aqueous solution with different concentrations of Fe³⁺; inset shows the PL intensity of the aqueous solution of C-dots against the concentration of Fe³⁺. (d) PL lifetime values of C-dots with different concentration of Fe³⁺. The excitation and emission wavelengths were 330 nm and 410 nm, respectively in (a)–(d), the concentration of C-dots was 0.05 mg mL⁻¹ in (b)–(d), and the concentration of Fe³⁺ was 1 mM in (b).

Time correlated single photon counting (TCSPC) was used to reveal the PL quenching mechanism of C-dots in the “off–on” process. The PL lifetimes of C-dots with different concentrations of Fe³⁺ are shown in Fig. 1(d). The fluorescence lifetime of the C-dots was 2.57 ns when fitted with a mono-exponential function, which reflects a fast electron–hole recombination. In addition, the lifetime of the aqueous solution of C-dots decreased from 2.57 ns to 2.38 ns with the increase of the Fe³⁺ concentration from 0 to 1 mM. This phenomenon supports a dynamic quenching mechanism, because the decrease of the PL lifetime with the addition of quencher is a phenomenon that is unique to the dynamic quenching process.²⁴

In the “on” step of the “off–on” process, AA was added to the PL quenched C-dots/Fe³⁺ system, and the PL intensity increased with AA concentration, as shown in Scheme 1. The PL intensity of C-dots/ferric ions with 1 mM AA was studied to obtain the optimal incubation time for AA detection (Fig. 2(a)). It was found that a stable PL intensity signal was obtained within a reaction time of 2 min. Therefore, an incubation time of 2 min is sufficient for the PL intensity measurement. The quenched PL of the C-dots/Fe³⁺ system was recovered gradually as the AA concentration increased (Fig. 2(b)). In this process, Fe³⁺ was reduced by AA to Fe²⁺, which has no quenching effect on the PL of C-dots; thus, the PL intensity of C-dots/ferric ions was recovered.²⁵ As indicated in Fig 2(c), some sigmoidal fitting is suitable for the range of 0–1000 μM. In addition, a good linear relationship between the PL intensity and the concentration of AA from 0.2 to 284 μM was obtained, with a linear equation of I = 3.414 + 0.02024 [AA] (μM) (R² = 0.993) and a limit of detection (LOD) of 0.05 μM.

Fig. 2 (a) Kinetic behaviour of the PL intensity of the C-dots/Fe³⁺ system with 1 mM AA. (b) The PL spectra of the C-dots/Fe³⁺ mixture with different concentrations of AA. (c) The plot of the fluorescence intensity against the AA concentration within 0.2–280 μM, according to (b); the inset shows the complete relationship between the PL intensity and AA from 0–1000 μM. (d) PL lifetime values of the C-dots/Fe³⁺ mixture with different concentrations of AA. The excitation wavelength is 330 nm in (a)–(d).

TCSPC experiments for the C-dots/ferric ions with different concentrations of AA were used to further explore the mechanism of the PL intensity recovery. When the concentration of AA was increased from 0 to 284 μM, the lifetime of the C-dots/ferric ions complex increased from 2.38 ns to 2.42 ns as shown in Fig. 2(d). The increase of the PL lifetime indicated that the dynamic quenching process between the C-dots and Fe³⁺ was decreased by the addition of AA, because the dynamic quenching process decreased the PL lifetime.²³ Therefore, the PL intensity recovery could be ascribed to the suppression of the dynamic quenching process, which originates from the reduction of Fe³⁺. Additionally, the changes in PL lifetime with the addition of analyte provides a new idea for sensing based on PL lifetime changes, if this change correlates linearly with the variation in the analyte concentration.

Table 1 shows a performance comparison between this method and other methods for AA detection, using the aspects of detection range and LOD. The results show that the C-dots/Fe³⁺ system for AA detection possesses the widest linear range (0.2–284 μM), and has almost the lowest detection limit.

Table 1 Comparison of previously reported fluorescent methods for AA detection with the present method

Sensor	Linear range (μM)	LOD (μM)	Reference
C-dots/Cr(VI)	30–100	—	9
CuInS2 quantum dots	0.25–200	0.05	10
LaF3 : Ce, Tb nanoparticles	8–100	2.4	11
Au nanoparticles	1.5–10	0.2	12
C-dots/Fe^3+	0.2–284	0.05	This work

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In order to investigate the selectivity of this C-dots/Fe³⁺ sensor for the detection of AA, the interference of common physiological molecules and ions, such as citric acid (Cit), glucose (Glu), glycine (Gly), lysine (Lys), bovine serum albumin (BSA), Na⁺, K⁺, Mg²⁺, and Ca²⁺ was studied under the optimum experimental conditions; the results are shown in Table 2. The results reveal that most of the molecules/ions could be allowed to coexist at a concentration of 50 times of AA within a relative error of 3.0% for AA detection. Therefore, these molecules/ions caused little interference with this sensor for AA detection. In summary, this method is highly selective, and it could be applied for AA detection in liposomes.

Table 2 Effect of co-existing substances on the PL intensity of C-dots/Fe³⁺ with 0.1 mM AA

Coexisting substances	Concentration/mM	ΔF/F0^a (%)
a ΔF = F − F0, where F and F0 are the PL intensity of AA–C-dots/Fe^3+ in the presence and absence of the co-existing substance.
Citric acid	5.0	+3.0
Glucose	5.0	+2.7
Glycine	5.0	+2.0
Lysine	5.0	+2.8
BSA	5.0	+2.0
Na^+	5.0	+1.4
K^+	5.0	−0.9
Mg^2+	5.0	−0.5
Ca^2+	5.0	−0.8

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To further explore the potential utility of this sensor, the C-dots/Fe³⁺ system was studied as a probe to detect AA in liposomes. The AA encapsultated liposome was prepared by a membrane evaporation method, and the details are presented in the ESI.† To measure the entrapment efficiency (EE) of AA in liposome solution, the dissociative AA was separated by a dialysis process and the dialysate was collected for AA determination (see ESI† for the detailed process). The fluorescence off–on nanosensor was used to determine the AA concentration in the dialysate solution. The EE% of AA in the liposomes was then calculated using the following equation: EE (%) = (1 − CV_dialysate/m_AA) × 100%. C, V and m_AA in the equation represent the concentration of AA in dialysate, the volume of dialysate and the total mass of AA, respectively. To verify the accuracy of the results obtained by this off–on assay, a traditional HPLC analysis assay was used to determine the EE of AA as a control. The results of the two methods for EE detection of AA in the liposomes are listed in Table S1† for comparison. The EE obtained by the traditional HPLC assay was in accordance with that obtained by the off–on assay. Compared with the HPLC analysis assay, the off–on fluorescence assay is more suitable for determining the EE of AA in liposomes, because it possesses the advantage of a wider linear detection range (0.2–284 μM vs.5–70 μM) and a lower detection limit.

In conclusion, an “off–on” approach for the detection of AA using C-dots as a fluorescent probe is presented, based on the ability of AA to recover the PL of the quenched carbon dots/Fe³⁺ complex. The quenching and recovery mechanisms were investigated. It was found that the quenching of fluorescence by Fe³⁺ is a dynamic quenching process, and the fluorescence was recovered after adding AA due to the reduction of Fe³⁺ to Fe²⁺, which has no quenching effect on C-dots. In addition to its wide detection range, low detection limit and high selectivity, this C-dots/Fe³⁺ sensor also has the advantages of low cost and facile fabrication. Therefore, it could be concluded that this sensor is excellent for AA detection. This off–on sensor system was successfully used to determine the encapsulation efficiency of ascorbic acid in a liposome formulation.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (21473055, 21273073 and 21073063), and the National High-Tech R & D (863) Program of China (2011AA06A107).

Notes and references

1. O. Arrigoni and M. C. De Tullio, Biochim. Biophys. Acta, Gen. Subj., 2002, 1569, 1–9.
2. N. Pakpayat, F. Nielloud, R. Fortuné, C. Tourne-Peteilh, A. Villarreal, I. Grillo and B. Bataille, Eur. J. Pharm. Biopharm., 2009, 72, 444–452.
3. M. Szymula and J. Narkiewicz-Michałek, Colloid Polym. Sci., 2003, 281, 1142–1148.
4. E. Pimentel and D. M. Lonard, US20140141066A1, 2014.
5. Y.-Q. Dai, G. Qin, S.-Y. Geng, B. Yang, Q. Xu and J.-Y. Wang, RSC Adv., 2012, 2, 3340–3346.
6. L. Suntornsuk, W. Gritsanapun, S. Nilkamhank and A. Paochom, J. Pharm. Biomed. Anal., 2002, 28, 849–855.
7. R. Koncki, T. Lenarczuk and S. Głąb, Anal. Chim. Acta, 1999, 379, 69–74.
8. J. Yan, S. Liu, Z. Zhang, G. He, P. Zhou, H. Liang, L. Tian, X. Zhou and H. Jiang, Colloids Surf., B, 2013, 111, 392–397.
9. M. Zheng, Z. Xie, D. Qu, D. Li, P. Du, X. Jing and Z. Sun, ACS Appl. Mater. Interfaces, 2013, 5, 13242–13247.
10. S. Liu, J. Hu and X. Su, Analyst, 2012, 137, 4598–4604.
11. C. Mi, T. Wang, P. Zeng, S. Zhao, N. Wang and S. Xu, Anal. Methods, 2013, 5, 1463–1468.
12. X. Wang, P. Wu, X. Hou and Y. Lv, Analyst, 2013, 138, 229–233.
13. X.-R. Liu, J.-H. Li, Y. Zhang, Y.-S. Ge, F.-F. Tian, J. Dai, F.-L. Jiang and Y. Liu, J. Membr. Biol., 2011, 244, 105–112.
14. H. Li, Z. Kang, Y. Liu and S.-T. Lee, J. Mater. Chem., 2012, 22, 24230–24253.
15. S. N. Baker and G. A. Baker, Angew. Chem., Int. Ed., 2010, 49, 6726–6744.
16. P. Anilkumar, L. Cao, J.-J. Yu, K. N. Tackett, P. Wang, M. J. Meziani and Y.-P. Sun, Small, 2013, 9, 545–551.
17. A. B. Bourlinos, M. A. Karakassides, A. Kouloumpis, D. Gournis, A. Bakandritsos, I. Papagiannouli, P. Aloukos, S. Couris, K. Hola, R. Zboril, M. Krysmann and E. P. Giannelis, Carbon, 2013, 61, 640–643.
18. C. Hu, C. Yu, M. Li, X. Wang, J. Yang, Z. Zhao, A. Eychmüller, Y. P. Sun and J. Qiu, Small, 2014, 10, 4926–4933.
19. J. C. G. Esteves da Silva and H. M. R. Gonçalves, TrAC, Trends Anal. Chem., 2011, 30, 1327–1336.
20. Y.-L. Zhang, L. Wang, H.-C. Zhang, Y. Liu, H.-Y. Wang, Z.-H. Kang and S.-T. Lee, RSC Adv., 2013, 3, 3733–3738.
21. X. Teng, C. Ma, C. Ge, M. Yan, J. Yang, Y. Zhang, P. C. Morais and H. Bi, J. Mater. Chem. B, 2014, 2, 4631–4639.
22. J. Gong, X. An and X. Yan, New J. Chem., 2014, 38, 1376–1379.
23. J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer US, New York, 3rd edn, 2006, pp. 277–330.
24. Y. Song, S. Zhu, S. Xiang, X. Zhao, J. Zhang, H. Zhang, Y. Fu and B. Yang, Nanoscale, 2014, 6, 4676–4682.
25. S. Zhu, Q. Meng, L. Wang, J. Zhang, Y. Song, H. Jin, K. Zhang, H. Sun, H. Wang and B. Yang, Angew. Chem., Int. Ed., 2013, 52, 3953–3957.

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Footnote

† Electronic supplementary information (ESI) available: Including details of synthetic of C-dots and sensing of AA. See DOI: 10.1039/c4ra13576h

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