b University of Chinese Academy of Sciences, Beijing 100049, China;
c Beijing Institute of Petro-chemical Technology, Beijing 102617, China
L-Lysine (L-Lys) is an essential amino acid, which can help the body make collagen . Diets excessive in L-Lys disturb the balance of nutrition and metabolism in the living body, resulting in depressing effect . Reduced L-Lys concentrations leads to failures in liver functions, which further causes increased serum cholesterol levels . Therefore, development of high sensitivity and selectivity assay for analysis of L-Lys in biological fluids is important and useful in disease diagnoses
Chemiluminmetry , capillary electrophoresis , high performance liquid chromatography , UV–vis spectrometry [6, 7] and fluorometry  have become the customary analytical methods for L-Lys detection. However, some of the tools are burdensome in terms of time and complicated operation requirements. With advantages of size-tunable emission, broad excitation bands, long fluorescence lifetime, good biocompatibility, facile synthesis and strong photoluminescence, gold nanoclusters (AuNCs) have attracted great attention for detection of fluid L-Lys .
Usually, AuNCs are synthesized by reduction of gold precursor to Au atoms in the presence of stabilizing and reducing agents. Various proteins, peptides, DNA oligonucleotides and thiolates are commonly used as the stabilizing agents  in the preparation of AuNCs. Recently, to economic synthesize gold nanoparticles, the eco-friendly, non-toxic and multifunctional reactants (having both reducing and stabilizing activities) derived from nature sources, e.g., from fruit juices [11-17], have been explored as the promising alternatives. For example, citrus juice , punica granatum juice , longan juice  and sugar cane juice  have been utilized for the synthesis of gold nanoparticles. The major components of fruits are naturally occuring compounds (such as proteins, organic acids, polyphenols), therefore, these organic compounds of fruit juices could act both as reducing agents as well as the stabilizing agents in the preparation of gold nanoparticles . Although the previous studies indicated that the fruit juices indeed had both reducing and stabilizing activities, none of the fruit juices capped gold nanoparticles was applied for detecting fluids L-Lys. Importantly, there is no report for synthesis of AuNCs using fruit juices till now. Therefore, digging out simple synthetic methods for AuNCs using nature fruit juices is meaningful and desirable.
Herein, a rapid and green strategy for preparation of papaya juice capped AuNCs (P-AuNCs) was demonstrated for the first time. Furthermore, a good selectivity and sensitivity fluorescence protocol was established and applied for sensing L-Lys contents in urines with the P-AuNCs based fluorescent probe.
P-AuNCs was prepared with a simple "one pot" method. Briefly, 0.5 mL HAuCl4 (10.0 mmol/L), 2.5 mL of papaya juice were mixed under vigorous stirring at room temperature for 5 min. The mixture solution was stirred and incubated at 120 ℃ for 2 min. Then, 0.2 mL NaOH (1.0 mol/L) was added into the reaction system and reacted with the mixture solution at 120 ℃ for 3 min. After the reaction finished, the color of the mixture solution changed from orange to brown (Fig. 1a, inset). The final P-AuNCs was yielded and stored at 4 ℃ before use.
|Fig. 1. (a) Fluorescence spectra of the prepared P-AuNCs; (b) UV–vis absorption spectra of aqueous HAuCl4, papaya juice and P-AuNCs; Inset: photograph of the synthesized P-AuNCs under the visible light (left) and UV light (right).|
Fig. S1 (Supporting information) exhibits the schematic illustration of the P-AuNCs synthesis process. It has been reported that papain , chymopapain A and chymopapain B , could be purified from papaya fruit, which contain different classes of amino acid residues, including cysteine, methionine, histidine, etc. . It is assumed that the abundant amino acids, organic acids, proteins of different enzymes in papaya juice should be good candidates of stabilizing and reducing agents for synthesis of fluorescent AuNCs. In this study, Au3+ ions was reduced to form Au0 atoms based on the strong reducibility of the organic acids, the amino acids, papain and chymopapain in papaya juice. Curves b and c in Fig. S2 (Supporting information) display the fluorescence spectra of the reactants. Curve a (Fig. S2) depicts the fluorescence spectra of P-AuNCs with an emission maximum at 440 nm, indicating that the P-AuNCs was successfully synthesized using papaya juice as the capping and reducing agents.
Various synthetic parameters were optimized to improve the yield of P-AuNCs. Fig. S3 (Supporting information) shows that formation of P-AuNCs started within 2 min. The fluorescence intensity of P-AuNCs obviously increased as the synthetic time increased from 2 min to 3 min. Then, it dramatically reduced when the synthetic time was prolonged from 3 min to 15 min. Therefore, 3 min was selected as the best synthetic time for obtaining the maximum fluorescence intensity of P-AuNCs. Then, different synthetic temperatures from 90 ℃ to 130 ℃ for forming P-AuNCs were studied. Fig. S4 (Supporting information) describes that the increase of temperature greatly boosted up the fluorescence intensity of the P-AuNCs, and thereafter resulted in a maximum value at 120 ℃.
Fig. S5 (Supporting information) displays that the concentration of HAuCl4 increasing from 0 mmol/L to 10.0 mmol/L enhanced the fluorescence intensity of the P-AuNCs, while a further increase triggered decrease of the fluorescence intensity of the fluorescent probe. Moreover, it has been reported that NaOH can significantly accelerate the formation of gold nanoparticles , therefore, the effect of NaOH concentration was investigated. Fig. S6 (Supporting information) exhibits that the fluorescence intensity of the P-AuNCs climbed up gradually with the NaOH concentration increased from 0.1 mol/L to 1.0 mol/L. While it decreased in the range of 1.0–2.0 mol/L because of the strong alkali-induced protein unfolding and denaturation . The results indicated that alkali solution is important in the formation of P-AuNCs.
Next, the effect of acidity of the buffer solution on the fluorescence intensity of P-AuNCs was investigated. The results revealed that the prepared P-AuNCs was stable in the pH range of 2.0–12.0 (Fig. S7 in Supporting information). To avoid the interference of metal ions in selective analysis of biological fluids, the buffer solution at pH 2.0 was selected for further sensing study.
Under the optimized conditions, P-AuNCs were synthesized within 3 min at 120 ℃. As Fig. 1a demonstrated, the fluorescence intensity of the resultant P-AuNCs showed an excitation and emission maximum wavelength at 360 nm and at 440 nm, respectively. Fig. 1b displays the UV–vis spectra of HAuCl4, papaya juice and P-AuNCs in the wavelength range 200–800 nm. It should be mentioned that the characteristic surface plasmon absorption of gold nanoparticles could not be observed in the UV–vis spectra of P-AuNCs solution. Instead, it exhibited absorptions wavelength at around 280 nm, which demonstrated that the P-AuNCs was synthesized successfully .
The stability during long standing would be beneficial to the storage and applications of P-AuNCs. Fig. S8 (Supporting information) reveals that due to the functional groups on the surface of P-AuNCs, the resultant fluorescent probe remained very stable for storage at least for 2 months. Moreover, the proposed P-AuNCs also maintained well stability even in 200.0 mmol/L NaCl solution (Fig. S9 in Supporting information), which can provide the possibility for selective analysis in high salty biological fluids.
The FT-IR measurements of papaya juice and P-AuNCs were carried out to identify the interaction between proteins of papaya juice and the P-AuNCs. Fig. S10 (Supporting information) shows that the strong vibration absorption band located at 3380 cm-1 position in the spectra corresponded to the N-H stretch. While, the strong vibration absorption peaks represented at 1596 cm-1 and 1406 cm-1 indicated the presence of C——N stretch and O——H stretch. Furthermore, the presence of C——O stretch and C——C stretch was confirmed by the 1260 cm-1 and 1061 cm-1 stretch in the FT-IR spectra [15, 24]. It could be deduced that the proteins in papaya juice bound through the amino groups to the Au atoms and were responsible for the preparation and stabilization of AuNCs. These proteins intertwined on the surface of the AuNCs and served as the surface-coatings to prevent agglomeration of the AuNCs. Therefore, stabilized P-AuNCs was observed, which corroborated with the results of UV–vis spectra (Fig. 1b).
Although it has been proved that AuNCs could be used as the fluorescent probes to detect L-Lys , whether the prepared PAuNCs is specific for sensing L-Lys remains unknown. Moreover, as is known to all, L-amino acids are the predominant compounds for living beings, only trace amount of free D-amino acids are found in human physiological fluid . Therefore, the fluorescence response of the sensing system to common L-amino acids (L-Ala, L-Arg, L-Cys, L-Glu, L-His, L-Ile, L-Leu, L-Lys, L-Met, L-Phe, L-Thr, L-Try and L-val) and metal ions (Mg2+, Ca2+, Mn2+, Na+, K+, Fe3+, Zn2+, Pb2+, Fe2+) have been investigated. It has been found that addition of common metal ions into the P-AuNCs solution was not led to great changes in the fluorescence intensity of the P-AuNCs (Fig. S11 in Supporting information). While, as exhibited in Fig. 2, we observed that only L-Lys could cause obvious change in the value of I-I0, indicating a good selectivity of the P-AuNCs. Further, an assay based on fluorescence "turn on" strategy was constructed for high selective sensing L-Lys with the proposed fluorescent probe (Fig. S12 in Supporting information).
|Fig. 2. The relative fluorescence (Ⅰ-Ⅰ0) of P-AuNCs in the presence of 5.0 mmol/L of LAla, L-Arg, L-Cys, L-Glu, L-His, L-Ile, L-Leu, L-Lys, L-Met, L-Phe, L-Thr, L-Try, L-Val, at pH 2.0.|
As displayed in Fig. 3, the principle of P-AuNCs fluorescence "turned on" by L-Lys was explored. The DLS data was recorded and showed that the average size of the P-AuNCs was 6.9 ± 1.1 nm (Fig. S13a in Supporting information). In the presence of L-Lys, it was found to be 7.2 ±1.4 nm (Fig. S13b in Supporting information). Furthermore, the TEM image (Fig. S13c in Supporting information) exhibited the morphology of the P-AuNCs, which was uniform particle shapes and well dispersed in the solution. Fig. S13d (Supporting information) indicates that the size of the P-AuNCs almost did not change after adding L-Lys. The results revealed that the L-Lys could not induce aggregation of the P-AuNCs but enclosed the surface of the P-AuNCs. It has been reported that the electronrich functional groups (such as—NH2, —COOH) attached on the surface of AuNCs  would increase its fluorescence intensity. In this work, papaya juice was used as the stabilizer of the P-AuNCs. In the presence of L-Lys, the formed P-AuNCs-L-Lys composites increased the surface electron density because L-Lys possesses the electron-rich functional groups (such as—NH2, —COOH). Thus, it further "turned on" the fluorescence of the P-AuNCs (Fig. S12). Moreover, to test whether the L-Lys is formed on the surface of the P-AuNCs, the zeta potential measurement has been carried out. A slight increase of 3.5 mV in the zeta potential for P-AuNCs-L-Lys (-19.2 mV) in comparison to P-AuNCs (-22.7 mV) was observed. The increase in zeta potential  might be attributed to the formation of the P-AuNCs-L-Lys composites, which further revealed that the L-Lys indeed attached on the surface of the PAuNCs.
Fig. 4a depicts the fluorescence intensity increased gradually with the concentration of L-Lys increase. The prepared P-AuNCs was further applied to sensitive detection of L-Lys by "turn on" fluorescence strategy. The relationship between L-Lys and the fluorescence efficiency (Ⅰ-Ⅰ0, where Ⅰ0 and Ⅰ are the fluorescence intensities in the absence and presence of L-Lys, respectively) could be expressed (Fig. 4b) as Ⅰ–Ⅰ0 = 251.6 [L-Lys] + 15.5 in the range of 10.0–1000.0 mmol/L (R2 = 0.969) with the detection limit as low as 6.0 mmol/L (3 kb/k) . It indicates that the P-AuNCs based system had a favorable ability for sensing L-Lys.
|Fig. 4. (a) The fluorescence response of the P-AuNCs upon addition of various concentrations of L-Lys; (b) relative fluorescence intensity (Ⅰ–Ⅰ0) of P-AuNCs contrast to the L-Lys concentration.|
In addition, the prepared P-AuNCs supplied a preferable selectivity towards L-Lys. Therefore, the fluorescent probe was further applied in detection of L-Lys in real biological examples . The amount of the L-Lys contents in the human urinary samples was determined by the established assay. The amount of the L-Lys contents in the three urines was 286.2 mmol/L, 239.4 mmol/L and 322.1 mmol/L, respectively, which was comparable to the data reported [27, 28].
Moreover, the study on the recovery of L-Lys was realized. As shown in Table 1, the proposed assay for monitoring urine L-Lys displayed an acceptable recovery and relative standard deviation (RSD). It should be mentioned that the present protocol does exhibit the advantage in rapid preparation of P-AuNCs, which was comparable to the reported ones (Table S1 in Supporting information), promoting future practical application of P-AuNCs in real samples analysis.
In summary, the "onepot"protocolwasfoundtobefacile, rapidand green synthesis of P-AuNCs with papaya juice as the reducing and capping agent. The resultant P-AuNCs was monodispersed and stable. The fluorescent probe showed strong L-Lys sensing activity over other common amino acids due to the surface electron density increaseinduced "turn on" fluorescence principle. In addition, the proposed strategy was successfully applied to the analysis of urine L-Lys. It is believed that fruit juices stabilized AuNCs has a great potential for high selective sensing analytes in real biological samples.Acknowledgments
The authors are grateful for the financial support from the National Natural Science Foundation of China (Nos. 21575144, 91732103, 21874138, 21727809, 21635008, 21621062) and Chinese Academy of Sciences (No. QYZDJ-SSW-SLH034). We also would like to thank Mr. Han Wu for his kind help.Appendix A. Supplementary data
Supplementary material related to this article can be found, in theonlineversion, atdoi:https://doi.org/10.1016/j.cclet.2018.10.001.
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