Ratiometric Fluorescence Detection of 6-Mercaptopurine Based on the Nanohybrid of Fluorescence Carbon Dots and Gold Nanoclusters

The development of a simple and accurate quantitative method for the determination of 6-mercaptopurine (6-MP) is of great importance because of its serious side effects. Ratiometric fluorescence (RF) sensors are not subject to inter-ference from environmental factors, and exhibit enhanced precision and accuracy. Therefore, a novel RF sensor for the selective detection of 6-MP was developed. The present work reports a sensitive and selective RF sensor for the detection of 6-mercaptopurine, by hybridizing carbon nanodots (CDots) and gold nanoclusters (AuNCs) capped with bovine serum albumin (BSA). The CDots serve as the reference signal and the AuNCs as the reporter. On addition of the 6-MP, AuNCs formed aggregates, because the existing cross-links within the AuNCs and BSA structure were broken in favour of the Au-S bonds, which can enhance the fluorescence of AuNCs, while the fluorescence of CDots is stable against 6-MP, leading to distinct ratiometric fluorescence changes when exposed to 6-MP. 6-MP could be detected in the range of 0 - 30.22 μM with a detection limit of 54 nM. The developed sensor was applied for the determination of 6-MP in human serum samples and satisfactory results were obtained.


Introduction
6-Mercaptopurine (6-MP), a sulfur analogue of adenine, is a kind of conventional chemotherapy anticancer drug [1], and is widely used in the therapy of acute lymphoblastic leukemia [2]. But as a cytotoxic anti-tumor drug, 6-MP always brings about serious side effects [3]. Moreover, the concentration of 6-MP in the plasma of a recipient varies, and it depends on individual differences [4] [5] [6]. The individual dosage regimens instead of standardized treatment regimens for some patients would make the drug concentration maintain at an optimal plasma level [4]. Therefore, it is still challenging to develop a simple and accurate quantitative method for 6-MP in order to monitor the concentrations of 6-MP in human serum.
In order to increase the selectivity and sensitivity, ratiometric fluorescent (RF) sensors are utilized, in which analyte concentrations are determined by measuring the ratios of the emission at two wavelengths [28]. Compared with singlechannel detection methods, RF sensors can avoid many problems, such as the drifts of the optoelectronic system (lamps and detectors), the probe concentration, autofluorescence in complicated biosystems, which are prone to disturbance in Table 1. Comparison of published fluorescent nanosensors for the detection of 6-MP.
In the present work, we have built a RF sensor for the detection of 6-MP by combining CDots and AuNCs. The CDots serve as the reference signal and the AuNCs as the reporter. On addition of the 6-MP, AuNCs formed aggregates, because the existing cross-links within the AuNCs and BSA structure were broken in favour of the thiol-Au bond, which can decrease the fluorescence of AuNCs, while the fluorescence of CDots is stable against 6-MP, leading to distinct ratiometric fluorescence changes when exposed to 6-MP. A limit of detection of 54 nM for 6-MP in aqueous solution was estimated. Thus, we applied the sensor for the detection of 6-MP in human serum.
Preparation for stock solution of 6-MP [25]. N 2 was bubbled through an aqueous solution of 0.1 M NaOH until saturation to remove dissolved oxygen. 86 mg 6-MP was added into 500 μL of the aforementioned solution. 4.5 mL boiled ultrapure deionized water was added after the solid had dissolved completely. Then 100 mM 6-MP stock solution was prepared. The stock solution was diluted 5 times and stored as 100 μL per batch independent solution. The as-prepared solutions were stored in a −20˚C refrigerator. 1.90 mL boiled ultrapure deionized water per batch was added into the thawed solution before use.
The fluorescence spectra were recorded on an RF-6000 spectrofluorometer (Shimadzu, Japan) with 1 cm quartz cells. The light source used in the spectrofluorometer was a 150 W Xe arc lamp (Ushio Inc, Japan), and the emitted power density was approximately 20 -32 mW•cm −2 in its wavelength range. The slits for excitation and emission monochromators width were both 5 nm. The transmission electron microscopy (TEM) images were recorded using a JEOL 2010 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were acquired from a Thermo Fisher Nicolet iS10 FT-IR spectrometer.

Synthesis of Water-Soluble Cdots
The CDots were prepared by a simple one-pot hydrothermal method with minor modifications [41]. Typically, 4.204 g of citric acid and 1.34 mL of 1,2-ethylenediamine were mixed and dissolved into 40 mL of water to form a clear solution.
The mixture was put into a 50 mL poly(tetrafluoroethylene) Teflon-lined autoclave tube and the solution was sealed and treated at 200˚C for 4 h. The resulting brown solution was cooled to room temperature naturally and filtered through 0.45 μM Suporfilters to remove the large or agglomerated particles. Then the CDots solution was purified by dialyzing against pure water using a membrane (M W = 3.5 kDa) for 12 h and then storing at 4˚C for further use.

Synthesis of BSA-Au Nanoclusters
All the glasswares were first washed with aqua regia and then rinsed with ultrapure water, several times before use. AuNCs were synthesized and purified according to the literature [42]. In a typical experiment, HAuCl 4 solution (10 mL, 10 mM) was added to the BSA solution (10 mL, 50 mg/mL) under vigorous stirring. After 5 min, suitable NaOH (1 M) solution was introduced to the mixture to adjust the pH to 11.0 and then the mixture was kept under stirring for 12 h at 37˚C. The solution color changed from pale yellow to brown. Then the resulting brown solution was purified by dialyzing against pure water using a membrane (M W = 12 kDa) for 24 h and then storing at 4˚C for further use.

Determination of 6-MP Using the Nanohybrid Probe
The nanohybrid system was prepared by the following procedure. First, in order to adjust the fluorescent intensity ratio of CDots and AuNCs to be 1:1, suitable CDots solution was added to 10 mL AuNCs solution. The mixture was kept under vigorous stirring for surface hybridization through reaction and interaction.
The fluorescence spectra were recorded from 375 to 775 nm under excitation at 365 nm. This nanohybrid probe shows a good physical stability [38].
To evaluate the sensitivity of the RF probe for the 6-MP, in 1.5 mL centrifuge tube, 100 μL nanohybrid solution and 800 μL PB buffer (pH 8.0, 50 mM) were added, then 100 μL various concentrations of 6-MP were also added. The mixture was stirred. Subsequently, the fluorescence spectra were recorded from 375 to 775 nm under excitation at 365 nm.
To further study the specificity of the sensing system towards detecting 6-MP, the interferences of co-existing foreign substances were tested under the aboveselected conditions, spiked with different substances of a known concentration individually.

Design Strategy
A schematic illustration of the ratiometric fluorescence bioassay platform for the detection of 6-MP based on the nanohybrid of fluorescence carbon dots and gold nanoclusters was shown in Figure 1. In brief, we first prepared the CDots and AuNCs. In the presence of 6-MP, the sulfhydryl group of 6-MP was preferentially bound with AuNCs through thiol-Au bond [23] and caused the fluorescence signal of AuNCs can be effectively quenched, whereas the fluorescence intensity of CDots is unaffected, which can serve as a better reference signal for 6-MP assay. By combining the two fluorescence behaviors, the nanohybrid represented an ideal platform for the ratiometric determination of 6-MP, with the AuNCs serving as the 6-MP recognition component and the CDots acting as the reference fluorophore.

Characterization of the As-Prepared AuNCs and CDots
The properties of the as-prepared Au NCs and CDots were investigated by fluorescence spectroscopy, TEM, and FT-IR spectra. Figure 2 shows the maximum emission center of AuNCs at 656 nm (Figure 2(a)), and the CDots emission band at 450 nm (Figure 2(b)). The fluorescence spectra of Au NCs and CDots were further recorded every 5 min for 1 h under ultraviolet irradiation at 365 nm, and the fluorescence intensities exhibit no distinct change, implying that both the AuNCs and CDots exhibit good stability against photobleaching in aqueous solutions. The nanohybrid solution emits two emission bands at 450 and 656 nm. The morphologies and the sizes of the two components were characterized by TEM as shown in Figure 3 The diameter of CDots was estimated to be ~4 nm with a good dispersity (Figure 3(a)). The AuNCs were readily dispersed in water and possessed a good monodispersity with a particle size of about ~3 nm (Figure 3(b)). Additionally, CDots, AuNCs and CDots-AuNCs hybrid were characterized by FT-IR as shown in Figure 4 CDots have many characteristic peaks, such as −OH and N−H (3100 -3500 cm −1 ), C−H (2930 cm −1 ), C=ONR   For the CDots-AuNCs hybrid, three absorption bands at 3457 cm −1 , 1661 and 1524 cm −1 were assigned to AuNCs, the characteristic peak at 1290 cm −1 could be assigned to the stretching vibration of the C−N groups and 1084 cm −1 was related to C−O stretching vibration from the CDots [39]. The results revealed that the CDots-AuNCs hybrid showed signals of both CDots and AuNCs.    can form. The formation of such bonds removed the protection effect of the BSA and neutralized the surface charge of the AuNCs. Without this protection effect of the BSA, the detached AuNCs coated with thiols, could aggregate, because there would be an increase in the van der Waals attraction forces between them [23]. In this study, precision was measured by intra-day and inter-day variability and expressed as the RSD, which was calculated from three replicate determinations of reference standard solution concentration of 6-MP at three concentrations (5.5, 22.5, 30.2 μM) within one day (intra-day precision) and three replicates over three days (inter-day precision). The RSDs for the intra-day and inter-day precision were 2.3% -5.4% and 2.5% -6.8%, respectively.

Effect of Ion Strength
Ion strength is always a key factor that influences a spectral method. In this work, we studied the effect of ion strength on detection of 6-MP using NaCl to regulate the ion intensity of the analytical system. The results were shown that the addition of NaCl strongly influences the analytical systems. So in the detection procedure, there is no need to add other electrolytes to adjust the ion strength of the detection system.

Effect of pH Value
The synthesis of AuNCs and in the present work was completed in a relative high pH environment. In order to study the effect of acidity on the ratiometric fluorescence of the analytical system, the effect of pH value was investigated from 4.5 to 10. During the work, small aliquots of 0.1 M NaOH or HCl was used to adjust the pH value. The results were shown that the fluorescence of nanohybrid probe strongly suffered by pH. The fluorescence of nanohybrid probe was strongly decreased when the pH < 7.0, then almost unchanged from 7.5 to 10.0. Therefore, pH value 8.0 was determined as the optimal incubation pH value during the experiments.

Dynamic Fluorescence Process of the Analytical System
As to a spectral system, its stability affects its sensitivity and repeatability and thus we studied the time-dependent fluorescence of the analytical system by synchronous fluorescence with excitation at 365 nm and emission at 656 nm. The results shown that the quenching effect of the 6-MP on the fluorescence intensity of AuNCs was quite fast in the first 10 min, and the level of quenching remained unchanged for the next time; Therefore, the detection of 6-MP was measured after 10 min.

Selectivity of the Method for 6-MP Detection
The interferences of co-existing foreign substances were tested under the aboveselected conditions, spiked with different substances of a known concentration individually. An error of ±5% in the relative fluorescence intensity was considered tolerable. The results are summarized in Table 2. It can be seen from Table 2 that most of the tested substances including those of metal ions, some protein-forming amino acids, uracil, dextrose and glucosuria scarcely interfere with the determination at high tolerance levels. It is concluded that the method is free from many interferences of foreign substances. However, the thiol-containing compounds, cystine, thiophenol, glutathione, which were similar in structure to 6-MP, could be tolerated only at relative low levels. This suggested that the sensor responded differently with different thiol compounds. These compounds often have other different functional groups attached. Such as Yu et al. constructed a FRET assembly by using gold nanoclusters and carbon dots and their application as a ratiometric probe for cysteine [40]. Though AuNCs have been success-  [46], these metal ions did not affect the analytical results, the reason was that all these metal ions did not exist at alkaline solutions (pH 8.0), they could form the corresponding precipitation of hydroxide with OH -. Thus, satisfactory analytical selectivity may be expected for different bio-thiols, and hence, the novel method has satisfactory selectivity for the analysis of 6-MP.

Detection of 6-MP in Human Serum
The proposed method was applied to the determination of 6-MP in spiked human serum. The serum samples, obtained from healthy volunteers, were spiked with 6-MP at different concentrations, and treated as the recommended procedure. The concentrations of 6-MP were calculated from the calibration graph. The results obtained for the determination of 6-MP in spiked human serum are given in Table 3. The satisfactory recoveries obtained with such a simple sample

Conclusion
In conclusion, we designed a ratiometric fluorescence probe by hybridizing the CDots and the AuNCs. The nanohybrid probe exhibited dual emissions at 450 and 656 nm under a single excitation. The fluorescence at 450 nm was inert to 6-MP, while the fluorescence at 656 nm showed good specificity to 6-MP, leading to distinct ratiometric fluorescence changes. 6-MP could be detected in the range of 0 -30.22 μM with a detection limit of 54 nM. The proposed method was satisfactorily applied for analysis of 6-MP in human serum.