Spectrofluorometric determination of berberine using a novel Au nanocluster with large Stokes shift
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Berberine hydrochloride (BHC), a natural isoquinoline alkaloid, is widely applied as a an agent in traditional Chinese medicine. Almost all the traditional methods for BHC detection require complicated preprocessing steps or expensive instruments. In this article, we report a simple, rapid, sensitive, and selective method for BHC detection using fluorescent gold nanoclusters (F-AuNCs) as the fluorescent probe with a large Stokes shift of 237 nm. The F-AuNCs prepared with citrate-stabilized stannous chloride and hydrogen tetrachloroaurate(III) as raw materials in an aqueous medium display strong and stable fluorescence at 566 nm. When F-AuNCs are mixed with BHC, the fluorescence of F-AuNCs is effectively quenched. Under optimized conditions, this method allows sensitive and selective measurements of BHC in a concentration ranging from 1.0 × 10-6 to 1.0 × 10-4 mol L-1 with a detection limit of 7.5 × 10-8 mol L-1, which is relatively low among reported spectral methods. This method provides excellent selectivity for the detection of BHC against inorganic anions and natural amino acids. In addition, the BHC content in two different types of berberine tablets was successfully determined by this method and the results showed high accuracy.
KeywordsBerberine hydrochloride Gold nanoclusters Fluorescent Stokes shift
Berberine hydrochloride (BHC) is an isoquinoline alkaloid isolated from Rhizoma coptidis, Berberis aquifolium, Berberis vulgaris, and other Chinese herbs . Because of BHC’s antibacterial, antilipid peroxidation, antiatherosclerotic, and neuroprotective properties as well as its property of relieving polycystic ovary syndrome [2, 3, 4, 5, 6], its quantification is crucial in the fields of clinic medical assay and optical probes [7, 8, 9]. Many analytical methods, including chromatography [10, 11], mass spectrometry , capillary electrophoresis , chemiluminescence , electrochemical analysis [15, 16], light scattering spectrometry [17, 18], colorimetric assay [19, 20] fluorescence spectra [21, 22, 23], and optical fiber sensing [24, 25, 26], for the quantification of BHC have been established. These methods have certain advantages, such as sensitivity and selectivity, but require expensive instruments or complicated procedures. On the basis of these methods, sensing methods using fluorescence, scattering, and the fluorescence spectrum have been rapidly developed in recent years [27, 28, 29], possessing simplicity in approach, shortened response time, and cost-effectiveness.
Recently, fluorescent probes using metal nanoclusters (NCs) with small particle size have drawn considerable research interest in the fields of analytical chemistry owing to their special characteristics, such as good biocompatibility, large surface area, low toxicity, and exceptional fluorescent properties [30, 31, 32]. By choosing different capping and stabilizing agents, researchers have developed various approaches to synthesize NCs and have applied them in chemosensing/biosensing . However, the development of high-quality NCs (e.g., relatively narrow size distributions, high quantum yield, and large Stokes shift) remains a challenge . A large Stokes shift is beneficial in reducing spectral overlap  and mitigating reabsorption losses , as well as for signal detection in fluorescence imaging . More importantly, these benefits are helpful in reducing interference when the analyte is being determined. Therefore, many researchers are trying to develop new probes with a large Stokes shift for use in spectrofluorometric determination.
In this work, we demonstrate a novel fluorescent probe using fluorescent gold NCs (F-AuNCs) with a Stokes shift as large as 237 nm to detect BHC. Because BHC can strongly quench the fluorescence of F-AuNCs, BHC can be determined at low levels. Furthermore, the decrease in the fluorescence intensity shows a good linear relationship with the concentration of BHC. Thus, a sensitive and selective method for the determination of BHC has been established, and its analytical application has been investigated in compound berberine tablets and BHC tablets.
All the fluorescence spectra and fluorescence intensities were obtained with an RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan) equipped with a 1-cm quartz cell. The absorption spectra were measured with a Lambda 35 UV–vis spectrophotometer (PerkinElmer, USA). Transmission electron microscopy (TEM) images were obtained with a Tecnai G20 electron microscope (FEI, USA). A ZF-7 (black box-type) three-wavelength UV analyzer (JiaPeng, China) at 365 nm as the light source and a Nikon Coolpix 4500 digital camera were used to obtain images of the F-AuNCs. Resonance light scattering (RLS) intensities were measured with the same fluorescence spectrophotometer. The sample was scanned synchronously with the same excitation and emission wavelengths from 240 to 650 nm with use of the fluorescence spectrophotometer. The BHC content was characterized by high-performance liquid chromatography (HPLC) using a liquid chromatograph (LC2010AT, Japan) equipped with a UV detector (TSP, USA). The detection wavelength was 265 nm and the chromatographic column was an AcclaimTM 120 C18 column (4.6 mm × 250 mm, 5 μm). The separation was performed at 30 °C, and the isocratic elution at a flow rate of 1.0 mL/min used a mobile phase of 30:70 acetonitrile and KH2PO4 (0.03 mol L-1). The injection volume was 20 μL.
All reagents were of analytical grade, including stannous chloride (SnCl2; AR, Sigma-Aldrich), hydrogen peroxide (H2O2; AR, Xilong Chemical Co.), phosphoric acid (H3PO4; AR, Xihuang Chemical Co.), trisodium citrate (C6H5Na3O7·2H2O; AR, J&K Scientific), hydrogen tetrachloroaurate(III) tetrahydrate (HAuCl4·4H2O; AR, Aladdin), and BHC (E. Merck). BHC tablets and compound berberine tablets were purchased from a local drugstore, and were manufactured by Jiangsu Aipusen Pharmaceutical Co. Britton–Robinson buffer solution was used to control the acidity of the system, and double-deionized and double-distilled water was used to prepare all solutions. The Sn2+ stock solution was prepared by addition of solid SnCl2 to 1.2 × 10-2 mol L-1 trisodium citrate solution. The trisodium citrate was dissolved in double-distilled water and then deoxidized with nitrogen gas for 30 min before addition of SnCl2 to prevent oxidation of Sn(II). Trisodium citrate acts as a stabilization reagent to prevent the hydrolysis of Sn2+.
Synthesis of F-AuNCs
F-AuNCs were synthesized according to our previously reported hydrothermal method with minor modification . First, 30.0 mL of citrate-stabilized SnCl2 (1.2 × 10-2 mol L-1), 30.0 mL of 0.005% HAuCl4·4H2O, 315 μL of 3% H2O2 and 560 μL of H3PO4 (1.0 mol L-1) were mixed and heated for 2 h at 150 °C until a pale yellow solution of F-AuNCs was obtained. Then the solution was naturally cooled to room temperature and kept in a refrigerator (about 4 °C). The solution was stable for at least 3 months.
Spectral detection procedures
Typically, 1.0 mL F-AuNC solution, 200 μL Britton–Robinson buffer (pH 2.87), and a certain volume of BHC solution or sample solution were successively added to a 2.5-mL test tube. Then the mixture was diluted to 2.0 mL with water and maintained for 2 min at room temperature. The fluorescence spectra were measured with slit widths of 5 nm at the excitation and emission maxima of 329 and 566 nm, respectively. The degree of fluorescence quenching of F-AuNCs was determined by the relative fluorescence intensity ratio Q: Q = (IFo − IF)/IF, where IFo and IF are the fluorescence intensities of F-AuNCs in the absence and presence of BHC, respectively. For the detection of BHC in tablets, BHC tablets and compound berberine tablets were pretreated as follows: 20 slices of tablets were ground to a uniform powder after removal of the sugar coating from the tablets. Then 40 mg powder was weighed accurately and then dissolved in boiling water in a 100-mL beaker, and the resulting mixture was cooled to room temperature. The resulting solution was then diluted to 100 mL.
Results and discussion
Characterization of F-AuNCs
The F-AuNCs exhibit UV–vis absorption in the wavelength range from 200 to 400 nm (Fig. 1, spectrum c). The absence of a surface plasmon peak at approximately 520 nm, which is the typical absorption wavelength of large gold nanoparticles as reported in the literature , indicates the relatively small diameter of the as-prepared F-AuNCs. Moreover, it is fascinating that the F-AuNCs have low-energy emission at 566 nm when the excitation wavelength is 329 nm. The Stokes shift is 237 nm, which can be very attractive for sensor applications.
Interaction between F-AuNCs and BHC
As seen from the fluorescence spectra, the interaction between BHC and F-AuNCs can result in great quenching of the fluorescence of F-AuNCs. The different quenching mechanisms are usually classified as either dynamic or static. To distinguish these two mechanisms, observation of the absorption spectra of the fluorophore is a basic method [46, 47, 48]. Dynamic quenching affects only the excited fluorophore because of the interaction between the quencher and the excited fluorophore, and thus no changes in the absorption spectra are expected. In contrast, static quenching involves the perturbation of the absorption spectra caused by the nonluminous complex generated from the interaction between the quencher and the ground-state fluorophore. To confirm the mechanism of fluorescence quenching of F-AuNCs by BHC, the system’s absorption spectra were measured.
Curve 4 in Fig. S1 was obtained by our subtracting curve 2 (the absorption spectrum of F-AuNCs) from curve 1 (the absorption spectrum of the mixture of F-AuNCs and BHC). From comparison with the absorption spectrum of BHC (curve 3 in Fig. S1), it is obvious that a hypochromic effect  appears in the absorption spectrum of BHC in the presence of F-AuNCs, and the wavelength range for this is from 240 to 500 nm.
Optimization of the detection conditions for F-AuNCs
The optimum conditions of the reaction, the effect of foreign substances, and the analytical application of F-AuNCs were investigated. We found that the fluorescence intensity of the mixture of F-AuNCs and BHC changed with the reaction time (within 16 min); the results are shown in Fig. S2a. The fluorescence intensity of the mixture of F-AuNCs and BHC decreased with increase of the reaction time to 200s. The fluorescence intensity reached a minimum and the fluorescence intensity difference (IFo − IF) reached a maximum after reaction for 400s. Thus the following determination was performed after the reaction had occurred for 2 min.
The effect of pH on the fluorescence intensity of F-AuNCs was examined with the concentration of BHC fixed at 5.0 × 10-5 mol L-1. As shown in Fig. S2b, the fluorescence intensity difference (IFo − IF) (Fig. S2b, curve 3) reached a maximum at pH 2.87. Therefore, 2.87 was chosen as the most appropriate pH.
The influence of the incubation temperature on the fluorescence intensity of F-AuNCs was also examined with the concentration of BHC fixed at 5.0 × 10-5 mol L-1. As also shown in Fig. S2c, BHC caused strong fluorescence quenching of F-AuNCs (Fig. S2c, curve 3). Furthermore, with the rise of temperature, the fluorescence of F-AuNCs decreased obviously. This shows that the fluorescence intensity difference (IFo − IF) (Fig. S2c, curve 3) is relatively high when the temperature is in the range from 0 to 20 °C, but decreases evidently when the temperature is above 25 °C. Considering both the accuracy and the convenience, the following test was conducted at 20 °C.
Effect of coexisting substances
Sensitivity for BHC detection
BHC detection in a composite sample
Results obtained from fluorescence (n = 3)
Detection value (mg/slide)
Average value (mg/slide)
Reference value (mg/slide)
Compound berberine tablets
Berberine hydrochloride tablets
Results obtained by high-performance liquid chromatography (n = 3)
Detection value (mg/slide)
Average value (mg/slide)
Reference value (mg/slide)
Compound berberine tablets
Novel water-soluble F-AuNCs with a large Stokes shift were developed as a spectrofluorometric probe for the determination of berberine. The F-AuNCs were prepared with citrate-stabilized stannous chloride and hydrogen tetrachloroaurate(III) tetrahydrate as raw materials. The properties of the F-AuNCs were characterized by their fluorescence, their absorption spectra, and TEM. With the F-AuNCs used as a probe, a simple and rapid method was developed for BHC detection based on the strong fluorescence quenching of F-AuNCs. The fluorescence intensity had a good linear relationship with the concentration of BHC, with a relatively low detection limit (7.5 × 10-8 mol L-1). Moreover, the method was further applied in the determination of two different tablets (compound berberine tablets and BHC tablets) and displayed outstanding feasibility and precision. This novel strategy is expected to be a good choice in future pharmaceutical analysis.
This work was supported by the National Natural Science Foundation of China (nos 51502088, 21275047, 21445008), the Hunan Provincial Natural Science Foundation of China (nos 2016JJ3058, 2016JJ5005), the Research Foundation of Education Department of Hunan Province (no. 17B091), the Graduate Innovation Project of the Hunan University of Science and Technology (CX2017B619), and the Foundation of Science and Technology on Transient Impact Laboratory (No. 614260601010317).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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