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Self-Referenced Ratiometric Detection of Sulfatase Activity with Dual-Emissive Urease-Encapsulated Gold Nanoclusters Hao-Hua Deng, Hua-Ping Peng, Kaiyuan Huang, Shao-Bin He, QiaoFeng Yuan, Zhen Lin, Ruiting Chen, Xing-Hua Xia, and Wei Chen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01130 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019
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Self-Referenced Ratiometric Detection of Sulfatase Activity with Dual-Emissive Urease-Encapsulated Gold Nanoclusters Hao-Hua Deng,a Hua-Ping Peng,a Kai-Yuan Huang,a Shao-Bin He,a Qiao-Feng Yuan,a Zhen Lin,a RuiTing Chen,a Xing-Hua Xia,b Wei Chena* a Higher
Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China b State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ABSTRACT: In this study, on the basis of the biomineralization capability of urease, a facile, one-step, and green synthetic method has been proposed for the fabrication of gold nanoclusters (AuNCs). The prepared urease-encapsulated AuNCs (U-AuNCs) exhibited strong red fluorescence emission (λem = 630 nm) with a quantum yield as high as 17%. Interestingly, at a low concentration, the U-AuNC solution was found to be a dual-emissive system with the blue emission of the dityrosine (diTyr) residues of urease and the red emission of the embedded AuNCs. Further experiments demonstrated that p-nitrophenol (PNP) can selectively suppress the 410 nm emission of the diTyr residues of U-AuNCs without affecting the red emission of the U-AuNCs. The fluorescence quenching mechanism between U-AuNCs and PNP was systematically studied, and the leading role of the inner filter effect (IFE) was identified. Additionally, based on the sulfatase-catalyzed hydrolysis of p-nitrophenyl sulfate (PNPS) to release PNP, a self-referenced ratiometric detection method for sulfatase, which plays a crucial role in sulfur cycling, degradation of sulfated glycosaminoglycans and glycolipids, and extracellular remodeling of sulfated glycosaminoglycans, was developed by using dual-emissive U-AuNCs as the signal readout, in which the diTyr residues served as the probe and the AuNCs functioned as the internal reference. This IFE-based ratiometric sensing strategy showed a good linear relationship over the range of 0.01-1 U/mL (R2 = 0.997). The detection limit for sulfatase activity was 0.01 U/mL. The developed protocol was successfully used to detect sulfatase activity in human serum samples. The simplicity, rapidness, low cost, high credibility, good reproducibility, and excellent selectivity of the detection platform serve as an inspiration for further applications of fluorescent AuNCs in chemo/biosensing. KEYWORDS: ratiometric detection, gold nanocluster, dityrosine, inner filter effect, sulfatase
In the past few decades, fluorescence based sensing strategies for the detection of biological and chemical analytes have attracted considerable attention due to advantages including high sensitivity, excellent selectivity, operational simplicity, rapid signal response time, low invasiveness, good portability, high spatial and temporal resolution. The sensing principle is of crucial importance for the development and design of fluorescent sensors. To date, various fluorescence sensing mechanisms have been proposed based on the different photophysical processes occurring between the particular targets and the fluorophores, including fluorescence resonance energy transfer (FRET), photo-induced electron transfer (PET), electronic energy transfer (EET), intramolecular charge transfer (ICT), metal-ligand charge transfer (MLCT), and twisted intramolecular charge transfer (TICT).1-3 Generally, sensor fabrication based on the above mechanisms involves either intermolecular interaction and recognition between the sensor and target analyte, or covalent linking between the receptor and fluorophore. These conditions make sensor fabrication complicated, expensive, and extraordinarily timeconsuming, which inevitably restricts their further practical applications.1 The inner filter effect (IFE) is another significant phenomenon in the field of spectrofluorometry and can be categorized as a non-irradiation energy conversion model.4 IFE results from the absorption of the excited and/or emitted light of fluorophores by the absorbers in the detection system,
and subsequently induces a decline in the fluorescence intensity.5 IFE has been utilized as a powerful means for constructing sensitive and selective sensors by transforming the analytical absorption signals into fluorescence signals.6-11 The IFE-based methods provide great flexibility and are simple to implement because there is no need for any link between the fluorophore and the quencher molecule or for complex modifications of sensors.12 Typically, the design strategies for IFE-based fluorescent sensors can be classified into three major categories: switch-off mode, switch-on mode, and ratiometric sensing mode. In the switch-off sensing approach, the absorbance of the absorber increases with increasing analyte concentration and leads to reduced fluorescence of the sensing system. In contrast, in the switchon sensing approach, fluorescence is first quenched by the absorber due to IFE, and then restored by the analyte through various chemical/biochemical reactions such as complexation, dissolution of the absorber, and oxidation/reduction. To date, the vast majority of the IFE-based methods for the detection and monitoring of analyte concentrations and molecular events operate in the above two modes. Unfortunately, one of the biggest defects of these single-channel-based methods is their absolute intensity-dependent signal readout, which results in inaccurate measurement data owing to the presence of a variety of target concentration-independent experimental or physiological factors that can cause undulation of their absolute fluorescence intensity.4 On the other hand, the
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ratiometric sensing approach relies on the built-in selfcalibration of the fluorescence signal by determining the ratios of the photoluminescence (PL) intensities at two wavelengths. It can remove false signals stemming from the impact of the surrounding environment and eliminate the fluctuations in probe concentration and excitation light intensity, enabling a more sensitive, accurate, and reliable quantitation.13,14 Nevertheless, so far, the relevant information on IFE-based sensors developed by this new strategy is quite limited.15-18 Moreover, most of the IFE-based ratiometric fluorescence probes reported to date have some of the following disadvantages: (1) there is a small difference in the emission peak wavelength between the response and reference signal labels that leads to interference between the two emission peaks; (2) not all of the fluorescence probes are biocompatible and environment-friendly, which greatly hinders their practicability; (3) typically, external fluorophores need to be introduced, which increases complexity because of preconjugation or pre-assembly that take place by physical or chemical methods. It is therefore highly desirable to seek a new ratiometric probe with outstanding optical properties, low toxicity, and a facile design procedure for establishing novel IFE-based sensing platforms. Due to the discrete energy levels, gold nanoclusters (AuNCs) possess distinctly different optical, chemical, and electrical properties from those of larger nanoparticles.19 One of the most fascinating features is their strong PL. The numerous reports published in recent years have confirmed the enormous potential of few-atom AuNCs as a new generation of luminescent nanomaterials for both practical application and fundamental research.20-30 Of particular interest are AuNCs that are protected by biomolecules, such as proteins, primarily because of their simple, one-pot, and “green” synthesis routes. Since Xie et al. firstly prepared AuNCs employing bovine serum albumin (BSA) as a capping agent and reductant,31 numerous proteins have been used as efficient bioscaffolds for the formation of fluorescent AuNCs.32-36 The intense red emission of protein-AuNCs bioconjugates together with their excellent stability under harsh conditions, good biocompatibility, and easy post-synthetic surface modifications make them particularly attractive in the field of biomedicine. In addition, the protein coating layer on the AuNCs offers great opportunities for constructing multifunctional bio-nano hybrid systems. Owing to these fascinating features, fluorescent protein-AuNCs have been used extensively to fabricate diverse chemo/biosensors on the basis of PL quenching or recovery. However, very few ratiometric fluorescence assays have been reported using protein-AuNCs that are worthy of further explorations.37-40 Sulfatases (EC 3.1.6.X) represent a class of enzymes that can specifically catalyze the hydrolytic desulfonation of sulfate esters (CO–S) and sulfamates (CN–S). These enzymes play vital roles in numerous cellular functions, such as cell signaling, cellular degradation, and hormone regulation.41 An abnormal concentration of sulfatase implicates the pathophysiological procedure of several diseases, including developmental abnormalities, bacterial pathogenesis, lysosomal storage disorders, and hormone-dependent cancers.42 Therefore, the development of a sulfatase assay is of critical importance, especially in disease diagnosis and drug discovery. Herein, a facile method for the preparation of AuNCs in the urease scaffold through biomimetic mineralization was described. Urease was utilized for the first time to reduce gold ions and protect AuNCs. The generated urease-encapsulated
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AuNCs (U-AuNCs) displayed strong red emission at 630 nm with a high quantum yield (QY) of 17%. Interestingly, at a low U-AuNC concentration (1.11 mg/mL), an emission peak centered at 410 nm corresponding to the dityrosine (diTyr) residues of urease could be observed besides the characteristic red fluorescence originating from the gold core. As these two emission bands were situated in the red and blue regions, respectively, and did not interfere with each other, they satisfied an ideal pre-condition for the development of ratiometric fluorescence sensors. Furthermore, p-nitrophenol (PNP) was discovered to be capable of effectively quenching the fluorescent emission of the diTyr residues via IFE, while exhibiting only a slight influence on the red fluorescence of UAuNCs. In addition, based on the fact that sulfatase can hydrolyze p-nitrophenyl sulfate (PNPS) to release PNP, a selfreferenced ratiometric fluorescent method for sensing sulfatase activity was readily achieved, in which the diTyr residues were adopted as the probe and the AuNCs served as the endogenous reference. As far as we know, this is the first report about applying an IFE-based ratiometric sensing strategy to photoluminescent AuNCs for enzyme activity determination, which has immense potential for application in clinical diagnosis and drug screening. EXPERIMENTAL SECTION Chemicals and Materials. Urease (from Canavalia ensiformis), HAuCl4·4H2O, NaOH, PNP, cysteamine, tris(hydroxymethyl)aminomethane (tris), HCl, KCl, Na2HPO4, NaHCO3, MgSO4, KNO3, CaCl2, Zn(OAc)2, creatinine, creatine, sarcosine, glucose, lactic acid, acetylcholine, urea, proteinase K, horseradish peroxidase, and glucose oxidase were purchased from Aladdin Reagent Co. (Shanghai, China). Potassium 4-nitrophenyl sulfate, sulfatase (EC 3.1.6.X from Helix pomatia), catalase, tyrosinase, sarcosine oxidase, superoxide dismutase, choline oxidase, acetylcholinesterase, lysozyme, pyrophosphatase, α-glucosidase, acid phosphatase, and alkaline phosphatase were bought from Sigma-Aldrich Chemical Co. (Shanghai, China). Doubly distilled water and analytical grade reagents were used for preparation of all aqueous solutions. Apparatus and Characterization. Ultraviolet-visible (UVVis) absorption spectra were recorded on a UV-2450 UV-Vis spectrophotometer (Shimadzu, Japan) with a cell of path length 1 cm. Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent, USA). The PL lifetime was counted on an F900 time-correlated singlephoton-counting fluorescence lifetime spectrometer (Edinburgh Analytical Instruments, Edinburgh, U.K.). Absolute QY measurements were conducted on an integrating sphere (Edinburgh Instruments). Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were taken using a JEM-2100 microscope (JEOL, Japan). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250 XI electron spectrometer (Thermo). Zeta potential measurements were performed using a particle analyzer Litesizer 500 (Anton Paar, Graz, Austria). Synthesis of U-AuNCs and diTyr-Urease. All glassware was cleaned with aqua regia solution (1:3 HNO3-HCl, v/v) and rinsed with water prior to use. Highly fluorescent U-AuNCs were synthesized in basic aqueous solution using urease as the reducing and protecting agent. Aqueous NaOH (5 mL, 1M) and HAuCl4 (20 mL, 10 mM) solutions were added in turn to urease solution (20 mL, 50 mg/mL) and the mix solution was incubated at 37 °C for 8 h. The prepared U-AuNC solution (22.22 mg/mL) could be stored in the dark at 4 °C for at least one
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month with no obvious changes in its fluorescent property. The diTyr-urease solution was obtained according to a previous approach with some modifications.43 One milliliter of the assynthesized U-AuNC solution was introduced into 3 mL of 50 mM cysteamine (pH = 10.0) and the mixture was incubated in a 55 °C bath for 1 h. The resultant mixture was purified by ultrafiltration (Millipore, 10 kDa). The purification process was repeated three times. Subsequently, the retentates were collected and resuspended in 4 mL of tris-HCl buffer solution (50 mM, pH 7.4), defined as the diTyr-urease solution. Fluorescence Quenching of U-AuNCs by PNP. For optimization of the incubation time for fluorescence quenching by PNP, 200 μL of the U-AuNC solution (5.55 mg/mL) was added into 800 μL of tris-HCl buffer solution (50 mM, pH = 7.40) containing 50 μM PNP. The fluorescence of the mixture was recorded after different incubation time durations at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min after the introduction of U-AuNC solution. To avoid the interference from second-order Rayleigh scattering of the exciting light, the excitation wavelength was set as 340 nm. This experiment was conducted at room temperature. Ratiometric Fluorescent Detection of Sulfatase Activity. The IFE-based ratiometric fluorescence detection of sulfatase activity was performed as follows. Different concentrations of sulfatase were diluted with tris-HCl buffer solution (50 mM, pH = 7.40). Then, 100 μL of the sulfatase solutions with activities ranging from 0 to 1 U/mL were added to 700 μL of tris-HCl buffer solution (50 mM, pH = 7.40) containing 0.1 mM PNPS. The mixture was incubated at 37 °C for 1 h, followed by the addition of 200 μL of the U-AuNCs solution (5.55 mg/mL). Subsequently, the PL emission spectra under 340 nm excitation were measured. The selectivity of the UAuNC-based ratiometric sensor toward sulfatase was tested by adding other enzymes, small molecules or ions solutions instead of sulfatase in a similar manner. Human Serum Sample Detection. Human serum samples were collected and spiked with different concentrations of sulfatase. Then, 10 μL of the spiked sample was added to 790 μL of tris-HCl buffer solution (50 mM, pH = 7.40) containing 0.1 mM PNPS. The mixture was incubated at 37 °C for 1 h, followed by the addition of 200 μL of U-AuNC solution (5.55 mg/mL). Subsequently, the PL emission spectra under 340 nm excitation were recorded.
RESULTS AND DISCUSSION Preparation and Characterization of U-AuNCs. The highly photoluminescent U-AuNCs were synthesized by mixing urease and HAuCl4 under alkaline conditions and incubating the mixture at 37 °C. Au(III) in HAuCl4 was reduced to Au(0) by the amino acid residues such as tyrosine (Tyr) in urease at high pH, leading to the formation of the red luminescent U-AuNCs. At a constant Au(III) precursor concentration of 10 mM, the other synthesis conditions for UAuNCs were optimized and employed as follows: a urease concentration of 50 mg/mL, a NaOH concentration of 1 M, and an incubation time of 8 h (Figure S1). The light brown solution of U-AuNCs (Figure 1A inset) emitted intense red fluorescence (Figure 1B inset) under UV light. Figure 1A displayed a typical absorption spectrum of aqueous U-AuNC solution. It can be seen that apart from a weak absorption peak at ~280 nm, which was assigned to the absorbance of aromatic amino acid residues of the protein, there was no absorption peak corresponding to gold surface plasmon resonance at ~520 nm. This suggested the preparation
of the U-AuNC bioconjugates was successful. The product exhibited excitation and emission peaks at 490 and 630 nm (Figure 1B), respectively, quite similar to the fluorescence spectra of BSA-AuNCs.31 The absolute QY of the U-AuNCs was measured to be 17%, which was much higher than that of most of the reported protein-protected AuNCs (usually
