Self-Referenced Ratiometric Detection of Sulfatase Activity with Dual-Emissive Urease-Encapsulated Gold Nanoclusters

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 (λ em = 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 IFEbased ratiometric sensing strategy showed a good linear relationship over the range of 0.01−1 U/mL (R 2 = 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, rapidity, 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.

Strategies of Luminescent Gold Nanoclusters for Chemo-/Bio-Sensing

“…The stabilizers represented by proteins are the classic method to prepare luminescent AuNCs. Besides BSA, a series of proteins, such as urease, [44] catalase, [45] lysozyme, [46] plant proteins, [47] egg white, [48] goose feathers, [49] and globulin, [50] have been applied as stabilizers for the synthesis of luminescent AuNCs using a ‘‘one-pot’’ fashion method. The protein cages act as excellent scaffolds, which can regulate the growth of AuNCs within their well-defined ultrasmall 3D structures.…”

“…After thoroughly investigating the luminescence properties of the duel emissive system, we developed a ratiometric sensor for p-nitrophenol (pNP), which was able to effectively quench the emission of diTyr by IFE rather than that of AuNCs (Figure 10) [78]. Further, Deng et al developed this response for enzyme analysis [44]. They used the selective quenching of diTyr emissions by pNP to design an analytical ratiometric approach for sulfatase, which could catalyze p-nitrophenyl sulfate and then release pNP.…”

Rare-Earth Eu3+/Gold Nanocluster Ensemble-Based Fluorescent Photoinduced Electron Transfer Sensor for Biomarker Dipicolinic Acid Detection

“…Gold nanoclusters (AuNCs) have become a hot topic for the current nanoscience research because they have a broad application prospect in many fields including sensing, biological imaging, catalysis, and electronic devices. − AuNCs typically comprise several to hundreds of atoms with sizes approaching the Fermi wavelength of the conduction electrons, leading to the emergence of unexpected strong photoluminescence. , As a new kind of fluorescent nanomaterials, AuNCs have many distinct features such as simple synthesis, easy modification, good water dispersibility, low toxicity, good compatibility, and excellent stability. Based on their unique optical properties, AuNCs as robust and promising fluorescent probes have been frequently used for the determination of numerous analytes like ions, , small molecules, , enzymes, − nucleic acids, cancer cells, and so on. However, most of the AuNC-related sensors were fabricated via fluorescence quenching (turn-off), which are comparatively less sensitive than turn-on sensors.…”

Charge Neutralization Strategy to Construct Salt-Tolerant and Cell-Permeable Nanoprobes: Application in Ratiometric Sensing and Imaging of Intracellular pH

“…Water-soluble gold nanoclusters (AuNCs) are a class of promising fluorescent nanomaterials consisting of several to tens of gold atoms and ligand shells with multiple charges. − They have attracted increasing interest as effective nanocarriers for the construction of ratiometric fluorescent sensing platforms due to their ultrasmall sizes, good biocompatibility, excellent photostability, and easy modification. − For pH sensing, the pH-insensitive AuNCs are primarily used as a reference, − resulting in a low correlation between the pH response and the sensitivity of AuNC emission. It is also a challenge for the pH-insensitive AuNCs to help extend the pH measurement range.…”