Protein-stabilized Gold Nanoclusters Research Articles

Machine learning-assisted photoluminescent sensor array based on gold nanoclusters for the discrimination of antibiotics with test paper

Antibiotic residues accumulation in the environment endangers ecosystems and human health. There is an urgent need for a facile and efficient strategy to detect antibiotics. Here, we report a photoluminescent sensor array based on protein-stabilized gold nanoclusters (AuNCs) for the detection of two families of antibiotics, tetracyclines and quinolones. The nanoclusters were synthesized with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. They had different interactions with seven kinds of antibiotics and exhibited diverse photoluminescence (PL) responses, which were analyzed by linear discriminant analysis and ExtraTrees algorithms. The sensor array performed well in both classification and quantification of seven antibiotics. And the quantitative results of all antibiotics obtained R2 of no less than 0.99 at 0–100 μM when using suitable regression models. Additionally, the sensor array was able to distinguish antibiotic mixtures and multiple interfering substances, and it also kept 100% classification accuracy in river water samples. Moreover, test paper assisted by a smartphone was applied for quick detection of antibiotics, with good performance in both HEPES buffer and river water. These studies reveal great potential for the point-of-use analysis of antibiotics in environmental monitoring.

Horseradish Peroxidase-Encapsulated Fluorescent Bio-Nanoparticle for Ultra-Sensitive and Easy Detection of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) has been a fascinating target in various chemical, biological, clinical, and industrial fields. Several types of fluorescent protein-stabilized gold nanoclusters (protein-AuNCs) have been developed for sensitive and easy detection of H2O2. However, its low sensitivity makes is difficult to measure negligible concentrations of H2O2. Therefore, to overcome this limitation, we developed a horseradish peroxidase-encapsulated fluorescent bio-nanoparticle (HEFBNP), comprising bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and horseradish peroxidase-stabilized gold nanoclusters (HRP-AuNCs). The fabricated HEFBNP can sensitively detect H2O2 owing to its two properties. The first is that HEFBNPs have a continuous two-step fluorescence quenching mechanism, which comes from the heterogenous fluorescence quenching mechanism of HRP-AuNCs and BSA-AuNCs. Second, the proximity of two protein-AuNCs in a single HEFBNP allows a reaction intermediate (•OH) to rapidly reach the adjacent protein-AuNCs. As a result, HEFBNP can improve the overall reaction event and decrease the loss of intermediate in the solution. Due to the continuous quenching mechanism and effective reaction event, a HEFBNP-based sensing system can measure very low concentrations of H2O2 up to 0.5 nM and show good selectivity. Furthermore, we design a glass-based microfluidic device to make it easier use HEFBNP, which allowed us to detect H2O2 with the naked eye. Overall, the proposed H2O2 sensing system is expected to be an easy and highly sensitive on-site detection tool in chemistry, biology, clinics, and industry fields.

Thermostable protein-stabilized gold nanoclusters as a peroxidase mimic

By using a genetically modified thermostable protein (KTQ5C), we have synthesized protein-stabilized goldnanoclusters (AuNC@KTQ5C) with advantageous properties, such as heat stable fluorescent emission and heat resistant peroxidase-like activity.

Fabrication of avidin-stabilized gold nanoclusters with dual emissions and their application in biosensing

Protein-stabilized gold nanoclusters (Prot-Au NCs) have been widely used in biosensing and cell imaging owing to their excellent optical properties and low biotoxicity. However, several Prot-Au NCs reported in the literature do not retain the biological role of the protein, which greatly limits their ability to directly detect biomarkers. This study demonstrated for the first time the successful synthesis of dual-function avidin-stabilized gold nanoclusters (Av–Au NCs) using a one-pot method. The resulting Av–Au NCs exhibited intense blue and red emissions under 374 nm excitation. Furthermore, the Av–Au NCs retained the native functionality of avidin to bind to biotin. When DNA strands modified with biotin at both ends (i.e., linker chains) were mixed with Av–Au NCs, large polymers were formed, indicating that Av–Au NCs could achieve fluorescence signal amplification by interacting with biotin. Taking advantage of the aforementioned properties, we constructed a novel enzyme-free fluorescent biosensor based on the Av–Au NCs-biotin system to detect DNA. The designed fluorescent biosensor could detect target DNA down to 0.043 nM, with a wide line range from 0.2 nM to 20 µM. Thus, these dual-functional Av–Au NCs were shown to be an excellent fluorescent material for biosensing.Graphic abstract

Blood Plasma Stabilized Gold Nanoclusters for Personalized Tumor Theranostics

Simple SummaryCancer is a disease that has a high fatality rate over the world. Nanotechnology is one of the most promising current approaches for developing novel diagnostic and treatment methods in accomplishing more personalized medicine. Personalized gold nanoclusters have potential to be used in cancer theranostics. We demonstrate that biocompatible gold nanoclusters could be synthesized directly in human blood plasma. Such gold nanoclusters have a wide photoluminescence band in the optical tissue window and generate reactive oxygen species under irradiation with visible light, thus are suitable for cancer theranostics.Personalized cancer theranostics has a potential to increase efficiency of early cancer diagnostics and treatment, and to reduce negative side-effects. Protein-stabilized gold nanoclusters may serve as theranostic agents. To make gold nanoclusters personalized and highly biocompatible, the clusters were stabilized with human plasma proteins. Optical properties of synthesized nanoclusters were investigated spectroscopically, and possible biomedical application was evaluated using standard cell biology methods. The spectroscopic investigations of human plasma proteins stabilized gold nanoclusters revealed that a wide photoluminescence band in the optical tissue window is suitable for cancer diagnostics. High-capacity generation of singlet oxygen and other reactive oxygen species was also observed. Furthermore, the cluster accumulation in cancer cells and the photodynamic effect were evaluated. The results demonstrate that plasma proteins stabilized gold nanoclusters that accumulate in breast cancer cells and are non-toxic in the dark, while appear phototoxic under irradiation with visible light. The results positively confirm the utility of plasma protein stabilized gold nanoclusters for the use in cancer diagnostics and treatment.

Boosting the Photoluminescent Properties of Protein-Stabilized Gold Nanoclusters through Protein Engineering.

This work reports on the use of protein engineering as a versatile tool to rationally design metal-binding proteins for the synthesis of highly photoluminescent protein-stabilized gold nanoclusters (Prot-AuNCs). The use of a single repeat protein scaffold allowed the incorporation of a set of designed metal-binding sites to understand the effect of the metal-coordinating residues and the protein environment on the photoluminescent (PL) properties of gold nanoclusters (AuNCs). The resulting Prot-AuNCs, synthesized by two sustainable procedures, showed size-tunable color emission and outstanding PL properties. In a second stage, tryptophan (Trp) residues were introduced at specific positions to provide an electron-rich protein environment and favor energy transfer from Trps to AuNCs. This modification resulted in improved PL properties relevant for future applications in sensing, biological labeling, catalysis, and optics.

Synthesis of gold nanoclusters-loaded lysozyme nanoparticles for ratiometric fluorescent detection of cyanide in tap water, cyanogenic glycoside-containing plants, and soils

The modification of protein-stabilized gold nanoclusters with fluorophores has been intensively applied for the ratiometric detection of biomolecules, metal ions, and anions. This study developed a straightforward strategy to prepare lysozyme nanoparticle-encapsulated gold nanoclusters (LysNP-AuNCs) as a dual-emission probe for the ratiometric sensing of cyanide through fluorescence resonance energy transfer (FRET) without the conjugation of additional fluorophores. The reduction of gold ion precursors with lysozyme generated lysozyme-stabilized AuNCs under an alkaline pH, which were demonstrated to self-assemble into nanoaggregates during the formation of AuNCs. The aggregated lysozyme molecules on the AuNCs were treated with glutaraldehyde, triggering the conversion of the aggregated lysozymes into blue-emitting lysozyme nanoparticles. As a result, the AuNCs were well distributed inside a single lysozyme nanoparticle, as demonstrated by transmission electron microscopy. The presence of cyanide triggered the etching of the AuNCs in the LysNP-AuNCs, leading to the suppression of FRET from lysozyme nanoparticle to AuNCs. The LysNP-AuNC probe was implemented for FRET detection of cyanide with a linear range of 3–100 μM. Additionally, the selectivity of the LysNP-AuNC probe for cyanide toward other anions was remarkably high. The practicality of the proposed probe was evaluated by quantifying cyanide in tap water and soils and monitoring the liberation of hydrogen cyanide from cyanogenic glycoside-containing foods.

Novel paper-based sensing platform using photoluminescent gold nanoclusters for easy, sensitive and selective naked-eye detection of Cu2+

Herein, we developed a novel, cheap, fast and simple portable sensing platform based on the photoluminescence (PL) quenching effect of paper-incorporated bovine serum albumin protein stabilized gold nanoclusters (BSA-AuNCs) when interacting with Cu ions (Cu2+) from real water samples. First, we demonstrated the sensibility and specificity of the BSA-AuNCs-based sensor for the detection of Cu2+ in solution via PL quenching effect, using a portable fluorescence spectrometer, under 502 nm LED excitation. The limit of detection (LOD) was found to be as low as 0.83 µM. For improved portability and accessibility, the sensing performance of BSA-AuNCs was successfully combined with the unique properties of Whatman paper substrates in order to develop a portable and ready-to-use sensor for the detection of Cu2+ from real water samples. The obtained BSA-AuNCs incorporated paper-based sensing substrate was successfully used for naked-eye detection of Cu2+ levels from water, under UV light, based on the visual detection of PL quenching. We obtained a LOD of 5 µM which was confirmed even for 14 days aged paper sensing substrates. The as-prepared BSA-AuNCs-paper-based sensor was validated for the detection of Cu2+ in different real water samples through the visual examination of the PL quenching on the paper substrates under UV light illumination and relevantly dangerous levels for humans or subaquatic organisms were successfully identified. The newly prepared BSA-AuNCs-incorporated paper-based sensing platform is easy-to-use and presents great promise in the fast, selective, qualitative and semi-quantitative naked-eye detection of Cu2+ in real water samples.

The surface-dependent biological effect of protein-gold nanoclusters on human immune system mimetic cells

In this paper, the comprehensive toxicity mechanism of four well-defined protein-stabilized gold nanoclusters (Au NCs) is firstly highlighted against promyelocytic leukaemia cells (HL-60). For this purpose, bovine serum albumins (BSA) and human serum albumins (HSA), as well as lysozyme (LYZ) and gamma globulin (γG) immunoproteins were applied as reducing and stabilizing agents of the Au nanohybrids. The selected cell line served as an innate human immunity mimetic to detect nonspecific immune responses after the cluster treatments. The surface-dependent comparative study of the synthesized Au-containing systems was determined by several biochemical assays to measure the changes in mitochondrial activity, membrane integrity, and the level of secretion of inflammatory, as well as apoptosis mediators. Besides, the mechanical action and the penetration of the Au NCs were also investigated by confocal microscopy. Based on these experiments, it was proved that the protein-stabilized Au NCs showed a minor toxic effect on HL-60 cells. Although, the clusters did not induce proinflammatory processes but caused noticeable oxidative stress and the apoptosis was also enhanced. In comparison to the surface properties of the Au NCs, it was recognized that the LYZ-stabilized Au NCs had the most negative effect on the HL-60 cells.

Cytotoxicity studies of protein-stabilized fluorescent gold nanoclusters on human lymphocytes

In this work, we clearly focus on the comparative cytotoxicity investigations of several protein-stabilized gold nanoclusters (Au NCs) towards lymphocytes B (COLO-720 L) and lymphocytes T (HUT-78) cells. For synthesis, the one-pot template-assisted method was carried out using lysozyme (LYZ), human (HSA) and bovine (BSA) serum albumins, and gamma globulin (γG) as stabilizing agents. Regardless of the type of proteins, all synthesized Au NCs possess intense red emission (λem ∼ 650 nm) and have similar size of a metal core (ca. 1.4 nm) with negative surface charge at pH = 7.4. During the treatment of cells with clusters, changes in mitochondrial activity, membrane integrity, secretion of inflammatory and apoptosis mediators of the lymphocytes were studied to determine the potential effect of protein layers on the toxicity of clusters. It was found that γG-Au NCs induced the highest disorders in mitochondrial activity, but the influence of other NCs on the cell viability was minor. Besides, all Au NCs caused oxidative stress by peroxidation of membrane lipids. The secretion of malonic dialdehyde (MDA) was enhanced by LYZ- and γG-Au NCs. Apart from LYZ-Au NCs, the clusters did not exhibit strong proinflammatory and apoptotic properties. The enhanced secretion of tumor necrosis factor (TNF-α) by lymphocytes B, in comparison to control, was independent of the clusters type. Despite the lack of significant influence of the Au NCs on the viability of the lymphocytes, they can stimulate undesirable cellular processes, which clearly depends on the stabilizing proteins.

Fluorescent Labeling of Hyaluronic Acid-Chitosan Nanocarriers by Protein-Stabilized Gold Nanoclusters

In medical research the visualization of drug carrier accumulation and release of the loaded drugs in vivo is an important field. In this work, two protein-stabilized gold nanoclusters (Au NCs) as effective fluorescent reporters (FRs) were investigated for labeling of biocompatible chitosan-modified hyaluronic acid based nanocarriers having two different structures. The colloid stability of the labeled carriers was studied by dynamic light scattering and Zeta potential measurements, while the changes in the fluorescence of the lysozyme- (LYZ) and bovine serum albumin (BSA)-stabilized Au NCs were analyzed by spectrofluorimetry and confocal fluorescent microscopy. We found that the labeling was effective with a wide range of marker:carrier mass ratios, and the fluorescence of the NCs and the colloid stability of the complexes were retained. Labeling during preparation and subsequent labeling were compared, and based on composition (nanocluster:carrier mass ratio) and structure of the complex systems we preferred the latter method, as it left the Au NCs free for further modifications. Considering both marker:carrier mass ratios and emission intensities, the LYZ-stabilized Au NCs proved to be better labels. The core-shell type carrier formulations showed increased fluorescence with LYZ-stabilized NCs, presumably from aggregation induced emission.

Protein-stabilized gold nanoclusters for PDT: ROS and singlet oxygen generation

Suitable properties as well as eco-friendly synthesis of photoluminescent Au nanoclusters (NCs) make them promising compounds for biomedical diagnostics and visualization applications. However, the potential photochemical activity of such agents on cancerous cells is largely unknown. The nanoclusters (BSA-Au NCs) were synthetized in the presence of BSA (an average hydrodynamic diameter was about 9.4 nm, while the size of the metal cluster was <1.3 nm according to atomic force microscopy measurements) and possessed a broad photoluminescence band at 680 nm in buffered (pH 7.2) aqueous medium. The photochemical activity was studied by adding two fluorescent probes (dihydrorhodamine or Singlet Oxygen Sensor Green) for detection of reactive oxygen species in samples irradiated at 405 nm to minimize direct excitation of the probes. The photoluminescence measurements evidenced the capability of BSA-Au NCs to generate reactive oxygen species upon light exposure, while the observed sensitivity of the photoluminescence properties might be used to indicate photooxidative processes in the medium. The viability test performed on breast cancer cells after incubation with BSA-Au NCs and subsequent irradiation revealed notable difference in induced phototoxicity between two cell lines, which was not the case after the corresponding treatment using the photosensitizer chlorin e6.

Advances in biomedical and pharmaceutical applications of protein-stabilized gold nanoclusters.

The interest in using gold nanoclusters (AuNCs) as imaging probes is growing, covering wide ranges of applications. The stabilization of AuNCs with protein ligands enhances their biomedical and pharmaceutical applications. This is due to the biocompatibility, water solubility and bioactivity of proteins. Different factors can control the optical properties of AuNCs such as protein size, amino acids content and conformational structure. Controlling the synthesis conditions can result in tuning the AuNCs excitation, emission, fluorescence intensity and physicochemical properties to fulfill different applications. NIR-emitting protein-stabilized AuNCs are promising as imaging agents for targeting and visualization of cancer in vitro and in vivo. They are promising to be included as an important part of multifunctional theranostic nanosystems, due to their potential dual functions as imaging and photosensitizing agent for photodynamic therapy. Additionally, the protein around AuNCs represents a rich environment of active functional groups that are susceptible for conjugation with various biomolecules. Protein-AuNCs can act as fluorescent probes for rapid and selective analysis of different analytes in solution, cells or biological fluids. In conclusion, the variability of protein-AuNC applications can advance research in different biomedical and pharmaceutical fields.

Revisiting the conformational state of albumin conjugated to gold nanoclusters: A self-assembly pathway to giant superstructures unraveled.

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin’s secondary structure and conjugate’s luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native β-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.

Gold nanocluster-based ratiometric fluorescent probes for hydrogen peroxide and enzymatic sensing of uric acid

A method is described for ratiometric fluorometric assays of H2O2by using two probes that have distinct response profiles. Under the catalytic action of ferrous ion, the 615nm emission of protein-stabilized gold nanoclusters (under 365nm photoexcitation) is quenched by H2O2, while an increased signal is generated with a peak at 450nm by oxidizing coumarin with the H2O2/Fe(II) system to form a blue emitting fluorophore. These decrease/increase responses give a ratiometric signal. The ratio of the fluorescences at the two peaks are linearly related to the concentration of H2O2 in the range from 0.05 to 10μM, with a 7.7nM limit of detection. The detection scheme was further coupled to the urate oxidase catalyzed oxidation of uric acid which proceeds under the formation of H2O2. This method provides an simple and effective means for the construction of ratiometric fluorometric (enzymatic) assays that involve the detection of H2O2. Graphical abstract Under catalysis byferrous ion, hydrogen peroxide quenches the luminescence of gold nanoclusters (AuNCs) and oxidizes coumarin into a fluorescent derivative, which rendered fluorescence ON and OFF at two distinct wavelengths for ratiometric measurements.

Nanohybrids consisting of magnetic nanoparticles and gold nanoclusters as effective peroxidase mimics and their application for colorimetric detection of glucose.

Although protein-stabilized gold nanoclusters (AuNCs) have gathered recent attention as biocompatible peroxidase mimics, their practical utility has been critically limited by the low catalytic activity. Here, the authors have developed a nanohybrid material to significantly enhance the catalytic activity of AuNCs by combining them with other inorganic enzyme mimetics, Fe3O4 magnetic nanoparticles (MNPs), through electrostatic attraction. Owing to the synergistic effect by incorporating AuNCs and MNPs, the constructed nanohybrids yielded highly enhanced catalytic activity and enabled rapid catalytic oxidation of 3,3',5,5'-tetramethylbenzidine substrate to produce a blue-colored solution in proportional to the amount of H2O2. Moreover, a highly sensitive and selective glucose biosensing strategy was developed based on the coupled catalytic action between glucose oxidase and the nanohybrids. Using this method, target glucose was successfully detected in a linear concentration range from 150 to 750 μM with a detection limit as low as 100 μM. Along with excellent linearity, high precision and reproducibility were achieved by employing real human blood serums, which enables its use for the reliable quantification of glucose in practical use. Based on these results, the authors anticipate that the nanohybrids consisting of MNPs and AuNCs can serve as potent peroxidase mimics for the detection of clinically important target molecules.

The mechanism and application of the protein-stabilized gold nanocluster sensing system.

Protein-stabilized gold nanoclusters (protein-Au NCs) have been an attractive frontier of nanoparticle research. Due to their unique fluorescence properties, high stability, environmentally friendly synthetic routes and nontoxicity, protein-Au NCs could find applications in highly sensitive and selective detection of metal ions, inorganic ions and biomolecules in food, soil, water and biological samples. The past few years have witnessed the development of many successful strategies for the preparation of numerous protein-Au NC-based sensing systems. In this review, we focus on a number of sensing systems based on protein-Au NCs for the detection of various analytes of interest with a special emphasis on their corresponding sensing mechanisms.

Turn-on fluorescence detection of ascorbic acid with gold nanolcusters

In this report, a sensitive fluorescent detection of ascorbic acid was developed with protein-stabilized gold nanoclusters. The fluorescence signal from the gold nanoclusters could be effectively quenched by elemental iodine. This kind of quenching could be inhibited by ascorbic acid through a corresponding reduction process, thus a turn-on response toward ascorbic acid was obtained. The validity and performances of the detection scheme were evaluated. The fluorescence responses were linearly related to ascorbic acid in the concentration range 0.1–10μM, and the method was capable of detecting ascorbic acid down to 22nM. The high performances of the developed scheme greatly simplified the sample preparation step, and the method was successfully applied for real sample analyses.

Designed Modular Proteins as Scaffolds To Stabilize Fluorescent Nanoclusters.

Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.

Ratiometric Fluorescent Polymeric Thermometer for Thermogenesis Investigation in Living Cells.

Intracellular temperature has a fundamental effect on cellular events. Herein, a novel fluorescent polymer ratiometric nanothermometer has been developed based on transferrin protein-stabilized gold nanoclusters as the targeting and fluorescent ratiometric unit and the thermosensitve polymer as the temperature sensing unit. The resultant nanothermometer could feature a high and spontaneous uptake into the HeLa cells and the ratiometric temperature sensing over the physiological temperature range. Moreover, the precise temperature sensing for intracellular heat generation in HeLa cells following calcium ions stress has been achieved. This practical intracellular thermometry could eliminate the interference of the intracellular surrounding environment in cancer cells without a microinjection procedure, which is user-friendly. The prepared new nanothermometer can provide tools for unveiling the intrinsic relationship between the intracellular temperature and ion channel function.