Formation and Stabilization of Fluorescent Gold Nanoclusters Using Small Molecules

Synthesis, characterization, and bioconjugation of fluorescent gold nanoclusters (FGNCs) are reported. The gold nanocluster is synthesized by etching gold nanoparticle. When the gold nanocluster is capped with reduced dipoic acid (DHLA, dihydrolipoic acid) that the nanocluster has the fluorescent property. The fluorescent gold nanoclusters have the emission in the range of 600 800 nm that is suitable for cell labeling, and its size and hydrodynamic radius are around 1.8 nm and 1.8 3.4 nm. The FGNCs can also be conjugated with desired biomolecules, such as biotin-PEG and avidin. In the preliminary data, the FGNCs can be taken nonspecifically by human aortic endothelial cells and the FGNCs conjugated with avidin can label specifically endogenous biotin within human hepatoma cells. Compare the FGNCs with semiconductor quantum dots and conventional organic dyes, the FGNCs are more biocompatible than quantum dots and more stable than organic dyes. So we believe that the FGNCs have great potential for biomedical applications.

Biosensor applications often require the simultaneous detection of multiple analytes, with a clear need to go beyond the traditional multiplexing relying on distinct fluorescent dyes across the visible spectrum. Fluorescence lifetime correlation spectroscopy (FLCS) is a powerful approach taking advantage of the fluorescence lifetime information to separate the contributions of different fluorescent species with overlapping emission spectra. However, so far FLCS detection has been demonstrated only on binary mixtures of two fluorescent dyes, limiting its multiplexing capabilities. Here, we report the first quantitative FLCS measurements within a ternary mixture composed of three different fluorescent emitters with near-identical emission spectra. Two organic fluorescent dyes, Alexa Fluor 647 and CF640R, are combined with water-soluble Au18(SG)14 gold nanoclusters. Our experimental data establish that FLCS allows to accurately determine individual concentrations within intricate ternary mixtures. Another major aspect of interest concerns the assessment of the suitability of gold nanoclusters for FLCS multiplexing applications. With their microsecond lifetime and stable emission characteristics, gold nanoclusters add a valuable new aspect to the array of FLCS probes. Extending FLCS multiplexing beyond binary mixtures paves the way for further progress in the simultaneous highly parallel biosensing of multiple species.

Multifunctional nanoparticles (NPs) combining the superparamagnetism of Mn−Zn ferrite and the fluorescence property of gold nanoclusters (NCs) have been prepared by wet chemistry. Magnetic NPs synthesized by co-precipitation method were coated several times with oppositely charged polyelectrolytes (PEs) using the layer-by-layer technique. Common techniques (Fourier transform infrared spectroscopy, electron microscopy, zeta potential, etc.) indicated the monodispersity and the stability of the coated NPs providing a positive charged surface. Fluorescent gold NCs bound to a standard protein bovine serum albumin were adsorbed on the surface of the magnetic NPs. Structural investigations proved the presence of small gold clusters (~2 nm) in a shell surrounding the magnetic nanomaterial. The stable nanocomposite kept the original fluorescence property of the metal clusters with 211-fold increase of the red emission (λ = 690 nm) compared to the uncoated NPs. These NPs can be moved with a permanent magnet despite a 72-wt% increase of the non-magnetic fraction due to the PE coating and the protein adsorption.

We introduce a general approach to make colorful fluorescent gold nanoclusters which are protected by dihydrolipoic acid, mercaptoundecanoic acid and polyethylenimine. The fluorescent gold nanoclusters can perform a variety of bioconjugation processes such as PEGylation, biotinylation as well as forming complex nanobioconjugates with streptavidins. The brightening effects under proper surface modification are also reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Nonspecific uptake by human aortic endoth elial cells is demonstrated. Keywords: fluorescent gold nanoclusters, bioconjugation, near infrared, cell labeling, PEGylation, biotinylation, lipoic acid 1. INTRODUCTION Gold nanoclusters with size smaller than 2 nm have been gained great attention in the last decade for their unique role in bridging the link between atomic and nanoparticle behavior 1 . Researches have focused on their quantum physical properties including photoluminescence

Formation of gold nanoclusters in MgO by ion implantation at elevated temperatures

ABSTRACTGlucose oxidase (GOx) catalyses oxidation of glucose accompanied with the generation of hydrogen peroxide. With the addition of Fe2+, hydroxyl radical produced by Fenton reaction between hydrogen peroxide and Fe2+ may quench the fluorescence of gold nanoclusters. In this work, a fluorescent enzyme-linked immunosorbent assay with gold nanoclusters was designed with a straightforward signal output, in which the fluorescence of gold nanoclusters was quenched by GOx-triggered Fenton reaction. Olaquindox was selected as a target analyte. Gold nanoclusters capped with bovine serum albumin and GOx-linked olaquindox conjugates were successfully prepared. Olaquindox in samples directly competed with the GOx-linked olaquindox conjugates for binding immobilized antibody. Consequently, the fluorescence signal increased with the amount of olaquindox. Under optimal conditions, the fluorescent enzyme-linked immunosorbent assay exhibited a favorable performance to detect olaquindox in swine feeds, demonstrating a good linear range from 1.0 µg kg−1 to 150 µg kg−1 with a reliable correlation coefficient (R2 = 0.9918); the limit of detection was 0.68 µg kg−1. Average recoveries in spiked samples were 85.3% to 113.5%. The proposed strategy is a promising approach for the detection of olaquindox and other harmful small molecules.

Oligonucleotide-templated rapid formation of fluorescent gold nanoclusters and its application for Hg2+ ions sensing

As the most abundant protein in plasma, human serum albumin plays a vital role in physiological processes, such as maintaining blood osmotic pressure and carrying small-molecule ligands. Since the content of albumin in the human serum can reflect the status of liver and renal function, albumin quantitation is significant in clinical diagnosis. In this work, fluorescence turn-on detection of human serum albumin (HSA) had been performed based on the assembly of gold nanoclusters and bromocresol green. Gold nanoclusters (AuNCs) capped by reduced glutathione (GSH) were assembled with bromocresol green (BCG), and the assembly was used as a fluorescent probe for HSA. After BCG assembling, the fluorescence of gold nanoclusters was nearly quenched. In acidic solution, HSA can selectively bind to BCG on the assembly and recover the fluorescence of the solution. Based on this turn-on fluorescence, ratiometric HSA quantification was realized. Under optimal conditions, HSA detection by the probe possessed agood linear relationship in the range of 0.40-22.50mg·mL-1, and the detection limit was 0.27 ± 0.04mg·mL-1 (3σ, n = 3). Common coexisting components in serum and blood proteins did not interfere with the detection of HSA. This method has the advantages of easy manipulation and high sensitivity, and the fluorescent response is insensitive to reaction time.

Fluorescent protein-encapsulated gold nanoclusters (AuNCs) offer a non-toxic means of sensing and imaging biological phenomena on the nanoscale. However, the biofunctionality of proteins encapsulating AuNCs has not been fully elucidated to date. Here we studied the biofunctionality of the second major drug binding site (Sudlow II) of Human Serum Albumin (HSA) encapsulated AuNCs after AuNC synthesis. L-Dopa, a fluorescent drug molecule associated with the clinical treatment of Parkinson’s disease, which commonly binds to the Sudlow II site, was used to study the availability of the site before and after AuNC synthesis through changes to its fluorescence characteristics. L-Dopa was observed using its intrinsic fluorescence to readily bind to HSA-AuNCs complexes. Interestingly, the fluorescence emission intensity of AuNCs linearly increased with L-Dopa concentration while exciting the AuNC directly at 470 nm, Using a 400 nM HSA-AuNC solution, L-Dopa was rapidly detected at a limit of 300 pM, indicating that HSA-AuNCs fluorescence is extremely sensitive to molecular binding at the Sudlow II binding site. Future research may be able to utilize this sensitivity to improve the fluorescence characteristics of AuNCs within HSA-AuNCs for imaging and sensing including drug binding studies.

We present a general method for converting colloidal nanomaterials into microbubbles as ultrasound contrast agents. Protein-caged nanomaterials, made either by self-assembled nanoparticles' protein corona or by fluorescent gold nanoclusters, can be rapidly transformed into microbubbles via a sonochemical route, which promote disulfide cross-linking of cysteine residues between protein-caged nanomaterials and free albumin during acoustic cavitation. The proposed methods yielded microbubbles with multiple functions by adjusting the original nanoparticle/protein mixture. We also showed a new dual-modal imaging agent of fluorescent gold microbubbles in vitro and in vivo, which can hold many potential applications in medical diagnostics and therapy.

The fluorescence of metal nanoclusters provides an amusing optic feature to be applied in various fields. However, rational design of dual functional fluorescent metal nanoclusters directed by active enzyme with targeted application remains little explored. In this work, we report a new strategy to construct enzyme functionalized fluorescent gold nanoclusters via a biomineralization process for the detection of hydrogen peroxide. Horseradish peroxidase (HRP) was used as a model functional template to direct the synthesis of fluorescent gold nanoclusters (Au NCs) at physiological conditions to form HRP-Au NCs bioconjugates. We found that the fluorescence of HRP-Au NCs can be quenched quantitatively by adding H(2)O(2), indicating that HRP enzyme remains active and enables catalytic reaction of HRP-Au NCs and H(2)O(2). Upon the addition of H(2)O(2) under optimal conditions, the fluorescence intensity quenched linearly over the range of 100 nM to 100 μM with high sensitivity (LOD = 30 nM, S/N = 3). This study would be potentially extended to other functional proteins to generate dual functional nanoclusters and applied to real time monitoring of biologically important targets in living cells.

Sensitive and selective nitrite assay based on fluorescent gold nanoclusters and Fe2+/Fe3+ redox reaction

Inositol directed facile “green” synthesis of fluorescent gold nanoclusters as selective and sensitive detecting probes of ferric ions

Confinement of gold nanoclusters on hydroxy double salt for versatile fluorescent sensing of bio-enzymes

BackgroundImmune checkpoint inhibitor therapy addressing the PD-1/PD-L1 axis is a promising approach in cancer treatment. A clinically suitable radiotracer would allow molecular imaging of the temporospatial changes in tumor PD-L1 expression. This could enable the clinicians to select eligible patients for checkpoint therapy and monitor therapeutic efficacy.ResultsFour biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route, with a linear sequence of up to eleven steps. Two candidates were covalently labeled with 18F via either an azido glycosyl or PEG2 moiety, while the other two were modified with a RESCA chelator for Al[18F]F2+-labeling. The lipophilicity was assessed through determination of log D7.4 values. In vitro binding affinities (inhibition constant, Ki) toward PD-L1 were determined in competition with one of our previously published biphenyl-based small-molecule (KD = ~ 21 nM). Compared to this compound, both covalently labeled 18F-ligands exhibited decreased water solubility (log D7.4 ~ − 2.5 and − 2.7), along with a markedly reduced (Ki = 200‒500 nM) affinity. This was in line with in vivo small animal PET, where both compounds were characterized by a negligible tumor uptake, lack of contrast between target-positive/negative tumors and exclusively unfavorable hepatobiliary excretion. Similar results were observed for the chelator-modified ligands with slightly increased hydrophilicity (log D7.4 ~ − 2.8 and − 2.9), showing a binding affinity of 150 nM for one compound, while binding was lost completely for the other. Again, a poor in vivo performance was observed, characterized by hepatobiliary clearance and lack of specific tumor uptake in the PD-L1 positive tumor.ConclusionFour biphenyl-based, 18F-labeled PD-L1 radioligands were developed using prosthetic groups (azido glycosyl or PEG2) for covalent fluorination and Al[18F]F2⁺-complexation with the RESCA chelator. Despite limited in vitro and in vivo performance, these fluorination approaches offer a foundation for developing improved PD-L1 radioligands after increasing the hydrophilicity and the spacing between the radiolabel and binding motif.