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Gold Nanoparticle-Based Photoluminescent Nanoswitch Controlled by Host-Guest Recognition and Enzymatic Hydrolysis for Arginase Activity Assay
Abstract
The development of simple yet powerful methods for monitoring enzyme activity is of great significance. Herein, a facile, convenient, cost-effective, and continuous fluorescent method for the detection of arginase and its inhibitor has been reported based on a host-guest interaction- and enzymatic hydrolysis-controlled luminescent nanoswitch. The fluorescence intensity of 6-aza-2-thiothymine-stabilized gold nanoparticle (ATT-AuNP) is enhanced by L-arginine, owing to the formation of supramolecular host-guest assembly between the guanidine group of L-arginine and ATT molecules capped on AuNP surface. However, hydrolysis of L-arginine, catalyzed by arginase, leads to a decrease of fluorescence intensity of L-arginine/ATT-AuNPs hybrids. Upon incorporation of the arginase inhibitor L-norvaline, the fluorescence of the ATT-AuNP-based detecting system is restored. The linear range of arginase activity determination is from 0.0625 to 1.15 U/mL and the limit of detection is 0.056 U/mL. The half-maximal inhibition value IC50 of L-norvaline is determined to be 5.6 mM. The practicability of this luminescent nanoswitch is validated by assaying arginase activity in rat liver and monitoring the response of rat liver arginase to pharmacological agent. Compared to the existing fluorescent method of arginase activity assay, the approach demonstrated here does not involve any complicated technical manipulation, thereby greatly simplifying the detection steps. We propose that this AuNP-based luminescent nanoswitch would find wide applications in the field of life sciences and medicine.
... The prepared ATT-AuNCs were dialyzed against 500 mL of deionized water for 24 h using a dialysis membrane with a molecular weight cutoff of 3500. 21 2.2. Antibacterial Activity of ATT-AuNCs. ...
Multidrug-resistant bacterial infections, especially those caused by multidrug-resistant Escherichia coli (E. coli) bacteria, are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Therefore, it is urgently needed to develop a kind of novel, long-term antibacterial agent effectively overcome resistant bacteria. Herein, we present a novel designed antibacterial agent—6-Aza-2-thiothymine-capped gold nanoclusters (ATT-AuNCs), which show excellent antibacterial activity against multidrug-resistant E. coli bacteria. The prepared AuNCs could permeabilize into the bacterial cell membrane via binding with a bivalent cation (e.g., Ca²⁺), followed by the generation of reactive oxygen species (e.g., •OH and •O2–), ultimately resulting in protein leakage from compromised cell membranes, inducing DNA damage and upregulating pro-oxidative genes intracellular. The AuNCs also speed up the wound healing process without noticeable hemolytic activity or cytotoxicity to erythrocytes and mammalian tissue. Altogether, the results indicate the great promise of ATT-AuNCs for treating multidrug-resistant E. coli bacterial infection.
... Recently, it also has been reported to improve vascular function in diabetics by decreasing arginase activity in cavernous tissue and raising NO levels [168]. L-norvaline has minimal side effects, but because of its high water solubility and high half-maximal inhibitory concentration (IC50 of 5.6 mM on rat arginase), its application in blocking the arginase pathway is still unsatisfactory [169]. High water solubility can lead to burst or uncontrolled release, while high IC50 requires high drug loading content of L-norvaline to satisfy high dosage. ...
Arginase is a ubiquitous enzyme in the urea cycle (UC) that hydrolyzes L-arginine to urea and L-ornithine. Two mammalian arginase isoforms, arginase1 (ARG1) and arginase2 (ARG2), play a vital role in the regulation of β-cell functions, insulin resistance (IR), and vascular complications via modulating L-arginine metabolism, nitric oxide (NO) production, and inflammatory responses as well as oxidative stress. Basic and clinical studies reveal that abnormal alterations of arginase expression and activity are strongly associated with the onset and development of diabetes mellitus (DM) and its complications. As a result, targeting arginase may be a novel and promising approach for DM treatment. An increasing number of arginase inhibitors, including chemical and natural inhibitors, have been developed and shown to protect against the development of DM and its complications. In this review, we discuss the fundamental features of arginase. Next, the regulatory roles and underlying mechanisms of arginase in the pathogenesis and progression of DM and its complications are explored. Furthermore, we review the development and discuss the challenges of arginase inhibitors in treating DM and its related pathologies.
... For example, the drug delivery system formed by PPase-nanodiamond is expected to treat calcium pyrophosphate crystal deposition diseases and related pathologic diseases [32][33][34]. Fluorescent biosensors made by monitoring the activity of PPases can be utilized to screen for potential inhibitors of PPases, which are associated with many clinical diseases (lung cancer and colorectal cancer, etc.) [35][36][37]; In laboratory studies, using pyrophosphatase-coupled assays to monitor enzymatic activity is a potential application of PPases, such as assays of the activity of arginase, cyclic GMP-AMP synthase, and santalene synthases [38][39][40]; PPases can also be applied in industry to increase the yield of UDP-sugar synthesis and the amplification efficiency of PCR. In this work, Ton1914 exhibited excellent stability, with half-lives of 7 h and 2.5 h at 80 °C and 90 °C, respectively. ...
Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32–41%, while that of short-chain DNA was only improved by 9.5–15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.
... synthesized highly luminescent AuNCs (Arg/ATT/AuNCs) with QY of approximately 65% via a host− guest recognition assembly after the addition of L-arginine. Using the synthesized AuNCs as fluorescent probes, the same group successfully detected arginase activity with a LOD of 0.056 U/mL, thus implying the great possibility of using this AuNC for highly sensitive determination (Deng et al., 2018). Encouraged by these results (Zheng et al., 2021), directly conjugated Arg/ATT/AuNCs with antibodies as a signal probe to establish fluorescent ICTS nanosensor for the sensitive determination of four pyrrolizidine alkaloids (Fig. 3C). ...
Background
Immunochromatographic test strip (ICTS) has become one of the most widely used rapid diagnostics platforms for the point-of-care testing of various contaminant residues in food. However, traditional gold nanoparticle (AuNP)-based ICTSs suffer limited sensitivity ranged from ng/mL to μg/mL, thus severely hindering them from meeting the increasing demand for trace target detection.
Scope and approach
The use of novel nanomaterials with high signal strength to replace AuNPs as signal probes is considered as the most effective strategy to enhance the sensitivity of ICTS nanosensor. Among them, highly luminescent nanomaterials have attracted increasing interest in improving the detection performance of ICTS due to its advantages of visualization, quantitation, multiplexing, anti-jamming capability, and high sensitivity. Currently available nanomaterials include dye-based fluorescent nanomaterials, semiconductor quantum dots and its nanobeads, up-conversion nanoparticles, time-resolved fluorescence nanomaterials, aggregation-induced emission nanomaterials, noble metal nanoclusters, and magnetic fluorescent nanomaterials, and they have been successfully applied for detecting various hazardous substances, including pesticide and veterinary drug residues, food additives, mycotoxins, heavy metal ions, allergens, and microorganisms. In this review, thus we comprehensively summarize the application potentials and critical roles of such nanomaterials as signal reporters in current ICTS systems to ensure food safety.
Key findings and conclusions
A detailed classification of signal probes with emphasis on the inherent advantage, synthesis, signal amplification strategy, and further improvement of each luminescent nanomaterial was discussed and the state-of-the-art of commercial fluorescent ICTS kits and readers for food safety was summarized. Further improvement should focus on the design and development of high-quality luminescent nanomaterials, ultra-sensitive detection, multiplexing and multimode sensing, accurate quantification, and intelligent signal reading.
... Degradation of L-arginine leads to the reduced expression of the CD3 ζ chain, resulting in inactivating T cells responsiveness and greatly diminishing immunotherapeutic efficacy [7][8][9] . Although Lnorvaline, an ARG1 pathway inhibitor [10][11][12][13] , is very cheap with negligible side effects, the application of L-norvaline in the block- age of ARG1 pathway is still unsatisfactory because of its highwater solubility and high half-maximal-inhibitory concentration (IC50, 5.6 m M ) [14] . High-water solubility leads to burst or uncontrolled release while high IC50 requires high drug loading content of L-norvaline for satisfying high dosage. ...
Arginase 1 (ARG1) inactivates T cells by degrading L-arginine, severely reducing the immunotherapeutic efficacy. Effectively blocking the ARG1 pathway remains a challenge. L-norvaline is a very cheap and negligible side effects inhibitor of ARG1. However, its blockage efficacy for ARG1 is impeded by its high half-maximal-inhibitory concentration (IC50) requiring high drug loading content of L-norvaline in carriers. Moreover its high water solubility results in bursting and uncontrolled release. Herein we reported an injectable hydrogel strategy via an L-norvaline-based immunomodulating gelator that could effectively block ARG1 pathway. The designed gelator was a diblock copolymer containing L-norvaline-based polypeptide block, which could construct a thermally responsive injectable hydrogel by its self-gelation in tumor microenvironments. The hydrogel not only ensures high drug loading of L-norvaline, but also ensures controlled release of L-norvaline through responsive peptide bond cleavage, thereby solving the problems encountered by L-norvaline. The injectable hydrogel in combination with doxorubicin hydrochloride demonstrated a potent immunotherapy for removal of primary tumors, suppression of abscopal tumors and inhibition of pulmonary metastasis by combining the blockage of ARG1 pathway and the immunogenic cell death. Our immunomodulating gelator strategy provides a robust injectable hydrogel platform to efficiently reverse ARG1 immunosuppressive environments for amplified immunotherapy.
Statement of Significance
We designed an injectable hydrogel via an L-norvaline-based immunomodulating gelator. The designed gelator, a diblock copolymer containing an L-norvaline-based polypeptide block, enabled a thermally responsive injectable hydrogel by its self-gelation in tumor microenvironments. The injectable hydrogel not only guarantees high drug loading of L-norvaline, but also ensures controlled release of L-norvaline through responsive peptide bonds cleavage, thereby solving the problems encountered by L-norvaline. By further introducing doxorubicin hydrochloride in the hydrogel for inducing immunogenic cell death, the hydrogel showed remarkable immunotherapeutic efficacy towards ablation of primary tumors, suppression of abscopal tumors and inhibition of pulmonary metastasis. Our immunomodulating gelator strategy provides a new concept to efficiently reverse Arginase 1 immunosuppressive environments for amplified immunotherapy.
... The use of photoluminescent gold nanoparticles coated with L-arginine exhibited efficacy to monitor arginase activity, as there is a decrease of the photoluminescence signal in the presence of this enzyme due to the hydrolysis of L-arginine from the coating [217]. As a proof-of-concept, this imaging agent was tested in rats with and without triptolide-induced liver injury and showed great promise for arginase-targeted biomedical applications [218]. However, being vectorized by L-arginine, this probe is also subject to NOS activity and may lack specificity to arginase. ...
Arginase is a widely known enzyme of the urea cycle that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The action of arginase goes beyond the boundaries of hepatic ureogenic function, being widespread through most tissues. Two arginase isoforms coexist, the type I (Arg1) predominantly expressed in the liver and the type II (Arg2) expressed throughout extrahepatic tissues. By producing L-ornithine while competing with nitric oxide synthase (NOS) for the same substrate (L-arginine), arginase can influence the endogenous levels of polyamines, proline, and NO•. Several pathophysiological processes may deregulate arginase/NOS balance, disturbing the homeostasis and functionality of the organism. Upregulated arginase expression is associated with several pathological processes that can range from cardiovascular, immune-mediated, and tumorigenic conditions to neurodegenerative disorders. Thus, arginase is a potential biomarker of disease progression and severity and has recently been the subject of research studies regarding the therapeutic efficacy of arginase inhibitors. This review gives a comprehensive overview of the pathophysiological role of arginase and the current state of development of arginase inhibitors, discussing the potential of arginase as a molecular imaging biomarker and stimulating the development of novel specific and high-affinity arginase imaging probes.
... In our previous study, we found that the restriction of ligand motion can enhance the fluorescence quantum yield of 6-aza-2-thio-thymine protected gold nanoclusters (ATT-AuNCs) [8,26]. Inspired from this fact, we here test the influence of protein absorption on the fluorescence characteristics of ATT-AuNCs. ...
This study puts forward an efficient method for protein detection in virtue of the tremendous fluorescence enhancement property of 6-aza-2-thio-thymine protected gold nanoclusters (ATT-AuNCs). In-depth studies of the protein-induced photoluminescence enhancement mechanism illustrate the mechanism of the interaction between ATT-AuNCs and protein. This new-established probe enables feasible and sensitive quantification of the concentrations of total protein in real samples, such as human serum, human plasma, milk, and cell extracts. The results of this proposed method are in good agreement with those determined by the classical bicinchoninic acid method (BCA method).
... ATT/Arg/AuNCs have been synthesized in our previous work [40], and the results of characterization are presented in Figure S9. during the preparation of ATT/Arg/AuNCs [40,41]. However, Arg would be disengaged from the surface of ATT/Arg/AuNCs with the addition of Mn(II), which thanks to the higher binding affinity of Arg for Mn(II) [42], resulting in the fluorescence quenching of the system. ...
Research Highlights The flower-like SiO2@MnO2 nanocomposites were synthesized with a rapid and simple method. Based on the theory ATT/Arg/AuNCs can be effectively quenched by Mn 2+ , a platform was successfully developed for GSH detection. GSH and antibiotics can be detected rapidly through two developed detection platforms with the same nanomaterials. ABSTRACT In 2-(N-morpholino)ethanesulfonic acid buffer (0.1 M, pH 6.0), manganese dioxide-modified silicon dioxide (SiO2@MnO2) nanocomposites were facilely synthesized under ultrasonic conditions. Given their good biocompatibility and evident brown color, SiO2@MnO2 nanocomposites were used as labels in immunochromatography sensor for high-throughput screening of antibiotic residues in milk within 10 min. Meanwhile, J o u r n a l P r e-p r o o f 3 excessive amounts of antibiotic residues might cause certain diseases and inhibit the activities of glutathione reductase in human body through the food chain, altering glutathione (GSH) levels in the human serum. GSH can reduce MnO2 to Mn(II). This phenomenon can effectively quench the fluorescence of Au nanoclusters. Thus, a fluorescent platform based on SiO2@MnO2 nanocomposites and Au nanoclusters has been designed for sensitive detection of GSH in the human serum with a limit of detection of 1.23 nM. After the parameters were optimized, the developed detection platforms based on SiO2@MnO2 nanocomposites have been used to detect antibiotics and GSH in real samples (milk and human serum) with satisfactory results. Results demonstrated that the proposed SiO2@MnO2 nanocomposites hold great potential for applications in food safety and clinical diagnosis simultaneously.
... Since the guanidine group of Arg binds with ATT through hydrogen bonding and significantly rigidifies the ligand shell, the resultant Arg/ATT/AuNCs exhibited good water solubility and strong green luminescence (QY ¼ 65%) under UV irradiation. Arg/ATT/AuNCs have been used to detect arginase activity based on fluorescence alterations caused by arginase-catalyzed hydrolysis [20]. ...
h i g h l i g h t s g r a p h i c a l a b s t r a c t Highly luminescent green-emitting AuNCs were applied to LFIA for the first time. AuNCs-MLFIA sensor was developed for simultaneous and quantitative determination of Clen and RAC in swine urine. The developed AuNCs-MLFIA sensor revealed superior in sensitivity than other LFIA sensors. a b s t r a c t A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and L-arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/ AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 mg L À1. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 mg L À1 , respectively, with detection limits of 0.003 and 0.023 mg L À1. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatographyetandem mass spec-trometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection.
... Few-atom gold nanoclusters (AuNCs) have attracted intensive interest because they are fascinating materials for both fundamental studies and practical applications [7][8][9][10][11][12][13][14]. Emerging as a new type of fluorescent material, AuNCs possess distinct characteristics that include good water dispersibility, low toxicity, good biocompatibility, tunable emission, large Stokes shift, excellent photostability, and a long decay lifetime [15][16][17][18]. The photoluminescence (PL) properties of AuNCs are closely associated with the size of the gold core and the ligands capped on the AuNC surface, and are sensitive to the surrounding conditions. ...
Doxorubicin (DOX), an anthraquinone-based anticancer drug, is effective for the treatment of a variety of human tumors. However, due to its narrow therapeutic window, serum levels need to be closely monitored. Herein, we report the development of a novel fluorescence probe based on gold nanoclusters co-modified with dithiothreitol (DTT) and carboxylated chitosan (CC) (CC/DTT-AuNCs) for the determination of DOX. The emission from the CC/DTT-AuNCs is significantly suppressed by DOX. The fluorescence-quenching mechanism involving DOX was systematically explored, and photoinduced electron transfer was identified to play a dominant role. DOX readily binds to the CC/DTT-AuNC surface through electrostatic attraction, after which it accepts the electron from the photoexcited CC/DTT-AuNC and quenches its fluorescence. We found that other anthraquinone structures (e.g., alizarin and emodin) devoid of –NH2 groups exhibited almost no CC/DTT-AuNCs fluorescence-quenching effect due to weak electrostatic interactions between these anthraquinones and the NCs. A linear relationship between photoluminescence suppression and the concentration of DOX was observed in the 0.05–2 μM range under the optimal experimental conditions, with a detection limit as low as 5 nM. This new sensing approach was also used to successfully measure DOX in human serum samples with satisfactory results obtained.
... Since the guanidine group of Arg binds with ATT through hydrogen bonding and significantly rigidifies the ligand shell, the resultant Arg/ATT/AuNCs exhibited good water solubility and strong green luminescence (QY ¼ 65%) under UV irradiation. Arg/ATT/AuNCs have been used to detect arginase activity based on fluorescence alterations caused by arginase-catalyzed hydrolysis [20]. ...
A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and L-arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 μg L⁻¹. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 μg L⁻¹, respectively, with detection limits of 0.003 and 0.023 μg L⁻¹. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatography–tandem mass spectrometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection.
Progress in syntheses and understanding of the intriguing properties of chiral noble metal nanoclusters sparks interest to extend investigations of their chiroptical response to the nonlinear optics regime. We present a quantitative determination of two-photon circular dichroism of chiral gold nanoclusters with ATT and L- or D-Arg ligands (ATT = 6-aza-2-thiotymine and Arg = arginine). Introduction of arginine ligands enables the formation of two enantiomers of the nanoclusters, with strong chiroptical effects in both linear and nonlinear regime. We present two-photon absorption and luminescent properties measured in a wide range of wavelengths, with the two-photon absorption cross section reaching 1743 GM and two-photon brightness ∼1102 GM at 825 nm. We report strong, 245-fold enhancement of the two-photon circular dichroism of nanoclusters with respect to the one-photon absorption counterpart - the dissymmetry factor. The presence of multiple advantages of nanoclusters: high fluorescence quantum yield, strong nonlinear optical properties and well-controlled chirality is a powerful combination for applications of such clusters in multiphoton microscopy.
The rational design of ice recrystallization inhibition (IRI) materials is challenging due to the poor understanding of the IRI mechanism at the molecular level. Here we report several new findings about IRI. (1) A dense hydroxyl monolayer of small molecules, e.g. 6-aza-2-thiothymine (ATT), adsorbed on a nanogold surface was demonstrated, for the first time, to have IRI activity. Five structural analogues adsorbed on groups nanogold with outward hydroxyl or methyl were created to evidence the origin of IRI activity. (2) Their IRI mechanism is closely related to the density of hydroxyls on a nanogold surface. However, the hydrophobic interaction in our model is not essential for macroscopic IRI activity. (3) A molecular dynamics simulation elucidates the hydroxyl density dependent IRI trajectories underlying the experimental observations, and the radial distribution function reveals that the methyl even slightly hinders the formation of hydrogen bonding due to a hydrophobic interaction. This work sheds more light on the IRI mechanism that should help in the customization of novel IRI materials.
A novel lateral flow assay (LFA) was developed based on the aggregation induced emission (AIE) of gold nanoclusters (AuNCs) for the detection of melamine. We compared the interaction between melamine and AuNCs prepared with 6-Aza-2-thiothymine (ATT) and its four structural analogs as ligands and found that the ATT-AuNCs showed the best AIE induced by melamine. Then the ATT-AuNCs were directly immobilized on the test line of LFA strips as reporters. The sensing platform exhibited a linear response to melamine from 1 to 100 μM (R²=0.997) with a detection limit of 217 nM. Moreover, this LFA strip could discriminate 1.0 μM melamine clearly by the naked eyes within 3 min. The recoveries of 91.6-101.5% were attained in milk and baby formula samples. This strip inherited the superior characteristics of low background interference of aggregation-induced fluorescence recognition and LFA technology, thus effectively avoiding the use of antibodies, aptamers, and other biological elements.
The cytidine 5'-monophosphate (CMP)-protected gold nanoclusters ([email protected]) was developed to be a fluorescent nanosubstrate for direct ratiometric determination of alkaline phosphatase (ALP). The binding mode investigations revealed that the ligand CMP binds to the gold core via two oxygen atoms of ribose and cytosine and leaves the phosphate group freely outside, promoting [email protected] to be the direct nanosubstrate of ALP. [email protected] emits at 570 nm while the hydrolysis product of [email protected] emits at 485 nm; the large emission gap between them provided a ratiometric approach for ALP determination. Moreover, a natural and biofunctional oligomer, chitosan oligosaccharide (COS), was introduced to the system, which significantly amplified the fluorescence signals of [email protected] and improved the limit of detection (LOD) down to 0.00026 U·L⁻¹. Furthermore, the intrinsic mechanism of [email protected] hydrolysis by ALP and the COS amplification on [email protected] were studied in-depth, which indicated the emission enhancement was attributed to the aggregation-induced emission enhancement (AIEE) property of the produced [email protected] Finally, the developed approach was successfully applied to determine ALP in human serum and the evaluation of ALP inhibitors. Therefore, the present study develops a novel nanosubstrate for ALP determination in a range of 0.0050–0.25 U·L⁻¹ and provides a straightforward way to amplify the fluorescence signal by employing available polymer. The work will stimulate and encourage the structural promotion of metal nanoclusters, and extend their biological applications broadly.
Herein, a novel fluorescence quenching immunochromatographic test strip (FQICTS) for simultaneous detection of chloramphenicol (CAP) and amantadine (AMD) was developed on the basis of inner filter effect (IFE), with the combination of gold nanoparticles (AuNPs) and highly luminescent green-emitting gold nanoclusters (AuNCs) as the IFE quencher/donor pair. The AuNPs could quench the excitation light and emission light of AuNCs and achieve a high IFE efficiency due to dual spectral overlapping. Under optimal conditions, the “turn-on” mode of the AuNCs-based dual-readout FQICTS showed good linearity for CAP detection in chicken samples from 0.05 ng/g to 10 ng/g, with a limit of detection (LOD) of 0.043 ng/g. The linear range of AMD is 0.5-50 ng/g, with LOD of 0.45 ng/g. The visual LODs of CAP and AMD in “turn-on” mode were 200 and 10 times lower than that in “turn-off” mode, respectively. The “turn-on” mode of FQICTS showed high recovery for detecting CAP (82.5%–94.5%) and AMD (81.9%–110.7%) spiked into chicken samples. The performance and practicability of the established method were verified with commercial enzyme-immunoassay kits, and good correlations were observed. Overall, the newly developed AuNCs-based dual-readout FQICTS is a promising on-site screening tool for rapid, high-sensitivity detection of multiple food contaminants in practical applications.
Metal nanoclusters (MNCs) have ultrasmall size (≤3 nm), precise molecular structure, strong luminescence, size-sensitive physicochemical properties, engineerable metallic composition, and rich yet tailorable surface chemistry, enabling them to be promising for the construction of luminescent sensors for diversified sensing applications in fields of environmental protection, food safety, agriculture, disease diagnosis, biomarker identification, etc. However, a systematic summary is urgently needed to correlate the physicochemical properties of MNCs with their influences on sensing applications, to summarize the synthetic strategies of highly luminescent MNCs towards sensor construction, and to discuss the general configurations and sensing mechanisms of MNCs-based luminescent probes. In this review, we first discuss relevant physicochemical properties of MNCs that may have major influences on the sensing mechanisms/performance of MNCs-based luminescent sensors, and then summarize the main synthetic strategies of highly luminescent MNCs. Then, we briefly illustrate the general configurations of MNCs-based sensors, and elaborately clarify their sensing mechanisms with the exemplification of representative sensing applications. We conclude the review with our perspectives on the opportunities/challenges of MNCs-based luminescent sensors. It is believed that this review will shed light on the design of MNCs-based luminescent sensors for future sensing applications.
The authors here developed a stearic acid-doped liquid crystal (LC)-based sensor for the label-free and real-time detection of arginase (ARG1). This sensor was based on the orientational transition of LCs due to the adsorption of stearic acid at the aqueous/LC interface. The shift in the optical image of LCs from bright to dark was obtained after transferring an aqueous solution of arginase, ʟ-arginine, and urease onto the aqueous/stearic acid-doped LC interface. Arginase catalyzes ʟ-arginine hydrolysis to produce urea, which is further catalyzed by urease to produce ammonium, which in turn increases the pH of the aqueous bulk. This increase in pH causes the deprotonation and adsorption of stearic acid and the formation of a stearate self-assembled monolayer at the interface, which induces a change in the orientation of the LC film from planar to homeotropic anchoring. ʟ-norvaline can inhibit the enzymatic activity of arginase; therefore, the LC optical image stayed bright after adding a mixed solution of arginase and ʟ-norvaline to the optical cell incubated with ʟ-arginine and urea, indicating no orientation change of LCs. ARG1 could be quantitatively detected by the proposed sensor in the range from 1 ng/mL to 100 ng/mL, with the detection limit as low as 0.6 ng/mL. The effect of the human serum matrix on the performance of proposed sensors was also investigated to confirm the applicability in ARG1 detection. The proposed sensor shows great potential for the development of pH-sensitive instruments for the monitoring of enzymatic reactions.
Metal nanoclusters (MNCs) are ultrasmall metal‐organic aggregates, composed of a metal core less than 2 nm and a protecting shell of metal‐organic ligand motifs. The controlled aggregation of metal atoms (in the cluster core) and metal‐organic ligand motifs (around the cluster core) renders MNCs with numerous molecule‐like properties, among which strong and bright luminescence has attracted extensive basic and applied interests. It has now known that aggregation‐induced emission is a feasible mechanism for controlling luminescence of MNCs, which makes it particularly useful in biosensing and bioimaging applications. Although the luminescence fundamentals and design principles largely determine the practicality and effectiveness of MNCs in biosensing and bioimaging applications, a systematic summary of this topic is lacking in the current literature. In this review, we aim to provide a concise discussion of the latest developments in biosensing and bioimaging applications of luminescent MNCs, highlighting their luminescence mechanisms, biosensing principles, and bioimaging strategies. Specifically, we first introduce the recent advances in the synthetic chemistry of MNCs, and then briefly discuss the luminescence fundamentals of MNCs. Then the design strategy and practicality of luminescent MNCs in biosensing and bioimaging applications are exemplified. We conclude the review with our perspectives on the further development of MNC‐based optical probes in biosensing and bioimaging applications. Our review is expected to provide guidance for the future practice of designing and synthesizing luminescent MNCs for biomedical and other applications.
In recent years, gold nanoclusters (AuNCs) have received considerable attention as optical transducers in chemo/biosensors. Herein, a facile and efficient assay for NO2- has been successfully developed based on the fluorescence quenching of AuNCs co-modified by bovine serum albumin and 3-mercaptopropionic acid (BSA/MPA-AuNCs). In the presence of NO2- under acidic conditions, Fe2+ can be readily oxidized and transformed to Fe3+, which can significantly suppress the fluorescence of BSA/MPA-AuNCs via non-radiative electron-transfer mechanism. The linear range and detection limit for this system were found to be 5–30 μM (r = 0.9975) and 0.7 μM, respectively. Other common anions and cations showed only very minor interference with the NO2- detection. Furthermore, the effectiveness of the proposed sensing strategy was validated by the demonstration of good performance in the determination of the amount of NO2- in ham samples, rendering it a powerful tool for the assessment of food security and water quality.
Donor-linker-acceptor (D-L-A)-based photoinduced electron transfer (PET) has been frequently used for the construction of versatile fluorescent chemo/biosensors. However, sophisticated and tedious processes are generally required for the synthesis of these probes, which leads to poor design flexibility. In this work, by exploiting a Schiff base as a linker unit, a covalently bound D-L-A system was established and subsequently utilized for the development of a PET sensor. Cysteamine (Cys) and N-acetyl-L-cysteine (NAC) co-stabilized AuNC (Cys/NAC-AuNC) was synthesized and adopted as an electron acceptor, and pyridoxal phosphate (PLP) was selected as an electron donor. PLP can form a Schiff base (an aldimine) with the primary amino group of Cys/NAC-AuNC through its aldehyde group and thereby suppresses the fluorescence of Cys/NAC-AuNC. Rehm-Weller-formula results and a HOMO-LUMO orbital study revealed that a reductive PET mechanism is responsible for the observed fluorescence quenching. Since the pyridoxal (PL) produced by the acid phosphatase (ACP)-catalyzed cleavage of PLP has a weak interaction with Cys/NAC-AuNC, a novel turn-on fluorescent method for selective detection of ACP was successfully realized. To the best of our knowledge, this is the first example of the development of a covalently bound D-L-A system for fluorescent PET sensing of enzyme activity based on AuNC nanoprobes, using a Schiff base.
In 2-(N-morpholino)ethanesulfonic acid buffer (0.1 M, pH 6.0), manganese dioxide-modified silicon dioxide (SiO2@MnO2) nanocomposites were facilely synthesized under ultrasonic conditions. Given their good biocompatibility and evident brown color, SiO2@MnO2 nanocomposites were used as labels in immunochromatography sensor for high-throughput screening of antibiotic residues in milk within 10 min. Meanwhile, excessive amounts of antibiotic residues might cause certain diseases and inhibit the activities of glutathione reductase in human body through the food chain, altering glutathione (GSH) levels in the human serum. GSH can reduce MnO2 to Mn(II). This phenomenon can effectively quench the fluorescence of Au nanoclusters. Thus, a fluorescent platform based on SiO2@MnO2 nanocomposites and Au nanoclusters has been designed for sensitive detection of GSH in the human serum with a limit of detection of 1.23 nM. After the parameters were optimized, the developed detection platforms based on SiO2@MnO2 nanocomposites have been used to detect antibiotics and GSH in real samples (milk and human serum) with satisfactory results. Results demonstrated that the proposed SiO2@MnO2 nanocomposites hold great potential for applications in food safety and clinical diagnosis simultaneously.
Gold(I)-alkanethiolates could assemble themselves into highly ordered nanostructures, however, they could not be dissolved or dispersed in various common solvents. Here, a novel and facile method of synthesizing water-dispersed luminescent Au(I)-alkanethiolate nanoparticles via aggregation-induced emission is reported for the first time. With the aid of two surfactants (didodecyldimethylammonium bromide (DDAB) and tween 80), the luminescent gold nanoparticles possess excellent water dispersion stability. Besides, they exhibit a large Stokes shift (24278 cm-1), red emission, high quantum yield (50.7%) and extraordinary stability towards pH, salts, redox agents (NaBH4, Na2S, cysteine and H2O2) and light irradiation. The luminescent gold nanoparticles are also used for cell imaging successfully.
In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of a bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely; 1) the ability to isolate mycobacterial TOP1A from crude sample, and 2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU)/mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.
Metal nanoclusters, composed of several to a few hundred metal atoms, have received worldwide attention due to their extraordinary physical and chemical characteristics. Recently, great efforts have been devoted to the exploration of the potential diagnostic and therapeutic applications of metal nanoclusters. Here we focus on the recent advances and new horizons in this area, and introduce the rising progress on the use of metal nanoclusters for biological analysis, biological imaging, therapeutic applications, DNA assembly and logic gate construction, enzyme mimic catalysis, as well as thermometers and pH meters. Furthermore, the future challenges in the construction of biofunctional metal nanoclusters for diagnostic and therapeutic applications are also discussed. We expect that the rapidly growing interest in metal nanocluster-based theranostic applications will certainly not only fuel the excitement and stimulate research in this highly active field, but also inspire broader concerns across various disciplines.
Two main types of macrophage functions are known: classical (M1), producing nitric oxide, NO, and M2, in which arginase activity is primarily expressed. Ornithine, the product of arginase, is a substrate for synthesis of polyamines and collagen, important for growth and ontogeny of animals. M2 macrophages, expressing high level of mitochondrial arginase, have been implicated in promoting cell division and deposition of collagen during ontogeny and wound repair. Arginase expression is the default mode of tissue macrophages, but can also be amplified by signals, such as IL-4/13 or transforming growth factor-β (TGF-β) that accelerates wound healing and tissue repair. In worms, the induction of collagen gene is coupled with induction of immune response genes, both depending on the same TGF-β-like pathway. This suggests that the main function of M2 “heal” type macrophages is originally connected with the TGF-β superfamily of proteins, which are involved in regulation of tissue and organ differentiation in embryogenesis. Excretory–secretory products of metazoan parasites are able to induce M2-type of macrophage responses promoting wound healing without participation of Th2 cytokines IL-4/IL-13. The expression of arginase in lower animals can be induced by the presence of parasite antigens and TGF-β signals leading to collagen synthesis. This also means that the main proteins, which, in primitive metazoans, are involved in regulation of tissue and organ differentiation in embryogenesis are produced by innate immunity. The signaling function of NO is known already from the sponge stage of animal evolution. The cytotoxic role of NO molecule appeared later, as documented in immunity of marine mollusks and some insects. This implies that the M2-wound healing promoting function predates the defensive role of NO, a characteristic of M1 macrophages. Understanding when and how the M1 and M2 activities came to be in animals is useful for understanding how macrophage immunity, and immune responses operate.
Fluorescent metal nanoclusters (NCs) as a new class of fluorophores have attracted more and more attention due to their unique electronic structures and the subsequent unusual physical and chemical properties. The size of metal NCs approaches the Fermi wavelength of electrons, between metal atoms and nanoparticles, resulting in molecule-like properties including discrete energy levels, size-dependent fluorescence, good photostability and biocompatibility. These excellent properties make them ideal fluorescent probes for biological application. Up to now, significant efforts have been devoted to the synthesis, property and application studies of gold and silver NCs. Recently, a growing number of studies on copper and other metal clusters have also been reported. In this review article, we focus on summarizing recent advances in controllable synthesis strategies, chemical and optical properties, and sensing and imaging applications of metal NCs (mainly including Au, Ag, Cu, etc.). Finally, we conclude with a look at the future challenges and prospects of the future development of metal NCs.
Recent advances in nanotechnology have given rise to a new class of fluorescent labels, fluorescent metal nanoclusters, e.g., Au and Ag. These nanoclusters are of significant interest because they provide the missing link between atomic and nanoparticle behavior in metals. Composed of a few to a hundred atoms, their sizes are comparable to the Fermi wavelength of electrons, resulting in molecule-like properties including discrete electronic states and size-dependent fluorescence. Fluorescent metal nanoclusters have an attractive set of features, such as ultrasmall size, good biocompatibility and excellent photostability, making them ideal fluorescent labels for biological applications. In this review, we summarize synthesis strategies of water-soluble fluorescent metal nanoclusters and their optical properties, highlight recent advances in their application for ultrasensitive biological detection and fluorescent biological imaging, and finally discuss current challenges for their potential biomedical applications.
A class of 0-dimensional/2-dimensional (0D/2D) nanoheterostructures based on carbon quantum dots (CQDs) and graphitic carbon nitride (g-C3N4) was designed as the signal-generation tags for the sensitive photoelectrochemical (PEC) immunoassay of prostate-specific antigen (PSA) coupling with the copper nanoclusters (CuNCs). Combination of CQDs with g-C3N4 promoted the photoexcited electron/hole separation, and largely increased the photocurrents of the nanoheterostructures. Initially, a sandwich-type immunoreaction was carried out on monoclonal anti-PSA antibody-coated microplate by using PSA aptamer linked with CuNCs as the tracer. Accompanying the immunocomplex, the carried CuNCs were dissolved under acidic conditions. The as-released copper ions from the CuNCs could be captured onto the CQDs/g-C3N4 nanoheterostructures via the amino-group on the CQD surface as well as the -NHx (x = 1,2,3) of g-C3N4 nanosheets. The strong coordination of the Lewis basic sites on the CQDs/g-C3N4 with Cu2+ decreased the photocurrent of the nanoheterostructures. Under optimal conditions, CQDs/g-C3N4 nanoheterostructures displayed good photocurrent responses for the detection of PSA within the dynamic linear range of 0.02 – 100 ng mL-1 and a limit of detection (LOD) of 5.0 pg mL-1. This method was also evaluated for quantitative screening of human PSA serum specimens by using the referenced electrochemiluminescent enzyme-linked immunoassay (ECL-ELIA), and gave good matched results between two methods. Additionally, this system is beneficial to explore the charge-separation and photo-induced electron transfer mechanism in the photoelectrochemical sensing protocols.
Ultrasmall noble metal and especially gold nanoclusters (AuNCs, ≤2 nm diameter in size) display a range of unique quantum confined and photophysical properties which are far different from their larger-sized nanoparticle counterparts or that of the bulk parent material. Amongst these properties, the photoluminescence of gold AuNCs has stimulated much interest for biological applications due to a combination of their small size, high photostability and range of emissions depending on size and surface stabilizing ligands, including near-IR emission. The dearth of robust Förster resonance energy transfer (FRET) donors and acceptors available to populate the latter spectral range is also driving strong interest in applying AuNCs for similar utility and especially for biosensing. However, the exact mechanism of how AuNCs engage in this type of energy transfer (ET) is still not defined and accumulating evidence indicates that it is not by a classical Förster process although, interestingly, many of the same characteristics and photophysical requirements seem to be present and even many-times required. Here, we summarize the state of the art in AuNC ET studies with a special emphasis on relevance to biological utility ranging from diagnostics to distance measurements along with describing the different ET mechanisms that have been ascribed with their use. Due to its corresponding importance in this discussion, we provide a brief overview of how these materials are synthesized, the current understanding of how their photoluminescence originates, and some related information on silver nanocluster (AgNC) ET along with related processes such as chemically-induced ET. A perspective and outlook on how this area will develop in the future is also provided.
A direct fluorescence turn-on method for simple and sensitive acetylcholinesterase (AChE) activity assay and AChE inhibitor screening has been developed by first using low-fluorescence glutathione-capped gold nanoclusters (GSH-AuNCs). The thiocholine produced by AChE-catalyzed hydrolysis of S-acetylthiocholine iodide could effectively enhance the fluorescence of GSH-AuNCs via AuS bond formation. In the presence of inhibitors, AChE activity was suppressed and thus fluorescence enhancement decreased. Therefore, AChE activity assay and inhibitor screening could be performed by measuring the fluorescence intensity of the system. The linear range of the AChE activity assay was 0–30 mU mL⁻¹ with a limit of detection of 0.03 mU mL⁻¹ (S/N = 3). The IC50 values of two inhibitors (tacrine and neostigmine bromide) were 42.92 nM and 37.04 nM, respectively. The developed protocol provides a simple and sensitive platform for assaying AChE activity and screening its inhibitors.
Unique aggregation-induced emission (AIE) property has been found and widely applied in chemo/biosensors for thiolated gold nanoclusters and copper nanoclusters; however, little is known about this property of thiolate-protected silver nanoclusters. In this work, specific aggregation-induced emission enhancement (AIEE) of glutathione-capped silver nanoclusters (AgNCs) was verified via its solid-state luminescence and enhanced emission in poor solvent, three stimuli responsive nanoswitches were constructed based on its AIEE property, and a reliable and sensitive PPase assay was developed via ion-triggered luminescence switch. Glutathione-capped AgNCs from a facile one-pot synthesis were found to possess bright red luminescence and aggregation-induced emission enhancement property. This AIEE feature enables AgNCs in sensitive response to pH and temperature in a reversible way, allowing the two nanoswitches to precisely monitor the change of environmental pH and temperature. Complexation reactions among AgNCs, aluminum cation and PPi were also designed for an ion-triggered luminescence nanoswitch, which allows selective response to aluminum cation or PPi in luminescence. This ion-driven luminescence switch is further utilized to design a novel detection strategy for PPase activity through competitive coordination reactions. Our method illustrates a novel detection strategy mediated by complexation reaction between Al3+ and AgNCs avoiding the involvement of copper cations in the detection, and this developed assay performed well in detection of PPase level in fresh rat serum. This work confirms unique aggregation-induced emission enhancement property of glutathione-capped AgNCs, constructs multiple luminescence switches based on its multi-stimuli responsive behaviors, and demonstrates an example of Al3+-mediated detection strategy for PPase assay.
Metal nanoclusters (NCs) containing a few to a few hundreds of atoms bridge the gap between nanoparticles and molecular compounds. The last decade evidenced impressive developments of noble metal NCs such as Au and Ag. Copper is an earth abundant, inexpensive metal from the same group of the periodic table, which is increasingly coming into focus for NC research. This review specifically addresses wet chemical synthesis methods, optical properties and some emerging applications of Cu NCs. As surface protecting templates/ligands play an important role in the stability and properties of Cu NCs, we classified the synthetic methods by the nature of the capping agents. The optical properties of Cu NCs are discussed from the point of view of the effects of the metal core, surface ligands and environment (solvents and aggregation) on the emission of the clusters. Applications of luminescent Cu NCs in biological imaging and light emitting devices are considered.
By using bovine serum albumin (BSA) as the enzyme substrate, we propose a label-free, convenient, reliable and highly sensitive fluorescence “turn-on” assay for trypsin activity and inhibitor screening by means of fluorescent gold nanoclusters capped with 11-mercaptoundecanoic acid (namely AuNCs@11-MUA) for the first time. The principle for the assay depends on the following features: (1) Cu²⁺-triggered fluorescence quenching of AuNCs owing to the binding between Cu²⁺ and the carboxyl groups, (2) the cleavage of BSA into amino acids/peptide fragments catalyzed by trypsin, and (3) the stronger binding ability between amino acids/peptide fragments and Cu²⁺ than that between AuNCs and Cu²⁺, accompanied by the conversion from fluorescence off-state to on-state. Under the optimized conditions, the proposed sensing assay shows a good linear relationship from 0.01 to 2 μg/mL and provides an inspiring detection limit of 0.004 μg/mL, which possesses adequate sensitivity for practical samples of human urine. In addition, our proposed trypsin bioassay can be expanded into the inhibitor evaluation by using trypsin inhibitor from soybean as a model. Such unprecedented AuNCs-based fluorescence “turn-on” analytical proposal for assessing trypsin activity gives a new sight to develop several other potential bioassays or enzyme activity assays with the help of different kinds of substrates in the near future.
Metal nanoclusters (NCs) as a new type of fluorescent materials have been extensively explored owing to their attractive set of features such as ultrafine size, low toxicity, and excellent photostability. However, little progress has been made in producing water-soluble, homogeneous, and ultrabright metal NCs. In this study, gold NCs (AuNCs) with photoluminescence quantum yield (QY) as high as 65% are synthesized in water through a simple blending route. Weak emission is observed from the 6-aza-2-thiothymine-protected AuNCs (ATT-AuNCs), however, the fluorescent intensity can be prominently enhanced by introducing L-arginine (Arg) into the capping layer. The fluorescence enhancement mechanism is systematically investigated by the measurements of UV-vis absorption spectroscopy, photoluminescence spectroscopy, fluorescence lifetime spectroscopy, transmission electron microscopy, dynamic light scattering, X-ray photoelectron spectroscopy, 1H NMR, and as well as calculations from density functional theory, with results isolating the vital role of ligand shell and ruling out the effect of gold core. The supramolecular host-guest assemblies formed between ATT capped on the gold core and guanidine group of Arg make the capping ligands of ATT rigid. Subsequently, the intramolecular vibration and rotation of ATT are greatly suppressed, which reduce the non-radiative relaxation of excited states and, as a result, predominantly raise the luminescence QY of ATT-AuNCs. Further experiments demonstrate that small change in guanidine substituents can arouse obvious changes in NCs photoluminescence features. We envision that this work will substantively contribute to the process of developing efficient synthetic routes to obtain high quality metal NCs.
In this work, a novel approach for simple and sensitive determination of alkaline phosphatase (ALP) is developed based on an inner filter effect of p-nitrophenylphosphate (PNPP) on the fluorescence of gold nanoclusters (AuNCs). AuNCs with a high quantum yield of 12% were synthesized by one-pot strategy and were directly applied as fluorescent substance. When AuNCs were mixed with PNPP, the fluorescence of the AuNCs was remarkably quenched or decreased via the inner filter effect since the absorption spectrum of PNPP overlaps well with the excitation spectrum of the AuNCs . While in the presence of ALP, PNPP was catalytically hydrolyzed into p-nitrophenol, which has different absorption characteristics to those of PNPP, resulting in the recovery of the AuNCs fluorescence. Thus, a novel “turn on” fluorescent sensor for detecting ALP was established with a detection limit as low as 0.002 U/L (signal-to-noise ratio of 3). The “turn on” fluorescent sensor exhibit many merits such as high sensitivity, excellent selectivity and high signal output because of the low background signals. In addition, the developed sensing method was successfully applied to investigate ALP inhibitors and ALP determination in serum samples. A good linear relationship was obtained in the range from 0.02 to 50 U/L and satisfactory recoveries at four spiking levels of ALP ranged from 95% to 106% with precision below 5%. The very simple sensing approach proposed here should promote the development of fluorescence “turn on” chemosensors for chemo/biodetection.
A novel label-free system for the sensitive fluorescent detection of deoxyribonuclease I (DNase I) activity has been developed by utilizing DNA-templated silver nanocluster/graphene oxide (DNA-AgNC/GO) nanocomposite. AgNC is first synthesized around C-rich template DNA and the resulting DNA-AgNC binds to GO through the interaction between the extension DNA and GO. The resulting DNA-AgNC/GO would show quite reduced fluorescence signal because the fluorescence from DNA-AgNCs is quenched by GO. In the presence of DNase I, however, it degrades the DNA strand within DNA/RNA hybrid duplex probe employed in this study, consequently releasing RNA which is complementary to the extension DNA. The released free RNA then extracts DNA-AgNC from GO by hybridizing with the extension DNA bound to GO. This process would restore the quenched fluorescence, emitting highly enhanced fluorescence signal. By employing this assay principle, DNase I activity was reliably identified with a detection limit of 0.10 U/ml which is lower than those from previous fluorescence-based methods. Finally, the practical capability of this assay system was successfully demonstrated by its use to determine DNase I activity in bovine urine.
Herein, gold-silver bimetallic nanoclusters (Au-Ag NCs) with the high fluorescent intensity were first synthesized successfully and utilized for the fabrication of sensitive and specific sensing probes toward inorganic pyrophosphatase (PPase) activity with the help of copper ion (Cu2+) and inorganic pyrophosphate ion (PPi). Cu2+ was used as the quencher of fluorescent Au-Ag NC, whilst PPi was employed as the hydrolytic substrate of PPase. The system consisted of PPi, Cu2+ ion and bovine serum albumin (BSA)-stabilized Au-Ag NC. The detection was carried out by enzyme-induced hydrolysis of PPi to liberate copper ion from the Cu2+-PPi complex. In the absence of target PPase, free copper ions were initially chelated with inorganic pyrophosphate ions to form the Cu2+-PPi complexes via the coordination chemistry, thus preserving the natural fluorescent intensity of the Au-Ag NCs. Upon addition of target PPase into the detection system, the analyte hydrolyzed PPi into phosphate ions, and released Cu2+ ion from the Cu2+-PPi complex. The dissociated copper ions readily quenched the fluorescent signal of Au-Ag NCs, thereby resulting in the decrease of fluorescent intensity. Under optimal conditions, the detectable fluorescent intensity of the as-prepared Au-Ag NCs was linearly dependent on the activity of PPase within a dynamic linear range of 0.1 - 30 mU/mL, and allowed the detection at a concentration as low as 0.03 mU/mL at the 3sblank criterion. Good reproducibility (CV < 8.5% for the intra-assay and interassay), high specificity and long-term stability (90.1% of the initial signal after a storage period of 48 days) were also received by using our system toward target PPase activity. In addition, good results with the inhibition efficiency of sodium fluoride were obtained in the inhibitor screening research of pyrophosphatase. Importantly, this system based on highly enhanced fluorescent Au-Ag NCs offer promise for simple and cost-effective screening of target PPase activity without the needs of sample separation and multiple washing steps.
Thiolate-protected metal nanoclusters (or thiolated metal NCs) have recently emerged as a promising class of functional materials due to their well-defined molecular structures and intriguing molecular-like properties. Recent developments in the NC field have aimed at exploring metal NCs as novel luminescent materials in biomedical field due to their inherent biocompatibility and good photoluminescence (PL) properties. From the fundamental perspectives, recent advances in the field have also aimed at addressing the fundamental aspects of PL properties of metal NCs, shedding some light on developing efficient strategies to prepare highly luminescent metal NCs. In this Perspective, we discuss physical chemistry of a recently discovered aggregation-induced emission (AIE) phenomenon and show the significance of AIE in understanding the PL properties of thiolated metal NCs. We then explore the unique physicochemical properties of thiolated metal NCs with AIE characteristics, and highlight some recent developments in synthesizing the AIE-type luminescent metal NCs. We finally discuss perspectives and directions for future development of the AIE-type luminescent metal NCs.
Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05mU/mL within a good linear range from 0.05 to 2.5mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes.
Copyright © 2015 Elsevier B.V. All rights reserved.
A green method was employed for synthesizing peptide-templated nanoclusters without strong reducing agents. Using the synthetic peptide-gold nanoclusters as fluorescence probe, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion.
Arginase has roots in early life-forms. It converts L-arginine to urea and ornithine. The former provides protection against NH3; the latter serves to stimulate cell growth and other physiological functions. Excessive arginase activity in mammals has been associated with cardiovascular and nervous system dysfunction and disease. Two relevant aspects of this elevated activity may be involved in these disease states. First, excessive arginase activity reduces the supply of L-arginine needed by nitric oxide (NO) synthase to produce NO. Second, excessive production of ornithine leads to vascular structural problems and neural toxicity. Recent research has identified inflammatory agents and reactive oxygen species (ROS) as drivers of this pathologic elevation of arginase activity and expression. We review the involvement of arginase in cardiovascular and nervous system dysfunction, and discuss potential therapeutic interventions targeting excess arginase.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Herein, we reported for the first time a facile synthetic process of gold nanoclusters (AuNCs) by using N-acetyl-L-cysteine as both reducing agent and protection ligand. Based on the pH stimuli-responsive property of the as-prepared AuNCs, we constructed a pH-sensing platform for the detection of urea, urease, and urease inhibitor.
A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.
Fluorescent gold nanoclusters (Au NCs) or nanodots (NDs) with sizes smaller than 3 nm are a specific type of gold nanomaterials. In this review, Au NCs are used to represent fluorescent Au nanomaterials with sizes smaller than 3 nm. Unlike the most popular and well-known spherical, large gold nanoparticles, Au NCs do not exhibit surface plasmon resonance (SPR) absorption in the visible region, but have fluorescence in the visible to near-infrared (NIR) region. With advantages of long lifetime, large Stokes shift, and biocompatibility, Au NCs have become interesting sensing and imaging materials.
Recent progress in nanotechnology has given rise to a new class of fluorescent nanomaterials: fluorescent noble metal nanoclusters. The noble metal nanoclusters include Ag, Au, Cu and Pt and typically consist of a ligand shell and a metal core, which incorporates various numbers of atoms ranging from only a few to several hundred, with diameters <2 nm. Au nanoclusters are the most intensely studied of these materials because of their attractive features, including convenient synthesis, ultra-small size, fluorescent properties, excellent optical/colloidal stability and biocompatibility. This paper summarizes the common synthesis strategies for obtaining water-soluble, highly fluorescent Au nanoclusters using different capping agents, and highlights recent advances in their fluorescent sensing applications with an emphasis on the sensing mechanisms. We also consider the prospects, opportunities and challenges for the synthesis and application of Au nanoclusters.
Luminescent gold nanoclusters (AuNCs), composed of a few to about 100 gold atoms, have attracted considerable attention due to their molecule-like properties. These include the discrete electronic states and size-dependent fluorescence resulting from their size, which is comparable to the Fermi wavelength of conduction electrons. AuNCs have proved to be ideal fluorescence labels for biological applications and environmental monitoring and surveillance, thanks to an attractive set of features (e.g., ultra-small size, good biocompatibility and excellent photostability). This article covers in detail the synthesis strategies and optical properties, and highlights recent advances in analytical and biological applications of water-soluble luminescent AuNCs. We also discuss the potential challenges facing luminescent AuNCs in making breakthroughs in synthesis and biological applications.
The luminescence property of thiolated gold nanoclusters (Au NCs) is thought to involve the Au(I)-thiolate motifs on the NC surface; however, this hypothesis remains largely unexplored due to the lack of precise molecular composition and structural information of highly luminescent Au NCs. Here we report a new red-emitting thiolated Au NC, which has a precise molecular formula of Au22(SR)18 (SR here is glutathiolate) and exhibits intense luminescence. Interestingly, this new Au22(SR)18 species shows distinctively different absorption and emission features from the previously reported Au22(SR)16, Au22(SR)17, and Au25(SR)18. In stark contrast, Au22(SR)18 luminesces very intensely at ~665 nm with a high quantum yield (QY) of ~8%, while the other three Au NCs show very weak luminescence. Our results indicate that the luminescence of Au22(SR)18 originates from the long Au(I)-thiolate motifs on the NC surface via the aggregation-induced emission pathway. Structure prediction by density functional theory suggests that Au22(SR)18 has two RS-[Au-SR]3 and two RS-[Au-SR]4 motifs, interlocked and capping on a prolate Au8 core. This predicted structure is further verified experimentally by Au L3-edge X-ray absorption fine structure analysis. The precise molecular formula and structural model of luminescent Au NCs lend critical insights into the luminescence mechanism of thiolated Au NCs, towards unravelling the origin of the luminescence and the key motif structures that could lead to high QYs.
Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent AuNCs and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs' fluorescence can be dynamically decreased with increasing concentration of the enzymes, and sub-picomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.
We report the synthesis of fluorescent 11-mercaptoundecanoic acid-gold nanodot-liposome (11-MUA-Au ND/Lip) hybrids by incorporation of gold nanoparticles (~3 nm) and 11-MUA molecules in hydrophobic phospholipid membranes that self-assemble to form small unilamellar vesicles. A simple and homogeneous fluorescence assay for phospholipase C (PLC) was developed based on control of the fluorescence quenching of 11-MUA-Au ND/Lip hybrids in aqueous solution. The fluorescence of the 11-MUA-Au ND/Lip hybrids is quenched by oxygen (O2) molecules in solution, and quenching is reduced in the presence of PLC. PLC catalyzes the hydrolysis of phosphatidylcholine units from Lip to yield diacylglycerol (DAG) and phosphocholine (PC) products, leading to the decomposition of Lip. The diacylglycerol further interacts with 11-MUA-Au NDs via hydrophobic interactions, leading to inhibition of O2 quenching. The 11-MUA-Au ND/Lip probe provides a limit of detection (at a signal-to-noise ratio of 3) of 0.21 nM for PLC, with high selectivity over other proteins, enzymes, and phospholipases. We have validated the practicality of using this probe for the determination of PLC concentration in breast cancer cells (MCF-7 and MDA-MB-231 cell lines) and nontumor cells (MCF-10A cell line), revealing that the PLC activity in the first two is at least 1.5-fold higher than that in the third. An inhibitory assay using 11-MUA-Au ND/Lip hybrids demonstrated that tricyclodecan-9-yl potassium xanthate (D609) inhibits PLC (10 nM) with an IC50 value of 3.81 ± 0.22 μM. This simple, sensitive, and selective approach holds great potential for detection of PLC in cancer cells and for screening of anti-PLC drugs.
In the past few years, highly luminescent noble metal nanoclusters (e.g., Au and Ag NCs or Au/Ag NCs in short) have emerged as a class of promising optical probes for the construction of high-performance optical sensors because of their ultrasmall size (<2 nm), strong luminescence, good photostability, excellent biocompatibility, and unique metal-core@ligand-shell structure. In this Focus Review, we briefly summarize the common syntheses for water-soluble highly-luminescent thiolate- and protein-protected Au/Ag NCs and their interesting luminescence properties, highlight recent progress in their use as optical sensors with an emphasis on the mechanisms underlying their selectivity, and finally discuss approaches to improving their sensitivity. The scope of the works surveyed is confined to highly luminescent thiolate- and protein-protected Au/Ag NCs.
A fundamental understanding of the luminescence of Au–thiolate nanoclusters (NCs), such as the origin of emission and the size effect in luminescence, is pivotal to the development of efficient synthesis routes for highly luminescent Au NCs. This paper reports an interesting finding of Au(I)–thiolate complexes: strong luminescence emission by the mechanism of aggregation-induced emission (AIE). The AIE property of the complexes was then used to develop a simple one-pot synthesis of highly luminescent Au–thiolate NCs with a quantum yield of 15%. Our key strategy was to induce the controlled aggregation of Au(I)–thiolate complexes on in situ generated Au(0) cores to form Au(0)@Au(I)–thiolate core–shell NCs where strong luminescence was generated by the AIE of Au(I)–thiolate complexes on the NC surface. We were able to extend the synthetic strategy to other thiolate ligands with added functionalities (in the form of custom-designed peptides). The discovery (e.g., identifying the source of emission and the size effect in luminescence) and the synthesis protocols in this study can contribute significantly to better understanding of these new luminescence probes and the development of new synthetic routes.
Arginase catalyzes the conversion of arginine to urea and ornithine in the liver of ureotelic animals. Higher activity of this enzyme is found in tumors as well as in the sera of patients with hepatic diseases. We have developed a simple colorimetric method for its determination. This is based on the determination of residual arginine, after its conversion with p-nitrophenyl glyoxal (PNPG) at pH 9.0 in the presence of sodium ascorbate. The reaction product obeys Beer's law in the range of 0.01–0.20 mmol/L arginine with an arginine-equivalent molar extinction coefficient of 0.65 × 104 M−1cm−1. The decrease in absorbance in the presence of arginase correlates with the enzyme activity. Color development as well as termination of enzyme activity is achieved by addition of a single reagent, thereby obviating the use of many chemicals necessary in other methods. The sensitivity of this method is equivalent to those of currently available procedures but has the added advantages of greater convenience.
Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.
Several analytical methods have been developed for the determination of arginase (l-arginine amidinohydrolase) activity in physiological samples. These methods are limited by the considerable effort and time required to obtain reliable and reproducible measurements. Here we describe a simple high-throughput colorimetric assay for the determination of arginase activity based on the ornithine-ninhydrin reaction. This method is an improvement over the original single cuvette assay developed by Chinard in that no boiling step is required. The turnaround time has been reduced, with improved precision and reproducibility. The method was extended to the determination of arginase activity in human leukemic (K562) cells and sickle erythrocytes. We believe that the method will find applications for routine analysis as well as for characterizing the action of novel and potent inhibitors on arginase activity.
The mechanism of inhibition of urea synthesis by L-norvaline was investigated in vitro with perfused rat liver and purified enzymes of the urea cycle, and in vivo. L-Norvaline caused potent inhibition of urea synthesis from ammonium chloride in the perfused liver and increases in citrulline and arginine in the liver. The increased ratio of the concentration of arginine to ornithine suggested that the main inhibition step of the urea cycle with L-norvaline is arginase. Kinetic studies with purified enzymes showed that L-norvaline inhibits arginase as well as ornithine transcarbamylase and argininosuccinate synthetase competitively with respect to their amino acid substrates, arginine, ornithine, and citrulline, respectively. The inhibition of arginase by L-norvaline was much stronger at pH 7.5 than at pH 9.7. An in vivo experiment was performed to determine whether the same inhibition mechanism operates in the rat. Judging from a decrease in urea and an increase in ammonia in the liver, intraperitoneal injection of L-norvaline caused inhibition of urea synthesis in vivo. The same conclusion, that L-norvaline inhibits urea synthesis mainly through the inhibition of arginase, was also obtained in the in vivo experiment showing an increased ratio of the concentration of arginine to ornithine. The concentration of acetylglutamate was increased by the addition of L-norvaline both in the perfused liver and in the liver in vivo, probably as a result of secondary effects of the increase in arginine.
A dual and sequential enzyme catalyzed reaction is utilized for the determination of arginase, an important metabolic enzyme. The progress of the enzymic reaction is monitored by a cation selective electrode responsive to NH4+, a product of the reaction. Experiments indicate a precision of about 3% with a detection range of about 1.6 to 16 units of arginase and an average time requirement of less than 10 minutes per analysis. The change in potential with time is measured, and the amount of arginase is determined from a previously prepared calibration curve. The utility of this procedure is exemplified by its use to study several parameters which affect the catalytic efficiency of arginase. The results of studies on the effect of pH, temperature, substrate concentration, and immobilized enzyme concentration using this new potentiometric method for arginase are presented. The possibility of extended application is likewise discussed.
An assay for arginase is described that uses l-[guanido-14C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [14C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.
We describe a microfluorometric method for determination of arginase activity in dried blood spots on filter paper. The arginase in discs punched from such dried blood specimens is activated by preincubation with Mn2+ at 37 degrees C. After incubation with substrate at the same temperature, urea is determined fluorometrically by oxidation of NADH to NAD+ in a coupled kinetic reaction. We compare the results of this method with those of a colorimetric method involving liquid blood samples, and assess the stability of the enzyme in dried blood on filter paper. The presence of serum has no effect on the activity. This method may be useful in the early detection of arginase deficiency and certain hematological disorders.
The activity of guanidino compounds as alternate substrates for rat liver arginase is critically dependent on the length of the amino acid side chain and the substituents about C-alpha. In addition to L-arginine, the enzyme catalyzes the hydrolysis of L-argininamide, L-canavanine, L-homoarginine, L-argininic acid, and agmatine. The kcat values for these substrates are 15- to 5000-fold slower than the kcat for L-arginine. Guanidobutyrate, D-arginine, and NG-methyl-L-arginine are not substrates. Competitive inhibition by the products L-ornithine and urea indicates a rapid-equilibrium random mechanism for the enzyme. Despite the requirement for added divalent cations in the activation of the enzyme, metal chelators such as EDTA and citrate do not inhibit the enzyme. These results suggest that the metal site is not readily accessible to solvent. Multiple inhibition experiments with the noncompetitive inhibitor borate demonstrate that borate and urea bind in a mutually exclusive manner, while L-ornithine and borate can bind simultaneously to the enzyme. Borate inhibition is proposed to arise from chelation of Mn(II) in the binuclear Mn(II) center, thus displacing a metal-bound water molecule that is responsible for nucleophilic attack on the guanidium carbon.
Each individual excretes roughly 10 kg of urea per year, as a result of the hydrolysis of arginine in the final cytosolic step of the urea cycle. This reaction allows the disposal of nitrogenous waste from protein catabolism, and is catalysed by the liver arginase enzyme. In other tissues that lack a complete urea cycle, arginase regulates cellular arginine and ornithine concentrations for biosynthetic reactions, including nitric oxide synthesis: in the macrophage, arginase activity is reciprocally coordinated with that of NO synthase to modulate NO-dependent cytotoxicity. The bioinorganic chemistry of arginase is particularly rich because this enzyme is one of very few that specifically requires a spin-coupled Mn2+-Mn2+ cluster for catalytic activity in vitro and in vivo. The 2.1 angstrom-resolution crystal structure of trimeric rat liver arginase reveals that this unique metal cluster resides at the bottom of an active-site cleft that is 15 angstroms deep. Analysis of the structure indicates that arginine hydrolysis is achieved by a metal-activated solvent molecule which symmetrically bridges the two Mn2+ ions.
Human arginase was purified from liver and two monoclonal antibodies (MAbs), HA1 and HA2, were produced by fusion of spleen cells from an arginase-immunized BALB/c mouse and the NS-1 myeloma cell line. Both MAbs were of the IgG3 subclass and contained the kappa light chain. HA1 inhibited arginase activity, suggesting that it binds to the arginase catalytic site. HA1 and a horseradish peroxidase-conjugated polyclonal rabbit anti-human arginase antibody were used to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for the quantification of human arginase, which can be used in the 1 to 300 ng/mL range. Because of its sensitivity and specificity, this MAb can be successfully applied to the ELISA quantification of arginase in serum and culture supernatants.
Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method, we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC.
Nanoheterostructures-Based Signal-Generation Tags for Photoelectrochemical Immunoassay of Cancer Biomarkers Coupling with Copper Nanoclusters
Nanoheterostructures-Based Signal-Generation Tags for Photoelectrochemical Immunoassay of Cancer Biomarkers Coupling with
Copper Nanoclusters. ACS Appl. Mater. Interfaces 2017, 9, 38336−
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- Nanocluster
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