Effect of phosphodiester composition in polyphosphoesters on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells.

Osteoporosis is a common bone disorder characterized by reduced bone density and increased risk of fractures. The modulation of bone cell functions, particularly the inhibition of osteoclastic differentiation, plays a crucial role in osteoporosis treatment. Polyphosphoesters (PPEs) have shown the potential in reducing the function of osteoclast cells, but the effect of their chemical structure on osteoclastic differentiation remains largely unexplored. In this study, we evaluated the effect of PPE's chemical structure on the inhibition of osteoclastic differentiation of murine bone marrow mononuclear cells (BMNCs). PPEs containing phosphotriester and phosphodiester units at varying compositions were synthesized. Cytotoxicity testing confirmed the biocompatibility of the copolymers at concentrations below 0.5 mg/mL. Isolated from long bones, BMNCs were cultured in a differentiation medium supplemented with different PPE concentrations. Osteoclast formation was assessed through tartrate-resistant acid phosphatase and phalloidin staining. A significant decrease in the size of osteoclast cells formed upon BMNC contact with PPEs was observed, with a more pronounced effect observed at higher PPE concentrations. In addition, an increased composition of phosphodiester units in the PPEs yielded a decreased density of differentiated osteoclasts. Furthermore, real-time PCR analysis of major osteoclastic markers provided gene expression data that correlated with microscopic observations, confirming the effect of phosphodiester units in suppressing osteoclast differentiation of BMNCs from the early stages. These findings highlight the potential of PPEs as polymers are capable of modulating bone cell functions through their chemical structures.

A Machine Learning-Based Image Segmentation Method to Quantify In Vitro Osteoclast Culture Endpoints.

Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods. These approaches are labour intensive, time consuming and result in substantial inter- and intra-user variability. This study aimed to develop and validate an automated workflow to robustly quantify in vitro osteoclast cultures. Using ilastik, a machine learning-based image analysis software, images of tartrate resistant acid phosphatase-stained mouse osteoclasts cultured on dentine discs were used to train the ilastik-based algorithm. Assessment of algorithm training showed that osteoclast numbers strongly correlated between manual- and automatically quantified values (r = 0.87). Osteoclasts were consistently faithfully segmented by the model when visually compared to the original reflective light images. The ability of this method to detect changes in osteoclast number in response to different treatments was validated using zoledronate, ticagrelor, and co-culture with MCF7 breast cancer cells. Manual and automated counting methods detected a 70% reduction (p < 0.05) in osteoclast number, when cultured with 10 nM zoledronate and a dose-dependent decrease with 1-10 µM ticagrelor (p < 0.05). Co-culture with MCF7 cells increased osteoclast number by ≥ 50% irrespective of quantification method. Overall, an automated image segmentation and analysis workflow, which consistently and sensitively identified in vitro osteoclasts, was developed. Advantages of this workflow are (1) significantly reduction in user variability of endpoint measurements (93%) and analysis time (80%); (2) detection of osteoclasts cultured on different substrates from different species; and (3) easy to use and freely available to use along with tutorial resources.

Bimetallic AuPt alloy/rod-like CeO2 nanojunctions with high peroxidase-like activity for colorimetric sensing of organophosphorus pesticides.

Organophosphorus pesticides (OP) have extensive applications in agriculture, while their overuse causes inevitable residues in food, soil, and water, ultimately being harmful to human health and even causing diverse dysfunctions. Herein, a novel colorimetric platform was established for quantitative determination of malathion based on peroxidase mimic AuPt alloy decorated on CeO2 nanorods (CeO2@AuPt NRs). The synthesized nanozyme oxidized colorless 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Besides, the oxidized TMB was inversely reduced by ascorbic acid (AA), which were originated from hydrolysis of L-ascorbic acid-2-phosphate (AA2P) with the assistance of acid phosphatase (ACP). Based upon this observation ACP analysis was explored by colorimetry, showing a wid linear range of 0.2 ~ 3.5 U L-1 and a low limit of detection (LOD = 0.085 U L-1, S/N = 3). Furthermore, malathion present in the colorimetric system inhibited the activity of ACP and simultaneously affected the generation of AA, in turn promoting the recovery of the chromogenic reaction. Based on this, the LOD was decreased to 1.5 nM (S/N = 3) for the assay of malathion with a wide linear range of 6 ~ 100 nM. This simple colorimetric platform provides some informative guidelines for determination of other pesticides and disease markers.

Sensitive acid phosphatase assay based on light-activated specific oxidase mimic activity.

Acid phosphatase (ACP) is a key marker in the diagnosis of many diseases. However, exploiting a simple and sensitive sensor for the real-time quantitative analysis of ACP is still challenging. Herein, we attempted to develop a sensitive colorimetric sensing strategy for the detection of ACP based on light-activated oxidase mimic property of carbon dots (CDs). The synthesized CDs were proved to be capable of intrinsic light-activated oxidase mimic activity, which could generate reactive oxygen species to oxidize chromogenic substrate under ultraviolet light stimulation. Interestingly, this light-activated oxidase mimic behavior would be effectively suppressed by the antioxidant ascorbic acid (AA), a product from the hydrolysis of 2-phospho-L-ascorbic acid trisodium (AAP) mediated by ACP. Based on the above property, a facile and sensitive colorimetric sensing method for ACP was developed. Under the optimal conditions, the linear range for ACP 0.1-5.5 U/L, and the detection limit was 0.056 U/L. Compared with conventional nanozyme based ACP assay systems, the catalytic activity of light-activated nanozyme could be conveniently regulated by switching the light on and off, which made it easier to precisely control the extent of the reaction and ensured the accuracy of the assay. In addition, the proposed sensing system would be readout directly by the naked eye or smartphone-based RGB analysis system, and have been successfully applied to analyze diluted in diluted fetal bovine serum and urine samples spiked with ACP. All these results indicated that this approach holds good promise for future applications in clinical analysis and point-of-care (POC) biosensor platforms.

Morphology and cytochemical patterns of peripheral blood cells of tiger frog (Rana rugulosa).

Background: Tiger frog (Rana rugulosa) is a national second-class protected amphibian species in China with an important ecological and economic value. In recent years, due to excessive human hunting, pollution and habitat loss, the wild population of tiger frog has declined sharply. To protect wildlife resources, the artificial breeding of tiger frogs has rapidly developed in China. Diseases are increasing and spreading among tiger frogs due to the increasing scale of artificial farming. The blood examination is the most straightforward and less invasive technique to evaluate the animal health condition. Thus, it is essential to obtain the normal hematological indicators of tiger frogs. The objective of this study was to investigate the morphometry, microstructure and cytochemical patterns of peripheral blood cells in tiger frogs. Methods: The number of blood cells in tiger frogs was counted on a blood count board, and the cell sizes were measured by a micrometer under light microscope. The morphology and classification of blood cells were studied by Wright-Giemsa staining, and the cytochemical pateerns was investigated by various cytochemical staining including periodic acid-Schiff (PAS), Sudan black B (SBB), peroxidase (POX), alkaline phosphatase (AKP), acid phosphatase (ACP), chloroacetic acid AS-D naphthol esterase (CAE) and α-naphthol acetate esterase (ANAE) staining. Results: Besides erythrocytes and thrombocytes, five types of leukocytes were identified in tiger frogs: neutrophils, eosinophils, basophils, lymphocytes and monocytes. The mean erythrocyte, leukocyte and thrombocyte counts were 1.33 ± 0.15 million/mm3, 3.73 ± 0.04 × 104/mm3 and 1.7 ± 0.01 × 104/mm3, respectively. Small lymphocytes were the most abundant leukocytes, followed by large lymphocytes, Neutrophils, eosinophils and monocytes, basophils were the fewest. Eosinophils were strongly positive for PAS, positive for SBB, POX, ACP, CAE, ANAE, while weakly positive for AKP staining; basophils were strongly positive for PAS, ACP, positive for SBB, CAE, weakly positive for ANAE, negative for AKP, POX staining; neutrophils were strongly positive for ACP, SBB, positive for PAS, POX, weakly positive for AKP, CAE and ANAE staining; monocytes were positive for PAS, SBB, ANAE, weakly positive for ACP, AKP, POX, CAE staining; large lymphocytes and thrombocytes were positive for PAS, ACP, weakly positive for ANAE, while negative for SBB, POX, AKP, CAE; small lymphocytes were similar to large lymphocytes, except for strongly positive for PAS and ACP staining. Conclusions: The blood cell types and morphology of tiger frogs were generally similar to those of other amphibians, while their cytochemical patterns had some notable species specificity.Our study could enrich the knowledge of peripheral blood cell morphology and cytochemistry in amphibians, and provide baseline data for health condition evaluation and disease diagnosis of tiger frogs.

Comparison of the Validity of Enzymatic and Immunohistochemical Detection of Tartrate-resistant Acid Phosphatase (TRAP) in the Context of Biocompatibility Analyses of Bone Substitutes.

BACKGROUND/AIM: Macrophages and biomaterial-induced multinucleated giant cells (BMGCs) are central elements in the tissue reaction cascade towards bone substitute materials (BSM). The enzymatic detection of the lytic enzyme tartrate-resistant acid phosphatase (TRAP) has manifoldly been used to examine the so-called "bioactivity" of BSM. The present study aimed to compare the detection validity and expression pattern of the TRAP enzyme using enzymatic and immunohistochemical detection methods in the context of biocompatibility analyses of BSM. PATIENTS AND METHODS: Biopsies from 8 patients were analyzed after sinus augmentation with a xenogeneic bone substitute. Analysis of both macrophage and BMGC polarization were performed by histochemical TRAP detection and immunohistochemical detection of TRAP5a. Histomorphometrical analysis was used for comparison of the TRAP detection of BMGCs. RESULTS: The enzymatic TRAP detection method revealed that in 7 out of 8 biopsies only single cells were TRAP-positive, whereas most of the cells and especially the BMGCs were TRAP-negative. The immunohistochemical detection of TRAP5a showed moderate numbers of stained mononuclear cells, while the majority of the BMGCs showed signs of TRAP5a-expression. The enzymatic TRAP detection was comparable to the results obtained via immunohistochemistry only in one case. The histomorphometrical analysis showed that significantly more mononuclear and multinucleated TRAP-positive cells were found using immunohistochemical TRAP5a-staining compared to the enzymatic TRAP detection method. Also, significantly more TRAP-negative BMGCs were found using the enzymatic TRAP detection. CONCLUSION: The immunohistochemical detection of TRAP is more accurate for examination of the bioactivity and cellular degradability of BSM.

Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity.

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

Soil enzyme responses to land use change in the tropical rainforest of the Colombian Amazon region.

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.

A dual-signal fluorescent probe for detection of acid phosphatase.

Acid phosphatase has become a significant indicator of prognostic and medical diagnosis, and its dysfunction may lead to a series of diseases. A novel dual-signal fluorescence method for acid phosphatase detection based on europium polymer (europium-pyridine dicarboxylicacid-adenine) and pyridoxal phosphate (PLP) was proposed. PLP coordinated with europium polymer via Eu3+ and P-O bonds, and the fluorescence of europium polymer was quenched due to the photoinduced electron transfer (PET) effect between aldehyde and europium polymer. Upon addition of acid phosphatase, the PLP was transformed to phosphate (Pi) and pyridoxal (PL). The PL was released from the surface of europium polymer, and the blue emission was enhanced due to the formation of internal hemiacetal, while the fluorescence of europium polymer recovered. The blue (PL) and red emission (Eu3+) were positively correlated with acid phosphatase activity; thus the sensitive assay of acid phosphatase was effectively achieved. The two signals were applied to determine the acid phosphatase with limits of detection (LOD) of 0.04 mU/mL and 0.38 mU/mL, and the linear ranges were 0.13-5.00 mU/mL and 1.25-20.00 mU/mL, respectively. The probe can be used to trace the acid phosphatase in biological systems and holds promise for use in clinical diagnosis and early prevention.

Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for KD are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (KD = 1.48 nM) while Pep2856 had a good affinity for PPG (KD 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL.

Sugarcane/peanut intercropping system improves the soil quality and increases the abundance of beneficial microbes.

Sugarcane/peanut intercropping is a highly efficient planting pattern in South China. However, the effects of sugarcane/peanut intercropping on soil quality need to be clarified. This study characterized the soil microbial community and the soil quality in sugarcane/peanut intercropping systems by the Illumina MiSeq platform. The results showed that the intercropping sugarcane (IS) system significantly increased the total N (TN), available N (AN), available P (AP), pH value, and acid phosphatase activity (ACP), but it had little effect on the total P (TP), total K (TK), available K (AK), organic matter (OM), urease activity, protease activity, catalase activity, and sucrase activity, compared with those in monocropping sugarcane (MS) and monocropping peanut (MP) systems. Both intercropping peanut (IP) and IS soils contained more bacteria and fungi than soils in the MP and MS fields, and the microbes identified were mainly Chloroflexi and Acidobacteria, respectively. Intercropping significantly increased the number of unique microbes in IS soils (68 genera), compared with the numbers in the IP (14), MS (17), and MP (16) systems. The redundancy analysis revealed that the abundances of culturable Acidobacteriaceae subgroup 1, nonculturable DA111, and culturable Acidobacteria were positively correlated with the measured soil quality in the intercropping system. Furthermore, the sugarcane/peanut intercropping significantly increased the economic benefit by 87.84% and 36.38%, as compared with that of the MP and MS, respectively. These results suggest that peanut and sugarcane intercropping increases the available N and P content by increasing the abundance of rhizospheric microbes, especially Acidobacteriaceae subgroup 1, DA111, and Acidobacteria.

NKX3.1 expression in cervical 'adenoid basal cell carcinoma': another gynaecological lesion with prostatic differentiation?

Adenoid basal cell carcinoma (ABC) is considered a rare cervical neoplasm which when present in 'pure' form, uniquely amongst apparently malignant cervical tumours, has never been reported to metastasise or lead to fatal patient outcome. We recently encountered a case of ABC that was morphologically reminiscent of prostatic differentiation, more specifically basal cell hyperplasia of the prostate. Immunohistochemistry was strongly positive for the prostate related marker NKX3.1 in the glandular cells, but there was no expression of prostate specific antigen (PSA) or prostatic acid phosphatase (PAP). However, subsequent review of five additional cervical ABCs demonstrated focal PAP expression in two of four tested cases, and all were NKX3.1 positive. NKX3.1 expression was also demonstrated in the glandular epithelium of 10 additional gynaecological lesions considered to show prostatic differentiation including five cases of cervical ectopic prostatic tissue, three ovarian teratomas with prostatic differentiation, and two vaginal tubulosquamous polyps. We suggest that some lesions traditionally classified as ABC may in fact represent a variant of prostatic differentiation within the cervix, possibly analogous to basal cell hyperplasia of the prostate.

Do methanotrophs drive phosphorus mineralization in soil ecosystem?

Experiments were carried out to elucidate linkage between methane consumption and mineralization of phosphorous (P) from different P sources. The treatments were (i) no CH4 + no P amendment (absolute control), (ii) with CH4 + no P amendment (control), (iii) with CH4 + inorganic P as Ca3(PO4)2, and (iv) with CH4 + organic P as sodium phytate. P sources were added at 25 µg P·(g soil)-1. Soils were incubated to undergo three repeated CH4 feeding cycles, referred to as feeding cycle I, feeding cycle II, and feeding cycle III. CH4 consumption rate k (µg CH4 consumed·(g soil)-1·day-1) was 0.297 ± 0.028 in no P amendment control, 0.457 ± 0.016 in Ca3(PO4)2, and 0.627 ± 0.013 in sodium phytate. Rate k was stimulated by 2 to 6 times over CH4 feeding cycles and followed the trend of sodium phytate > Ca3(PO4)2 > no P amendment control. CH4 consumption stimulated P solubilization from Ca3(PO4)2 by a factor of 2.86. Acid phosphatase (µg paranitrophenol released·(g soil)-1·h-1) was higher in sodium phytate than the no P amendment control. Abundance of 16S rRNA and pmoA genes increased with CH4 consumption rates. The results of the study suggested that CH4 consumption drives mineralization of unavailable inorganic and organic P sources in the soil ecosystem.

Impact of Ag2S NPs on soil bacterial community - A terrestrial mesocosm approach.

Soils might be a final sink for Ag2S nanoparticles (NPs). Still, there are limited data on their effects on soil bacterial communities (SBC). To bridge this gap, we investigated the effects of Ag2S NPs (10 mg kg-1 soil) on the structure and function of SBC in a terrestrial indoor mesocosm, using a multi-species design. During 28 days of exposure, the SBC function-related parameters were analysed in terms of enzymatic activity, community level physiological profile, culture of functional bacterial groups [phosphorous-solubilizing bacteria (P-SB) and heterotrophic bacteria (HB)], and SBC structure was analysed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis. The SBC exposed to Ag2S NPs showed a significative decrease of functional parameters, such as ß-glucosidase activity and L-arginine consumption, and increase of the acid phosphatase activity. At the structural level, significantly lower richness and diversity were detected, but at later exposure times compared to the AgNO3 treatment, likely because of a low dissolution rate of Ag2S NPs. In fact, stronger effects were observed in soils spiked with AgNO3, in both functional and structural parameters. Changes in SBC structure seem to negatively correlate with parameters related to phosphorous (acid phosphatase activity) and carbon cycling (abundance of HB, P-SB, and ß-glucosidase activity). Our results indicate a significant effect of Ag2S NPs on SBC, specifically on parameters related to carbon and phosphorous cycling, at doses as low as 10 mg kg-1 soil. These effects were only observed after 28 days, highlighting the importance of long-term exposure experiments for slowly dissolving NPs.

Developing robust protein analysis profiles to identify bacterial acid phosphatases in genomes and metagenomic libraries.

Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups of acid phosphatases. These sequence-based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.

Polydopamine coated copper nanoclusters with aggregation-induced emission for fluorometric determination of phosphate ion and acid phosphatase activity.

The preparation of aggregation-induced emission-type copper nanoclusters (CuNCs) capped with polydopamine (PDA) is described. PDA was formed via in situ polymerization of dopamine in the presence of alkaline polyethylenimine. The PDA-capped CuNCs (PDA-CuNCs) exhibit orange fluorescence with maximal emission at 580 nm upon excitation at 340 nm, a storage stability of at least 2 weeks, and a quantum yield (QY) of 2.54% in aqueous solution. The QY is 28-fold higher than that of sole CuNCs. The fluorescence of the PDA-CuNCs is quenched by Fe3+ ion while it is recovered by PO43- due to its stronger affinity for Fe3+. On this basis, a fluorometric phosphate assay was developed that has a 1.5 nM detection limit and a linear range over 0.003-70 µM. The method was satisfactorily applied to the determination of phosphate in local tap water and human sera, and the results agreed well with those obtained by a colorimetric method. In the presence of acid phosphatase (ACP), PO43- is produced by the catalytic hydrolysis of adenosine triphosphate (ACP substrate). Thus, a fluorogenic assay for screening ACP activity was established. Response is linear over the activity range 0.0012-25 U L-1, with a detection limit of 0.001 U L-1 (at S/N = 3). Graphic abstract We proposed an effective polydopamine-templating strategy for the in situ synthesis of highly emissive and stable CuNCs and demonstrated its use as an ion-driven fluorescence switch for the determination of phosphate and acid phosphatase activity.

Dual-emission carbon dots for ratiometric detection of Fe3+ ions and acid phosphatase.

We have developed a simple and convenient route to prepare fluorescent carbon dots with dual emission peaks respectively at 470 and 570 nm. The prepared dual-emission carbon dots can be used for ratiometric detection of Fe3+ ions in the range from 0 to 50 µmol·L-1 with 0.8 µmol·L-1 detection limit based on the fluorescence quenching at 570 nm. The quenched fluorescence induced by Fe3+ ions could be recovered by pyrophosphate. We further used the carbon dots-Fe3+ ions-pyrophosphate mixed system for ratiometric detection of acid phosphatase in the range from 0.08 to 6.75 µg·mL-1 with 0.01 µg·mL-1 detection limit.

Colorimetric acid phosphatase sensor based on MoO3 nanozyme.

Nanozymes, or nanomaterials that mimic the behaviors of enzymes, are highly promising materials for biomedical applications because of their excellent chemical stability under harsh conditions, simple preparation method and lower costs compared with natural enzymes. We herein report the intrinsic oxidase-mimicking activity of molybdenum oxide nanoparticles (MoO3 NPs). MoO3 NPs catalyzed the oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to green product. The catalytic mechanism of the oxidase-mimicking activity of the MoO3 NPs was investigated in detail using electron spin resonance and a radical inhibition method. The oxidation of ABTS stems from 1O2 generated from the interaction between MoO3 NPs and dissolved oxygen in the solution. Acid phosphatase (ACP) catalyzes the hydrolysis of the ascorbic acid 2-phosphate (AAP) substrate to produce ascorbic acid (AA). AA was found to fade the coloration process of the MoO3 NP-mediated ABTS oxidation. By combining the oxidase-mimicking property of the MoO3 NPs and the ACP-catalyzed hydrolysis of AAP, a novel and simple colorimetric method for detecting ACP was established. The linear range for ACP determination is 0.09-7.3 U/L with a detection limit of 0.011 U/L. This new colorimetric method was successfully applied to the detection of ACP in diluted human serum samples and screening of ACP inhibitors. The present study proposes MoO3 NPs as a new oxidase mimic for establishing various biosensing method.

A lysosome-targeted near-infrared fluorescent probe for imaging of acid phosphatase in living cells.

Fluorescent probes for the detection of acid phosphatases (ACP) are important in the investigation of the pathology and diagnosis of diseases. We reported a lysosome-targeted near-infrared (NIR) fluorescent probe SHCy-P based on a novel NIR-emitting thioxanthene-indolium dye for the detection of ACP. The probe showed a long wavelength fluorescence emission at λem = 765 nm. Due to the ACP-catalyzed cleavage of the phosphate group in SHCy-P, the probe exhibited high selectivity and sensitivity for the 'turn-on' detection of ACP with a limit of detection as low as 0.48 U L-1. The probe SHCy-P could also be used to detect and image endogenous ACP in lysosomes. In light of these prominent properties, we envision that SHCy-P will be an efficient optical imaging approach for investigating the ACP activity in disease diagnosis.

Enzyme Activity Triggered Blocking of Plasmon Resonance Energy Transfer for Highly Selective Detection of Acid Phosphatase.

Plasmon resonance energy transfer (PRET), as a new form of energy transfer first discovered in 2007, has been widely applied for the biomolecular recognition, detection of ions, cellular physiological status monitoring, and energy conversion. It occurs between noble metal nanoparticles (donor) and conjugated molecules or nanoparticles (acceptor). In this study, we used urchin-like gold nanoplasmonics (UGPs) and oxTMB as a new donor-acceptor pair to establish a novel PRET coupling system, avoiding trivial modification. PRET from UGPs to conjugated redox-active oxTMB leads to resonant quenching in the localized surface plasmon resonance (LSPR) spectra. However, when the acid phosphatase (ACP) was introduced, the hydrolyzate ascorbic acid (AA) converted from 2-phospho-l-ascorbic acid trisodium salt (AAP) could be capable of reducing oxTMB into TMB, thereby preventing the occurrence of PRET. The recovery of the scattering spectral intensity of UGPs was linearly related to the concentration of ACP in the range of 0.1 to 5.0 U/L, and the ACP with a detection limit of 0.076 U/L could be measured. In addition, this method also showed good selectivity attributed to the substrate specificity of enzyme.