Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis.

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO.

To meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett-Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L-1, and µ = 0.0943 h-1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

Boosting toxic protein biosynthesis: transient in vivo inactivation of engineered bacterial alkaline phosphatase.

BACKGROUND: The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts' metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host's viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors' background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. RESULTS: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3'-protruding). CONCLUSIONS: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

Alkaline Phosphatase.

Several types of alkaline phosphatases (or alkaline phosphomonoesterase) are commonly used in molecular cloning, including bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CIP, CIAP, or CAP). Similar enzymes isolated from more esoteric cold-blooded organisms (e.g., SAP from shrimp) have become available in recent years and have the advantage of being easier to inactivate than BAP or CIP at the end of dephosphorylation reactions. The uses and properties of these enzymes are introduced here.

Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody-Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B.

In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody-alkaline phosphatase (Nb-ALP) fusion protein. The SEB-binding nanobodies were obtained from a naïve phage-display library and the Nb-ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37-ALP, was 3.12-50.0 ng mL-1 with SC50 = 8.59 ± 0.37 ng mL-1. The limit of detection was 1.44 ng mL-1 according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB-Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class Ⅱ molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb-ALP appears to have the potential to replace chemically labeled probes for the detection of SEB.

A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296.

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S-1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S-1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

A ratiometric multicolor fluorescence biosensor for visual detection of alkaline phosphatase activity via a smartphone.

Herein we designed a selective and smartphone-based strategy for visual detection of alkaline phosphatase (ALP) by utilizing the property of amino-functionalized copper (II)-based metal-organic frameworks (NH2-Cu-MOFs) with oxidase mimic property and fluorescence property. Surprisingly, the oxidase mimic property of NH2-Cu-MOFs can work well at a high pH value 8.0. Thus, a cascade reaction between ALP and NH2-Cu-MOFs was realized for the construction of a ratiometric multicolor sensing platform through the controllable catalytic activity of NH2-Cu-MOFs by pyrophosphate (PPi) and ALP. The catalytic activity of NH2-Cu-MOFs was greatly inhibited because of the binding ability of Cu2+ with PPi. When the ALP was added, the catalytic activity of NH2-Cu-MOFs was restored and then further catalyzed the o-phenylenediamine to form the 2, 3-diaminophenazine due to the hydrolysis function of ALP towards PPi into orthophosphates. RGB analysis of the fluorescent sample images was adopted for ALP quantitative analysis. Besides, a hydrogel test kit and mobile app for ALP detection were designed as conceptual products for point-of-care. The LODs of the fluorescence sensing platform was 0.078 mU mL-1 and 0.35 mU mL-1 by solution analysis and hydrogel test kit analysis, respectively. This fluorescent visual method was applied to ALP detection in serum samples with satisfying results, which opened a promising horizon for the diagnosis of other biomarkers in clinical serum samples based on ALP-mediated enzyme-linked immunosorbent assay for the development of biomedicine and clinical diagnosis.

Biochemical characterization of digestive membrane-associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis.

In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.

A ratiometric fluorescent assay for the detection and bioimaging of alkaline phosphatase based on near infrared Ag2S quantum dots and calcein.

Herein, a ratiometric fluorescent method was developed for alkaline phosphatase (ALP) detection based on near-infrared (NIR) Ag2S quantum dots (QDs) and calcein through the competitive approach. The system based on Ag2S QDs and calcein shows green (maximum emission at 512 nm from calcein) and near infrared (NIR) fluorescence (maximum 798 nm from Ag2S QDs) under the same excitation wavelength (468 nm). In the presence of Ce3+, the fluorescence intensity of calcein is decreased due to static quenching, while the fluorescence intensity of Ag2S QDs is enhanced through aggregation induced emission (AIE). The p-nitrophenyl phosphate is hydrolyzed by ALP, and the yield phosphate ions bind with Ce3+ with higher affinity than these of Ag2S QDs and calcein. Therefore, the green fluorescence from calcein is recovered while NIR fluorescence from Ag2S QDs is decreased. On the basis of these findings, a ratiometric fluorescence assay was developed for the measurement of ALP activity. The ratio of fluorescence intensity at 512 and 798 nm (F512/F798) was well associated with the ALP concentration ranging from 2 to 100 mU/mL with the detection limit of 1.28 mU/mL. The method was successfully applied for detecting ALP in human serum with an acceptable recovery and bioimaging intracellular ALP with good performance. In addition, the approach was also employed for the screening ALP inhibitor.

Sludge: next paradigm for enzyme extraction and energy generation.

Excess of sludge generated during activated sludge process by various industries is causing sludge disposable problem worldwide. Sludge contains organic materials of a highly complex mixture of molecules with some toxic compounds. Thus, the harmful effect of sludge could be managed by processing of sludge and recovery of useful products. Sludge could undergo biodegradation and used for the recovery of enzymes and energy. Enzymes (amylase, alkaline phosphatase, lipase, proteases) are produced by microorganisms and released into the media to degrade organic matters of sludge. These enzymes can be extracted by means of different physical and chemical methods from activated sludge for the multifarious application. Current manuscript discussed all the methods utilized for the extraction of enzyme and their utilization in terms of energy generation. Extracted industrial enzymes are used in agriculture, dairy, detergents, pulp, paper, cosmetics, and pharmaceutics. Apart from enzyme extraction, production of energy (biofuels, electricity) is also done by key treatment of sludge. It is a compilation of current knowledge and expected competitiveness with respect to existing methods already applied in practice for enzyme recovery. This paper presents an overview of the production of valuable enzymes and various forms of energy from sludge, which is toxic and unwanted for life.

Paper-based miniaturized immunosensor for naked eye ALP detection based on digital image colorimetry integrated with smartphone.

Alkaline phosphatase (ALP) is a metalloprotein found naturally in raw milk samples and is considered as an important biomarker in quality control of milk. Its easy, personalized, as well as instrument-less detection is important to ensure the pasteurization and its differentiation form raw milk. In view of such importance of APL, we have developed an office punching machine crafted paper biosensor for naked eye detection of ALP in milk samples. The quantitative estimation is done by digital image colorimetry (DIC) based technique integrated with smartphone. The sensor-probe was developed by the covalent immobilization of ALP antibody (anti-ALP) onto the functionalized paper surface. The fabrication of the biosensing probe was characterized using DIC, Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The detection was based on immunocomplexation between the sensor-probe and ALP, which generates blue-green precipitate as an analytical signal by exploiting the catalytic activity of ALP towards 5-bromo-4-chloro 3-indolyl phosphate (BCIP). The dose dependent appearance of the blue-green complex was captured using smartphone camera and DIC was employed using Red, Green, and Blue (RGB) profiling system, where the maximum sensitivity was obtained for the red color channel. Based on the DIC analysis, a wide dynamic range for the ALP detection is obtained from 10 to 1000 U/mL with the detection limit of 0.87 (±â€¯0.07) U/mL. The designed paper-based biosensor is successfully applied to detect ALP in commercial and raw milk samples. Interferences due to components present in the milk samples was evaluated and the long-term stability of the designed biosensor was examined. Based on the detection principle, a miniaturized kit [20.0 mm (L) × 20.0 mm (W) × 2.15 mm (H)] was developed and applied for the ALP detection to demonstrate the instrument-free direct in-kitchen applicability.

Magnetic iron oxide nanoparticles modified with vanadate and phosphate salts for purification of alkaline phosphatase from the bovine skim milk.

Modified Fe3O4 magnetic nanoparticles (magnetic nanocarrier) technology have found the proper place in separation and purification techniques, such as protein and enzyme purification, mostly due to its easy and fast operational procedure by using an external permanent magnet. Herein, Fe3O4 magnetic nanoparticles were prepared, and surface modification was performed with vanadate and phosphate salts to yield four various model of magnetic nanocarriers. Affinity ligands which are used for immobilization on the nanocarriers leading to the development of appropriative nanocarriers for the affinity separation of alkaline phosphatase from the bovine milk. The findings showed that the use of sodium hexametaphosphate affinity ligand attached to the carrier with an 18-atom linker leads to better separation of alkaline phosphatase from the bovine milk with 14.1-fold purification efficiency. All results confirmed that our designed nanocarriers can purify alkaline phosphatase using a fast and low-cost approach.

Identification of midgut membrane proteins from different instars of Helicoverpa armigera (Lepidoptera: Noctuidae) that bind to Cry1Ac toxin.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes.

A novel feather-degrading bacterium named CA-1 was isolated from the gut of the spider Chilobrachys guangxiensis, which degrades native whole chicken feathers within 20 h. The CA-1 was confirmed to belong to Stenotrophomonas maltophilia based on morphologic and molecular analysis. Maximum feather degradation activity of the bacterium was observed at 37 °C in basal feather medium (NaCl 0.5 g/L, KH2PO4 0.3 g/L, K2HPO4 0.4 g/L, feather powder 10.0 g/L, pH 8.0), which was inhibited when glucose and ammonium nitrate were added in the medium. Furthermore, the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC-MS/MS. Three enzymes, namely alkaline serine protease (29.1 kDa), ABC transporter permease (27.5 kDa), and alkaline phosphatase (40.8 kDa), were isolated and identified from the supernatant of the optimal culture and were considered to play principal roles. On the other hand, the potential synergic effects of the three proteins in S. maltophilia CA-1 feather degradation system were analyzed theoretically. CA-1 may product outer-membrane vesicles comprised of membranes and periplasmic proteins in the feather medium. The newly identified CA-1 and its synergic enzymes provide a new insight into further understanding the molecular mechanism of feather degradation by microbes. They also have potential application in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the three newly identified enzymes.

Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii.

To gain insight into the effect of Cu2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii, the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography. We studied Cu2+-mediated inhibition and aggregation of ALP, and found that Cu2+ significantly inactivated ALP activity with an IC50 of 1.47 ±â€¯0.02 mM. We further revealed that Cu2+ reversibly inhibited ALP in a mixed-type manner with Ki = 0.41 ±â€¯0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii.

Identificación de hipofosfatasia en la práctica clínica: manifestaciones clínicas y recomendaciones diagnósticas en pacientes adultos / Identification of hypophosphatasia in a clinical setting: Clinical manifestations and diagnostic recommendations in adult patients

No disponible

Arsanilic acid modified superparamagnetic iron oxide nanoparticles for Purification of alkaline phosphatase from hen's egg yolk.

Recent studies of magnetic carrier technology have focused on its applications in separation and purification technologies, due to easy separation of the target from the reaction medium by applying an external magnetic field. In the present study, Fe3O4 superparamagnetic nanoparticles were prepared to utilize a chemical co-precipitation method, then the surfaces of the nanoparticles were modified with arsanilic acid derivatives which were used as the specific nanocarriers for the affinity purification of alkaline phosphatase from the hen's egg yolk. The six different types of magnetic nanocarriers with varied lengths of the linkers were obtained. All samples were characterized step by step and validated using FTIR, SEM, EDX, VSM and XRD analysis methods As the results were shown, the use of inflexible tags with long linkers on the surface of the nanocarrier could lead to better results for separation of alkaline phosphatase from the hen's egg yolk with 76.2% recovery and 1361.7-fold purification. The molecular weight of the purified alkaline phosphatase was estimated to be 68kDa by SDS-PAGE. The results of this study showed that the novel magnetic nanocarriers were capable of purifying alkaline phosphatase in a practically time and cost effective way.

Lysophosphatidylcholine elicits intracellular calcium signaling in a GPR55-dependent manner.

The GPR55 signaling is fertile ground for drug discovery, however despite considerable research progress during the past 10 years, many open questions remain. The GPR55 pharmacology remains controversial, as many ligands have been reported with inconsistent results. Here, we show that various molecular species of lysophosphatidylcholine (LPC) elicit intracellular Ca2+ mobilization in GPR55-expressing PC-3 human prostate carcinoma cells. The response was even stronger than [Ca2+]i flux evoked by endogenous (OEA) and synthetic (Abn-CBD) agonists. Treatment with GPR55 antagonists CID16020046 and ML193 as well as the lipid raft disrupter methyl-ß-cyclodextrin strongly blunted LPC-induced calcium signal. Additionally, molecular modeling analysis revealed that LPC 16:0 and LPC 18:1 interact stronger with the receptor than to OEA. Identified electrostatic interactions between GPR55 residues and the ligands overlap with the binding site identified previously for lysophosphatidylinositol. Therefore, we prove that LPC is another GPR55-sensitive ligand. This finding is relevant in understanding lysophospolipids-mediated signaling and opens new avenues to develop therapeutic approach based on GPR55 targeting.

Label-free upconversion nanoparticles-based fluorescent probes for sequential sensing of Cu2+, pyrophosphate and alkaline phosphatase activity.

An efficient near-infrared fluorescence probe has been developed for the sequential detection of Cu2+, pyrophosphate (P2O74-, PPi), and alkaline phosphatase (ALP), which is based on the "off-on-off" fluorescence switch of branched polyethyleneimine (PEI)-capped NaGdF4:Yb/Tm upconversion nanoparticles (UCNPs). The fluorescence is quenched via energy transfer from UCNPs to Cu2+ for the coordination of PEI with Cu2+. The strong affinity between Cu2+ and PPi leads to the formation of Cu2+-PPi complex and results in the detachment of Cu2+ from the surface of UCNPs, thus the fluorescence is triggered on. ALP-directed hydrolysis of PPi causes the disassembly of Cu2+-PPi complex and re-conjugation between Cu2+ with PEI, which leads to the switch-off fluorescence of UCNPs. The system allows sequential analysis of Cu2+, PPi, and ALP by modulating the switch of the fluorescence of UCNPs with detection limits of 57.8nM, 184nM, and 0.019U/mL for Cu2+, PPi, and ALP, respectively. By virtue of the NIR feature and excellent biocompatibility, the UCNPs-based probes are suitable for bioimaging. Taking Cu2+ visualization as a model, the nanoprobes have been successfully applied for intracellular imaging of Cu2+ in living cells.