Alkaline Phosphatase Substrate Research Articles

Metal-Organic Framework-Based Nanovaccine for Relieving Immunosuppressive Tumors via Hindering Efferocytosis of Macrophages and Promoting Pyroptosis and Cuproptosis of Cancer Cells.

Current cancer vaccines face challenges due to an immunosuppressive tumor microenvironment and their limited ability to produce an effective immune response. To address the above limitations, we develop a 3-(2-spiroadamantyl)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane (alkaline phosphatase substrate) and XMD8-92 (extracellular signal-regulated kinase 5 inhibitor)-codelivered copper-tetrahydroxybenzoquinone (Cu-THBQ/AX) nanosized metal-organic framework to in situ-generate therapeutic vaccination. Once inside the early endosome, the alkaline phosphatase overexpressed in the tumor cells' membrane activates the in situ type I photodynamic effect of Cu-THBQ/AX for generating •O2-, and the Cu-THBQ/AX catalyzes O2 and H2O2 to •O2- and •OH via semiquinone radical catalysis and Fenton-like reactions. This surge of ROS in early endosomes triggers caspase-3-mediated proinflammatory pyroptosis via activating phospholipase C. Meanwhile, Cu-THBQ/AX can also induce the oligomerization of dihydrolipoamide S-acetyltransferase to trigger tumor cell cuproptosis. The production of •OH could also trigger the release of XMD8-92 for effectively inhibiting the efferocytosis of macrophages to convert immunosuppressive apoptosis of cancer cells into proinflammatory secondary necrosis. The simultaneous induction of pyroptosis, cuproptosis, and secondary necrosis effectively converts the tumor microenvironment from "cold" to "hot" conditions, making it an effective antigen pool. This transformation successfully activates the antitumor immune response, inhibiting tumor growth and metastasis.

A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development

Botulinum neurotoxins (BoNTs) serotypes A and B are the two most common of the four BoNTs that cause the high mortality botulism disease in individuals consuming contaminated foods. The gold standard assay for BoNT detection is the live mouse bioassay, which has several major disadvantages, including tedious procedures and animal sacrifice requirements. In this study, we developed an immuno-based assay using magnetic streptavidin nanoparticles (mSNP) functionalized with specific synthetic biotinylated, 6xHis-tagged peptide substrates (Peptides A, PA and B, PB) designed for BoNT/A and BoNT/B proteolytic reactions, respectively. In the presence of active toxins that possess endopeptidase activity, upon cleavage, the released fragments with His-tag were dotted on a blotting membrane, ultimately producing color signals after incubation with anti-His antibody, alkaline phosphatase (AP)-conjugated antibody, and then AP substrates. The results showed that the efficiency of peptide-mSNP complex formation reached up to 81%, and the dot blot immunoassay allowed peptide detection from 10 ng of His-tagged peptides. Preliminary testing with the extracellular extracts from the isolated Clostridium botulinum strains indicated that the botulinum toxin in the 2020 botulism outbreak in Vietnam belonged to serotype A, the most potent BoNT. The established assay could be applied to construct a portable biosensor for BoNT detection and a high throughput device to screen potential BoNT inhibitors for drug development.

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

A Ratiometric Biosensor Containing Manganese Dioxide Nanosheets and Nitrogen-Doped Quantum Dots for 2,4-Dichlorophenoxyacetic Acid Monitoring.

Nanomaterials are desirable for sensing applications. Therefore, MnO2 nanosheets and nitrogen-doped carbon dots (NCDs) were used to construct a ratiometric biosensor for quantification of 2,4-dichlorophenoxyacetic acid. The MnO2 nanosheets drove the oxidation of colorless o-phenylenediamine to OPDox, which exhibits fluorescence emission peaks at 556 nm. The fluorescence of OPDox was efficiently quenched and the NCDs were recovered as the ascorbic acid produced by the hydrolyzed alkaline phosphatase (ALP) substrate increased. Owing to the selective inhibition of ALP activity by 2,4-D and the inner filter effect, the fluorescence intensity of the NCDs at 430 nm was suppressed, whereas that at 556 nm was maintained. The fluorescence intensity ratio was used for quantitative detection. The linear equation was F = 0.138 + 3.863·C 2,4-D (correlation coefficient R2 = 0.9904), whereas the limits of detection (LOD) and quantification (LOQ) were 0.013 and 0.040 μg/mL. The method was successfully employed for the determination of 2,4-D in different vegetables with recoveries of 79%~105%. The fluorescent color change in the 2,4-D sensing system can also be captured by a smartphone to achieve colorimetric detection by homemade portable test kit.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Specializing Carbon Nanozyme Active Sites for Sensitive Alkaline Phosphatase Activity Metal-Free Detection.

As biological enzymes regulate metabolic processes, alkaline phosphatase (ALP) is a critical diagnostic indicator associated with many diseases. To accurately measure the enzyme activity, nanozymactic materials can offer sensitive strategies for ALP detection. However, nanozymes often lack specific target binding sites, and the presence of common co-components, e. g., metal ions, may cause false-positive or false-negative results in enzyme activity determination. Herein, we developed a colorimetric assay for ALP detection using metal-free nanozymatic carbon dots (CDs). The ALP hydrolysis of pyrophosphate ions (PPi) to phosphate ions (Pi) induces a "turn-on" response based on the nanozyme activity. This PPi-induced inhibition mechanism is extensively studied via the Michaelis-Menten model, revealing that PPi acts as a noncompetitive inhibitor for CDs at a binding site distinct from the common nanozyme active site. With superior responses to ALP substrates, a highly sensitive and selective method is established for sensing ALP activity with a linear range of 0.010-0.200 U/L and a detection limit of 0.009 U/L. This finding explores the recognition and binding behavior of nanozymes, allowing for precise and reliable measurements even in complex samples, and represents a significant breakthrough for nanozyme-based assays in biological analysis.

Evaluating Alkaline Phosphatase-Instructed Self-Assembly of d-Peptides for Selectively Inhibiting Ovarian Cancer Cells.

Cancer is a major public health concern requiring novel treatment approaches. Enzyme-instructed self-assembly (EISA) provides a unique approach for selectively inhibiting cancer cells. However, the structure and activity correlation of EISA remains to be explored. This study investigates new EISA substrates of alkaline phosphatase (ALP) to hinder ovarian cancer cells. Analogues 2-8 were synthesized by modifying the amino acid residues of a potent EISA substrate 1 that effectively inhibits the growth of OVSAHO, a high-grade serous ovarian cancer (HGSOC) cell line. The efficacy of 2-8 against OVSAHO was assessed, along with the combination of substrate 1 with clinically used drugs. The results reveal that substrate 1 displays the highest cytotoxicity against OVSAHO cells, with an IC50 of around 8 μM. However, there was limited synergism observed between substrate 1 and the tested clinically used drugs. These findings indicate that EISA likely operates through a distinct mechanism that necessitates further elucidation.

First Report of Pectobacterium versatile Causing Stem Rot of Tobacco in China.

Tobacco is one of the most significant non-food cash crops (Lu et al. 2020). In March 2022, cigar tobacco plants showing characteristic symptoms of vascular discoloration, stem rotting, leaf wilting and rotting were observed in Tengchong city (N 25°3'26″, E 98°25'6″) of Yunnan province, China (Fig. S1). The disease incidence was about 5% on cultivar Yunxue 6 in a 33-ha field. Infected stems were collected from Tengchong for pathogen isolation and 16S rDNA sequence analysis was performed as previously described (Lu et al. 2021). Sequence analysis showed that tobacco isolates (GenBank accession numbers: ON795108, ON795107 and ON795106) had an identical sequence with that of the species type strain of Pectobacterium versatile CFBP 6051T and shared the sequence identities of 99.55% and 99.47% with P. carotovorum DSM 30168T and P. parvum s0421T, respectively. Furthermore, phylogenetic analysis showed that tobacco strains were clustered with Pectobacterium versatile CFBP 6051T (Fig. S2a). In API assays, strain 22TC1 was positive for β-galactosidase activity, reduction of nitrates to nitrites, fermentation of glucose, hydrolysis of esculin and gelatin, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, malic acid and trisodium citrate; positive for the enzymatic substrates of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase and α-glucosidase. Furthermore, the average nucleotide identity (ANI) analysis (Richter et al. 2015) showed that strain 22TC1 (GenBank accession number: JAMWYQ000000000) had the highest ANIb score of 96.76% and ANIm value of 97.19% with P. versatile CFBP 6051T. Similarly, in silico DNA-DNA hybridization (isDDH) value was 74.5% compared to P. versatile CFBP 6051T, isDDH values were 35.5-63.7% with the other Pectobacterium species, which below the 70% threshold value for species delineation (Meier-Kolthoff et al. 2021). The phylogenomic analysis also showed that strain 22TC1 was clustered with the species type strain of P. versatile CFBP 6051T. For pathogenicity tests, cell suspension with ten-fold dilution (approx. 1 x 108 CFU/ml) was injected into the leaf axils of two 2-month-old tobacco stems (cv. Yunyan 87). As a control, tobacco seedlings were inoculated with sterile distilled water. The plants were sealed in plastic bags and maintained in a growth chamber at 28°C for 2 d. The symptoms of water-soaked decay were observed within 24 h of inoculation. Whole-plant decay was at 2 days after injection. No symptoms were developed in the controls. Reisolation was performed on diseased stems and the identity of isolated bacteria was confirmed by PCR and sequencing of 16S rRNA. Similar results were obtained in two independent experiments. Based on the above-described data, the causal pathogen of stem rot on cigar tobacco in Tengchong was identified as P. versatile. To our knowledge, this is the first time that P. versatile is found to cause stem rot on tobacco. Pectobacterium species have been reported to cause seed-borne diseases on tobacco seedlings in the floating tray system and soil-borne diseases in tobacco fields (Wang et al. 2017; Xia and Mo 2007). Therefore, studying the possible transmission of the P. versatile to tobacco plants is necessary.

A label-free ThT-assisted fluorescence detection strategy of alkaline phosphatase activity based on MnO2 nanosheets

Alkaline phosphatase (ALP) is a metalloenzyme, the level of which is clinically significant as an abnormality of ALP activity results in several diseases. In the present study, we introduced a MnO2 nanosheet-based assay for ALP detection employing the adsorption and reduction characteristics of G-rich DNA probes and ascorbic acid (AA), respectively. Ascorbic acid 2-phosphate (AAP) was utilized to act as a substrate for ALP which hydrolyzes AAP generating AA. In the absence of ALP, MnO2 nanosheets adsorb the DNA probe destructing the G-quadruplex formation and showing no fluorescence emission. On the contrary, being present in the reaction mixture ALP hydrolyzes AAP yielding AA, then the AA reduce the MnO2 nanosheets into Mn2+, hence, the probe is free to react with a dye, thioflavin T (ThT), and synthesizes ThT/G-quadruplex to spark high fluorescence intensity. Therefore, under optimized conditions (250 nM DNA probe, 8 μM ThT, 96 μg/mL MnO2 nanosheets, and 1 mM AAP) the sensitive and selective measurement of ALP activity can be achieved through the change of fluorescence intensity, with a linear range and a limit of detection of 0.1–5 U/L and 0.045 U/L. Our assay exhibited its potential to assess the ALP inhibitor when in an inhibition assay Na3VO4 inhibited ALP with an IC50 value of 0.137 mM and also was validated in clinical samples.

A rare case of adult hypophosphatasia masked with primary hyperparathyroidism

Abstract Introduction Hypophosphatasia (HPP) is a rare inherited disorder of bone and mineral metabolism due to mutations in the Alkaline Phosphatase-Liver (ALPL) gene encoding the tissue nonspecific alkaline phosphatase (TNSALP). Clinical features of HPP in adults include early loss of teeth, muscle weakness, musculoskeletal pain, osteoporosis and fragility fractures with delayed healing. The most typical biochemical manifestation of the disease is persistently low level of alkaline phosphatase (ALP). Primary hyperparathyroidism (PHPT) is a relatively common endocrine disorder leading to increase in bone turnover markers including ALP. Coexistence of HPP and PHPT is a very rare condition. Herein we present an adult HPP patient who presented with primary hyperparathyroidism and normal serum ALP levels, which decreased significantly after parathyroidectomy. Clinical Case A 33-year-old female with a past medical history of Hashimoto's thyroiditis and acne rosacea admitted to our outpatient clinic because of gradually increasing bone pain, fatigue and weakness. She had early teeth loss and dental problems, but she denied history of fractures and kidney stones. Her family history was remarkable for nephrolithiasis in both parents. On physical examination the patient had facial rash and telangiectasia. She also had teeth loss and many dental cavities. Her vital signs were within normal limits. In biochemical investigations creatinine was 0.45 mg/dL (0.7–1.4), ALP 81 U/L (35–105), PTH 531 pg/mL (15–65), calcium 10.84 mg/dL (8.5–10.5), albumin 4.9 g/dL (3.2–5.5), phosphorus 2.25 mg/dL (2.7–4.5), Vitamin D 5 ng/mL and urine calcium excretion 613 mg/day. Laboratory results were compatible with normocalcemic hyperparathyroidism and severe vitamin D deficiency. Bone mineral density of the lumbar spine and the femoral neck revealed osteopenia and 1/3 distal radius revealed osteoporosis. There was no evidence of nephrolithiasis and nephrocalcinosis on abdominal ultrasound. Neck ultrasound and CT scan and Tc-99m MIBI scanning of the patient revealed a parathyroid adenoma in the posteroinferior of thyroid gland's left lobe. A successful parathyroidectomy was performed. During follow-up the patient's serum ALP levels decreased significantly to 29 U/L with a repeat being 26 U/L. Bone-specific ALP was 4.2 μg/L (3–14 μg/L). Urine phosphoethanolamine was elevated to 68.6 μmol/g.Cr (0–48), which supported the diagnosis of HPP. Genetic analysis was performed and heterozygous p.N190del (c.568_570delAAC/c.566_568delACA) mutation was detected. This mutation was previously reported as pathogenic and has been associated with adult hypophosphatasia. Conclusion Although persistently low level of ALP is characteristic of HPP, disorders that increase bone turnover may result in an increase in serum ALP levels, which may mask underlying HPP. Acknowledgements We are thankful to Alexion Pharmaceuticals, Inc for providing fund for ALP substrates and genetic analysis of the patient.

A new fluorescent biosensor based on inner filter effect and competitive coordination with the europium ion of non-luminescent Eu-MOF nanosheets for the determination of alkaline phosphatase activity in human serum

The abnormal level of alkaline phosphatase (ALP) in human blood stream indicates health problem. A novel fluorescent sensing system based on competitive coordination with europium ions (Eu3+) integrated in a non-luminescent lanthanide-metal organic framework nanosheets (NO2-Eu-MOF NS) was developed for ALP analysis in human serum. The fluorescence signal is turned-on by the antenna ligand (1,4-phenylenediphosphonic acid (PEPP)) that competitively coordinates with Eu3+ ions from the nanosheet to produce emissive species (Eux(PEPP)y(NO2BDC)z) in-situ. 4-Nitrophenyl phosphate (PNPP) is a substrate of ALP and specifically hydrolyzed into 4-nitrophenol. PNPP exhibits a strong absorption in the UV-Vis region (250–400 nm), which covers the excitation wavelength (280 nm) of Eux(PEPP)y(NO2BDC)z. Therefore, the existing of PNPP causes Eux(PEPP)y(NO2BDC)z non-emissive and, after hydrolysis with ALP, Eux(PEPP)y(NO2BDC)z can be excited and give a detectable fluorescence signal. Based on the fluorescence change, quantitative analysis of ALP activity is thus achievable. This biosensing system was demonstrated in the determination of ALP activity with good linear relationship in the range of 5–1000 U L−1. The limit of detection was 1.1 U L−1 (S/N = 3). The result reveals that the biosensor exhibits excellent sensing performance and anti-interference ability. It could be potentially utilized for clinical routine analysis of ALP activity in human serum.

A novel and sensitive ratiometric fluorescent quantum dot-based biosensor for alkaline phosphatase detection in biological samples via the inner-filter effect.

Alkaline phosphatase (ALP) is an important biomarker whose abnormal level in activity is associated with hepatobiliary, skeletal, and renal diseases as well as cancer. Herein, we synthesized ZnSe@ZnS quantum dots (ZnSe@ZnS QDs) and Mn-doped ZnS quantum dots (Mn:ZnS QDs) as fluorophores to establish the ratiometric fluorescent assay for ALP activity detection in biological samples. p-Nitrophenyl phosphate (PNPP) was used as a substrate for ALP, and the overlaps between absorption spectra of PNPP and excitation spectra of QDs resulted in sharp fluorescence quenching. Under the catalysis of ALP, PNPP was hydrolyzed into p-nitrophenol (PNP), which caused a red shift of absorption band of PNPP and fluorescence recovery of Mn:ZnS QDs (585 nm). However, the overlaps between absorption spectra of PNP and emission spectra of ZnSe@ZnS QDs led a further quenching of ZnSe@ZnS QDs (405 nm). Therefore, the ratiometric fluorescent signals (F 585/F 405) were associated with activity of ALP based on bidirectional responses of QDs to the concentration of PNPP. Under the optimum conditions, the method exhibited a good linear relationship from 4 to 96 U per L (R 2 = 0.9969) with the detection limit of 0.57 U per L. Moreover, the method was successfully applied for detecting the ALP activity in a complex biological matrix (human serum and HepG2 cells) with impressive specificity. In particular, the complicated chemical modifications of QDs and pretreatments of biological samples were not required in the whole detection procedures. Therefore, it not only provided a sensitive, specific and simple approach to clinical ALP activity detection, but it also provided support for early diagnosis of diseases.

Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho=0.2). There was also no significant difference between the sexes. These preliminary reference intervals will help assess the health of seahorses.

Diagnostic Approach to Patients with Low Serum Alkaline Phosphatase.

Increased serum levels of alkaline phosphatase (ALP) are widely recognized as a biochemical marker of many disorders affecting the liver or bone. However, the approach for patients with low ALP phosphatase is not well-established. Low serum ALP is an epiphenomenon of many severe acute injuries and diseases. Persistently low serum ALP may be secondary to drug therapy (including antiresorptives) or a variety of acquired disorders, such as malnutrition, vitamin and mineral deficiencies, endocrine disorders, etc. Hypophosphatasia, due to pathogenic variants of the ALPL gene, which encodes tissue non-specific ALP, is the most common genetic cause of low serum ALP. Marked bone hypomineralization is frequent in severe pediatric-onset cases. However, adult forms of hypophosphatasia usually present with milder manifestations, such as skeletal pain, chondrocalcinosis, calcific periarthritis, dental problems, and stress fractures. The diagnostic approach to these patients is discussed. Measuring several ALP substrates, such as pyrophosphate, pyridoxal phosphate, or phosphoethanolamine, may help to establish enzyme deficiency. Gene analysis showing a pathogenic variant in ALPL may confirm the diagnosis. However, a substantial proportion of patients show normal results after sequencing ALPL exons. It is still unknown if those patients carry unidentified mutations in regulatory regions of ALPL, epigenetic changes, or abnormalities in other genes.

Dual-product synergistically enhanced ratiometric fluorescence assay for alkaline phosphatase activity using core-shell lanthanide-based nanoprobe

We developed a new core-shell ratiometric fluorescent nanoprobe simply prepared by a seeded growth method. The strong luminescence arising from the shell formed by coordination self-assembly of an alkaline phosphatase (ALP) substrate, l-ascorbic acid 2-phosphate (AAP), and Tb3+, together with the weak fluorescence of the core-a metal-organic framework, UiO-66-NH2, constitutes an ideal dual-emission characteristic. Since AAP can be specifically cleaved by ALP, the well-formulated core-shell nanostructure was destroyed upon exposure to ALP. In this case, the luminescence of Tb3+ was quenched due to the inefficient antenna effect, while the fluorescence of UiO-66-NH2 was strengthened by the synergistical enhancement of dual hydrolysates to inhibit the ligand-to-metal charge transfer (LMCT) process. Using the dual signal response, this nanoprobe was employed for ratiometric fluorescence detection of ALP activity in the range of 0.05–0.6 U mL−1 with a detection limit of 0.018 U mL−1, accompanied by a discernible fluorescence color evolution from turquoise to blue. In virtue of good properties in accuracy, sensitivity, selectivity and simplicity, this assay enabled quantitative detection of ALP activity in human serum and efficient screening of ALP inhibitors. More Importantly, a smartphone-assisted paper-based sensing platform was designed for on-site visual analysis of ALP activity in human serum, which may be very promising as a facile, portable, and robust format to facilitate relevant biological and clinical applications.

A novel electrochemical platform for assay of alkaline phosphatase based on amifostine and ATRP signal amplification.

Alkaline phosphatase (ALP), an important hydrolase involved in dephosphorylation, is a common clinical indicator of many diseases. In the present study, we constructed a novel electrochemical sensor using amifostine as the substrate of ALP and activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) as a signal amplification strategy for sensitive determination of ALP activity. In particular, in the presence of ALP, the phosphate group of amifostine was hydrolyzed to form a sulfhydryl group, which could attach to a gold electrode via a sulfur-gold bond. Then, the initiator α-bromophenylacetic acid (BPAA) was linked to the hydrolysis product of amifostine through an amide bond, resulting in the production of electroactive polymer chainson the gold electrode by the monomer ferrocenylmethyl methacrylate (FMMA) via ARGET ATRP. Under optimal parameters, the electrochemical sensor demonstrated a limit of detection (LOD) of 1.71 mU mL-1 with a linear range of 5-100 mU mL-1. In addition to satisfactory selectivity, the potential application of this approach for ALP activity detection in human serum samples was demonstrated. Due to its efficiency, simplicity of operation, and cost-effectiveness, the proposed electrochemical sensor has great promise as a universal method for ALP assays and inhibitor screening.

In situ reaction-based ratiometric fluorescent assay for alkaline phosphatase activity and bioimaging

Alkaline phosphatase (ALP) is an important biomarker, it is of great significance to develop a sensitive and efficient analytical method for ALP. In this study, an in situ reaction based ratiometric fluorescence assay for ALP was proposed. l-ascorbic acid-2-phosphate (AA2P) was used as a substrate for ALP, and Cu2+/o-phenylenediamine (OPD) were involved in this system. Cu2+ can oxidize OPD to 2,3-diaminophenazine (OPDox) with an emission centered at 566 nm. The presence of ALP can catalyze the hydrolysis of AA2P to ascorbic acid (AA), which will inhibit the production of OPDox and reduce the corresponding fluorescence intensity, and AA will react with OPD to generate 3-(dihydroxyethyl)furan[3,4-b]quinoxalin-1-one (DFQ) with an emission peak at 447 nm. The fluorescence ratio of F447/F566 has a linear relationship with ALP activity. The proposed method is highly sensitive, finely selective, cost efficiency and easy to operate, it exhibits good linearity in the range of 0.5–22 and 22–40 mU·mL−1, with a detection limit as low as 0.06 mU·mL−1. The excellent applicability of this strategy in human serum samples and MCF-7 cells imaging suggests that this method has promising prospects for biomedical research.

Dental manifestations in adult hypophosphatasia and their correlation with biomarkers.

Hypophosphatasia (HPP) is a genetic condition with broad clinical manifestations caused by alkaline phosphatase (ALP) deficiency. Adults with HPP exhibit a wide spectrum of signs and symptoms. Dental manifestations including premature tooth loss are common. Much of the published literature reporting dental manifestations consists of case reports and series of symptomatic patients, likely biased towards more severe dental manifestations. The objective of this study was to systematically explore the dental manifestations among adults with HPP by conducting a comprehensive dental evaluation. To minimize bias, the study explored dental manifestations in an unselected cohort of adults with HPP. Participants were identified searching electronic health record (EHR) data from a rural health system to discover adults with persistent ALP deficiency. Heterozygotes with pathogenic (P), likely pathogenic (LP), or uncertain variants (VUS) in ALPL and at least one elevated ALP substrate were defined as adults with HPP and underwent genetic, dental, oral radiographic, and biomarker evaluation. Twenty‐seven participants completed the study. Premature tooth loss was present in 63% (17/27); 19% (5/27) were missing eight or more teeth. Statistically significant associations were found between premature permanent tooth loss and HPP biomarkers ALP (p = 0.049) and bone‐specific ALP (p = 0.006). Serum ALP (ρ = −0.43, p = 0.037) and bone‐specific ALP (ρ = −0.57, p = 0.004) were negatively correlated with number of teeth lost prematurely. As noted with tooth loss, periodontal breakdown was associated with bone‐specific ALP. An inverse association between periodontal breakdown and bone‐specific ALP was observed (p = 0.014). These findings suggest a role for ALP in maintenance of dentition.

3D origami paper-based ratiometric fluorescent microfluidic device for visual point-of-care detection of alkaline phosphatase and butyrylcholinesterase

On-site multiplex enzyme detection is crucial for diagnosis, therapeutics and prognostic. To date, it is still a daunting challenge to develop portable, low-cost, and efficient multi-enzyme detection methods. Herein, a novel sample-in-result-out platform integrating ratiometric fluorescent assays with 3D origami microfluidic paper-based device (μPAD) was developed for simultaneous visual point-of-care testing (POCT) of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Cascade catalytic reaction with the same two fluorescent signal indicators was rationally designed to ratiometric fluorescent detection of ALP and BChE: substrate of ALP (pyrophosphate) and product of BChE (thiocholine) can strongly complex with Cu2+, Cu2+ oxidizes o-phenylenediamine to fluorescent 2,3-diaminophenazine (oxOPD) (emission, 565 nm), oxOPD quenches the fluorescence of carbon dots (CDs, emission at 445 nm) via inner filter effect, thus oxOPD/CDs values are relevant to ALP and BChE activities. Then 3D origami μPAD composing of four layers and two parallel channels was fabricated and simply prepared by one-step plotting with black oil-based marker and specific metal molds. After simple folding and unfolding neighboring layers to sequentially initiate reactions of pre-loaded reagents, fluorescent images on the detection zone can be captured by smartphone and analyzed by red-green-blue software for quantitative analysis. Under optimal conditions, the proposed platform was successfully performed to detect ALP and BChE with activity difference at 3 orders of magnitude in human serum samples without any pretreatment procedures. Excellent selectivity, good precision, favorable linear range, and high accuracy were exhibited. Importantly, the platform opens a promising horizon for high-throughput POCT of multiplex biomarkers.

Screening for hypophosphatasia: does biochemistry lead the way?

Patients with childhood hypophosphatasia (HPP) often have unspecific symptoms. It was our aim to identify patients with mild forms of HPP by laboratory data screening for decreased alkaline phosphatase (AP) within a pediatric population. We conducted a retrospective hospital-based data screening for AP activity below the following limits: Girls:≤12 years: <125U/L; >12 years: <50U/L Boys:≤14 years: <125U/L; >14 years: <70U/L. Screening positive patients with otherwise unexplained hypophosphatasemia were invited for further diagnostics: Re-test of AP activity, pyridoxal 5'-phosphate (PLP) in hemolyzed whole blood, phosphoethanolamine (PEA) in serum and urine, and inorganic pyrophosphate in urine. Sequencing of the ALPL gene was performed in patients with clinical and/or laboratory abnormalities suspicious for HPP. We assessed a total of 14,913 samples of 6,731 patients and identified 393 screening-positive patients. The majority of patients were excluded due to known underlying diseases causing AP depression. Of the 30 patients who participated in the study, three had a decrease in AP activity in combination with an increase in PLP and PEA. A heterozygous ALPL mutation was detected in each of them: One patient with a short stature was diagnosed with childhood-HPP and started with enzyme replacement therapy. The remaining two are considered as mutation carriers without osseous manifestation of the disease. A diagnostic algorithm based on decreased AP is able to identify patients with ALPL mutation after exclusion of the differential diagnoses of hypophosphatasemia and with additional evidence of increased AP substrates.