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Nanoceria have demonstrated a wide array of catalytic activity similar to natural enzymes, holding considerable significance in the colorimetric detection of alkaline phosphatase (ALP), which is a biomarker of various biological disorders. However, the issues of physiological stability and formation of protein corona, which are strongly related to their surface chemistry, limit their practical application. In this work, CeO 2 nanoparticles characterized by enhanced dimensional uniformity and specific surface area were synthesized, followed by encapsulation with various polymers to further increase catalytic activity and physiological stability. Notably, the CeO 2 nanoparticles encapsulated within each polymer exhibited improved catalytic characteristics, with PAA-capped CeO 2 exhibiting the highest performance. We further demonstrated that the PAA-CeO 2 obtained with enhanced catalytic activity was attributed to an increase in surface negative charge. PAA-CeO 2 enabled the quantitative assessment of AA activity within a wide concentration range of 10 to 60 μM, with a detection limit of 0.111 μM. Similarly, it allowed for the evaluation of alkaline phosphatase activity throughout a broad range of 10 to 80 U/L, with a detection limit of 0.12 U/L. These detection limits provided adequate sensitivity for the practical detection of ALP in human serum., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Intermittent and continuous mechanical loads are known to influence osteogenic activity. The present study examines the effects of matched intermittent and continuous load in vitro on bone formation markers. MC3T3 (mouse pre-osteoblasts) were cultured and placed in a bioreactor to undergo continuous, intermittent, or unloading for 1, 3 and 12 days. Loading conditions were matched for magnitude, duration and frequency. Each time point was analysed for alkaline phosphatase (ALP) activity, procollagen 1 N-terminal propeptide (PINP) and alizarin red staining (ARS). Intermittent load caused an increase in ALP activity across all time points compared to continuous loading (↑30%-59%) and unloaded conditions (↑70%-90%). PINP concentrations from intermittent load were lower than continuous load (↓112%) on day 3. However, no differences were observed in PINP concentrations between loading conditions at other time points. No differences were observed for ARS between loading conditions. Intermittent load caused an increase in bone formation marker ALP, but not PINP, when compared to continuous loading and unloaded conditions. These findings further our knowledge in bone formation response and provide additional tools for the analysis of osteogenesis in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The present study aimed to investigate the effects of infrared diode laser irradiation on the proliferation and differentiation capacity of periodontal ligament stem cells (hPDLSCs), which are optimal cell sources for periodontal regeneration., Methods: hPDLSCs were isolated and characterized by flow cytometric analysis of mesenchymal stem cell markers, and their trilineage differentiation capacity was tested. hPDLSCs were then cultured and irradiated with infrared diode laser (970 nm) at a power of 200 mW and a fluence of 4 J/cm 2 for 3 s. MTT assay was performed to assess cellular proliferation. Cell cycle analysis was performed, and the impact of infrared diode laser irradiation on the stemness and osteogenic differentiation potential of hPDLSCs was evaluated via RT‒PCR., Results: Infrared diode laser application enhanced the stemness, viability, proliferation, and differentiation of PDLSCs. Stem cell markers (OCT4, SOX2, and NANOG) were significantly upregulated in hPDLSCs exposed to laser irradiation. There was significant overexpression of RUNX2, ALP, OPN, and OCN on day 14 after laser application., Conclusions: These findings provide valuable insights into the specific applications of infrared diode lasers to effectively regenerate periodontal tissues. The results can aid in the development of precise clinical protocols aimed at enhancing osseointegration and promoting tissue regeneration. Ultimately, the combination of infrared diode laser with hPDLSCs is promising for stimulating periodontal regeneration., (© 2024. The Author(s).)

Enzyme-induced in-situ fluorescence is crucial for the development of biosensing mechanisms and correlative spectroscopic analysis. Inspired by simple p-aminophenol (AP)-controlled synthesis and the specific catalytic reaction of 4-aminophenyl phosphate (APP) triggered by alkaline phosphatase (ALP), our research proposed a strategy to prepare carbon dots (CDs) as fluorescent signals for ALP detection using AP and 3-aminopropyltrimethoxysilane (APTMS) as the precursors. The further constructed ratiometric fluorescence sensor reduced the detection limit of ALP to 0.075 μU/mL by a significant margin. Considering the need for point-of-care testing (POCT), we chose agarose for the preparation of portable hydrogel sensors so that even untrained personnel can quickly achieve semi-quantitative visual detection of ALP using colorimetric cards. These results demonstrate the practical applicability of ratiometric fluorescence sensing hydrogel pillar arrays, which are important for high-sensitivity, visualization, and portable rapid enzyme activity assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Temperature sensing is commonly used in point-of-care (POC) detection technologies, yet the portability and convenience of use are frequently compromised by the complexity of thermosensitive processes and signal transduction. Especially, multi-step target recognition reactions and temperature measurement in the reaction vessel present challenges in terms of stability and integration of detection devices. To further combine photothermal reaction and signal readout in one assay, these two processes enable to be integrated into miniaturized microfluidic chips, thereby facilitating photothermal sensing and achieving a simple visual temperature sensing as POC detection., Results: A copper ion (Cu 2+ )-catalyzed photothermal sensing system integrated onto a microfluidic distance-based analytical device (μDAD), enabling the visual, portable, and sensitive quantitative detection of multiple targets, including ascorbic acid, glutathione, and alkaline phosphatase (ALP). The polydopamine nanoparticles (PDA NPs) were synthesized by the regulation of free Cu 2+ through redox or coordination reactions, facilitating the transduction of distinct photothermal response signals and providing the versatile Cu 2+ -responsive sensing systems. Promoted by integration with a photothermal μDAD, the system combines PDA's photothermal responsiveness and thermosensitive gas production of ammonium bicarbonate for improved sensitivity of ALP detection, reaching the detection limit of 9.1 mU/L. The system has successfully achieved on-chip detection of ALP with superior anti-interference capability and recoveries ranging from 96.8 % to 104.7 %, alongside relative standard deviations below 8.0 %., Significance and Novelty: The μDAD design accommodated both the photothermal reaction of PDA NPs and thermosensitive gas production reaction, achieving the rapid sensing of visual distance signals. The μDAD-based Cu 2+ -catalyzed photothermal sensing system holds substantial potential for applications in biochemical analysis and clinical diagnostics, underscored by the versatile Cu 2+ regulation mechanism for a broad spectrum of biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 μg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 μg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 μg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.

This study introduces an innovative approach for the real-time and efficient detection of alkaline phosphatase (ALP) activity, using a calcein fluorescence probe and leveraging the static quenching properties of calcein fluorescence by Ce 3+ metal ions. In this method, calcein serves as the signal element, with its fluorescence effectively preserved through energy transfer or charge transfer when coordinated with Ce 3+ . Conversely, ALP catalyzes the phosphopeptide substrate to generate a substantial amount of Pi, preventing calcein fluorescence quenching due to the higher affinity between Pi and Ce 3+ compared with that between calcein and Ce 3+ . The fluorescence intensity ratio (F-F 0 /F 0 ) exhibited excellent linearity, facilitating sensitive ALP detection. The proposed ALP detection method covers a range from 0 to 1.4 mU/mL (R 2  = 0.9942), with the limit of detection at 0.069 mU/mL (S/N = 3). Additionally, this method was successfully applied for detecting ALP in serum samples and studying its inhibitors. This research introduces a novel clinical diagnosis approach for ALP sensing while broadening the potential applications of calcein., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Alkaline phosphatase (ALP) is abnormally expressed in some cancers and promotes the growth, metastasis, and invasion of cancer cells. The detection of ALP is of great significance for both pathological study and clinical detection. In this work, a europium (Eu)-based fluorescence detection sensor was prepared in a mild reaction condition. LaF 3 :Eu nanoparticles was mixed with ethylene imine polymer (PEI) and Ag + ions. PEI was used as stabilizer and reducing agent, and Ag + ions were reduced as molecular-like silver clusters (ML-Ag NCs). The fluorescence of LaF 3 :Eu nanoparticles was enhanced by ML-Ag NCs through energy transfer. When ascorbic acid 2-phosphate (AAP) was hydrolyzed to ascorbic acid (AA) in the presence of ALP, AA reduced Ag + ions to silver nanoparticles (Ag NPs) and quenched the fluorescence of LaF 3 :Eu/PEI/Ag. The activity of ALP was detected by measuring the fluorescence intensity of Eu 3+ at 618 nm. In the concentration range from 2.0 to 16.0 U/L, the fluorescence intensity ratio ((F 0 -F)/F 0 ) had a linear relationship with the logarithm of ALP concentration. The limit of detection (LOD) was 1.3 U/L. Moreover, the ALP activity was detected successfully in cancer cells by this method. The sensing platform has application potential in the detection of ALP activity in biological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The synthesis of graphene quantum dots-like enriched with specific oxygenated groups (o-GQDs) exhibiting great catalytic performance offers a promising tool for diagnosis and biomedicine, but introducing specific oxygen groups remains a challenge. Here, we propose a mild synthetic protocol for producing regulated fluorescence emission (from blue to yellow) carbonyl functionalized GQDs with double catalytic function through Fe 3 O 4 -catalyzed hydroxyl radical (·OH) oxidation the precursors like graphene oxide, polyaniline (PANI) and polydopamine (PDA). The method can be carried out at room temperature than the traditional high-temperature oxidation in concentrated acid. Interestingly, o-GQDs exhibit excellent peroxidase (POD)- and ascorbate oxidase-like activity. XPS characterization showed a significant increase in carbonyl content in o-GQDs compared to the precursor, even a 14-fold increase in blue-emitting iron-doped GQDs (b-Fe-GQDs). The introduction of Fe 3 O 4 during the synthesis process results in a minor degree of Fe doping, which enhances the catalytic activity of b-Fe-GQDs through coordination with N. Based on this feature, highly sensitive single-signal and ultra-selective dual-signal methods for alkaline phosphatase detection were developed. This low cost and safe synthesis strategy paves the way for practical usage of o-GQDs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Objectives : Enhanced osteoblast differentiation may be leveraged to prevent and treat bone-related diseases such as osteoporosis. No-ozone cold plasma (NCP) treatment is a promising and safe strategy to enhance osteoblast differentiation. Therefore, this study aimed to determine the effectiveness of direct and indirect NCP treatment methods on osteoblast differentiation. Mouse osteoblastic cells (MC3T3-E1) were treated with NCP using different methods, i.e., no NCP treatment (NT group; control), direct NCP treatment (DT group), direct NCP treatment followed by media replacement (MC group), and indirect treatment with NCP-treated media only (PAM group). Materials and Methods : The MC3T3-E1 cells were subsequently assessed for cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and ALP and osteocalcin mRNA expression using real-time polymerase chain reaction. Results : Cell proliferation significantly increased in the NCP-treated groups (DT and PAM; MC and PAM) compared to the NT group after 24 h ( p < 0.038) and 48 h ( p < 0.000). ALP activity was increased in the DT and PAM groups at 1 week ( p < 0.115) and in the DT, MC, and PAM groups at 2 weeks ( p < 0.000) compared to the NT group. Calcium deposition was higher in the NCP-treated groups than in NT group at 2 and 3 weeks ( p < 0.000). ALP mRNA expression peaked in the MC group at 2 weeks compared to the NP group ( p < 0.014). Osteocalcin mRNA expression increased in the MC group at 2 weeks ( p < 0.000) and was the highest in the PAM group at 3 weeks ( p < 0.000). Thus, the effects of direct (DT and MC) and indirect (PAM) treatment varied, with MC direct treatment showing the most significant impact on osteoblast activity. Conclusions : The MC group exhibited enhanced osteoblast differentiation, indicating that direct NCP treatment followed by media replacement is the most effective method for promoting bone formation.

Cupric ions (Cu 2+ ), pyrophosphate (PPi), and alkaline phosphatase (ALP) are involved in a variety of biochemical processes such as DNA replication, cellular metabolism and play an important role in human growth and development. It is of great significance to establish a method for the sensitive detection of Cu 2+ , PPi and ALP. In this work, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were successfully synthesized by a one-pot method using hydrazine sulfate as reductant, exhibiting a unique strong fluorescence emission in the near-ultraviolet region at ∼339 nm. Since the fluorescence of PEI-AgNCs can be quenched by Cu 2+ through inner filtering effect (IFE), then recovered by competitive binding of pyrophosphate and Cu 2+ , and later weakened again by catalytic hydrolysis of alkaline phosphatase, a sensitive and selective strategy based on the changes of fluorescence "ON" or "OFF" was established to detect Cu 2+ , PPi and ALP. The LODs of these three analytes were 36 nM, 0.2 μM, and 0.14 U L -1 at a S/N ratio of 3, respectively. A series of logic gate circuits for sensing cupric ions, pyrophosphate, and alkaline phosphatase were successfully constructed. The established methods have the potential for biosensing and environmental analysis and the specific UV-A fluorescence property of PEI-AgNCs may be helpful in photonic and optical areas., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The rapid epidemic around the world of coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, proves the need and stimulates efforts to explore efficient diagnostic tests for the sensitive detection of the SARS-CoV-2 virus. An aggregation-induced electrochemiluminescence (AIECL) sensor was developed for the ultrasensitive detection of the SARS-CoV-2 nucleocapsid (N) protein in this work. Tetraphenylethylene doped in zeolite imidazole backbone-90 (TPE-ZIF-90) showed highly efficient aggregation-induced emission (AIE) to endow TPE-ZIF-90 with high ECL intensity. Upon the capture of the SARS-CoV-2 N protein by immune recognition, an alkaline phosphatase (ALP)-modified gold nanoparticle (AuNP)-decorated zinc oxide (ZnO) nanoflower (ALP/Au-ZnO) composite was introduced on the sensing platform, which catalyzed L-ascorbate-2-phosphate trisodium salt (AA2P) to produce PO 4 3- and ascorbic acid (AA). Based on a multiquenching of the ECL signal strategy, including resonance energy transfer (RET) between TPE-ZIF-90 and Au-ZnO, disassembly of TPE-ZIF-90 triggered by the strong coordination between PO 4 3- and Zn 2+ , and RET between TPE-ZIF-90 and AuNPs produced in situ by the AA reductive reaction, the constructed AIECL sensor achieved highly sensitive detection of the SARS-CoV-2 N protein with a low limit of detection of 0.52 fg/mL. With the merits of high specificity, good stability, and proven application ability, the present RET- and enzyme-triggered multiquenching AIECL sensor may become a powerful tool in the field of SARS-CoV-2 virus diagnosis.

Alkaline phosphatase is an important biomarker for medical diagnosis. An enzymatic fluorescence supramolecular hydrogel with AIE properties was developed and used for sensing alkaline phosphatase in vitro and in living cells. In the presence of ALP, K(TPE)EFYp was partially converted to the hydrogelator K(TPE)EFY and self-assembled into nanofibers to form Hydrogel. With the sol-gel transition and the AIE effect, the fluorescence emission was turned on. The linear concentration range of ALP activity in vitro quantified by this method was determined as 0-3 U/L with aLODat 0.02 U/L. In addition, cell imaging and serum experiment showed that K(TPE)EFYp could also be used to detect ALP activity in living cells and biological samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Algal growth depends strongly on phosphorus (P) as a key nutrient, underscoring the significance of monitoring P levels. Algal species display a sensitive response to fluctuations in P availability, notably through the expression of alkaline phosphatase (AP) when challenged with P-depletion. As such, alkaline phosphatase activity (APA) serves as a valuable metric for P availability, offering insights into how algae utilize and fix available P resources. However, current APA quantification methods lack single cell resolution, while also being time- and reagent consuming. Microfluidics offers a promising cost-effective solution to these limitations, providing a platform for precise single-cell analysis. In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, Chlamydomonas reinhardtii , when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1-41 mM), C. reinhardtii cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of C. reinhardtii cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses.

The combination of closed bipolar electrodes (cBPE) with electrochemiluminescence (ECL) imaging has demonstrated remarkable capabilities in the field of bioanalysis. Here, we established a cBPE-ECL platform for ultrasensitive detection of alkaline phosphatase (ALP) and two-dimensional imaging of epidermal growth factor receptor (EGFR). This cBPE-ECL system consists of a high-density gold nanowire array in anodic aluminum oxide (AAO) membrane as the cBPE coupled with ECL of highly luminescent cadmium selenide quantum dots (CdSe QDs) luminophores to achieve cathodic electro-optical conversion. When an enzyme-catalyzed amplification effect of ALP with 4-aminophenyl phosphate monosodium salt hydrate (p-APP) as the substrate and 4-aminophenol (p-AP) as the electroactive probe is introduced, a significant improvement of sensing sensitivity with a detection limit as low as 0.5 fM for ALP on the cBPE-ECL platform can be obtained. In addition, the cBPE-ECL sensing system can also be used to detect cancer cells with an impressive detection limit of 50 cells/mL by labeling ALP onto the EGFR protein on A431 human epidermal cancer cell membranes. Thus, two-dimensional (2D) imaging of the EGFR proteins on the cell surface can be achieved, demonstrating that the established cBPE-ECL sensing system is of high resolution for spatiotemporal cell imaging.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (μPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this μPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO 2 ) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H 2 O 2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3'-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A study of an integrated OPECT biosensor gate and the EC color-changing region on the same chip was carried out, achieving sensitive detection through bioetching-induced signal changes. Enzymatic bioetching enables specific alkaline phosphatase (ALP) detection by catalyzing the production of CdS, which modulates the channel current and generates a visual signal.

Background: The utilization of inner filter effect (IFE) brings more opportunities for construction of fluorescence immunoassays but remains a great challenge, especially how to select best donor in the face of extensive fluorescent nanomaterials. Aflatoxin B1 possesses high toxicity among mycotoxins and is frequently found in agricultural products that may significantly threaten to human health. Therefore, with the help of signal transduction mechanism of IFE to develop a convenient and sensitive approach for AFB1 detection is of great significance in ensuring food safety., Results: Herein, the classical alkaline phosphatase (ALP) catalyzes hydrolysis of p-nitrophenylphosphate to produce p-nitrophenol (PNP) was employed as a model reaction, which intends to explore tunable multicolor fluorescence of gold nanoclusters (AuNCs) for matching PNP to maximize IFE efficiency. The luminescent green-emitting AuNCs were selected as an optimal donor in terms of excellent spectral overlap, high photoluminescence, and adequate system adaptability, thus achieving a 22-fold increase in sensitivity improvement compared to colorimetric method for ALP detection. The fluorescence quenching mechanism between PNP and AuNCs was validated as IFE by studying ultraviolet absorption, zeta potentials and fluorescence lifetime. In light of this, we integrated a highly specific antibody-antigen recognition system, efficient enzymatic reaction and excellent optical characteristics of AuNCs to develop dual-mode immunoassay for AFB1 monitoring. The sensitivity of fluorometric immunoassay was lower to 0.06 ng/mL, which obtained a 3.5-fold improvement compared to "gold standard" ELISA. Their practicability and applicability were confirmed in the tap water, corn, wheat and peanuts samples., Significance: This work provides an easy-to-understand screening procedure to select optimal donor-acceptor pairs in IFE analysis. Furthermore, we expect that integration of IFE-based signal conversion strategy into mature immunoassay not only extends the signal types, simplifies signal amplification steps, and reduces the false-positive/false-negative rates, but also provides a simple, convenient, and versatile strategy for monitoring of trace other contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Fabrication of a multi-signal readout assay with high sensitivity and selectivity is highly desirable for clinical and biochemical analysis, but remains a challenge due to laborious procedures, large-scale instruments, and inadequate accuracy. Herein, a straightforward, rapid, and portable detection platform based on palladium(II) methylene blue (MB) coordination polymer nanosheets (PdMBCP NSs) was unveiled for the ratiometric dual-mode detection of alkaline phosphatase (ALP) with temperature and colorimetric signal readout properties. The sensing mechanism is the ALP-catalyzed generation of ascorbic acid for competitive binding and etching PdMBCP NSs to release free MB in a quantitive means for detection. Specifically, ALP addition led to the decrease of temperature signal readout from the decomposed PdMBCP NSs under 808 nm laser excitation, and simultaneous increase of the temperature from the generated MB with a 660 nm laser, together with the corresponding absorbance changes at both wavelengths. Notably, this ratiometric nanosensor exhibited a detection limit of 0.013 U/L (colorimetric) and 0.095 U/L (photothermal) within 10 min, respectively. The reliability and satisfactory sensing performance of the developed method were further confirmed by clinic serum samples. Therefore, this study provides a new insight for the development of dual-signal sensing platforms for convenient, universal, and accurate detection of ALP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study investigated the hormetic responses of soil alkaline phosphatase (ALP) to exogenous Cd under five different vegetation cover types in a typical coastal wetland, including mudflat (Mud), Phragmites australis (PA), Spartina alterniflora (SA), Metasequoia glyptostroboides (MG), and Cinnamomum camphora (CC). The results showed that the activity of soil ALP was significantly enhanced by exogenous 0.3-1.0, 0.2-0.8, 0.05-0.3, 0.05-0.6, and 0.05-0.60 mg Cd /kg in Mud, PA, SA, MG, and CC, respectively. Moreover, the Hor zone (an integrated indicator of the stimulation phase) of Mud and PA was significantly higher than that of SA, MG, and CC. Multiple factor analysis revealed that soil chemical properties and soil bacteria community play an important role in the hormetic effect of soil ALP to Cd stress. Soil electric conductivity (EC) and the relative abundance of Gammaproteobacteria were also identified as key drivers of the hormetic effects of Cd on soil ALP under five vegetation cover types. These findings suggest that the soil ecosystem had better resistance to exogenous Cd stress under mudflat and native species (PA) than invasive species (SA), and artificial forests (MG and CC) when soil ALP activity was the test endpoint. Consequently, this study is beneficial for future ecological risk assessment of soil Cd contamination under divergent vegetation covers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rapid and accurate identification of foodborne pathogens improves public health. Currently employed methods are time-consuming, sensitive to environmental factors, and complex. This study develops a colorimetric sensor for detecting multiple bacteria with one probe using double-enzyme-induced colorimetry. Based on alkaline phosphatase (ALP) in bacteria decomposes L-ascorbic acid 2-magnesium phosphate salt hydrate into ascorbic acid (AA). Manganese dioxide flowers (MnO 2 NFs) can oxidize TMB to etch gold nanorods (Au NRs), which can be inhibited by AA reduction to produce rich colors. Bacteria with varying ALP levels can be identified based on color changes and plasmon resonance wavelength signals produced from Au NRs. Furthermore, the conversion of RGB signals to digital signals and the use of linear discriminant analysis (LDA) allowed 99.57% accuracy in identifying multiple bacteria. It can simultaneously identify five foodborne pathogens across diverse environments (shrimp, meat, milk, etc.). This method may be useful for the rapid and simple identification of foodborne illnesses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Herein, a novel magnetic relaxation sensing strategy based on the change in Fe 3+ content has been proposed by utilizing the conversion of Fe 3+ ions to Prussian blue (PB) precipitates. Compared with the common detection approach based on the valence state change of Fe 3+ ions, our strategy can cause a larger change in the relaxation time of water protons and higher detection sensitivity since PB precipitate can induce a larger change in the Fe 3+ ion concentration and has a weaker effect on the relaxation process of water protons relative to Fe 2+ ions. Then, we employ alkaline phosphatase (ALP) as a model target to verify the feasibility and detection performance of the as-proposed strategy. Actually, ascorbic acid (AA) generated from the ALP-catalyzed L-ascorbyl-2-phosphate hydrolysis reaction can reduce potassium ferricyanide into potassium ferrocyanide, and potassium ferrocyanide reacts with Fe 3+ to form PB precipitates, leading to a higher relaxation time. Under optimum conditions, the method for ALP detection has a wide linear range from 5 to 230 mU/mL, and the detection limit is 0.28 mU/mL, sufficiently demonstrating the feasibility and satisfactory analysis performance of this strategy, which opens up a new path for the construction of magnetic relaxation sensors. Furthermore, this strategy has also been successfully applied to ascorbic acid oxidase detection, suggesting its expansibility in magnetic relaxation detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Exosomes are small extracellular vesicles that can be utilized as noninvasive biomarkers for diagnosis and treatment of cancer and other diseases. This study reports on a strategy involving a hybridized chain reaction-amplified chain coupled with an alkaline phosphatase-induced Ag-shell nanostructure for the ultrasensitive and rapid surface-enhanced Raman scattering immunoassay of exosomes. Exosomes from prostate cancer were captured using prostate-specific membrane antigen aptamer-modified magnetic beads; then, the hybridized chain reaction-amplified chain was released, incorporating a large number of functional moieties with signal amplification effects. Moreover, the steps of traditional immunoassay were simplified using magnetic materials, and the rapid, sensitive, and accurate detection of exosomes was achieved. Results could be obtained within 40 min with a detection limit of 19 particles/μL. Furthermore, the sera of human prostate cancer patients could be easily distinguished from those of healthy controls, highlighting the potential use of exosome analysis in clinical diagnostics.

Numerous studies have reported the clinical value of alkaline phosphatase (ALP) and its bone-specific isoforms (bone-specific alkaline phosphatase (BAP)) in breast cancer. The purpose of this meta-analysis was to summarize the prognostic value of serum ALP and BAP in breast cancer, especially focused on bone metastasis and survival. PRISMA guidelines were followed to conduct this review. Observational studies were searched in PubMed, Cochcrane Library and EMBASE to January 1, 2022. Data were extracted to explore the prognostic value of ALP and BAP. The quality of the included studies was assessed and the outcome effects were evaluated. Subgroup and sensitivity analyses were performed to explore the potential sources of heterogeneity. Publication bias was assessed. There was a total of 53 studies with 22,436 patients included. For the primary outcome of survival, high levels of both ALP and BAP were associated with short survival time. The hazard ratio of high ALP level on overall survival was 1.72 (95% CI 1.37, 2.16, P  < 0.001). For the secondary outcomes, a high ALP level (not BAP) was detected in breast cancer compared with healthy controls, and high levels of both ALP and BAP were risk factors for bone metastasis, while ALP (not BAP) was a risk factor for non-bone metastasis. This study showed that high levels of both serum ALP and BAP were associated with metastasis (BAP was associated with bone metastasis) and survival in breast cancer. The biomarkers could provide useful information for the early diagnostic assessment and monitoring in the follow-up of breast cancer patients.

Alkaline phosphatase (ALP) and interleukin-1beta (IL-1β) are crucial salivary biomarkers for the diagnosis of periodontal disease that harms the periodontal tissue along with tooth loss. However, there has been no way of sensitive and portable detection of both biomarkers in saliva with multivariate signal readout. In this work, we design the multicolorimetric ALP and IL-1β sensing platform based on geometrical transformation of silver nanoplate transducer. By utilizing enzymatic activity of ALP that dephosphorylates p -aminophenol phosphate ( p -APP) to p -aminophenol ( p -AP), localized surface plasmon resonance properties of silver nanoplate vary with ALP and show a distinct color change from blue to yellow based on a controlled seed transformation from triangular to hexagonal, rounded pentagonal, and spherical shape. The multicolor sensor shows an ALP detection range of 0-25 U/L with a limit of detection (LOD) of 0.0011 U/L, which is the lowest range of LOD demonstrated to date for state-of-the-art ALP sensor. Furthermore, we integrate the sensor with the conventional ELISA to detect IL-1β for multicolor signaling and it exhibits a linear detection range of 0-250 pg/mL and an LOD of 0.066 pg/mL, which is 2 orders of magnitude lower than the monochromic conventional ELISA (LOD of 3.8 pg/mL). The ALP multicolor sensor shows high selectivity with a recovery of 100.9% in real human saliva proving its reliability and suitability for the readily accessible periodontal diagnosis with multivariate signal readout.

Herein, a simple and sensitive ratiometric fluorescence sensing platform to detect alkaline phosphatase (ALP) activity is developed on the basis of yellow fluorescent nitrogen-doped carbon quantum dots (YNCDs). The hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) is catalyzed by ALP. Then, AA will react with o-phenylenediamine (OPD) to form 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) which is a blue fluorescent quinoxaline derivative with emission at 435 nm in the presence of Cu 2+ . YNCDs have yellow fluorescence emission at 555 nm, and can maintain stable in QXD reaction system. Therefore, by utilizing the fluorescence of YNCDs at 555 nm as reference signal and the fluorescence of QXD at 435 nm as report signal, we can detect the ALP activity by monitoring the fluorescence ratio (F 435 /F 555 ). The linear range is 0.5-5 U/L, and the limit of detection is 0.14 U/L. An application of this method for the analysis of ALP in human serum has given satisfactory results. A ratiometric fluorescent nanoprobe for ascorbic acid and alkaline phosphatase detection with excellent biocompatible and high sensitivity was successfully constructed based on YNCDs and QXD., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Alkaline phosphatase is one of the most important tool enzymes and diseases indicator, monitoring ALP activity with convenient, precise, efficient and sensitive methods plays a fundamental role in modern life and healthcare industries. In this study, we described a novel method for ALP analysis based on Pb 2+ dependent DNAzyme. By modifying DNAzyme sequence with terminal phosphate group and introducing exonuclease I (exo I), we managed to analyze ALP by utilizing its causal function of DNAzyme probe from exo I mediated degradation and function of triggering the subsequent cleavage of the hairpin reporting probe. Other than one amplificative strategy by DNAzyme mediated cleavage and cycle, this system also involved an exo I mediated degradation to further reduce the background noise. Combining stepwise fluorimetry and electrophoresis, we verified the detective mechanism of this proposed method. Further, after selectivity demonstration, this method achieved a considerable LOD of 0.0017 U L -1 and linear range of 0.0025 U L -1 to 250 U L -1 . For potential of practical application, this method also exhibited excellent performances in inhibitor screening and intracellular ALP assay, both with a linear fitting equation. Based on these results, this method should be highly committed for improving ALP analysis in modern life industry., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The determination of pyrophosphate and alkaline phosphatase activity plays a significant role in medical diagnosis. In this work, a label-free "ON-OFF-ON" fluorescence strategy is developed for the analysis of pyrophosphate and alkaline phosphatase activity. Using PolyT single strand DNA as templates to synthesize fluorescent copper nanoparticles, the coordination effect of pyrophosphoric acid on Cu 2+ inhibited the generation of fluorescence. Afterwards, the addition of alkaline phosphatase into hydrolyze pyrophosphoric acid resulted in the release of Cu 2+, whereby the fluorescence intensity could be recovered. Thereupon enhanced-sensitivity for alkaline phosphatase was obtained (0.1 mU/L), much better than previously reported methods. Meanwhile, it could be performed directly in homogeneous solution, which was very close to the actual activity level of alkaline phosphatase under physiological conditions. Likewise, satisfactory results were also obtained in specificity assessment, which demonstrated its potential application in clinical diagnosis. Notably, a new, sensitive, low-cost, short-time, and high-sensitivity platform for alkaline phosphatase detection was constructed, and the design of biosensor using DNA-templated Copper nanoclusters (CuNCs) was instructed in this study., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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/mm 3 , 3.73 ± 0.04 × 10 4 /mm 3 and 1.7 ± 0.01 × 10 4 /mm 3 , 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., Competing Interests: The authors declare there are no competing interests., (©2022 Chen et al.)

Introduction: To evaluate the prognostic value of albumin-to-alkaline phosphatase ratio (AAPR) on recurrence and survival in patients with non-metastatic renal cell carcinoma (RCC) treated with radical or partial nephrectomy., Patients and Methods: Between June 1994 and December 2018, 491 patients with RCC who underwent radical or partial nephrectomy at 2 institutions were enrolled in this study. Recurrence-free survival (RFS) and cancer-specific survival (CSS) analyses were performed to distinguish the differences in postoperative recurrence and survival between patients stratified by an optimal cut-off value of AAPR. Multivariable Cox proportional hazards regression models were established to determine the independent prognostic factors after propensity score weighting., Results: Of the total 491 patients, 51 patients (10.4%) developed local recurrence or distant metastasis and 26 patients (5.3%) died of disease during the follow-up period. Patients with AAPR<0.41 had significantly lower rates of RFS and CSS than those of patients with AAPR≥0.41 in multivariate analysis (P < .001 and P = .027, respectively). After propensity scroe matching analyses, this difference was still remained for RFS (P < .001). However, AAPR was not an independent prognostic factor for CSS but the value was almost pregnant (HR = 2.674; 95%CI = 0.872-8.203; P = .086)., Conclusion: AAPR can serve as a novel and useful tool to refine prognosis in patients with non-metastatic RCC treated with partial or radical nephrectomy. These findings suggest that AAPR could be a promising prognostic factor for prediction of recurrence and survival in patients with non-metastatic RCC who undergo nephrectomy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To explore the diagnostic value of the preoperative liver function for occult pancreaticobiliary reflux (OPBR) in patients with gallstones. Methods: Patients with gallstones in Shanghai East Hospital were enrolled from December 2020 to June 2021. Their intraoperative bile and clinical data were collected. According to the gallbladder bile amylase level, patients were divided into the OPBR group (bile amylase>110 U/L) and the control group (bile amylase ≤ 110 U/L). Preoperative liver function levels of the two groups were compared, and the differential parameters were accessed by the receiver operating characteristic (ROC) curve. And the risk factors for OPBR were tested by multiple logistic regression analysis. Results: Among 249 patients, 83 were male and 166 were female, aged 50 (37, 62) years; There were 218 cases in control group, including 70 males and 148 females, aged 49 (36, 61) years; There were 31 patients in the OPBR group, including 13 males and 18 females, aged 58 (51, 65) years. For preoperative liver function, gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) in the OPBR group were higher than those in the control group [35 (18, 59) vs 19 (13, 34) U/L, 80 (71, 97) vs 69 (57, 83) U/L; both P <0.01]. ROC indicated that preoperative GGT and ALP had important predictive values for OPBR in gallstone patients. Their respective optimal cut-off value and area under the ROC curve [AUC (95% CI )] were GGT ≥ 30 U/L, 0.656 (0.542-0.770), P= 0.005; ALP≥70 U/L, 0.693 (0.613-0.773), P= 0.001, respectively. In addition, multivariate logistic regression analysis showed that the levels of GGT [ OR (95% CI )=2.856 (1.260-6.473), P =0.012] and ALP [OR (95% CI )=3.685 (1.314-10.333), P =0.013] were independent-related factors for OPBR in patients with gallstones. Conclusion: Preoperative liver function assessment is of great significance for patients with gallstones, while GGT and ALP are important for predicting OPBR in patients with gallstones.

Coordination polymers (CPs) with tunable structures and properties have been extensively explored in a variety of fields. In this work, we demonstrated the potential of stimuli-responsive CPs as a host of integrating enzymes to construct a portable immunoassay. By employing terbium ion (Tb 3+ ) as a metal node and guanine monophosphate (GMP) as a bridge ligand, an alkaline phosphatase (ALP)-responsive Tb/GMP CPs was fabricated, which allows amyloglucosidase (GA) to be integrated to form GA@Tb/GMP composite. Owing to the size-selectivity of Tb/GMP CPs as a host, the loaded GA was physically isolated from its substrate starch. However, Tb/GMP CPs is highly sensitive to ALP, which can hydrolyze the phosphate group of GMP to destroy the structure of Tb/GMP CPs, leading to the release of GA from GA@Tb/GMP composite. The released GA can digest starch to produce glucoses and give a measured signal by personal glucose meter (PGM). This finding leads to a PGM-based portable immunoassay for the quantitative analysis of carcinoembryonic antigen (CEA), and satisfactory results with a detection limit of 0.28 ng/mL have been achieved. The successful determination of CEA in serum samples demonstrates its potential in practical application. We believe that this work can provide a remarkable insight for the rational design of stimuli-responsive CPs for a wide of applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Alkaline phosphatase (ALP), an essential hydrolase with crucial roles in living organisms, has widely been regarded as a biomarker for various human diseases in clinical diagnoses. Herein, taking advantage of cobalt oxyhydroxide (CoOOH) nanoflakes and nonenzymatic cascade recycling amplification (CRA), a highly sensitive and label-free fluorescence biosensing strategy for the determination of ALP activity is introduced. In our design, ALP can promote the dephosphorylation of l-ascorbic acid 2-phosphate (AAP) to reduce ascorbic acid (AA), which is then able to decompose CoOOH in a nucleic acids@CoOOH nanocomplex into Co 2+ cofactors. Further, enzyme-free CRA was rapidly initiated by integrating DNAzyme recycling amplification and catalytic hairpin assembly, resulting in the generation of an abundance of G-quadruplex structure-contained DNA duplexes. In the presence of thioflavin T (ThT), analytical target ALP was converted in an amplified and activatable fluorescence signal. The experimental results show that this method can be applied for the quantitative analysis of ALP activity with a low detection limit of 0.027 mU/mL. Moreover, this developed biosensing approach exhibits excellent specificity, and the evaluation of ALP activity in the complex human serum samples was successfully realized, indicating that it can afford a reliable, robust, and cost-effective nanoplatform for an ALP-based clinical diagnosis and for biomedical research.

A catalyst-free co-reaction luminol-H 2 O 2 -K 2 S 2 O 8 chemiluminescence (CL) system was developed, with long-life and high-intensity emission, and CL emission lasting for 6 h. A possible mechanism of persistent and intense emission in this CL system was discussed in the context of CL spectra, cyclic voltammetry, electron spin resonance (ESR), and the effects of radical scavengers on luminol-H 2 O 2 -K 2 S 2 O 8 system. H 2 O 2 and K 2 S 2 O 8 co-reactants can promote each other to continuously generate corresponding radicals (OH • , 1 O 2 , O 2 •- , SO 4 •- ) that trigger the CL emission of luminol. H 2 O 2 can also be constantly produced by the reaction of K 2 S 2 O 8 and H 2 O to further extend the persistence of this CL system. CL emission can be quenched via ascorbic acid (AA), which can be generated through hydrolysis reaction of L-ascorbic acid 2-phosphate trisodium salt (AAP) and alkaline phosphatase (ALP). Next, a CL-based method was established for the detection of ALP with good linearity from 0.08 to 5 U·L -1 and a limit of detection of 0.049 U·L -1 . The proposed method was used to detect ALP in human serum samples., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Alkaline phosphatase (ALP) activity is a diagnostic indicator for a variety of clinical diseases. In this study, an electrochemical method for detecting ALP activity through activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) was developed. Specifically, 3-mercaptopropionic (MPA) was firstly fixed on the electrode through sulfur-gold bonding. Subsequently, α-bromophenylacetic acid (BPAA) as initiator was attached to MPA through the recognized carboxylate-Zr 4+ -phosphate chemistry. Finally, in the existence of ALP, L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAPS) was hydrolyzed to produce ascorbic acid (AA) which participated in the ARGET ATRP reaction, grafting polymer containing plenty of ferrocene electroactive probes on the surface of electrode. Under optimal experimental conditions, this method had a linear scope of 20-200 mU mL -1 , and a limit of detection (LOD) of 1.64 mU mL -1 . In addition, the proposed method had good selectivity as well as anti-interference capability, with satisfactory results in inhibition rate and human serum experiments. By merits of good analytical performance, easy operation, and low cost, such a method for ALP activity detection has promising applications in ALP-related disease detection and inhibitor screening., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ratiometric electrochemical assays have been demonstrated to be more sensitive and selective in various sensing events, mainly due to their affordable built-in correction and good self-reference capability. But it is known that complicated modification and labeling operations usually are necessary for the construction of ratiometric electrochemical assays, therefore is a hot and important issue needing consideration carefully. We herein report a new yet simple bare electrode-based ratiometric electrochemical bioassay to achieve sensitive and selective analysis of alkaline phosphatase (ALP), using a liquid phase system that contains CoOOH nanozymes and commercially available indicator substrate. This proposed bioassay works based on the ratiometric change of dual electrochemical signals, arising from an exclusive target ALP-triggered hydrolysis of electrochemical substrate p-nitrophenyl phosphate (PNPP). In this design, the two hydrolyzed products of electrochemically active p-nitrophenol (PNP) and electrochemically inactive phosphate anion (PO 4 3- ) are responsible together for the ratiometric electrochemical analysis of ALP. PNP exhibits a straightforward current response toward ALP content; however, PO 4 3- cannot show a direct electrochemical signal thus is rationally designed to offer an alternative response by linking it with the specific CoOOH nanozyme-catalyzed reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and H 2 O 2 , in which the nanozyme-catalyzed product oxTMB shows a direct reduction current at the GCE, and significantly decreases with increasing PO 4 3- species due to the good inhibition of PO 4 3- toward CoOOH nanozyme activity. As a result, a ratiometric electrochemical strategy for ALP analysis with a low limit of detection of 0.366 U/L (S/N = 3) was successfully achieved by integrating the above direct and indirect dual electrochemical responses. This developed bioassay can allow the quantitative diagnosis of ALP activity especially with a label-free and modification-free merit, therefore paving the way for simple, convenient, and portable electroanalytical tools in biosensing design and application., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Monitoring the liver status in a convenient and low-cost way is significant for obtaining a warning about drug-indued liver diseases promptly. Herein, we designed a novel colorimetric point-of-care (POC) platform for the determination of three liver-related biomarkers─aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP). This platform integrated agarose hydrogels into a portable device, where hydrogels were loaded with nanozymes and different reaction substances for triggering specific reactions and generating colorimetric signals. Typically, Au-decorated CoAl-layered double oxide (Au/LDO) was for the first time developed as the nanozyme with peroxidase (POD) mimic activity, which can accelerate the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxTMB with the coexistence of hydrogen peroxide (H 2 O 2 ). The detection mechanism of AST and ALT is based on the fact that they can cause individual cascade reactions to generate H 2 O 2 , and H 2 O 2 further activates the Au/LDO nanozyme to catalyze the chromogenic reaction of TMB. As for ALP, it can catalytically hydrolyze l-ascorbic acid-2-phosphate to ascorbic acid. The latter then discolored the oxTMB that was produced with the assistance of Au/LDO. Teaming up with a smartphone, the color information of hydrogels can be converted to hue values, which allow quantitative analysis of ALT, AST, and ALP with detection limits of 15, 10, and 5 U/L, respectively. Moreover, the simple and cost-effective platform was successfully applied for the simultaneous determination of the three analytes in human plasma. Additionally, since the hydrogel is disposable and can be replaced by new ones loaded with different reaction regents, the platform is expected to serve the POC testing of various chem/bio targets.

Results: A strong positive correlation was found between CA and cervical stages ( r = 0.836, P < 0.001). Based on the regression model analysis, the model which combined IGF-1, ALP, and CA provided the best prediction at P < 0.001 with McFadden's pseudo R 2 value of 0.552 for cervical stage prediction and 0.646 for growth phase prediction. In particular, its predictive ability for the prepubertal, pubertal, and postpubertal growth phases was 95%, 80%, and 90%, respectively., Conclusions: A new model with the combination of salivary IGF-1 and ALP with CA can be used as a less invasive method to determine the cervical stage and also growth phase with high predictive ability in healthy subjects., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Asma Sookhakian et al.)

The ever-increasing attention on the highly sensitive biosensors pushes people to explore functional nanomaterials for signal amplification. To endow inert metal-organic frameworks (MOFs) with enzyme mimicking activity, a simple strategy of introducing Cu 2+ via coordination with 2,2'-bipyridine ligands of Zr-MOF, just like "Midas touch," is proposed. More details on the coordination environment of Cu active sites in Zr-MOF-Cu are disclosed via electron paramagnetic resonance and synchrotron-radiation-based X-ray absorption fine structure analyses. The as-prepared Zr-MOF-Cu exhibits unparalleled catalytic ability, which can catalyze ascorbic acid (AA) to dehydroascorbic acid and further stimulate the reaction with o -phenylenediamine to produce fluorescent signal probes with 8-fold signal amplification. On the basis of catalyzing the dephosphorylation process of l-ascorbic acid-2-phosphate to yield AA via alkaline phosphatase (ALP) and AA-dependent signal responses, a universal fluorescent system has been successfully constructed for quantitative measurement of the activity of ALP and the ALP-related enzyme-linked immunosorbent assay with carcinoembryonic antigen as a model. Moreover, the stable loading of Cu active sites endows the sensing platform with anti-inference capacity and enables its reuse without loss of catalytic activity after 6 months.

Enzymes can be used as biomarkers for a variety of diseases. However, profiling enzyme activity in clinical samples is challenging due to the heterogeneity in enzyme activity, and the low abundance of the target enzyme in biofluids. Single-molecule methods can overcome these challenges by providing information on the distribution of enzyme activities in a sample. Here, we describe the concept of using the single-molecule enzymology (SME) method to analyze enzymatic activity in clinical samples. We present recent work focused on measuring alkaline phosphatase isotypes in serum samples using SME. Future work will involve improving and simplifying this technology, and applying it to other enzymes for diagnostics., (© 2021 Wiley-VCH GmbH.)

Purpose: The study aimed to define the clinical, biochemical and genetic features of adult patients with osteopenia/osteoporosis and/or bone fragility and low serum alkaline phosphatase (sALP)., Methods: Twenty-two patients with at least two sALP values below the reference range were retrospectively enrolled after exclusion of secondary causes. Data about clinical features, mineral and bone markers, serum pyridoxal-5'-phosphate (PLP), urine phosphoethanolamine (PEA), lumbar and femur bone densitometry, and column X-ray were collected. Peripheral blood DNA of each participant was analyzed to detect ALPL gene anomalies., Results: Pathogenic ALPL variants (pALPL) occurred in 23% and benign variants in 36% of patients (bALPL), while nine patients harbored wild-type alleles (wtALPL). Fragility fractures and dental anomalies were more frequent in patients harboring pALPL and bALPL than in wtALPL patients. Of note, wtALPL patients comprised women treated with tamoxifen for hormone-sensitive breast cancer. Mineral and bone markers were similar in the three groups. Mean urine PEA levels were significantly higher in patients harboring pALPL than those detected in patients harboring bALPL and wtALPL; by contrast, serum PLP levels were similar in the three groups. A 6-points score, considering clinical and biochemical features, was predictive of pALPL detection [P = 0.060, OR 1.92 (95% CI 0.972, 3.794)], and more significantly of pALPL or bALPL [P = 0.025, OR 14.33 (95% CI 1.401, 14.605)]., Conclusion: In osteopenic/osteoporotic patients, single clinical or biochemical factors did not distinguish hypophosphatasemic patients harboring pALPL or bALPL from those harboring wtALPL. Occurrence of multiple clinical and biochemical features is predictive of ALPL anomalies, and, therefore, they should be carefully identified. Tamoxifen emerged as a hypophosphatasemic drug., (© 2021. The Author(s).)

Background: Serum alkaline phosphatase (ALP) is a useful bone turnover marker to diagnose metabolic bone disease in preterm infants. In Japan, serum ALP levels were generally measured using the Japan Society of Clinical Chemistry (JSCC) method. It is problematic that ALP levels measured using the JSCC method tend to be higher in people with blood types B and O regardless of the disease. For international standardization, since 2020, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) method has been used as a reference method for ALP measurement instead of the JSCC method. However, no report has investigated the correlation between these two methods in neonates. We therefore aimed to compare the JSCC and IFCC methods and demonstrate a conversion formula in neonates., Methods: In this retrospective study, we used a total of 402 samples in 49 preterm and 38 term infants. Serum ALP levels were measured using the JSCC and IFCC methods., Results: Alkaline phosphatase measured using the JSCC method strongly correlated with that measured using the IFCC method in all blood types in preterm and term infants (P < 0.01 for all)., Conclusions: We found that the serum ALP levels measured using the IFCC method could be calculated as 0.34 times the ALP levels measured using the JSCC method in preterm and term infants with any blood type: ALP levels (IFCC method) = 0.34 × ALP levels (JSCC method)., (© 2021 Japan Pediatric Society.)

Aim: The aim of this study was to compare dental pulp tissue in human exfoliated deciduous teeth (SHEDs) and dental pulp stem cells (DPSCs) in response to ascorbic acid as the sole osteoblast inducer., Background: A cocktail of ascorbic acid, β-glycerophosphate, and dexamethasone has been widely used to induce osteoblast differentiation. However, under certain conditions, β-glycerophosphate and dexamethasone can cause a decrease in cell viability in stem cells., Objectives: This study aims to determine the cytotoxic effect and potential of ascorbic acid as the sole inducer of osteoblast differentiation., Methods: Cytotoxicity analyses in the presence of 10-500 μg/mL ascorbic acid were performed in both cell types using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The concentrations below the IC 50 (i.e., 10-150 μg/mL) were used to determine osteoblast differentiation potential of ascorbic acid using the alkaline phosphatase (ALP) assay, von Kossa staining, and reverse transcription-polymerase chain reaction., Results: SHEDs and DPSCs proliferated for 21 days, expressed a Mesenchymal Stem Cell (MSC) marker (CD73+), and did not express Hematopoietic Stem Cell (HSC) markers (CD34- and SLAMF1-). SHEDs had a higher range of IC50 values (215-240 μg/mL ascorbic acid), while the IC50 values for DPSCs were 177-211 μg/mL after 24-72 hours. SHEDs treated with 10-100 μg/mL ascorbic acid alone exhibited higher ALP-specific activity and a higher percentage of mineralisation than DPSCs. Both cell types expressed osteoblast markers on day 21, i.e., RUNX2+ and BSP+, in the presence of ascorbic acid., Conclusions: SHEDs survive at higher concentrations of ascorbic acid as compared to DPSC. The cytotoxic effect was only exhibited at ≥250 μg/mL ascorbic acid. In addition, SHED exhibited better ALP and mineralization activities, but lower osteoblast marker expression than DPSC in response to ascorbic acid as the sole inducer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

2,2',4,4'-Tetrabromodiphenyl ether (BDE47) poses potential risks to reproduction and development, but the mechanism of its toxicity has not yet been elucidated. To explore the developmental toxicity of BDE47, mouse embryonic stem cells (mESCs), which are ideal models for testing the developmental toxicity of environmental contaminants in vitro, were exposed to BDE47 (0.04 μM, 1 μM, 25 μM, or 100 μM) for 24 h or 48 h in this study. Our results indicated that BDE47 treatment changed the morphology of mESCs, inhibited cell viability and increased apoptosis. In addition, alkaline phosphatase (AP) staining in mESCs was significantly decreased after BDE47 treatment (25 μM and 100 μM), indicating that BDE47 treatment affected the pluripotency of mESCs. Through a cell immunofluorescence assay, we found that the fluorescence intensities of Oct4, Sox2 and Nanog were all significantly lower in the group treated with the highest BDE47 concentration (100 μM) than in the control group, consistent with the qRT-PCR and Western blot results. The levels of miR-145 and miR-34a, which regulate genes related to cell differentiation, were significantly increased in BDE47-treated mESCs, further clarifying the potential mechanism. Overall, our findings demonstrate that BDE47 exposure upregulates the expression of miR-145 and miR-34a and in turn downregulates the expression of Oct4, Sox2 and Nanog, thereby affecting apoptosis and pluripotency and causing toxicity during embryonic development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The study aims to investigate the role of microRNA-149-3p (miR-149-3p) in regulating osteogenic differentiation of human adipose-derived stem cells (hADSCs) by targeting v-akt murine thymoma viral oncogene homolog 1 (AKT1). Bioinformatics websites and a dual luciferase reporter assay were used to predict and verify the targeting relationship between miR-149-3p and AKT1. The hADSCs were divided into the blank, negative control (NC), mimic, control siRNA, AKT1 siRNA, and miR-149-3p inhibitors + AKT1 siRNA groups and then subjected to Alizarin Red staining, Alkaline phosphatase (ALP) staining, ALP activity detections, MTT assay, and EdU cell proliferation assay. Gene or protein expression was quantified using quantitative real-time PCR (qRT-PCR) or Western blotting, respectively. The miR-149-3p expression increased gradually and AKT1 expression decreased gradually during osteogenic differentiation of hADSCs. The prediction of bioinformatics websites miRTarBase and TargetScan and the dual luciferase reporter assay indicated that miR-149-3p can directly target AKT1. After hADSCs were transfected with miR-149-3p mimic, AKT1 expression was significantly downregulated. However, transfection with AKT1 siRNA did not have an impact on miR-149-3p in hADSCs. In comparison with the AKT1 siRNA group, the miR-149-3p inhibitors + AKT1 siRNA group showed decreased miR-149-3p expression but increased AKT1 expression. In addition, AKT1 siRNA enhanced the cell viability and proliferation of hADSCs and increased mineral calcium deposition and ALP activity, resulting in higher expression of osteogenic differentiation-related genes, which was reversed by miR-149-3p inhibition. The miR-149-3p can increase the expression of osteogenic differentiation-related genes by targeting AKT1 and thereby enhance the osteogenic differentiation of hADSCs., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Placental alkaline phosphatase (PLAP) is commonly expressed at high levels in testicular germ cell tumors. PLAP immunohistochemistry (IHC) is thus often used to confirm this diagnosis, especially in cases of putative metastasis. However, other tumors can also express PLAP. To comprehensively determine PLAP expression in normal and tumor tissue, a tissue microarray containing 16,166 samples from 131 different tumor types and subtypes as well as 608 samples from 76 different normal tissue types was analyzed by IHC. Moderate to strong PLAP positivity was found in 27 (21%) of 131 different tumor types including seminoma (96%), embryonal carcinoma (85%), and yolk sac tumors of the testis (56%); endometrioid carcinoma of the endometrium (28%) and the ovary (20%); gastric adenocarcinoma (22%); serous carcinoma (not otherwise specified) of the ovary (17%) and the uterus (11%); adenocarcinoma of the ampulla of Vater (15%); carcinosarcoma of the ovary (11%) and the uterus (8%); esophageal adenocarcinoma (10%); invasive urothelial carcinoma (4%); cholangiocarcinoma (2%); and adenocarcinoma of the lung (1%). Low-level PLAP immunostaining, often involving only a small fraction of tumor cells, was seen in 21 additional tumor entities. The clinical significance of PLAP expression may vary between tumor types as high PLAP expression was linked to advanced pathological tumor stage (p = 0.0086), nodal metastasis (p = 0.0085), and lymphatic (p = 0.0007) and blood vessel invasion (p = 0.0222) in colorectal cancer, but to low pathological tumor stage in endometrial cancer (p = 0.0043). In conclusion, our data identify several tumor entities that can show PLAP expression at comparable levels to testicular germ cell tumors. These tumor entities need to be considered in cases of PLAP-positive metastasis. Low-level PLAP expression can be found in various other tumor entities and should generally not be viewed as a strong argument for germ cell neoplasia., (© 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)