Detection Of Alkaline Phosphatase Research Articles

Detection of alkaline phosphatase activity with a CsPbBr3/Y6 heterojunction-based photoelectrochemical sensor.

An ultra-sensitive photoelectrochemical (PEC) sensor based on perovskite composite was developed for the determination of alkaline phosphatase (ALP) in human serum. In contrast toCsPbBr3 or Y6 that generated anodic current, the heterojunction of CsPbBr3/Y6 promoted photocarriers to separate and generated cathodic photocurrent. Ascorbic acid (AA) was produced by ALP hydrolyzing L-ascorbic acid 2-phosphate trisodium salt (AAP), which can combine with the holes on the photoelectrode surface, accelerating the transmission of photogenerated carriers, leading to enhanced photocurrent intensity. Thus, the enhancement of PEC current was linked to ALP activity. The PEC sensor exhibits good sensitivity for detection of ALP owing to the unique photoelectric properties of the CsPbBr3/Y6 heterojunction. The detection limit of the sensor was 0.012 U·L-1 with a linear dynamic range of 0.02-2000 U·L-1. Therefore, this PEC sensing platform shows great potential for the development of different PEC sensors.

Hydrogel-Embedded Platinum–Ruthenium Alloy Peroxidase Mimic as a Visual Sensor for “Signal-on” Alkaline Phosphatase Detection

Hydrogel-Embedded Platinum–Ruthenium Alloy Peroxidase Mimic as a Visual Sensor for “Signal-on” Alkaline Phosphatase Detection

Paper Analytical Device for the Colourimetric Detection of Alkaline Phosphatase in Serum and Saliva

Paper Analytical Device for the Colourimetric Detection of Alkaline Phosphatase in Serum and Saliva

A Novel Approach Using Reduced Graphene Oxide for the Detection of ALP and RUNX2 Osteogenic Biomarkers

In this work, we propose a new technique involving the modification of commercial screen-printed carbon electrodes with electrochemically reduced graphene oxide to serve as the starting point of a future electrochemical biosensor for the detection of two osteogenic biomarkers: alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). The electrodes were characterized after each modification by cyclic voltammetry and electrochemical impedance spectroscopy, showing the appropriate electrochemical characteristics for each modification type. The results obtained from scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and contact angle measurements are well correlated with each other, demonstrating the successful modification of the electrodes with graphene oxide and its subsequent reduction. The bioreceptors were immobilized on the electrodes by physical adsorption, which was confirmed by electrochemical methods, structural characterization, and contact angle measurements. Finally, the functionalized electrodes were incubated with the specific target analytes and the detection relied on monitoring the electrochemical changes occurring after the hybridization process. Our results indicated that the pilot platform has the ability to detect the two biomarkers up to 1 nM, with increased sensitivity observed for RUNX2, suggesting that after further optimizations, it has a high potential to be employed as a future biosensor.

Polymer-enhanced peroxidase activity of ceria nanozyme for highly sensitive detection of alkaline phosphatase.

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, CeO2 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 CeO2 nanoparticles encapsulated within each polymer exhibited improved catalytic characteristics, with PAA-capped CeO2 exhibiting the highest performance. We further demonstrated that the PAA-CeO2 obtained with enhanced catalytic activity was attributed to an increase in surface negative charge. PAA-CeO2 enabled the quantitative assessment of AA activity within a wide concentration range of 10 to 60μM, with a detection limit of 0.111μM. 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.

Hollow Cu/CoS2 Nanozyme with Defect-Induced Enzymatic Catalytic Sites and Binding Pockets for Highly Sensitive Fluorescence Detection of Alkaline Phosphatase.

Along with an ever-deepening understanding of the catalytic principle of natural enzymes, the rational design of high-activity biomimetic nanozymes has become a hot topic in current research. Inspired by the active centers of natural enzymes consisting of catalytic sites and binding pockets, a Cu-doped CoS2 hollow nanocube (Cu/CoS2 HNCs) nanozyme integrating substitution defects and vacancies is developed through a defect engineering strategy. It is shown that the vacancies and substitution defects in the developed Cu/CoS2 HNC nanozymes serve as binding pockets and catalytic sites, respectively. The construction of this key active center and the accelerated electron transfer from the Co/Cu redox cycle significantly improve the substrate affinity and catalytic efficiency of the Cu/CoS2 HNCs nanozymes, which results in the excellent catalytic performance of the Cu/CoS2 HNC nanozymes. Using the superior enzymatic activity of Cu/CoS2 HNCs, a fluorescence detection platform for alkaline phosphatase (ALP) is established, which is a wider detection range and lower limit of detection (LOD) than previous work. This work broadens the family of nanozymes and provide a new idea for the development of novel nanozymes with high enzyme activity, as well as a guideline for the construction of highly sensitive fluorescent sensors.

Mn2+-Mediated Modulation of PfAgo Activity for Biosensing.

Argonaute (Ago) as a powerful enzyme has provided new insights into biosensing due to its programmability, high sensitivity, and user-friendly operation. However, current strategies mainly rely on phosphorylated guide DNA to modulate the cleavage activity of Ago, which is limited in versatility and simplicity. Herein, the authors report the Mn2+-enhanced cleavage activity of Ago and employ Mn-ions with variable valence to regulate the activity of Pyrococcus furiosus Ago (PfAgo) for biosensing applications. The conversion of Mn ions with different valence states through MnO2 nanoflowers enables the sensitive detection of ascorbic acid, alkaline phosphatase, and arsenic with limits of detection of 2.5 nmol L-1, 0.009 U L-1, and 0.4 ng mL-1, respectively. A PfAgo-based immunoassay is further developed that allows for the detection of diverse targets, thus providing a promising toolbox to broaden PfAgo-based sensors into versatile bioanalytical and biomedical applications.

A novel NiO/CeO2-mSiO2 nanocages with peroxidase-mimetic activity for ultra-sensitive biosensing

A novel NiO/CeO2−mSiO2 nanocages with superior peroxidase−like activities due to the synergetic effect between NiO and CeO2 were fabricated by a simple etching−precipitation method with core−shell structured Cu2O@mSiO2 as a sacrificial template. Based on the peroxidase−like activity of the NiO/CeO2−mSiO2 nanocages, an enzyme cascade−triggered colorimetric sensing platform was exploited for alkaline phosphatase (ALP) detection, owing to the fact that ascorbic acid (AA) that is the ALP enzymatic hydrolysis product of ascorbic acid 2−phosphate (AAP) can efficiently reduce the oxidation product of TMB (oxTMB) generated under the catalysis of the NiO/CeO2−mSiO2. For the sensing of ALP, a good linear response was observed in the range of 0.0005–1.25 mU mL−1 with the limit of detection (LOD) as low as 0.00048 mU mL−1. Furthermore, the proposed enzyme cascade−triggered colorimetric sensing platform could be further explored for sensing of tyrosinase, according to the fact that dopamine (DA) the catalytic product of tyramine (Tyr) by tyrosinase could initiate the redox reaction with the oxTMB acquired under the catalysis of the NiO/CeO2−mSiO2. A wide linear response was found for tyrosinase detection from 0.02 mU mL−1 to 3.4 U mL−1 with a LOD as low as 0.002 U mL−1. This work not only offers a facile and effective approach to fabricate nanozymes with excellent catalytic activity, but also provides a promising enzyme cascade−triggered colorimetric method for the ultra−sensitive detection of biological enzymes.

Polyacrylic Acid-Ca2+(Ce3+) Nanoclusters as Fluorescent “Off–On–Off” Probes for Quantitative Detection of Pyrophosphate and Alkaline Phosphatase

Polyacrylic Acid-Ca²⁺(Ce³⁺) Nanoclusters as Fluorescent “Off–On–Off” Probes for Quantitative Detection of Pyrophosphate and Alkaline Phosphatase

Fabrication and properties of PLA/β-TCP scaffolds using liquid crystal display (LCD) photocuring 3D printing for bone tissue engineering.

Introduction: Bone defects remain a thorny challenge that clinicians have to face. At present, scaffolds prepared by 3D printing are increasingly used in the field of bone tissue repair. Polylactic acid (PLA) has good thermoplasticity, processability, biocompatibility, and biodegradability, but the PLA is brittle and has poor osteogenic performance. Beta-tricalcium phosphate (β-TCP) has good mechanical properties and osteogenic induction properties, which can make up for the drawbacks of PLA. Methods: In this study, photocurable biodegradable polylactic acid (bio-PLA) was utilized as the raw material to prepare PLA/β-TCP slurries with varying β-TCP contents (β-TCP dosage at 0%, 10%, 20%, 30%, 35% of the PLA dosage, respectively). The PLA/β-TCP scaffolds were fabricated using liquid crystal display (LCD) light-curing 3D printing technology. The characterization of the scaffolds was assessed, and the biological activity of the scaffold with the optimal compressive strength was evaluated. The biocompatibility of the scaffold was assessed through CCK-8 assays, hemocompatibility assay and live-dead staining experiments. The osteogenic differentiation capacity of the scaffold on MC3T3-E1 cells was evaluated through alizarin red staining, alkaline phosphatase (ALP) detection, immunofluorescence experiments, and RT-qPCR assays. Results: The prepared scaffold possesses a three-dimensional network structure, and with an increase in the quantity of β-TCP, more β-TCP particles adhere to the scaffold surface. The compressive strength of PLA/β-TCP scaffolds exhibits a trend of initial increase followed by decrease with an increasing amount of β-TCP, reaching a maximum value of 52.1MPa at a 10% β-TCP content. Degradation rate curve results indicate that with the passage of time, the degradation rate of the scaffold gradually increases, and the pH of the scaffold during degradation shows an alkaline tendency. Additionally, Live/dead staining and blood compatibility experiments suggest that the prepared PLA/β-TCP scaffold demonstrates excellent biocompatibility. CCK-8 experiments indicate that the PLA/β-TCP group promotes cell proliferation, and the prepared PLA/β-TCP scaffold exhibits a significant ability to enhance the osteogenic differentiation of MC3T3-E1 cells in vitro. Discussion: 3D printed LCD photocuring PLA/β-TCP scaffolds could improve surface bioactivity and lead to better osteogenesis, which may provide a unique strategy for developing bioactive implants in orthopedic applications.

Icariin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating USP47/SIRT1/Wnt/β-catenin.

Icariin has been shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanism by which Icariin regulates osteogenic differentiation needs to be further revealed. The viability of BMSCs was assessed by cell counting kit 8 assay. BMSC osteogenic differentiation ability was evaluated by detecting alkaline phosphatase activity and performing alizarin red S staining. The protein levels of osteogenic differentiation-related markers, sirtuin 1 (SIRT1), ubiquitin-specific protease 47 (USP47), and Wnt/β-catenin-related markers were determined using western blot. SIRT1 mRNA level was measured using quantitative real-time PCR. The regulation of USP47 on SIRT1 was confirmed by ubiquitination detection and co-immunoprecipitation analysis. Icariin could promote BMSC osteogenic differentiation. SIRT1 expression was enhanced by Icariin, and its knockdown suppressed Icariin-induced BMSC osteogenic differentiation. Moreover, deubiquitinating enzyme USP47 could stabilize SIRT1 protein expression. Besides, SIRT1 overexpression reversed the inhibiting effect of USP47 knockdown on BMSC osteogenic differentiation, and USP47 knockdown also restrained Icariin-induced BMSC osteogenic differentiation. Additionally, Icariin enhanced the activity of the Wnt/β-catenin pathway by upregulating SIRT1. Icariin facilitated BMSC osteogenic differentiation via the USP47/SIRT1/Wnt/β-catenin pathway.

A two-dimensional iron-doped carbon-based nanoenzyme with catalase-like activity for the detection of alkaline phosphatase and ascorbate oxidase

Herein, we successfully synthesized two-dimensional iron-doped carbon-based nanosheets (Fe–N800 CS) with catalase-like activity through doping Fe into Zn MOF and introducing graphitic C3N4 (g-C3N4). The interaction of the Fe–N800 CS with hydrogen peroxide could generated abundant reactive oxygen species (ROS) and further oxidize o-Phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) which has constant fluorescence at 560 nm. Ascorbic acid (AA) could be generated via the hydrolysis reaction between alkaline phosphatase (ALP) and ascorbic acid 2-phosphate (AAP). AA can be oxidized to dehy-droascorbic acid (DHA) by ROS, and then combined with OPD to generate 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) with fluorescence at 440 nm, which could increase as the concentration of AA enhanced. DHA could also be generated through oxidation of AA by ascorbate oxidase (AAO). Thus, by monitoring the fluorescence ratio (I560/I440), a ratiometric fluorescence biosensing platform for ALP and AAO was established with the linear ranges in 0.2–10 U/L and 1–60 U/L, respectively. The limit of detection for ALP and AAO were 0.12 U/L and 0.59 U/L. Furthermore, the biosensing platform was successfully applied for the detection of ALP and AAO activity in human serum samples. This work provides a potential tool for future biomedical diagnostics.

Revisiting the Chemistry and Photophysics of 3‐(N‐Methylpyridinium‐4‐yl)Coumarins for Designing “Covalent‐Assembly” and “Molecular Disassembly” Fluorescent Probes**

AbstractThe constant need for high‐performance aniline‐ or phenol‐based fluorophores suitable for the construction of activity‐based fluorescent probes, led us to study both synthesis and photophysics of C3‐N‐methylpyridinium‐4‐yl substituted 7‐(dialkylamino)/7‐hydroxycoumarins. Indeed, in the field of photoactive organic molecules, the positively charged N‐alkylpyridinium‐4‐yl groups are often used as acceptor units to dramatically impact spectral features through promoting intramolecular charge transfer (ICT) processes. They are also known as effective water‐solubilizing and mitochondria targeting moieties. The poor fluorescence efficiency of cationic 7‐hydroxycoumarin derivatives in aqueous physiological conditions was highlighted and rationalized by the predominance of a neutral quinonoid form in such buffer medium. The ability of the excited singlet state (S1) of this neutral species to undergo intersystem crossing (ISC) to triplet state (T1) was partly supported by phosphorescence measurements of singlet oxygen. We also took advantage of green‐emissive properties of 7‐(diethylamino)‐3‐(N‐methylpyridinium‐4‐yl)coumarin to successfully design and validate a novel small‐molecule fluorescent probe for the detection of alkaline phosphatase (ALP), based on the “covalent‐assembly” principle. A practical use of ortho‐formylated 7‐hydroxy‐3‐(N‐methylpyridinium‐4‐yl)coumarin was next considered with the synthesis of a Fe(III)‐salen complex whose the potential as a “molecular disassembly” probe for fluorogenic sensing of pyrophosphate (PPi) anion was assessed.

Colorimetric detection of alkaline phosphatase based on the off-on effect of light-responsive oxidase mimicking activity of covalent organic framework (Cu-TpBpy-COF) under near-neutral condition.

A colorimetric strategy has beendeveloped for the detection of alkaline phosphatase (ALP) activity based on the off-on effect of the catalytic activity of light-responsive oxidase mimics covalent organic framework (Cu-TpBpy-COF) in near-neutral condition. Cu-TpBpy-COF can effectively catalyze the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (oxTMB) with an absorption peak at 652 nm. Cu2+ is the active center of Cu-TpBpy-COF and pyrophosphate (PPi) can form a complex with Cu2+ to weaken the catalytic activity of Cu-TpBpy-COF. In the presence of ALP, PPi is hydrolyzed into orthophosphates (Pi) with low affinity to Cu2+, thus resulting in absorbance restoration. The absorbance at 652 nm is related to ALP activity in the linear range 10-150 U·L-1 with a detection limit of 7.17 U·L-1. The recoveries of ALP in serum samples are in the range 94.7~107.0% with relative standard deviations (RSD) lower than 5%. The decisive role of Cu2+ on the enhancing catalytic activities of Cu-TpBpy-COF in neutral condition was verified by TpBpy-COF and TpBD-COF as controls, in which the main difference between them is that TpBpy-COF contains pyridine nitrogen. Upon Cu2+ modification, Cu-TpBpy-COF has better catalytic activity than TpBpy-COF in a broader pH range because of the in situ generation of Cu+ under irradiation.

Stabilizer-free MnO2 nanozyme with singlet-oxygen conducted high oxidase-like activity and fast catalysis capability for improving alkaline phosphatase assay

Oxidase (OXD) nanozyme is a type of artificial enzyme that can directly oxidize reducible substrates. By virtue of enzyme catalysis, it is often designed as a sensor for alkaline phosphatase (ALP) assays. MnO2 nanozymes have received extensive attention due to their low cost and ease of preparation. To enhance its sensing capability, further development of MnO2-based OXD nanozyme with higher activity and faster catalysis is necessary. In this study, we synthesized Fe3O4 @polydopamine supported MnO2 (IOPM) nanoparticles, which exhibited excellent OXD activity with a Vmax of 19.67 × 10-8 M·s-1 and a catalysis balance period of less than 1 min. Further investigation confirms that IOPM's good performance is associated with its stabilizer-free interface, magnetic purification method, and singlet-oxygen-conducted catalysis mechanism. More importantly, IOPM is demonstrated to possess the ability of improving ALP assay, with lower limit of detection (0.045 U/L) and wider linear range (0.05 – 40 U/L). Finally, it is successfully used to evaluate ALP activity in clinical blood samples. This study aims to develop a more efficient MnO2-based OXD nanozyme. It emphasizes the positive impact of magnetic purification and a stabilizer-free interface in enhancing OXD activity and improving the ALP detection capability of the nanozyme.

Ti3C2@UiO−TCPP Schottky junction photoelectrochemical sensor for detecting alkaline phosphatase through the steric hindrance effect of phosphopeptide

Alkaline phosphatase (ALP) is a major biomarker for clinical diagnosis, but detection methods of ALP are limited in sensitivity and selectivity. In this paper, a novel method for ALP determination is proposed. A photoelectrochemical (PEC) sensor was prepared by growing UiO−tetratopic tetrakis (4-carbox-yphenyl) porphyrin (TCPP) in situ between layered Ti3C2 through a one-pot hydrothermal method. The obtained Schottky heterojunction photoelectric material Ti3C2@UiO−TCPP not only has a large light absorption range but also greatly improves the efficiency of photogenerated electron hole separation and thereby enhances sensitivity for PEC detection. The phosphate group on the phosphorylated polypeptide was utilized to form a Zr–O–P bond with the zirconium ion on UiO-66, and then photocurrent decreases due to the steric hindrance effect of phosphorylated polypeptides, that is, the hindrance of electron transfer between the photoelectric material and a solution. The specific interaction between ALP and phosphorylated polypeptides shears the bond between phosphate and zirconium ion on UiO-66 in the peptides then weakens the hindrance effect and increases the photocurrent, thus realizing ALP detection. The linear range of ALP is 0.03–10,000 U·L−1, and the detection limit is 0.012 U·L−1. The method is highly sensitive and selective, and has been applied in detection of ALP in serum samples.

A bioetching-induced visualized-organic photoelectrochemical transistor dual-signal mode sensor for alkaline phosphatase detection.

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.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

A sensitive immunosensor for the detection of alkaline phosphatase as a biomarker for fracture healing

Fractures occur at all ages and are highly prevalent. At the same time, there is a problem of delayed healing after fracture, and drugs are often taken to promote healing. Alkaline phosphatase (ALP) is an important indicator for assessing fracture healing, and achieving its quantification would be helpful in evaluating fracture recovery in patients on medication. Here, we present an ultrasensitive immunosensor based on NH2-MIL-101(Cr)@AuNPs/rGO nanocomposites. The poor conductivity of metal–organic skeletons (MOF) limits their electrochemical applications. In this study, NH2-MIL-101(Cr) was used as a carrier for the in situ reductive growth of gold nanoparticles (AuNPs) on its surface. Then NH2-MIL-101(Cr)@AuNPs were synergized with reduced graphene oxide (rGO) to amplify the electrochemical signals. The obtained NH2-MIL-101(Cr)@AuNPs/rGO nanocomposites exhibited electrical conductivity and stability. Mercaptosuccinic acid introduces a sufficiently large number of carboxyl groups by binding to AuNPs, providing abundant binding sites for ALP antibodies. The immunosensor used differential pulse voltammetry (DPV) for electrochemical analysis, and could capture the concentration response in the range of 10-12 −10-5 g mL−1 with a relatively low detection limit of 1.64 pg mL−1. In addition, the sensor has been successfully applied to the detection of ALP in massively diluted serum of fracture patients before and after drug administration.

MWCNTs/CeO2-NRs-Functionalized WaveFlex Biosensor for Alkaline Phosphatase Detection in Agricultural Food Crops

MWCNTs/CeO₂-NRs-Functionalized WaveFlex Biosensor for Alkaline Phosphatase Detection in Agricultural Food Crops