Detection Of Alkaline Phosphatase Research Articles

Hofmeister Effect Promoted the Introduction of Tunable Large Mesopores in MOFs at Low Temperature for Femtomolar ALP Detection.

In addressing the demand for hierarchically mesoporous metal-organic frameworks (HMMOFs) with adjustable large mesopores, a method based on the synergistic effects of low-temperature microemulsions and Hofmeister ions is developed. Low temperaturedramatically enhanced the solubility of hydrophobic solvent in the microemulsion core, enlarging the mesopores in HMMOFs replica. Meanwhile, Hofmeister salt-in ions continuously controlled mesopore expansion by modulating the permeability of swelling agent into the microemulsion core. The large mesopores up to 33nm provided sufficient space for the alkaline phosphatase (ALP) enrichment, and retained the remaining channel to facilitate the free mass diffusion. Leveraging these advantages, a colorimetric sensor is successfully developed using large-mesopore HMMOFs for femtomolar ALP detection based on the enrichment and cycling amplification principles. The sensor exhibited a linear detection range of 100 to 7500fm and a limit of detection of 42fm, presenting over 4000 times higher sensitivity than classic para-nitrophenyl phosphate colorimetric methods. Such high sensitivity highlights the importance of adjustable mesoporous structures of HMMOFs in advanced sensing applications, and prefigures their potential for detecting large biomolecules in diagnostics and biomedical research.

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

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

Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Recyclable magnetic nanoparticles combined with TiO2 enrichment and “Off” to “On” SERS assay for sensitive detection of alkaline phosphatase

As an important enzyme in human tissues, abnormally alkaline phosphatase (ALP) is strongly associated with many diseases, particularly breast and prostate cancer. In this study, we proposed a rapid and sensitive surface-enhanced Raman scattering (SERS)-based biosensor for ALP detection. The biosensor utilized Fe3O4@Au@TiO2 nanoparticles (FAT NPs) as magnetic separation and enrichment materials to enrich grafted mediator Ag-4-mercaptophenylboronic acid (4-MPBA)-ascorbic acid 2-phosphate (AAP) NPs to form FAT-(AAP-4-MPBA-Ag) NPs, and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was used as the internal standard tag molecule to prepare Au@DTNB@Ag as the signal source. The phosphate group in AAP was cleaved by ALP to form ascorbic acid (AA), which acts as a receptor for the boric acid to adsorb the 4-MPBA-Au@DTNB@Ag NPs. When the switch was “Off” to “On”, the Au@DTNB@Ag NPs were used to significantly improve the sensitivity of the proposed SERS platform, enabling detection as low as 1.17U/L of target ALP with a good linear range (R 2 = 0.991) of 2–10 U/L. Furthermore, the platform successfully detected ALP from 2 % human serum. The recoveries were 99.5–106 %. In addition, FAT-(AAP-4-MPBA-Ag) NPs could be transformed into FAT NPs to achieve recyclability by ultrasonication in acidic environments. Overall, this sensitive and fast SERS platform has promising potential for accurate and efficient identification of ALP from human serum.

Controllable formation of polydopamine on carbon dots for ultrasensitive detection of alkaline phosphatase and ratiometric fluorescence immunoassay of benzocaine.

Sensing alkaline phosphatase (ALP) activity with high sensitivity and accuracy is critical for both ALP-related health and food safety supervision and the development of ALP-triggered immunoassay platforms. Herein, an ultrasensitive ratiometric fluorescence (RF) sensing system based on the controllable formation of luminescent polydopamine and efficient quenching of carbon dots was proposed for the ALP activity assay, achieving quantitative detection in the range of 0.01-100mU/L. Furthermore, this RF sensing system was integrated with an ALP-based ELISA platform to construct an RF-ELISA for benzocaine, a potentially abused anesthetic in edible fish, and ultrasensitive assay at the level of fg/mL was realized. This ratiometric strategy-based platform effectively shields various interferences through the self-calibration effect, thus providing more accurate and reliable quantification results. This study not only offers an efficient method for ultratrace detection of ALP and benzocaine but also proposes a universal platform for ultrasensitive detection of diverse targets in food analysis by replacing the recognition unit.

A point-of-care solid-phase colorimetric sensor based on the enzyme-induced metallization for ALP detection

Alkaline phosphatase (ALP) is a crucial biomarker for clinical diagnosis, which is closely related to the physiological homeostasis regulation process of human body. And the abnormal level of ALP is associated with numerous diseases, such as liver dysfunction, bone diseases, diabetes, and so on. In order to meet the demand of personalized healthcare, it is particularly important to develop a miniaturized point-of-care testing (POCT) device for ALP detection. Herein, a portable solid-phase colorimetric sensor based on enzyme-induced metallization signal amplification strategy was constructed for ALP detection. The AuNPs modified on the glass slides acted as crystal seeds, allowing Ag+ in the solution to be reduced and deposited on the surface of AuNPs, which further formed the gold core and silver shell (Au@Ag) complex and generated visual signals. The visual signals were recorded by a smartphone and quantified using open-source ImageJ software. Under the optimal conditions, the proposed method exhibited a good linear relationship from 2.0 to 16.0 pM, and the detection limit was as low as 0.9 pM. In addition, it was further successfully applied for ALP detection in non-transparent and complex samples (milk, different types of cells). A sensitive, low cost, rapid and convenient solid-phase sensor was developed for ALP detection, which was expected to provide a promising strategy for POCT devices.

Self-Calibrating Lanthanide Infinite Coordination Polymer Constructs Fluorescent Probes: A Sensitive Approach for Early Diagnosis of Hepatocellular Carcinoma and Environmental Analysis.

The development of a self-calibrating ratio fluorescence probe without the need for additional substrates is a major advancement in biosensing. In this study, at room temperature, a self-calibrating infinite coordination polymer (SSA-Tb-ATP ICPs) has been proposed by self-assembling adenosine triphosphate (ATP) with 5-sulfosalicylic acid (SSA) and Tb3+. Due to the antenna effect, SSA-Tb-ATP ICPs exhibited strong green fluorescence emission of Tb3+ (at 547 nm) and blue fluorescence emission of SSA (at 407 nm). This material offers several advantages over existing detection methods, including simplicity of synthesis and exceptional sensitivity. Our self-calibrating SSA-Tb-ATP ICPs demonstrated excellent performance in detecting alkaline phosphatase (ALP) and phosphate (Pi) in both serum and environmental samples with detection limits of 0.076 U/L and 0.025 μM, respectively. Moreover, we successfully employed the SSA-Tb-ATP ICPs to perform cellular imaging of ALP in both hepatocellular carcinoma cells (HepG2) and normal liver cells (LO2), representing a significant advancement in ALP detection and imaging. The simplicity of the synthesis and high sensitivity make this probe a promising tool for early diagnosis of hepatocellular carcinoma in clinical settings and environment analysis.

A Ratiometric Fluorescence Biosensor for Detection of Alkaline Phosphatase Via an Advanced Chemometric Model.

In this paper, a ratiometric fluorescence biosensor was introduced for alkaline phosphatase (ALP) detection based on 2-aminopurine (2-Amp) and thioflavin T (ThT)-G-quadruplex system. We designed a special DNA (5'-AGGGTTAGGGTTAGGGTTAGGGAAA/i2-Amp/AAAA-PO4-3', AP) modified with a phosphate moiety at the 3'-end, G-quadruplex at the 5'-end, and a fluorophore (2-Amp) in the middle. In the absence of ALP, the G-rich AP strand could be prone to fold into G-quadruplex structures in the presence of K+. Then, ThT combined with G-quandruplex, resulting in the enhancement of fluorescence emission peak at 485 nm. However, ALP-mediated hydrolysis of the 3'-phosphoryl end promoted the cleavage of AP by the exonuclease I (Exo I), releasing 2-Amp which displayed a strong fluorescence emission peak at 365 nm. Moreover, the quantitative fluorescence model (QFM) was derived for the analysis of the fluorescence measurements obtained by the proposed ratiometric fluorescent biosensor. With the aid of the advanced model, the proposed ratiometric fluorescent biosensor possessed satisfactory results for the detection of ALP in the human serum samples, with accuracy comparable to that of the reference method-the commercial ALP assay kit. Under the optimized experimental conditions, this method exhibited good selectivity and higher sensitivity, and the detection limit was found to be as low as 0.017 U/L. Therefore, it is reasonable to expect that the method had a great potential to detect ALP quantitatively in clinical diagnosis.

Carbon Vacancy-Enhanced Activity of Fe-N-C Single Atom Catalysts toward Luminol Chemiluminescence in the Absence of H2O2.

The classic luminol-H2O2 chemiluminescence (CL) systems suffer from easy self-decomposition of H2O2 at room temperature, hindering the practical applications of the luminol-H2O2 CL system. In this work, unexpectedly, we found that the carbon vacancy-modified Fe-N-C single atom catalysts (VC-Fe-N-C SACs) can directly trigger a luminol solution to generate strong CL emission in the absence of H2O2. The Fe-based SACs were prepared through the conventional pyrolysis of zeolitic imidazolate frameworks. The massive carbon vacancies were readily introduced into Fe-N-C SACs through a tannic acid-etching process. Carbon vacancy significantly enhanced the catalytic activity of Fe-N-C SACs on the CL reaction of luminol-dissolved oxygen. The VC-Fe-N-C SACs performed a 13.4-fold CL enhancement compared with the classic luminol-Fe2+ system. It was found that the introduction of a carbon vacancy could efficiently promote dissolved oxygen to convert to reactive oxygen species. As a proof of concept, the developed CL system was applied to detect alkaline phosphatase with a linear range of 0.005-1 U/L as well as a detection limit of 0.003 U/L. This work demonstrated that VC-Fe-N-C SAC is a highly efficient CL catalyst that can promote the analytic application of the luminol CL system.

Distinguishable Colorimetric Biosensor for Diagnosis of Prostate Cancer Bone Metastases.

Castration-resistant prostate cancer (PCa) causes severe bone metastasis (BM), which significantly increases mortality in men with PCa. Imaging tests and radiometric scanning require long analysis times, expensive equipment, specialized personnel, and a slow turnaround. New visualization technologies are expected to solve the above problems. Nonetheless, existing visualization techniques barely meet the urgency for precise diagnosis because the human eyes cannot recognize and capture even slight variations in visual information. By using dye differentiated superposition enhancement colorimetric biosensors, an effective method to diagnose prostate cancer bone metastases (PCa-BM) with excellent accuracy for naked-eye quantitative detection of alkaline phosphatase (ALP) is developed. The biomarker ALP specific hydrolytic product ascorbic acid can be detected by rhodamine derivatives (Rd) as gold nanobipyramids (Au NBPs) are deposited and grown. Color-recombining enhancement effects between Rd and Au NBPs significantly improved abundance. The 150UL-1 threshold between normal and abnormal can be identified by color. And with color enhancement effect and double signal response, the ALP index is visually measured to diagnose PCa-BM and provide handy treatment recommendations. Additionally, the proposed colorimetric sensing strategy can be used to diagnose other diseases.

An accurate and sensitive sensor based on the internal filtration effect for the detection of alkaline phosphatase and ascorbic acid by the Fenton reaction and dual readout signal

Ascorbic acid (AA) and alkaline phosphatase (ALP) in human serum are indispensable substances in the process of biological metabolism, and their abnormal contents are strongly associated with a range of diseases. Therefore, it is meaningful to develop a simple, accurate and sensitive assay for the detection of AA and ALP. Up-conversion nanoparticles (UCNPs) have some unique characteristics, especially, the potential for background-free excitation by the near-infrared region (NIR) significantly improves the limits of detection (LODs). Moreover, sensors based on the inner filter effect (IFE) do not require covalent linkage between receptors and fluorophores or surface modification, thus the probe fabrication is simple and flexible UCNP-based. Therefore, a simple, selective, sensitive, and flexible sensing system for the detection of AA and ALP is proposed by combining the IFE and the cyclic signal amplification strategy. Under ideal circumstances, the LODs of ALP through the colorimetric and fluorescence method were 0.192 U/L and 0.013 U/L. The established sensing platform was applied to assessing ALP activity in human serum, and the detection results are close to those of commercially available kits, indicating its immense potential as an effective and convenient tool for monitoring ALP activity in clinical settings.

Effects of tension on mitochondrial autophagy and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to explore the osteogenic ability and mitochondrial autophagy of periodontal ligament stem cells (PDLSCs) under cyclic tensile stress (CTS). Primary PDLSCs were isolated from the periodontal membrane and cultured by passage. Alizarin red staining, alkaline phosphatase detection, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the osteogenic differentiation level of PDLSCs. Mitochondrial autophagy in PDLSCs after CTS was measured using a mitochondrial autophagy detection kit, and the expression levels of autophagy-related proteins LC3B, LAMP1 and Beclin1 were measured using cellular immunofluorescence technology, RT-PCR and Western blot. After applying CTS, the osteogenic differentiation ability of PDLSCs was significantly improved, and the expression of alkaline phosphatase on the surface of the cell membrane and the formation of calcium nodules in PDLSCs were significantly increased respectively. We also studied the relevant mechanism of action and found that applying CTS can promote the osteogenic differentiation of PDLSCs and is related to the activation of mitochondrial autophagy. This study provides new insights into the mechanism of increased osteogenic differentiation on the tension side of orthodontic teeth and provides new experimental evidence for the involvement of mitochondrial autophagy in the regulation of osteogenic differentiation.

A controllable design of ZIF-67 based magnetic carbon nanotubes as efficient oxidase mimetics for sensitive detection of alkaline phosphatase in serum

The activity of alkaline phosphatase (ALP) is related to various pathological processes. Therefore, developing an ALP activity detection technology with high sensitivity and accuracy is of great significance for clinical diagnosis and cancer screening. To solve uncontrollable carbon nanotubes (CNTs) structure and lack of solubility, FeCo hollow nanocages (HNCs)@CNTs catalysts were prepared by facile pyrolysis of ZIF-67 precursor mixed with Fe-Co catalyst, displaying an oxidase-like activity. From the characterization, formation of CNTs was observed on the surface of FeCo HNCs@CNTs with the mole ratio of Fe3+ to Co2+ of 3:1. Density functional theory calculations demonstrated that the performed catalytic activity of FeCo HNCs@CNTs derived from active orbital of Co-3d band, which enabled the activation and decomposition of O2 with a feasible thermodynamic process. ·O2− and 1O2 were the main reactive oxygen species as electron acceptors. ALP catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) to convert it into ascorbic acid (AA), which could effectively reduce the FeCo HNCs@CNTs-mediated chromogenic reaction of TMB oxidation. The performance of the FeCo HNCs@CNTs-linked biosensing assay showed a broad detection range from 0.1 U/L to 150 U/L and a relatively low limit of detection of 0.083 U/L. The ALP activity of the mouse serum sample obtained by the proposed biosensing assay was highly consistent with that via the clinical method, indicating a promising technology in biomolecule detection.

Osteoblast-derived exosomes promote osteogenic differentiation of osteosarcoma cells via URG4/Wnt signaling pathway

Osteosarcoma is a primary malignant bone tumor. Although surgery and chemotherapy are the main treatment methods, the overall curative effect remains unsatisfactory. Therefore, there is an urgent need to develop new therapeutic options for osteosarcoma. In this study, the effect and molecular mechanism of osteoblast-derived exosomes on the treatment of osteosarcoma were evaluated. Human primary osteoblasts were cultured to observe the effects of osteoblast-derived exosomes on the osteogenic differentiation of osteosarcoma cells both in vitro and in vivo. Alizarin red staining and alkaline phosphatase detection were used to evaluate the degree of osteogenic differentiation, and immunofluorescence and Western blotting were used to detect protein expression. The results showed that osteoblast-derived exosomes effectively inhibited the proliferation of osteosarcoma cells and promoted their mineralization in vitro. The exosomes also significantly inhibited tumor growth and promoted tumor tissue mineralization in vivo. Osteoblast-derived exosomes upregulated the expression of bone sialoprotein, osteonectin, osteopontin, runt-related transcription factor 2, and Wnt inhibitory factor 1, downregulated the expression of cyclin D1, and suppressed the nuclear accumulation of β-catenin and promoted its phosphorylation in vitro and in vivo. However, these effects were significantly reversed by upregulated gene (URG) 4 overexpression. These findings suggest that osteoblast-derived exosomes could activate the osteogenic differentiation process in osteosarcoma cells and promote their differentiation by targeting the URG4/Wnt signaling pathway.

Target-Induced Photocurrent-Polarity-Switching PEC Sensing Platform Based on In Situ Generation of Oxygen Vacancy-Modulated Energy Band Structures.

The polarity of the photocurrent can be modulated by tunable bipolar photoelectrochemical (PEC) behavior, which is anticipated to address the issues of high background signal caused by traditional unidirectional increasing/decreasing response and false-positive/false-negative problems. Here, a new approach is suggested for the first time, which employs a target-induced enzyme-catalyzed reaction and in situ oxygen vacancy (OV) generation to achieve heterojunction photocurrent switching for highly sensitive detection of alkaline phosphatase (ALP). Among them, the ALP can catalyze the decomposition of ascorbic acid phosphate to produce ascorbic acid, which not only acts as an electron donor to change the redox environment but also acts as a reducing agent to introduce OVs into BiOBr semiconductors in cooperation with illumination. The introduction of vacancies can effectively modulate the energy band structure of BiOBr, while with the change of redox conditions, the transfer path of photogenerated carriers is changed, thus realizing the switching of photocurrents, which leads to its use in the construction of a negative-background anti-interference PEC sensing platform, achieving a wide linear range from 0.005 to 500 U·L-1 with a low detection limit of 0.0017 U·L-1. In conclusion, the photocurrent switching operation of this system is jointly regulated by chemistry, optics, and carrier motion, which provides a new idea for the construction of a PEC sensing platform based on photocurrent polarity switching.

Ligand‐induced Assembly of Copper Nanoclusters with Enhanced Electrochemical Excitation and Radiative Transition for Electrochemiluminescence**

AbstractCopper nanoclusters (CuNCs) are emerging electrochemiluminescence (ECL) emitters with unique molecule‐like electronic structures, high abundance, and low cost. However, the synthesis of CuNCs with high ECL efficiency and stability in a scalable manner remains challenging. Here, we report a facile gram‐scale approach for preparing self‐assembled CuNCs (CuNCsAssy) induced by ligands with exceptionally boosted anodic ECL and stability. Compared to the disordered aggregates that are inactive in ECL, the CuNCsAssy shows a record anodic ECL efficiency for CuNCs (10 %, wavelength‐corrected, relative to Ru(bpy)3Cl2/tripropylamine). Mechanism studies revealed the unusual dual functions of ligands in simultaneously facilitating electrochemical excitation and radiative transition. Moreover, the assembly addressed the limitation of poor stability of conventional CuNCs. As a proof of concept, an ECL biosensor for alkaline phosphatase detection was successfully constructed with an ultralow limit of detection of 8.1×10−6 U/L.

Ligand-induced Assembly of Copper Nanoclusters with Enhanced Electrochemcial Excitation and Radiative Transition for Electrochemiluminescence.

Copper nanoclusters (CuNCs) are emerging electrochemiluminescence (ECL) emitters with unique molecule-like electronic structures, high abundance, and low cost. However, the synthesis of CuNCs with high ECL efficiency and stability in a scalable manner remains challenging. Here, we report a facile gram-scale approach for preparing self-assembled CuNCs (CuNCsAssy ) induced by ligands with exceptionally boosted anodic ECL and stability. Compared to the disordered aggregates that are inactive in ECL, the CuNCsAssy shows a record anodic ECL efficiency for CuNCs (10 %, wavelength-corrected, relative to Ru(bpy)3 Cl2 /tripropylamine). Mechanism studies revealed the unusual dual functions of ligands in simultaneously facilitating electrochemical excitation and radiative transition. Moreover, the assembly addressed the limitation of poor stability of conventional CuNCs. As a proof of concept, an ECL biosensor for alkaline phosphatase detection was successfully constructed with an ultralow limit of detection of 8.1×10-6 U/L.

Deciphering the Mechanisms of Photo-Enhanced Catalytic Activities in Plasmonic Pd-Au Heteromeric Nanozymes for Colorimetric Analysis.

The growing demand for highly active nanozymes in various fields has led to the development of several strategies to enhance their activity. Plasmonic enhancement, a strategy used in heterogenous catalysis, represents a promising strategy to boost the activity of nanozymes. Herein, Pd-Au heteromeric nanoparticles (Pd-Au dimers) with well-defined heterointerfaces have been explored as plasmonic nanozymes. As a model system, the Pd-Au dimers with integrated peroxidase (POD)-like activity and plasmonic activity are used to investigate the effect of plasmons on enhancing the activity of nanozymes under visible light irradiation. Mechanistic studies revealed that the generation of hot electron-hole pairs plays a dominant role in plasmonic effect, and it greatly enhances the decomposition of H2 O2 to the reactive oxygen species (ROS) intermediates (•OH, •O2 - and 1 O2 ), leading to elevated POD-like activity of the Pd-Au dimers. Finally, the Pd-Au dimers are applied in the plasmon-enhanced colorimetric method for the detection of alkaline phosphatase, exhibiting broad linear range and low detection limit. This study not only provides a straightforward approach for regulating nanozyme activity through plasmonic heterostructures but also sheds light on the mechanism of plasmon-enhanced catalysis of nanozymes.

Construction of Metal Organic Framework-Derived Fe-N-C Oxidase Nanozyme for Rapid and Sensitive Detection of Alkaline Phosphatase.

Alkaline phosphatase (ALP) is a phosphomonoester hydrolase and serves as a biomarker in various diseases. However, current detection methods for ALP rely on bulky instruments, extended time, and complex operations, which are particularly challenging in resource-limited regions. Herein, we synthesized a MOF-derived Fe-N-C nanozyme to create biosensors for the coulometric and visual detection of ALP. Specifically, we found the Fe-N-C nanozyme can efficiently oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue-colored tetramethyl benzidine (TMBox) without the need for H2O2. To construct the biosensor, we incorporated the ALP enzymatic catalytic reaction to inhibit the oxidation of TMB by Fe-N-C oxidase nanozyme. This biosensor showed rapid and highly sensitive detection of ALP in both buffer and clinical samples. The limit of detection (LOD) of our approach could be achieved at 3.38 U L-1, and the linear range was from 5 to 60 U L-1. Moreover, we also developed a visual detection for ALP by using a smartphone-based assay and facilitated practical and accessible point-and-care testing (POCT) in resource-limited areas. The visual detection method also achieved a similar LOD of 2.12 U L-1 and a linear range of 5-60 U L-1. Our approach presents potential applications for other biomarker detections by using ALP-based ELISA methods.

Ultrasensitive alkaline phosphatase activity assay based on controllable signal probe production coupled with the cathodic photoelectrochemical analysis.

A highly sensitive and selective split-type perovskite-based photoelectrochemical (PEC) platform was developed for measuring alkaline phosphatase (ALP) activity in milk and serum samples. ALP in the test sample hydrolyzed 2-phosphate sesquimagnesium salt hydrate (AAPS) in a 96-microwell plate to produce ascorbic acid (AA), a PEC electron donor. The resulting AA, which could preferentially annihilate the photogenerated holes, indirectly reflects ALP activity. The PEC used a cetyltrimethylammonium bromide (CTAB)-functionalized CH3NH3PbI3 (CTAB@CH3NH3PbI3) film as the cathode to monitor the controlled AA production. Due to the excellent photoelectric characteristics of the CH3NH3PbI3 perovskite and the split-type assay, excellent sensitivity and selectivity for ALP detection were obtained. Under the optimum experimental conditions, ALP activity with a limit of detection (LOD) of 2.6×10-4 U/L in a linear dynamic range of 10-3∼102 U/L was obtained. With its sensitive, rapid, and high-throughput detection capabilities, this split-type and label-free PEC platform has great potential for use in food and biomedical analysis.