Detection Of Alkaline Phosphatase Research Articles

Pyrophosphate-Mediated On–Off–On Oxidase-Like Activity Switching of Nanosized MnFe2O4 for Alkaline Phosphatase Sensing

Developing reliable and facile approaches for alkaline phosphatase (ALP) sensing is important due to its role as a clinical biomarker for many diseases. In this study, we proposed a new and convenient colorimetric assay based on the pyrophosphate (PPi)-mediated oxidase-mimicking activity switching of nanosized MnFe2O4 for the detection of ALP. The synthesized MnFe2O4 exhibited high oxidase-like activity to catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to its blue product TMBox in the presence of dissolved O2, leading to a color reaction rapidly and remarkably; PPi could significantly inhibit the activity of the MnFe2O4 nanozyme via the strong interaction between PPi and the Fe(III) species in MnFe2O4, resulting in the suppression of the TMB color reaction; when ALP was added, it hydrolyzed the PPi substrate to phosphate (Pi) that had no obvious effect on the MnFe2O4 activity, and such that the TMB color reaction catalyzed by the nanozyme could be observed again. With the above principle, linear colorimetric determination of ALP in the scope of 0.6–55 U L−1 was achieved, giving the limit of detection down to 0.27 U L−1. Besides, the developed assay could provide selective response toward ALP against other co-existing biological species. Furthermore, reliable detection of ALP in human serum samples was verified by our assay, revealing its great promise as an effective and facile tool for ALP monitoring in clinical practice.

A pilot study of human mesenchymal stem cells from visceral and sub-cutaneous fat tissue and their differentiation to osteogenic phenotype.

To evaluate the different behavior of two different human adult adipocytes derived stem cells (hASCs) during proliferation and osteogenic differentiation. Human adult adipocytes stem cells (hAT-SCs) from visceral (hAV-SCs) and subcutaneous (hAS-SCs) sites were obtained after surgery procedures of seven patients. All samples were fully investigated and the different proliferation rates were evaluated. All MSCs clusters were cultured with an osteogenic and adipogenic differentiation medium. Homogeneous pools of Mesenchymal Stem Cells (MSCs) were confirmed by Flow-Cytometry Analysis (FACS) and Spectrophotometric Assay. The differentiated cells were eventually assessed for the expression of Alkaline Phosphatase (ALP), Alizarin Red (AR) and Oil Red-O (OR-O) detection, and analyzed by the Spectrophotometric Assay. After osteogenic differentiation, the cell clusters were incubated and analyzed with Real Time-Polymerase Chain Reaction (qRT-PCR) and fluorescence microscopy. The FACS analysis performed on hAT-SCs confirmed the homogenous presence of MSCs in all samples. The ALP, AR stain confirmed the osteogenic differentiation capacity of MSCs towards osteoblast-like-cells. The colorimetric cell metabolic activity (MTS) assay showed an increase in the proliferation rate with different values in both sets hAS-SCs vs. hAV-SCs. These in vitro findings of both hAS-SCs and hAV-SCs suggested an important role of these stem cells for future clinical use in bone regeneration. Indeed, the final outcomes suggested a better performance of cells coming from subcutaneous adipose tissue vs. those from visceral fat tissue.

Detecting Alkaline Phosphatase-Labeled Cells.

Different substrates are available for detection of cells labeled with alkaline phosphatase. Naphthol-AS-BI-phosphate (NABP)/New Fuchsin produces an intense red product that is insoluble in alcohols as well as aqueous solutions and is compatible with a range of histochemical stains. Bromochloroindoyl phosphate/nitro blue tetrazolium (BCIP/NBT) is the most commonly used of the chromogenic substrates for alkaline phosphatase. It is somewhat more sensitive than NABP/New Fuchsin but gives less contrast during microscopic observation.

A sensitive ratiometric fluorescent probe for quantitive detection and imaging of alkaline phosphatase in living cells

We report a sensitive fluorescence probe Ratio-ALP for in vivo ratiometric imaging of alkaline phosphatase (ALP). The probe was designed by conjugating a naphthalimide with a phosphate group as the responsive moiety for enzymatic reactions. The fluorescence intensity ratio displayed a linear relationship against the concentration of ALP in the concentration range from 0 to 200 U/L, its LOD is as low as 0.25 U/L. Ratio-ALP has good selectivity and sensitivity, high cell permeability, and low cytotoxicity. Furthermore, probe Ratio-ALP was also successfully applied to image endogenous ALP activity in living cells.

Facile colorimetric assay of alkaline phosphatase activity using polydiacetylene liposomes with calcium ions and pyrophosphate

The detection of alkaline phosphatase (ALP) activity has received great attention since its activity in serum is highly associate with various human diseases. Herein, we describe a simple yet effective colorimetric assay for ALP activity based on polydiacetylene (PDA) and competitive binding of pyrophosphate (PPi) with calcium ions (Ca2+). PDA liposomes prepared from 10,12-pentacosadiynoic acid (PCDA) display the distinct blue-to-red color transition towards Ca2+, which can be effectively blocked by PPi due to the ultra-strong binding ability of Ca2+ with PPi. Since PPi can be specifically hydrolyzed into orthophosphate (Pi) by ALP, the negative effect of PPi could be removed when ALP is introduced into the system, thereby producing a colorimetric signal relying on ALP activity. Based on this strategy, ALP was sensitively and selectively detected with a limit of detection of 5.4 U/L. Besides, the proposed sensing method was successfully applied to evaluate the inhibitor efficiency. Detection of ALP in real human serum samples could also be achieved by this system with acceptable accuracy. As the proposed PDA liposome sensor is simple and straightforward, this strategy holds great potential as a promising alternative for ALP-based clinic diagnosis or biological research.

Controllably Prepared Aptamer-Molecularly Imprinted Polymer Hybrid for High-Specificity and High-Affinity Recognition of Target Proteins.

Molecularly imprinted polymers (MIPs) and aptamers, as effective mimics of antibodies, can overcome only some drawbacks of antibodies. Since they have their own advantages and disadvantages, the combination of MIPs with aptamers could be an ideal solution to produce hybrid alternatives with improved properties and desirable features. Although quite a few attempts have been made in this direction, a facile and controllable approach for the preparation of aptamer-MIP hybrids still remains lacking. Herein, we present a new approach for facile and controllable preparation of aptamer-MIP hybrids for high-specificity and high-affinity recognition toward proteins. An aptamer that can bind the glycoprotein alkaline phosphatase (ALP) with relative weak affinity and specificity was used as a ligand, and controllable oriented surface imprinting was carried out with an in-water self-polymerization system of dopamine. A thin layer of polydopamine was formed to cover the template to an appropriate thickness. After removing the template from the polymer, an aptamer-MIP hybrid with apparently improved affinity and specificity toward ALP was obtained, giving cross-reactivity of 3.2-5.6% and a dissociation constant of 1.5 nM. With this aptamer-MIP hybrid, a plasmonic immunosandwich assay (PISA) was developed. Reliable detection of ALP in human serum by the PISA was demonstrated.

Determination of alkaline phosphatase activity based on enzyme-triggered generation of a thiol and the fluorescence quenching of silver nanoclusters.

A fluorimetric method is described for the determination of alkaline phosphatase (ALP) activity. It is based on the use of polyethyleneimine-coated silver nanoclusters (AgNCs), which display an intense blue fluorescence peaking at 450nm (under 375nm excitation). ALP catalyzes the dephosphorylation of the thiophosphate amifostine to generate a thiol that binds to the AgNCs and causes its fluorescence to be quenched. Under the optimal experimental conditions, fluorescence linearly drops in the 0.08-2.0UL-1 ALP activity range, and the limit of detection is 0.02UL-1. The method was successfully applied to the determination of ALP activity in spiked human serum samples. Graphical abstract Alkaline phosphatase (ALP) catalyzes the degradation of amifostine with a generation a thiol product. The thiol quenches the fluorescence of silver nanoclusters, and a method for the detection of ALP down to 0.02UL-1 was developed.

The Emerging Role of Bone Markers in Diagnosis and Risk Stratification of Patients With Coronary Artery Disease.

Molecules that govern bone metabolism, such as osteoprotegerin (OPG) and osteopontin (OPN), have been isolated from other tissues, including blood vessels. Atherosclerosis and coronary artery disease (CAD) are leading causes of mortality worldwide. Despite novel biochemical and imaging techniques, early detection of CAD is still unsatisfactory. Experimental data indicate that bone turnover markers (BTMs) contribute to the development of atherosclerosis. This finding has sparked interest in their clinical use. This narrative review analyzed information from >50 human studies, which strongly suggest that OPG, OPN, and alkaline phosphatase (ALP) serum concentrations are altered in patients with CAD. Osteoprotegerin seems to be more useful for the detection of early disease, while OPN and ALP are recruited in vessels after the establishment of disease. Osteocalcin may be used as a flow cytometry marker for endothelial progenitor cells and can constitute a marker to monitor response to interventional treatments and risk of restenosis. However, most data derive from observational studies. Incorporation of BTMs in multifactorial computational algorithms could further determine their role in CAD diagnosis and prognosis together with other imaging techniques and biochemical markers.

Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus aureus.

The spread of antibiotic-resistant bacteria poses a global threat to public health. Conventional bacterial detection and identification methods often require pre-enrichment and/or sample preprocessing and purification steps that can prolong diagnosis by days. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread antibiotic-resistant bacteria and is the leading cause of hospital-acquired infections. Here, we have developed a method to specifically capture and detect MRSA directly from patient nasal swabs with no prior culture and minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. Bacteria are captured based on antibody recognition of a membrane-bound protein marker that confers β-lactam antibiotic resistance. MRSA identification is then achieved by the use of a strain-specific antibody functionalized with alkaline phosphatase for electrochemical detection. This approach ensures that only those bacteria of the targetstrain and resistance profile are measured. The method has a limit of detection of 845 CFU/mL and excellent discrimination against high concentrations of common nontarget nasal flora with a turnaround time of under 4.5 h. This detection method was successfully validated using clinical nasal swab specimens ( n = 30) and has the potential to be tailored to various bacterial targets.

Detection of alkaline phosphatase activity with a functionalized nanopipette

In this study, a label-free sensing method based on a functionalized nanopipette is proposed for detection of alkaline phosphatase (ALP) activity. This method is based on the unique charge transport properties of nanopores in which the ionic current can be modulated by variation in the charge on the inner surface of the nanopipette. A phosphorylated peptide, O-phospho-l-tyrosine (p-Tyr), was used as a model substrate and to functionalize the nanopipette. The covalent functionalization procedures were characterized by measuring the current-voltage (IV) curves and extracting the ionic current rectification status. On ALP catalysis, the removal of the p-Tyr phosphate group induces a decrease in the negative surface charge, subsequently leading to a sensitive response in the ionic current change. An ANOVA test confirmed a linear relationship between the normalized ionic current change and ALP activity. The functionalized nanopipettes demonstrated a directly measured detection limit of 0.1 mU/mL, and excellent selectivity against four common interfering proteins. Furthermore, this nanopore-based method has potential for use in characterizing or sensing the activities of other enzymes with a similar biochemical process to ALP.

Photoelectrochemical determination of the activity of alkaline phosphatase by using a CdS@graphene conjugate coupled to CoOOH nanosheets for signal amplification.

A method is described for photoelectrochemical determination of the activity of alkaline phosphatase (ALP). It employs an indium tin oxide (ITO) electrode modified a CdS quantum dots@graphene (CdS@GR) composite and hexagonal cobalt oxyhydroxide (CoOOH) nanosheets. The CdS@GR nanocomposite was synthesized by assembling the CdS quantum dots onto a GO film to receive a basic photocurrent response of the ITO. This is further improved by covering it with CoOOH nanosheets. Secondly, 2-phospho-L-ascorbic acid (AAP) is added as a substrate for ALP. Its hydrolysis yields ascorbic acid which reduces CoOOH to form cobalt(II) ion. As a result, the CoOOH nanosheets decompose. This is accompanied by a reduction of the photocurrent. The effect was used to design a selective and sensitive assay of determination of the activity of ALP. Under the optimized experimental conditions, response is linear in the 10 to 300U·L-1ALP activity range. The detection limit is 1.5U·L-1 at a signal-to-noise ratio of 3. Graphical abstract Indium tin oxide (ITO) was coted withCdS@graphene andCoOOH to obtain a material with superior photoelectrochemical properties. The detection of alkaline phosphatase (ALP) was accomplished by using 2-phospho-L-ascorbic acid (AAP) which ishydrolyzed by ALP to releaseascorbic acid (AA) which reduces CoOOH to Co2+.

Detection of alkaline phosphatase activity and inhibition with fluorescent hydroxyapatite nanoparticles

Fluorescence detection of alkaline phosphatase (ALP) activity and inhibition based on fluorescent hydroxyapatite nanoparticles.

An ALP-activatable and mitochondria-targeted probe for prostate cancer-specific bimodal imaging and aggregation-enhanced photothermal therapy.

Tumor-derived alkaline phosphatase (ALP) is over-expressed in metastatic prostate cancer. The development of selective probes for ALP detection is therefore critical for early diagnosis and therapy of metastatic prostate cancer. Herein, we develop a mitochondria-targeted near-infrared activatable fluorescent/photoacoustic (NIR FL/PA) probe for the selective detection of prostate cancer-derived ALP and aggregation-enhanced photothermal therapy. Upon dephosphorylation, the probes are activated and they provide a red-shifted strong absorption and emission in the NIR window and thus enable NIR FL and PA imaging of ALP activity in tumor tissues. Particularly, the activated probes self-assemble in situ into a supramolecular network structure which induces cell apoptosis and significantly enhances the photothermal therapy efficacy.

Rapid and simple detection of ascorbic acid and alkaline phosphataseviacontrolled generation of silver nanoparticles and selective recognition

Ascorbic acid (AA) and alkaline phosphatase (ALP) serve as an important coenzyme and enzyme in multiple biological metabolism reactions, respectively, and abnormal levels of these substrates have been associated with several diseases. Herein, a new and simple fluorescence strategy has been developed for AA and ALP sensing by exploiting CdTe quantum dots (QDs) as an effective signal indicator. This method is mainly based on the selective fluorescence-quenching reaction between Ag+ and CdTe QDs, as opposed to silver nanoparticles (Ag NPs); Ag+ can be reduced to Ag NPs by AA. Furthermore, by taking advantage of AA as a mediator, this strategy is further exploited for ALP assay given that ALP can cause the hydrolysis of l-ascorbic acid-2-phosphate (AAP), which yields AA. Under optimal conditions, controlled generation of Ag NPs and the selective recognition-based sensing system exhibit high sensitivity toward AA and ALP with limits of detection (LODs) of 3 μM and 0.25 U L-1 and linear ranges of detection from 0 to 800 μM and 1 to 1000 U L-1, respectively. Moreover, the sensor was successfully used for assaying AA in fruit juice and ALP in human serum. The results demonstrate that the proposed fluorescence strategy has significant advantages, such as its simplicity, cost-effectiveness, and rapid runtime, and the operational convenience of this label-free method further demonstrates its potential for constructing effective sensors with biochemical and clinical applications.

Mineralisation Influence of Betamethasone on Lipopolysaccharide-Stimulated Dental Pulp Cells.

To evaluate the mineralisation response of lipopolysaccharide (LPS)-induced dental pulp cells (DPCs) to betamethasone and the potential benefit of betamethasone application on the recovery of injured dental pulp. The proliferation influence of betamethasone on DPCs was analysed through the cell counting kit-8 assay. To assess the anti-inflammatory effects of betamethasone, the expression levels of inflammatory factors IL-6, IL-1ß and TNF-∂ were determined by real-time polymerase chain reaction (PCR). Mineralisation was investigated through the detection of the mineralisation-related biomarkers alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and osteocalcin (OCN) through the ALP activity assay, immunohistochemistry staining, Alizarin Red and tissue nonspecific alkaline phosphatase (TNAP) staining, the reverse transcriptase PCR technique and western blot. A low concentration of betamethasone (1 µ/mL) promoted the proliferation of DPCs. The real-time PCR results demonstrated that inflammatory cytokines were downregulated by betamethasone treatment. The mineralisation outcome in DPCs treated with betamethasone was better than in those treated without betamethasone. Betamethasone promoted the proliferation of DPCs. Betamethasone enhanced mineralisation in LPS-stimulated DPCs.

A "turn-on" sensor based on MnO2 coated UCNPs for detection of alkaline phosphatase and ascorbic acid.

Alkaline phosphatase (ALP), an extraordinary enzyme, can catalyze the dephosphorylation of small molecules, proteins and nucleic acids. ALP has been confirmed as a crucial serum diagnostic indicator, since the abnormal level of ALP is closely related to a variety of pathological processes, especially in the early diagnosis and screening of cancer. Herein, we designed a "turn-on" sensor to detect ALP and ascorbic acid (AA) based on the redox reaction between manganese dioxide (MnO2) coated upconversion nanoparticles (UCNPs) and AA. Firstly, 2 nm sized NaYF4:Yb3+,Tm3+ UCNPs were synthesized by a facile one-pot hydrothermal method. Then MnO2 coated UCNPs were prepared via electrostatic interactions between MnO2 nanosheets and UCNPs. MnO2 nanosheets could absorb blue light emitted by UCNPs near 471 nm under laser excitation at 980 nm, and so the luminescence of UCNPs was quenched based on luminescence energy transfer (LET). In the presence of AA, the luminescence was recovered again by the redox reaction between AA and MnO2 coated UCNPs. MnO2 nanosheets were reduced to Mn2+ and UCNPs were released. Furthermore, the "turn-on" sensor was applied to monitor ALP since the phosphate group of 2-phospho-l-ascorbic acid (AAP) was removed by ALP to yield AA. The bio-assay showed a good linear relationship between the restored luminescence intensities (ΔI = I-I0) and ALP concentrations ranging from 0.25 to 150 mU mL-1 with a limit of detection (LOD) of 0.045 mU mL-1 and between ΔI and AA concentrations ranging from 0.5 to 250 μM with an LOD of 0.29 μM. The luminescent sensor was also successfully applied for detection of ALP and AA in human serum samples with recoveries from 94.9% to 104.6% and 99.4% to 104.9%.

Ligase amplification reaction-catalyzed assembly of a single quantum dot-based nanosensor for sensitive detection of alkaline phosphatase.

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.

A light-up near-infrared probe with aggregation-induced emission characteristics for highly sensitive detection of alkaline phosphatase.

Developing activatable near-infrared (NIR) probes to specifically monitor and visualize the activities of cancer-related enzymes is highly significant yet challenging in early cancer diagnosis. Taking advantage of the unique photophysical characteristics of aggregation-induced emission (AIE) fluorophores, here we design and synthesize a novel activatable probe QMTP by conjugating an AIE fluorophore quinolone-malononitrile to a hydrophilic phosphate-modified phenol group. The probe was initially non-fluorescent in aqueous solution due to its good water solubility, but was readily activated to generate a strong NIR fluorescence upon treatment with alkaline phosphatase (ALP), which enables specific detection of ALP activity. Furthermore, we have employed QMTP to monitor and spatially map the activity of endogenous ALP both in cancer cells and in drug-treated zebrafish larvae. The experimental results reveal that the QMTP probe has great specificity and sensitivity for ALP detection. We thus believe that our work offers a promising tool for accurate detection of ALP-associated diseases in preclinical applications.

Pyrophosphate ion-responsive alginate hydrogel as an effective fluorescent sensing platform for alkaline phosphatase detection.

A pyrophosphate ion (PPi)-responsive alginate hydrogel was fabricated by using Cu2+ as a crosslinker. Benefiting from the ultrahigh affinity of Cu2+ to PPi, which can discriminate PPi from phosphate ions and other anions, the incorporation of carbon dots enables the alginate hydrogel to be an effective fluorescent sensing platform for the detection of alkaline phosphatase.

Bifunctional MIL-53(Fe) with pyrophosphate-mediated peroxidase-like activity and oxidation-stimulated fluorescence switching for alkaline phosphatase detection

Alkaline phosphatase (ALP) is extensively used as a clinical biomarker because of its close relevance with a variety of diseases. Thus, exploring reliable and practical methods for ALP analysis is of great significance. In the present work, we explored MIL-53(Fe) as a bifunctional platform with pyrophosphate (PPi)-mediated peroxidase-like activity and oxidation-stimulated fluorescence switching for ALP sensing. The proposed MIL-53(Fe) could exhibit favorable peroxidase-mimicking activity to catalytically decompose H2O2 to hydroxyl radicals, which had strong oxidizing ability to oxidize the terephthalic acid bridging ligand, resulting in the oxidation-stimulated turn-on fluorescence of MIL-53(Fe) itself. Due to the strong coordination interaction between PPi and Fe3+, the former with a relatively large molecular structure was able to inhibit the catalytic activity of MIL-53(Fe) via capping active Fe3+ sites, leading to the suppression of its self-fluorescence response. When ALP was present, it could hydrolyze the PPi inhibitor and restore the dual functions of MIL-53(Fe) to provide fluorescence again. With the above principle, highly sensitive and selective determination of ALP with a linear scope of 2-80 U L-1 and a detection limit down to 0.7 U L-1 was achieved. The MIL-53(Fe) was also demonstrated to be very reliable in measuring the target in human serum, indicating its great promise as an integrated tool for ALP detection in clinical practice.