Detection Of Alkaline Phosphatase Activity Research Articles

Restoring the Oxidase-Like Activity of His@AuNCs for the Determination of Alkaline Phosphatase.

In this paper, we propose a simple colorimetric method for the sensitive and selective detection of alkaline phosphatase (ALP) activity based on the turn off/turn on oxidase mimic activity of His@AuNCs. His@AuNCs/graphene oxide hybrids (His@AuNCs/GO) were easily obtained using the self-assembly method with poly (diallyldimethylammonium chloride) (PDDA)-coated GO and showed high oxidase-like activity compared with His@AuNCs. We found that the pyrophosphate ion (P2O74−, PPi) could effectively inhibit the oxidase mimic activity of His@AuNCs/GO, and the hydrolysis of PPi by ALP restored the inhibited activity of His@AuNCs/GO, enabling them to efficiently catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate the blue oxidized product oxTMB. The intensity of the color showed a linear dependency with the ALP activity. ALP was detected in the linear range of 0–40 mU/mL with a low detection limit (LOD) of 0.26 mU/mL (S/N = 3). The proposed method is fast, easy, and can be applied to monitor the ALP activity in serum samples accurately and effectively, which suggests its practicability and reliability in the detection of ALP activity in clinical practice.

Thermo-responsive chitosan/silk fibroin/amino-functionalized mesoporous silica hydrogels with strong and elastic characteristics for bone tissue engineering

Amino-functionalized mesoporous silica nanoparticles with radially porous architecture were optimally synthesized, and they were used together with silk fibroin and chitosan to produce a type of covalently crosslinked composite hydrogel using genipin as a crosslinker. The optimally achieved composite gels were found to be thermo-responsive at physiological temperature and pH with well-defined injectability. They were also detected to have mechanically strong and elastic characteristics. In addition, these gels showed the ability to release bioactive Si ions suited to an effective dose range in approximately linear manners for a few weeks. Studies on the cell-gel constructs revealed that the composite gels well supported the growth of seeded MC3T3-E1 cells, and the deposition of matrix components. Results obtained from the detection of alkaline phosphatase activity and the matrix mineralization in the cell-gel constructs confirmed that these composite gels had certain osteogenic capacity. The obtained results suggest that these composite gels have promising potential in bone repair and regeneration.

Adrenomedullin 2 improves bone regeneration in type 1 diabetic rats by restoring imbalanced macrophage polarization and impaired osteogenesis

BackgroundBoth advanced glycation end products (AGEs) and AGE-mediated M1 macrophage polarization contribute to bone marrow mesenchymal stem cell (BMSC) dysfunction, leading to impaired bone regeneration in type 1 diabetes mellitus (T1DM). Adrenomedullin 2 (ADM2), an endogenous bioactive peptide belonging to the calcitonin gene-related peptide family, exhibits various biological activities associated with the inhibition of inflammation and reduction of insulin resistance. However, the effects and underlying mechanisms of ADM2 in AGE-induced macrophage M1 polarization, BMSC dysfunction, and impaired bone regeneration remain poorly understood.MethodsThe polarization of bone marrow-derived macrophages was verified using flow cytometry analysis. Alkaline phosphatase (ALP) staining, ALP activity detection, and alizarin red staining were performed to assess the osteogenesis of BMSCs. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and immunofluorescence staining were used to assess polarization markers, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and osteogenic markers. In vivo, a distraction osteogenesis (DO) rat model with T1DM was established, and tibia samples were collected at different time points for radiological, biomechanical, and histological analyses, to verify the effects of ADM2 on bone regeneration and M2 polarization under diabetic conditions.ResultsADM2 treatment reversed AGE-induced M1 macrophage polarization towards the M2 phenotype, which was partially achieved by the peroxisome proliferator-activated receptor γ (PPARγ)-mediated inhibition of NF-κB signaling. The PPARγ inhibitor GW9662 significantly attenuated the effects of ADM2. Besides, ADM2 treatment improved the AGE-impaired osteogenic potential of BMSCs in vitro. Furthermore, ADM2 accelerated bone regeneration, as revealed by improved radiological and histological manifestations and biomechanical parameters, accompanied by improved M2 macrophage polarization in diabetic DO rats, and these effects were partially blocked by GW9662 administration.ConclusionsThese results indicate that ADM2 enhances diabetic bone regeneration during DO, by attenuating AGE-induced imbalances in macrophage polarization, partly through PPARγ/NF-κB signaling, and improving AGE-impaired osteogenic differentiation of BMSCs simultaneously. These findings reveal that ADM2 may serve as a potential bioactive factor for promoting bone regeneration under diabetic conditions, and imply that management of inflammation and osteogenesis, in parallel, may present a promising therapeutic strategy for diabetic patients during DO treatment.

MnO2 shell-isolated SERS nanoprobe for the quantitative detection of ALP activity in trace serum: Relying on the enzyme-triggered etching of MnO2 shell to regulate the signal

• A MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 ) is prepared for quantitative detection of ALP activity. • Due to the efficient physical isolation of MnO 2 shell, the nanoprobe demonstrates high stability against salt and thiol. • The sensing performance is achieved by enzyme-triggered etching of MnO 2 shell. • With MnO 2 shell as Raman reference, this system effectively eliminates the experimental interferences. • Integrating with in-capillary technique, this SERS system is capable of direct quantitative detection in trace serum. Establishing a stable, reliable and sensitive assay for the direct analysis in trace serum is of significance for the medical diagnosis and disease monitoring. We develop a MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 , AMAM) for the quantitative detection of alkaline phosphatase (ALP) in trace serum. Due to the physical isolation of MnO 2 shell, AMAM is equipped with high stability, rarely happening the accidental aggregation. Particularly, the SERS signal is regulated by the etching of MnO 2 shell, requiring not to change the local electromagnetic field or the molecular structure of Raman reporter (4-mercaptopyridine (Mpy)). Designating MnO 2 shell as the Raman reference and the ratio of I Mpy /I MnO2 as the response signal, AMAM system can obviously eliminate the experimental interference and achieve highly reliable SERS quantification for ALP. This ratiometric SERS system exhibits good reproducibility, high stability and sensitivity for the ALP analysis. AMAM system is able to perform the colorimetric and SERS dual-readout detection, which displays a wide linear dynamic range (0.1–70 U/L) for ALP with a detection limit of 0.079 U/L. Additionally, integrating with in-capillary SERS technique, our proposed nanoprobe is also applicable of direct SERS analysis in trace undiluted human serum (2 μL).

A novel electronic assay based on a sol-gel transition reaction and a thin-film transistor of supramolecular hydrogels to detect alkaline phosphatase activity

Abstract Herein, we developed an extended floating-gate thin-film transistor (EFG-TFT) together with various sol-gel phases to determine alkaline phosphatase (ALP) activity. The EFG-TFT was fabricated with ZnO as the channel material and YOX as the dielectric layer, which provided an extended area for sensing the analyte interacting with the designed hydrogelator. A peptide-based supramolecular hydrogel material, i.e., 2-naphthylacetic acid- l -phenylalanine- l -phenylalanine- l -O-phosphor tyrosine (named Nap-FF-Yp) was synthesized as our sensing platform to detect ALP activity and the sol-gel state. Nap-FF-Yp could be cleaved by ALP during hydrolysis, leading to π-π stacking, and finally changed to a hydrogel. Due to the difference in the potential drop between the liquid phase and gel phase of the hydrogel, the sol-gel transition reaction could be monitored by the electrical signal. The greater the extent of hydrogelation, the smaller the threshold voltage (Vth) variation, and the threshold voltage shifted to the left. Under the device’s optimal conditions with oxygen plasma treatment, the limit of detection (LOD) reached 0.024 U/L. The development of this novel sol-gel state for a high-speed screening sensing platform with an excellent electrical response demonstrates the potential capabilities of future high-speed response sensors.

A fluorometric and optical signal dual-readout detection of alkaline phosphatase activity in living cells based on ATP-mediated porphyrin MOFs

Alkaline phosphatase (ALP), as an important biomarker, is closely associated with various diseases. Therefore, detecting the ALP activity is of considerable importance to diagnosis and treatment of diseases. In this study, we have first exploited porphyrin-adenosine triphosphate-based nanoscale chiral metal-organic framework (denoted as NPCN-222-ATP) for the quantitative determination the activity of ALP through fluorometric and optical signal dual-readout mode. In this detection system, the ALP-catalyzed hydrolysis of chiral ATP molecules leads to the recovery of the fluorescence of porphyrin, accompanied by the gradual decrease of the circular dichroism (CD) signal. This probe showed excellent selectivity and sensitivity for ALP activity with the limits of detection of 0.0240 mU/mL and 0.0225 mU/mL for the fluorometric and optical signal readout mode, respectively, which were significantly better than the current ALP detection system. More importantly, such chiral probe successfully realized the monitoring and imaging of ATP activity in living cells, providing a new way for chiral MOFs to be used in biological analysis and detection platform.

Target-induced transcription amplification to trigger the trans-cleavage activity of CRISPR/Cas13a (TITAC-Cas) for detection of alkaline phosphatase

Herein, an ultra-sensitive alkaline phosphatase (ALP) sensing strategy is developed by target-induced transcription amplification to trigger the trans-cleavage activity of Cas13a (TITAC-Cas). A double-stranded DNA duplex integrating a T7 promoter with 5′-phosphate and a transcription template (5′P-dsDNA) serves as the ALP substrate. In the absence of ALP, 5′P-dsDNA can be degraded by the λexo, leading to the subsequent transcription failure. In the presence of ALP, dephosphorylation reaction converts the 5′P-dsDNA to 5′OH-dsDNA and provides the protection for T7 promoter against the λexo-digestion. The intact T7 promoter of 5′OH-dsDNA can activate T7 transcription to produce a mass of single-stranded RNA (ssRNA). The ssRNA products possess a full complementarity to the spacer of crRNA and activate the ssRNase activity of CRISPR/Cas13a. As a result, Cas13a exhibits the indiscriminate cleavage of collateral FQ-reporter to release significant fluorescence signal, realizing the ultra-sensitive detection of ALP. Due to the triple signal amplification (ALP self-catalysis, T7 transcription amplification, and trans-cleavage of CRISPR/Cas13a), TITAC-Cas assay shows the ultra-sensitive detection of ALP activity with a wide linear range from 0.008 to 250 U∙L−1). The LOD is calculated to be 6 ± 0.52 mU∙L−1. TITAC-Cas assay is also successfully applied for analysis of ALP activity in HepG2 cell lysate with high fidelity. In addition, this method is employed to screen ALP inhibitor.

Development of a new chromogenic medium for the enumeration of Bacillus cereus in various ready-to-eat foods

Enumeration of Bacillus cereus in ready-to-eat food with high background flora has been challenging due to the low selectivity and recoverability of the selective media. In this study, a new medium for detection of B. cereus was developed. This medium, B. cereus chromogenic agar (BCCA), contains an egg yolk emulsion for detection of lecithinase activity, a chromogenic substrate specific for detecting alkaline phosphatase activity, and three antibiotics for selectivity. The aim of this study was to compare the performance of BACCA with that of two conventional selective media (MYPA and PEMBA) and two commercialized chromogenic media (CBC agar and Bacara medium). The newly developed media has been evaluated for its inclusivity/exclusivity and recoverability in pure culture and in artificially inoculated ready-to-eat foods, respectively. BCCA exhibited similar or better recoverability and inclusivity/exclusivity for enumeration of B. cereus and B. thuringiensis from pure cultures than two conventional [mannitol yolk polymyxin B agar (MYPA) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA)] and two commercial chromogenic media [chromogenic B. cereus (CBC) agar and Bacara medium]. Furthermore, BCCA yielded similar or enhanced levels of recoverability and superior selectivity of B. cereus and B. thuringiensis from ready-to-eat food products, particularly radish sprouts and a sprout mix, compared with the other media tested. These findings indicate that BCCA comprises a useful option for the isolation and enumeration of B. cereus from commercial foods.

Rational Design of Dual-Emission Lanthanide Metal-Organic Framework for Visual Alkaline Phosphatase Activity Assay.

The alkaline phosphatase (ALP) activity assay is very significant for disease diagnosis and biomedical research. Lanthanide metal-organic framework (Ln-MOF) based fluorescence sensors have great application potential in ALP activity assays. However, it is critical but challenging to investigate the emission law of Ln-MOFs for revealing rational design principles and selecting an appropriate MOF. Here, we describe a reasonable design strategy for dual-emission Ln-MOFs based on theoretical calculations. This strategy combines Reinhoudt empirical rule, intramolecular charge transfer theory, and aggregation/coordination-induced emission theory; reveals the luminescence law of Ln-MOFs; and provides theoretical guidance for the rational design of dual-emission Ln-MOFs. On the basis of this strategy, we create a dual-emission Tb-MOF fluorescent probe used for ALP activity assay and investigate the detection mechanism. The probe shows ultrasensitive (limit of detection 0.002 mU mL-1) and selective response to ALP, and it suits for point-of-care visual detection coupled with a self-designed portable enzyme activity assay kit and smartphone-assisted visual device. The kit-based visual assay method can accurately quantify the activity of ALP in real serum samples (recovery >93%, and relative error is less than 6.8% compared with the results of fluorescence spectrometer-based method) and consumes only 25 μL of serum. In addition, a logical decoder based on the "dual-key unlocking strategy" is designed, providing a feasible solution for the development of intelligent ALP activity detection equipment. As far as we know, this is the first report of a theoretical calculation-guided versatile design strategy for dual-emission Ln-MOFs and a portable enzyme activity assay kit for visual detection.

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

BackgroundLong non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).MethodsHealthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-β-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).ResultsHPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3′-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.ConclusionThis study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

Alkaline phosphatase-regulated in situ formation of chromogenic probes for multicolor visual sensing of biomarkers

Alkaline phosphatase (ALP), as an immunological label, is widely used in biochemical assays. Here, a simple yet effective strategy for ALP activity detection was proposed on the basis of in situ formation of Prussian blue nanoparticles and polychromatic superposition effect. Firstly, ascorbic acid, a product from ALP-catalyzed hydrolysis of 2-phospho-l-ascorbic acid (AAP), converted yellow ferricyanide into ferrocyanide. Then, the specific reaction between ferrocyanide and ferric ions (Fe3+) initiated the generation of Prussian blue nanoparticles in situ. Meanwhile, the residual AAP chelated with Fe3+, and a stable Fe3+-AAP complex was quickly formed. When Prussian blue nanoparticles mixed with brown Fe3+-AAP complex and yellow ferricyanide at different ratios, a distinct color variation was presented. Therefore, a sensitive multicolor assay of ALP activity with a detection limit of 1.0 U/L was realized by simply blending commercially available reagents. Furthermore, magnetic sandwich and competitive sensing platforms for multiple biomarkers detection were constructed by combining the ALP-regulated multicolor system with the well-developed aptasensor. The feasibility of the sensors was convincingly demonstrated by using thrombin and prostate specific antigen as model targets. In addition, the proposed multicolor strategy was employed for evaluating inhibition efficiency, and shows potential in visual screening of enzyme inhibitors. Such a facile, sensitive and low-cost sensing strategy provides a new perspective to develop universal platforms of point-of-care testing.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100mU/mL with the limit of detection (LOD) of 1.48mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Obesity regulates miR-467/HoxA10 axis on osteogenic differentiation and fracture healing by BMSC-derived exosome LncRNA H19.

This study explored the therapeutic effect of bone marrow mesenchymal stem cell‐derived exosomes on the treatment of obesity‐induced fracture healing. Quantitative real‐time PCR was used to detect the expression of lncRNA H19, miR‐467 and Hoxa10 and combined with WB detection to detect osteogenic markers (RUNX2, OPN, OCN). Determine whether exosomes have entered BMSCs by immunofluorescence staining. Alkaline phosphatase (ALP) and alizarin red staining (ARS) staining were used to detect ALP activity and calcium deposition. We found that high‐fat treatment can inhibit the secretion of BMSCs‐derived exosomes and affect the expression of H19 carried by them. In vivo and in vitro experiments show that high‐fat or obesity factors can inhibit the expression of osteogenic markers and reduce the staining activity of ALP and ARS. The treatment of exosomes from normal sources can reverse the phenomenon of osteogenic differentiation and abnormal fracture healing. Further bioinformatics analysis found that miR‐467 as a regulatory molecule of lncRNA H19 and Hoxa10, and we verified the targeting relationship of the three through dual luciferase report experiments. Further, we found similar phenomena in ALP and ARS staining. Bone marrow mesenchymal stem cell‐derived exosomes improve fracture healing caused by obesity.

Ratiometric fluorescence determination of alkaline phosphatase activity based on dual emission of bovine serum albumin-stabilized gold nanoclusters and the inner filter effect.

A novel and convenient method for the ratiometric fluorescence detection of alkaline phosphatase (ALP) activity was proposed based on dual emission of bovine serum albumin-templated gold nanoclusters (BSA-AuNCs) and the mechanism of the inner filter effect between BSA-AuNCs and p-nitrophenol (PNP). First, ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate (PNPP) to produce PNP. PNP effectively quenched the emission peak of BSA-AuNCs at 410 nm because of the overlap in absorbance feature of PNP and the fluorescence spectrum of BSA-AuNCs, and the peak at 650 nm was almost unaffected. Thus, a sensitive ratiometric method for detection of ALP activity was developed using the fluorescence intensity of BSA-AuNCs at 650 nm as a reference signal. ALP activity versus the ratio of fluorescence intensities at 410 and 650 nm showed good linearity between 0.2 and 5 mU mL-1 (R2 = 0.9931) and high sensitivity with a detection limit of 0.03 mU mL-1 (S/N = 3). The developed sensing method was successfully applied to investigate ALP inhibitors and detect ALP in serum samples.

A redox modulated fluorescence nanoplatform for the detection of alkaline phosphatase activity with fluorescent polydopamine nanoparticles.

Herein, we simply synthesized intrinsic fluorescent polydopamine nanoparticles (PDA NPs) in sodium hydroxide solution (NaOH, pH 11), and constructed a new fluorescence nanoplatform for the detection of alkaline phosphatase (ALP) using PDA NPs as an effective signal reporter. The nanoplatform was constructed by the combination of enzymatic hydrolysis of ALP to the substrate l-ascorbic acid-2-phosphate (AA2P) and the chemical redox reaction between l-ascorbic acid (AA) and mercury ion (Hg2+). The fluorescence of PDA NPs could be effectively quenched by Hg2+ through the coordination effect between Hg2+ and the functional groups on the surface of PDA NPs. However, the quenching effect was greatly inhibited by the addition of AA into the solution. Based on this point, the activity of ALP could be monitored by hydrolysis of the substrate AA2P to AA and the fluorescence output of PDA NPs. The nanoplatform exhibited high sensitivity and desirable selectivity for ALP detection. With a wide linear range of 0 to 18 U L-1, a detection limit of 0.4 U L-1 was achieved using the developed nanosensor. The proposed method could not only be used to screen the inhibitor of ALP but also be used to detect ALP activity in human serum samples successfully. Moreover, the strategy can easily be expanded to determining other kinds of enzymes participating in AA-generation reactions.

A carbon dot doped lanthanide coordination polymer nanocomposite as the ratiometric fluorescent probe for the sensitive detection of alkaline phosphatase activity

The development of sensitive methods for alkaline phosphatase (ALP) activity analysis is an important analytical topic. Based on the stimulus-responsive lanthanide coordination polymer, a simple ratiometric fluorescence sensing strategy was proposed to detect ALP activity. A carbon dot (CD) doped fluorescent supramolecular lanthanide coordination polymer (CDs@Tb-GMP) was prepared with Tb3+ and the ligand guanine single nucleotide (GMP). To construct a ratiometric fluorescence biosensor, the fluorescence of Tb-GMP was used as a response signal, and the fluorescence of CDs was used as a reference signal due to its good stability. When excited at 290 nm, the polymer network Tb-GMP emits characteristic fluorescence at 545 nm, while the CDs encapsulated in the polymer network emit fluorescence at 370 nm. After adding ALP to the system, the substrate GMP can be hydrolyzed by ALP, resulting in the destruction of the polymer network. Accordingly, the fluorescence of Tb-GMP significantly decreased, while the fluorescence of CDs slightly increased due to their release from the polymer network. By comparing the relationship between the fluorescence intensity ratio of the two signals and the concentration of ALP, sensitive detection of ALP could be achieved with the linear range from 0.5 to 80 U L-1 and a detection limit of 0.13 U L-1. Furthermore, the proposed ratiometric sensing system was applied to the detection of ALP in human serum samples with desirable results, indicating potential application in clinical diagnosis.

A novel aggregation-induced dual emission probe for in situ light-up detection of endogenous alkaline phosphatase

Abnormal level of alkaline phosphatase (ALP) activity has been linked to many diseases in human. The development of fluorescent molecular probes that can report the expression and activity of ALP in various biological systems will be extremely valuable. However, the in vivo monitoring for ALP in living cells and more complex biological systems remains a great challenge. The excited-state intramolecular proton transfer (ESIPT) probe with proportional fluorescence has low background noise, while the aggregation induced emission (AIE) probe has the advantages of signal amplification and good light stability. Herein, an “AIE + ESIPT” fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenyl dihydrogen phosphate (THP) was constructed for the highly selective and sensitive detection of ALP. By introducing a phosphate ester at the hydroxyl position of the solid fluorophore 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenol, ESIPT was hindered and the probe present a faint blue fluorescence in DMSO solution. While ALP was introduced, causing the phosphate in THP hydrolyzed, and the ESIPT process was restored to yield a yellow fluorescence at 550 nm, thereby achieving proportionality detection. THP exhibited high selectivity and sensitively to ALP with low limit of detection (1.21228 U/L), and the reaction completed within 20 min. In addition, with its outstanding advantages of low biological toxicity and enzyme conversion characteristics, THP has been successfully applied to ALP imaging in living cells (Hela cells, A549 cells and Hek293 cells), and can provide in situ information on the reaction site. Therefore, THP has the potential for detecting ALP activity in biomedical application.

Hydroxyapatite-Bovine Serum Albumin-Paclitaxel Nanoparticles for Locoregional Treatment of Osteosarcoma.

Osteosarcoma is the most primary type of bone tumor occurring in the pediatric and adolescent age groups. In order to obtain the most appropriate prognosis, both tumor recurrence inhibition and bone repair promotion are required. In this study, a ternary nanoscale biomaterial/antitumor drug complex including hydroxyapatite (HA), bovine serum albumin (BSA) and paclitaxel (PTX) is prepared for post-surgical cancer treatment of osteosarcoma in situ. The HA-BSA-PTX nanoparticles, about 55 nm in diameter with drug loading efficiency (32.17 wt%), have sustained release properties of PTX and calcium ions (Ca2+ ) and low cytotoxicity to human fetal osteoblastic (hFOB 1.19) cells in vitro. However, for osteosarcoma (143B) cells, the proliferation, migration, and invasion ability are significantly inhibited. The in situ osteosarcoma model studies demonstrate that HA-BSA-PTX nanoparticles have significant anticancer effects and can effectively inhibit tumor metastasis. Meanwhile, the detection of alkaline phosphatase activity, calcium deposition, and reverse transcription-polymerase chain reaction proves that the HA-BSA-PTX nanoparticles can promote the osteogenic differentiation. Therefore, the HA-BSA-PTX nanodrug delivery system combined with sustained drug release, antitumor, and osteogenesis effects is a promising agent for osteosarcoma adjuvant therapy.

Development of a HPLC Fluorescence Method for Determining Efficacy of Milk Pasteurization

Alkaline phosphatase (ALP) is an active enzyme present in the raw milk. Due to its unique heat-sensitive properties, it is employed as an indicator to evaluate efficiency of the pasteurization treatment. In the present study, a method comprising of HPLC with fluorescence detector was developed for the quantification of ALP enzyme activity in the pasteurized milk. The method is based on the detection and quantification of 4-methylumbelliferone (4MU) liberated by the action of phosphatase enzyme (in the milk) on the substrate 4-methylumbelliferyl phosphate (4MUP). Extraction and purification of 4MU was done by clarification, centrifugation, and solid phase extraction processes. The target compound separation was achieved with the help of reversed-phase C18 column using gradient elution consisting of a binary mobile phase: HPLC grade water and methanol. The fluorescence detection was accomplished with the excitation and emission wavelengths being 365 and 460 nm, respectively. The method validation experiments clearly showed high specificity, good linearity (r2 > 0.9901), low limits of detection (0.349 mg/L) and quantification (LOQ, 0.432 mg/L), accuracy (> 100%), and precision (%RSD < 4.7652). Comparative evaluation against Lovibond comparator method revealed that the HPLC method has an advantage of detecting very low levels of ALP activity and raw milk contamination. This is the first report of detecting such low levels of ALP in the milk by HPLC-fluorescence detector technique. This new chromatographic method can be considered as a substitute to the existing standard methods that employ fluorometric technique for detecting ALP activity in the pasteurized milk. Our method could certainly enhance the quality assurance standards currently employed in the dairy industry.

Colorimetric alkaline phosphatase activity detection by integrating phosphorylation-mediated sulfydryl protection/deprotection and fluorosurfactant stabilized gold nanoparticles

Protection/deprotection of thiolates are widely used for the design of sensing systems by producing free sulfydryl (–SH) groups. However, most conventional protection/deprotection strategies reported so far suffer poor protection and/or difficult release of –SH group. In this study, phosphorylation/dephosphorylation strategy that exhibits efficient protection and facile liberation of –SH group via the formation of a phosphate-thiol ester, was utilized to develop colorimetric probe for alkaline phosphatase (ALP) activity evaluation. The proposed probe was composed of a cysteamine S-phosphate as a model thiolate substrate and Zonyl FSN-100 functionalized gold nanoparticles (FSN-AuNPs) as reporters. In the presence of ALP, the cysteamine S-phosphate hydrolyzed to generate cysteamine, which aggregated FSN-AuNPs with detectable spectroscopic changes through nanointerface interactions. The changes in UV–vis absorption spectroscopy allowed a detection limit of ALP at 0.1 unit/L. The specific enzyme catalytic hydrolysis provided high selectivity toward ALP over other proteins, small molecules, and metal ions. The practicability of the sensor was validated by accurate examination of ALP in blood samples. In addition, by integrating the conventional ELISA technique and the proposed phosphorylation tactic, sensitive immunoglobulin G detection was achieved.