Detection Of Alkaline Phosphatase Research Articles

Rapid aqueous synthesis of CuInS/ZnS quantum dots as sensor probe for alkaline phosphatase detection and targeted imaging in cancer cells

Early diagnosis of chronic, critical diseases improves clinical outcomes, and biomarkers play an important role as an indicator of severity or presence of a disease. Alkaline phosphatase (ALP) is one such vital biomarker in the diagnosis of several diseases. Herein we introduce a facile, sensitive fluorescent assay, based on the inner filter effect (IFE), for ALP activity determination in serum and in living cells. It is well known that the key to maximize the sensitivity of an IFE-based fluorescence assays is to broaden the overlap between the absorption of an absorber and the excitation/emission of a fluorophore. We employed CuInS/ZnS quantum dots (CIS/ZnS QDs) and p-nitrophenylphosphate (PNPP) as the fluorescent indicator and the substrate, respectively, for ALP activity assessment. Due to the CIS/ZnS QDs have an efficient excitation at 405 nm, meanwhile with a large Stokes shift emission at 588 nm, p-nitrophenol (PNP) with absorption peak at 405 nm, the hydrolyzed produce of PNPP and ALP, can act as a competitive absorber to absorb the excitation light of CIS/ZnS QDs, resulting in noticeable quenching of CIS/ZnS QDs. The proposed sensor detects ALP activity in human serum samples (sample consumption: 20 μL) with detection limit of 0.01 U L-1. Excellent biocompatibility of CIS/ZnS QDs enables the sensor to monitor endogenous ALP in living cells. Furthermore, because the surface modification or the linking between the receptor and the fluorophore is no longer required, this fluorescent sensing system has the potential to simplify ALP clinical measurement, thereby improving diagnostics of relevant diseases.

Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165

Prereduction-promoted enhanced growth of silver nanoparticles for ultrasensitive colorimetric detection of alkaline phosphatase and carbohydrate antigen 125

A highly sensitive colorimetric method for detection of alkaline phosphatase (ALP) and carbohydrate antigen (CA125) was developed, which was achieved by ascorbic acid (AA) - mediated enhanced growth of silver nanoparticles (AgNPs) promoted with NaBH4 as pre-reducing agent. With prereduction by NaBH4, the AA controlled growth of AgNPs was promoted and the localized surface plasmon resonance absorbance was much higher compared with that without prereduction. ALP could hydrolyze L-ascorbic acid 2-phosphate trisodium salt (AAP) to form AA, which could then reduce Ag+ to AgNPs resulting in the enhancement of localized surface plasmon resonance peak of AgNPs and accompanying the color change from colorless to bright yellow. Benefiting from the highly sensitive response of Ag+ to AA, the detection limit for ALP could be lowered to 0.003 U L−1, and the proposed plasmonic ELISA could achieve a low detection limit of 1.75 U mL−1 for CA125. Moreover, this method was validated for the analysis of ALP and CA125 in human serum samples, giving results matched well with conventional method, demonstrating the potential application of the developed method in clinical diagnosis.

An ultrasensitive and simple method for alkaline phosphatase assay and targeted natural compound screening in vitro.

As an essential phosphate group hydrolase, alkaline phosphatase (ALP), whose level in serum is correlated with bone disease, liver dysfunction, and cancer, could be used as a biomarker for clinical diagnosis and biomedical studies. Hence, developing a convenient and sensitive method for ALP assay has importance in disease diagnosis, drug treatment, and prognosis assessment. In this work, using a hairpin DNA strand as the substrate, we developed an ultrasensitive and simple fluorescence method for quantitative ALP assay based on the binding difference of reduced graphene oxide (rGO) with different DNA strands coupled with λ exonuclease (λ exo) cleavage. Under the optimal conditions, the limit of detection (LOD) of ALP is estimated to be 0.01U/L with the linear region from 0.5U/L to 70U/L. Furthermore, the proposed assay was used to detect ALP in complicated cell-free extracts and evaluate the inhibitory effects of two well-known inhibitors of ALP activity. Finally, the method was used to investigate the effect of natural compounds on ALP activity and five compounds with different inhibitory capability were screened. In summary, we propose that the new method for ALP assay can be applied for therapeutic drug monitoring (TDM) and high-throughput compound screening in combination with multiwell plate technology.

A new copper mediated on-off assay for alkaline phosphatase detection based on MoOx quantum dots

We present a new on-off assay for alkaline phosphatase (ALP) detection using molybdenum oxide quantum dots (MoOx QDs) based on three favorable properties: the quenching ability of Cu2+ on MoOx QDs, the strong binding affinity between Cu2+ and PPi, and the catalysis ability of ALP to hydrolyze PPi to orthophosphate (Pi). Benefiting from the strong affinity of Cu2+ and PPi, the effective quenching of MoOx QDs by Cu2+ was hampered when PPi was added into the system containing MoOx QDs and Cu2+, leading to a significant photoluminescence enhancement (“ON”). Further addition of ALP, PPi was catalytically hydrolyzed into Pi, which would disable the formation of Cu2+-PPi complex and release free Cu2+, resulting in photoluminescence quenching (“OFF”). The on-off assay allows the analysis of PPi and ALP by measuring the photoluminescence intensity, and quantitative detection of ALP in a linear range from 0.1 to 5.0 U/L with the detection limit of 0.02 U/L (3σ/K) is realized. Moreover, the present assay was successfully applied in ALP inhibitor screening and ALP detection in diluted human serum samples, and satisfactory recoveries between 99.6% and 103.0% were obtained. The on-off photoluminescence assay shows many merits, including convenient operation, cost-saving, high sensitivity and superior selectivity, which allow the assay for ALP detection in real human serum samples.

Fluorometric determination of the activity of alkaline phosphatase based on the competitive binding of gold nanoparticles and pyrophosphate to CePO4:Tb nanorods.

A fluorometric method is described for the determination of the activity of alkaline phosphatase (ALP). It relies on the competition between gold nanoparticles (AuNPs) and pyrophosphate (PPi) for the coordination sites on the surface of CePO4:Tb nanorods. The green fluorescence of the CePO4:Tb is reduced in the presence of AuNPs due to fluorescence resonance energy transfer (FRET), but can be restored on addition of PPi due to the stronger affinity of PPi to the CePO4:Tb. In the presence of ALP, PPi is hydrolyzed to form phosphate which has much weaker affinity for the CePO4:Tb. Hence, the AuNPs will reassemble on the CePO4:Tb, and fluorescence is reduced. Fluorescence drops linearly in the 0.2 to 100U·L-1 activity range, and the detection limit is 60mU·L-1 (at S/N = 3). The method does not require any modification of the surface of the CePO4:Tb and is highly sensitive and selective. The inhibition of ALP activity by Na3VO4 was also studied. In our perception, the method may find application in the diagnosis of ALP-related diseases, in screening for inhibitors, and in studies on ALP-related functions in biological systems. Graphical abstract A assay for the detection of alkaline phosphatase is proposed based on the fluorescence resonance energy transfer between CePO4:Tb and AuNPs. It relies on the competitive binding of AuNPs and pyrophosphate (PPi) to CePO4:Tb and the hydrolysis of PPi by ALP.

Metal-to-Ligand Charge-Transfer-based Visual Detection of Alkaline Phosphatase Activity

The ability to directly detect alkaline phosphatase (ALP) activity in undiluted serum samples is of great importance for clinical diagnosis. In this work, we report the use of the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of the Cu(BCA)2+ (BCA = bicinchoninic acid) reporter for the visual detection of ALP activity. In the presence of ALP, the substrate ascorbic acid 2-phosphate (AAP) can be enzymatically hydrolyzed to release ascorbic acid (AA), which in turn reduces Cu2+ to Cu+. Subsequently, the complexation of Cu+ with the BCA ligand generates the chromogenic Cu(BCA)2+ reporter, accompanied by a color change of colorless-to-purple of the solution with a sharp absorption band at 562 nm. The underlying MLCT-based mechanism has been demonstrated on the basis of density functional theory (DFT) calculations. Needless of any sequential multistep operations and elaborately designed colorimetric probe, the proposed MLCT-based method allows for a fast and sensitive visual detection of ALP activity within a broad linear range of 20 - 200 mU mL-1 (R2 = 0.999), with a detection limit of 1.25 mU mL-1. The results also indicate that it is highly selective and has great potential for the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum samples. Owing to the prominent simplicity and practicability, it is reasonable to conclude that the proposed MLCT-based method has a high application prospect in clinical diagnosis.

Dynamics of male canine germ cell development.

Primordial germ cells (PGCs) are precursors of gametes that can generate new individuals throughout life in both males and females. Additionally, PGCs have been shown to differentiate into embryonic germ cells (EGCs) after in vitro culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (Canis lupus familiaris). Here, we elucidated the dynamics of the expression of pluripotent (POU5F1 and NANOG), germline (DDX4, DAZL and DPPA3), and epigenetic (5mC, 5hmC, H3K27me3 and H3K9me2) markers that are important for the development of male canine germ cells during the early (22–30 days post-fertilization (dpf)), middle (35–40 dpf) and late (45–50 dpf) gestational periods. We performed sex genotype characterization, immunofluorescence, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. Furthermore, in a preliminary study, we evaluated the capacity of canine embryo PGCs (30 dpf) to differentiate into EGCs. To confirm the canine EGCs phenotype, we performed alkaline phosphatase detection, immunohistochemistry, electron and transmission scanning microscopy and RT-qPCR analyses. The PGCs were positive for POU5F1 and H3K27me3 during all assessed developmental periods, including all periods between the gonadal tissue stage and foetal testes development. The number of NANOG, DDX4, DAZL, DPPA3 and 5mC-positive cells increased along with the developing cords from 35–50 dpf. Moreover, our results demonstrate the feasibility of inducing canine PGCs into putative EGCs that present pluripotent markers, such as POU5F1 and the NANOG gene, and exhibit reduced expression of germinative genes and increased expression of H3K27me3. This study provides new insight into male germ cell development mechanisms in dogs.

Label-free fluorescent detection of alkaline phosphatase with vegetable waste-derived green carbon probes

The level of alkaline phosphatase (ALP) is regarded as an important biomarker in medical diagnosis. In this study, a carbon-based fluorescent probes (C-probes) to detect ALP was prepared by modified-ultrasound irradiation from vegetable waste as a starting carbon source. The C-probes are water soluble, non-cytotoxic, biocompatible, and exhibits high fluorescence, which diminishes depending on the amount of ALP. The developed assay relies on Cu2+ induced fluorescence quenching of the C-probes and its inhibition by the very selective coordination of pyrophosphate (PPi) with Cu2+ ions. In the presence of PPi, Cu2+ reacts preferentially with them rather than forming complexes with the C-probes. This phenomenon disappears when PPi undergoes ALP-catalyzed hydrolysis to generate phosphate (Pi), which does not bind to Cu2+ ions. The ALP inhibits the coordination of PPi with Cu2+ and thus causes fluorescence quenching of the C-probes by free Cu2+. Using this strategy, ALP could be analyzed successfully down to 0.25 nM, with a dynamic linear range of 0.5–10 nM. This sensing strategy also revealed a high selectivity for ALP over other protein molecules. Furthermore, the method successfully detected biological ALP in human serum, which verifies the potential of the method for diagnostic and practical purposes.

Fluorescence Immunoassay Based on the Phosphate-Triggered Fluorescence Turn-on Detection of Alkaline Phosphatase.

A simple and cost-effective fluorescence immunoassay for the sensitive quantitation of disease biomarker α-fetoprotein (AFP) has been developed based on the phosphate-triggered fluorescence turn-on detection of alkaline phosphatase (ALP), with the reversible binding between calcein and Ce3+ as a signaling element. In this immunoassay, fluorescent calcein is readily quenched by Ce3+ via a coordination process. The ALP-catalyzed hydrolysis of p-nitrophenyl phosphate leads to the formation of p-nitrophenol and inorganic orthophosphate, and the newly formed orthophosphate could potently combine with Ce3+ due to the higher affinity, thus, recovering the fluorescence of calcein. The corresponding fluorescence signal triggered by phosphate is related to ALP activities labeled on antibody, and thus could be applied to detect target antigen in an enzyme-linked immunosorbent assay (ELISA) platform. The fluorescence intensity correlated well to the AFP concentration ranges of 0.2-1.0 and 1.0-4.0 ng/mL, with a detection limit of 0.041 ng/mL. The proposed fluorescence ELISA possesses convincing recognition mechanism and exhibits excellent assay performance in the evaluation of the AFP level in serologic test, which unambiguously reveals great application potential in the clinic diagnosis of disease biomarkers.

Magnesium prevents vascular calcification in vitro by inhibition of hydroxyapatite crystal formation

Magnesium has been shown to effectively prevent vascular calcification associated with chronic kidney disease. Magnesium has been hypothesized to prevent the upregulation of osteoblastic genes that potentially drives calcification. However, extracellular effects of magnesium on hydroxyapatite formation are largely neglected. This study investigated the effects of magnesium on intracellular changes associated with transdifferentiation and extracellular crystal formation. Bovine vascular smooth muscle cells were calcified using β-glycerophosphate. Transcriptional analysis, alkaline phosphatase activity and detection of apoptosis were used to identify transdifferentiation. Using X-ray diffraction and energy dispersive spectroscopy extracellular crystal composition was investigated. Magnesium prevented calcification in vascular smooth muscle cells. β-glycerophosphate increased expression of osteopontin but no other genes related to calcification. Alkaline phosphatase activity was stable and apoptosis was only detected after calcification independent of magnesium. Blocking of the magnesium channel TRPM7 using 2-APB did not abrogate the protective effects of magnesium. Magnesium prevented the formation of hydroxyapatite, which formed extensively during β-glycerophosphate treatment. Magnesium reduced calcium and phosphate fractions of 68% and 41% extracellular crystals, respectively, without affecting the fraction of magnesium. This study demonstrates that magnesium inhibits hydroxyapatite formation in the extracellular space, thereby preventing calcification of vascular smooth muscle cells.

Fast and sensitive near-infrared fluorescent probes for ALP detection and 3d printed calcium phosphate scaffold imaging in vivo

Alkaline phosphatase (ALP) is a critical biological marker for osteoblast activity during early osteoblast differentiation, but few biologically compatible methods are available for its detection. Here, we describe the discovery of highly sensitive and rapidly responsive novel near-infrared (NIR) fluorescent probes (NIR-Phos-1, NIR-Phos-2) for the fluorescent detection of ALP. ALP cleaves the phosphate group from the NIR skeleton and substantially alters its photophysical properties, therefore generating a large “turn-on” fluorescent signal resulted from the catalytic hydrolysis on fluorogenic moiety. Our assay quantified ALP activity from 0 to 1.0UmL−1 with a 10−5−10−3UmL−1 limit of detection (LOD), showing a response rate completed within 1.5min. A potentially powerful approach to probe ALP activity in biological systems demonstrated real-time monitoring using both concentration- and time-dependent variations of endogenous ALP in live cells and animals. Based on high binding affinity to bone tissue of phosphate moiety, bone-like scaffold-based ALP detection in vivo was accessed using NIR probe-labeled three-dimensional (3D) calcium deficient hydroxyapatite (CDHA) scaffolds. They were subcutaneously implanted into mice and monitored ALP signal changes using a confocal imaging system. Our results suggest the possibility of early-stage ALP detection during neo-bone formation inside a bone defect, by in vivo fluorescent evaluation using 3D CDHA scaffolds.

Colorimetric determination of the activity of alkaline phosphatase based on the use of Cu(II)-modulated G-quadruplex-based DNAzymes.

A colorimetric detection scheme is introduced for the determination of alkaline phosphatase (ALP) activity based on Cu(II)-modulated G-quadruplex-based DNAzymes. It is exploiting the strong affinity of Cu(II) for pyrophosphate (PPi) upon which the cofactor PPi is trapped by Cu(II). Hence, the activity of the DNAzyme is inhibited. ALP catalyzes the hydrolysis of PPi, causing the release of Cu(II). DNAzyme, in turn, is activated and catalyzes the cleavage of the DNA probe substrate. The released G-rich sequence folds into the G-quadruplex, which can bind hemin and catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonate (ABTS), and this leads to an increase in absorbance at 420nm. Absorbance increases linearly with increasing ALP activity in 0.07 to 300U.L-1 range, with a 70mU.L-1 detection limit. The method was applied in ALP inhibition tests and to the determination of ALP activity in spiked serum samples where it gave satisfactory results. Graphical abstract A colorimetric method has been developed for the detection of alkaline phosphatase based on the use of Cu(II)-modulated G-quadruplex-based DNAzymes.

Fluorescence assay for alkaline phosphatase based on ATP hydrolysis-triggered dissociation of cerium coordination polymer nanoparticles.

Alkaline phosphatase (ALP) is a significant biomarker for diagnostics. Simple, selective and sensitive detection of ALP activity is thus of critical importance. In this study, an artful fluorescence assay for ALP is proposed based on adenosine triphosphate (ATP) hydrolysis-triggered disassociation and fluorescence quenching of cerium coordination polymer nanoparticles (CPNs). ATP, a recognized natural substrate of phosphatase, can serve as a superb "antenna" to sensitize the luminescence of Ce3+ with the aid of tris(hydroxymethyl) aminomethane (Tris), forming Ce3+-ATP-Tris CPNs. In the presence of ALP, ATP will be catalytically converted into adenosine and inorganic orthophosphate, however neither of them can sensitize Ce3+ in alkaline media. As a result, the obtained CPNs are disassociated, inducing the quenching of the fluorescence. On this basis, a straightforward fluorescence assay for ALP activity is rationally developed. The fluorescence quenching efficiency shows a linear relationship for ALP within the activity range from 0.1 to 10 mU mL-1 with a detection limit of 0.09 mU mL-1 under the optimal experimental conditions. Moreover, this facile yet effective fluorescence method featured simplicity, cost-effectiveness, high sensitivity and high selectivity and can be successfully utilized for the quantitative detection of ALP in human serum samples.

Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase.

Alkaline phosphatase (ALP) is an important diagnostic indicator for various human diseases including bone diseases, liver dysfunction, diabetes, breast and prostatic cancers. However, the conventional methods for ALP assay are usually cumbersome and time-consuming with low sensitivity. Here, we develop a new fluorescent method for ultrasensitive detection of ALP activity on the basis of primer dephosphorylation-initiated isothermal circular exponential amplification. We design two dual-functional hairpin probes (HP1 and HP2), which function as both the templates for exponential amplification reaction (EXPAR) and the generators for signal output. In the presence of ALP, the 3'-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3' protruding end of HP1 to initiate the first strand displacement amplification (SDA), producing trigger 1 and fluorescence signal. The released trigger 1 is complementary to the 3' protruding end of HP2 for the initiation of the second SDA, producing trigger 2 and fluorescence signal. Notably, trigger 2 is complementary to the 3' protruding end of HP1 and may subsequently initiate two consecutive SDAs, enabling circular EXPAR to generate an amplified fluorescence signal. This method exhibits high sensitivity with a detection limit of 2.0 × 10-10 U μL-1 and a large dynamic range of 5 orders of magnitude from 1.0 × 10-9 to 1.0 × 10-4 U μL-1, and it can measure ALP at the single-cell level. Importantly, this method can be applied for the measurement of kinetic parameters and the screening of potential inhibitors, providing a powerful tool for ALP-related biomedical research and clinical diagnosis.

Water-soluble MoS2 quantum dots for facile and sensitive fluorescence sensing of alkaline phosphatase activity in serum and live cells based on the inner filter effect.

We report a facile and sensitive method for the detection of alkaline phosphatase (ALP) activity in serum and live cells using molybdenum disulfide quantum dots (MoS2 QDs) based on the Inner Filter Effect (IFE). In the present work, water soluble MoS2 QDs with bright green fluorescence were synthesized through direct ultrasonic exfoliation of MoS2 powder in 85 vol% aqueous ethanol solution. p-Nitrophenylphosphate (PNPP) was employed to act as an ALP substrate, and its enzyme catalytic product (p-nitrophenol (PNP)) functioned as a powerful absorber in the IFE to influence the excitation of MoS2 QDs. PNPP was transformed into PNP in the presence of ALP, leading to the transition of the absorption peak from 310 nm to 405 nm and therefore resulted in a complementary overlap between the absorption of PNP and the excitation of MoS2 QDs. The fluorescence of MoS2 QDs was quenched due to the significant weakening of the excitation of MoS2 QDs by competitive absorption between QDs and PNP. A good linear relationship was obtained from 0 to 5 U L-1 (R2 = 0.9919) using the present IFE based sensing strategy with the lowest detection activity of 0.1 U L-1. The proposed sensing approach was successfully applied to ALP sensing in serum samples and ALP inhibitor investigation, as well as in ALP cell imaging.

Sensitive detection of alkaline phosphatase by dephosphorylation-initiated transcription reaction-mediated dual signal amplification.

We develop a new fluorescence method for the sensitive detection of alkaline phosphatase (ALP) based on dephosphorylation-initiated transcription reaction-mediated dual signal amplification. This research demonstrates for the first time a significant improvement in ALP assay performance by using a nucleic acid amplification strategy, which enables sensitive detection of ALP activity in vitro and in cell extracts. Importantly, this method can be further applied for kinetic analysis and ALP inhibition assay.

Eu/Tb luminescence for alkaline phosphatase and β-galactosidase assay in hydrogels and on paper devices.

Simple technologies for efficient detection of important (bio)molecules are always in great demand. We now report the detection and assay of two biologically important enzymes, alkaline phosphatase and β-galactosidase, in Eu- or Tb-based cholate hydrogels, respectively, and on filter paper discs coated with such hydrogels. Pro-sensitizers derived from 1-hydroxypyrene and 2,3-dihydroxynaphthalene were incorporated into Eu or Tb cholate hydrogels, respectively. Upon enzyme action, these artificial substrates liberate free sensitizers both in the gel and on gel-coated discs, resulting in turn-on luminescence, red/magenta for Eu, and green for Tb. The detection of enzymes was also demonstrated in natural/biological samples using low-cost systems.

A turn-on fluorescence assay of alkaline phosphatase activity using a DNA–silver nanocluster probe

A label-free fluorescence assay has been developed for the detection of alkaline phosphatase based on DNA–silver nanocluster probes.

A sensitive fluorescent probe for alkaline phosphatase and an activity assay based on the aggregation-induced emission effect.

In this work, we report a fluorescent probe (TPEQN-P) for detecting alkaline phosphatase (ALP) with high sensitivity and monitoring its activity based on the specific aggregation-induced emission (AIE) effect. TPEQN-P can be constructed by conjugating the phosphate moiety into an AIE molecule (TPE-QI) through a reactive p-hydroxybenzyl group, which exhibits very weak emission in aqueous media due to its good water solubility. In the presence of ALP, TPEQN-P undergoes dephosphorylation and releases the hydrolysis product TPE-QI, which is intensely fluorescent because of its poor water solubility. The detection limit for ALP using TPEQN-P can be as low as 0.0077 U L−1 with a linear range of 0–30 mU mL−1 in solution. TPEQN-P also shows excellent applicability in serum samples, demonstrating potential applications in clinical diagnosis and biomedical research. TPEQN-P also can be applied for the determination of ALP activity and in ALP inhibitor screening.