4-Methylumbelliferyl phosphate (4-MUP)

Kinetics study on recombinant alkaline phosphatase and correlation with the generated fluorescent signal

Alkaline phosphatase (AP) (EC 3.1.3.1) is one of the most commonly used enzymes in immunoassays. In VIDAS? assays (bioMérieux, Marcy l'Etoile, France), AP catalyzes the hydrolysis of 4-methylumbelliferyl phosphate (4-MUP) in 4-methylumbelliferone (4-MU) producing a fluorescent signal. This work introduces an original method of characterization of the kinetic parameters Km, Vmax, and Kcat of AP embedded in VIDAS? assays. Assessment of such constants allows us to predict the fluorescent signal generated for given amounts of enzyme and its associated substrate; in the particular case of VIDAS?, it has been estimated that 0.06 nmol/L of AP produces 3144 Relative Fluorescent Values (RFV). Abbreviations: 4-MUP, 4-Methylumbelliferyl phosphate; 4-MU, 4-Methylumbelliferone; RFV, Relative Fluorescent Values; RFU, Relative Fluorescent Units; QDs, Quantum Dots; LoD, Limit of Detection.

Use of fluorescent substrate 4-MUP in the detection of biotin-labeled DNA probes

To improve the sensitivity of detecting biotin-labeled DNA Probes, a new fluorescent substrate of alkaline phosphatase, 4-methylum belliferylphosphate (4-mup) was studied instead of conventional BCIP-NBT. The result of dot-blot hybridization demonstrates that this new substrate can be used for the colorimetric detection of biotin-labeled probes after hybridization to immobilized nucleic acids. The sensitivity is about one order of magnitude higher than that of BCIP-NBT system, and the time required for color development is very short, only about five min. It is suggested that the Bio-SA-Bio-AP-4-MUP colorimetric detection system can be widely used in gene diagnosis.

Serological diagnosis of Toxoplasmosis disease using a fluorescent immunosensor with chitosan-ZnO-nanoparticles

This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T. gondii antigens. Bound antibodies were quantified by the addition of anti-IgG antibodies labeled whit alkaline phosphatase (ALP). ALP enzymatically converts the non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to soluble fluorescent methylumbelliferone that was measured using excitation at 355 nm and emission at 440 nm. The relative fluorescent response of methylumbelliferone is proportional to the concentration of anti-T. gondii IgG antibodies. The coefficients of variation are less than 4.73% for within-day assays and less than 6.34% for between-day assays. Results acquired by LIF immunosensor agree with those obtained by enzyme-linked immunosorbent assay method, suggesting that the designed sensor represents a promising tool for the quantitative determination of anti-T. gondii IgG antibodies of clinical samples.

Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline phosphatase in cultured skin fibroblasts

We explored the biochemical basis for the disorder pseudohypophosphatasia (PsHYPT) in one patient by examining the substrate specificity and localization of alkaline phosphatase (ALP) in cultured dermal fibroblasts. Despite substantial ALP activity, in cell homogenates, toward the artificial substrate 4-methyl-umbelliferyl phosphate (4-MUP), there was a marked deficiency in ALP activity toward the natural substrates pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), indicating altered substrate specificity. Furthermore, although 4-MUP phosphatase (4-MUP-P'tase) activity was predominantly localized as an ecto-enzyme, the small amount of PLP phosphatase (PLP-P'tase) activity was intracellular. This differential localization was apparent in intact cells, since (1) brief acidification of the medium at 4 degrees C inactivated a majority of the 4-MUP-P'tase activity but only 15% of the PLP-P'tase activity (in contrast to greater than 85% inactivation of both in homogenates), (2) greater than 70% of the 4-MUP-P'tase activity but only 30% of the PLP-P'tase activity was released by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, and (3) degradation of extracellular PLP was less than 35% of that of disrupted cells. Both 4-MUP- and PLP-P'tase activities were predominantly in a lipid-anchored form that could be converted to a soluble, lipid-free form by PI-PLC digestion. Our findings suggest that the clinical and biochemical presentation of this PSHPT patient results from the production of two aberrant ALP species. One form of ALP has appropriate ectoorientation but is preferentially deficient in activity toward natural substrates; the other ALP species has appropriate substrate specificity but is sequestered from substrates by its intracellular location.

Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10(-18) mol l(-1) for t-DNA has been achieved.