4-methylumbelliferyl Phosphate Research Articles

Dissolved organic phosphorus bond-class utilization by Synechococcus.

Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus-carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5'-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5'-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus-carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5'-triphosphate, and lastly adenosine 5'-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains' exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5'-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems.

Simple hydrated electron-induced electrochemiluminescence determination of alkaline phosphatase with novel time-resolved internal standard strategy at boron doped diamond electrodes

Hydrated electrons were generated at boron doped diamond electrodes in fully aqueous solutions during cathodic pulse polarization and utilized for the electrochemiluminescence (ECL) determination of alkaline phosphatase (ALP) by adapting one of the most common fluorescent methods. In this approach, highly fluorescent 4-methylumbelliferyl (MU) is enzymatically generated from non-fluorescent 4-methylumbelliferyl phosphate (MUP) substrate and chemically excited in the presence of oxidizing radicals and reducing hydrated electrons. A novel time-resolved internal standard strategy is presented to counter matrix effects to certain degree and to lower signal variation among concurrent measurements. In this unique approach, the cathodic ECL signals of MU and Tb(III)-chelate were separated on the basis of their exceptionally different excited state lifetimes. This enabled more precise determination of ALP by simply measuring the ratio of ECL emissions during and after cathodic excitation pulse with simple two-electrode, single photomultiplier tube setup. The ratiometric ECL signals in the absence and presence of 1.0 % serum (v/v) were interchangeable whereas ratiometric signal recovery in the presence of 10.0 % serum was ∼85 %.

Phosphomonoesterase and phosphodiesterase activities in the eastern Mediterranean in two contrasting seasonal situations

Abstract. Hydrolysis of dissolved organic phosphorus by marine planktonic microorganisms is a key process in the P cycle, particularly in P-depleted, oligotrophic environments. The present study assessed spatiotemporal variations in phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea in two contrasting situations: the end of winter (including a small bloom period) and autumn. The distribution and regulation of the maximum hydrolysis rate (Vm) and half-saturation constant (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached its maximum activities (Vm) after the addition of 1 µM MUF-P (4-methylumbelliferyl phosphate), whereas, for PDE, it was necessary to add up to 50 µM bis(4-methylumbelliferyl)phosphate (bis-MUF-P) to reach saturation state. On average, the Km of PDE was 33 ± 25 times higher than that of PME. The Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm values were on the same order of magnitude for both ectoenzymes, with their ratio (Vm PME : Vm PDE) ranging between 0.2 and 6.3. Dissolved organic phosphorus (DOP) and the phosphomonoesterase hydrolysable fraction of DOP explained most of the lack of variability in Vm PME and Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to the concentration of dissolved inorganic phosphorus. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 µM, high Km, high turnover times) are discussed with respect to the possible unequal distribution of PDE and PME among the size continuum of organic material and accessibility of phosphodiesters.

Regulating the redox and non-redox enzyme-mimicking activities of Ce-UiO-66-NO2 nanozyme for dual-mode sensing of phosphate

Nanozymes with multi-enzyme mimicking activities have received a great deal of attention, which can mimic peroxidase, oxidase, superoxide dismutase, catalase, and alkaline phosphatase, etc. However, the current research focuses on regulating the multi-enzyme-like activity of redox types. Regulating the catalytic activity of different types of nanozyme such as redox and non-redox types is still full of challenges. Here, Ce-UiO-66-NO2 with hydrolase-like activity and oxidase-like activity was taken as the model to regulate its dual enzyme-like activities. Interestingly, a distinct response in oxidase-like and hydrolase-like activities of Ce-UiO-66-NO2 was observed towards the addition of phosphate (PO43-). When PO43- was present, the oxidase mimicking activity of Ce-UiO-66-NO2 was enhanced and the hydrolase mimicking activity was inhibited. With X-ray photoelectron spectroscopy, Fourier transform infrared spectrometry, X-ray diffraction and zeta potential characterizations, the regulatory mechanism was recognized as the coordinated impact of the reactive oxygen species, electrostatic adsorption and competitive inhibition. As a proof of concept, a colorimetric/fluorometric dual-mode sensing method for detecting PO43- through regulating the dual enzyme mimicking activities of Ce-UiO-66-NO2 was constructed. PO43- can be quantitated with 3,3′,5,5′-tetramethylbenzidine (TMB) and 4-methylumbelliferyl phosphate (4-MUP) as substrates for colorimetric and fluorescent sensing, respectively. This study will deepen the understanding of nanozymes with multi-enzyme mimicking activities and accelerate the development of efficient sensing.

Ratiometric fluorescent detection of total phosphates in frozen shrimp samples using catalytic active Zr(IV) modified gold nanoclusters.

Phosphate salts are important food additives in a variety of foods. In this study, the Zr(IV) modified gold nanoclusters (Au NCs) were prepared for ratiometric fluorescent sensing of phosphate additives in seafood samples. Compared with bare Au NCs, the synthesized Zr(IV)/Au NCs showed stronger orange fluorescence at 610nm. On the other hand, the Zr(IV)/Au NCs retained the phosphatase-like activity of Zr(IV) ions and could catalyze the hydrolysis of fluorescent substrate 4-methylumbelliferyl phosphate to produce blue emission at 450nm. The addition of phosphate salts could effectively inhibit the catalytic activity of Zr(IV)/Au NCs, resulting the fluorescence decrease at 450nm. However, the fluorescence at 610nm almost unchanged upon the addition of phosphates. Based on this finding, the ratiometric detection of phosphates using the fluorescence intensity ratio (I450/I610) was demonstrated. The method has been further applied for sensing total phosphates in frozen shrimp samples with satisfactory results.

Ratiometric fluorescence sensor and smartphone-based microfluidic sensing platform based on oxidation induced Ce(III)/Ce(IV) phosphatase-like activity and complexation effect activation for sarcosine detection

Prostate cancer (PCa) has seriously affected men's quality of life and even endangered their lives. Thus, precise and reliable determination of related biomarkers in clinical samples is extremely important for early diagnosis and treatment of PCa. Sarcosine (Sar) is widely regarded as a potential biomarker for non-invasive diagnosis of PCa owing to its specific high-level expression in the development and metastasis of PCa. With the aid of Ce(III) as a bridge between 4-Methylumbelliferyl Phosphate (4-MUP) and orange emitted carbon dots (o-CDs), here we have constructed a new ratiometric fluorescence sensor for Sar determination by reasonably combining the phosphatase-like activity and complexation reaction characteristics of Ce(IV). This sensor indicates high sensitivity, exceptional selectivity, good reliability, and wide linear range of 0.2–120 μM with a detection limit of 89 nM. Besides, our proposed sensor demonstrates excellent precise and stability even in complex actual urine samples. Interestingly, an inexpensive, portable but reliable smartphone-based microfluidic sensing platform has been fabricated and successfully employed for precise determination of Sar in urine samples, which offers a powerful and convenient tool for the early diagnosis of PCa, and even inspires the research and development of other practical optical sensing devices for cancer home screening.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation – analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics

Biomarkers in the Atacama Desert along the moisture gradient and the depth in the hyperarid zone: Phosphatase activity as trace of microbial activity

AbstractMicrobiological activities can be detected in various extreme environments on Earth, which suggest that extraterrestrial environments, such as on Mars, could host life. There have been proposed a number of biomarkers to detect extant life mostly based on specific molecules. Because terrestrial organisms have catalytic proteins (enzymes), enzymatic activity may also be a good indicator to evaluate biological activities in extreme environments. Phosphatases are essential for all terrestrial organisms because phosphate esters are ubiquitously used in genetic molecules (DNA/RNA) and membranes. In this study, we evaluated microbial activity in soils of the Atacama Desert, Chile, by analysing several biomarkers, including phosphatase activity. Phosphatases extracted with Tris buffer were assayed fluorometrically using 4-methylumbelliferyl phosphate as a substrate. The horizontal distribution of phosphatase activity and other parameters in soils from the Atacama Desert showed that phosphatase activity was positively correlated with amino acid concentration and colony-forming units and negatively correlated with precipitation amount. We found consistent that biochemical indicators including phosphatase significantly decreased in the extreme hyper-arid zone where rainfall of <25 mm year−1. The results were compared with phosphatase activities detected in extreme environments, such as submarine hydrothermal systems and Antarctic soils, as well as soils from ordinary environments. Overall, our results suggested that phosphatase activity could be a good indicator for evaluating biological activities in extreme environments.

Dual-channel fluorescent imaging of reactive oxygen species in living cells based on Ce(III) modified quantum dots with oxidation triggered phosphatase-like activity

The two-wavelength fluorescent sensing and imaging of reactive oxygen species (ROS) was demonstrated using Ce(III) ions modified fluorescent quantum dots (QDs). After the addition of ROS, the Ce(III) on the surface of QDs was transformed to Ce(IV) which exhibits phosphatase-like catalytic activity. The Ce(IV)/QDs can catalyze the hydrolysis of 4-methylumbelliferyl phosphate (4-MUP) to generate blue emission. On the other hand, ROS had only slight effect on the red fluorescent signal of QDs. Based on this phenomenon, the ratiometric fluorescent detection of ROS (ClO-, H2O2, and ONOO-) was successfully performed by using Ce(III)/QDs/4-MUP system. The two fluorescent emission peaks (450 nm and 620 nm) were well-separated, which holds the advantages such as high selectivity and accuracy. Finally, the Ce(III)/QDs/4-MUP system was successfully used for dual-channel fluorescent ROS imaging in HeLa and RAW264.7 cells.

Assessing the microcystins concentration through optimized protein phosphatase inhibition assay in environmental samples

Protein phosphatase (PPase) inhibition assay (PPIA) is widely used to analyze the concentration of microcystins (MCs) because it is comparatively less expensive and faster than other assays. This study aimed to optimize the PPIA by determining a suitable reaction terminator and an optimal methanol concentration in the sample. The most suitable reaction time was 90 min, with the corresponding methanol concentration in the sample being 15% or less. When p-nitrophenyl phosphate (pNPP) was used as a substrate, copper chloride solution was suitably used as a reaction terminator, and when 4-methylumbelliferyl phosphate (MUP) was used, a glycine buffer not only increased the measurement sensitivity of the reaction product but also terminated the enzymatic reaction. When PPase 1 and MUP were used as an enzyme and a substrate, respectively, the limit of quantitation for MC-leucine/arginine (LR) was 0.02 µg/L, whereas it was 0.1 µg/L when pNPP was used as a substrate. The proposed method facilitated the measurement of MC-LR concentration without additional pretreatments, such as concentration or purification; therefore, this method was suitable and feasible for the continuous monitoring of MCs in drinking water.

Extraction methods for untargeted metabolomics influence enzymatic activity in diverse soils

Soil organic matter (SOM) is the largest carbon pool in terrestrial ecosystems and underpins the health and productivity of soil. Accurate characterization of its chemical composition will improve our understanding of biotic and abiotic processes regulating its stabilization. Our purpose in this study was to estimate the loss of SOM by microbial and exoenzymatic activity that might occur when soil is extracted for analysis of representative low molecular weight mass features using untargeted metabolomics. Two mined clays (kaolinite, montmorillonite) and three diverse soils (varying in texture, specific surface area and cation exchange capacity) were used to assess the extraction efficiency and loss of three enzymatic activity indicators (2,6-dichloroindophenol sodium salt hydrate [DCIP], 4-methylumbelliferyl phosphate [MUBph] and 3,4-dihydroxy-L-phenylalanine [LDOPA]) during extraction with two different solvents (water and methanol). Losses of the indicators were attributed to extraction method (ultrasonication, shaking, or shaking following chloroform fumigation), physical properties associated with the soil/clay type, and microbial activity. Soil/clay type strongly influenced indicator recovery and hence, SOM recovery. Choice of extraction method strongly influenced the composition and recovery of representative SOM mass features, while the choice of solvent determined whether the soil type or extraction method had a greater influence of compositional differences in the SOM mass features extracted. Extraction following chloroform fumigation had the greatest loss of the indicators, due to enzymatic activity and/or adsorption onto the soil matrix. Minimal variation in composition and loss of SOM mass features occurred during extraction by shaking for the soils tested; we therefore recommend it as the method of choice for untargeted SOM extraction studies.

Biochemical inhibition of acid phosphatase activity in two mountain spruce forest soils

The product inhibition of the 4-methylumbelliferyl phosphate (MUB-P) decay rate, a measure of potential acid phosphatase activity, has not been considered in most of the published kinetic studies. The aim of this study was to determine the type and strength of the product inhibition in order to better define reaction conditions at which the acid phosphatase activity assay produces unbiased results. The MUB-P decay rate was measured in the forest floor and topsoil organic horizons of two spruce forest catchments at different initial MUB-P and P-PO43− concentrations. The type and strength of the inhibition was analyzed by non-linear regression. We found that MUB-P decay was competitively inhibited by the P-PO43− but also by dissolved organic P. By using estimated kinetic parameters, we calculated an underestimation of potential acid phosphatase activity by up to ~ 20% at concentrations of added MUB-P as high as 300 µmol g−1 dry weight and an incubation time one hour due to background inorganic and organic P concentrations in range of units of µmol per gram of dry soil. We discuss approaches that can be used to minimize the underestimation and show that the previously defined recommendations might not be always sufficient to avoid the bias. Therefore, we recommend to analyze progress curves at a wide range of initial MUB-P concentrations using non-linear methods when the enzyme assay is optimized for a given soil. When changes in inorganic and/or organic P concentrations are expected, we further recommend measuring background inorganic and organic P concentrations.

Formaldehyde preservation for deferred measurements of alkaline phosphatase activities in marine samples

Alkaline phosphatases are the main enzymes required by microorganisms to hydrolyse organic phosphorus into available phosphate in aquatic environments. The investigations of alkaline phosphatase activity (APA) usually generate numerous samples (size fractionation, Michaelis-Menten kinetics). Therefore, convenient and reliable preservation of incubated samples for a deferred analysis would be very useful when measurements cannot be performed right away. The APA of marine pond waters was measured using 4-Methylumbelliferyl phosphate (MUF-P) as the fluorogenic substrate modelling natural organic phosphorus compounds. Where typical inhibitors of other enzymatic activities, such as 1% sodium dodecyl sulfate, mercuric chloride, or buffered solutions of ammonium and glycine, failed to stop APA, the addition of formaldehyde efficiently inhibited APA. The effect of formaldehyde was the strongest with the highest concentration tested (4% final concentration) and in buffered (pH 8) solutions. Since a slow and gradual increase in APA may persist with time, the combination of the addition of 4% buffered formaldehyde with immediate freezing is the best method to entirely inhibit APA. The maximal rate of hydrolysis (Vmax) and the Michaelis constant (Km) of formaldehyde (4%)-inhibited samples did not significantly change during storage at -20 °C for 11 days. The method was successfully tested on samples with extremely high values of APA (15000–40000 nM h−1) that were preserved for 1 month at -20 °C (98% inhibition). This method is a reliable and useful means of preserving incubated samples, and it provides convenient controls for background fluorescence of water and substrate, without provoking abiotic hydrolysis of the substrate.

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.

Selective Autonomous Molecular Transport and Collection by Hydrogel-Embedded Supramolecular Chemical Gradients.

Selective transport and concentration of molecules to specified regions on a substrate both enhances the potential to detect such molecules and provides a path to spatially localize such molecules prior to initiation of subsequent chemical reactions. Here, we first embed radially symmetric α-, β-, and γ-cyclodextrin gradients in a hydrogel matrix. Driven by host-guest interactions between the cyclodextrins and the target molecule, we observe these gradients can serve to direct 2D molecular transport. Using xanthene dyes and organophosphates as target molecules, we found the transport metrics, e.g., selectivity, rate, and concentration limits, are strongly dependent on the specific cyclodextrin forming the gradient. In all cases, as the concentrating power of the gradient increased, the rate of target concentration slowed, which we hypothesize is because stronger interactions between the target and the cyclodextrin decrease the rate of target diffusion. The concentration enhancement for the nerve agent simulant 4-methylumbelliferyl phosphate (15.8) is the greatest when the gradient is formed using β-cyclodextrin while directed concentration of cyanomethyl phosphonate, a smaller non-aromatic organophosphate, is observed only for the smaller α-CD. To provide a near real-time read-out of the concentration of the analyte, we used an array of IR resonant metallic nanoantennas tuned to a specific IR absorption band of the analyte to enhance the IR signal generated by the analyte.

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.

Sensor system based on flexible screen-printed electrodes for electrochemical detection of okadaic acid in seawater

The monitoring of marine dinophysistoxin okadaic acid in seawater can serve as an early alert system for preventing the potential negative effects this toxin can have on the food industry and human health in general. A disposable sensor system for electrochemical detection of this toxin has been developed using screen-printed electrodes. The method described is based on the inhibition of protein phosphatase type 2A by okadaic acid, evaluating the enzyme activity. p-Nitrophenyl phosphate, 4-methylumbelliferyl phosphate and phenyl phosphate have been tested as substrates achieving good limits of detection of 2.7·10–12 M of okadaic acid. The proposed method has been successfully applied to okadaic acid determination in seawater samples. A study of divalent cations present in seawater that can interfere with the measurement has been carried out. The electrode systems were printed on both rigid and textile backing materials to observe the influence of those materials on the final performance of the sensor.

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

ABSTRACTAlkaline 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

In-Gel Protein Phosphatase Assay Using Fluorogenic Substrates.

Protein phosphorylation plays important roles in regulating a variety of biological processes in animals, plants, and microorganisms. Therefore, it is important to use appropriate techniques to detect and analyze protein kinases and protein phosphatases. In this chapter, we describe the method to detect protein phosphatase activities using fluorogenic substrates such as 4-methylumbelliferyl phosphate (MUP) after separating proteins by one-dimensional or two-dimensional polyacrylamide gel electrophoresis.

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.