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#26419907   2015/10/20 Save this To Up

Versatile and Amplified Biosensing through Enzymatic Cascade Reaction by Coupling Alkaline Phosphatase in Situ Generation of Photoresponsive Nanozyme.

The alkaline phosphatase (ALP) biocatalysis followed by the in situ enzymatic generation of a visible light responsive nanozyme is coupled to elucidate a novel amplification strategy by enzymatic cascade reaction for versatile biosensing. The enzymatic hydrolysis of o-phosphonoxyphenol (OPP) to catechol (CA) by ALP is allowed to coordinate on the surface of TiO2 nanoparticles (NPs) due to the specificity and high affinity of enediol ligands to Ti(IV). Upon the stimuli by CA generated from ALP, the inert TiO2 NPs is activated, which demonstrates highly efficient oxidase mimicking activity for catalyzing the oxidation of the typical substrate of 3,3',5,5'-tetramethylbenzidine (TMB) under visible light (λ ≥ 400 nm) irradiation utilizing dissolved oxygen as an electron acceptor. On the basis of the cascade reaction of ALP and the nanozyme of CA coordinated TiO2 (TiO2-CA) NPs, we design exquisitely colorimetric biosensors for probing ALP activity and its inhibitor of 2, 4-dichlorophenoxyacetic acid (2,4-DA). Quantitative probing of ALP activity in a wide linear range from 0.01 to 150 U/L with the detection limit of 0.002 U/L is realized, which endows the methodology with sufficiently high sensitivity for potentially practical applications in real samples of human serum (ALP level of 40-190 U/L for adults). In addition, a novel immunoassay protocol by taking mouse IgG as an example is validated using the ALP/nanozyme cascade amplification reaction as the signal transducer. A low detection limit of 2.0 pg/mL is attained for mouse IgG, which is 4500-fold lower than that of the standard enzyme-linked immuno-sorbent assay (ELISA) kit. Although only mouse IgG is used as a proof-of-concept in our experiment, we believe that this approach is generalizable to be readily extended to other ELISA systems. This methodology opens a new horizon for amplified and versatile biosensing including probing ALP activity and following ALP-based ELISA immunoassays.

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#21090120   2010/11/22 Save this To Up

[Membrane transfer-based colorimetric DNA detection using enzyme modified gold nanoparticles].

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.

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#9168313   1997/07/17 Save this To Up

Comparison of a protein phosphatase inhibition assay, HPLC assay and enzyme-linked immunosorbent assay with the mouse bioassay for the detection of diarrhetic shellfish poisoning toxins in European shellfish.

Consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates can lead to diarrhetic shellfish poisoning (DSP). UK legislation necessitates toxin detection by mouse bioassay but this method is non-specific and lacks sensitivity. As an alternative method, an HPLC technique has been optimized, with detection limits of 0.26 micrograms of toxin/g of shellfish hepatopancreas for both Okadaic Acid (OA) and Dinophysistoxin-1 (DTX-1). A colorimetric protein phosphatase inhibition (PPI) assay has also been optimized. This assay detects inhibition of protein phosphatase 1 (PPI gamma) by OA and DTX-1 with detection limits of 1.5 ng of total toxin/g of hepatopancreas. Contaminated shellfish from several European sources, the UK monitoring programmes and mussels associated with an outbreak of DSP poisoning in the UK, have been analyzed and assessed using the two alternative methods and a commercially available enzyme-linked immunosorbent assay (ELISA) kit. The results indicate that both the HPLC and PPI assays correlate well with each other and with the UK standard mouse bioassay. In contrast, and not withstanding its advantages of rapidity and ease, the ELISA kit did not accurately and consistently detect low toxin concentrations, although it may be useful as a screening tool.

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