Authors: Joanna Kosman YuTang Wu Agata Gluszynska Bernard Juskowiak
Publish Date: 2014/09/12
Volume: 406, Issue: 28, Pages: 7049-7057
Abstract
Characterization and optimization studies of Nmethyl4hydrazino7nitrobenzofurazan MNBDH as a new fluorogenic substrate in the peroxidation reaction catalyzed by DNAzyme are reported The effects of pH H2O2 concentration metalcation type and the concentration and type of surfactant on the fluorescence intensity were investigated The optimized reaction was subsequently used for the development of an assay for DNA detection based on a molecularbeacon probe The use of a fluorogenic substrate enabled the detection of a singlestranded DNA target with a 1 nmol L−1 detection limitDNA is one of the most important molecules in living organisms In addition to its biological function of storing genetic information these macromolecules have proved to also be a useful material for nanotechnology 1 medicine 2 biotechnology 3 and the development of sensing devices 4 One of the DNA systems that have attracted great attention is DNAzyme which has peroxidasemimicking activity 5 To have catalytic activity a DNA oligonucleotide must first adopt a Gquadruplex structure and then form a complex with a molecule of hemin The peroxidasemimicking DNAzyme catalyzes the reaction between hydrogen peroxide and an organic substrate Through the selection of a proper substrate this system can be successfully incorporated into new bioassays Most of the developed assays are based on chemiluminescence with luminol as a substrate or on a colorimetric approach with 22ʹazinobis3ethylbenzthiazoline6sulfonic acid ABTS as a substrate 6 7 8 9 10 11 12 Using these substrates several analytical systems have been designed for the detection of analytes including metal ions 6 7 DNA sequences 8 9 proteins 10 enzymes 11 and others 12 13Compared with protein enzymes DNAzymes have several advantages that enable the development of simpler bioassays First they are thermally stable do not require strict refrigerator storage and can be heated even to 90 °C and cooled to room temperature with no effect on their catalytic activity Second DNA oligonucleotides are simple and cheap regarding synthesis multiplication by PCR and purification One of the most important advantages of deoxyribozymes however is their ability to hybridize This makes it possible to design systems with selected features A good example of such a system is a molecularbeacon probe MB Xiao et al reported a system for DNAtarget detection based on a molecularbeacon probe which after DNAtarget hybridization to the MB loop formed a peroxidasemimicking DNAzyme that catalyzed ABTS oxidation 14 This system enabled DNA detection at a 02 μmol L−1 concentrationEnhancement of the analytical signal through DNAzyme action and use of a colorimetric detection technique enables the determination of analytes in the submicromolar concentration range However medicine and molecular biology are still seeking detection systems that enable the detection of molecules at nano or even picomolar levels One solution that provides higher sensitivity is the use of fluorogenic substrates which emit fluorescence after oxidation by a DNAzyme Two fluorogenic substrates have been used in the development of peroxidase DNAzyme sensors Zhang et al reported the use of thiamine in the detection of thrombin with a detection limit of 1 pmol L−1 15 Other research groups focused on use of Amplex Red in DNAzyme systems for the detection of thrombin 16 glucoseoxidase activity 16 or lead ions 17 With the use of this substrate which oxidizes to the strongly fluorescent molecule resorufine it was possible to develop methods for the analytes mentioned above with detection limits in the nanomolar range However new substrates are required for the development of sensitive biosensorsIn this paper we report a new fluorogenic substrate Nmethyl4hydrazino7nitrobenzofurazan MNBDH which can be oxidized to a fluorescing amino derivative Nmethyl4amino7nitrobenzofurazan MNBDA in the presence of a DNAzyme with peroxidasemimicking activity MNBDH has several advantages over other fluorogenic substrates which often require an alkaline pH and have a short excitation maximum wavelength Use of MNBDH to develop a new sensitive assay for DNA detection by a molecularbeacon probe is also presented
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