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Substrate-free electrochemical biosensor for detecting uracil-DNA glycosylase based on non-enzymatic nano-material signal amplification

A biosensor, glycosylase technology, applied in the field of electrochemical detection, can solve problems such as increasing operational uncertainty and affecting sensitivity, and achieves the effects of universality, enhanced sensitivity, and reduced detection cost

Active Publication Date: 2019-10-18
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, traditional peroxidase-catalyzed amplification strategies usually require 2 o 2 conditions exist, but H 2 o 2 The instability of the will increase the uncertainty of the operation and affect the sensitivity

Method used

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  • Substrate-free electrochemical biosensor for detecting uracil-DNA glycosylase based on non-enzymatic nano-material signal amplification
  • Substrate-free electrochemical biosensor for detecting uracil-DNA glycosylase based on non-enzymatic nano-material signal amplification
  • Substrate-free electrochemical biosensor for detecting uracil-DNA glycosylase based on non-enzymatic nano-material signal amplification

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Experimental program
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preparation example Construction

[0050] As introduced in the background technology, in view of the complex operation steps, poor stability and poor sensitivity of traditional electrochemical biosensors for detecting UDG activity, this application provides a new substrate-free biosensor based on non-enzyme nanomaterial signal amplification A preparation method for an electrochemical biosensor, comprising the following steps:

[0051] (1) Preparation of porphyrin-based nanomaterials (OAPS-Por) and signal probes (OAPS-Por / Thi@AuNPs-ssDNA);

[0052] (2) Establish an electrochemical biosensor to detect uracil-DNA glycosylase (UDG).

[0053] The preparation process of porphyrin-based nanomaterials (OAPS-Por) and signal probes of the present invention comprises the following steps:

[0054] (1) Preparation of porphyrin-based nanomaterials (OAPS-Por):

[0055] First, 52.75mg of Fe(III)tetracarboxybenzoporphyrin was added to 100mL EDC / NHS (EDC: 34Mm; NHS: 34mM) mixed solution, stirred at room temperature for 12 hour...

Embodiment 1

[0078] The preparation of embodiment 1 electrode

[0079] (1) Use 1.0, 0.3 and 0.05μm Al respectively 2 o 3 Powder-polished glassy carbon electrode (d=3mm), then ultrasonicated in water-ethanol-water for 5 minutes, and finally dried with nitrogen gas for later use, recorded as GCE;

[0080] (2) Dip the treated electrode into 8mL of 0.8% HAuCl 4 In the solution, at a constant potential (-0.2V), deposit for 30s, rinse the electrode with ultrapure water 3 times, and record it as AuNPs / GCE;

[0081] (3) Drop 10 μL of annealed hairpin DNA (hDNA, 0.5 μM) on the electrode, incubate overnight at 4°C and ensure 100% humidity, then rinse the electrode several times with 10 mM Tris-HCl (pH=7.4), record for hDNA / AuNPs / GCE;

[0082] (4) Drop 10 μL of 1 mM 6-mercapto 1-hexanol (MCH) on the surface of the electrode treated in step 3, incubate at room temperature for 30 min, rinse the electrode several times with 10 mM Tris-HCl (pH=7.4), record as MCH / hDNA / AuNPs / GCE;

Embodiment 2

[0083] Embodiment 2 Preparation of porphyrin-based nanomaterials (OAPS-Por):

[0084] First, 52.75mg of Fe(III)tetracarboxybenzoporphyrin was added to 100mL EDC / NHS (EDC: 34Mm; NHS: 34mM) mixed solution, stirred at room temperature for 12 hours, and then 36mg of octaaminophenyl oligomerized sesqui Siloxane (OAPS) was added to the above solution and stirred for 24 hours. After the reaction was completed, a gray-black powder was obtained by filtration, and finally vacuum-dried at 80°C for 24 hours to obtain OAPS-Por, and a series of tests were carried out on it. representation, such as figure 2 .

[0085] The electrocatalytic activity of OAPS-Por on Thi was evaluated by measuring CV and DPV of different modified electrodes in 0.1M HAc-NaAc (pH5.0) solution containing 35 μM thionine ( image 3 ). 10μLOAPS-Por (1mg mL -1 ) and 10 μL of OAPS and FeTCPP at concentrations equivalent to those in OAPS-Por were dropped onto the GCE and measured after drying under infrared light. S...

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Abstract

The present invention relates to a substrate-free electrochemical biosensor for detecting uracil-DNA glycosylase based on non-enzymatic nano-material signal amplification. The non-enzymatic nano material (OAPS-Por) used in the sensor not only can adsorb a large amount of thionine molecules serving as an electroactive substance, but also can catalyze reduction of thionine on an electrode under thecondition that H2O2 does not participate, so that the signal intensity is greatly enhanced. On the basis of the material, a signal probe (OAPS-Por / Thi@AuNps-ssDNA) is prepared. In the presence of UDG,uracil bases in hairpin DNA on the electrode are removed, a hairpin structure is expanded, and the signal probe is connected to the surface of the electrode through DNA base complementary pairing, sothat an amplified thionine reduction peak is generated, and the constructed biosensor shows a relatively wide linear range (0.005-1U / ml), and the detection limit is 0.000697U / ml. In addition, the biosensor can also be used for detecting the activity of UDG in a UDG activity inhibitor and a HeLa cell lysis solution, and has huge potential in clinical diagnosis and biomedical research.

Description

technical field [0001] The invention relates to an electrochemical biosensor and its application, in particular to a novel substrate-free electrochemical biosensor based on signal amplification of non-enzyme nanomaterials, which is used to detect uracil-DNA glycosylase, and belongs to electrochemical detection field. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Maintaining the integrity of the cellular genome is essential for all organisms. However, DNA is easily damaged by X-rays, reactive oxygen species (ROS), and ultraviolet radiation, among others. If DNA damage is not repaired in time, it will induce various diseases and even cancer. Base excision repair (BER) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/30
CPCG01N27/308G01N27/3276G01N27/3277G01N27/3278
Inventor 张晓梅柳婷婷
Owner SHANDONG UNIV
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