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Biosensor for detecting uracil glycosylase (UDG) and application thereof

A uracil glycosylase and biosensor technology, applied in the field of biosensors, can solve the problems of complex operation, high cost, low specificity and sensitivity, etc., and achieve the effects of good repeatability, simple operation and good specificity

Active Publication Date: 2018-08-14
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of relatively low specificity and sensitivity, high cost and complicated operation of the method for detecting uracil glycosylase in the above prior art, the present invention provides a method with high specificity and sensitivity, low cost and fast detection speed A biosensor for detecting uracil glycosylase based on Exo III-assisted cycle amplification and silver cluster fluorescence intensity changes

Method used

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  • Biosensor for detecting uracil glycosylase (UDG) and application thereof
  • Biosensor for detecting uracil glycosylase (UDG) and application thereof
  • Biosensor for detecting uracil glycosylase (UDG) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of HAP2-AgNCs.

[0034] Configure PB buffer solution (concentration is 20mM), PB buffer solution is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, weigh 0.7163g disodium hydrogen phosphate and 0.3120g sodium dihydrogen phosphate respectively, and make 100ml solution each , and then take a part of disodium hydrogen phosphate and mix a part of sodium dihydrogen phosphate, adjust the pH value of the mixed solution to 6.5 and then set aside.

[0035] Formulating AgNO 3 Concentration of 2mM, volume of 1mL, AgNO 3 Ready to use, store away from light.

[0036] Prepare NaHBO 4 Concentration 2mM, volume 1mL, NaHBO 4 It is ready-to-use and prepared with ice water at 0°C.

[0037] Take a 1 mL centrifuge tube, add 76 μL of PB (20 mM), add 15 μL of HAP2 (100 μM), add 4.5 μL of AgNO 3 (2mM), shake for 1min, put in 4℃ refrigerator for 30min; then add 4.5μL NaHBO 4 (2mM) in the reaction system, shake for 1min, and place in a refrig...

Embodiment 2

[0039] Example 2 The change of fluorescence intensity with the concentration of ExoIII.

[0040] Mix 2 μL of S chain (100 μM), 2 μL of HAP1 (100 μM), and 2 μL of NEBuffer2.1, and let it react at 37°C for 2 hours to obtain S-HAP1 hybrid double strands for use;

[0041] Mix 2 μL of UDG template (1 μM), 3 μL of ExoIII (1U / μL, 5U / μL, 10U / μL, 15U / μL, 20U / μL), 4 μL of NEBuffer 2.1, 2 μL of S-HAP1 hybrid double strand (5 μM) , 8 μL of HAP2-AgNCs (15 μM) and ultrapure water (21 μL) were mixed to measure the fluorescence intensity, then 1 μL of UDG (50 U / mL) was added, reacted at 37 ° C for 2 hours, and the fluorescence intensity was detected;

[0042] The result is as figure 2 As shown, among them, "-S" represents the fluorescence intensity when there is no free S chain in the system, that is, the fluorescence intensity when UDG is not added; "+S" represents the fluorescence intensity when there is a free S chain in the system, That is, the fluorescence intensity after adding UDG, ...

Embodiment 3

[0043] Example 3 Fluorescence intensity changes with the concentration of S-HAP1 hybrid duplex.

[0044]Mix 2 μL of S chain (100 μM), 2 μL of HAP1 (100 μM), and 2 μL of NEBuffer2.1, and let it react at 37°C for 2 hours to obtain S-HAP1 hybrid double strands for use;

[0045] Mix 2 μL of UDG template (1 μM), 3 μL of ExoIII (10 U / μL), 4 μL of NEBuffer 2.1, 2 μL of S-HAP1 hybrid double-stranded (0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM), 8 μL of HAP2-AgNCs (15 μM) and ultrapure water (21 μL) were mixed, and the fluorescence intensity was measured, then 1 μL of UDG (50 U / mL) was added, reacted at 37°C for 2 hours, and the fluorescence intensity was detected.

[0046] The result is as image 3 , the detected fluorescence signal intensity gradually decreases with the concentration of S-HAP1 hybrid duplex in the range of 0.01-5 μM. When the concentration of S-HAP1 hybrid duplex in the reaction system is 5 μM, the fluorescence intensity value is large.

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Abstract

The invention provides a biosensor for detecting uracil glycosylase. The biosensor comprises an UDG template, an S-HAP1 hybridization chain, HAP2-AgNCs and ExoIII enzyme, fluorescence detection is employed for detecting the activity of the uracil glycosylase, an excitation wavelength of the fluorescence detection is 560 nm, the emission wavelength is 625 nm, and a detection wave band is 575-750 nm. The biosensor has the advantages of good specificity, high sensitivity, mild reaction condition, and fast reaction speed; silver cluster detection is employed, operation is simple, a detection period is short, and carrying is easy; the technology cost is low, and the technology is suitable for low-cost requirement in industrialization; the preparation method is simple, the performance is stable,the repeatability is good, and the method is suitable for detecting UDG in field of medical treatment and public health.

Description

technical field [0001] The invention belongs to the technical field of biosensors, and relates to a biosensor for detecting uracil glycosylase based on ExoIII-assisted cycle amplification and silver cluster fluorescence intensity changes. Background technique [0002] UDG (uracil glycosylase) is the primary DNA excision repair enzyme in various mammalian base mismatches, responsible for the removal of uracil. It mainly cuts the N-glycosidic bond between the wrongly inserted uracil (U) and the sugar group in DNA, removes U, generates an abasic site (AP site), and then is activated by AP endonuclease (AP endonucleases1 , APE1) cuts the DNA single strand, and finally the DNA polymerase and DNA ligase recognize and repair the break site, thereby completing the repair of the mismatched DNA. [0003] Currently reported detection methods for UDG include radioimmunoassay, chemiluminescence assay, chemiluminescence enzyme immunoassay, electrochemiluminescence immunoassay, etc. These...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/64
Inventor 王玉张雪刘素黄加栋
Owner UNIV OF JINAN
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