CN108398406A - A kind of detection uracil glycosylase enzyme（UDG）Biosensor and its application - Google Patents

A kind of detection uracil glycosylase enzyme（UDG）Biosensor and its application Download PDF

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CN108398406A
CN108398406A CN201810029772.6A CN201810029772A CN108398406A CN 108398406 A CN108398406 A CN 108398406A CN 201810029772 A CN201810029772 A CN 201810029772A CN 108398406 A CN108398406 A CN 108398406A
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udg
hap1
hap2
biosensor
solution
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CN108398406B (en
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王玉
张雪
刘素
黄加栋
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University of Jinan
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University of Jinan
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Abstract

The present invention provides a kind of biosensors of detection uracil glycosylase enzyme, hybridize chain including UDG templates, S HAP1, HAP2 AgNCs and ExoIII enzymes, the activity that fluoroscopic examination measures uracil glycosylase enzyme can be used, the excitation wavelength of fluoroscopic examination is 560nm, launch wavelength is 625nm, and detection wave band is 575 750nm.The present invention biosensor specificity is good, high sensitivity, reaction condition is mild, reaction speed is fast；It is easy to operate, detection cycle is short, portable using silver-colored cluster fluoroscopic examination；Process costs are low, the inexpensive requirement suitable for industrialization；Preparation method is simple, and performance is stablized, reproducible, is suitable for detection of the medicine and hygiene fields to UDG.

Description

A kind of detection uracil glycosylase enzyme（UDG）Biosensor and its application
Technical field
The invention belongs to biosensor technology field, it is related to a kind of cycle amplification assisted based on ExoIII and silver-colored cluster is glimmering Intensity variation detects the biosensor of uracil glycosylase enzyme.
Background technology
UDG（Uracil glycosylase enzyme）It is that DNA excision repair enzymes primary when base mispairing occur for various mammals, bears Blame the removal of uracil.It mainly by cutting off N glycosidic bond of the uracil (U) being erroneously inserted in DNA between glycosyl, is moved U is removed, abasic site (AP site) is generated, is then cut off by AP endonucleases (AP endonucleases1, APE1) DNA is single-stranded, finally identifies and repair the broken site by archaeal dna polymerase and DNA ligase again, to complete to mismatched dna It repairs.
The detection method of the UDG reported at present includes radio immunoassay, chemo-immunity luminesceence analysis, chemiluminescence enzyme Often there is expensive equipment, complicated for operation, price in immuno analytical method, Electrogenerated chemiluminescent immunoassay technology etc., these methods Costliness, sensitivity is low, has the problems such as radioactivity to human body.Therefore, it is badly in need of establishing one kind at present quickly, accurately, sensitive and Gao Te Anisotropic detection method detects uracil glycosylase enzyme.
Invention content
In order to solve it is above in the prior art detection uracil glycosylase enzyme method specificity and sensitivity it is all relatively low, Problem of high cost, complicated for operation, the present invention provides a species specificity and high sensitivity, base at low cost, detection speed is fast The biosensor of uracil glycosylase enzyme is detected in the cycle amplification of Exo III auxiliary and silver-colored cluster fluorescence intensity change.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of detection uracil glycosylase enzyme（UDG）Biosensor, including UDG templates, S-HAP1 hybridize chain, HAP2-AgNCs（The silver-colored cluster of probe containing hair fastener 2）With ExoIII enzymes（Exonuclease III）；
The sequence of the UDG templates is as shown in SEQ No. 1；
The sequence of the S chains is as shown in SEQ No. 2；
The sequence of the HAP1 is as shown in SEQ No. 3；
The sequence of the HAP2 is as shown in SEQ No. 4.
The S-HAP hybridization chains are obtained using following preparation method：Buffer solution, S chains solution, HAP1 solution are mixed equal It is even, 37 DEG C of isothermal reaction 2h.
The HAP2-AgNCs is obtained using following preparation method：By buffer solution, the solution of HAP2, AgNO3Mix postposition The 15-30min at 4 DEG C；Then cold NaHBO is added4Solution, 4 DEG C stand 4h or more to get.
Described HAP2, AgNO3With NaHBO4Molar ratio be 1:6:6.
A method of UDG being detected using above-mentioned biosensor, is included the following steps：
（1）S chains and HAP1 are hybridized to S-HAP1 heteroduplexes；
（2）By UDG templates, ExoIII, S-HAP1 heteroduplex, HAP2-AgNCs mixings in buffer solution, fluorescence intensity is measured, Then UDG or prepare liquid is added, 2h, fluorescence intensity are reacted at 37 DEG C；
（3）Standard curve is done according to the fluorescence intensity of series concentration UDG standard solution, regression equation is calculated, according to determinand Fluorescence intensity, calculate contained UDG content.
A concentration of 1-20U/ μ L of ExoIII, preferably 1-10 U/ μ L；
A concentration of 0.01-5 μM of the S-HAP1 heteroduplexes；
The UDG template concentrations are 50-100 nM.
The fluoroscopic examination condition is：Excitation wavelength is 560nm, and launch wavelength 625nm, detection wave band is 575- 750nm。
The operation principle of this biosensor is as follows：
S chains and HAP1 number of base complementary pairings, can be hybridized to double-strand S-HAP1, UDG template in object UDG and ExoIII 2 sections are cut under the conditions of existing at " U " base, generates Trigger sequences（5’-GCAAGAGTG ACATCATAGAC AAAAA-3’）.5 ' the ends of Trigger can be with 3 ' the end hybridization of HAP1 in the S-HAP1 hybridized in advance so that the 3 ' of HAP1 at this time End is flat end, and in the presence of ExoIII, ExoIII can be from 3 ' end cutting HAP1 chains, finally so that S chains and Trigger Chain is released from double-strand system, in addition can leave 5 ' the end part bases of HAP1, have in the part 9 bases and 9 bases at 5 ' ends are identical in Trigger chains, therefore the part is the equal of a secondary Trigger, to realize The cycle of Trigger chains is amplified.5 ' 12 bases in end of HAP2 are to close the sequence of silver-colored cluster, and 3 ' ends include the sequence rich in G.When When HAP2 is hairpin structure, in advance by the synthetic silver-colored clusters of HAP2, since the 5 ' ends of HAP2 are silver-colored cluster, the ends 5' silver cluster and 3 ' ends are rich in The sequence of G is close, to generate strong fluorescence, since 5 ' ends of the S chains generated in the system can be combined with the 3 ' ends of HAP2 To open HAP2, after S chains open HAP2, its fluorescence intensity rapid drawdown will be made, simultaneously because S chains and the mutual of HAP2 recruit Pair so that the 3 ' ends of HAP2 are flat end, in the presence of ExoIII, ExoIII can be from 3 ' ends of hair clip 2 rich in G Sequence starts to cut so that S chains separate out again is discharged into system, is carried out new and HAP2 hybridization reaction, is realized The amplification of signal.By measuring the fluorescence intensity change detected before and after determinand is added, the content of contained UDG is measured.
The present invention has the following advantages：
The present invention biosensor specificity is good, high sensitivity, reaction condition is mild, reaction speed is fast；Using silver-colored cluster fluorescence Detection, it is easy to operate, detection cycle is short, portable；Process costs are low, the inexpensive requirement suitable for industrialization；Preparation method Simply, performance is stablized, reproducible, is suitable for detection of the medicine and hygiene fields to UDG.
Description of the drawings
Fig. 1 is the fundamental diagram of this biosensor；
Fig. 2 is variation diagram of the fluorescence intensity ratio with ExoIII concentration；
Fig. 3 is variation of the fluorescence intensity with S-HAP1 heteroduplex concentration；
Fig. 4 is variation of the fluorescence intensity with UDG template concentrations；
Fig. 5 is variation of the fluorescence intensity with UDG concentration；
Fig. 6 is the standard curve for detecting UDG.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not limited by following embodiments System.
The preparation of 1 HAP2-AgNCs of embodiment.
Configure PB buffer solutions（A concentration of 20mM）, PB buffer solutions are made of disodium hydrogen phosphate and sodium dihydrogen phosphate, The disodium hydrogen phosphate of 0.7163g and the sodium dihydrogen phosphate of 0.3120g are weighed respectively, are respectively made into 100ml solution, are then taken a part Disodium hydrogen phosphate is mixed with a part of sodium dihydrogen phosphate, be mixed solution pH value be adjusted to 6.5 then it is spare.
Prepare AgNO3A concentration of 2mM, volume 1mL, AgNO3Matching while using is protected from light storage.
Prepare NaHBO4A concentration of 2mM, volume 1mL, NaHBO4Matching while using is prepared with 0 DEG C of ice water.
The centrifuge tube of 1mL is taken, the PB of 76 μ L is added（20mM）, 15 μ L HAP2 are added（100μM）, it is added 4.5 μ L's AgNO3（2mM）, 1min is shaken, 4 DEG C of refrigerator 30min are put in；Add 4.5 μ L NaHBO4（2mM）In reaction system, concussion 1min is placed on 4 DEG C of refrigerator 4h or more, obtains HAP2-AgNCs solution.
It takes the HAP2-AgNCs that 30 μ L are prepared in centrifuge tube, 120 μ L ultra-pure water mixings is added, 150 μ are taken with liquid-transfering gun L solution is scanned it in microcolorimetric ware, using fluorescence analyser, exciting light 560nm, measures its emission peak and exists 625nm, illustrates to have in solution that there are HAP2-AgNCs.
2 fluorescence intensity of embodiment with ExoIII concentration variation.
By the S chains of 2 μ L（100μM）With the HAP1 of 2 μ L（100μM）, 2 μ L NEBuffer2.1 be uniformly mixed, make it 37 2h is reacted at DEG C, and it is spare to obtain S-HAP1 heteroduplexes；
By the UDG templates of 2 μ L（1μM）, 3 μ L ExoIII（1U/μL、5U/μL、10U/μL、15U/μL、20U/μL）, 4 μ L The S-HAP1 heteroduplexes of NEBuffer 2.1,2 μ L（5μM）、8μL HAP2-AgNCs（15μM）And ultra-pure water（21μL）It is mixed It is even, fluorescence intensity is measured, the UDG of 1 μ L is then added（50U/mL）, 2h, fluorescence intensity are reacted at 37 DEG C；.
The results are shown in Figure 2, wherein fluorescence intensity when what "-S " was represented is the S chains not dissociated in system does not add Enter fluorescence intensity when UDG；"+S " represents fluorescence intensity when there is free S chains in system, that is, the fluorescence being added after UDG is strong It spends, number is fluorescent value before fluorescent value/addition after addition in column diagram.As seen from the figure, the fluorescence signal intensity detected with The concentration of ExoIII continuously decreases in the sections 1-20U/ μ L, and the concentration of ExoIII is in 10U/ μ L, fluorescence when in reaction system Intensity is than minimum.
3 fluorescence intensity of embodiment with S-HAP1 heteroduplex concentration variation.
By the S chains of 2 μ L（100μM）With the HAP1 of 2 μ L（100μM）, 2 μ L NEBuffer2.1 be uniformly mixed, make it 37 2h is reacted at DEG C, and it is spare to obtain S-HAP1 heteroduplexes；
By the UDG templates of 2 μ L（1μM）, 3 μ L ExoIII（10U/μL）, the NEBuffer 2.1 of 4 μ L, 2 μ L S-HAP1 hybridization Double-strand（0.01μM、0.1μM、0.5μM、1μM、5μM）、8μL HAP2-AgNCs（15μM）And ultra-pure water（21μL）Mixing is surveyed Determine fluorescence intensity, the UDG of 1 μ L is then added（50U/mL）, 2h, fluorescence intensity are reacted at 37 DEG C.
As a result such as Fig. 3, the fluorescence signal intensity detected is as the concentration of S-HAP1 heteroduplexes is in 0.01-5 μM of section It is inside gradually reduced, when a concentration of 5 μM of S-HAP1 heteroduplexes in reaction system, fluorescence intensity level is big.
4 fluorescence intensity of embodiment with UDG template concentrations variation.
By the S chains of 2 μ L（100μM）With the HAP1 of 2 μ L（100μM）, 2 μ L NEBuffer2.1 be uniformly mixed, make it 37 2h is reacted at DEG C, and it is spare to obtain S-HAP1 heteroduplexes；
By the UDG templates of 2 μ L（50nM、100nM、500nM、1μM）, 3 μ L ExoIII（10U/μL）, 4 μ L NEBuffer 2.1, the S-HAP1 heteroduplexes of 2 μ L（5μM）、8μL HAP2-AgNCs（15μM）And ultra-pure water（21μL）Mixing measures glimmering Then the UDG of 1 μ L is added in luminous intensity（50U/mL）, 2h, fluorescence intensity are reacted at 37 DEG C.
The results are shown in Figure 4, the fluorescence signal intensity detected with the concentration of UDG templates in 50nM-1 μM of section by Gradually decline, when a concentration of 1 μM of UDG templates in reaction system, fluorescence intensity level is maximum.
The detection of 5 UDG of embodiment.
By the S chains of 2 μ L（100μM）With the HAP1 of 2 μ L（100μM）, 2 μ L NEBuffer2.1 be uniformly mixed, make it 37 2h is reacted at DEG C, and it is spare to obtain S-HAP1 heteroduplexes；
By the UDG templates of 2 μ L（1μM）, 3 μ L ExoIII（10U/μL）, the NEBuffer 2.1 of 4 μ L, 2 μ L S-HAP1 hybridization Double-strand（5μM）、8μL HAP2-AgNCs（15μM）And ultra-pure water（21μL）Mixing measures fluorescence intensity, is then added 1 μ L's UDG（0.0005U/mL、0.005U/mL、0.05U/mL、0.5U/mL、5U/mL、50U/mL）Or prepare liquid, it is reacted at 37 DEG C 2h, fluorescence intensity.
Testing result as shown in figure 5, fluorescence signal intensity with UDG concentration in the sections 0.0005U/ml- 50U/ml by Gradually decline；Standard curve is done according to the fluorescence intensity of series concentration UDG standard solution, as shown in Figure 6；Calculate regression equation is Y=- 161.4logC+436.92, related coefficient 0.998.
<110>University Of Ji'nan
<120>A kind of detection uracil glycosylase enzyme（UDG）Biosensor and its application
<130> 20180112
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> UDG T
<400> 1
gtctaugcaa gagtgacatc atagacaaaa a 31
<210> 2
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> HAP1
<400> 2
gcaagagtga tataagtctg aatgagcggg tggggtgggg tggggcactc ttgc 54
<210> 3
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> HAP2
<400> 3
cccttaatcc ccgctcatat gcgtactgaa tgagcgggtg gggtggggtg ggg 53
<210> 4
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> S
<400> 4
ccccacccca ccccacccgc tcattcagac taaaaaa 37

Claims (6)

1. a kind of detection uracil glycosylase enzyme（UDG）Biosensor, which is characterized in that it is miscellaneous including UDG templates, S-HAP1 Interlinkage, HAP2-AgNCs and ExoIII enzymes；
The sequence of the UDG templates is as shown in SEQ No. 1；
The sequence of the S chains is as shown in SEQ No. 2；
The sequence of the HAP1 is as shown in SEQ No. 3；
The sequence of the HAP2 is as shown in SEQ No. 4.
2. biosensor according to claim 1, which is characterized in that S-HAP1 hybridization chains are obtained using following preparation method ：Buffer solution, S chains solution, HAP1 solution are uniformly mixed, 37 DEG C of isothermal reaction 2h.
3. biosensor according to claim 1, which is characterized in that HAP2-AgNCs is obtained using following preparation method ：By buffer solution, the solution of HAP2, AgNO3Mixing is placed on 15-30min at 4 DEG C；Then cold NaHBO is added4Solution, 4 DEG C quiet Set 4h or more to get.
4. biosensor according to claim 3, which is characterized in that described HAP2, AgNO3With NaHBO4Molar ratio It is 1:6:6.
5. a kind of method detecting UDG using biosensor as described in claim 1, which is characterized in that including following step Suddenly：
（1）Synthesize HAP2-AgNCs；
（2）S chains and HAP1 are hybridized to S-HAP1 heteroduplexes；
（3）By UDG templates, ExoIII, S-HAP1 heteroduplex, HAP2-AgNCs mixings in buffer solution, fluorescence intensity is measured, Then UDG or prepare liquid is added, 2h, fluorescence intensity are reacted at 37 DEG C；
（4）Standard curve is done according to the fluorescence intensity of series concentration UDG standard solution, regression equation is calculated, according to determinand Fluorescence intensity, calculate contained UDG content.
6. according to the method described in claim 5, it is characterized in that, a concentration of 1-20U/ μ L of the ExoIII；The S-HAP1 A concentration of 0.01-5 μM of heteroduplex；The UDG template concentrations are 50-100 nM.
CN201810029772.6A 2018-01-12 2018-01-12 Biosensor for detecting uracil glycosylase (UDG) and application thereof Active CN108398406B (en)

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CN109444105A (en) * 2018-12-28 2019-03-08 济南大学 A kind of biological sensor and preparation method thereof detecting DNA glycosylase UDG
CN109459423A (en) * 2018-10-22 2019-03-12 济南大学 Active biosensor of a kind of detection uracilase (UDG) and preparation method thereof
CN109752362A (en) * 2019-01-10 2019-05-14 济南大学 A kind of biosensor and preparation method thereof detecting uracil-DNA glycosylase
CN110734961A (en) * 2019-11-29 2020-01-31 福州大学 enzyme-free biosensors for detecting uracil-DNA glycosylase activity

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109459423A (en) * 2018-10-22 2019-03-12 济南大学 Active biosensor of a kind of detection uracilase (UDG) and preparation method thereof
CN109459423B (en) * 2018-10-22 2021-06-15 济南大学 Biosensor for detecting uracil glycosidase (UDG) activity and preparation method thereof
CN109444105A (en) * 2018-12-28 2019-03-08 济南大学 A kind of biological sensor and preparation method thereof detecting DNA glycosylase UDG
CN109752362A (en) * 2019-01-10 2019-05-14 济南大学 A kind of biosensor and preparation method thereof detecting uracil-DNA glycosylase
CN109752362B (en) * 2019-01-10 2021-06-15 济南大学 Biosensor for detecting uracil-DNA glycosylase and preparation method thereof
CN110734961A (en) * 2019-11-29 2020-01-31 福州大学 enzyme-free biosensors for detecting uracil-DNA glycosylase activity
CN110734961B (en) * 2019-11-29 2021-10-29 福州大学 Enzyme-free biosensor for detecting activity of uracil-DNA glycosylase

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