CN108398406B - Biosensor for detecting uracil glycosylase (UDG) and application thereof - Google Patents
Biosensor for detecting uracil glycosylase (UDG) and application thereof Download PDFInfo
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Abstract
The invention provides a biosensor for detecting uracil glycosylase, which comprises a UDG template, an S-HAP1 hybrid chain, HAP2-AgNCs and ExoIII enzymes, and can be used for detecting the activity of the uracil glycosylase by adopting fluorescence detection, wherein the excitation wavelength of the fluorescence detection is 560nm, the emission wavelength is 625nm, and the detection wavelength band is 575-750 nm. The biosensor has the advantages of good specificity, high sensitivity, mild reaction conditions and high reaction speed; the silver cluster fluorescence detection is adopted, so that the operation is simple and convenient, the detection period is short, and the carrying is easy; the process cost is low, and the method is suitable for the requirement of low price in industrialization; the preparation method is simple, stable in performance and good in repeatability, and is suitable for detecting the UDG in the field of medical treatment and health.
Description
Technical Field
The invention belongs to the technical field of biosensors, and relates to a biosensor for detecting uracil glycosylase based on ExoIII-assisted cyclic amplification and silver cluster fluorescence intensity change.
Background
UDG (uracil glycosylase) is the primary DNA excision repair enzyme responsible for uracil removal in a variety of mammals when a base mismatch occurs. It mainly cuts off the N glycosidic bond between the incorrectly inserted uracil (U) and glycosyl in DNA, removes U, generates abasic site (AP site), cuts off DNA single strand by AP endonuclease (AP endonulases 1, APE1), finally recognizes and repairs the broken site by DNA polymerase and DNA ligase, thus completing the repair of mismatched DNA.
The currently reported detection methods of UDG include radioimmunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, electrochemiluminescence immunoassay and the like, and the methods often have the problems of expensive instruments, complex operation, high price, low sensitivity, radioactivity to human bodies and the like. Therefore, a rapid, accurate, sensitive and high-specificity detection method for detecting uracil glycosylase is urgently needed to be established.
Disclosure of Invention
In order to solve the problems of low specificity and sensitivity, high cost and complex operation of the method for detecting uracil glycosylase in the prior art, the invention provides the biosensor for detecting uracil glycosylase based on Exo III-assisted cyclic amplification and silver cluster fluorescence intensity change, which has high specificity and sensitivity, low cost and high detection speed.
In order to achieve the purpose, the invention adopts the following technical scheme.
A biosensor for detecting uracil glycosylase (UDG) comprises a UDG template, a S-HAP1 hybrid double strand, HAP2-AgNCs (silver cluster containing hairpin probe 2) and an ExoIII enzyme (exonuclease III);
the sequence of the UDG template is shown as SEQ number 1;
the sequence of the S chain is shown as SEQ number 4;
the sequence of the HAP1 is shown as SEQ number 2;
the sequence of the HAP2 is shown as SEQ number 3.
The S-HAP hybrid double strand is obtained by adopting the following preparation method: the buffer solution, the S chain solution and the HAP1 solution are mixed evenly and reacted for 2 hours at the constant temperature of 37 ℃.
The HAP2-AgNCs are obtained by adopting the following preparation method: mixing buffer solution, HAP2 solution, AgNO3Mixing, and standing at 4 deg.C for 15-30 min; then adding cold NaHCO4Standing the solution at 4 deg.C for more than 4 hr.
The HAP2 and AgNO3With NaHCO4In a molar ratio of 1:6: 6.
A method for detecting UDG using the above biosensor, comprising the steps of:
(1) hybridizing the S chain and HAP1 into an S-HAP1 hybrid double chain;
(2) uniformly mixing the UDG template, the ExoIII, the S-HAP1 hybrid double strand and the HAP2-AgNCs in a buffer solution, measuring the fluorescence intensity, adding a UDG standard solution or a solution to be measured with a series of concentrations, reacting for 2 hours at 37 ℃, and detecting the fluorescence intensity;
(3) and (3) making a standard curve according to the fluorescence intensity of the UDG standard solution with the series of concentrations, calculating a regression equation, and calculating the content of the contained UDG according to the fluorescence intensity of the object to be detected.
The concentration of the ExoIII is 1-20U/mu L, preferably 1-10U/mu L;
the concentration of the S-HAP1 hybrid chain is 0.01-5 mu M;
the concentration of the UDG template is 50-100 nM.
The fluorescence detection conditions are as follows: the excitation wavelength is 560nm, the emission wavelength is 625nm, and the detection wavelength band is 575-750 nm.
The working principle of the biosensor is as follows:
the S strand, base-complementary paired with part of the HAP1, can hybridize to double-stranded S-HAP1, the UDG template is cleaved into 2 pieces at the "U" bases in the presence of the target UDG and ExoIII, yielding the Trigger sequence (5'-GCAAGAGTG ACATCATAGAC AAAAA-3'). At the moment, the 5 'end of the Trigger is hybridized with the 3' end of HAP1 in S-HAP1 hybridized in advance, so that the 3 'end of HAP1 is a flat end, in the presence of ExoIII, ExoIII cuts a HAP1 chain from the 3' end, and finally the S chain and the Trigger chain are released from a double-stranded system, and in addition, the 5 'end part base of the HAP1 is left, 9 bases in the part are identical to 9 bases in the 5' end of the Trigger chain, so that the part is equivalent to a secondary Trigger, and the circulation amplification of the Trigger chain is realized. The 5 'end 12 bases of HAP2 is a silver-binding cluster sequence, and the 3' end comprises a G-rich sequence. When the HAP2 is a hairpin structure, silver clusters are synthesized in advance by HAP2, the 5' end of HAP2 is silver clusters, the 5' end silver clusters are close to a G-rich sequence at the 3 ' end, so strong fluorescence is generated, the 5' end of an S chain generated in the system is combined with the 3 ' end of HAP2 so as to open HAP2, the fluorescence intensity of the HAP2 is suddenly reduced after the HAP2 is opened by the S chain, meanwhile, the 3 ' end of HAP2 is a flat end due to the complementary pairing of the S chain and HAP2, and in the presence of ExoIII, ExoIII is cut from the G-rich sequence at the 3 ' end of hairpin 2, so that the S chain is released again and released into the system to perform a new hybridization reaction with HAP2, and the amplification of signals is realized. The content of UDG contained in the sample is determined by measuring the change in fluorescence intensity before and after the addition of the sample.
The invention has the following advantages:
the biosensor has the advantages of good specificity, high sensitivity, mild reaction conditions and high reaction speed; the silver cluster fluorescence detection is adopted, so that the operation is simple and convenient, the detection period is short, and the carrying is easy; the process cost is low, and the method is suitable for the requirement of low price in industrialization; the preparation method is simple, stable in performance and good in repeatability, and is suitable for detecting the UDG in the field of medical treatment and health.
Drawings
FIG. 1 is a schematic diagram of the biosensor;
FIG. 2 is a graph showing the change of fluorescence intensity ratio with the concentration of ExoIII;
FIG. 3 shows the fluorescence intensity as a function of the concentration of S-HAP1 hybrid duplex;
FIG. 4 shows the variation of fluorescence intensity with concentration of UDG template;
FIG. 5 shows the change in fluorescence intensity with UDG concentration;
FIG. 6 is a standard curve for detecting UDG.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 preparation of HAP 2-AgNCs.
Preparing a PB buffer solution (the concentration is 20 mM), wherein the PB buffer solution is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, 0.7163g of disodium hydrogen phosphate and 0.3120g of sodium dihydrogen phosphate are respectively weighed to prepare 100ml of solutions, then, a part of disodium hydrogen phosphate and a part of sodium dihydrogen phosphate are mixed, and the pH value of the mixed solution is adjusted to 6.5 for later use.
Preparation of AgNO3Concentration of 2mM, volume of 1mL, AgNO3It is prepared at present and stored in dark place.
Preparation of NaHPO4Concentration of 2mM, volume of 1mL, NaHBO4The preparation is carried out on site and is carried out with ice water at 0 ℃.
A1 mL centrifuge tube was charged with 76. mu.L PB (20 mM), 15. mu.L HAP2 (100. mu.M), and 4.5. mu.L AgNO3(2 mM), shaking for 1min, and placing in a refrigerator at 4 ℃ for 30 min; then adding 4.5 mu L of NaHPO4(2 mM) in the reaction system, shaking for 1min, and placing in a refrigerator at 4 ℃ for more than 4h to obtain HAP2-AgNCs solution.
And (3) putting 30 mu L of prepared HAP2-AgNCs into a centrifuge tube, adding 120 mu L of ultrapure water, uniformly mixing, putting 150 mu L of solution into a microcuvette by using a pipette, scanning the microcuvette by using a fluorescence analyzer, exciting light at 560nm, and detecting that the emission peak is 625nm, wherein HAP2-AgNCs exist in the solution.
Example 2 fluorescence intensity as a function of ExoIII concentration.
Mixing 2 μ L S chain (100 μ M), 2 μ L HAP1 (100 μ M) and 2 μ L NEBuffer2.1, reacting at 37 deg.C for 2h to obtain S-HAP1 hybrid double chain;
mu.L of UDG template (1. mu.M), 3. mu.L of ExoIII (1U/. mu.L, 5U/. mu.L, 10U/. mu.L, 15U/. mu.L, 20U/. mu.L), 4. mu.L of NEBuffer2.1, 2. mu.L of S-HAP1 hybrid duplex (5. mu.M), 8. mu.L of HAP2-AgNCs (15. mu.M) and ultrapure water (21. mu.L) were mixed well and the fluorescence intensity was measured, and then 1. mu.L of UDG (50U/mL) was added and reacted at 37 ℃ for 2 hours to measure the fluorescence intensity.
The results are shown in FIG. 2, wherein "-S" represents the fluorescence intensity when there is no free S chain in the system, i.e., the fluorescence intensity when UDG is not added; "+ S" represents the fluorescence intensity in the presence of free S strands in the system, i.e.after addition of UDG, and the number in the bar graph is the fluorescence value after addition/before addition. As can be seen from the figure, the intensity of the detected fluorescence signal gradually decreased with the concentration of ExoIII in the interval of 1-20U/. mu.L, and the ratio of the fluorescence intensity was the smallest when the concentration of ExoIII was 10U/. mu.L in the reaction system.
Example 3 fluorescence intensity as a function of the concentration of S-HAP1 hybridized duplex.
Mixing 2 μ L S chain (100 μ M), 2 μ L HAP1 (100 μ M) and 2 μ L NEBuffer2.1, reacting at 37 deg.C for 2h to obtain S-HAP1 hybrid double chain;
mu.L of UDG template (1. mu.M), 3. mu.L of ExoIII (10U/. mu.L), 4. mu.L of NEBuffer2.1, 2. mu.L of S-HAP1 hybrid duplex (0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M), 8. mu.L of HAP2-AgNCs (15. mu.M) and ultrapure water (21. mu.L) were mixed well and the fluorescence intensity was measured, and then 1. mu.L of UDG (50U/mL) was added and reacted at 37 ℃ for 2 hours to measure the fluorescence intensity.
As a result, as shown in FIG. 3, the intensity of the detected fluorescence signal gradually decreased with the concentration of the S-HAP1 hybrid duplex in the range of 0.01 to 5. mu.M, and the fluorescence intensity was large when the concentration of the S-HAP1 hybrid duplex in the reaction system was 5. mu.M.
Example 4 fluorescence intensity as a function of UDG template concentration.
Mixing 2 μ L S chain (100 μ M), 2 μ L HAP1 (100 μ M) and 2 μ L NEBuffer2.1, reacting at 37 deg.C for 2h to obtain S-HAP1 hybrid double chain;
mu.L of UDG template (50 nM, 100nM, 500nM, 1. mu.M), 3. mu.L of ExoIII (10U/. mu.L), 4. mu.L of NEBuffer2.1, 2. mu.L of S-HAP1 hybrid duplex (5. mu.M), 8. mu.L of HAP2-AgNCs (15. mu.M) and ultrapure water (21. mu.L) were mixed well, the fluorescence intensity was measured, and then 1. mu.L of UDG (50U/mL) was added and reacted at 37 ℃ for 2h, and the fluorescence intensity was measured.
As a result, as shown in FIG. 4, the intensity of the detected fluorescence signal gradually decreased with the concentration of the UDG template in the range of 50nM to 1. mu.M, and the fluorescence intensity was maximized when the concentration of the UDG template in the reaction system was 1. mu.M.
Example 5 detection of UDG.
Mixing 2 μ L S chain (100 μ M), 2 μ L HAP1 (100 μ M) and 2 μ L NEBuffer2.1, reacting at 37 deg.C for 2h to obtain S-HAP1 hybrid double chain;
mu.L of UDG template (1. mu.M), 3. mu.L of ExoIII (10U/. mu.L), 4. mu.L of NEBuffer2.1, 2. mu.L of S-HAP1 hybrid duplex (5. mu.M), 8. mu.L of HAP2-AgNCs (15. mu.M) and ultrapure water (21. mu.L) were mixed well, and the fluorescence intensity was measured, followed by addition of 1. mu.L of UDG (0.0005U/mL, 0.005U/mL, 0.05U/mL, 0.5U/mL, 5U/mL, 50U/mL) or a test solution, reaction at 37 ℃ for 2 hours and detection of the fluorescence intensity.
As shown in FIG. 5, the fluorescence signal intensity gradually decreased with the UDG concentration in the range of 0.0005U/ml to 50U/ml; making a standard curve according to the fluorescence intensity of the UDG standard solution with the series of concentrations, as shown in FIG. 6; the regression equation was calculated as Y = -161.4logC +436.92 with a correlation coefficient of 0.998.
<110> university of Jinan
<120> biosensor for detecting uracil glycosylase (UDG) and application thereof
<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 biosensor for detecting uracil glycosylase, which is characterized by comprising a UDG template, S-HAP1 hybrid double chains, HAP2-AgNCs and ExoIII enzyme;
the sequence of the UDG template is shown as SEQ number 1;
the sequence of the S chain is shown as SEQ number 4;
the sequence of the HAP1 is shown as SEQ number 2;
the sequence of the HAP2 is shown as SEQ number 3.
2. The biosensor according to claim 1, wherein the S-HAP1 hybrid duplex is obtained by the following preparation method: the buffer solution, the S chain solution and the HAP1 solution are mixed evenly and reacted for 2 hours at the constant temperature of 37 ℃.
3. The biosensor according to claim 1, wherein HAP2-AgNCs are obtained by the following preparation method: mixing buffer solution, HAP2 solution, AgNO3Mixing, and standing at 4 deg.C for 15-30 min; then adding cold NaHCO4Standing the solution at 4 deg.C for more than 4 hr.
4. Biosensor according to claim 3, wherein the HAP2, AgNO3With NaHCO4In a molar ratio of 1:6: 6.
5. A method for detecting uracil glycosylase using the biosensor according to claim 1, comprising the steps of:
(1) synthesizing HAP 2-AgNCs;
(2) hybridizing the S chain and HAP1 into an S-HAP1 hybrid double chain;
(3) uniformly mixing a UDG template, an ExoIII, an S-HAP1 hybrid double strand and HAP2-AgNCs in a buffer solution, measuring the fluorescence intensity, adding a uracil glycosylase standard solution or a solution to be measured with a series of concentrations, reacting for 2 hours at 37 ℃, and detecting the fluorescence intensity;
(4) and (3) making a standard curve according to the fluorescence intensity of the uracil glycosylase standard solution with the series of concentrations, calculating a regression equation, and calculating the content of the uracil glycosylase according to the fluorescence intensity of the substance to be detected.
6. The method of claim 5, wherein the ExoIII concentration is 1-20U/μ L; the concentration of the S-HAP1 hybrid double strand is 0.01-5 mu M; the concentration of the UDG template is 50-100 nM.
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| CN109459423B (en) * | 2018-10-22 | 2021-06-15 | 济南大学 | Biosensor for detecting uracil glycosidase (UDG) activity and preparation method thereof |
| CN109444105B (en) * | 2018-12-28 | 2021-03-30 | 济南大学 | Fluorescent biosensor for detecting DNA glycosylase UDG and preparation method thereof |
| CN109752362B (en) * | 2019-01-10 | 2021-06-15 | 济南大学 | Biosensor for detecting uracil-DNA glycosylase and preparation method thereof |
| 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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| CN105506078B (en) * | 2015-12-18 | 2019-07-09 | 山东大学 | One kind is for being measured in parallel uracil-DNA glycosylase and the active method and its application of restriction endonuclease IV and kit |
| CN106929563B (en) * | 2017-02-24 | 2018-10-12 | 山东师范大学 | The two active methods of step series signals amplification detection UDG mediated by enzyme are repaired based on excision |
| CN106995840B (en) * | 2017-03-20 | 2020-05-05 | 山东师范大学 | Method for detecting activity of thymine DNA glycosylase based on double-signal amplification strategy mediated by cyclic enzyme repair |
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