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

Abstract

The invention provides a biosensor for detecting uracil glycosylase, which includes UDG template, S-HAP1 hybrid chain, HAP2-AgNCs and ExoIII enzyme, and can use fluorescence detection to measure the activity of uracil glycosylase. Fluorescence detection The excitation wavelength is 560nm, the emission wavelength is 625nm, and the detection wavelength is 575-750nm. The biosensor of the present invention has good specificity, high sensitivity, mild reaction conditions, and fast reaction speed; it adopts silver cluster fluorescence detection, which is easy to operate, short detection cycle, and easy to carry; the process cost is low, and it is suitable for low-cost requirements in industrialization; The preparation method is simple, the performance is stable and the repeatability is good, and the method is suitable for detecting UDG in the medical and health field.

Description

A biosensor for detecting uracil glycosylase (UDG) and its application

technical field

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

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.

Currently reported detection methods for UDG include radioimmunoassay, chemiluminescence assay, chemiluminescence enzyme immunoassay, electrochemiluminescence immunoassay, etc. These methods often have the disadvantages of expensive instruments, complicated operation, high price, low sensitivity, and The human body has problems such as radioactivity. Therefore, there is an urgent need to establish a rapid, accurate, sensitive and highly specific detection method to detect uracil glycosylase.

Contents of the invention

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.

In order to achieve the above object, the present invention adopts the following technical solutions.

A biosensor for detecting uracil glycosylase (UDG), including UDG template, S-HAP1 hybrid chain, HAP2-AgNCs (silver cluster containing hairpin probe 2) and ExoIII enzyme (exonuclease III);

The sequence of the UDG template is shown in SEQ No.1;

The sequence of the S chain is shown in SEQ No. 2;

The sequence of the HAP1 is shown in SEQ No. 3;

The sequence of HAP2 is shown in SEQ No. 4.

The S-HAP hybrid chain is obtained by the following preparation method: uniformly mix buffer solution, S chain solution, and HAP1 solution, and react at a constant temperature of 37° C. for 2 hours.

The HAP2-AgNCs are obtained by the following preparation method: mix the buffer solution, HAP2 solution, and AgNO ₃ and place it at 4°C for 15-30 minutes; then add cold NaHBO ₄ solution and let it stand at 4°C for more than 4 hours.

The molar ratio of HAP2, _AgNO3 and _NaHBO4 is 1:6:6.

A method for utilizing the above-mentioned biosensor to detect UDG, comprising the following steps:

(1) Hybridize the S strand and HAP1 to form a S-HAP1 hybrid double strand;

(2) Mix the UDG template, ExoIII, S-HAP1 hybrid double strands, and HAP2-AgNCs in the buffer to measure the fluorescence intensity, then add UDG or the test solution, react at 37°C for 2 hours, and measure the fluorescence intensity;

(3) Make a standard curve according to the fluorescence intensity of the serial concentration UDG standard solution, calculate the regression equation, and calculate the content of UDG according to the fluorescence intensity of the analyte.

The ExoIII concentration is 1-20 U/μL, preferably 1-10 U/μL;

The S-HAP1 hybrid duplex concentration is 0.01-5 μM;

The UDG template concentration is 50-100 nM.

The fluorescence detection conditions are as follows: the excitation wavelength is 560nm, the emission wavelength is 625nm, and the detection waveband is 575-750nm.

The working principle of this biosensor is as follows:

The base pairing between the S chain and HAP1 can be hybridized into a double-stranded S-HAP1. The UDG template is cut into two segments at the "U" base in the presence of the target UDG and ExoIII to generate a Trigger sequence (5 '-GCAAGAGTG ACATCATAGACAAAAA-3'). At this time, the 5' end of the Trigger will hybridize with the 3' end of HAP1 in the pre-hybridized S-HAP1 so that the 3' end of HAP1 is blunt. In the presence of ExoIII, ExoIII will cut the HAP1 chain from the 3' end, Finally, both the S chain and the Trigger chain are released from the double-stranded system, and some bases at the 5' end of HAP1 are left, and 9 bases in this part are the same as the 9 bases at the 5' end of the Trigger chain Therefore, this part is equivalent to a secondary Trigger, thus realizing the cyclic amplification of the Trigger chain. The 12 bases at the 5' end of HAP2 are the sequence of the silver cluster, and the 3' end includes the G-rich sequence. When HAP2 has a hairpin structure, the silver clusters of HAP2 are synthesized in advance. Since the 5' end of HAP2 is a silver cluster, the silver cluster at the 5' end is close to the G-rich sequence at the 3' end, thereby generating strong fluorescence. The 5' end of the S chain generated in the system will bind to the 3' end of HAP2 to open HAP2. After the S chain opens HAP2, its fluorescence intensity will drop sharply. At the same time, due to the complementary pairing of the S chain and HAP2, the HAP2 The 3' end of the hairpin is a blunt end. In the presence of ExoIII, ExoIII will start to cut the G-rich sequence at the 3' end of hairpin 2, so that the S chain will be released again and released into the system for new and HAP2 The hybridization reaction achieves signal amplification. The content of UDG is determined by measuring the change of fluorescence intensity before and after adding the test substance.

The present invention has the following advantages:

The biosensor of the present invention has good specificity, high sensitivity, mild reaction conditions, and fast reaction speed; adopts silver cluster fluorescence detection, is easy to operate, short detection cycle, easy to carry; low process cost, and is suitable for low-cost requirements in industrialization; The preparation method is simple, the performance is stable, and the repeatability is good, and it is suitable for detecting UDG in the medical and health field.

Description of drawings

Fig. 1 is the working principle diagram of this biosensor;

Fig. 2 is the change figure of fluorescence intensity ratio with ExoIII concentration;

Fig. 3 is the variation of fluorescence intensity with the concentration of S-HAP1 hybrid duplex;

Fig. 4 is the change of fluorescence intensity with the concentration of UDG template;

Fig. 5 is the variation of fluorescence intensity with UDG concentration;

Figure 6 is a standard curve for detecting UDG.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited by the following embodiments.

Example 1 Preparation of HAP2-AgNCs.

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.

Prepare AgNO ₃ with a concentration of 2mM and a volume of 1mL. AgNO ₃ is ready-to-use and stored in the dark.

Prepare NaHBO ₄ with a concentration of 2mM and a volume of 1mL. NaHBO ₄ is ready-to-use and prepared with ice water at 0°C.

Take a 1mL centrifuge tube, add 76μL of PB (20mM), add 15μL of HAP2 (100μM), add 4.5μL of AgNO ₃ (2mM), shake for 1min, put in a 4°C refrigerator for 30min; then add 4.5μL of NaHBO ₄ (2mM) In the reaction system, shake for 1 min, and place in a refrigerator at 4°C for more than 4 h to obtain a HAP2-AgNCs solution.

Take 30 μL of the prepared HAP2-AgNCs in a centrifuge tube, add 120 μL of ultrapure water and mix well, take 150 μL of the solution with a pipette gun into a micro cuvette, scan it with a fluorescence analyzer, the excitation light is 560nm, and measure Its emission peak is at 625nm, indicating that there are HAP2-AgNCs in the solution.

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

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;

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;

The results are shown in Figure 2, where "-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 presence of free S chain in the system The fluorescence intensity of , that is, the fluorescence intensity after adding UDG, and the numbers in the histogram are the fluorescence value after addition/fluorescence value before addition. It can be seen from the figure that the detected fluorescence signal intensity gradually decreases with the concentration of ExoIII in the range of 1-20U/μL. When the concentration of ExoIII is 10U/μL in the reaction system, the fluorescence intensity ratio is the smallest.

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

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;

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.

The results are shown in Figure 3. The detected fluorescence signal intensity gradually decreases with the concentration of S-HAP1 hybrid duplex in the range of 0.01-5 μM. .

Example 4 Fluorescence intensity changes with UDG template concentration.

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;

Mix 2 μL of UDG template (50 nM, 100 nM, 500 nM, 1 μM), 3 μL of ExoIII (10 U/μL), 4 μL of NEBuffer2.1, 2 μL of S-HAP1 hybrid duplex (5 μM), 8 μL of HAP2-AgNCs (15 μM) and Mix ultrapure water (21 μL) and measure the fluorescence intensity, then add 1 μL of UDG (50 U/mL), react at 37°C for 2 hours, and measure the fluorescence intensity.

The results are shown in Figure 4. The detected fluorescence signal intensity gradually decreases with the concentration of UDG template in the range of 50nM-1μM. When the concentration of UDG template in the reaction system is 1μM, the fluorescence intensity value is the largest.

Example 5 Detection of UDG.

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;

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 duplex (5 μM), 8 μL of HAP2-AgNCs (15 μM) and ultrapure water (21 μL) Evenly, measure the fluorescence intensity, then add 1 μL of UDG (0.0005U/mL, 0.005U/mL, 0.05U/mL, 0.5U/mL, 5U/mL, 50U/mL) or the solution to be tested, and react at 37°C 2h, detect the fluorescence intensity.

The detection result is shown in Figure 5, and the fluorescence signal intensity gradually decreases with the UDG concentration in the interval of 0.0005U/ml-50U/ml; the standard curve is made according to the fluorescence intensity of the serial concentration UDG standard solution, as shown in Figure 6; the calculated The regression equation is Y= -161.4logC+436.92, and the correlation coefficient is 0.998.

<110> Jinan University

<120> A biosensor for detecting uracil glycosylase (UDG) 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 ccccaccccgc 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.