CN108398406B - A biosensor for detecting uracil glycosylase (UDG) and its application - Google Patents

Abstract

The present invention provides a biosensor for detecting uracil glycosylase, including UDG template, S-HAP1 hybrid chain, HAP2-AgNCs and ExoIII enzyme, the activity of uracil glycosylase can be determined by fluorescence detection, and fluorescence detection The excitation wavelength is 560nm, the emission wavelength is 625nm, and the detection band is 575‑750nm. The biosensor of the invention has good specificity, high sensitivity, mild reaction conditions and fast reaction speed; adopts silver cluster fluorescence detection, which is easy to operate, has a short detection period and is easy to carry; the process cost is low, and it is suitable for the requirement of low price in industrialization; The preparation method is simple, the performance is stable and the repeatability is good, and the invention is suitable for the detection of 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 cyclic amplification and changes in the fluorescence intensity of silver clusters.

Background technique

UDG (uracil glycosylase) is the primary DNA excision repair enzyme responsible for the removal of uracil when base mismatches occur in various mammals. It mainly cuts off the N-glycosidic bond between the wrongly inserted uracil (U) and the sugar group in DNA, removes U, and generates an abasic site (AP site), which is then processed by AP endonucleases (AP endonucleases1). , APE1) cuts the DNA single strand, and finally is recognized and repaired by DNA polymerase and DNA ligase to complete the repair of mismatched DNA.

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

SUMMARY OF THE INVENTION

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

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

A biosensor for the detection of uracil glycosylase (UDG), including UDG template, S-HAP1 hybrid duplex, HAP2-AgNCs (silver clusters 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. 4;

The sequence of the HAP1 is shown in SEQ No. 2;

The sequence of the HAP2 is shown in SEQ No. 3.

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

The HAP2-AgNCs are obtained by the following preparation method: mixing buffer, HAP2 solution, and AgNO3, and placing at ₄ °C for 15-30 min; then adding cold NaHBO4 solution and standing at ₄ °C for more than 4 hours.

The molar ratio of the HAP2, AgNO ₃ and NaHBO ₄ is 1:6:6.

A method for detecting UDG using the above-mentioned biosensor, comprising the following steps:

(1) Hybridize S-chain and HAP1 into S-HAP1 hybrid duplex;

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

(3) Make a standard curve according to the fluorescence intensity of the UDG standard solution with a series of concentrations, calculate the regression equation, and calculate the content of UDG contained according to the fluorescence intensity of the analyte.

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

The concentration of the S-HAP1 hybrid chain is 0.01-5 μM;

The UDG template concentration was 50-100 nM.

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

The working principle of this biosensor is as follows:

The S chain is complementary to the HAP1 part, which 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, resulting in a Trigger sequence (5 '-GCAAGAGTGACATCATAGAC AAAAA-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 a blunt end. 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 the bases at the 5' end of HAP1 are left, and the 9 bases in this part are the same as the 9 bases at the 5' end of the Trigger chain. , so this part is equivalent to a secondary Trigger, thus realizing the circular 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 is a hairpin structure, HAP2 is synthesized into a silver cluster in advance. Since the 5' end of HAP2 is a silver cluster, the silver cluster at the 5' end and the G-rich sequence at the 3' end are close to each other, resulting in strong fluorescence. The 5' end of the S chain generated in the system will combine with 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, HAP2 The 3' end of the hairpin is blunt-ended. In the presence of ExoIII, ExoIII will cut from the G-rich sequence at the 3' end of hairpin 2, so that the S chain is freed again and released into the system for new and HAP2 The hybridization reaction achieves signal amplification. The content of UDG contained was determined by measuring the change of fluorescence intensity before and after adding the analyte.

The present invention has the following advantages:

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

Description of drawings

Figure 1 is a schematic diagram of the working principle of the biosensor;

Fig. 2 is a graph showing the change of fluorescence intensity ratio with ExoIII concentration;

Figure 3 is the change of fluorescence intensity with the concentration of S-HAP1 hybridized duplex;

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

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

Figure 6 is a standard curve for detecting UDG.

Detailed ways

The present invention will be further described below with reference to the embodiments and the 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 of disodium hydrogen phosphate and 0.3120g of sodium dihydrogen phosphate, respectively, to make 100ml of solution. , and then mix a part of disodium hydrogen phosphate with a part of sodium dihydrogen phosphate, adjust the pH value of the mixed solution to 6.5 and then use it for later use.

Prepare AgNO ₃ with a concentration of 2mM and a volume of 1mL. AgNO ₃ is prepared for immediate use and stored in the dark.

The concentration of NaHBO ₄ is 2mM and the volume is 1mL. The NaHBO ₄ is prepared as it is, and prepared with ice water at 0°C.

Take a 1 mL centrifuge tube, add 76 μL PB (20 mM), add 15 μL HAP2 (100 μM), add 4.5 μL AgNO ₃ (2 mM), shake for 1 min, and place in a 4°C refrigerator for 30 min; then add 4.5 μL NaHBO ₄ (2 mM) 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, use a pipette to take 150 μL of the solution into a microcuvette, scan it with a fluorescence analyzer, and measure the excitation light at 560 nm. Its emission peak is at 625 nm, indicating that there are HAP2-AgNCs in solution.

Example 2 Variation of fluorescence intensity with ExoIII concentration.

Mix 2 μL of S chain (100 μM), 2 μL of HAP1 (100 μM), and 2 μL of NEBuffer 2.1 evenly, and let them react at 37°C for 2 h to obtain S-HAP1 hybridized duplex for use;

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 hybridization duplex (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, and the reaction was carried out at 37 °C for 2 h to detect the fluorescence intensity.

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 when there is free S chain in the system The fluorescence intensity of , that is, the fluorescence intensity after adding UDG, the number in the bar graph is the fluorescence value after adding/the fluorescence value before adding. It can be seen from the figure that the detected fluorescence signal intensity gradually decreased with the concentration of ExoIII in the range of 1-20U/μL. When the concentration of ExoIII in the reaction system was 10U/μL, the fluorescence intensity ratio was the smallest.

Example 3 Variation of fluorescence intensity with the concentration of S-HAP1 hybridized duplex.

Mix 2 μL of S chain (100 μM), 2 μL of HAP1 (100 μM), and 2 μL of NEBuffer 2.1 evenly, and let them react at 37°C for 2 h to obtain S-HAP1 hybridized duplex for use;

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 hybridization duplex (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) and mixed well to measure the fluorescence intensity, then add 1 μL of UDG (50 U/mL), react at 37°C for 2 h, and measure the fluorescence intensity.

The results are shown in Figure 3. The detected fluorescence signal intensity gradually decreased 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 was 5 μM, the fluorescence intensity value was large. .

Example 4 Variation of fluorescence intensity with UDG template concentration.

Mix 2 μL of S chain (100 μM), 2 μL of HAP1 (100 μM), and 2 μL of NEBuffer 2.1 evenly, and let them react at 37°C for 2 h to obtain S-HAP1 hybridized duplex for use;

2 μL of UDG template (50 nM, 100 nM, 500 nM, 1 μM), 3 μL of ExoIII (10 U/μL), 4 μL of NEBuffer 2.1, 2 μL of S-HAP1 hybridization duplex (5 μM), 8 μL of HAP2-AgNCs (15 μM) and ultra Pure water (21 μL) was mixed, and the fluorescence intensity was measured, then 1 μL of UDG (50 U/mL) was added, and the reaction was carried out at 37 °C for 2 h to measure the fluorescence intensity.

The results are shown in Figure 4. The detected fluorescence signal intensity gradually decreased with the concentration of UDG template in the range of 50nM-1μM. When the concentration of UDG template in the reaction system was 1μM, the fluorescence intensity value was 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 NEBuffer 2.1 evenly, and let them react at 37°C for 2 h to obtain S-HAP1 hybridized duplex 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). uniform, 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, the fluorescence intensity was detected.

The detection results are shown in Figure 5. The fluorescence signal intensity gradually decreases with the concentration of UDG in the range of 0.0005U/ml- 50U/ml; the standard curve is made according to the fluorescence intensity of the UDG standard solution of a series of concentrations, as shown in Figure 6; 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

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Claims (6)

1. a biosensor for detecting uracil glycosylase, is characterized in that, comprises UDG template, S-HAP1 hybridization duplex, HAP2-AgNCs and ExoIII enzyme; The sequence of the UDG template is shown in SEQ No. 1; The sequence of the S chain is shown in SEQ No. 4; The sequence of the HAP1 is shown in SEQ No. 2; The sequence of the HAP2 is shown in SEQ No. 3. 2 . The biosensor according to claim 1 , wherein the S-HAP1 hybrid duplex is obtained by the following preparation method: mixing the buffer solution, the S chain solution and the HAP1 solution evenly, and reacting at a constant temperature of 37° C. for 2 hours. 3 . 3. The biosensor according to claim 1, wherein the HAP2-AgNCs are obtained by the following preparation method: the buffer solution, the solution of HAP2 and AgNO are mixed and placed at ₄ °C for 15-30min; then cold NaHBO is added ₄ Solution, stand at 4°C for more than 4h, and then get it. _4. The biosensor according to claim ₃ , wherein the molar ratio of HAP2, AgNO3 and NaHBO4 is 1:6:6. 5. a method utilizing the biosensor as claimed in claim 1 to detect uracil glycosylase, is characterized in that, comprises the following steps: (1) Synthesis of HAP2-AgNCs; (2) Hybridize the S-chain and HAP1 into an S-HAP1 hybrid double-strand; (3) Mix the UDG template, ExoIII, S-HAP1 hybrid double-stranded, HAP2-AgNCs in the buffer, measure the fluorescence intensity, then add a series of concentrations of uracil glycosylase standard solution or test solution, at 37 ℃ The reaction was carried out for 2h, and the fluorescence intensity was detected; (4) Make a standard curve according to the fluorescence intensity of the standard solution of uracil glycosylase with a series of concentrations, calculate the regression equation, and calculate the content of uracil glycosylase contained according to the fluorescence intensity of the analyte. 6. The method according to claim 5, wherein the ExoIII concentration is 1-20U/μL; the S-HAP1 hybrid duplex concentration is 0.01-5 μM; the UDG template concentration is 50-100 nM.

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