阅读说明：本技术 一种检测尿嘧啶糖基化酶的化学发光生物传感器及其制备方法与应用 (Chemiluminescence biosensor for detecting uracil glycosylase, and preparation method and application thereof ) 是由 王玉 张雪 刘素 黄加栋 宋晓蕾 李莎莎 王敬锋 王海旺 孙文玉 王业茹 于 2019-09-20 设计创作，主要内容包括：本发明涉及生物传感器技术领域,特别涉及基于三通路结构驱动链置换反应及DNA walker技术驱动球形核酸酶的化学发光技术检测尿嘧啶糖基化酶。为了解决以上现有技术中检测尿嘧啶糖基化酶的方法存在操作复杂、灵敏度比较低、成本高的问题,一种基于三通路结构和DNA walker两种纳米技术的生物传感器利用球形核酸酶催化鲁米诺发生化学发光反应进行检测。制备方法：纳米金的制备；球形核酸的制备；均相中形成球形核酸酶用于催化鲁米诺的化学发光反应。利用了尿嘧啶糖基化酶对U碱基的特异性识别和切除,实现目标的特异性检测；同时采用DNA walker纳米技术实现目标的快速、高灵敏性检测。(The invention relates to the technical field of biosensors, in particular to a method for detecting uracil glycosylase by using a chemiluminescence technology based on a three-way structure driving strand displacement reaction and a DNA walker technology driving spherical nuclease. In order to solve the problems of complex operation, low sensitivity and high cost of the method for detecting uracil glycosylase in the prior art, the biosensor based on the three-way structure and the DNA walker nanotechnology is used for detecting uracil glycosylase by utilizing the spherical nuclease to catalyze luminol to generate chemiluminescence reaction. The preparation method comprises the following steps: preparing nano gold; preparing spherical nucleic acid; the formation of spherical nucleases in homogeneous phase is used to catalyze the chemiluminescent reaction of luminol. The specific recognition and excision of the U basic group by uracil glycosylase are utilized to realize the specific detection of the target; meanwhile, the DNA walker nanotechnology is adopted to realize the rapid and high-sensitivity detection of the target.)

1. A chemiluminescence biosensor for detecting uracil glycosylase is characterized by comprising the following raw materials: SA chain, UP probe, BS chain, 1 XCutsmart buffer solution, endonuclease IV, spherical nucleic acid SNA marked with G-HP and linker chain, uracil glycosylase UDG, heme, luminol and hydrogen peroxide;

the UP base series is shown as SEQ No. 1; the middle of the fourteenth base and the fifteenth base at the 5' end in the UP sequence is a uracil base U;

the SA base series is shown as SEQ No. 2;

the BS base series is shown as SEQ No. 3;

the linker base series is shown as SEQ No. 4;

the G-HP base series is shown as SEQ No. 5; and the eighteenth base at the 5' end in the G-HP sequence is a tetrahydrofuran abasic site.

2. The chemiluminescent biosensor for detecting uracil glycosylase of claim 1, wherein the G-HP and linker chain labeled spherical nucleic acid is V_G-HP:VlinkerAnd (4) adding a PB buffer solution and a PBS buffer solution into the mixture of the =20:1 and the nanogold, wherein the concentration of NaCl in the final solution is 0.3M.

3. The method of preparing a chemiluminescent biosensor of claim 1 comprising the steps of:

(1) preparing nano gold;

(2) preparing marked G-HP and linker chain spherical nucleic acid SNA;

(3) and (3) carrying out chemiluminescence reaction.

4. The preparation method according to claim 3, wherein the nano-gold in the step (1) is prepared by a process of reducing chloroauric acid by sodium citrate, the size of the nano-gold is 20nm, and the molar extinction coefficient is 0.878X 109M^-1·cm^-1。

5. The method according to claim 3, wherein the method for preparing the labeled G-HP and linker chain spherical nucleic acid SNA of step (2) comprises the following steps:

s1, adjusting the concentration of the nano-gold solution to 5 nM;

s2 the G-HP and the linker chain are arranged according to V_G-HP:VlinkerAdding the solution with a ratio of 20:1 into the nanogold solution obtained in the step S1, adding a PB buffer solution, adding PBS in batches, and adjusting the concentration of NaCl in the solution to be 0.3M;

s3 centrifuging to remove the DNA chain not marked;

s4 the precipitate after centrifugation is redissolved in ultrapure water and kept at 4 ℃ for later use.

6. The method according to claim 3, wherein the step (3) comprises the steps of:

j1 adding the SA chain, the UP probe and the BS chain into a 1 XCutsmart buffer solution to form a three-channel system;

j2 adding endonuclease IV, spherical nucleic acid marked with G-HP and linker chain, and uracil glycosylase UDG into J1 three-way system;

j3 adding heme and luminol into J2 to form G-quadruplex spherical nuclease;

j4 addition of hydrogen peroxide to J3 was immediately used for detection of chemiluminescent signal with a chemiluminescent signal acquisition time interval of 1.5s and a chemiluminescent spectral measurement range of 350nm to 550 nm.

7. The chemiluminescent biosensor of claim 1 is used for the early detection of uracil glycosylase.

Technical Field

The invention relates to the technical field of biosensors, in particular to a chemiluminescence biosensor for detecting uracil glycosylase based on a three-way structure and a DNA walker nanotechnology, a preparation method thereof and a spherical nuclease technology.

Background

The integrity of DNA is critical to maintaining the function of an organism, and when spontaneous damage occurs to DNA, a base excision repair mechanism (BER) is initiated to repair the DNA damage. The BER repair pathway is initiated primarily by DNA glycosylase, which forms AP sites by specifically recognizing and removing uracil U deaminated from DNA, cleaves the DNA strand under the action of the apurinic/pyrimidine endonuclease 1 (APE 1) or endonuclease iv (endo iv) to produce strand breaks, and completes the DNA strand repair by the subsequent co-action of DNA polymerase and ligase. Uracil glycosylase plays a very important role in maintaining the integrity of genes, and in addition, abnormal expression of the enzyme is related to the occurrence of various diseases, including immunodeficiency diseases, neurodegenerative diseases, cancers and the like. Therefore, early detection of uracil glycosylase plays an important role in the research of some biological processes and early diagnosis of diseases.

The uracil glycosylase detection technologies reported at present include enzyme-linked immunosorbent assay, colorimetric method, gel electrophoresis coupling radioactive labeling method and the like, and some of the methods have antibody functionalization, antibody enzyme labeling and other processes, so that the detection process is very complex, and in addition, the problems of high cost, poor detection sensitivity, poor reproduction and the like exist. Therefore, a platform with high sensitivity, simple operation, high efficiency and reliability needs to be constructed for detecting the uracil glycosylase.

Disclosure of Invention

Aiming at the problem that a method for efficiently, sensitively and conveniently detecting uracil glycosylase is lacked at present, the invention provides a method for detecting uracil glycosylase based on a three-way structure driving strand displacement reaction and a DNA walker technology driving spherical nuclease chemiluminescence technology, which mainly comprises the steps of constructing two nano structures of a three-way structure and spherical nucleic acid, specifically identifying and excising U base by utilizing uracil glycosylase, and realizing the change of the three-way structure and the driving of the DNA walker reaction with the assistance of endonuclease IV, so that the formed spherical nuclease is used for catalyzing luminol to react to generate a chemiluminescence signal, the detection sensitivity and the detection speed are obviously improved, and the detection of uracil glycosylase in an actual sample is facilitated.

The invention is obtained by the following steps:

in the invention, 5 DNA chains are used in total, and the sequences are respectively as follows:

UP：AGT CAG TAT GCA CUC GTG TTA AGC GTG SA：TCT TTC GGC CGC GTT CAC GAG TGC ATC CGC GCT TGG G(T)₅₀ G CTC GCC CAA GCG CGG BS：TTTCAC GCT TCG CGG CCG AAA GAT TT linker：SH-(T)₁₀CA AGC GCG GAT GCA C G-HP：SH-(T)₁₀GG GTA GGG CGG G TT GGG XGA GC T TTT CCC TAC。

in the UP strand, there is a uracil base U, and the italic base in the UP strand is complementary to the italic base in the SA strand, thereby blocking the toehold portion and the strand-migrating portion of the SA strand, and the bold base in the UP strand is used for complementary pairing with the bold base in the BS strand. In the hairpin SA strand, underlined bases are complementarily paired with underlined bases in the BS strand, bases in the italic bold portion of the SA strand and linker bold bases are complementarily paired, there are consecutive 50 thymine bases in the SA strand as a spacer for flexible control of the wobble arm, and italic underlined bases in the SA strand are complementarily paired with italic underlined bases in the G-HP, and a portion of the italic underlined bases is blocked at the stem end of the SA strand. In the G-HP chain, X represents the AP site, bold base is the sequence capable of forming G-quadruplex DNase, underlined base in the bold base is blocked at the stem end in hairpin G-HP, reducing background signal.

The system comprises two types of nanostructures: one is a three-way structure (TWJ) and the other is a spherical nucleic acid Structure (SNA) for performing the function of DNA walker. The Spherical Nucleic Acid (SNA) takes nanogold as a core, two strands are modified on the surface through sulfydryl, one strand is a DNA hairpin (G-HP) which comprises an AP site and can form G-quadruplex DNase in a closed mode, and the other strand is a linker strand which can be subjected to base complementation with a Swing Arm (SA) strand in a three-way structure. The three-way structure (TWJ) consists of a Swing Arm (SA) chain which plays a role of DNA walker, an UP probe chain which is specifically identified by a target substance uracil glycosylase and a base chain BS chain. The UP strand contains a uracil base U and contains a sequence complementary to the BS strand, the 3' end of the SA strand contains a sequence complementary to the base of the loop end portion of the G-HP hairpin, a spacer end containing 50 thymines (polyT 50), a sequence complementary to the linker strand and a sequence complementary to the BS strand. The SA chain is designed into a hairpin configuration, the 3' end of the SA chain and the complementary base pairs of the ring loop end in the G-HP hairpin have 10 pairs, 5 pairs of the 10 pairs are blocked at the stem end of the hairpin, the AP site is arranged in the middle, the hybridization temperature of the 10 pairs of the base pairs is 23.1 ℃, so the SA chain cannot exist stably in a 37 ℃ reaction system, and the DNA walker reaction cannot be carried out.

In the presence of the target uracil glycosylase, the target removes U bases in the UP probe to form an AP site, at the moment, under the assistance of endonuclease IV (endo IV), the UP probe is cut at the AP site to be broken and released from a three-channel system, a toehold end which is closed by the UP probe and is positioned in an SA chain is exposed, at the moment, linkerDNA in spherical nucleic acid is hybridized with the SA chain, and the toehold mediates the hybridization through the toeholdThe SA strand is opened by the strand displacement reaction of (1), a part for hybridizing with the G-HP is released, 10 pairs of bases of the SA and the G-HP are adjacent due to the hybridization of the linker sequence and the SA sequence, the local concentration is remarkably increased, unstable intermolecular hybridization is changed into stable intramolecular hybridization, and the hybridization temperature is 55.1 ℃. In this case, endonuclease IV acts on AP site to cut off G-HP at AP site, so that hairpin structure of G-HP is destroyed, stem end hybridization is not stable at this time, so that only sulfhydryl-bound chain of G-HP on the surface of nano gold remains, at this time, SA chain is released, and can be hybridized with next G-HP to make subsequent reaction until all G-HP on the surface of nano gold is cut off, so that all the originally blocked part capable of forming G-quadruplex is released to form G4-SNA structure, and K is added into the system⁺And heme, which can form a G-quadruplex spherical nuclease (G4-SNAzyme), and G4-SNAzyme can catalyze chemiluminescence of luminol in the presence of hydrogen peroxide. As the whole system starts the toehold mediated strand displacement reaction and the subsequent DNA walker reaction based on the existence of the uracil glycosylase, a G-quadruplex spherical nuclease structure is further formed, compared with the mode of directly forming the G-quadruplex DNA enzyme on the surface of spherical nucleic acid in advance, the method effectively reduces background signals and improves the signal to noise ratio of the reaction. In addition, the DNA walker nanotechnology and the spherical nuclease technology are adopted, so that the chemiluminescence signal is remarkably amplified. Therefore, a chemiluminescence biosensor with high efficiency, high sensitivity and high signal-to-noise ratio is designed, and an effective technical platform is provided for detection of uracil glycosylase and subsequent clinical research.

The detection of the uracil glycosylase is realized in a homogeneous solution, and the three-way mediated strand displacement reaction and the formation of DNA walker driven spherical nuclease are used for catalyzing luminol to generate a chemiluminescent signal, so that the high-sensitivity detection of the uracil glycosylase is realized, and a lower detection lower limit is obtained.

A chemiluminescence biosensor for detecting uracil glycosylase comprises the following raw materials: SA chain, UP probe, BS chain, 1 XCutsmart buffer solution, endonuclease IV, spherical nucleic acid SNA marked with G-HP and linker chain, uracil glycosylase UDG, heme, luminol and hydrogen peroxide;

the UP base series is shown as SEQ No. 1; the middle of the fourteenth base and the fifteenth base at the 5' end in the UP sequence is a uracil base U;

the SA base series is shown as SEQ No. 2; the BS base series is shown as SEQ No. 3; the linker base series is shown as SEQ No. 4; the G-HP base series is shown as SEQ No. 5; and the eighteenth base at the 5' end in the G-HP sequence is a tetrahydrofuran abasic site.

The spherical nucleic acid marked with G-HP and a linker chain is V_G-HP:VlinkerAnd (4) adding a PB buffer solution and a PBS buffer solution into the mixture of the =20:1 and the nanogold, wherein the concentration of NaCl in the final solution is 0.3M.

The preparation method of the chemiluminescence biosensor comprises the following steps:

(1) preparing nano gold;

(2) preparing marked G-HP and linker chain spherical nucleic acid SNA;

(3) carrying out chemiluminescence reaction;

the nano-gold in the step (1) is prepared by adopting a process of reducing chloroauric acid by sodium citrate, the size of the nano-gold is 20nm, and the molar extinction coefficient is 0.878 multiplied by 109M^-1·cm^-1。

The preparation method of the G-HP and linker chain spherical nucleic acid SNA marked in the step (2) comprises the following steps:

s1, adjusting the concentration of the nano-gold solution to 5 nM;

s2 the G-HP and the linker chain are arranged according to V_G-HP:VlinkerAdding the solution with a ratio of 20:1 into the nanogold solution obtained in the step S1, adding a PB buffer solution, adding PBS in batches, and adjusting the concentration of NaCl in the solution to be 0.3M;

s3 centrifuging to remove the DNA chain not marked;

s4 the precipitate after centrifugation is redissolved in ultrapure water and kept at 4 ℃ for later use.

The process of the step (3) is as follows:

j1 adding the SA chain, the UP probe and the BS chain into a 1 XCutsmart buffer solution to form a three-channel system;

j2 adding endonuclease IV, spherical nucleic acid marked with G-HP and linker chain, and uracil glycosylase UDG into J1 three-way system;

j3 adding heme and luminol into J2 to form G-quadruplex spherical nuclease;

j4 addition of hydrogen peroxide to J3 was immediately used for detection of chemiluminescent signal with a chemiluminescent signal acquisition time interval of 1.5s and a chemiluminescent spectral measurement range of 350nm to 550 nm.

The detection method of the invention is to detect uracil glycosylase by generating a chemiluminescent signal, wherein the uracil glycosylase can specifically recognize and excise U base, under the assistance of endonuclease IV, the three-way structure is changed, the toehold end is exposed by the change of the three-way structure to initiate strand displacement reaction, after adding spherical nucleic acid, DNA walker reaction is carried out under the action of endonuclease IV, finally, the surface of the nano-gold only contains a DNA sequence (G4-SNA) capable of forming G-quadruplex, and K is added⁺And heme, spherical nuclease (G4-SNAzyme) can be formed. The spherical nuclease can catalyze luminol to generate chemiluminescence reaction in the presence of hydrogen peroxide.

The sensor has the advantages of high efficiency, high sensitivity and high specificity, and can make up for the defects of the existing detection method of uracil glycosylase by only using endonuclease IV which is one enzyme, thereby realizing the rapid and accurate quantitative detection of the uracil glycosylase.

The invention has the beneficial effects that:

1. the method utilizes the specificity recognition and excision of the uracil glycosylase to the U basic group, and can cut off the DNA chain containing the U basic group with the assistance of the endonuclease IV so as to initiate the subsequent reaction, and has the characteristic of high specificity;

2. the invention can realize the orderly assembly of DNA chains by means of a three-way structure, and can skillfully perform the migration of the DNA chains by means of the toehold mediated chain displacement reaction, thereby accelerating the speed of the DNA chain migration;

3. by means of the DNA walker nanotechnology, the reaction efficiency can be obviously improved, signals can be obviously enlarged, and the monitoring sensitivity is improved;

4. according to the invention, the spherical nuclease is utilized to realize the high enrichment effect of G-quadruplet DNA enzyme on the surface of the nanogold, so that the enrichment of signals is realized, the signals can be expanded, and the sensitivity is improved;

5. compared with the method that G-quadruplet DNA chains are directly marked on the surface of the nanogold to directly form the spherical nuclease, the method has the advantages that the background signal of the reaction can be obviously reduced, and the signal to noise ratio of the reaction is improved;

6. the sensor has mild reaction conditions and high reaction speed.

7. The main processes of the detection principle of the invention are realized in homogeneous phase, thus improving the reaction speed, reducing the complexity of operation and realizing the rapid, simple and sensitive detection of the target;

8. the preparation method is simple, has stable performance, and is suitable for the detection of uracil glycosylase in the field of medical health, laying a foundation for the treatment of subsequent tumors and the practical application of biosensor industrialization;

9. the process for manufacturing the biosensor has low cost and is suitable for the requirement of low price in industrialization.

Drawings

FIG. 1 is a schematic diagram of the experiment;

FIG. 2 is a graph showing the results of the optimized detection of the concentration of endonuclease IV in example 1;

FIG. 3 is a graph showing optimized hemoglobin detection results of example 2;

FIG. 4 is a graph of the results of luminol concentration optimization assays of example 3;

FIG. 5 is a graph showing the results of the hydrogen peroxide concentration optimization assay of example 4;

FIG. 6 is a calibration curve of the sensor detection of example 5;

FIG. 7 is a linear relationship of concentration detected by the sensor of example 5.

Detailed Description

The present invention is further illustrated by the following specific examples.

The preparation method of the biosensor comprises the following steps:

(1) preparing nano gold;

(2) preparing spherical nucleic acid;

(3) spherical nucleases are formed in homogeneous phase to catalyze the chemiluminescent reaction of luminol;

in the preparation method, the preparation of the nano gold comprises the following steps:

the preparation of the nano-gold is realized according to a method for reducing chloroauric acid by sodium citrate. mu.L of chloroauric acid (0.04 g/mL) was added to 200mL of ultrapure water, heated to boiling with stirring, and 3mL of sodium citrate (1%) was added to the boiled solution quickly. Subsequently, it can be observed that the solution changes color from light yellow to black and finally to wine red. Heating was continued for 15min after the color turned to wine red to ensure completion of the reaction. Then, the nano gold solution is cooled to room temperature and then is placed at 4 ℃ for standby. The nano gold with the wavelength of 20nm has an ultraviolet absorption peak of about 520nm and a molar extinction coefficient of 0.878X 109M^-1•cm^-1。