A biosensor for detecting ovarian cancer exosomes by chemiluminescence based on logic gate response

Abstract

The present invention belongs to the field of sensor technology, and relates to a biosensor for detecting ovarian cancer exosomes by chemiluminescence method based on logic gate response. A chemiluminescence biosensor is constructed based on the specific recognition of aptamers corresponding to ovarian cancer exosome proteins, rolling circle amplification reaction, and the catalytic activity of G4 / heme DNA enzyme. The sensor has the advantages of wide detection range, low detection limit, high specificity, etc., which can make up for the defects and deficiencies of existing detection methods in early diagnosis of ovarian cancer and realize rapid and accurate quantitative detection of ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of sensors and relates to a biosensor for detecting ovarian cancer exosomes by a chemiluminescence method based on logic gate response. Background Art

[0002] Ovarian cancer (OC) is the second most common cause of gynecological cancer death worldwide. Timely diagnosis, appropriate surgery, and systemic treatment are challenging due to the numerous subtypes of ovarian cancer tissue. Exosomes carry a wealth of important components, including proteins, DNA, RNA, and other bioactive substances. They play an important role in various diseases and pathophysiological processes, and the number of cancer exosomes in body fluids is closely related to the progression of cancer. Therefore, cancer exosomes have been regarded as potential biomarkers for non-invasive and early detection of tumor carcinogenesis.

[0003] At present, various methods for detecting cancer exosomes have been established, including traditional nanoparticle tracking analysis (NTA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, etc. However, due to the complex operation steps and expensive analytical instruments, these methods still face some problems and are difficult to adapt to the requirements of convenience, speed, sensitivity, etc. of cancer exosome detection. At present, it is urgent to establish a fast, accurate, sensitive and highly specific detection method to detect ovarian cancer exosomes. Summary of the invention

[0004] In order to achieve more sensitive and specific detection of ovarian cancer exosomes, the present application proposes a biosensor for detecting ovarian cancer exosomes by specifically binding an ovarian cancer exosome protein to a DNA ternary complex with an adaptor to expose a single strand connected to a rolling circle amplification primer. The single strand is cut by phi29 DNA enzyme to expose the primer, thereby initiating a rolling circle amplification reaction to obtain a large amount of G4, which combines with hemin chloride in the solution to form a G4 / heme DNA enzyme that can catalyze chemiluminescence, and finally catalyzes hydrogen peroxide to produce reactive oxygen to oxidize luminol to detect ovarian cancer exosomes.

[0005] The technical solution of the present invention is as follows:

[0006] A biosensor for detecting ovarian cancer exosomes by chemiluminescence based on logic gate response, comprising ovarian cancer exosomes, C1, C2, CT, phi29 DNA enzyme, dNTP, hydrogen peroxide, luminol, and hemin;

[0007] C1 is composed of the ovarian cancer exosome-specific aptamer CA24A-5, the primer chain primer that triggers rolling circle amplification, and cSYL3C complementary to Apt2; the nucleotide sequence of C1 is shown in SEQ ID No. 1; specifically: 5'-TATGTGGGTGGGTGGGTGGTTATGCTGAGTCAGCCTTGCTTTTTTTTTGCTAGAAGGAAACAGTTACACCTCTGTAGTG-3';

[0008] CT is a rolling circle amplification template obtained from the padlock probe and the linker sequence;

[0009] The nucleotide sequence of the padlock probe in CT is shown in SEQ ID No. 2; specifically: 5'-TAGCACGGACATTTTCCCAACCCGCCCTACCCTTTTTTTTTTCCCAACCCGCCCTACCCTTTTGTAACTGTTTCCTTC-3';

[0010] The nucleotide sequence of the linker sequence in CT is shown in SEQ ID No. 3; specifically: 5'-TGTCCGTGCTAGAAGGAAACAGTTAC-3';

[0011] C2 is the aptamer SYL3C, and its nucleotide sequence is shown in SEQ ID No. 4; specifically: 5'- CAC TAC AGAGGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG -3'.

[0012] Furthermore, ovarian cancer exosomes specifically bind to C1 and C2; cSYL3C is cleaved by phi29 DNA enzyme to expose the primer, triggering the rolling circle amplification of CT to obtain G4, which combines with hemin chloride to form G4 / heme DNA enzyme, catalyzing hydrogen peroxide to oxidize luminol to obtain a chemiluminescent signal.

[0013] Furthermore, the 3' end of the C2 chain is modified with inVDT, which can prevent the chain from reversing.

[0014] Furthermore, in the method for preparing the rolling circle amplification template CT, the padlock probe and the connection probe are mixed in a buffer, and T4 DNA enzyme and nuclease exonucleases I and III are added and heated and incubated to obtain the synthesis.

[0015] Another object of the present invention is to provide a method for preparing the above-mentioned biosensor, comprising the following steps:

[0016] (1) Preparation of DNA ternary complex: C1, CT and C2 are mixed in a buffer solution, and phi29 DNA enzyme is added and heated and incubated;

[0017] (2) After incubation of the DNA ternary complex with exosomes, rolling circle amplification was performed to obtain G4;

[0018] (3) Preparation of G4 / heme DNA enzyme: adding hemin chloride to the G4 obtained in step (2), and heating and incubating to obtain G4 / heme DNA enzyme;

[0019] (4) Chemiluminescent reaction: The G4 / heme DNA enzyme obtained in step (3) is mixed with hydrogen peroxide and luminol to generate a chemiluminescent reaction to produce a chemiluminescent signal.

[0020] Furthermore, the buffer in step (1) includes 0.05 mM NaCl, 0.05 mM MgCl 2 and 100 mM Tris-HCl.

[0021] Furthermore, in step (1), C1, CT and C2 are added in equal concentrations.

[0022] Furthermore, the process of step (2) is: incubate and identify the DNA ternary complex of step (1) and ovarian cancer exosomes, and then add dNTP, phi29 DNA enzyme and 10×phi29 DNA buffer to perform rolling circle amplification reaction to obtain G4.

[0023] Another object of the present invention is to protect the biosensor prepared by the above preparation method.

[0024] Beneficial effects of the present invention

[0025] The biosensor for detecting ovarian cancer exosomes by chemiluminescence based on logic gate response of the present invention utilizes the aptamer corresponding to the ovarian cancer exosome protein for specific response, thereby exposing the primer before G4 amplification through the cutting of the single strand by phi29 DNA enzyme, and then inducing CT to perform rolling circle amplification reaction to obtain a large amount of G4, and after incubation with hemin chloride, a G4 / heme DNA enzyme with a strong catalytic effect is formed, which finally catalyzes hydrogen peroxide to produce reactive oxygen, oxidizes the luminescent substrate luminol, and realizes the amplification of the chemiluminescent signal. The sensor uses the chemiluminescence method to perform bispecific detection of ovarian cancer exosomes. Compared with other methods such as fluorescence method, electrochemical method, surface Raman, etc., this method is simple to operate, has weak false positive signals, fast reaction speed, and has rapid detection, accurate quantitative determination, and high sensitivity when detecting ovarian cancer exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of a sensor according to an embodiment of the present invention;

[0027] Figure 2 is a diagram of DNA volume optimization in Example 2 of the present invention;

[0028] Figure 3 is a graph for optimizing the hemin concentration in Example 3 of the present invention;

[0029] Figure 4 This is a graph showing the concentration gradient of ovarian cancer exosomes in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1

[0032] Preparation of rolling circle amplification template CT: 1.8 μL padlock probe and 5.4 μL linker sequence were mixed and reacted at 85°C for 5 min, then slowly cooled to room temperature, 12 μL T4 DNA enzyme, 5 μL 10×T4 buffer, 10 μL 0.5 mg / mL BSA, and 15.8 μL DEPC water were added, reacted at 4°C overnight, heated to 65°C for 10 min, and stored in a -20°C refrigerator.

[0033] Preparation of DNA ternary complex: Take 4 μL C1, 8 μL C2, 8 μL CCT and 17 μL buffer (0.05 mM NaCl, 0.05 mM MgCl) at equal concentrations (10 μM). 2 and 100 mM Tris-HCl) were mixed and heated at 95 °C for 5 min, then slowly cooled to room temperature, 2 μL phi29 DNA enzyme and 4.7 μL 10× phi DNA enzyme buffer were added, reacted at 30 °C for 60 min, then heated to 80 °C for 10 min, and stored in a -20 °C refrigerator.

[0034] The preparation steps of the chemiluminescent sensor are as follows:

[0035] Pipette the 10-fold diluted DNA ternary complex (12 μL, 14 μL, 16 μL, 20 μL) and 2 μL of 5×10 9The ovarian cancer exosomes of 1000 μg / mL were mixed and incubated at 37 °C for 1 h. 1 μL dNTP, 2 μL 10× phi29 DNA enzyme buffer, and 1 μL phi29 DNA enzyme were added. The reaction was carried out at 30 °C for 2 h and heated at 80 °C for 10 min to obtain the rolling circle amplification product G4. 6 μL hemin (50 μM) was added to the G4 obtained by the above reaction and incubated at 37 °C for 10 min to obtain G4 / heme DNA enzyme. 25 μL 10 mM hydrogen peroxide and 50 μL 10 mM luminol were taken and the chemiluminescence signal was detected using an enzyme plate reader.

[0036] Preparation method of the solution used in the above process:

[0037] 1. Ultrapure water must be sterilized at high temperature. The specific method is to place the ultrapure water in blue-capped bottles and seal them with tin foil. Sterilize in an autoclave at 120°C for 20 minutes.

[0038] 2. The brand of 10×phi29DNA enzyme buffer is Bio-Tech, and the product number is D7053M.

[0039] Results Figure 2 ,As can be seen from the figure, the detected chemiluminescent signal intensity increases with the ,increase of the volume of the DNA ternary complex, and the chemiluminescent ,signal reaches the highest when the volume of the DNA ternary complex is 14 ,μL. Therefore, the optimal volume of the DNA ternary complex in this ,sensor is 14 ,μL.

[0040] Example 2

[0041] Preparation of rolling circle amplification template CT: 1.8 μL padlock probe and 5.4 μL linker sequence were mixed and reacted at 85°C for 5 min, then slowly cooled to room temperature, 12 μL T4 DNA enzyme, 5 μL 10×T4 buffer, 10 μL 0.5 mg / mL BSA, and 15.8 μL DEPC water were added, reacted at 4°C overnight, heated to 65°C for 10 min, and stored in a -20°C refrigerator.

[0042] Preparation of DNA ternary complex: Take 4 μL C1, 8 μL C2, 8 μL CCT of equal concentration (10 μM) and 17 μL buffer (0.05 mM NaCl, 0.05 mM MgCl 2 and 100 mM Tris-HCl) were mixed and heated at 95 °C for 5 min, then slowly cooled to room temperature, 2 μL phi29 DNA enzyme and 4.7 μL 10× phi29 DNA enzyme buffer were added, reacted at 30 °C for 60 min, then heated to 80 °C for 10 min, and stored in a -20 °C refrigerator.

[0043] The preparation steps of the chemiluminescent sensor are as follows:

[0044] Pipette 12 μL of the 10-fold diluted DNA ternary complex and 2 μL of 5×10 9 The ovarian cancer exosomes of 1000 μg / mL were mixed and incubated at 37 °C for 1 h. 1 μL dNTP, 2 μL 10× phi29 DNA enzyme buffer, and 1 μL phi29 DNA enzyme were added. The reaction was carried out at 30 °C for 2 h and heated at 80 °C for 10 min to obtain the rolling circle amplification product G4. 6 μL hemin (concentrations of 5 μM, 50 μM, 100 μM, 200 μM, and 500 μM, respectively) was added to the G4 obtained in the above reaction and incubated at 37 °C for 10 min to obtain G4 / heme DNA enzyme. 25 μL 10 mM hydrogen peroxide and 50 μL 10 mM luminol were taken and the chemiluminescence signal was detected using an enzyme plate reader.

[0045] Preparation method of the solution used in the above process:

[0046] 1. Ultrapure water must be sterilized at high temperature. The specific method is to place the ultrapure water in blue-capped bottles and seal them with tin foil. Sterilize in an autoclave at 120°C for 20 minutes.

[0047] 2. The brand of 10×phi29DNA enzyme buffer is Bio-Tech, and the product number is D7053M.

[0048] Results Figure 3 As can be seen from the figure, the intensity of the detected chemiluminescent signal increases with the increase of the concentration of hemin chloride. When the concentration of hemin chloride is 50 μM, it is 4 times higher than the signal before rolling circle amplification, and the sensitivity is highest at this concentration. Therefore, 50 μM hemin chloride is selected as the optimal concentration.

[0049] Example 3

[0050] Preparation of rolling circle amplification template CT: 1.8 μL padlock probe and 5.4 μL linker sequence were mixed and reacted at 85°C for 5 min, then slowly cooled to room temperature, 12 μL T4 DNA enzyme, 5 μL 10×T4 buffer, 10 μL 0.5 mg / mL BSA, and 15.8 μL DEPC water were added, reacted at 4°C overnight, heated to 65°C for 10 min, and stored in a -20°C refrigerator.

[0051] Preparation of DNA ternary complex: Take 4 μL C1, 8 μL C2, 8 μL CCT of equal concentration (10 μM) and 17 μL buffer (0.05 mM NaCl, 0.05 mM MgCl 2 and 100 mM Tris-HCl) were mixed and heated at 95 °C for 5 min, then slowly cooled to room temperature, 2 μL phi29 DNA enzyme and 4.7 μL 10× phi29 DNA enzyme buffer were added, reacted at 30 °C for 60 min, then heated to 80 °C for 10 min, and stored in a -20 °C refrigerator.

[0052] The preparation steps of the chemiluminescent sensor are as follows:

[0053] Pipette 12 μL of the 10-fold diluted DNA ternary complex and 2 μL of ovarian cancer exosomes (concentration of 5×10 4 Particles / mL, 5×10 5 Particles / mL, 5×10 6 Particles / mL, 5×10 7 Particles / mL, 5×10 8 Particles / mL, 5×10 9 Particles / mL) were mixed, incubated at 37 °C for 1 h, 1 μL dNTP, 2 μL 10× phi29 DNA enzyme buffer, 1 μL phi29 DNA enzyme were added, reacted at 30 °C for 2 h, and heated at 80 °C for 10 min to obtain the rolling circle amplification product G4. 6 μL hemin (50 μM) was added to the G4 obtained in the above reaction, and incubated at 37 °C for 10 min to obtain G4 / heme DNA enzyme. 25 μL 10mM hydrogen peroxide and 50 μL 10 mM luminol were taken, and the chemiluminescent signal was detected using an enzyme reader.

[0054] Preparation method of the solution used in the above process:

[0055] 1. Ultrapure water must be sterilized at high temperature. The specific method is to place the ultrapure water in blue-capped bottles and seal them with tin foil. Sterilize in an autoclave at 120°C for 20 minutes.

[0056] 2. The brand of 10×phi29DNA enzyme buffer is Bio-Tech, and the product number is D7053M.

[0057] Results Figure 4 As can be seen from the figure, the detected chemiluminescent signal increases with the increase of the concentration of ovarian cancer exosomes, and the chemiluminescent signal is linearly related to the concentration of exosomes. The linear equation is y=-0.84+0.184x, R 2 It is 0.996.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A biosensor for detecting ovarian cancer exosomes by chemiluminescence based on logic gate response, characterized in that: Including ovarian cancer exosomes, C1, CT, C2, phi29 DNA enzyme, dNTP, hydrogen peroxide, luminol, hemin; C1 is composed of the ovarian cancer exosome-specific aptamer CA24A-5, the primer chain primer that triggers rolling circle amplification, and cSYL3C complementary to Apt2; the nucleotide sequence of C1 is shown in SEQ ID No. 1; specifically: 5'-TATGTGGGTGGGTGGGTGGTTATGCTGAGTCAGCCTTGCTTTTT TTTTGCTAGAAGGAAACAGTTACACCTCTGTAGTG-3'; CT is a rolling circle amplification template obtained from the padlock probe and the linker sequence; C2 is the specific aptamer SYL3C; wherein, The nucleotide sequence of the padlock probe is shown in SEQ ID No. 2; specifically: 5'-TAGCACGGACATTTTCCCAACCCGCCCTACCCTTTTTTTTTTCCCAACCCGCCCTACCCTTTTGTAACTGTTTCCTTC-3'; The nucleotide sequence of the linker sequence is shown in SEQ ID No. 3; specifically: 5'-TGTCCGTGCTAGAAGGAAACAGTTAC-3'; The nucleotide sequence of the aptamer SYL3C is shown in SEQ ID No. 4; specifically: 5'- CAC TAC AGA GGT TGCGTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG -3'.

2. The biosensor according to claim 1, characterized in that Ovarian cancer exosomes specifically bind to C1 and C2; cSYL3C is cut by phi29 DNA enzyme to expose the primer, triggering the rolling circle amplification of CT to obtain G4, which combines with hemin chloride to form G4 / heme DNA enzyme, catalyzing hydrogen peroxide to oxidize luminol to obtain a chemiluminescent signal.

3. The biosensor according to claim 1, characterized in that The 3' end of the C2 chain is modified with inVDT.

4. The biosensor according to claim 1, characterized in that The method for preparing the rolling circle amplification template CT comprises the following steps: mixing the padlock probe and the connection sequence in a buffer, adding T4 DNA enzyme and nuclease exonucleases I and III, and heating and incubating to obtain the synthesis.

5. A method for preparing the biosensor according to claim 1, characterized in that: The following steps are involved: (1) Preparation of DNA ternary complex: C1, CT and C2 are mixed in a buffer solution, and phi29 DNA enzyme is added and heated and incubated; (2) After incubation of the DNA ternary complex with exosomes, rolling circle amplification was performed to obtain G4; (3) Preparation of G4 / heme DNA enzyme: adding hemin chloride to the G4 obtained in step (2), and heating and incubating to obtain G4 / heme DNA enzyme; (4) Chemiluminescent reaction: The G4 / heme DNA enzyme obtained in step (3) is mixed with hydrogen peroxide and luminol to generate a chemiluminescent reaction to produce a chemiluminescent signal.

6. The preparation method according to claim 5, characterized in that: The buffer solution in step (1) includes NaCl, MgCl2, and Tris-HCl.

7. The preparation method according to claim 5, characterized in that: In step (1), C1, CT and C2 are added in equal concentrations.

8. The preparation method according to claim 5, characterized in that: The process of step (2) is: incubate and identify the DNA ternary complex of step (1) and ovarian cancer exosomes, and then add dNTP, phi29 DNA enzyme and phi29 DNA enzyme buffer to perform rolling circle amplification reaction to obtain G4.

9. A biosensor prepared by the preparation method according to any one of claims 5 to 8.

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