Sensor for rapid activation of CRISPR-Cas12a signal switch by spherical nucleic acid self-assembly triggered by the target

Abstract

The present invention discloses a sensor for rapidly activating a CRISPR-Cas12a signal switch by target-triggered self-assembly of spherical nucleic acids, which relates to the technical field of biochemical analysis sensors, and includes a luminescent material and a sensor. The luminescent material is AuAgNCs@MOF-5, and a quenching probe modified with dopamine is on the surface of the material AuAgNCs@MOF-5. The sensor includes spherical nucleic acid A and spherical nucleic acid B. Both spherical nucleic acid A and spherical nucleic acid B are composed of a hairpin and a Y-shaped DNA nanostructure. The hairpin not only contains the recognition site of a DNAzyme dependent on Zn 2+ , but also contains an activator of CRISPR / Cas12a. The Y-shaped DNA nanostructure is composed of an auxiliary strand (APs), DNAzyme strands and an AMACR aptamer. The auxiliary strands of spherical nucleic acid A and spherical nucleic acid B are AP A and AP B respectively. The present invention can be used to detect the prostate cancer marker AMACR in serum samples, and can achieve highly sensitive and accurate detection of AMACR; it can solve the problems of low detection sensitivity and long time for detecting the prostate cancer marker AMACR in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and particularly to a sensor for a target-triggered spherical nucleic acid self-assembly to rapidly activate a CRISPR-Cas12a signal switch. Background Art

[0002] Existing technologies have problems of low specificity and low early diagnosis rate in the diagnosis of prostate cancer. Early diagnosis of prostate cancer can greatly improve the survival rate of patients, and most early-stage prostate cancer patients have no obvious symptoms. At present, the diagnosis of prostate cancer still relies on pathological tissue biopsy, which is time-consuming and more painful for patients. Human α-methylacyl-CoA racemase (AMACR) has been confirmed as a reliable biomarker for PCa. The expression of AMACR in needle biopsy has 97% sensitivity and 100% specificity for PCa detection. Some existing methods have low sensitivity for AMACR detection. For example, imaging methods such as X-ray, CT, and MRI can only diagnose from the morphological aspect; for the detection of serum samples, since the content of AMACR in serum is low, the detection is relatively difficult.

[0003] Therefore, the present invention aims to provide a sensor for a target-triggered spherical nucleic acid self-assembly to rapidly activate a CRISPR-Cas12a signal switch, so as to solve the problems of low sensitivity and long time for the detection of the prostate cancer biomarker AMACR, and achieve highly sensitive and accurate detection of AMACR. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide a sensor for a target-triggered spherical nucleic acid self-assembly to rapidly activate a CRISPR-Cas12a signal switch.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows: A sensor for a target-triggered spherical nucleic acid self-assembly to rapidly activate a CRISPR-Cas12a signal switch, the sensor comprising a luminescent material and a sensor, the luminescent material being a material AuAgNCs@MOF-5 with a strong and stable ECL signal, and a quenching probe modified with dopamine being connected to the surface of the material AuAgNCs@MOF-5;

[0006] The sensor comprises spherical nucleic acid A and spherical nucleic acid B, both the spherical nucleic acid A and the spherical nucleic acid B are composed of a hairpin and a Y-shaped DNA nanostructure, wherein the hairpin not only contains Zn 2+the recognition site of the DNAzyme and also contains an activator of CRISPR / Cas12a. The Y-shaped DNA nanostructure is composed of auxiliary strands (APs), DNAzyme strands, and the AMACR aptamer. The auxiliary strands of the spherical nucleic acid A and the spherical nucleic acid B are AP A and AP B , AP A and AP B complementary base pairing.

[0007] Furthermore, the construction method of the sensor includes the following steps:

[0008] Step 1: Synthesize the material AuAgNCs@MOF-5 with strong and stable ECL signals, and connect a quenching probe modified with dopamine on the surface of the material to reduce the background signal;

[0009] Step 2: Construction of the sensor:

[0010] 1) Construct two different spherical nucleic acids A and B, both of which are composed of a hairpin and a Y-shaped DNA nanostructure; among them, the hairpin not only contains the recognition site of the DNAzyme dependent on Zn 2+ but also contains an activator of CRISPR / Cas12a; the Y-shaped DNA nanostructure is composed of auxiliary strands (APs), DNAzyme strands, and the AMACR aptamer; the auxiliary strands of the spherical nucleic acid A and the spherical nucleic acid B are AP A and its complementary AP B ;

[0011] 2) In the presence of the target AMACR, AMACR binds to the AMACR aptamer, and the Y-shaped DNA nanostructure disintegrates, resulting in the dissociation of the DNAzyme strands. AP A and AP B complementary base pair and connect the spherical nucleic acids into a network structure;

[0012] 3) The free DNAzyme strands are complementary base paired with the hairpin to form a DNA enzyme dependent on Zn 2+ ; when Zn 2+ exists, the DNA enzyme recognizes the specific site and cleaves the hairpin, so that the activator of CRISPR / Cas12a is cleaved off;

[0013] 4) Collect the supernatant containing the activator by magnetic separation;

[0014] 5) The activator of CRISPR / Cas12a activates the CRISPR / Cas12a protein, exerts trans-cleavage ability, cleaves the quenching probe on the surface of the material AuAgNCs@MOF-5, restores the signal, constructs a sensor, amplifies the amount of a small amount of AMACR in serum, and realizes accurate and sensitive detection of the target.

[0015] The present invention also provides an application of the sensor for rapidly activating the CRISPR-Cas12a signal switch by target-triggered spherical nucleic acid self-assembly in the detection of the prostate cancer marker AMACR in serum.

[0016] Compared with the prior art, the beneficial effects of the present solution are as follows:

[0017] In the present invention, the sensor of the present invention can be used to detect the prostate cancer marker AMACR in a serum sample, and can realize highly sensitive and accurate detection of AMACR; the network structure formed by target triggering in the solution of the present invention can improve the reaction efficiency through the confinement enhancement effect, and solve the problems of low detection sensitivity and long time for detecting the prostate cancer marker AMACR in the prior art. Description of the Drawings

[0018] Figure 1 is the flowchart of the construction method of the sensor in the embodiment of the present invention;

[0019] Figure 2 is the characterization of the luminescent material in the embodiment of the present invention;

[0020] Figure 3 is the feasibility verification (gel electrophoresis, ECL) of the sensor in the embodiment of the present invention;

[0021] Figure 4 is the network confinement enhancement verification of the sensor in the embodiment of the present invention;

[0022] Figure 5 is the performance detection of the sensor in the embodiment of the present invention. Detailed Embodiments

[0023] In order to enable those skilled in the art of the present technology to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Embodiment:

[0026] As Figure 1 shown, the target triggers a sensor that rapidly activates the CRISPR-Cas12a signal switch through the self-assembly of spherical nucleic acids. The sensor includes a luminescent material and a sensor. The luminescent material is the material AuAgNCs@MOF-5 with strong and stable ECL signals, and a quenching probe modified with dopamine is connected to the surface of the material AuAgNCs@MOF-5;

[0027] The sensor includes spherical nucleic acid A and spherical nucleic acid B. Both spherical nucleic acid A and spherical nucleic acid B are composed of a hairpin and a Y-shaped DNA nanostructure. The hairpin not only contains the recognition site of the DNAzyme dependent on Zn 2+ but also contains the activator of CRISPR / Cas12a. The Y-shaped DNA nanostructure consists of auxiliary strands (APs), DNAzyme strands, and AMACR aptamers. The immobilization strands of spherical nucleic acid A and spherical nucleic acid B are AP A and AP B respectively, and AP A and AP B are complementary to each other.

[0028] The construction method of the sensor includes the following steps:

[0029] Step 1: Synthesize the material AuAgNCs@MOF-5 with strong and stable ECL signals, and connect a quenching probe modified with dopamine to the surface of the material to reduce the background signal (as Figure 1 shown in B);

[0030] Step 2: Construction of the sensor (as Figure 1 shown in A):

[0031] 1) Construct two different spherical nucleic acids A and B. Both spherical nucleic acid A and spherical nucleic acid B are composed of a hairpin and a Y-shaped DNA nanostructure; among them, the hairpin not only contains the recognition site of the DNAzyme dependent on Zn 2+ but also contains the activator of CRISPR / Cas12a; the Y-shaped DNA nanostructure consists of auxiliary strands (APs), DNAzyme strands, and AMACR aptamers; the auxiliary strands of spherical nucleic acid A and spherical nucleic acid B are AP A and AP A complementary to AP B respectively;

[0032] 2) In the presence of the target AMACR, AMACR binds to the AMACR aptamer, and the Y-shaped DNA nanostructure disassembles, resulting in the release of DNAzyme strands, and AP A and AP B complementary pair and connect spherical nucleic acids into a network structure;

[0033] 3) The released DNAzyme strands complementary pair with the hairpin to form a Zn 2+ -dependent DNA enzyme; when Zn 2+ is present, the DNA enzyme recognizes the specific site and cleaves the hairpin, so that the activator of CRISPR / Cas12a is cleaved off;

[0034] 4) The supernatant containing the activator is collected by magnetic separation;

[0035] 5) The activator of CRISPR / Cas12a activates the CRISPR / Cas12a protein, exerts trans-cleavage ability, cleaves the quenching probe on the surface of the material AuAgNCs@MOF-5, and restores the signal. By constructing a sensor, a small amount of AMACR in serum is converted and amplified to achieve accurate and sensitive detection of the target.

[0036] Since human α-methylacyl-CoA racemase (AMACR) has been confirmed as a reliable biomarker for PCa, and the expression of AMACR in needle biopsy has 97% sensitivity and 100% specificity for PCa detection, the sensor of the present invention that the target triggers the self-assembly of spherical nucleic acids to rapidly activate the CRISPR-Cas12a signal switch can be applied to the detection of the prostate cancer biomarker AMACR in serum.

[0037] In this embodiment, the method for synthesizing the material AuAgNCs@MOF-5 with strong and stable ECL signals in Step 1 is as follows: Dissolve 144 mg of Zn(NO3)2·6H2O in 5 mL of N,N-dimethylformamide (DMF) and mix it with 10 mL of the prepared AuAgNC solution at room temperature for 10 min. After dissolving 30 mg of 1,4-benzenedicarboxylic acid (H2BDC) in 5 mL of DMF, add it to the obtained solution and stir to mix. Then, according to the previous steps, inject 200 mg of polyvinylpyrrolidone (PVP) dissolved in 2 mL of methanol into the mixed solution and then stir. Then, the mixture is reacted in a stainless-steel autoclave with a Teflon liner at 120 °C for 12 hours to obtain AuAgNC@MOF-5. After cooling to room temperature, wash the product three times with DMF and centrifuge at 12,000 rpm for 10 minutes to collect. Finally, store the prepared AuAgNC@MOF-5 at 4 °C for subsequent use.

[0038] In Step 2, the method for constructing two different spherical nucleic acids A and B is as follows: First, reduce the auxiliary probe (APs) with 5 μL of 1 M Tris(2-carboxyethyl)phosphine (TCEP) for 30 minutes. Then, mix 10 μL of 10 μM AMACR aptamer, 12 μL of 10 μM DNAzyme strand, and 8 μL of 10 μM APs (AP A :AP B = 1:1) and heat and anneal at 95 °C for 10 minutes, and then hybridize at 4 °C for 2.5 hours to obtain a Y-shaped probe solution. Subsequently, add 40 μL of Au@Fe3O4 suspension and 20 μL of 20 μM hairpin annealed for 53 minutes to the Y-shaped probe solution. After stirring overnight at 4 °C on a shaker table, purify the prepared spherical nucleic acids A and B by magnetic separation and then store them at 4 °C for subsequent steps.

[0039] The following is the detection of the sensor performance of the present invention in this embodiment:

[0040] As Figure 5 shown, under the optimal conditions, the sensitivity, specificity, and stability of the sensor were investigated. First, the ECL responses of different concentrations of AMACR were applied to analyze the sensitivity of the proposed biosensor. As Figure 5 shown in A, when the AMACR concentration is in the range of 1 ng / mL to 100 μg / mL, the ECL signal gradually increases. It should be noted that Figure 5 a satisfactory linear relationship between the logarithm and the AMACR concentration is described in B, and the linear regression equation is expressed as:

[0041] I = 1514.50 lgc + 9257.75,

[0042] Among them, I refers to the ECL intensity, the correlation coefficient is 0.9942, and c refers to the concentration of AMACR. In addition, the limit detection is calculated to be 0.10 ng / ml. To evaluate the specificity of the biosensor constructed in the present invention, its performance was verified using potential interfering substances such as PTK-7, MUC1, CD63 and their mixtures with AMACR. As Figure 5 shown in C, only in the presence of AMACR, the constructed biosensor shows a high ECL signal, otherwise it shows a negligible ECL reaction, indicating that the biosensor has relatively high specificity for AMACR detection. Regarding stability, another important characteristic of the biosensor of the present invention was studied at an AMACR concentration of 10 μg / mL by continuous scanning for 14 cycles. As Figure 5 shown in D, the relatively stable ECL reaction indicates that the biosensor has good stability for the characteristic quantification of AMACR.

[0043] Experimental sample detection:

[0044] To evaluate the feasibility of the biosensor in clinical applications, the standard addition method was used to determine the recovery rate. As shown in the following table:

[0045]

[0046] Samples of different concentrations of AMACR were added to human serum solution, and then measured using the proposed biosensor. The experimental results show that the relative standard deviation and quantitative recovery rate are both acceptable.

[0047] In summary, in the above embodiments of the present invention, the sensor of the present invention can be used to detect the prostate cancer marker AMACR in serum. Compared with the existing methods for AMACR detection, the present invention provides a sensor that triggers the self-assembly of spherical nucleic acids to rapidly activate the CRISPR-Cas12a signal switch by the target substance, which can solve the problems of low sensitivity and long time of the existing detection methods and achieve highly sensitive and accurate detection of AMACR.

[0048] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A sensor that the target triggers the spherical nucleic acid self-assembly to rapidly activate the CRISPR-Cas12a signal switch, characterized in that: The sensor includes a luminescent material and a sensor. The luminescent material is the material AuAgNCs@MOF-5 with a strong and stable ECL signal, and a quenching probe modified with dopamine is present on the surface of the material AuAgNCs@MOF-5; The sensor includes spherical nucleic acid A and spherical nucleic acid B, both of which are composed of hairpin and Y-shaped DNA nanostructures. The hairpin contains not only the recognition site of the DNAzyme dependent on Zn 2+ , but also the activator of CRISPR / Cas12a. The Y-shaped DNA nanostructure is composed of auxiliary strands, DNAzyme strands and AMACR aptamers. The auxiliary strands of spherical nucleic acid A and spherical nucleic acid B are AP A and AP B respectively, and AP A and AP B are complementary to each other.

2. The sensor for rapidly activating the CRISPR-Cas12a signal switch by target-triggered spherical nucleic acid self-assembly according to claim 1, characterized in that: The construction method of the sensor includes the following steps: Step 1: Synthesize the material AuAgNCs@MOF-5 with a strong and stable ECL signal, and connect a quenching probe modified with dopamine on the surface of the material to reduce the background signal; Step 2: Construction of the sensor: Construct two different spherical nucleic acids A and spherical nucleic acids B, both of which are composed of hairpins and Y-shaped DNA nanostructures; among them, the hairpin not only contains the recognition site of the DNAzyme dependent on Zn 2+ , but also contains the activator of CRISPR / Cas12a; the Y-shaped DNA nanostructure is composed of auxiliary strands, DNAzyme strands, and AMACR aptamers; the auxiliary strands of spherical nucleic acids A and spherical nucleic acids B are AP A and its complementary pairing AP B ; When incubated with the target AMACR, AMACR binds to the AMACR aptamer, and the Y-shaped DNA nanostructure disassembles, resulting in the release of the DNAzyme strands, and AP A and AP B complementary pair and connect the spherical nucleic acids into a network structure; Free DNAzyme srands are complementary paired with the hairpin to form a zinc ion-dependent DNA enzyme; when zinc ions are present, the DNA enzyme recognizes specific sites to cleave the hairpin, and the activator of CRISPR / Cas12a is cleaved off; The supernatant containing the activator is collected by magnetic separation; The activator of CRISPR / Cas12a activates the CRISPR / Cas12a protein, which exerts trans-cleavage ability to cleave the quenching probe on the surface of the material AuAgNCs@MOF-5, restoring the signal; by constructing the sensor, a small amount of AMACR in serum is transformed and amplified, and finally accurate and sensitive detection of the target substance is achieved.

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