CN112378971A - CRISPR/Cas13 a-driven catalytic renewable electrochemical biosensor and application thereof - Google Patents
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Abstract
The invention discloses a CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor, which comprises a CRISPR/Cas13a system/hairpin DNA catalytic loop two-stage signal amplification system and a regenerable screen-printed electrode chip. The system comprises a Cas13a protein, crRNA, a promoter molecule and a DNA hairpin; the chip comprises a screen printing electrode and a soft pore plate for loading a sample, wherein the electrode comprises a gold working electrode, a platinum counter electrode and a silver/silver chloride indicating electrode, and the nucleic acid monomolecular layer is anchored on the surface of the gold working electrode. When RNA in blood is detected, Cas13a protein cleaves the promoter molecule, triggering CHDC to generate an intermediate product, and the nucleic acid strand captures the intermediate product giving an electrical signal. The sensor of the invention has the advantages of rapidness, high sensitivity and specificity, low cost and the like, and can be used for molecular detection in the fields of various diseases, food, environmental safety and the like.
Description
Technical Field
The invention relates to a CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor, in particular to a preparation method of the CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor and application of the catalytic regenerable electrochemical biosensor in disease detection.
Background
The earlier a disease is diagnosed, the more likely it is to be cured. The miniaturized device is adopted to rapidly detect nucleic acid molecules in situ, particularly small molecules of RNA (microRNA) and messenger RNA (mRNA), so that the diagnosis, monitoring and treatment can be realized in time, and the cure rate of patients can be improved. Given the important role of mRNA and microRNA in regulating gene expression and dysfunction of its mutants in human disease, they have become effective biomarkers for cancer detection. Traditional RNA detection methods, including qRT-PCR, microarray and RNA sequencing, often rely on large, expensive instruments, complex sample preparation procedures and lengthy detection cycles, which limit their widespread use. Therefore, the development of new rapid, portable, highly specific and sensitive RNA detection techniques and devices is urgently needed.
In the field of biosensors, electrochemical and optical signals are the most common signaling modes. Biosensors based on optical detection are simple and fast to operate, but have low sensitivity and rely on non-disposable, rather bulky optical density readers and controllers for signal amplification/output, poor device integration, which limits their application in point-of-care testing (POCT) and field diagnostics. In contrast, electrochemical biosensors provide powerful functions for point-of-care detection applications, including system integration, miniaturization of detection devices, signal amplification, and multi-analyte detection.
CRISPR/Cas is a powerful gene editing technology and is widely applied to gene function research and gene modification and treatment. A CRISPR/Cas13a system comprising Cas13a protein and CRISPR RNA (crRNA). Studies have shown that Cas13a can bind and cleave a specific site of a target RNA with high specificity and efficiency under the guidance of crRNA. After Cas13a/crRNA specifically recognizes the target RNA, one activated Cas13a can nonspecifically cleave thousands of nearby signal molecules within minutes, exhibiting exponential fluorescence signal amplification. Based on the "trans-cleavage" effect of Cas13a protein, the CRISPR-Cas13a system has been developed as a rapid, low-cost, and highly sensitive nucleic acid detection tool. The hairpin nucleic acid chain catalytic loop (CHDC) is an enzyme-free DNA loop, a base complementary region is embedded into a double-stranded stem of the hairpin nucleic acid chain, and a target nucleic acid chain is hybridized with an anchor base region of the hairpin nucleic acid chain to initiate a plurality of hybridization catalytic reactions, so that high-sensitivity detection is realized. Meanwhile, the CHDC catalytic loop characteristic can realize the combination with CRIPSR/Cas, electrochemical sensors and other components to achieve higher performance. At present, no technical report based on the combination of CRISPR/Cas13a and CHDC and an electrochemical sensor is found, and no report of the technology for early detection of cancer is found.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, one of the technical problems to be solved by the present invention is to provide a novel catalytic regenerable electrochemical biosensor driven by CRISPR/Cas13 a. In the invention, a ferrocene (Fc) modified nucleic acid chain is anchored on the surface of a solid substrate to form a monomolecular layer to provide a reference signal, wherein a recognition element comprises a CRISPR/Cas13a system/hairpin DNA catalytic loop (Cas-CHDC) two-stage signal amplification system. Compared with the traditional electrochemical biosensor, the catalytic regenerable electrochemical biosensor driven by the CRISPR/Cas13a has a two-stage electric signal amplification function, a pair of ratio-type probes consisting of two redox groups provides stable signal output, and the regenerable performance of the sensor can be reused. The catalytic reproducible electrochemical biosensor driven by CRISPR/Cas13a is used for detecting nucleic acid molecules in early stages of cancers or other diseases, and has high selectivity and sensitivity.
In a first aspect of the invention, a CRISPR/Cas13a driven catalytic regenerable electrochemical biosensor is provided, the CRISPR/Cas13a driven catalytic regenerable electrochemical biosensor comprises a CRISPR/Cas13a system/hairpin DNA catalytic loop two-stage signal amplification system and a regenerable screen printing electrode chip, the CRISPR/Cas13a system/hairpin DNA catalytic loop specifically recognizes a target nucleic acid chain and generates an intermediate product after two-stage signal amplification, and the product after signal amplification is captured by the nucleic acid chain anchored on the regenerable screen printing electrode chip gives an electric signal.
Preferably, the reproducible screen-printed electrode chip comprises a screen-printed electrode and a soft well plate loaded with a liquid sample. The solid substrate of the screen printing electrode is glass, a silicon wafer, polymethyl methacrylate, ceramic or other solid materials. The soft orifice plate for loading the liquid sample is Polydimethylsiloxane (PDMS), polyphenylene oxide (PPO) or other engineering plastics; the size of the holes is 5 to 15 mm.
Preferably, the CRISPR/Cas13a system comprises a Cas13a protein, crRNA and a promoter molecule. The promoter molecule is RNA modified hairpin DNA nucleic acid chain, which is characterized in that a hairpin structure of about 12 base pairs is formed at the 5 'or 3' end and the internal ring structure has 2 to 5 ribonucleotide-labeled oligonucleotides. Wherein the labeled ribonucleotides include adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or a combination thereof.
The CRISPR/Cas13a system is used for detecting nucleic acid molecules such as mRNA, microRNA and the like.
Preferably, the hairpin DNA catalytic loop (CHDC) consists of: 1) two hairpin DNAs (C-H1 and C-H2) that can potentially combine into a double strand; 2) one of the hairpin DNAs is labeled at the 5 'or 3' end with a redox group and contains 3-7 uracil bases at the 5 'or 3' end.
Preferably, the monomolecular layer of the nucleic acid chain anchored on the surface of the gold working electrode comprises thiolated DNA, amino-modified DNA, or RNA. The length of the monomolecular layer of the nucleic acid strand is between 5 and 50 nucleotides.
Preferably, the redox group includes, but is not limited to, ferrocene, methylene blue, anthraquinone, and the like.
The catalytic regenerable electrochemical biosensor driven by CRISPR/Cas13a is used for quantifying a target nucleic acid chain in a sample and comprises the following steps:
(1) the soft orifice plate is fixed on the screen printing electrode by a micro-contact pasting technology to form a chip.
(2) Adding a nucleic acid chain/competitive chain solution into the pore plate to form a self-assembled monolayer on the surface of the gold electrode;
(3) mixing a target nucleic acid strand with the CRISPR/Cas13a system and the hairpin DNA catalytic loop;
(4) and (4) dropwise adding the mixed solution in the step (3) into a soft pore plate filled with the liquid sample, and quantifying the target nucleic acid chain by analyzing an electric signal, wherein the electric signal is in direct proportion to the concentration of the target nucleic acid chain.
The competitor strand comprises a base sequence complementary to a nucleic acid strand.
To prevent non-specific nucleic acid molecule adsorption, we chose to protect the non-array region in the chip with terminally thiolated β -mercaptoethanol (β ME), and the nucleic acid monolayer forms the microarray region.
For reproducibility of the screen-printed electrode chip, we chose to site-cleave the uracil base at the end of the hairpin DNA (C-H1 or C-H2) with uracil DNA glycosylase and endonuclease, so that the intermediate was detached from the nucleic acid monolayer anchored to the gold electrode surface.
Compared with the conventional electrochemical biosensor, the catalytic regenerable electrochemical biosensor driven by CRISPR/Cas13a in the invention has the following advantages for detecting nucleic acid: 1) the catalytic and renewable electrochemical biosensor driven by CRISPR/Cas13a has a Cas-CHDC two-stage signal amplification system, can detect a small amount of target RNA at the early stage of cancer, and has high selectivity and sensitivity; 2) the chip has a reproducible function and can be recycled for many times; 3) different biomarkers can be continuously detected, and multi-element detection is realized; 4) the detection time is short and is less than 6 minutes.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic illustration of the preparation of a biosensor according to the present invention;
FIG. 2 is a reproducible atomic force electron microscope image of a nucleic acid monolayer on the surface of an electrode;
FIG. 3 is a graph of typical oxidation peaks for Methylene Blue (MB) and ferrocene (Fc) read by SWV for measurements 1, 20, and 45;
FIG. 4 is a graph of ratio correction (IMB/IFc) as a function of increasing number of times;
FIG. 5 is a graph of electrical signals for detection of MB and Fc at 6 minute time points with different concentrations of miR-17(0.5 fM-5 nM) and PBS as background signals;
FIG. 6 is a graph of IMB/IFC as a function of miR-17 concentration (0.5 fM-5 nM);
FIG. 7 is a diagram of MB oxidation peaks with miR-17 as the target chain;
FIG. 8 is a diagram showing the MB oxidation peak of miR-155 as a target chain;
FIG. 9 is a diagram showing MB oxidation peaks of TTF-1mRNA as a target strand;
FIG. 10 is a graph of the results of a sensor chip differentiating miR-17 from non-target RNAs of the same family;
FIG. 11 is a graph of risk assessment of miR-17, miR-155 and TTF-1mRNA crosstalk in a sample from the same patient;
FIG. 12 is a plot of risk assessment of crosstalk for miR-17 in four different patient samples.
Detailed Description
The invention will be further explained and illustrated with reference to specific examples, without however being restricted thereto.
Example 1: preparation and physical and chemical property characterization of CRISPR/Cas13 a-driven catalytic renewable electrochemical biosensor
Example 1 describes a simple method to prepare CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor (fig. 1A). And fixing the PDMS template on a screen printing electrode by a micro-contact pasting technology to prepare a chip. Adding thiolated DNA solution into the pore plate to form a self-assembled monolayer on the surface of the gold electrode. The Cas13a/crRNA complex specifically recognizes a target nucleic acid strand by base sequence complementarity and activates the trans-cleavage activity of Cas13a protein, cleaving the Trigger strand (Trigger), thereby initiating the hairpin DNA catalytic loop (H1, H2, and Reporter), producing a large amount of C-I2 complex of C-H1 and C-H2. The nucleic acid monomolecular layer anchored on the surface of the gold electrode specifically recognizes C-I2. The target nucleic acid strand is quantified by detecting an electrical signal by an electrochemical workstation. FIG. 1B depicts the reproducibility of nucleic acid monolayers on the surface of electrodes. Atomic force electron microscopy images show: the surface of the gold electrode (A), the surface of the gold electrode (B) modified by thiolated DNA, the surface of the electrode (C) hybridized with a complementary nucleic acid chain and the surface of the gold electrode (D) after enzyme digestion reaction.
Example 2 CRISPR/Cas13 a-driven reproducible Performance and sensitivity testing of catalytic reproducible electrochemical biosensors
In this example, in order to evaluate the reproducible performance of the sensor, 45 repeated target nucleic acid strand detection experiments were performed on a single chip. Fig. 2A shows typical oxidation peaks of Methylene Blue (MB) and ferrocene (Fc) read by SWV in 1 st, 20 th, and 45 th measurements. For MB alone, the oxidation peak gradually decreased with increasing number of measurements, similar to the electrochemical biosensor with only one reporter molecule, resulting in a wide difference of 34.8%. However, application of the ratio correction (IMB/IFc) showed an average response of 0.61 and reduced the variability to a large extent to 2.2% (fig. 2B). We observed that at least 37 consecutive and stable measurements could be made on a single sensor chip (fig. 2B; red highlighted area).
Example 3 CRISPR/Cas13 a-driven sensitivity testing of catalytic regenerable electrochemical biosensors
In this example, MB and Fc electrical signals were detected at 6 minute time points with different concentrations of miR-17(0.5 fM-5 nM) and PBS as background signals (FIG. 3A). FIG. 3A shows that the MB oxidation peak is proportional to the concentration of the target nucleic acid chain (miR-17), while the signal response of Fc shows no significant change at different miR-17 concentrations. Using direct electrochemical readings at room temperature, the sensor chip was able to detect miR-17 levels as low as 0.5fM, with a dynamic range extending to 5 nM. The calibration curve shows that IMB/IFC increases in proportion to miR-17 concentration (0.5 fM-5 nM) (FIG. 3B). By fitting the measured data points to a four-parameter sigmoidal curve, the limit of detection (LOD) for miR-17 can be determined to be 50aM, with a sample/reagent volume consumed per reaction of less than 10 μ L, and a detection time of no more than 6 minutes.
Example 4 CRISPR/Cas13 a-driven specific testing of catalytic regenerable electrochemical biosensors
In this example, the target RNA is distinguished from other RNAs having one to several base mismatches. Compared with the MB oxidation peak of 5pM miR-17, almost no peak is generated at the same concentration of miR-155 or TTF-1 mRNA. IMB/IFc of miR-17 was 7.8 and 6.8 fold higher than miR-155 and TTF-1mRNA, respectively (FIG. 4A). Similar results were obtained with miR-155 and TTF-1mRNA as the target strands, respectively (FIGS. 4B-C). And the sensor chip is able to distinguish miR-17 from non-target RNAs of the same family: miR-106a (1-base mismatch), miR-20a and miR-20b (2-base) are not matched) (p <0.0001) (FIG. 4D).
Example 5 CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor for multiobjective RNA detection in serum of NSCLC patients
The crosstalk risk assessment was performed on serum samples of non-small cell lung cancer (NSCLC) patients, miR-17, miR-155 and TTF-1mRNA (in the same sample (FIG. 5A)) and miR-17 (in four different samples, FIG. 5B), and the results indicated that there was no crosstalk in the measurements between the serum samples.
Claims (18)
1. A CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor, wherein the CRISPR/Cas13 a-driven catalytic regenerable electrochemical biosensor comprises:
(a) the reproducible silk-screen printing electrode chip comprises a silk-screen printing electrode and a soft pore plate loaded with a liquid sample, wherein the silk-screen printing electrode comprises a gold working electrode, a platinum counter electrode and a silver/silver chloride indicating electrode;
(b) the redox group modified nucleic acid chain monomolecular layer is anchored on the surface of the gold working electrode;
(c) a CRISPR/Cas13a system comprising a Cas13a protein, crRNA, and a promoter molecule;
(d) hairpin DNA catalytic loops with redox groups and uracil DNA modifications.
2. The CRISPR/Cas13a driven catalytic regenerable electrochemical biosensor of claim 1, comprising:
(a) a solid substrate of a screen printing electrode, wherein the solid substrate is glass, ceramic, a silicon wafer or polymethyl methacrylate;
(b) and the soft orifice plate is loaded with a liquid sample and is made of Polydimethylsiloxane (PDMS), polyphenylene oxide (PPO) or other engineering plastics.
3. The monomolecular layer of nucleic acid strands anchored to the surface of a gold working electrode according to claim 1 comprising thiolated DNA, amino-modified DNA, or RNA.
4. The redox group of claim 1 including but not limited to ferrocene, methylene blue, anthraquinone and the like.
5. The nucleic acid strand monolayer of claim 3 is between 5-50 nucleotides in length.
6. The CRISPR/Cas13a system of claim 1 that specifically recognizes a target nucleic acid strand.
7. The target nucleic acid strand of claim 6 comprising, but not limited to, small molecules RNA (miRNA) and messenger RNA (mRNA).
8. The promoter molecule of claim 1 which is an RNA-modified hairpin DNA nucleic acid strand characterized by an oligonucleotide forming a hairpin structure of about 12 base pairs at the 5 'or 3' end itself and having an internal loop structure of 2 to 5 ribonucleotide tags.
9. The RNA-modified hairpin DNA nucleic acid strand of claim 8 wherein the RNA modification comprises adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or a combination thereof.
10. The RNA-modified hairpin DNA nucleic acid strand of claim 8, the RNA modification site being cleaved by the activated Cas13a protein, thereby breaking and melting.
11. The hairpin DNA catalytic loop of claim 1 comprising:
(a) two hairpin DNAs (C-H1 and C-H2) that can potentially combine into a double strand;
(b) one of the hairpin DNAs is labeled at the 5 'or 3' end with a redox group and contains 3-7 uracil bases at the 5 'or 3' end.
12. The hairpin DNA catalytic circuit of claim 11 wherein C-H1 and C-H2 undergo a chain hybridization reaction in the presence of the target nucleic acid strand to produce C-I2.
13. The C-I2 of claim 12 bound to a nucleic acid monolayer on the surface of a gold electrode to form a complex.
14. The method as claimed in claim 13, wherein the complex formed by the binding of C-I2 and the nucleic acid monolayer on the surface of the gold electrode is used for detecting the electric signal, and then the complex is washed away by using a solvent.
15. The method of claim 14, wherein the nucleic acid monolayer on the surface of the gold electrode is regenerable.
16. The CRISPR/Cas13a driven catalytic regenerable electrochemical biosensor of any one of claims 1-15 for quantifying a target nucleic acid strand in a sample comprises the steps of:
(1) fixing the soft orifice plate on a screen printing electrode by a micro-contact pasting technology to prepare a chip;
(2) adding a nucleic acid chain/competitive chain solution into the pore plate to form a self-assembled monolayer on the surface of the gold electrode;
(3) mixing a target nucleic acid strand with the CRISPR/Cas13a system and the hairpin DNA catalytic loop;
(4) and (4) dropwise adding the mixed solution in the step (3) into a soft pore plate filled with the liquid sample, and quantifying the target nucleic acid chain by analyzing the output electric signal, wherein the electric signal is in direct proportion to the concentration of the target nucleic acid chain.
17. The competitor strand of claim 16, comprising a base sequence complementary to a nucleic acid strand.
18. The CRISPR/Cas13a driven catalytic regenerable electrochemical biosensor of claim 1, which detects body fluid samples comprising blood, serum, plasma, urine, saliva, etc.
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