Fluorescent biosensor, preparation method thereof, miRNA-21 detection kit and application

Abstract

The invention relates to the technical field of biology, in particular to a fluorescent biosensor, a preparation method of the fluorescent biosensor, a miRNA21 detection kit and application. The fluorescent biosensor comprises a Cas12a protein, a crRNA sequence of Cas12a, a closed chain, an activated chain and an ssDNA reporter probe modified with a fluorescence reporter group and a fluorescence quenching group, wherein the nucleotide sequence of the crRNA sequence is as shown in SEQ ID NO. 1. According to the application, cascade strand displacement isothermal amplification is combined with Cas nuclease, and one-step rapid detection of miRNA21 can be realized through fluorescence signal output; the miRNA21 can be rapidly, efficiently and accurately detected only by DNA and RNA sequence design and Cas12a protein and by utilizing the cleavage of the Cas protein to release a fluorescence signal, so that the detection difficulty and the detection cost are greatly reduced, and the miRNA21 detection kit is suitable for an environment with limited resources and on-site rapid detection.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a fluorescent biosensor, a preparation method thereof, a miRNA-21 detection kit and an application thereof. Background Art

[0002] In recent years, nucleic acid tumor markers have become a research hotspot, among which microRNA (miRNA) has attracted the most attention. miRNAs are a class of endogenous non-coding single-stranded nucleic acid molecules composed of 19-23 nucleotides. They play an important regulatory role in various physiological or pathological processes such as cell proliferation, metabolism, differentiation, gene expression, and immune response, and are closely related to the occurrence, development, and prognosis of tumors. MicroRNA-21 (miRNA-21) is a small RNA that can regulate gene expression and plays an important role in autophagy of tumor cells. Abnormal expression of miRNA-21 is considered to be a reliable biomarker for early diagnosis of cancer and specific diseases. MiRNA-21 is one of the earliest discovered oncogenic miRNAs and is closely related to the occurrence and development of various cancers such as bladder cancer, colorectal cancer, glioma, breast cancer, ovarian cancer, lung cancer, and cervical cancer. Therefore, miRNA-21 is an important tool for monitoring the progression of various cancers and diseases, and its accurate quantification is crucial for timely feedback on the patient's condition.

[0003] At present, the detection methods of miRNA include Northern Blot, electrochemical detection, reverse transcription polymerase chain reaction (RT-PCR), microarray technology, high-throughput sequencing (Next-Generation Sequencing, NGS), etc. These traditional detection methods are cumbersome, complex and time-consuming, require expensive instruments, and have low sensitivity and selectivity. Although the use of qRT-PCR to detect miRNA is considered to be a very reliable "gold standard" in many studies and clinical diagnosis, the whole process is complicated. qRT-PCR relies on the design of specific primers, has high requirements for the purity and quality of RNA, and the operation process is cumbersome and expensive, including isolating miRNA from the sample, extending miRNA, reverse transcribing to form cDNA, and finally amplifying the cDNA at different temperatures, so expensive instruments are required.

[0004] In summary, how to detect miRNA conveniently, quickly, efficiently and accurately remains a huge challenge. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a fluorescent biosensor, a preparation method thereof, a miRNA-21 detection kit and application thereof, so as to detect miRNA21 conveniently, quickly, efficiently and accurately.

[0006] To achieve the above objectives, the scheme of this application is as follows:

[0007] In the first aspect, the present invention provides a fluorescent biosensor, which includes a Cas12a protein, a crRNA sequence of Cas12a, a closed chain, an activation chain, and a ssDNA reporter probe modified with a fluorescent reporter group and a fluorescent quencher group, and the nucleotide sequence of the crRNA sequence is shown in SEQ ID NO.1.

[0008] Optionally, the nucleotide sequence of the closed chain is shown as SEQ ID NO.2.

[0009] Optionally, the nucleotide sequence of the activation chain is shown in SEQ ID NO.3.

[0010] Optionally, the nucleotide sequence of the ssDNA reporter probe is shown in SEQ ID NO.4.

[0011] Optionally, the molar ratio of the Cas12a protein, crRNA sequence, closed strand, activation strand and ssDNA reporter probe is 1-3:1-3:12-18:3-6:25-35.

[0012] In a second aspect, the present invention also provides a method for preparing the fluorescent biosensor as described above, the preparation method comprising the following steps:

[0013] The Cas12a protein is co-incubated with the activation chain, the closed chain and the ssDNA reporter probe are added, and incubated again to obtain the fluorescent biosensor.

[0014] Optionally, the co-incubation temperature is 35-40° C., and the co-incubation time is 20-40 min.

[0015] Optionally, the temperature of the re-incubation is 35-40° C., and the duration of the re-incubation is 50-70 min.

[0016] In a third aspect, the present invention also provides use of the fluorescent biosensor as described above or the fluorescent biosensor prepared according to the method as described above in the preparation of a miRNA-21 detection kit.

[0017] In a fourth aspect, the present invention further provides a miRNA-21 detection kit, which comprises the fluorescent biosensor as described above or the fluorescent biosensor prepared according to the method as described above.

[0018] Beneficial effects of the present invention:

[0019] This application combines cascade strand displacement isothermal amplification with Cas nuclease, and can achieve one-step rapid detection of miRNA21 through fluorescent signal output.

[0020] The cascade strand displacement reaction of the present application can realize the recycling of target miRNA and achieve sensitive detection at lower concentrations of target miRNA, and has a lower detection limit than traditional miRNA detection methods.

[0021] The fluorescent biosensor of the present application can detect miRNA21 in one step under low temperature conditions without the need for temperature change and is simple to operate. Compared with existing miRNA21 detection technologies, it reduces the requirements for experimental conditions and operators.

[0022] This application only requires DNA, RNA sequence design and Cas12a protein, and uses the cutting of Cas protein to release fluorescent signals, which greatly reduces the difficulty and cost of detection and is suitable for resource-limited environments and rapid on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0024] Figure 1 This is the detection principle diagram of the present application, release&cycle means release&cycle, trans-cleavage means trans-cleavage, and Reporter means ssDNA reporter probe;

[0025] Figure 2 This is the electrophoresis diagram and fluorescence signal comparison diagram in feasibility verification. Figure 2 A is the electrophoresis diagram, Figure 2 B is a fluorescence signal comparison chart, Wavelength indicates wavelength, Fluorescence intensity indicates fluorescence intensity, Blank indicates blank control, without indicates no addition, the same below;

[0026] Figure 33A is the detection result diagram during the parameter optimization process, 3B is the detection result diagram during the optimization process of the molar ratio of crRNA to A-RNA, 3C is the detection result diagram during the optimization process of the length of the ssDNA reporter probe, 3D is the detection result diagram during the optimization process of the incubation time, S / N ratio represents the S / N ratio, Length of reporter represents the length of the ssDNA reporter probe, and Time of incubation represents the incubation time;

[0027] Figure 4 4A is a diagram showing the detection results during the optimization of the activated chain B-DNA, and 4B is a diagram showing the detection results during the optimization of the closed chain A-RNA;

[0028] Figure 5 5A is the result of the enzyme reaction kinetics test before and after optimization. 5B is the curve obtained by Michaelis-Menten kinetic analysis. 5C is the result of the sensitivity test of the system after optimization. 5D is the result of Michaelis-Menten kinetic analysis. After optimization means after optimization, Time means time, substrate means substrate, and Before optimization means before optimization.

[0029] Figure 6 6A is a graph showing the results of the sensitivity test, 6B is a graph showing the results of the fluorescence intensity test, and 6C is a linear relationship graph. Concentration of miRNA21 indicates the concentration of miRNA21.

[0030] Figure 7 7A is a diagram showing the test results of the repeatability test, 7B is a diagram showing the test results of the stability test, this method indicates the method of the present application, group indicates the group, and day indicates the day;

[0031] Figure 8 Figure 8 is a graph of specificity test results. 8A is a histogram of fluorescence intensity at 520 nm for detecting different targets. 8B is a fluorescence spectrum graph for detecting different targets. Mix is ​​a mixed sample of 10 miRNAs. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] like Figure 1 As shown, the present application designs the crRNA sequence of Cas12a based on the RNA sequence of miRNA21:

[0034] When the target miRNA21 is present, the primary cascade strand displacement of the present application will start, and the target miRNA21 will start to exchange with the crRNA strand from the teohold at the 3' end to form a more stable double strand, displacing the sequence A-RNA strand;

[0035] The secondary cascade chain displacement is used to activate the single-stranded DNA of Cas12a: under the action of the activation chain B-DNA, the 5' end of the teohold is located at the relatively proximal end, and the base complementary pairing with the crRNA chain is carried out. The displaced target miRNA21 enters the primary cascade chain displacement process again, thereby achieving the amplification of the inner cycle;

[0036] On the basis of this method, the present application sets the closed chain A-RNA (A-3'-Remain n-Toehold m) and the activated chain B-DNA (B-3'-Remain i-Toehold l) sequences with different fulcrum and retention point lengths, which can not only adjust the SDR rate by changing the length of the fulcrum, but also prevent the entire system from undergoing a chain displacement process in the absence of a target, which is the key to achieving one-step isothermal amplification;

[0037] Finally, an ssDNA reporter probe Reporter modified with a fluorescent reporter group and a fluorescent quencher group was added to the reaction system and introduced into the detection system to further evaluate the trans-cleavage activity triggered by Cas12a in the one-step isothermal amplification of the inner cycle Cas12a, thereby realizing the detection of miRNA21.

[0038] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0039] Experimental materials: All oligonucleotides, RNase inhibitors and DEPC water purified by HPLC were purchased from Sangon Biotech (Shanghai) Co., Ltd., 6× loading buffer and 20-200bp marker were purchased from Takara Biotech (Dalian) Co., Ltd., Cas12a protein, Cas12a diluent, NEbuffer 3, NEbuffer r2.1, and NEbuffer 4 were purchased from New England Biotechnology (Beijing) Co., Ltd. All chemical reagents were of analytical grade, and all aqueous solutions were prepared using DEPC water.

[0040] Experimental instruments: Fluorescence spectra were measured on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, CA), real-time fluorescence detection was measured on a QuantStudioTm 1Plus real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA), and native polyacrylamide gel electrophoresis (PAGE) was performed on a Bio-Rad (USA) protein electrophoresis instrument.

[0041] (I) Construction of a one-step isothermal amplification system for the inner circulation Cas12a

[0042] The internal cycle Cas12a reaction system was constructed according to the following steps, and all sequences used were dissolved in DEPC water buffer.

[0043] S1. Blocking crRNA with closed chain A-RNA: Mix 2 μL crRNA (1 μM) with 2 μL A-RNA (2 μM, specifically SEQ ID NO. 21), place in a water bath at 75°C for 5 min, and slowly cool to room temperature to form complex Y;

[0044] S2. Preparation of the inner cycle Cas12a reaction system: Mix 4 μL of complex Y with 2 μL of Cas12a (1 μM), and incubate at 37°C for 30 min to obtain a Y+Cas12a complex, and then prepare the inner cycle Cas12a reaction system according to Table 1:

[0045] Table 1 Internal cycle Cas12a reaction system

[0046] Y+Cas12a 6μL miRNA-21 5μL(20nM) / 0μL(Blank) B-DNA 3μL(5μM) 10×NEBuffer3.0 10μL DEPC water 68μL / 73μL(Blank) Reporter is ssDNA reporter probe 6μL(5μM)

[0047] Note: In the table, B-DNA refers to SEQ ID NO.42, Reporter refers to Reporter (15nt) 68 μL / 73 μL, and the volume after the slash refers to the amount of the blank control group.

[0048] After mixing the components, they were incubated again at 37°C for 60 min and then inactivated at 65°C for 10 min to prepare a fluorescence detection system.

[0049] The nucleotide sequences involved are shown in Table 2. All nucleotide sequences were synthesized by biological companies.

[0050] Table 2 Nucleotide sequences

[0051]

[0052]

[0053] Several fluorescence detection systems were prepared, and the differences between each system and Table 1 are:

[0054] One of the fluorescence detection systems only adds Reporter (15nt), one of the fluorescence detection systems only adds activation chain B-DNA, one of the fluorescence detection systems only adds miRNA-21, one of the reaction systems only adds crRNA, one of the fluorescence detection systems only adds A-RNA, one of the fluorescence detection systems only adds crRNA and A-RNA, one of the fluorescence detection systems only adds Cas12a, crRNA, A-RNA and 15ntReporter (i.e. Without B and miRNA-21), one of the fluorescence detection systems only adds Cas12a, crRNA, A-RNA, B-DNA and 15ntReporter (i.e. Blank control (Blank), one of the fluorescence systems is Cas12a, crRNA, A-RNA, B-DNA, miRNA21 and 15ntReporter (i.e. With miRNA).

[0055] (II) Feasibility Verification

[0056] PAGE electrophoresis was used to verify each detection process in (I), and the results are as follows Figure 2 As shown in A, wherein the lane marker is a 20bp-500bp DNA Marker; lane 1 is a Reportor (15nt); lane 2 is a B-DNA; lane 3 is miRNA21; lane 4 is crRNA; lane 5 is A-RNA; lane 6 is crRNA+A-RNA; lane 7 is Cas12a+crRNA+A-RNA+Reportor (15nt); lane 8 is Cas12a+crRNA+A-RNA+B-DNA+Reportor (15nt); lane 9 is Cas12a+crRNA, A-RNA+B-DNA+miRNA21+15nt Reporter.

[0057] like Figure 2 As shown in A, lanes 1, 2, 3, 4, and 5 correspond to ssDNA, B-DNA, miRNA21, crRNA, and A-RNA, respectively. Lane 6 represents a double-stranded band formed after crRNA and A-RNA annealing. Lane 7 is Cas12a, crRNA and A-RNA double-stranded, and Reportor (15nt). The result shows that crRNA cannot activate Cas12a by binding to closed chain A-RNA. Lane 8 represents Cas12a, crRNA and A-RNA double-stranded, B-DNA, and Reportor (15nt), and the trans-cleavage activity of Cas12a is still not activated. The result shows that the activation chain B-DNA cannot open crRNA and closed chain A-RNA, and the closed chain A-RNA can play an excellent blocking role. Lane 9 shows that the double-stranded bands of crRNA and A-RNA become weaker. This result shows that miRNA21 can replace A-RNA through the chain, and B-DNA disappears, that is, B-DNA is also involved in the chain replacement, thereby generating an internal cycle. The disappearance of the Reportor (15nt) band indicates that the trans-cutting activity of Cas12a is activated.

[0058] 100uL of the corresponding fluorescence detection system of the blank control group (i.e., Blank), the positive control group (i.e., With miRNA21), and the corresponding fluorescence detection system of without B and miRNA-21 were added to the quartz cuvettes respectively, and the fluorescence spectrophotometer was used for measurement. The specific steps are as follows:

[0059] Fluorescence cuvette cleaning: Soak the fluorescence cuvette in alcohol and clean it with DEPC water;

[0060] Setting parameters: set the excitation wavelength to 492nm, the emission wavelength range to 500-600nm, and the voltage to 600V;

[0061] Zero adjustment: Add DEPC water to the fluorescence cuvette to adjust the zero;

[0062] Perform the test: Add the reaction solution into the fluorescent cuvette, click on the test button, and you can get the fluorescent signal. Figure 2 As shown in B.

[0063] like Figure 2 As shown in B, when the target miRNA21 is not added to the reaction system, the activation chain B-DNA cannot bind to the crRNA blocked by the blocking chain A-RNA to activate Cas12a. Only when the target miRNA21 is present can the signal of the reaction system be restored. This result shows that the fluorescence detection system of the present application is feasible.

[0064] (III) Optimization

[0065] In addition to adjusting the molar ratio of crRNA to A-RNA to 1:1, 1:1.5, 1:2, 1:2.5 and 1:3, several fluorescence detection systems were prepared under the same conditions as Table 1 to optimize the ratio of crRNA to A-RNA. Each group of experiments was repeated three times. The results are shown in Figure 3 As shown in A.

[0066] Depend on Figure 3 A shows that the optimal molar ratio of crRNA to A-RNA is 1:2.

[0067] Several fluorescence detection systems were prepared under the same conditions as in Table 1 except that the molar ratio of crRNA to A-RNA was 1:2 and NEbuffer r2.1, NEbuffer r3 and NEbuffer r4 were used respectively to optimize the specific selection of NEbuffer. Each group of experiments was repeated three times. The results are shown in Figure 3 As shown in B.

[0068] Depend on Figure 3 B shows that the best type of NEbuffer is NEbuffer 3.

[0069] Several fluorescence detection systems were prepared under the same conditions as in Table 1 except that the molar ratio of crRNA to A-RNA was 1:2, NEbuffer was NEbuffer 3, and Reporter was Reporter (5nt), Reporter (6nt), Reporter (11nt), and Reporter (15nt), respectively, to optimize the length of Reporter. Each group of experiments was repeated three times. The results are shown in Table 1. Figure 3 As shown in C.

[0070] Depend on Figure 3 C shows that the optimal length of Reporter is 6nt, that is, the best choice of Reporter is Reporter(6nt).

[0071] Several fluorescence detection systems were prepared under the same conditions as in Table 1, except that the molar ratio of crRNA to A-RNA was 1:2, NEbuffer was NEbuffer 3, Reporter (6nt) was selected, and the incubation time at 37°C was 0.5h, 1h, 1.5h, 2h, and 2.5h, respectively, to optimize the incubation time. Each group of experiments was repeated three times. The results are shown in FIG. Figure 3 As shown in D.

[0072] Depend on Figure 3 D shows that the optimal incubation time is 1h.

[0073] In summary, the inner cycle Cas12a one-step isothermal amplification detection system of the present application has a better fluorescence signal when the molar ratio of crRNA to A-RNA is 1:2, NEbuffer 3, Reporter (6nt) and the reaction time is 1h.

[0074] In order to evaluate the effect of the support and retention point length on the effectiveness of SDR, the present application designs the closed chain A-RNA and the activation chain B-DNA with different base numbers participating in the two reactions SDR, which have different support and retention point lengths and positions during the reaction. In the first step SDR, the support point length of the closed chain A-RNA varies from 4 to 10 nucleotides, represented by m, and the retention point length varies from 4 to 7 nucleotides, represented by n. In the second step SDR, the support point length of the activation chain B-DNA varies from 4 to 7 nucleotides, represented by l, and the retention point length varies from 4 to 7 nucleotides, represented by i, and they are named A-3'-Remain n-Toehold m and B-3'-Remain i-Toehold l, respectively.

[0075] In order to explore the optimal length and position of the fulcrum and retention point, the present application conducted a pass-through detection analysis. Specifically, in addition to the above-mentioned optimal parameter conditions and the selection of different activation chain B-DNA (see Table 2 for details), several fluorescent detection systems were prepared under the same conditions as Table 1 to optimize the activation chain B-DNA. Each group of experiments was repeated three times. The results are as follows Figure 3 D, a fluorescence detection system was prepared to optimize the activation chain B-DNA. Each group of experiments was repeated three times. The results are shown in Figure 4 As shown in A.

[0076] like Figure 4 As shown in A, with the increase of the branch length, the initial rate constant (V0) also shows an order of magnitude increase. The retention point has little effect on the primary chain displacement reaction, but in the cascade chain displacement reaction, the selection of the retention point can reduce the false positive rate. Finally, B-3'-Remain 6-Toehold 7 (i.e., 3'-B6-7) with a branch of 7nt and a retention point of 6nt was selected as the optimal B-DNA, i.e., the activation chain.

[0077] According to the above optimal parameter conditions, the best B-DNA and different activation chain A-RNA (see Table 2 for details), several fluorescence detection systems were prepared under the same conditions as Table 1 to optimize the activation chain A-RNA. Each group of experiments was repeated three times. The results are as follows: Figure 4 As shown in B.

[0078] like Figure 4As shown in Figure B, with the increase of the length of the fulcrum, the initial rate constant (V0) also shows an order of magnitude increase. The retention point has little effect on the primary chain displacement reaction, but in the cascade chain displacement reaction, the selection of the retention point can reduce the false positive rate. Finally, A-3'-Remain6-Toehold9 (i.e., the best closed chain A-RNA) with a fulcrum of 9nt and a retention point of 6nt (i.e., 3'-A6-9) was selected as the displacement substrate for cascade chain displacement.

[0079] (IV) Michaelis-Menten kinetic test

[0080] To further explore the enzyme activity of Cas12a under the optimal reaction parameters, the application performed a Michaelis-Menten kinetic experimental analysis, and the specific steps were as follows: in the above-mentioned optimal parameter conditions, the optimal B-DNA, the optimal activation chain A-RNA and the concentration of Reporter (6nt) were 1nM, 10nM, 100nM, 200nM, 350nM, 500nM, 1uM and 2uM, respectively, and several copies of fluorescence detection systems (i.e., optimized systems) were prepared under the same conditions as Table 1, and compared with the system before optimization (except 3'-B6-5, 3'-A5-6, NEBuffer r2.1 and Reporter (11nt), several copies of fluorescence detection systems were prepared in the same manner as the optimized reaction system), and the real-time fluorescence of the above-mentioned experimental group was recorded by a fluorescence spectrophotometer. Each group of experiments was repeated three times, and the results are as follows Figure 5 As shown in A and 5C;

[0081] Michaelis-Menten kinetic analysis was performed under the corresponding parameters before and after optimization. The results are as follows Figure 5 B and 5D.

[0082] like Figure 5 As shown, compared with the system before optimization (3'-B6-5, 3'-A5-6, NEBuffer r2.1 and Reporter (11nt) combined with Cas12a), the initial reaction rate of the optimized system (3'-B6-7, 3'-A6-9, NEBuffer 3 and Reporter (6nt) reporter gene combined with Cas12a) was significantly improved (as shown in Figure 2). Figure 5 A and 5C, the kcat / Km ratio increased by about 11 times (e.g. Figure 5 B and 5D).

[0083] (V) Sensitivity test

[0084] To analyze the sensitivity of the inner circulation Cas12a one-step isothermal amplification system of the present application to detect miRNA21, the present application performs a linear analysis, and the specific steps are as follows: in addition to the above-mentioned optimal parameter conditions, the optimal B-DNA, the optimal activation chain A-RNA and the concentration of Reporter (6nt) being 300nM, Blank (i.e., blank), 0.1pM, 0.5pM, 1pM, 5pM, 10pM, 50pM, 100pM, 500pM and 1000pM different concentrations of miRNA21 were set, and the fluorescence spectrum of the above-mentioned experimental group was recorded by a fluorescence spectrophotometer. Each group of experiments was repeated three times, and the results are as follows Figure 6 shown.

[0085] like Figure 6 As shown, the present application has significant advantages in detecting low-abundance miRNA. Specifically, the detection system has a good linear relationship from 500fM to 100pM, and the detection limit is as low as 145fM (e.g. Figure 6 A and Figure 6 B). The results show that the one-step isothermal amplification system of the inner cycle Cas12a of the present application is used to detect miRNA21 with high sensitivity.

[0086] (VI) Repeatability and stability test

[0087] In order to evaluate the repeatability and stability of the inner cycle Cas12a one-step isothermal amplification system of the present application in detecting miRNA21, so as to ensure that it can continue to provide accurate and reliable detection results in practical applications, the present application verifies the repeatability and stability of the present application according to the optimal system. Specifically, the repeatability experiment sets up five sets of repeated reaction systems, and detects the fluorescence signals of the five experimental groups at the same time. The stability experiment sets up a set of reaction systems to detect the fluorescence signals of the experimental groups at 1, 3, 5, 7, 15 and 30 days respectively. Each of the above experiments was repeated three times, and the results are as follows: Figure 7 shown.

[0088] like Figure 7 As shown in A, the test results between the five replicate groups were highly consistent, with no obvious fluctuations or deviations. This result shows that the one-step isothermal amplification system of the inner cycle Cas12a of the present application has good repeatability. Figure 7 As shown in B, in the first 15 days, the fluorescence value remained stable, with no obvious downward trend or fluctuation, and there was an obvious downward trend at 30 days. The results show that the inner circulation Cas12a one-step isothermal amplification system of the present application has excellent stability.

[0089] (VII) Specificity test

[0090] To verify the specificity of the one-step isothermal amplification system of the inner cycle Cas12a in detecting miRNA21, the present application set up two experimental groups. Specifically, the detection target was replaced with different miRNA sequences according to the optimal detection system, and the fluorescence signal was recorded by fluorescence spectrophotometry. Each group of experiments was repeated three times, and the results are shown in the figure. Figure 8 shown.

[0091] like Figure 8 As shown, even in a complex environment with multiple miRNAs, branched RCA can accurately identify and detect miRNA21, with almost no interference from other non-miRNA21. This result shows that the inner cycle Cas12a one-step isothermal amplification system of the present application shows excellent specificity in distinguishing different types of miRNAs.

[0092] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A fluorescent biosensor, characterized in that: The fluorescent biosensor includes a Cas12a protein, a crRNA sequence of Cas12a, a closed chain, an activation chain, and a ssDNA reporter probe modified with a fluorescent reporter group and a fluorescent quencher group, and the nucleotide sequence of the crRNA sequence is shown in SEQ ID NO.

1.

2. The fluorescent biosensor according to claim 1, wherein The nucleotide sequence of the closed chain is shown as SEQ ID NO.

2.

3. The fluorescent biosensor according to claim 1, wherein The nucleotide sequence of the activation chain is shown in SEQ ID NO.

3.

4. The fluorescent biosensor according to claim 1, wherein The nucleotide sequence of the ssDNA reporter probe is shown in SEQ ID NO.

4.

5. The fluorescent biosensor according to claim 1, characterized in that: The molar ratio of the Cas12a protein, crRNA sequence, closed chain, activated chain and ssDNA reporter probe is 1-3: 1-3: 12-18: 3-6: 25-35.

6. The method for preparing the fluorescent biosensor according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: The Cas12a protein is co-incubated with the activation chain, the closed chain and the ssDNA reporter probe are added, and incubated again to obtain the fluorescent biosensor.

7. The preparation method according to claim 6, characterized in that: The co-incubation temperature is 35-40° C., and the co-incubation time is 20-40 min.

8. The preparation method according to claim 6, characterized in that: The temperature of the second incubation is 35-40° C., and the duration of the second incubation is 50-70 min.

9. Use of the fluorescent biosensor according to any one of claims 1 to 5 or the fluorescent biosensor prepared according to the method according to any one of claims 6 to 8 in preparing a miRNA-21 detection kit.

10. A miRNA-21 detection kit, characterized in that: The miRNA-21 detection kit comprises the fluorescent biosensor according to any one of claims 1 to 5 or the fluorescent biosensor prepared according to the method according to any one of claims 6 to 8.

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