A fluorescent biosensor, a preparation method thereof, an miRNA-21 detection kit and application thereof

Abstract

The application relates to the field of biotechnology, in particular to a fluorescent biosensor, a preparation method thereof, an miRNA21 detection kit and application. The fluorescent biosensor comprises a Cas12a protein, a crRNA sequence of the Cas12a, a blocking chain, an activating chain and an ssDNA report probe modified with a fluorescent report group and a fluorescent quenching group, and the nucleotide sequence of the crRNA sequence is shown in SEQ ID NO. 1. The application combines cascade chain displacement isothermal amplification and Cas nucleases, and realizes one-step rapid detection of miRNA21 through fluorescent signal output; the application only needs DNA and RNA sequence design and Cas12a protein, and the cutting of the Cas protein is used to release the fluorescent signal, so that miRNA21 can be quickly, efficiently and accurately detected, the detection difficulty and cost are greatly reduced, and the application is suitable for resource-limited environments and on-site rapid detection.

Description

TECHNICAL FIELD

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

[0002] In recent years, nucleic acid tumor markers have become a research hotspot, among which microRNAs (miRNAs) have attracted the most attention. miRNAs are a class of endogenous non-coding single-stranded nucleic acid small molecules composed of 19-23 nucleotides, which play an important regulatory role in various physiological or pathological processes such as cell proliferation, metabolism, differentiation, gene expression, immune response, and are closely related to the occurrence, development and prognosis of diseases. 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 certain 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 accurate quantification is crucial for timely feedback on patient conditions.

[0003] Currently, the detection methods of miRNA include Northern Blot, electrochemical detection, reverse transcription polymerase chain reaction (RT-PCR), microarray technology (Microarray), high-throughput sequencing (Next-Generation Sequencing, NGS), etc. These traditional detection methods are complicated, 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 "gold standard" very reliable in many research and clinical diagnoses, the entire process is complex, qRT-PCR relies on the design of specific primers, has high requirements for the purity and quality of RNA, is complicated and expensive to operate, including isolation of miRNA from samples, extension of miRNA, reverse transcription to form cDNA, and finally amplification of the cDNA at different temperatures, thus requiring expensive instruments.

[0004] In summary, how to conveniently, efficiently and accurately detect miRNA remains a great challenge. SUMMARY

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

[0006] To achieve the above object, the present application is as follows:

[0007] In a first aspect, the present application provides a fluorescent biosensor, wherein the fluorescent biosensor comprises a Cas12a protein, a crRNA sequence of Cas12a, a blocking strand, an activating strand, 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 as SEQ ID NO. 1.

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

[0009] Optionally, the nucleotide sequence of the activating strand is shown as SEQ ID NO. 3.

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

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

[0012] In a second aspect, the present application further provides a preparation method of the fluorescent biosensor as described above, wherein the preparation method comprises the following steps:

[0013] Incubating the Cas12a protein with the activating strand, adding the blocking strand and the ssDNA reporter probe, incubating again, and obtaining the fluorescent biosensor.

[0014] Optionally, the temperature of the incubation is 35-40℃, and the duration of the incubation is 20-40 min.

[0015] Optionally, the temperature of the incubation is 35-40℃, and the duration of the incubation is 20-40 min.

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

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

[0018] The present application has the following beneficial effects:

[0019] The application combines the cascade strand displacement isothermal amplification with the Cas nuclease, and realizes one-step rapid detection of miRNA21 through fluorescence signal output.

[0020] The cascade strand displacement reaction of the application can realize the recycling of the target miRNA, realize sensitive detection under a lower concentration of the target miRNA, and has a lower detection limit than the traditional miRNA detection method.

[0021] The fluorescence biosensor of the application can realize one-step detection of miRNA21 under low temperature conditions, without temperature change, and has simple operation, and compared with the existing miRNA21 detection technology, the requirement for experimental conditions and operators is reduced.

[0022] The application only needs DNA, RNA sequence design and Cas12a protein, and releases the fluorescence signal by using the cutting of the Cas protein, greatly reduces the detection difficulty and detection cost, and is suitable for resource-limited environment and on-site rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the structures shown in the drawings.

[0024] Figure 1 The detection principle diagram of the application, release & cycle represents release & cycle, trans-cleavage represents reverse cleavage, and reporter represents ssDNA reporter probe.

[0025] Figure 2 The electrophoresis diagram and fluorescence signal comparison diagram in the feasibility verification, Figure 2 A is the electrophoresis diagram, Figure 2 B is the fluorescence signal comparison diagram, wavelength represents wavelength, fluorescence intensity represents fluorescence intensity, blank represents blank control, without represents not added, and the same below,

[0026] Figure 3The graphs show the results of the parameter optimization process. 3A shows the results of the crRNA to A-RNA molar ratio optimization process, 3B shows the results of the NEbuffer selection optimization process, 3C shows the results of the ssDNA reporter probe length optimization process, and 3D shows the results of the incubation time optimization process. 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 The images show the detection results during the optimization of the closed strand A-RNA and the activated strand B-DNA. 4A shows the detection results during the optimization of the activated strand B-DNA, and 4B shows the detection results during the optimization of the closed strand A-RNA.

[0028] Figure 5 The graphs show the results of enzyme reaction kinetics before and after optimization. 5A shows the sensitivity detection results of the system before optimization, 5B shows the curve obtained from Michaelis-Menten kinetic analysis, 5C shows the sensitivity detection results of the system after optimization, and 5D shows the results of Michaelis-Menten kinetic analysis. "After optimization" indicates the result after optimization, "Time" indicates the time, "substrate" indicates the substrate, and "Before optimization" indicates the result before optimization.

[0029] Figure 6 The graphs show the results of the sensitivity test, 6A shows the results of the fluorescence intensity test, 6B shows the linear relationship graph, and the concentration of miRNA21 represents the concentration of miRNA21.

[0030] Figure 7 The graphs show the results of repeatability and stability tests. 7A shows the results of repeatability tests, and 7B shows the results of stability tests. "this method" refers to the method of this application, "group" refers to the group, and "day" refers to the number of days.

[0031] Figure 8 The results of the specificity test are shown in Figure 8A, which is a bar chart of fluorescence intensity at 520 nm for different targets; Figure 8B is a fluorescence spectrum of different targets; and Mix is ​​a mixed sample of 10 miRNAs. Detailed Implementation

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present application.

[0033] As shown in Figure 1 The present application designs the crRNA sequence of Cas12a according to the RNA sequence of miRNA21:

[0034] When the target miRNA21 exists, the primary cascade chain displacement of the present application is opened, and the target miRNA21 exchanges with the crRNA chain from the 3' end toehold, forming a more stable double strand, and displacing the sequence A-RNA chain;

[0035] The secondary cascade chain displacement is used to activate the single-stranded DNA of Cas12a: under the action of the activating chain B-DNA, from the toehold located in the relatively proximal end: 5' end, the target miRNA21 displaced from the crRNA chain enters the primary cascade chain displacement process again, so as to achieve the amplification effect of internal circulation;

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

[0037] Finally, the ssDNA reporter probe Reporter modified with a fluorescent reporter group and a fluorescent quenching group is added to the reaction system to introduce the detection system, further evaluate the transcleavage activity triggered by Cas12a in the internal circulation Cas12a one-step isothermal amplification, and thus realize the detection of miRNA21.

[0038] In the following description, a large number of details are discussed to provide a more thorough explanation of the embodiments of the present application, however, it is obvious for those skilled in the art that the embodiments of the present application can be implemented without these specific details.

[0039] Experimental materials: All oligonucleotides purified by HPLC, RNAase inhibitor and DEPC water were purchased from Shengong Bioengineering (Shanghai) Co., Ltd., 6x loading buffer and 20-200bp marker were purchased from Baobioengineering (Dalian) Co., Ltd., Cas12a protein, Cas12a diluent, NE buffer 3, NE buffer r2.1, NE buffer 4 were purchased from New England Biolabs (Beijing) Co., Ltd., all chemical reagents were analytical grade, and all aqueous solutions were prepared using DEPC water.

[0040] Experimental instruments: Fluorescence spectrum was 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 one-step isothermal amplification system of internal cycle Cas12a

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

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

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

[0045] Table 1 Internal cycle Cas12a reaction system

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

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

[0048] After mixing the components, incubate at 37°C for 60 min, then inactivate at 65°C for 10 min to prepare the fluorescence detection system.

[0049] The nucleotide sequences involved are shown in Table 2, all of which were synthesized by a biological company.

[0050] Table 2 Nucleotide sequences

[0051]

[0052]

[0053] And prepare several fluorescence detection systems, each system differs from Table 1 in that:

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

[0055] (ii) Feasibility verification

[0056] The detection process in (i) is verified by PAGE electrophoresis, and the results are shown in Figure 2 A, wherein lane Marker is 20bp-500bp DNA Marker; lane 1 is Reportor (15nt); lane 2 is 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 Reportor.

[0057] AsFigure 2 As shown in A, lanes 1, 2, 3, 4, 5 correspond to ssDNA, B-DNA, miRNA21, crRNA, A-RNA respectively. Lane 6 represents the band of double-stranded formed after annealing of crRNA and A-RNA. Lane 7 is Cas12a, crRNA and A-RNA double strand, Reportor (15nt). The results show that the combination of crRNA and blocking chain A-RNA cannot activate Cas12a. Lane 8 represents Cas12a, crRNA and A-RNA double strand, B-DNA, Reportor (15nt), the trans-cleavage activity of Cas12a is still not activated, and the results show that the activating chain B-DNA cannot open the crRNA and the blocking chain A-RNA, and the blocking chain A-RNA can play an excellent blocking effect. Lane 9 is the weak band of crRNA and A-RNA double strand, and the results show that miRNA21 can replace A-RNA by strand displacement, and B-DNA disappears, that is, B-DNA also participates in strand displacement, thereby generating an internal cycle, and the disappearance of the Reportor (15nt) band indicates that the trans-cleavage activity of Cas12a is activated.

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

[0059] Fluorescence cuvette cleaning: the fluorescence cuvette was soaked with alcohol and washed with DEPC water;

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

[0061] Zero adjustment: add DEPC water to the fluorescence cuvette for zero adjustment;

[0062] Detection: add the reaction solution to the fluorescence cuvette, click detection, and the fluorescence signal can be obtained, and the results are shown in Figure 2 B.

[0063] As shown in Figure 2 B, when no target miRNA21 is added in the reaction system, the activating chain B-DNA cannot bind to the crRNA blocked by the blocking chain A-RNA to activate Cas12a, and only when the target miRNA21 exists, the signal of the reaction system can be restored. The results show that the fluorescence detection system of the application has feasibility.

[0064] (Three) 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 respectively, 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, and the results are shown in Figure 3 A.

[0066] As can be seen from Figure 3 A, the optimal molar ratio of crRNA to A-RNA is 1:2.

[0067] In addition to the molar ratio of crRNA to A-RNA being 1:2 and NEbuffer being NEbuffer r2.1, NEbufferr3 and NEbuffer r4 respectively, several fluorescence detection systems were prepared under the same conditions as Table 1 to optimize the specific selection of NEbuffer, each group of experiments was repeated three times, and the results are shown in Figure 3 B.

[0068] As can be seen from Figure 3 B, the optimal selection of NEbuffer is NEbuffer 3.

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

[0070] As can be seen from Figure 3 C, the optimal length of Reporter is 6nt, that is, the optimal selection of Reporter is Reporter(6nt).

[0071] In addition to the molar ratio of crRNA to A-RNA being 1:2, NEbuffer being NEbuffer 3, Reporter being Reporter(6nt) and the incubation time at 37℃ being 0.5h, 1h, 1.5h, 2h and 2.5h respectively, several fluorescence detection systems were prepared under the same conditions as Table 1 to optimize the incubation time, each group of experiments was repeated three times, and the results are shown in Figure 3 D.

[0072] As can be seen from Figure 3 D, the optimal incubation time is 1h.

[0073] In summary, the one-step isothermal amplification detection system of the internal circulation Cas12a proposed in this application exhibits better fluorescence signal when the molar ratio of crRNA to A-RNA is 1:2, NEbuffer is 3, Reporter (6nt) is used, and the reaction time is 1h.

[0074] To evaluate the impact of fulcrum and retention point lengths on the effectiveness of SDR, this application designed closed-strand A-RNA and activated-strand B-DNA with different base numbers participating in two SDR reactions. They had different fulcrum and retention point lengths and positions during the reaction. In the first step of SDR, the fulcrum length of the closed-strand A-RNA varied from 4 to 10 nucleotides, denoted as m, and the retention point length varied from 4 to 7 nucleotides, denoted as n. In the second step of SDR, the fulcrum length of the activated-strand B-DNA varied from 4 to 7 nucleotides, denoted as l, and the retention point length varied from 4 to 7 nucleotides, denoted as i. They were named A-3'-Remain n-Toehold m and B-3'-Remain i-Toehold l, respectively.

[0075] To determine the optimal length and location of the fulcrum and retention point, this application conducted a pass-through detection analysis. Specifically, in addition to using the optimal parameters and selecting different activating B-DNA strands (see Table 2 for details), several fluorescence detection systems were prepared under the same conditions as in Table 1 to optimize the activating B-DNA strand. Each experiment was repeated three times, and the results are as follows: Figure 3 As shown in Figure D, a fluorescence detection system was prepared to optimize the activated B-DNA strand. Each experiment was repeated three times, and the results are as follows: Figure 4 As shown in Figure A.

[0076] like Figure 4 As shown in Figure A, the initial rate constant (V0) increases by orders of magnitude with the increase of the fulcrum length. 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 fulcrum of 7nt and a retention point of 6nt was selected as the optimal B-DNA, i.e., the activation chain.

[0077] Following the optimal parameters, selecting the best B-DNA, and choosing different activating strand A-RNAs (see Table 2 for details), several fluorescence detection systems were prepared under the same conditions as in Table 1 to optimize the activating strand A-RNA. Each experiment was repeated three times, and the results are as follows: Figure 4 As shown in B.

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

[0079] (Four) Michaelis-Menten kinetics test

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

[0081] And the Michaelis-Menten kinetics analysis was performed under the corresponding parameters before and after optimization, and the results are shown in Figure 5 B and 5D.

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

[0083] (Five) Sensitivity test

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

[0085] As shown in Figure 6 , the present application has a significant advantage in detecting low-abundance miRNA, and specifically, the detection system has a good linear relationship from 500 fM to 100 pM, and the detection limit is as low as 145 fM (as shown in Figure 6 A and Figure 6 B). The results show that the internal circulation Cas12a one-step isothermal amplification system of the present application for detecting miRNA21 has high sensitivity.

[0086] (VI) Reproducibility and stability test

[0087] To evaluate the reproducibility and stability of the internal circulation Cas12a one-step isothermal amplification system of the present application for detecting miRNA21, so as to ensure that it can continuously provide accurate and reliable detection results in actual application, the reproducibility and stability of the present application were verified according to the optimal system, and specifically, five sets of repeated reaction systems were set in the reproducibility experiment, and the fluorescence signals of the five experimental groups were detected at the same time. In the stability experiment, a set of reaction system was set, and the fluorescence signals of the experimental group were detected at 1, 3, 5, 7, 15, and 30 days, respectively. Each of the above experiments was repeated three times, and the results are shown in Figure 7 .

[0088] As shown in Figure 7 A, the detection results of the five repeated groups were highly consistent, and there was no obvious fluctuation or deviation. The results show that the internal circulation Cas12a one-step isothermal amplification system of the present application has good reproducibility. As shown in Figure 7 B, the fluorescence value remained stable without obvious downward trend or fluctuation in the first 15 days, and there was an obvious downward trend at 30 days. The results show that the internal 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 internal circulation Cas12a one-step isothermal amplification system for detecting miRNA21, two experimental groups were set up, specifically, the detection target was changed to 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 Figure 8 . .

[0091] As shown in Figure 8 , even in a complex environment with multiple miRNAs, the branched RCA can accurately identify and detect miRNA21, and is almost not interfered by other non-miRNA21. The results show that the internal circulation Cas12a one-step isothermal amplification system exhibits excellent specificity in distinguishing different types of miRNAs.

[0092] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A fluorescent biosensor, characterized by, The fluorescent biosensor comprises a Cas12a protein, a crRNA sequence of Cas12a, a blocking strand, an activating strand, and a ssDNA reporter probe modified with a fluorescent reporter group and a fluorescent quencher group, wherein the nucleotide sequence of the crRNA sequence is shown as SEQ ID NO. 1, the nucleotide sequence of the blocking strand is shown as SEQ ID NO. 2, the nucleotide sequence of the activating strand is shown as SEQ ID NO. 3, and the nucleotide sequence of the ssDNA reporter probe is shown as SEQ ID NO.

4.

2. Application of the fluorescent biosensor of claim 1 in the preparation of an miRNA-21 detection kit.

3. A miRNA-21 detection kit, characterized by, The miRNA-21 detection kit comprises the fluorescent biosensor of claim 1.

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