CN114894753A - Aptamer biosensor based on CRISPR-Cas14 system and application thereof - Google Patents
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Abstract
The invention discloses an aptamer biosensor based on a CRISPR-Cas14 system and application thereof, wherein the biosensor comprises a CRISPR-Cas14 report solution and an adapter activator, and the CRISPR-Cas14 report solution comprises a Cas14a protein, sgRNAs and a fluorescent reporter; the adapter activator contains an adapter sequence and can be specifically bound with a target object, and the adapter activator is complementary to the sgRNA partial sequence and can activate Cas14 a. The biosensor prepared by the invention has the characteristics of high sensitivity, high specificity, wide detection range, economy, high efficiency and high speed, and can be used for quickly analyzing various target objects including ATP and Cd 2+ Histamine, aflatoxins and thrombin, have broad application potential in clinical testing, food safety analysis and monitoring of environmental pollutants.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to an aptamer biosensor based on a CRISPR-Cas14 system and application thereof.
Background
The development of aptamer biosensors by using CRISPR-associated protein (Cas) is an important research trend in the current molecular diagnosis industry, and compared with the traditional enzyme-linked immunosorbent assay, western blotting and the like, the aptamer biosensors based on the Cas have the characteristics of simplicity in operation, low cost, rapidness in reaction and the like. However, most of the prior art is based on the Cas12a protein, and it is difficult to specifically recognize the aptamer sequence, so that the formed aptamer biosensor has defects in both sensitivity and specificity, and the market promotion of related products is limited. Therefore, there is a need to explore the application of novel Cas proteins in aptamer biosensing and to construct sensors with higher performance to break through the drawbacks of the prior art.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides an aptamer biosensor based on a CRISPR-Cas14 system, which has the advantages of simple operation, low reagent consumption, low cost, short time consumption, high sensitivity and high specificity.
Another object of the present invention is to provide an application of the aptamer biosensor based on CRISPR-Cas14 system in detecting target non-nucleic acid targets.
The technical scheme is as follows: in order to achieve the above objects, the present invention provides an aptamer biosensor based on CRISPR-Cas14 system, the biosensor comprising a CRISPR-Cas14 reporter solution, an aptamer activator; the CRISPR-Cas14 reporter solution includes a Cas14a protein, sgrnas, and a fluorescent reporter; the adapter activator contains an adapter sequence and can be specifically bound with a target object, and the adapter activator is complementary with the sgRNA partial sequence and can activate Cas14 a; the sgrnas can bind tightly to Cas14a, inducing Cas14a to recognize the adapter activator.
Further, the CRISPR-Cas14 report solution is formed by mixing Cas14a and sgRNA in a reaction buffer solution, then incubating, adding a fluorescent reporter and placing in an ice-water bath for storage.
Further, the aptamer biosensor also comprises a reaction buffer solution, wherein the reaction buffer solution comprises Na 2 HPO 4 /NaH 2 PO 4 NaCl and MgCl 2 And the pH value of the reaction buffer solution is 6.8-7.5.
Preferably, the reaction buffer has a pH of 6.8.
Further, Na in the reaction buffer solution 2 HPO 4 /NaH 2 PO 4 NaCl and MgCl 2 The molar mass ratio of (a) to (b) is 1-4: 10-15: 1-4.
Preferably, Na is contained in the reaction buffer solution 2 HPO 4 /NaH 2 PO 4 NaCl and MgCl 2 The molar mass ratio of (a) to (b) is 1:10: 1.
Wherein the sequence of the adaptive activator is shown in any one of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO. 10.
Wherein the sgRNA sequence is shown in any one of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 9.
Further, the fluorescent reporter is FAM-TTATT-BHQ 1.
The invention provides an application of an aptamer biosensor based on a CRISPR-Cas14 system in detection of target non-nucleic acid targets.
Further, the non-nucleic acid target analyte comprises ATP, Cd 2+ Any one of histamine, aflatoxin B1 and thrombin.
Preferably, the detection process is as follows: and mixing the reaction buffer solution with the adapter activator, adding the target analyte for incubation, adding the CRISPR report solution for oscillation mixing, and carrying out fluorescence detection after incubation in a dark place.
According to the invention, the activation of the Cas14 nuclease activity depends on a single-stranded DNA complementary with the guide RNA, through sequence functionalization, the single-stranded DNA can be used as an aptamer to identify a target object to be detected while the Cas14 can be started, and after the target object to be detected is combined with the single-stranded DNA, the single-stranded DNA is prevented from starting the Cas14, so that the detection of the corresponding target object can be realized.
The principle of the invention is shown in figure 1, and Cas14 and sgRNA are self-assembled in solution to form a Cas14-sgRNA complex. Based on base complementary pairing of the sgRNA and the adapter activator, the adapter activator is specifically combined with the Cas14-sgRNA complex to start the nuclease activity of Cas14, and a fluorescent reporter is cut to generate a strong fluorescent signal. When the target of the assay is present, the assay target will specifically bind to the adapter activator, resulting in the adapter activator being unable to further bind to the Cas14-sgRNA complex, making nuclease activity of Cas14 difficult to initiate. In this case, the fluorescent reporter is not cleaved and the fluorescent signal is weak. In practical assays, the observed phenomenon is that the fluorescence of the system gradually decreases as the concentration of the target increases. Through DNA sequence design, the activation aptamer has the functions of binding with a target and activating Cas 14.
According to the invention, Cas14 is used to replace traditional Cas12a, and through specific DNA sequence design, the activation aptamer has the functions of combining with a target and activating Cas 14; compared to Cas12 a-based aptamer sensors, the sensitivity is greatly improved. Actually, the Cas14 and the Cas12 have different enzyme activities and substrates and larger difference in enzymological performance, the Cas14 is used for the first time, and the effect of the invention can be effectively realized only by simultaneously designing and constructing a specific activation aptamer and combining with the Cas 14.
Compared with the prior art, the invention has the following remarkable advantages:
(1) the aptamer biosensor based on the CRISPR-Cas14 system has the advantages of high sensitivity, high specificity and wide detection range, and can quickly detect non-nucleic acid targets.
(2) The activator of the Cas14a protein is functionalized for the first time, so that the activator has the function of starting Cas14a and can be combined with a target to be detected through an aptamer sequence contained in the activator.
(3) The aptamer biosensor based on the CRISPR-Cas14 system can be used for rapid analysis of various targets, has the advantages of economy, high efficiency and rapidness, and can be used for ATP and Cd by further improving the sequence 2+ High-performance detection of histamine, aflatoxin B1 and thrombin has wide application in clinical examination, food safety analysis and environmental pollutant monitoringThe application potential is high.
(4) Compared with the aptamer biosensor based on Cas12a, the sensitivity of the aptamer biosensor based on the CRISPR-Cas14 system is greatly improved, when the concentration of ATP reaches 50nM, the fluorescence signal difference can be observed by the aptamer biosensor based on CRISPR-Cas14, and in the traditional CRISPR-Cas12 aptamer biosensor detection technology, the fluorescence signal difference can be caused only when the concentration of ATP reaches 500 nM.
Drawings
Fig. 1 is a schematic diagram of an aptamer biosensor based on the CRISPR-Cas14 system;
FIG. 2 is a graph showing the linearity of fluorescence measurements with different concentrations of ATP added;
FIG. 3 shows the addition of Cd in different concentrations 2+ Detecting the linearity obtained by fluorescence;
FIG. 4 is a line of fluorescence measurements with the addition of various concentrations of histamine;
FIG. 5 is a graph showing the linearity of fluorescence measurements obtained by adding different concentrations of aflatoxin B1(AFB 1);
FIG. 6 is a graph showing the linearity of fluorescence measurements with different concentrations of thrombin added;
fig. 7 is a comparison of the present invention and CRISPR-Cas12 related detection techniques in terms of analytical sensitivity.
Detailed Description
The invention is further illustrated by the following figures and examples.
The experimental procedures described in the examples are, unless otherwise specified, conventional. Drugs and reagents: the DNA, sgRNA used in the experiment were both synthesized by biological engineering (shanghai, china) and purified by HPLC. Cas14a protein was purchased from Touhong harbor Biotechnology Ltd, ATP, CdSO 4 Histamine, aflatoxin B1, thrombin were purchased from alatin (shanghai, china). Fluorescence readings were obtained from a TECAN Infinite M200 multi-functional plate reader (switzerland). Other reagents were purchased from national medicine reagents (shanghai, china) and were all analytically pure.
Example 1
Aptamer biosensor for detecting ATP based on CRISPR-Cas14 system
(1) Configuring CRISPR-Cas14 reporter solution (CRM): cas14a (50nM, 20. mu.L) and sgRNA-ATP (50nM, 20. mu.L) were mixed in 60. mu.L reaction buffer (10 mMNa) 2 HPO 4 /NaH 2 PO 4 ,100mMNaCl，10mM MgCl 2 pH 6.8) in which the sgRNA-ATP sequence is shown in SEQ ID No.1 in table 1. And (3) incubating the mixed solution at 25 ℃ for 10min, adding a fluorescence reporter FAM-TTATT-BHQ1(F-Q reporter) with the sequence shown as SEQ ID NO.11 in Table 1, and placing the mixture in an ice water bath for preservation to obtain a CRISPR-Cas14 reporter solution CRM. In the CRM final solution, the final concentrations of Cas14a-sgRNA and F-Q reporter were 10nM and 1. mu.M, respectively.
(2) mu.L of reaction buffer (10mM Na) 2 HPO 4 /NaH 2 PO 4 ,100mMNaCl,10mM MgCl 2 pH 6.8) was mixed with an aptamer activator (Aptavator) (5 μ L,20nM) whose sequence is shown in SEQ ID No.2 in table 1, and 5 μ L of ATP solution (100,500,1000,1800,2000 nM in concentration) was added thereto, respectively, and incubated at 37 ℃ for 10 min.
(3) And (3) adding 20 mu L of CRM prepared in the step (1) into the mixed solution in the step (2), shaking and mixing, and incubating for 20min at 25 ℃ in the dark.
(4) Respectively carrying out fluorescence detection on the mixed solution obtained in the step (3), and detecting by adopting a Teacan M200 Pro multifunctional microplate reader under the following detection conditions: lambda [ alpha ] ex ＝480nm，λ em 525nm, 20 readings per well. As shown in FIG. 2, a standard curve chart for ATP detection of the detection method shows that the linear detection range of ATP is 100-4000 nM, R 2 0.9951, the limit of detection of ATP by the sensor was calculated to be 80 nM.
TABLE 1 DNA sequences referred to in the present invention
Example 2
Aptamer biosensor for detecting Cd based on CRISPR-Cas14 system 2+
By adopting the preparation method of example 1, sgRNA and Aptavator are respectively changed into sequences shown as SEQ ID No.3 and SEQ ID No.4 in Table 1, and the ATP solution obtained in the step (2) of example 1 is changed into CdSO 4 Solutions (5, 20,100,250,500,1000,1500nM) and the same assay and conditions, as shown in FIG. 3, were used for Cd assay for this assay 2+ The standard curve chart of the detection shows that Cd 2+ The linear detection range of (a) is 5-1500 nM, R 2 The sensor pair Cd is calculated as 0.9987 2+ The detection limit of (3) is 4 nM.
Example 3
Aptamer biosensor for detecting histamine based on CRISPR-Cas14 system
By adopting the preparation method of example 1, the sequence of sgRNA and Aptavator is changed to be shown as SEQ ID No.5 and SEQ ID No.6 in Table 1 respectively, the ATP solution of step (2) of example 1 is changed to be histamine solution (the concentration is 40,150,250,500,1000nM), the same detection method and conditions are adopted, as shown in FIG. 4, the standard curve chart of the detection method for histamine detection shows that the linear detection range of histamine is 40-1500 nM, and R is 40-1500 nM 2 0.9944, the limit of histamine detection by the sensor was calculated to be 30 nM.
Example 4
Aptamer biosensor for detecting AFB1 based on CRISPR-Cas14 system
By adopting the preparation method of example 1, the sgRNA and Aptavator are respectively changed into the sequences shown in SEQ ID No.7 and SEQ ID No.8 in Table 1, the ATP solution obtained in the step (2) of example 1 is changed into AFB1 solution (with the concentration of 20,100,500,1000,2000,3000nM), the same detection method and conditions are adopted, as shown in FIG. 5, and the standard curve chart of the detection method for histamine detection shows that the linear detection range of histamine is 40-3000 nM, and R is the linear detection range of histamine 2 0.9996, the limit of histamine detection by the sensor was calculated to be 16 nM.
Example 5
Aptamer biosensor for detecting thrombin based on CRISPR-Cas14 system
Preparation of example 1The preparation method comprises the steps of changing sgRNA and Aptavator into the sequences shown as SEQ ID No.9 and SEQ ID No.10 in Table 1 respectively, changing the ATP solution in the step (2) in the example 1 into a thrombin solution (the concentration is 40,100,200,500,1000,1500,2000nM), and carrying out the same detection method and conditions, as shown in FIG. 6, in order to obtain a standard curve chart of the detection method for histamine detection, the linear detection range of histamine is 40-2000, and R is shown to be 40-2000 2 The limit of histamine detection by this sensor was calculated to be 36nM, 0.9973.
Example 6
ATP and Cd were added to the samples obtained in examples 1, 2 and 4 2+ And replacing the AFB1 standard solution with human serum, lake water and peanut extract, and detecting the sample loading recovery rate. The standard curves shown in examples 1, 2 and 4 are used for detection, the obtained sample adding recovery rate detection results are shown in table 2, all sample adding recovery rates are between 90% and 110%, and the detected RSD is less than 5%, so that the method can be specifically, precisely and accurately used for detecting actual samples.
TABLE 2 Performance of the method in actual sample testing
Example 7
The invention has performance difference on ATP detection with similar technology based on CRISPR-Cas 12. Among them, prepared based on CRISPR-Cas12 ATP detection technology reference (Sensors and activators B: Chemical 2020,320,128164), the present invention utilizes the aptamer biosensor of example 1 and its method to detect ATP. As shown in FIG. 7, in the CRISPR-Cas14 detection analysis method developed by the present invention, when the concentration of ATP reaches 50nM, a statistically significant difference in fluorescence signal can be observed, whereas in the conventional CRISPR-Cas12 detection technique, the concentration of ATP needs to reach 500nM to cause a comparable difference in fluorescence signal. The result shows that the aptamer biosensor based on CRISPR-Cas14 constructed by the invention is significantly superior to the traditional method based on CRISPR-Cas12 in detection sensitivity.
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Claims (10)
1. An aptamer biosensor based on a CRISPR-Cas14 system, wherein the biosensor comprises a CRISPR-Cas14 reporter solution and an adapter activator; the CRISPR-Cas14 reporter solution includes a Cas14a protein, sgrnas, and a fluorescent reporter; the adapter activator contains an adapter sequence and can be specifically bound with a target object, and the adapter activator is complementary to the sgRNA partial sequence and can activate Cas14 a; the sgRNA can bind tightly to Cas14a, inducing Cas14a to recognize the adapter activator.
2. The aptamer biosensor based on CRISPR-Cas14 system according to claim 1, wherein the CRISPR-Cas14 report solution is incubated after Cas14a protein and sgRNA are mixed in reaction buffer, and then a fluorescent reporter is added to be placed in an ice water bath for preservation.
3. The aptamer biosensor based on CRISPR-Cas14 system according to claim 2, further comprising a reaction buffer solution in the aptamer biosensor, wherein the reaction buffer solution comprises Na 2 HPO 4 /NaH 2 PO 4 NaCl and MgCl 2 And the pH value of the reaction buffer solution is 6.8-7.5.
4. Aptamer biosensor of CRISPR-Cas14 system according to claim 3, wherein Na in the reaction buffer solution 2 HPO 4 /NaH 2 PO 4 NaCl and MgCl 2 The molar mass ratio of (a) to (b) is 1-4: 10-15: 1-4.
5. The aptamer biosensor of CRISPR-Cas14 system according to claim 1, wherein the adapter activator sequence is shown in any one of SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 and SEQ ID No. 10.
6. The aptamer biosensor of CRISPR-Cas14 system according to claim 1, wherein the sgRNA sequence is shown as any one of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 and SEQ ID No. 9.
7. The aptamer biosensor of the CRISPR-Cas14 system of claim 1, wherein the fluorescent reporter is FAM-TTATT-BHQ 1.
8. Use of an aptamer biosensor of the CRISPR-Cas14 system of claim 1 to detect a target non-nucleic acid target.
9. The use of claim 8, wherein the target analyte comprises ATP, Cd 2+ Any one of histamine, aflatoxin B1 and thrombin.
10. The use according to claim 8, wherein the detection process is: and mixing the reaction buffer solution with the adapter activator, adding the target analyte for incubation, adding the CRISPR report solution for oscillation mixing, and carrying out fluorescence detection after incubation in a dark place.
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| CN115901911A (en) * | 2023-01-06 | 2023-04-04 | 南京邮电大学 | Detection method for detecting cardiac troponin I based on CRISPR/Cas12a |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115901911A (en) * | 2023-01-06 | 2023-04-04 | 南京邮电大学 | Detection method for detecting cardiac troponin I based on CRISPR/Cas12a |
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