An aptamer biosensor based on the CRISPR-Cas14 system and its application

Abstract

This invention discloses an aptamer biosensor based on the CRISPR-Cas14 system and its applications. The biosensor includes a CRISPR-Cas14 reporter solution and an aptamer activator. The CRISPR-Cas14 reporter solution includes Cas14a protein, sgRNA, and a fluorescent reporter. The aptamer activator contains an aptamer sequence that can specifically bind to the target analyte. Furthermore, the aptamer activator is partially complementary to the sgRNA sequence, thereby activating Cas14a. The biosensor prepared by this invention exhibits high sensitivity, high specificity, and a wide detection range. It is also economical, efficient, and rapid, and can be used for the rapid analysis of various targets, including ATP and Cd. 2+ Histamine, aflatoxin, and thrombin have broad application potential in clinical testing, food safety analysis, and environmental pollutant monitoring.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, specifically to an aptamer biosensor based on the CRISPR-Cas14 system and its applications. Background Technology

[0002] Developing aptamer biosensors using CRISPR-associated proteins (Cas) is a significant research trend in the molecular diagnostics industry. Compared to traditional enzyme-linked immunosorbent assays (ELISA) and Western blotting, Cas-based aptamer biosensors offer advantages such as ease of operation, low cost, and rapid response. However, existing technologies are mostly based on the Cas12a protein, which struggles to specifically recognize aptamer sequences. This results in deficiencies in both sensitivity and specificity in the formed aptamer biosensors, limiting the market penetration of related products. Therefore, it is necessary to explore the application of novel Cas proteins in aptamer biosensing and construct higher-performance sensors to overcome the shortcomings of existing technologies. Summary of the Invention

[0003] Purpose of the invention: To address the problems existing in the prior art, the present invention provides an aptamer biosensor based on the CRISPR-Cas14 system that is simple to operate, consumes little reagent, is low in cost, has short processing time, and is highly sensitive and specific.

[0004] Another objective of this invention is to provide an application of an aptamer biosensor based on the CRISPR-Cas14 system in detecting non-nucleic acid targets.

[0005] Technical Solution: To achieve the above objectives, this invention provides an aptamer biosensor based on the CRISPR-Cas14 system. The biosensor includes a CRISPR-Cas14 reporter solution and an aptamer activator. The CRISPR-Cas14 reporter solution includes Cas14a protein, sgRNA, and a fluorescent reporter. The aptamer activator contains an aptamer sequence that can specifically bind to the target, and is partially complementary to the sgRNA sequence, thereby activating Cas14a. The sgRNA binds tightly to Cas14a, inducing Cas14a to recognize the aptamer activator.

[0006] Furthermore, the CRISPR-Cas14 reporter solution is prepared by mixing Cas14a and sgRNA in a reaction buffer and incubating, then adding a fluorescent reporter and storing in an ice-water bath.

[0007] Furthermore, the aptamer biosensor also includes a reaction buffer solution comprising Na2HPO4 / NaH2PO4, NaCl, and MgCl2, wherein the pH value of the reaction buffer solution is 6.8–7.5.

[0008] Preferably, the reaction buffer solution has a pH of 6.8.

[0009] Furthermore, the molar mass ratio of Na2HPO4 / NaH2PO4, NaCl, and MgCl2 in the reaction buffer solution is 1–4:10–15:1–4.

[0010] Preferably, the molar mass ratio of Na2HPO4 / NaH2PO4, NaCl, and MgCl2 in the reaction buffer solution is 1:10:1.

[0011] The adaptor 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.

[0012] 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, or SEQ ID NO.9.

[0013] Furthermore, the fluorescent reporter is FAM-TTATT-BHQ1.

[0014] This invention provides an application of an aptamer biosensor based on the CRISPR-Cas14 system in detecting non-nucleic acid target analytes.

[0015] Furthermore, the non-nucleic acid target analytes include ATP and Cd. 2+ Any one of histamine, aflatoxin B1, or thrombin.

[0016] Preferably, the detection process is as follows: the reaction buffer solution is mixed with the adaptor activator, the target analyte is added and incubated, then the CRISPR reporter solution is added and shaken to mix, and after incubation in the dark, fluorescence detection is performed.

[0017] In this invention, the activation of Cas14 nuclease activity depends on a single-stranded DNA complementary to the guide RNA. Through sequence functionalization, this single-stranded DNA can not only initiate Cas14, but also act as an aptamer to recognize the target analyte. After the target analyte binds to the single-stranded DNA, it prevents the single-stranded DNA from initiating Cas14, thereby enabling the detection of the corresponding target analyte.

[0018] The principle of this invention is shown in the appendix. Figure 1Cas14 and sgRNA self-assemble in solution to form the Cas14-sgRNA complex. Based on the complementary base pairing between sgRNA and the aptamer, the aptamer specifically binds to the Cas14-sgRNA complex, initiating the nuclease activity of Cas14, cleaving the fluorescent reporter, and generating a strong fluorescence signal. When the target analyte is present, it specifically binds to the aptamer, preventing the aptamer from further binding to the Cas14-sgRNA complex, thus hindering the initiation of Cas14 nuclease activity. In this case, the fluorescent reporter is not cleaved, resulting in a weak fluorescence signal. In actual detection, the observed phenomenon is that the fluorescence of the system gradually decreases with increasing target analyte concentration. Through DNA sequence design, the activating aptamer can be made to have both target analyte binding and Cas14 activation functions.

[0019] This invention uses Cas14 instead of the traditional Cas12a. Through specific DNA sequence design, the activating aptamer possesses both the function of binding to the target and activating Cas14. Compared with Cas12a-based aptamer sensors, the sensitivity is significantly improved. In fact, Cas14 and Cas12 have different enzyme activities, different substrates, and significantly different enzymatic properties. This invention is the first to use Cas14, and the specific activating aptamer designed and constructed to bind with Cas14 is necessary to effectively achieve the effects of this invention.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] (1) The aptamer biosensor based on the CRISPR-Cas14 system of this invention has high sensitivity, high specificity and wide detection range, and can quickly detect non-nucleic acid targets.

[0022] (2) This invention is the first to functionalize the activator of the Cas14a protein, so that it can bind to the target analyte through the aptamer sequence contained therein while having the function of activating Cas14a.

[0023] (3) The aptamer biosensor based on the CRISPR-Cas14 system of this invention can be used for rapid analysis of a variety of targets, with the advantages of being economical, efficient, and fast. With further sequence improvement, it can be used for ATP, Cd 2+ High-performance detection of histamine, aflatoxin B1, and thrombin has broad application potential in clinical testing, food safety analysis, and environmental pollutant monitoring.

[0024] (4) The aptamer biosensor based on the CRISPR-Cas14 system of this invention has a significantly improved sensitivity compared with the aptamer biosensor based on Cas12a. When the concentration of ATP reaches 50 nM, the CRISPR-Cas14 aptamer biosensor can observe the difference in fluorescence signal, while in the traditional CRISPR-Cas12 aptamer biosensor detection technology, the concentration of ATP needs to reach 500 nM to cause a considerable difference in fluorescence signal. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of an aptamer biosensor based on the CRISPR-Cas14 system.

[0026] Figure 2 The linearity was obtained by detecting fluorescence with different concentrations of ATP added;

[0027] Figure 3 It involves adding different concentrations of Cd. 2+ Linearity obtained from fluorescence detection;

[0028] Figure 4 The linearity was obtained by detecting fluorescence after adding different concentrations of histamine;

[0029] Figure 5 The linearity was obtained by detecting fluorescence after adding different concentrations of aflatoxin B1 (AFB1);

[0030] Figure 6 The linearity was obtained by detecting fluorescence after adding different concentrations of thrombin;

[0031] Figure 7 This invention compares the analytical sensitivity of the present invention with that of CRISPR-Cas12 related detection technology. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] Unless otherwise specified, the experimental methods described in these examples are conventional methods. Reagents and reagents: The DNA and sgRNA used in the experiments were synthesized by Sangon Biotech (Shanghai, China) and purified by HPLC. Cas14a protein was purchased from Tolo Biotech Ltd., and ATP, CdSO4, histamine, aflatoxin B1, and thrombin were purchased from Aladdin (Shanghai, China). Fluorescence readings were obtained using a TECAN Infinite M200 multi-mode microplate reader (Switzerland). All other reagents were purchased from Sinopharm Reagent (Shanghai, China) and were of analytical grade.

[0034] Example 1

[0035] Aptamer biosensors based on the CRISPR-Cas14 system for ATP detection

[0036] (1) Preparation of CRISPR-Cas14 reporter solution (CRM): Cas14a (50 nM, 20 μL) and sgRNA-ATP (50 nM, 20 μL) were mixed in 60 μL reaction buffer (10 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, 10 mM MgCl2, pH = 6.8). The sgRNA-ATP sequence is shown in SEQ ID NO.1 in Table 1. The mixture was incubated at 25 °C for 10 min, and then the fluorescent reporter FAM-TTATT-BHQ1 (FQ reporter) was added. The sequence is shown in SEQ ID NO.11 in Table 1. The mixture was then stored in an ice-water bath to obtain the CRISPR-Cas14 reporter solution CRM. In the final CRM solution, the final concentrations of Cas14a-sgRNA and FQ reporter were 10 nM and 1 μM, respectively.

[0037] (2) Mix 20 μL of reaction buffer (10 mM Na2HPO4 / NaH2PO4, 100 mM NaCl, 10 mM MgCl2, pH = 6.8) with 5 μL of the appropriate activator (20 nM). The sequence of the activator is shown in SEQ ID NO.2 in Table 1. Then add 5 μL of ATP solution (concentrations of 100, 500, 1000, 1800, and 2000 nM) and incubate at 37°C for 10 min.

[0038] (3) Add 20 μL of the CRM prepared in step (1) to the mixed solution in step (2), shake to mix, and incubate at 25°C in the dark for 20 min.

[0039] (4) Perform fluorescence detection on the mixed solution from step (3) using a Teacan M200 Pro multi-functional microplate reader. The detection conditions are: λ ex =480nm, λ em =525nm, 20 readings per well. For example... Figure 2 The figure shows the standard curve for ATP detection using this method, indicating that the linear detection range for ATP is 100–4000 nM, R0. 2 =0.9951, and the detection limit of the sensor for ATP is calculated to be 80 nM.

[0040] Table 1. DNA sequences involved in this invention.

[0041]

[0042]

[0043] Example 2

[0044] Cd detection using aptamer biosensors based on the CRISPR-Cas14 system 2+

[0045] Using the preparation method of Example 1, the sgRNA and Aptavator sequences were changed as shown in SEQ ID NO.3 and SEQ ID NO.4 in Table 1, respectively. The ATP solution in step (2) of Example 1 was replaced with CdSO4 solution (concentrations of 5, 20, 100, 250, 500, 1000, 1500 nM). The same detection method and conditions were used. Figure 3 As shown, this detection method is used for Cd. 2+ The standard curve plot of the detection shows that Cd 2+ The linear detection range is 5–1500 nM, R 2 =0.9987, the calculated value of this sensor for Cd is 0.9987. 2+ The detection limit is 4 nM.

[0046] Example 3

[0047] Histamine Detection Based on CRISPR-Cas14 System Aptamer Biosensor

[0048] Using the preparation method of Example 1, the sgRNA and Aptavator sequences were changed as shown in SEQ ID NO.5 and SEQ ID NO.6 in Table 1, respectively. The ATP solution in step (2) of Example 1 was replaced with histamine solution (concentration of 40, 150, 250, 500, 1000 nM). The same detection method and conditions were used. Figure 4 The figure shows the standard curve for histamine detection using this method, indicating that the linear detection range for histamine is 40–1500 nM, R0. 2 =0.9944, and the detection limit of the sensor for histamine is calculated to be 30 nM.

[0049] Example 4

[0050] Detection of AFB1 by an aptamer biosensor based on the CRISPR-Cas14 system

[0051] Using the preparation method of Example 1, the sgRNA and Aptavator sequences were changed as shown in SEQ ID NO.7 and SEQ ID NO.8 in Table 1, respectively. The ATP solution in step (2) of Example 1 was replaced with AFB1 solution (concentrations of 20, 100, 500, 1000, 2000, and 3000 nM). The same detection method and conditions were used. Figure 5The figure shows the standard curve for histamine detection using this method, indicating that the linear detection range for histamine is 40–3000 nM, R0. 2 =0.9996, and the detection limit of the sensor for histamine is calculated to be 16 nM.

[0052] Example 5

[0053] Thrombin Detection Based on CRISPR-Cas14 System Aptamer Biosensor

[0054] Using the preparation method of Example 1, the sgRNA and Aptavator sequences were changed as shown in Table 1 (SEQ ID NO. 9 and SEQ ID NO. 10, respectively). The ATP solution in step (2) of Example 1 was replaced with thrombin solution (concentrations of 40, 100, 200, 500, 1000, 1500, 2000 nM). The same detection method and conditions were used. Figure 6 The figure shows the standard curve for histamine detection using this method, indicating that the linear detection range for histamine is 40–2000, R0. 2 =0.9973, and the detection limit of the sensor for histamine is calculated to be 36 nM.

[0055] Example 6

[0056] Using the preparation methods of Examples 1, 2, and 4, ATP and Cd were... 2+ AFB1 and standard solutions were replaced with human serum, lake water, and peanut extract, respectively, for sample recovery testing. The standard curves shown in Examples 1, 2, and 4 were used for the detection. The results of the sample recovery tests are shown in Table 2. All sample recoveries were between 90% and 110%, and the RSD was less than 5%, proving that this method can be used specifically, precisely, and accurately for the detection of actual samples.

[0057] Table 2. Performance of this method in actual sample testing.

[0058]

[0059]

[0060] Example 7

[0061] The performance differences of this invention compared to similar CRISPR-Cas12-based technologies in ATP detection are discussed. The CRISPR-Cas12-based ATP detection technology is referenced in the literature (Sensors and Actuators B: Chemical 2020, 320, 128164). This invention utilizes the aptamer biosensor and method from Example 1 to detect ATP. Results are as follows... Figure 7As shown, in the CRISPR-Cas14 detection and analysis method developed in this invention, a statistically significant difference in fluorescence signal can be observed when the ATP concentration reaches 50 nM. In contrast, in the traditional CRISPR-Cas12 detection technology, an ATP concentration of 500 nM is required to elicit a comparable difference in fluorescence signal. This result demonstrates that the CRISPR-Cas14-based aptamer biosensor constructed in this invention significantly outperforms traditional CRISPR-Cas12-based methods in terms of detection sensitivity. sequence list <110> Nanjing Normal University <120> An aptamer biosensor based on the CRISPR-Cas14 system and its application <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 181 <212> RNA <213> Artificial Sequence <400> 1 uuccuccgca auacuccccg guugcauucc uucauucuuu caaaugaauu uguuucgagg 60 guuacuuucc gaagaaagca cuucucgaca uuaggcugau gcaagcagcc caccuucacu 120 caaguucuaa uccccuaagg gacagcuuuu ggugaagcgg uucuccacuu uaucagugaa 180 g 181 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <400> 2 tacctggggg agtattgcgg aggaaggta 29 <210> 3 <211> 181 <212> RNA <213> Artificial Sequence <400> 3 acaaccaaaa auaauaccag guugcauucc uucauucuuu caaaugaauu uguuucgagg 60 guuacuuucc gaagaaagca cuucucgaca uuaggcugau gcaagcagcc caccuucacu 120 caaguucuaa uccccuaagg gacagcuuuu ggugaagcgg uucuccacuu uaucagugaa 180 g 181 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <400> 4 gggagggaac tgttgtggta ttatttttgg ttgtgcagta gggcggg 47 <210> 5 <211> 181 <212> RNA <213> Artificial Sequence <400> 5 agcagaucgg aagaaccgcg guugcauucc uucauucuuu caaaugaauu uguuucgagg 60 guuacuuucc gaagaaagca cuucucgaca uuaggcugau gcaagcagcc caccuucacu 120 caaguucuaa uccccuaagg gacagcuuuu ggugaagcgg uucuccacuu uaucagugaa 180 g 181 <210> 6 <211> 99 <212> DNA <213> Artificial Sequence <400> 6 agctccagaa gataaattac agggaacgtg ttggttgcgg ttcttccgat ctgctgtgtt 60 ctctatctgt gccatgcaac taggatacta tgaccccgg 99 <210> 7 <211> 181 <212> RNA <213> Artificial Sequence <40​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​guuacuuucc gaagaaagca cuucucgaca uuaggcugau gcaagcagcc caccuucacu 120 caaguucuaa uccccuaagg gacagcuuuu ggugaagcgg uucuccacuu uaucagugaa 180 g 181 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <400> 10 aaaaaaaaag gttggtgtgg ttggaaaaaa aaa 33

Claims

1. An application of an aptamer biosensor based on the CRISPR-Cas14 system in detecting non-nucleic acid target analytes, characterized in that: The target substances include ATP and Cd. 2+ Any one of the following: histamine, aflatoxin B1, or thrombin; The biosensor includes a CRISPR-Cas14 reporter solution and an aptamer; the CRISPR-Cas14 reporter solution includes Cas14a protein, sgRNA, and a fluorescent reporter; the aptamer contains an aptamer sequence that can specifically bind to the target, and the aptamer is partially complementary to the sgRNA sequence, thereby activating Cas14a; the sgRNA can bind tightly to Cas14a, inducing Cas14a to recognize the aptamer. The detection of ATP uses sgRNA sequences such as SEQ ID NO.1 and aptamer activator sequences such as SEQ ID NO.2; the detection of Cd... 2 + The sgRNA sequence is shown in SEQ ID NO.3, and the aptor activator sequence is shown in SEQ ID NO.4; the histamine is detected using the sgRNA sequence shown in SEQ ID NO.5, and the aptor activator sequence is shown in SEQ ID NO.6; the AFB1 is detected using the sgRNA sequence shown in SEQ ID NO.7, and the aptor activator sequence is shown in SEQ ID NO.8; the thrombin is detected using the sgRNA sequence shown in SEQ ID NO.9, and the aptor activator sequence is shown in SEQ ID NO.

10.

2. The application of the CRISPR-Cas14-based aptamer biosensor according to claim 1 in detecting non-nucleic acid target analytes, characterized in that, The CRISPR-Cas14 reporter solution is prepared by mixing Cas14a protein and sgRNA in a reaction buffer, incubating the mixture, adding a fluorescent reporter, and then storing it in an ice-water bath.

3. The application of the aptamer biosensor based on the CRISPR-Cas14 system according to claim 2 in detecting non-nucleic acid target analytes, characterized in that, The aptamer biosensor also includes a reaction buffer solution comprising Na2HPO4 / NaH2PO4, NaCl, and MgCl2, with a pH value of 6.8–7.

5.

4. The application of the aptamer biosensor based on the CRISPR-Cas14 system according to claim 3 in detecting non-nucleic acid target analytes, characterized in that, The molar mass ratio of Na2HPO4 / NaH2PO4, NaCl, and MgCl2 in the reaction buffer solution is 1–4:10–15:1–4.

5. The application of the aptamer biosensor based on the CRISPR-Cas14 system according to claim 1 in the detection of non-nucleic acid target analytes, characterized in that, The fluorescent reporter is FAM-TTATT-BHQ1.

6. The application of the aptamer biosensor based on the CRISPR-Cas14 system as described in claim 1 in the detection of non-nucleic acid target analytes, characterized in that, The detection process is as follows: the reaction buffer solution is mixed with the appropriate activator, the target analyte is added and incubated, then CRISPR reporter solution is added and shaken to mix, and after incubation in the dark, fluorescence detection is performed.

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