Intelligent responsive Cas12a / crRNA-dna complex hydrogel, preparation method thereof and application thereof in ATP detection

Abstract

The application belongs to the technical field of DNA hydrogel and specifically relates to an intelligent responsive Cas12a / CrRNA-DNA composite hydrogel, a preparation method thereof and application thereof in ATP detection. The application constructs a crosslinking network through functionalized DNA chains, and the Acrydite-DNA (1) chain and the DNA (2) chain, the Acrydite-DNA (3) chain and the ATP aptamer chain constitute a polyacrylamide polymer with DNA as a crosslinking unit. The Cas12a / CrRNA complex is stably anchored in the hydrogel system, and the ATP aptamer-mediated gel conformation transition activates the Cas12a enzyme cutting activity, realizes the targeted recognition and signal amplification detection of ATP, and provides a novel and efficient technical solution for the instant detection (POCT) of ATP and other small molecule biomarkers.

Description

Technical Field

[0001] This invention belongs to the field of DNA hydrogel technology, specifically relating to a smart responsive Cas12a / CrRNA-DNA composite hydrogel, its preparation method, and its application in ATP detection. Background Technology

[0002] Adenosine triphosphate (ATP), as a core energy source for organisms, is widely involved in key life activities such as cell metabolism, proliferation, apoptosis, and signal transduction. Abnormal ATP concentrations are closely related to the occurrence and development of various major diseases, including tumors, inflammation, and cardiovascular diseases. It is also an important biomarker for microbial contamination and cell activity assessment. Achieving highly sensitive and specific accurate detection of ATP is of vital practical significance for early clinical disease screening, basic research in cell biology, food safety, and environmental monitoring. Currently, traditional ATP detection methods mainly include high-performance liquid chromatography (HPLC), fluorescence spectroscopy, electrochemical sensing, and enzyme-linked immunosorbent assay (ELISA). These methods generally suffer from limitations such as high instrument dependence, high detection costs, cumbersome procedures, and weak on-site real-time detection capabilities. Some methods also have problems such as insufficient sensitivity, poor anti-interference ability, and weak specificity, making it difficult to meet the practical needs for rapid, visualized, and accurate detection in complex biological samples.

[0003] In recent years, DNA hydrogels have emerged as novel functional materials in the fields of biosensing and targeted detection due to their excellent biocompatibility, designability, stimulus responsiveness, and biomolecule loading capacity. By introducing specific aptamers, enzymes, and other functional modules into the gel backbone, precise recognition and response to target substances can be achieved, providing a new approach for the detection of small molecule biomarkers. Meanwhile, the CRISPR-Cas12a system, with its specific recognition capabilities and highly efficient trans-cleavage activity, exhibits strong signal amplification advantages in nucleic acid and small molecule detection, significantly improving detection sensitivity. However, its poor stability in the free state, easy inactivation, and limited application in in vivo / complex environments restrict its further application. How to organically combine the intelligent response characteristics of DNA hydrogels with the signal amplification function of CRISPR-Cas12a to construct a novel sensing platform that combines high specificity, high sensitivity, visualization, and instrument-free detection has become a research hotspot in the field of biosensing.

[0004] Based on this, this study focuses on the practical needs of ATP detection, and designs and develops a smart responsive Cas12a / CrRNA-DNA composite hydrogel. By constructing a cross-linking network through functionalized DNA strands, the Cas12a / CrRNA complex is stably anchored in the hydrogel system. Relying on the ATP aptamer-mediated gel conformational change to activate the Cas12a enzymatic activity, the targeted recognition and signal amplification detection of ATP are realized, in order to break through the bottleneck of existing detection technologies and provide a new and efficient technical solution for point-of-care testing (POCT) of ATP and other small molecule biomarkers. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a smart responsive Cas12a / CrRNA-DNA composite hydrogel, its preparation method, and its application in ATP detection. By constructing a cross-linking network using functionalized DNA strands, the Cas12a / CrRNA complex is stably anchored in the hydrogel system. Relying on ATP aptamer-mediated gel conformational change and activation of Cas12a enzymatic activity, targeted recognition and signal amplification detection of ATP are achieved. This aims to overcome the bottlenecks of existing detection technologies and provide a novel and efficient technical solution for point-of-care testing (POCT) of ATP and other small molecule biomarkers.

[0006] A smart responsive Cas12a / CrRNA-DNA composite hydrogel includes a Cas12a / CrRNA complex protein and a DNA hydrogel; the DNA hydrogel includes a polyacrylamide polymer with DNA as a crosslinking unit.

[0007] The polyacrylamide polymer with DNA as the crosslinking unit is composed of Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, and ATP aptamer chain; the three are subjected to free radical polymerization reaction under the action of an initiator to form a polymer copolymer with DNA as the crosslinking point. The DNA-based copolymer can be further crosslinked under the mediation of DNA (2) chain to construct a three-dimensional network structure.

[0008] The Acrydite-DNA (1) strand sequence is shown in SEQ ID NO: 1, which is 5'-ACAATATGTAAA-Acrydite-3'; The DNA (2) strand sequence is shown in SEQ ID NO: 2, which is 5'-ACATATTGTACCTTCCTCCG-3'; The Acrydite-DNA (3) strand sequence is shown in SEQ ID NO: 3, which is 5'-TGGATGACTGAGCAG-Acrydite-3'; The sequence of the ATP aptamer is shown in SEQ ID NO: 4, which is 5'-Acrydite-AAACTCATCTCCTGGGGGAGTATTGCGGAGGAAGGT.

[0009] The specific process for preparing the above-mentioned intelligent responsive Cas12a / CrRNA-DNA composite hydrogel is as follows: (1) Disperse the Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, and ATP aptamer chain with acrylamide monomer in Tris-HCl buffer solution, and deoxygenate by bubbling with nitrogen to obtain solution A; (2) Initiator was added to solution A in sequence, nitrogen was bubbled to remove oxygen, and then polymerized at 4°C for 12 h to obtain DNA hydrogel intermediate; (3) The DNA hydrogel intermediate was purified by using a 10 kD ultrafiltration tube to remove unreacted small molecule impurities in the system. Then, 1 mM of DNA (2) chain and Cas12a / CrRNA complex solution (3 μM CrRNA, 2 μM Cas12a) were added to re-swell the DNA hydrogel. The ATP aptamer was used as the functional response unit and Cas12a / CrRNA was used as the signal amplification element to obtain Cas12a / CrRNA-DNA composite hydrogel.

[0010] Preferably, the volume fraction of acrylamide in the buffer solution in step (1) is 2%.

[0011] Preferably, the concentration of the Tris-HCl buffer solution in step (1) is (10 mM, pH 8.0).

[0012] Preferably, the nitrogen bubbling deoxygenation time in steps (1) and (2) is 5 min.

[0013] Preferably, the initiator in step (2) is 0.5% APS and 0.25% TEMED.

[0014] Preferably, in step (1), the mass concentrations of Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, and ATP aptamer chain are all 1 mM; Cas12a and CrRNA are incubated at 37°C for 10 min to obtain Cas12a / CrRNA complex, wherein the concentrations of Cas12a and CrRNA are 2 μM and 3 μM, respectively.

[0015] Preferably, the initiator in step (2) is 0.5% APS and 0.25% TEMED.

[0016] The present invention also provides the application of the above-mentioned intelligent responsive Cas12a / CrRNA-DNA composite hydrogel in ATP detection.

[0017] Preferably, the intelligent responsive Cas12a / CrRNA-DNA composite hydrogel is used at 37°C for ATP detection.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The intelligent responsive Cas12a / CrRNA-DNA composite hydrogel obtained in this invention combines the high specificity recognition and signal amplification capabilities of CRISPR-Cas12a with the sequence programmability, structural controllability, and intelligent response of DNA hydrogels, achieving precise response to ATP molecules and significantly improving detection specificity. Furthermore, relying on the three-dimensional network structure of the hydrogel, target molecules and signal probes can be effectively enriched, reducing background interference and greatly improving detection sensitivity, enabling low-concentration, high-sensitivity detection of ATP. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 To determine the molar ratio of acrylamide / acrydite DNA units in the intelligent responsive Cas12a / CrRNA composite hydrogel copolymer of this Example 1, wherein, Figure 1 A represents the UV-Vis absorption spectra of acrydite DNA and different concentrations of polyacrylamide; Figure 1 B is the calibration curve for the molar ratio of DNA monomers to acrylamide monomers in Figure A; Figure 2 This is a photograph of the smart responsive DNA hydrogel of Example 1. Figure 3 This is a scanning electron microscope image of the smart responsive DNA hydrogel of Example 1. Figure 4 This is a schematic diagram of the Cas12a activation output fluorescence signal of the smart responsive DNA hydrogel in Example 1 of this invention; Figure 5 The cleavage activity of Cas12a in the smart responsive DNA hydrogel of Example 1 at different temperatures; Figure 6The test was conducted to assess the responsiveness of the Cas12a of the smart responsive DNA hydrogel in Example 1 to different concentrations of ATP. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. These examples are merely illustrative and not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0022] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0023] Example 1 A method for preparing a smart responsive Cas12a / CrRNA-DNA composite hydrogel is as follows: 1 mM DNA (1), 1 mM DNA (3), 1 mM DNA (4), and 2% acrylamide are dispersed in Tris-HCl (10 mM, pH 8.0) buffer solution. Nitrogen gas is bubbled to remove oxygen for 5 min. Initiators (0.5% APS, 0.25% TEMED) are added sequentially, and nitrogen gas is bubbled to remove oxygen for another 5 min. The mixture is then polymerized at 4℃ for 12 h to obtain a DNA hydrogel intermediate. Subsequently, the gel is purified using a 10 kD ultrafiltration tube to remove unreacted small molecule impurities. Then, 1 mM DNA (2) strand and a Cas12a / CrRNA mixture (CrRNA 3 μM, Cas12a 2 μM; both purchased from New England Biolabs) are added to re-swell the DNA hydrogel, obtaining the Cas12a / CrRNA-DNA composite hydrogel. Figure 2 As shown. The Acrydite-DNA (1) strand sequence is SEQ ID NO: 1, 5'-ACAATATGTAAA-Acrydite-3'; the DNA (2) strand sequence is SEQ ID NO: 2, 5'-ACATATTGTACCTTCCTCCG-3'; the Acrydite-DNA (3) strand sequence is SEQ ID NO: 3, 5'-TGGATGACTGAGCAG-Acrydite-3'; the ATP aptamer sequence is SEQ ID NO: 4, 5'-Acrydite-AAACTCATCTCCTGGGGGAGTATTGCGGAGGAAGGT.

[0024] The molar ratio of acrylamide / acrydite DNA units in the copolymer was tested: To determine the molar ratio of acrylamide / acrydite-DNA units in the copolymer and to precisely control the grafting density of functionalized DNA on the polymer chain, we mixed different concentrations of polyacrylamide solutions (0 μM, 5 μM, 10 μM, 30 μM, 50 μM, 80 μM, 100 μM) with acrydite-DNA (1) (0.4 μM), acrydite-DNA (3) (0.2 μM), and ATP aptamer (0.4 μM), respectively. The UV absorbance of each solution was measured using a UV spectrophotometer, and the obtained UV absorption spectra are shown below. Figure 1 As shown in Figure A, the results indicate that the UV absorption peak at 200 nm gradually increases with increasing acrylamide monomer concentration. Since the DNA concentration remains constant, the absorption peak at 260 nm remains unchanged. Based on the UV absorption spectrum, a calibration curve for the molar ratio of DNA monomer to acrylamide monomer was obtained, as shown in Figure A. Figure 1 As shown in B, R 2 =0.998. Based on the calibration curve, the ratio of the two acrylamide strands to acrylamide in the DNA hydrogel was found to be 1:56.

[0025] Experimental Example 1 Scanning tests were performed on the intelligent Cas12a / CrRNA-DNA composite hydrogel of Example 1: Take 10 μL of Cas12a / CrRNA-DNA composite hydrogel sample, freeze it in liquid nitrogen for 30 min, and then transfer it to a freeze dryer for lyophilization. Sputter-coated gold to conduct the gel sample, and then observe its microstructure using a scanning electron microscope. The results are as follows: Figure 3 As shown in the figure, the SEM results show that the prepared DNA hydrogel has a highly uniform and interconnected three-dimensional network structure, and there is no obvious aggregation phenomenon in the system; this regular three-dimensional interconnected network structure can ensure the rapid exchange and transport of molecules and ions inside the gel.

[0026] Experimental Example 2 The cleavage activity of the Cas12a / CrRNA-DNA composite hydrogel obtained in Example 1 at different temperatures: A mixed solution containing ATP and the Reporter chain was added to the obtained Cas12a / CrRNA-DNA composite hydrogel, and then it was placed in a PCR amplification instrument for programmed real-time detection. The signal output was observed at 25℃, 37℃, and 55℃. Figure 5As shown, the Cas12a / CrRNA-DNA composite hydrogel exhibits the highest cleavage activity at 37°C within the temperature range investigated.

[0027] Experimental Example 3 Detection of the ATP response capability of the Cas12a / CrRNA-DNA composite hydrogel obtained in Example 1: The responsiveness of the DNA microgel to different concentrations of ATP was verified in vitro using the output of PCR fluorescence signals. For example... Figure 6 As shown, the results indicate that the response of DNA microgels to ATP is concentration-dependent, with signal output capability increasing with increasing ATP concentration.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A smart responsive Cas12a / CrRNA-DNA composite hydrogel, characterized in that, The composite hydrogel comprises a Cas12a / CrRNA complex protein and a DNA hydrogel; the DNA hydrogel comprises a polyacrylamide polymer using DNA as a crosslinking unit. The polyacrylamide polymer with DNA as the crosslinking unit is composed of Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, and ATP aptamer chain; the Acrydite-DNA (1) chain sequence is SEQ ID NO: 1; the Acrydite-DNA (3) chain sequence is SEQ ID NO: 3; the ATP aptamer sequence is SEQ ID NO: 4; The DNA hydrogel also includes a DNA (2) chain; the DNA (2) chain sequence is shown in SEQ ID NO:

2.

2. The method for preparing the intelligent responsive Cas12a / CrRNA-DNA composite hydrogel as described in claim 1, characterized in that, The preparation method steps are as follows: (1) Disperse the Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, ATP aptamer chain and acrylamide monomer in Tris-HCl buffer solution, and deoxygenate by bubbling with nitrogen to obtain solution A; (2) Initiators were added to solution A in sequence, nitrogen was bubbled to remove oxygen, and then the mixture was polymerized at 4°C for 12 h to obtain DNA hydrogel intermediate; (3) The DNA hydrogel intermediate was purified by using a 10 kD ultrafiltration tube, and then 1 mM of DNA (2) chain and Cas12a / CrRNA complex solution were added to re-swell the DNA hydrogel to obtain a smart responsive Cas12a / CrRNA-DNA composite hydrogel; the DNA (2) chain sequence is SEQ ID NO:

2.

3. The preparation method according to claim 2, characterized in that, In step (1), the volume fraction of acrylamide in the buffer solution is 2%; the mass concentrations of Acrydite-DNA (1) chain, Acrydite-DNA (3) chain, and ATP aptamer chain are all 1 mM.

4. The preparation method according to claim 2, characterized in that, In step (1), the concentration of the Tris-HCl buffer solution is 10 mM and the pH is 8.

0.

5. The preparation method according to claim 2, characterized in that, The nitrogen bubbling deoxygenation time in steps (1) and (2) is 5 min.

6. The preparation method according to claim 2, characterized in that, The initiator in step (2) is 0.5% APS and 0.25% TEMED.

7. The preparation method according to claim 2, characterized in that, The specific preparation method of the Cas12a / CrRNA complex solution in step (3) is to incubate Cas12a and CrRNA at 37°C for 10 min to obtain the Cas12a / CrRNA complex solution, wherein the concentrations of Cas12a and CrRNA are 2 μM and 3 μM, respectively.

8. The application of the intelligent responsive Cas12a / CrRNA-DNA composite hydrogel as described in claim 1 in ATP detection.

9. The application of the intelligent responsive Cas12a / CrRNA-DNA composite hydrogel obtained by the preparation method according to any one of claims 2-7 in ATP detection.

10. The application according to claim 8 or 9, characterized in that, The intelligent responsive Cas12a / CrRNA-DNA composite hydrogel is used at 37°C for ATP detection.

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