A fluorescence sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction, and a preparation method and application thereof in rapid miRNA detection

Abstract

This invention belongs to the field of biosensing technology, specifically relating to a fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction, its preparation method, and its application in rapid miRNA detection. Hairpin probes H1, H2, H3, and H4 are designed for the CHA-HCR cascade reaction; H1, with its terminal modified fluorescent group (FAM / T) and carboxyl group, is covalently linked to an amino-functionalized MOF to construct a miRNA capture unit; this capture unit is mixed with chitosan hydrogel and fabricated into a fluorescent sensing hydrogel array using 3D printing technology. This invention combines the enrichment effect of MOF with the dual signal amplification capability of CHA-HCR, and utilizes 3D printing to achieve high-throughput detection, exhibiting advantages such as high sensitivity, high specificity, and rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to a fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction, its preparation method, and its application in rapid miRNA detection. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNA molecules, approximately 18-24 nucleotides in length. Encoded by genomic DNA, they bind incompletely to the 3' untranslated region (3'-UTR) of target messenger RNA (mRNA) after transcription, thereby inducing mRNA degradation or translational repression. They participate in almost all important biological processes, including cell proliferation, differentiation, apoptosis, metabolism, and stress responses.

[0003] Recent studies have revealed that dysregulation of miRNA expression is closely related to various human diseases, particularly cancer and drug-induced liver injury. Specific miRNAs (such as miR-122) have become highly promising novel blood biomarkers, providing new directions for early diagnosis. However, miRNA detection faces significant challenges: their short sequences and extremely low abundance in blood necessitate extremely high detection sensitivity; furthermore, members of the same miRNA family exhibit high sequence homology, differing by only 1-2 bases, requiring methods to possess high specificity for distinguishing single bases. Therefore, developing a highly specific and rapid method for measuring miRNAs remains a current challenge for the academic community.

[0004] Currently, the most widely used technique in laboratories and clinics is quantitative reverse transcription polymerase chain reaction (qRT-PCR). However, due to the short length of miRNAs, they usually need to be reverse transcribed into longer cDNAs first using tailing or stem-loop primers before real-time fluorescent PCR amplification. Although this method has high sensitivity, the procedure involves multiple enzymatic reactions, is complex, and takes 3-4 hours in total. Furthermore, it is prone to false positives due to primer dimers or non-specific amplification. Therefore, developing a faster and more convenient assay method is a current challenge. Summary of the Invention

[0005] This invention provides a fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction for rapid, highly sensitive, high-throughput, and low-cost detection of miRNAs, along with its preparation method and applications, to address the technical problems of short miRNA sequences, high homology, and difficulty in detecting multiple indicators in existing technologies. The technical solution is as follows: A fluorescence sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction includes a capture unit and a hydrogel supporting the capture unit. The capture unit includes an amino-containing metal-organic framework material as a carrier and quencher, and a hairpin probe H1 supported on the carrier. The hairpin probe H1 contains carboxyl groups and fluorescent groups that can be covalently linked to amino groups.

[0006] Furthermore, the amino-containing metal-organic framework material is prepared by amino-containing organic ligands and zirconium ions; the molar ratio of the amino-containing organic ligands to zirconium ions is 1:1.1~1.5; the hairpin probe H1 includes a carboxyl group linking sequence and a target miRNA recognition sequence; the sequence of the hairpin probe H1 is shown in SEQ ID NO:1; the fluorescent group includes one or more of 5-FAM, 6-FAM, (5-FAM)2 or (6-FAM)2.

[0007] A method for preparing the above-mentioned fluorescence sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction includes the following steps: obtaining an amino-containing metal-organic framework material; designing a hairpin probe H1 containing a carboxyl group linking sequence and a target miRNA recognition sequence, such that the hairpin probe H1 contains a carboxyl group and a fluorescent group; activating the carboxyl group in the hairpin probe and then covalently linking it with the amino group in the metal-organic framework to obtain a capture unit; mixing the capture unit with the hydrogel uniformly to obtain a mixture, and then cross-linking and curing it to obtain the final product.

[0008] Furthermore, the mixture is 3D printed onto the bottom of a porous plate to form a hydrogel microarray.

[0009] A miRNA detection kit includes the above-mentioned fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction, and hairpin probes H2, H3 and H4 that are complementary to hairpin probe H1.

[0010] Furthermore, the card-spinning probe H2 sequentially includes a card-spinning probe H1 identification sequence and a card-spinning probe H3 trigger sequence from the 5' end to the 3' end; the card-spinning probe H3 sequentially includes a card-spinning probe H2 identification sequence and a card-spinning probe H4 trigger sequence from the 5' end to the 3' end; and the card-spinning probe H4 sequentially includes a card-spinning probe H3 identification sequence from the 5' end to the 3' end.

[0011] Furthermore, the sequence of the hairpin probe H2 is shown in SEQ ID NO:2; the sequence of the hairpin probe H3 is shown in SEQ ID NO:3; and the sequence of the hairpin probe H4 is shown in SEQ ID NO:4.

[0012] A method for detecting miRNA using the above-mentioned kit includes the following steps: adding the sample to be tested to a sensing hydrogel, sequentially adding hairpin probe H2, hairpin probe H3 and hairpin probe H4, and incubating; the miRNA in the sample triggers the self-assembly of the hairpin probes, which in turn cascades and initiates a hybridization chain reaction to form a long double-stranded DNA product, causing the fluorescent group on the hairpin probe H1 to move away from the surface of the metal-organic framework material, generating a fluorescent signal; and quantitatively analyzing the fluorescence intensity.

[0013] Furthermore, hairpin probes H2, H3, and H4 were prepared into a 0.1–10 μM reaction solution, heated at 90–95 °C for 3–5 min, cooled to room temperature at a rate of 0.1–1 °C / min, and held at this temperature for 2 h to complete the annealing process, followed by incubation.

[0014] Application of the above-mentioned fluorescence sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction in rapid miRNA detection.

[0015] By adopting the above scheme, the method of the present invention has the following advantages: 1. This invention enriches miRNA and immobilizes hairpin probes using metal-organic framework (MOF) materials to form reaction centers with high local concentrations; it utilizes an isothermal amplification strategy of catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) cascade to achieve recycling of target miRNA and dual signal amplification; finally, it uses 3D printing technology to precisely construct the sensing unit into a hydrogel array to achieve high-throughput and rapid fluorescence detection of multiple samples.

[0016] 2. When using the kit of this invention for detection, the target miRNA first triggers the CHA reaction. This reaction can cyclically utilize a single miRNA molecule to open multiple hairpin probes H1-H2. Subsequently, each opened H2 can trigger the HCR reaction, causing hairpin probes H3-H4 to undergo chain hybridization assembly, forming a long double-stranded DNA polymer. This transforms the input of a single miRNA into the output of a large amount of double-stranded DNA. Before the reaction, the fluorescent group is quenched due to its proximity to the MOF, and is in an "off" state. After the reaction, the formed double-stranded DNA product moves the fluorescent group away from the MOF surface, and the fluorescence returns to an "on" state, thereby achieving ultrasensitive fluorescence detection of miRNA.

[0017] 3. The CHA-HCR cascade reaction of the present invention has a dual recognition mechanism, which can effectively distinguish miRNAs with single-base differences. Furthermore, the enrichment effect of MOF combined with the dual signal amplification capability of CHA-HCR enables the detection limit to reach the fM or even aM level.

[0018] 4. The entire isothermal amplification and detection process of this invention can be completed within 90 minutes, which is superior to the traditional qRT-PCR method and has an extremely fast detection speed. This invention, combined with a hydrogel array constructed using 3D printing technology, enables simultaneous and parallel detection of 96-well plate samples, meeting the needs of high-throughput screening. Attached Figure Description

[0019] Figure 1 A schematic diagram of the hairpin structure design and free energy change during the CHA-HCR reaction.

[0020] Figure 2 A comparison chart showing the detection of free nucleic acid content using polyacrylamide gel electrophoresis; Figure 3 A comparison of fluorescence intensity between FAM-labeled nucleic acids and MOFs after incubation; Figure 4 A comparison chart showing the specificity of the miR-122 detection array; Figure 5 The results are an evaluation of the sensitivity and quantification capability of miRNA-122 detection. Figure 6 The results of toxicity assays for a liver injury cell model constructed using acetaminophen (APAP); Figure 7 The figure shows the actual effect of detecting the expression level of miRNA-122 in the liver injury model; Control is the negative control group and APAP is the drug injury experimental group. Detailed Implementation

[0021] Example: (1) Ligand 2-aminoterephthalic acid and metal ion donor zirconium chloride were taken at a molar ratio of 1:1.13 and dissolved in N,N-dimethylformamide (DMF). The reaction was carried out in a high-pressure reactor at 100°C for 8 hours by hydrothermal reaction. After the reaction was completed, the mixture was cooled, centrifuged, washed and dried to obtain MOF material. (2) According to Figure 1Four hairpin-shaped DNA probes for cascade signal amplification were designed and synthesized, named H1, H2, H3, and H4, respectively. Hairpin probe H1 consists of a hairpin structure from the 5' end to the 3' end containing a carboxyl linking sequence, an H2 trigger sequence, and a target miRNA recognition sequence, with 6-FAM and a reactive group -COOH modified at the end. Hairpin probe H2 consists of the hairpin probe H1 recognition sequence and the hairpin probe H3 trigger sequence from the 5' end to the 3' end. Hairpin probe H3 consists of the hairpin probe H2 recognition sequence and the hairpin probe H4 trigger sequence from the 5' end to the 3' end. Hairpin probe H4 consists of the hairpin probe H3 recognition sequence from the 5' end to the 3' end. The sequences of hairpin probes H1, H2, H3, and H4 are shown in Table 1, where the underlined part of H1 is the target recognition fragment, which exposes the H2 trigger sequence after recognition. The AAAAAA at the 5' end of the H1 hairpin is a carboxyl linking occupant sequence. Table 1: Sequences of hairpin probes H1, H2, H3, and H4

[0022] (3) Construction of miRNA capture unit: The carboxyl group at the end of H1 is activated by carbodiimide and N-hydroxysuccinimide, so that it is covalently linked to the amino group on the surface of MOF material, thereby fixing H1 onto MOF and constructing miRNA capture unit. At this time, the fluorescent group modified on hairpin H1 is very close to MOF material, and fluorescence resonance energy transfer (FRET) occurs, resulting in efficient quenching of fluorescence, and the system is in the "signal off" state; (4) Preparation of fluorescence sensing hydrogel array: Using chitosan hydrogel as a carrier, the miRNA capture unit constructed in step (3) was uniformly mixed with 2% chitosan hydrogel prepolymer solution to obtain a uniform fluorescence sensing hydrogel prepolymer solution; then, the mixture was precisely printed onto the bottom of the well of a 96-well plate using a high-precision 3D printing device to form a regular microarray. After cross-linking and curing, a fluorescence sensing hydrogel array was obtained. (5) Prepare a 1 μM reaction solution by hairpin probe H2, hairpin probe H3 and hairpin probe H4, heat at 95℃ for 5 min, cool to room temperature at a rate of 0.1~1℃ / min, and keep at the temperature for 2 h to complete the annealing; Hairpin probes H2, H3, and H4 were diluted to a working concentration of 50 nM. The sample to be tested was added to the sensing hydrogel. The sample to be tested and hairpin probe H2 were added to a well plate containing the sensing hydrogel and incubated at 37 °C for 45 min. Then hairpin probes H3 and H4 were added, with each probe added in a volume of 50 μL. The total volume of the sample to be tested and all probes was 200 μL. The samples were incubated together for another 45 min. The secondary structure stability of each hairpin probe was verified using nucleotide sequence analysis software to ensure that it could maintain its hairpin conformation at 37°C. Specifically, hairpin H1 has a stem-loop portion with a recognition sequence that is completely complementary to the target miRNA, such as... Figure 1 As shown, its free energy is -13.39 kcal / mol, which is the basis for triggering subsequent cascade reactions; (6) The miRNA in the test sample is triggered, first recognizing and hybridizing with the loop of H1 to form a complex (free energy -30.33 kcal / mol). This complex opens the stem-loop structure of H1 through a chain displacement reaction, exposing its sticky end. This end binds to H2, forming the H1+H2 complex (free energy -43.42 kcal / mol). After hybridization between H1 and H2, the target miRNA is displaced, which can cyclically trigger the next H1 molecule, achieving first-stage catalytic amplification (CHA reaction). The exposed end of the H1-H2 complex binds to H3, forming the H1+H2+H3 complex (free energy -65.05 kcal / mol). Subsequently, this complex binds to H4, ultimately forming the H1+H2+H3+H4 complex (free energy -84.64 kcal / mol). (kcal / mol), cascaded hybridization chain reaction to form long double-stranded DNA product; with the generation of ultra-long double-stranded DNA polymer, the fluorescent group on the hairpin probe H1 is carried away from the MOF surface, the FRET effect is destroyed, the fluorescence is restored, and the system is switched to the "signal on" (On) state. (7) High sensitivity and high specificity of the target miRNA can be achieved by detecting the fluorescence intensity at Ex 480 nm / Em 525 nm wavelength using a microplate fluorescence reader. In the presence of the target miRNA, the fluorescence signal reaches a plateau within 90 minutes, and its signal intensity is several times that of the negative control.

[0023] Sensitivity (enrichment) and specificity evaluation of the detection system: 1. MOF enrichment of nucleic acid molecules: Polyacrylamide gel electrophoresis: Nucleic acid molecules were incubated with equal volumes of 0, 0.1, 0.5, and 1.0 mg / mL MOF solution for 30 min, respectively. After incubation, the free nucleic acid content was detected by polyacrylamide gel electrophoresis. The results of polyacrylamide gel electrophoresis are shown below. Figure 2 As shown in the figure, the amount of free nucleic acid gradually decreases with the increase of MOF concentration. When the MOF concentration is 1.0 mg / mL, the intensity of the electrophoretic bands decreases significantly, indicating that MOF has a good enrichment effect on nucleic acid molecules.

[0024] Fluorescence: After incubating FAM-labeled nucleic acids with MOF for 30 minutes, the fluorescence intensity was detected. Three groups were established: FAM-labeled nucleic acid, MOF, and MOF+FAM-labeled nucleic acid. The fluorescence detection results are shown below. Figure 3 As shown in the figure, compared with the FAM-labeled nucleic acid group, the fluorescence intensity of the FAM-labeled nucleic acid (i.e., the MOF+FAM-labeled nucleic acid group) after incubation with MOF for 30 minutes was significantly reduced.

[0025] 2. Specificity Assessment: By introducing other miRNAs commonly found in the miR-122 sequence as potential interfering substances, the ability of the hydrogel array to distinguish between target and non-target signals was tested to evaluate the high reliability of the detection method. miR-193, miR-194, miR-483, and miR-885 were selected as interfering substances. Under the same experimental conditions, samples containing only the target miR-122 and samples containing equal amounts of each interfering miRNA were detected. The results are as follows: Figure 4 As shown, only the target miR-122 produced a high-intensity fluorescence signal, which fully demonstrates the high specificity of the miR-122 detection array constructed in this invention. The detection method of this invention can effectively distinguish the target miR-122 from other miRNAs with similar sequences, ensuring the accuracy and reliability of detection results in complex clinical samples (such as serum and tissue extracts).

[0026] 3. Quantitative detection capability evaluation: miRNA-122 standards with concentration gradients from 1 aM to 100 nM were prepared, and fluorescence assays were performed according to the aforementioned detection procedure. Results are as follows... Figure 5 As shown in the figure, the fluorescence intensity is linearly correlated with the logarithm of the miRNA concentration in the range of 1 aM-10 nM. This result indicates that the detection system exhibits excellent quantitative detection capability in the concentration range of 1-9 Log(fM), and the fluorescence signal increases with the increase of the target miRNA concentration, confirming that this method has reliable quantitative analysis performance in practical applications.

[0027] Detection of miRNA-122 expression in a liver injury cell model: Construction of a liver injury cell model and sample preparation: HepG2 cells were cultured to the logarithmic growth phase, and a liver injury model was constructed using acetaminophen (APAP). A control group (without APAP) and an APAP-treated group (containing 7.8 mM APAP) were set up. After 24 hours of treatment, total RNA was extracted from both groups of cells using a column extraction method. The extracted RNA was appropriately diluted with TE buffer and used as the test sample.

[0028] 1. Experimental results of constructing a HepG2 cell liver injury model using acetaminophen (APAP). The results are as follows: Figure 6 As shown, HepG2 cell viability decreased in a dose-dependent manner with increasing APAP concentration. The half-maximal inhibitory concentration (IC50) of APAP for HepG2 cells was calculated to be 7.8 mM using the dose-response curve. This concentration provides experimental basis for selecting an appropriate concentration for establishing a liver injury model. This result indicates that APAP can effectively induce HepG2 cell injury, successfully establishing a cell model of drug-induced liver injury.

[0029] 2. High-throughput detection performance verification: Detection of miRNA-122: Samples from the control group and the APAP-treated group were added to a 96-well hydrogel array according to the CHA-HCR reaction system shown in Table 1. After incubation at 37°C in the dark for 90 minutes, the fluorescence intensity was detected using a microplate reader (Ex = 480 nm, Em = 525 nm).

[0030] The results are as follows Figure 7 As shown, by comparing the fluorescence intensity of the control group and the APAP-treated group, a significant enhancement of the fluorescence signal in the APAP-treated group was observed. This result demonstrates that under APAP-induced liver injury conditions, the expression level of miRNA-122 in HepG2 cells is significantly upregulated, indicating that this method can sensitively detect changes in miRNA expression levels during drug-induced liver injury.

[0031] The above detection results indicate that this invention has successfully developed a 3D-printed fluorescence sensing hydrogel system based on MOF and CHA-HCR isothermal amplification, achieving highly sensitive, high-throughput, and rapid detection of miRNAs, and its detection efficacy has been verified in a drug-induced liver injury model.

[0032] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction, characterized in that, The invention includes a capture unit and a hydrogel supporting the capture unit; the capture unit includes an amino-containing metal-organic framework material as a carrier and quencher, and a hairpin probe H1 supported on the carrier; the hairpin probe H1 contains a carboxyl group and a fluorescent group that can be covalently linked to an amino group.

2. The fluorescence sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction according to claim 1, characterized in that, The amino-containing metal-organic framework material is prepared by amino-containing organic ligands and zirconium ions; the molar ratio of the amino-containing organic ligands to zirconium ions is 1:1.1~1.5; the hairpin probe H1 includes a carboxyl group linking sequence and a target miRNA recognition sequence; the sequence of the hairpin probe H1 is shown in SEQ ID NO:1; the fluorescent group includes one or more of 5-FAM, 6-FAM, (5-FAM)2 or (6-FAM)2.

3. A method for preparing a fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction as described in claim 1 or 2, characterized in that, Includes the following steps: A metal-organic framework material containing amino groups was obtained; a hairpin probe H1 containing a carboxyl group linking sequence and a target miRNA recognition sequence was designed, so that the hairpin probe H1 contains carboxyl groups and fluorescent groups; the carboxyl groups in the hairpin probe were activated and then covalently linked with the amino groups in the metal-organic framework to obtain a capture unit; the capture unit was mixed evenly with a hydrogel to obtain a mixture, which was then cross-linked and cured to obtain the final product.

4. The method for preparing the fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction according to claim 3, characterized in that, The mixture was 3D printed onto the bottom of a porous plate to form a hydrogel microarray.

5. A miRNA detection kit, characterized in that, Includes the fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction as described in claim 1 or 2, and hairpin probes H2, H3 and H4 that are complementary to hairpin probe H1.

6. The miRNA detection kit according to claim 5, characterized in that, The card-spinning probe H2, from its 5' end to its 3' end, sequentially includes the card-spinning probe H1 identification sequence and the card-spinning probe H3 trigger sequence; the card-spinning probe H3, from its 5' end to its 3' end, sequentially includes the card-spinning probe H2 identification sequence and the card-spinning probe H4 trigger sequence; the card-spinning probe H4, from its 5' end to its 3' end, includes the card-spinning probe H3 identification sequence.

7. The miRNA detection kit according to claim 5, characterized in that, The sequence of the hairpin probe H2 is shown in SEQ ID NO:2; the sequence of the hairpin probe H3 is shown in SEQ ID NO:3; and the sequence of the hairpin probe H4 is shown in SEQ ID NO:

4.

8. A method for detecting miRNA using the kit described in claim 5, characterized in that, Includes the following steps: The sample to be tested was added to the sensing hydrogel, and hairpin probes H2, H3 and H4 were added in sequence for incubation. The miRNA in the sample triggered the self-assembly of the hairpin probes, which in turn triggered a hybridization chain reaction to form a long double-stranded DNA product. This caused the fluorescent group on the hairpin probe H1 to move away from the surface of the metal-organic framework material, thus generating a fluorescent signal. Quantitative analysis of fluorescence intensity was performed.

9. The method for detecting miRNA according to claim 8, characterized in that, Hairpin probes H2, H3, and H4 were prepared into a 0.1–10 μM reaction solution, heated at 90–95 °C for 3–5 min, cooled to room temperature at a rate of 0.1–1 °C / min, and held at this temperature for 2 h to complete the annealing process, followed by incubation.

10. The application of the fluorescent sensing hydrogel based on MOF and CHA-HCR isothermal amplification reaction as described in claim 1 or 2 in the rapid detection of miRNA.

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