Biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA

Abstract

The invention discloses a biochemical sensing system based on a catalytic hairpin assembly circular reaction and a G quadruplex-heme DNA enzyme formation reaction. The biochemical sensing system is used for photoelectrochemical colorimetric detection of circular RNA (circRNA). After the nucleic acid probe H1 is combined with the nano magnetic beads (MNPs), the circRNA is specifically recognized, and an MNPs / H1 / circRNA compound is formed; the MNPs / H1 / H2 compound reacts with a probe H2 to form an MNPs / H1 / H2 compound; the H1 probe and the H2 probe release a part of sequences by utilizing restriction endonuclease cutting, and the unreleased sequences are combined with hemin to form G quadruplex-heme DNA enzyme with peroxidase-like activity; through cascade catalytic reaction, the color of a solution of a reaction system is changed, the linear relation between the absorbance of the solution and the concentration of circRNA in the solution is fitted, and an effective method for carrying out photoelectrochemical colorimetric detection on circRNA is developed.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical detection of circular RNA and preparation of biochemical sensing systems, and in particular to a method for preparing a biochemical sensing system based on a catalytic hairpin assembly cycle reaction and a G-quadruplex-heme DNA enzyme formation reaction. The biochemical sensing system is used for photoelectrochemical colorimetric detection of circular RNA. Background Art

[0002] Circular RNA (circRNA) is a type of non-coding RNA molecule that lacks a 5' cap and a 3' poly(A) tail and is covalently linked to form a circular structure. CircRNAs contain miRNA response elements, which can act as competing endogenous RNAs, binding to miRNAs and acting as miRNA sponges in cells, thereby relieving the inhibitory effect of miRNAs on their target genes and upregulating target gene expression. As a special type of non-coding RNA molecule with a closed circular structure, circRNAs possess three distinct characteristics: a circular structure, the absence of a poly(A) tail, and the stability of non-coding RNA. Compared to linear miRNA molecules, circRNAs are more biologically stable. CircRNAs are single-stranded, covalently closed RNA molecules and are ubiquitous in species from viruses to mammals. Most circRNAs are formed by the circularization of exons, while a smaller number are formed by the circularization of introns to form a lariat structure.

[0003] CircRNAs are primarily found in the cytoplasm, with some also stored in exosomes. This distribution allows circRNAs to exert important biological functions in the cytoplasm and to be transferred between cells via exosomes. The sequences of circRNAs overlap substantially with those of their linear RNA homologues. Due to their unique covalently closed circular structure and the presence of back-splice junctions (BSJs), circRNAs and their linear RNA homologues exhibit significant differences in their intracellular functions and stability. In particular, the covalently closed circular structure renders circRNAs less susceptible to degradation by RNA exonucleases, enabling their persistence and function within cells. CircRNAs exert their biological functions by acting as transcriptional regulators, miRNA sponges, and protein templates.

[0004] circRNAs have unique expression profiles and play a key role in various diseases, making them potential diagnostic biomarkers and therapeutic targets. By participating in the regulation of gene transcription and translation, circRNAs play an important role in various life processes, including cell proliferation, apoptosis, and aging. Furthermore, the specific expression of circRNAs in diseases such as cardiovascular disease, liver cancer, and breast cancer makes them potential biomarkers. Common circRNA detection methods include quantitative real-time PCR (qRT-PCR) for quantitative verification, Northern blotting, sequencing, and alignment. For example, Liu Jingxin et al. developed a method for detecting circular RNA based on microfluidic technology (Liu Jingxin; He Shiliang; Wen Jiancheng. National invention patent application publication number: CN117587103A), Wang Shanshan et al. designed a circular RNA detection method based on capillary gel electrophoresis (Wang Shanshan; Dong Jia; Cao Jinping; Wang Fan. National invention patent application publication number: CN117538404A), Zhu Libo et al. disclosed a quantitative and rapid detection method for the purity of circular RNA vaccines based on nanopores (Zhu Libo; Tan Shengwei; Yang Haiping; Gao Bing; Tan Zheng. National invention patent application publication number: CN117517427A), Liao Xun et al. provided a visual circular RNA rapid detection kit based on the CRISPR-Cas13a system and its application (Liao Xun; Peng Yong. National invention patent application publication number: CN115948405A).

[0005] Although qRT-PCR has high accuracy and short detection time, its accuracy may be affected by many factors, especially the design of primers needs to be very precise to avoid amplifying linear RNA. Imprint hybridization analysis is not sensitive enough for low-abundance circRNA, and the analysis process is time-consuming, which is not conducive to rapid detection and analysis. In the context of current analytical detection technology, it is urgent to develop a biochemical sensing system for efficient detection of circRNA that can detect low abundance, high sensitivity and simple operation. Based on this, the present invention has developed a biochemical sensing system based on a catalytic hairpin assembly cycle reaction (Catalytic Hairpin Assembly abbreviated as CHA) and a G-quadruplex-hemin DNAzyme formation reaction and a preparation method thereof. The biochemical sensing system is used for photoelectrochemical colorimetric detection of circular RNA. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for preparing a biochemical sensing system based on a catalytic hairpin assembly cycle reaction and a G-quadruplex-heme DNA enzyme formation reaction. The biochemical sensing system is used for highly selective, highly sensitive, simple and rapid photoelectrochemical colorimetric detection of circular RNA.

[0007] To achieve the above objectives, the present invention relates to a biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA, which has the following significant features:

[0008] (1) Using specific probes, circular RNA (circRNA) is specifically identified by catalyzing the hairpin assembly cycle reaction (CHA); the CHA product is cut by restriction endonucleases and then combined with hemin to form G-quadruplex-hemin DNAzyme to amplify the signal; finally, the circRNA is quantitatively determined by colorimetric detection.

[0009] (2) The probe body connected to the surface of the nanoparticles (MNPs) is a specific nucleic acid probe H1 that has a specific recognition function for the circRNA back splicing site (BSJ) sequence. Its nucleic acid sequence is 5'-CAC AAG CCT CCC TTT TGTTTT TCT GAA ATT GAG AGT CAG AAA AAC AAA AGG GCC GGG AGG GAT GGG TT-3'. The probe H1 is treated with 3'-amino group and then combined with carboxylated MNPs to form the probe MNPs / H1; the free probe H2 has a nucleic acid sequence of 5'-GTT TTT CTG ACT CTC AAT TTC AGA AAA ACA AAA GGG GAA ATT GAG AGT C-3'. In the presence of the target circRNA, the free probe H2 specifically recognizes the MNPs / H1, thereby forming an MNPs / H1 / H2 complex system.

[0010] (3) Preparation of probe MNPs / H1: MNPs were mixed with coupling agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). After activation of the carboxyl groups on the surface of MNPs, the mixture was mixed with H1 and incubated overnight to prepare probe MNPs / H1.

[0011] (4) CHA reaction conditions: The probe MNPs / H1, target detection circRNA, H2 dispersion and phosphate buffer saline (PBS) buffer were measured in sequence at a volume ratio of 1:1:1:1, mixed evenly and reacted at 37°C. The mixed system was treated by magnetic separation to obtain a CHA reaction product; the product had the following characteristics, namely, the probe MNPs / H1 and the BSJ sequence of the circRNA were combined through base complementary pairing to form an MNPs / H1 / BSJ complex, the hairpin structure of the probe H1 was thus opened, and the recognition sites of the probe H2 and the probe H1 were exposed. The two combined with each other, and then the circRNA was released, forming a complex of the CHA reaction product and MNPs / H1 / H2.

[0012] (5) G-quadruplex-hemin DNAzyme formation reaction: Under the action of restriction endonucleases, partial sequences of probes H1 and H2 in the MNPs / H1 / H2 complex are released, while the sequence near the 3' end of probe H1 remains on the MNPs and then combines with the hemin solution to form a G-quadruplex-hemin DNAzyme structure with peroxidase-like activity.

[0013] (6) Determination of circRNA: MNPs with a certain amount of G-quadruplex-hemin DNAzyme bound to their surface were mixed with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), and the target circRNA was added to react. Based on the absorbance of the mixed reaction system and the logarithm of the circRNA concentration therein, a standard curve was prepared by linear fitting to achieve photoelectrochemical colorimetric detection of circRNA.

[0014] The present invention discloses a biochemical sensing system based on a catalytic hairpin assembly cycle reaction and a G-quadruplex-heme DNA enzyme formation reaction and a preparation method thereof. The biochemical sensing system is used for photoelectrochemical colorimetric detection of circular RNA and has the advantages of high selectivity, high sensitivity, and convenient detection, which are mainly reflected in:

[0015] 1. CircRNA is specifically identified by a specific probe through the CHA reaction. The CHA product is cut by a restriction endonuclease and then binds to hemin to form a G-quadruplex-hemin DNAzyme, achieving signal amplification. CircRNA is detected by photoelectrochemical colorimetry.

[0016] 2. A specific nucleic acid probe H1 binds to MNPs and then specifically recognizes the target circRNA, forming an MNPs / H1 / circRNA complex. This complex then reacts with probe H2 to form the CHA product MNPs / H1 / H2. The released circRNA can bind to the newly formed MNPs / H1 complex, triggering the CHA reaction again, thereby producing more MNPs / H1 / H2 complexes.

[0017] 3. When the CHA reaction products MNPs / H1 / H2 accumulate to a certain level, they are cut using a specific restriction endonuclease (such as Nt.BstNBI), thereby releasing the H1 probe and part of the H2 probe. The unreleased nucleic acid sequence contains a G-rich region, which binds to hemin to form a G-quadruplex-hemin DNAzyme, and is separated using the magnetic properties of magnetic nanoparticles (MNPs).

[0018] 4. The peroxidase-like catalytic effect of G-quadruplex-hemin DNAzyme catalyzes the decomposition of hydrogen peroxide (H2O2) to produce free radicals, thereby further promoting the oxidation reaction of TMB and improving its sensitivity. The linear relationship between the absorbance of the mixed reaction system and the concentration of circRNA in the system was fitted, realizing the effective detection of low-concentration target circRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .A biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA, as well as a schematic diagram of its preparation process and detection principle.

[0020] Figure 2 .The secondary structure and free energy of RNA (probes H1 and H2) at 37°C based on the minimum free energy model (MFE) optimization algorithm.

[0021] Figure 3 Agarose gel electrophoresis was used to characterize the related products, including H1, H2, cSMARCA5, H1+cSMARCA5, H2+cSMARCA5, H1+H2, and H1+H2+cSMARCA5.

[0022] Figure 4 Transmission electron microscopy (TEM) imaging was used to characterize the related products, including (A) MNPs, (B) MNPs / H1, (C) MNPs / H1 / circRNA, and (D) MNPs / H1 / H2.

[0023] Figure 5(A) Characterization of the CHA reaction triggered by circRNA using UV-visible absorption spectroscopy; (B) Absorption spectra of the corresponding systems measured in the control group without the addition of target and in the experimental group with the addition of target; (C) Results of cSMARCA5 detection in four groups of human serum samples.

[0024] Figure 6 .(A) Verification of the specificity of the detection method of the present invention; (B) Results of anti-interference experimental research; (C) Experimental results of detection research on actual samples.

[0025] Figure 7 .The sensitivity of the detection method of the present invention was determined for BSJ (AB) and circRNA (CD), respectively. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below with reference to the accompanying drawings and through specific embodiments.

[0027] Example 1

[0028] This embodiment involves the use of specific probes to specifically identify circRNA through the CHA reaction, using restriction endonucleases to cut the CHA product, which then binds to hemin to form a G-quadruplex-hemin DNAzyme to achieve signal amplification, and using photoelectrochemical colorimetry to achieve convenient detection of circRNA. The present invention involves a biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA, its preparation process, and a schematic diagram of the detection principle are shown in FIG. Figure 1 As shown, the specific preparation steps of the biochemical sensing system are as follows:

[0029] 1. Preparation of probe MNPs / H1: 100 μL of a 0.5 wt% MNPs solution was added to a 1.5 mL centrifuge tube, and the MNPs were magnetically washed three times with phosphate buffer PBS (0.01 M, pH 7.4), and then dispersed into 200 μL of PBS buffer; then mixed with 100 μL of a coupling agent containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.01 M N-hydroxysuccinimide (NHS), and reacted at room temperature for 30 min to complete the carboxyl activation of the MNPs; the MNPs were magnetically washed a second time three times, and then mixed with 10 μL of a 10 μM nucleic acid probe H1, and reacted at 37°C for 6 h to prepare MNPs / H1; after a third magnetic washing three times, the MNPs were dispersed into 1 mL of PBS buffer and stored at 4°C until use.

[0030] 2. Preparation of standard concentration circRNA solutions: Select a representative circRNA, cSMARCA5, for experimental preparation. Use distilled water to gradually dilute and mix a 10 μM cSMARCA5 stock solution to form a series of circRNA standard solutions with different concentration gradients, such as 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, and 10 fM, for subsequent experiments.

[0031] 3. CHA reaction: Take 16 μL of probe MNPs / H1 in step 1, 16 μL of probe H2, 16 μL of different concentrations of cSMARCA5, and 16 μL of PBS buffer in a clean bench, add them to a 1.5 mL centrifuge tube in sequence, and incubate at 37°C for 60 min; treat the MNPs / H1 / H2 complex and CHA reaction product in the mixed system by magnetic separation, wash them magnetically with PBS three times, and then disperse them into 64 μL of PBS. Among them, the secondary structure and free energy of RNA (probes H1 and H2) at 37°C based on the minimum free energy model (MFE) optimization algorithm are as follows: Figure 2As shown, the nucleic acid sequences of the representative circRNA (cSMARCA5), the back-splicing site BSJ, probe H1 and probe H2 from 5' to 3' end are: 5'-GGA GGC UUG UGG AUC AGA AUC UGA ACA AAAUUG GGA AAG AUG AAA UGC UUC AAA UGA UUA GAC AUG GAG CAA CAC AUG UGU UUG CUUCAA AGG AAA GUG AGA UCA CUG AUG AAG AUA UCG AUG GUA UUU UGG AAA GAG GUG CAAAGA AGA CUG CAG AGA UGA AUG AAA AGC UCU CCA AGA UGG GCG AAA GUU CAC UUA GAAACU UUA CAA UGG AUA CAG AGU CAA GUG UUU AUA ACU UCG AAG GAG AAG ACU AUA GAGAAA AAC AAA AG-3', 5'-AGA GAA AAA CAA AAG GGA GGC TTG TGG AT-3', 5'-CAC AAG CCTCCC TTT TGT TTT TCT GAA ATT GAG AGT CAG AAA AAC AAA AGG GCC GGG AGG GAT GGGTT-NH2-C6-3', 5'-GTT TTT CTG ACT CTC AAT TTC AGA AAA ACA AAA GGG GAA ATT GAGAGT C-3'.

[0032] 4. G-quadruplex-hemin DNAzyme formation reaction: Add 5 μL of 0.5 U / mL restriction endonuclease Nt.BstNBI and 10 μL of NEBuffer r3.1 to the MNPs / H1 / H2 solution in step 3. After mixing, incubate at 55°C for 40 min and then at 80°C for 20 min to inactivate the enzyme. Next, cool to room temperature and perform magnetic separation. Wash three times with PBS, disperse into 80 μL of PBS, react with 25 μL of 0.2 mM hemin solution at room temperature for 40 min, and finally separate magnetically and wash three times with PBS.

[0033] When the restriction endonuclease Nt.BstNBI specifically cleaves the MNPs / H1 / H2 complex, the 5'-AGAAAAACAAAAGGGCCGGGAGGGATGGGTT-3' sequence is removed. Near the 3' end, four groups of GGG bases are present, which react with the hemin solution in the subsequent reaction in PBS buffer (0.01 M, pH 7.4) in the dark at room temperature to form a G-quadruplex-heme DNA enzyme. This is due to the possible π-π stacking and hydrophobic interactions between the G bases of the G-quadruplex and the porphyrin ring of hemin, which help stabilize the structure of the G-quadruplex-heme DNA enzyme. This enzyme catalyzes reactions with H2O2 and the substrate TMB.

[0034] Agarose gel electrophoresis experiments were used to characterize the related products, including H1, H2, cSMARCA5, H1+cSMARCA5, H2+cSMARCA5, H1+H2, and H1+H2+cSMARCA5, e.g. Figure 3 As shown in the figure, H1 binds to cSMARCA5, while H2 does not. Under conditions without target priming, probe H1 does not undergo strand leakage and does not bind to probe H2. Both probes H1 and H2 undergo CHA reactions with the target cSMARCA5 without strand leakage. Due to the addition of excess H1, cSMARCA5 displaced by probe H2 binds to the new probe H1, but H1 / cSMARCA5 formation persists. The appearance of the H1 / H2 band indicates that under conditions of target circRNA priming, probes H1 and H2 bind to form the H1 / H2 complex.

[0035] Transmission electron microscopy (TEM) imaging was used to characterize the related products, including (A) MNPs, (B) MNPs / H1, (C) MNPs / H1 / circRNA, and (D) MNPs / H1 / H2. Figure 4As shown. MNPs and H1 were added to (B), and the base number of H1 was 68bp. Compared with (A), there was a clear film layer on the outside of the magnetic beads in (B), with a thickness of 298.33nm, indicating that the probe MNPs / H1 was successfully prepared. MNPs, H1 and circRNA were added to (C), and the base number of circRNA was 269bp. The thickness of the outer membrane of the magnetic beads in (C) was 505.26nm, which was about 200nm thicker than the outer membrane of the magnetic beads in (B), indicating that MNPs / H1 / circRNA was successfully bound. MNPs, H1, circRNA and H2 were added to (C), and the base number of H2 was 49bp. After the addition of probe H2, it specifically bound to the recognition site of MNPs / H1 / circRNA, released circRNA, and formed an MNPs / H1 / H2 complex. The thickness of the outer membrane of the magnetic beads in (D) was 357.91nm, indicating that the MNPs / H1 / H2 complex system was successfully bound.

[0036] 5. Colorimetric detection: Pipette the liquid from the centrifuge tube in step 4, add 2 μL each of 12 mg / mL TMB, 0.75% H2O2, and 100 μL of acetic acid-sodium acetate buffer solution (pH 4.0), react at 37°C in the dark for 5 min, then perform magnetic separation, transfer 100 μL of the liquid to a 96-well plate, and detect at 652 nm using a microplate reader to obtain a series of absorbance values ​​corresponding to the circRNA concentration. A linear regression equation is constructed between the absorbance value and the logarithm of the target circRNA concentration to obtain a standard curve.

[0037] UV-visible absorption spectroscopy was used to characterize the CHA reaction triggered by circRNA. Figure 5(A). Different reactants were reacted in an acetic acid-sodium acetate solution for 5 minutes, and the absorption spectra were measured at wavelengths of 500-800 nm. Line a shows the circRNA-triggered CHA reaction, followed by Nt.BstNBI digestion, which then reacted with hemin to form a G-quadruplex-hemin DNAzyme, which catalyzed the reaction between TMB and H2O2. Line b shows the circRNA-triggered CHA reaction, which then reacted with hemin without Nt.BstNBI digestion to form a G-quadruplex-hemin DNAzyme, which then catalyzed the reaction between TMB and H2O2. Comparing the results in b and a, we can see that enzymatic digestion improves sensitivity. After digestion, the G-rich sequence at the 3' end of MNPs-H1 is separated, facilitating binding to hemin. The G-quadruplex-hemin DNAzyme formed in line a is greater than that in line b, catalyzing the reaction between H2O2 and the substrate TMB more quickly, resulting in a higher sensitivity than b. Line c represents the product of a CHA reaction initiated by circRNA, followed by digestion with Nt.BstNBI but without reaction with hemin, resulting in no G-quadruplex-hemin DNAzyme formation. This product catalyzes the reaction between TMB and H2O2. Comparison of line c with line a demonstrates that hemin is crucial for the formation of the G-quadruplex-hemin DNAzyme. Without hemin, the G-quadruplex-hemin DNAzyme does not form and cannot accelerate the reaction between H2O2 and the substrate TMB. Line d represents a CHA reaction initiated by the non-target circRNA. Following addition of probes H1 and H2, no CHA reaction occurs. Following digestion with Nt.BstNBI, the product reacts with hemin but does not form the G-quadruplex-hemin DNAzyme, resulting in the catalysis of the reaction between TMB and H2O2. Comparison of line d with line a demonstrates that the target circRNA is crucial for initiating the CHA reaction, confirming the selectivity of the inventive method. Line e shows the absence of target circRNA, probes H1 and H2, and no CHA reaction. Following Nt.BstNBI digestion, the enzyme reacts with hemin, but no G-quadruplex-hemin DNAzyme is formed to catalyze the reaction between TMB and H2O2. Comparison of line e with lines a and d indicates that H1 and H2 do not bind, resulting in no Nt.BstNBI digestion product. Line f shows the absorption spectrum measured after a 5-minute reaction of a specific amount of TMB with H2O2.

[0038] 6. Detection of actual samples: A solution spiked with a certain concentration of a representative circRNA (cSMARCA5) was measured and quantitatively detected using the method of the present invention. The obtained absorbance value was substituted into the above-mentioned standard curve to calculate the corresponding concentration of the circRNA.

[0039] Figure 5(B) shows the absorption spectra of the corresponding systems measured in the control group without the target and in the experimental group with the target added. The experimental group, with the target added at a concentration of 100 nM, had an absorbance of 0.4477 at 652 nm; the control group, with no target added, had an absorbance of 0.27545 at 652 nm. The difference between the two is significant, demonstrating that the method of the present invention can detect the target circRNA. Figure 5 (C) is the result of detecting cSMARCA5 in four groups of human serum samples. The experimental results confirm that the method of the present invention can obtain accurate and reliable detection results in actual samples.

[0040] Example 2

[0041] This embodiment involves the use of specific probes to specifically identify circRNA through the CHA reaction, using restriction endonucleases to cut the CHA products, which then bind to hemin to form a G-quadruplex-hemin DNAzyme to achieve signal amplification, and using photoelectrochemical colorimetry to achieve convenient detection of circRNA. The present invention relates to a biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA. The specific preparation steps of the biochemical sensing system are as follows:

[0042] Steps 1 to 5 of Example 2 are the same as those of Example 1, and step 6 is specifically as follows:

[0043] Detection of actual samples: cSMARCA5 was added to human serum samples, and human serum samples containing a certain concentration of the circRNA were taken for detection. The obtained absorbance value was substituted into the standard curve to calculate the concentration of the circRNA.

[0044] Figure 6 (A) is a validation experiment for the specificity of the detection method of the present invention. The relevant validation experiment was performed using the special sequence BSJ sequence of circRNA. BSJ sequences and sequences containing 1, 2, and 3 base mismatches were designed. The study showed that the absorbance of the 0 base mismatch group (0MUT) was the highest, and the absorbance of the 1 base mismatch group (1MUT) and the 2 base mismatch group (2MUT) was significantly lower than that of the 0MUT group. The absorbance of the 3 base mismatch group (3MUT) was basically the same as that of the control group, indicating that the method is highly specific for the target sequence. Figure 6(B) shows the results of an anti-interference experiment. At concentrations of 100 pM and 100 nM, the absorbance difference between the group containing only the homologous linear RNA and the control group was almost zero. When the target circRNA and the homologous linear RNA coexisted, the absorbance difference was almost the same as that when only the target circRNA was present. This result confirms that the detection method of the present invention can effectively distinguish the target circRNA and is not interfered with by the homologous linear RNA, demonstrating its high specificity and reliability in complex sample environments. Figure 6 (C) is the experimental result of the detection study on actual samples, and the serum samples were detected using the method of the present invention. The experiment was carried out on four groups of human serum to detect cSMARCA5 and obtain the corresponding cSMARCA5 concentration. First, the UV-visible absorption spectra of serum samples containing different concentrations of cSMARCA5 were measured, and the corresponding absorbance values ​​were recorded as 0.0054, 0.0116, 0.0033 and 0.0099 respectively. Then, according to the linear equation ΔAbs2=0.00864lgC circRNA -0.00614 calculated corresponding lgC circRNA The values ​​are 1.335, 2.053, 1.092 and 1.856 respectively, and then the corresponding C circRNA The concentration values ​​were 21.6fM, 112.8fM, 12.4fM and 71.7fM respectively.

[0045] Example 3

[0046] This embodiment involves the use of specific probes to specifically identify circRNA through the CHA reaction, using restriction endonucleases to cut the CHA products, which then bind to hemin to form a G-quadruplex-hemin DNAzyme to achieve signal amplification, and using photoelectrochemical colorimetry to achieve convenient detection of circRNA. The present invention relates to a biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA. The specific preparation steps of the biochemical sensing system are as follows:

[0047] Steps 1 to 5 of Example 3 are the same as those of Example 1, and step 6 is as follows:

[0048] Detection of actual samples: Serum from healthy individuals and serum from liver cancer patients were collected respectively, and the prepared photoelectrochemical sensing method was used for detection. The obtained absorbance values ​​were substituted into the standard curve to calculate the concentration of the circRNA.

[0049] Figure 7The results of the sensitivity experiment study were presented, and the sensitivity of the detection method of the present invention was measured for BSJ (Figure AB) and circRNA (Figure CD). Under the optimal experimental conditions, this method was used to detect different concentrations of BSJ sequences in PBS buffer at pH 7.4, and the detection wavelength range was 600-700nm, as shown in Figure (AB). The experimental results showed that in the concentration range of 10fM to 100nM, the absorbance change (ΔAbs) was well linearly correlated with the logarithm of the BSJ sequence concentration (logC). The linear equation is ΔAbs1=0.02134logC BSJ +0.00157(R 2 =0.99703), with a limit of detection (LOD) of 1.54 fM. Furthermore, the assay was used to detect different concentrations of cSMARCA5 under optimal conditions, with a detection wavelength range of 600-700 nm, as shown in Figures (CD). The circRNA detection method establishes two linear ranges between the absorbance change and the logarithm of cSMARCA5 concentration (logC). Within the concentration range of 10 fM to 1 pM, based on the linear equation ΔAbs2 = 0.00864 lgC circRNA -0.00614(R 2 =0.9978), achieving quantitative detection of cSMARCA5. In the concentration range of 1pM to 100nM, based on the linear equation ΔAbs3 = 0.02678lgC circRNA +0.05834(R 2 =0.9988), the quantitative detection of cSMARCA5 can also be achieved, and the LOD can reach the fM level of 2.01 fM, so this detection method has high sensitivity.

[0050] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A biochemical sensing system for photoelectrochemical colorimetric detection of circular RNA, having the following significant features: (1) Using specific probes, circular RNA (circRNA) is specifically identified by catalyzing a hairpin assembly cycle (CHA) reaction. The CHA product is cleaved by a restriction endonuclease and then combined with hemin to form a G-quadruplex-hemin DNAzyme to amplify the signal. Finally, colorimetric detection is used to achieve quantitative determination of circRNA. (2) The probe body connected to the surface of the nanoparticles (MNPs) is a specific nucleic acid probe H1 that has a specific recognition function for the circRNA back splicing site (BSJ) sequence. Its nucleic acid sequence is 5'-CAC AAG CCT CCC TTT TGT TTTTCT GAA ATT GAG AGT CAG AAA AAC AAA AGG GCC GGG AGG GAT GGG TT-3'. The probe H1 is treated with 3'-amino group and then combined with carboxylated MNPs to form the probe MNPs / H1; the free probe H2 has a nucleic acid sequence of 5'-GTTTTT CTG ACT CTC AAT TTC AGA AAA ACA AAA GGG GAA ATT GAG AGT C-3'. In the presence of the target circRNA, the free probe H2 specifically recognizes the MNPs / H1, thereby forming an MNPs / H1 / H2 complex system. (3) Preparation of probe MNPs / H1: MNPs were mixed with coupling agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups on the surface of MNPs. The mixture was then mixed with H1 and incubated overnight to prepare probe MNPs / H1. (4) CHA reaction conditions: The probe MNPs / H1, target detection circRNA, H2 dispersion, and phosphate buffer saline (PBS) buffer were measured in sequence at a volume ratio of 1:1:1:1, mixed evenly, and reacted at 37°C. The mixed system was treated by magnetic separation to obtain a CHA reaction product; the product had the following characteristics: the probe MNPs / H1 and the BSJ sequence of the circRNA were bound through base complementary pairing to form an MNPs / H1 / BSJ complex, the hairpin structure of the probe H1 was thus opened, and the recognition sites of the probe H2 and the probe H1 were exposed. The two bound to each other, and then the circRNA was released, forming a complex of the CHA reaction product and the MNPs / H1 / H2; (5) G-quadruplex-hemin DNAzyme formation reaction: Under the action of restriction endonucleases, partial sequences of probes H1 and H2 in the MNPs / H1 / H2 complex are released, while the sequence near the 3' end of probe H1 remains on the MNPs and then combines with the hemin solution to form a G-quadruplex-hemin DNAzyme structure with peroxidase-like activity; (6) Determination of circRNA: MNPs with a certain amount of G-quadruplex-hemin DNAzyme bound to their surface were mixed with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), and the target circRNA was added to react. Based on the absorbance of the mixed reaction system and the logarithm of the circRNA concentration therein, a standard curve was prepared by linear fitting to achieve photoelectrochemical colorimetric detection of circRNA.

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