Method for detecting double miRNA in urine based on multiple signal amplification and metal ion selective recognition phenomenon and application thereof

Abstract

The application provides a urine double-miRNA detection method based on multiple signal amplification and metal ion selective recognition phenomenon and application thereof, and relates to the technical field of biomedical diagnosis. 2+ and Ag + , which are respectively reacted with CDs and CdTe QDs, and finally, the fluorescence signals of CDs and the fluorescence signals of CdTe QDs are used for quantifying miRNA-375 and miRNA-148a. The application establishes a simple, rapid, sensitive and simultaneous homogeneous urine double-miRNA quantitative analysis strategy from the aspects of analysis instrument, analysis steps and cost, selection of signal reporting molecules, multiple nucleic acid signal amplification technology and application of selective recognition phenomenon.

Description

Technical Field

[0001] The present invention relates to the field of biomedical diagnostic technology, and in particular to a method for detecting dual miRNA in urine based on multiple signal amplification and metal ion selective recognition phenomena and its application. Background Art

[0002] Prostate cancer, a malignant epithelial tumor, is one of the most common urological tumors and the fifth leading cause of cancer death in men. Early screening and diagnosis are crucial for improving patient survival. Currently, the most commonly used clinical method for diagnosing prostate cancer is serum prostate-specific antigen (PSA) testing combined with prostate biopsy. However, the sensitivity and specificity of serum PSA testing are insufficient.

[0003] In recent years, liquid biopsy technology has attracted increasing attention for its ability to obtain tumor or genetic characteristics in body fluids by identifying markers such as tumor cells, nucleic acids, and exosomes. Compared with pathological biopsies, liquid biopsy technology has relatively high accuracy and does not require invasive puncture. Among them, the key to liquid biopsy technology is microRNA (miRNA). MiRNA is a short non-coding RNA with a length of 18-25 nucleotides. It can be detected and exists stably in urine and has been considered as an early specific biomarker. There is increasing evidence that the simultaneous detection of multiple miRNAs is more sensitive and specific for the diagnosis of prostate cancer.

[0004] For the detection of urine miRNA, traditional methods such as real-time quantitative polymerase chain reaction (qRT-PCR) still have limitations in detecting multiple miRNAs simultaneously. In addition, the detection time is long, strict temperature control is required, and the introduction of enzymes also increases costs.

[0005] In previous studies, the inventors used CdTe quantum dots (QDs) as signal molecules and combined them with Ag + By leveraging the fluorescence quenching characteristics of cation exchange reactions between proteins and proteins, and combining them with enzyme-free nucleic acid signal amplification technologies such as catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), along with various detection instruments, quantitative analysis of miRNA-141 was achieved. However, this strategy does not allow for the simultaneous detection of multiple miRNAs, and its detection sensitivity is only 10 fM, which cannot fully guarantee the detection of ultra-trace miRNAs in urine samples. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a method for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition, so as to solve the technical problems that the existing technology uses qRT-PCR, which still has limitations in detecting multiple miRNAs at the same time, and the detection time is long, strict temperature control is required, and the introduction of enzymes also increases the cost.

[0007] A second object of the present invention is to provide an application of a detection method for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition phenomena.

[0008] In order to achieve one of the above objectives, the present invention provides a method for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition phenomenon, the method comprising extracting miRNA-375 and miRNA-148a from urine, hybridizing the miRNA-375 and miRNA-148a with a hairpin structure HP, and triggering a catalytic hairpin assembly amplification reaction with an auxiliary chain, while releasing Hg 2+ and Ag + , and then selectively identify Hg based on CDs 2+ , and selective recognition of Ag based on CdTeQDs + Finally, the miRNA-375 and the miRNA-148a were quantified based on the fluorescence signals of CDs and CdTeQDs.

[0009] According to an optional embodiment, the hairpin structure HP includes a hairpin structure HP1, a hairpin structure HP2 and a hairpin structure HP3.

[0010] According to an optional embodiment, the hairpin structure HP1 is C-Ag + -C@T-Hg 2+ -T, the hairpin structure HP2 is a double C-Ag + -C, the hairpin structure HP3 is a double T-Hg 2+ -T.

[0011] According to an optional embodiment, the miRNA-375 and the miRNA-148a hybridize with the hairpin structure HP1 to form an HP1-miRNAs double-stranded structure.

[0012] According to an optional embodiment, the HP1-miRNAs structure binds to the probe Helper1 and the probe Helper2 to form a Helper1-HP1-Helper2 structure, and at the same time, the miRNA-375 and the miRNA-148a are released. The free miRNA-375 and the miRNA-148a hybridize with the hairpin structure HP1 again to release Hg 2+ and Ag + .

[0013] According to an optional embodiment, the hairpin structure HP2 and the hairpin structure HP3 are combined with the Helper1-HP1-Helper2 structure at the same time to release double Hg 2+ and Ag + , and at the same time form a stable HP2-Helper1-HP1-Helper2-HP3 structure.

[0014] According to an optional embodiment, the CDs and the CdTeQDs are added one after the other, and the CDs consumes Hg 2+ , the CdTeQDs consume Ag + .

[0015] In order to achieve the second of the above-mentioned purposes, the present invention provides an application of a method for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition phenomenon, wherein the application includes applying any of the above-mentioned methods for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition phenomenon to the clinical diagnosis of prostate cancer.

[0016] The present invention provides a method for detecting dual miRNAs in urine based on multiple signal amplification and metal ion selective recognition, and its application, which has the following technical effects:

[0017] This invention starts from the aspects of analytical instruments, analytical steps and costs, selection of signal reporter molecules, multiple nucleic acid signal amplification technology and application of selective recognition phenomenon, and establishes a simple, rapid, sensitive and homogeneous urine dual-miRNA quantitative analysis strategy. It does not require the use of expensive instruments or reagents, shortens the detection time, and realizes the simultaneous high-sensitivity detection of dual-miRNA in urine under room temperature conditions. It has been verified that it improves the diagnostic sensitivity and specificity of prostate cancer compared with serum PSA, enriches the medical diagnostic system, and lays the foundation for the rapid, simple and non-invasive diagnosis of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a schematic diagram of the simultaneous homogeneous fluorescence detection of dual miRNAs in urine based on multiple nucleic acid amplification and metal ion selective recognition.

[0020] Figure 2 It is the characterization of nanomaterials, verification of the selective recognition phenomenon and feasibility of dual-miRNA analysis;

[0021] Figure 3 It is the optimization of conditions for simultaneous analysis of dual miRNAs;

[0022] Figure 4 It is the performance of miRNAs analysis. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0024] 1. Principle of the Invention

[0025] The present invention constructs a strategy for simultaneous homogeneous fluorescence detection of miRNA-375 and miRNA-148a in prostate cancer urine samples based on multiple signal amplification of nucleic acids and nanomaterials and the selective recognition phenomenon of fluorescent materials.

[0026] The hairpin structure HP is a dumbbell structure, including hairpin structure HP1, hairpin structure HP2 and hairpin structure HP3. The hairpin structure HP1 is C-Ag + -C@T-Hg 2+ -T, the hairpin structure HP2 is a double C-Ag + -C, the hairpin structure HP3 is a double T-Hg 2+ -T.

[0027] like Figure 1As shown in A, the detection system includes hairpin structures HP1, HP2, HP3 and two stable hairpin structures Helper1 and Helper2. The hairpin structure HP1 can hybridize with the target miRNA.

[0028] When a solution containing miRNA-375 and miRNA-148a was added, these two miRNAs could be recognized by HP1 and release Ag. + and Hg 2+ , which will subsequently react with CdTeQDs and carbon dots (CDs) respectively.

[0029] Next, Helper1 and Helper2 were introduced to achieve CHA amplification. Specifically, these two probes can bind to the sticky ends of HP1 to form a stable "Helper1-HP1-Helper2" double-stranded DNA, while releasing miRNA-375 and miRNA-148a from HP1. The free miRNA can then release Ag from the remaining HP1. + and Hg 2+ , to achieve signal amplification, this is the first level of amplification.

[0030] In order to further improve the sensitivity, the present invention designed a second-stage amplification, introduced the hairpin structure HP2 and the hairpin structure HP3, including the double "C-Ag + -C" and double "T-Hg 2+ -T" structure. Compared with HP1 alone, HP2 and HP3 can bind to Helper1 and Helper2 and release twice as much Ag + and Hg 2+ , while forming a long and stable "HP2-Helper1-HP1-Helper2-HP3" double-stranded DNA. Compared with conventional CHA amplification, it uses the sticky ends of Helper1 and Helper2 hairpins to trigger the cascade reaction, thereby improving amplification efficiency.

[0031] Finally, CdTeQDs and CDs were selected as signal reporter molecules to selectively bind to the released Ag + and Hg 2+ At the same time, the introduction of fluorescent nanomaterials further improves the sensitivity, forming a third level of amplification.

[0032] It is worth mentioning that CdTeQDs can be + Can also be Hg 2+ After identifying and optimizing the conditions, the present invention adds CDs to consume Hg 2+ , realizing the CdTeQDs and Ag +Specific identification of miRNAs. The fluorescence signals of CdTeQDs and CDs correspond to the concentrations of miRNA-375 and miRNA-148a, respectively. Prostate cancer patients have higher levels of miRNA-375 and lower levels of miRNA-148a in their urine, resulting in lower signals from CdTeQDs and higher signals from CDs. By determining and calculating changes in fluorescence signals, simultaneous quantitative analysis of both miRNAs in urine is possible.

[0033] 2. Synthesis of CdTe QDs and CDs

[0034] Synthesis of CdTeQDs:

[0035] First, a 50 mL solution containing CdCl2 (0.5 mmol) and trisodium citrate (0.2 g) was prepared;

[0036] Then, MPA (52 μL) was immediately added to the above solution, and the pH of the solution was adjusted to 10.5 with NaOH;

[0037] Subsequently, Na2TeO3 (0.1 mmol) and KBH4 (50 mg) were added to the above solution and refluxed for 1 hour to obtain CdTeQDs. High-purity CdTeQDs were obtained by precipitation with n-propanol and centrifugation (11000 rpm). Before use, the purified yellow-green CdTeQDs were redispersed in ultrapure water.

[0038] Synthesis of CDs:

[0039] High-temperature dried corn bracts (1.0 g) and 20 mL of anhydrous ethanol were thoroughly mixed;

[0040] Then it was transferred to a 50 mL autoclave at 150 °C for 6 h;

[0041] After the reaction mixture was cooled to room temperature, the supernatant was filtered using a 0.22 μm membrane, and 1 mL of 0.6 mg / mL H2N-PEG-NH2 / anhydrous ethanol solution was added to 4 mL of the filtrate and mixed thoroughly in a beaker;

[0042] After standing in the dark at room temperature for 48 hours, 4.0 mL of 0.1 M Na2CO3 solution was added and mixed thoroughly. The mixture was allowed to stand in the dark at room temperature for 24 hours.

[0043] The solution was filtered using a 0.22 μm membrane, and the resulting filtrate was dialyzed against water and stored in a refrigerator at 4°C until use.

[0044] 3. miRNAs Analysis Steps

[0045] Mix 4μL HP1 (10μM), 4.8μL AgNO3 (50μM) and Hg2+ The standard solution (50 μM) was added to 25 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 2.5 mM Mg(NO3)2, 100 mM NaNO3, pH 7.4) and incubated at room temperature in the dark for 60 min to form “C-Ag + -C@T-Hg 2+ -T" structure. At the same time, 4 μL HP2 (10 μM), 9.6 μL AgNO3 (50 μM), 4 μL HP3 (10 μM) and 9.6 μL Hg 2+ The standard solution (50 μM) was incubated at room temperature in the dark for 60 minutes to form a double “C-Ag + -C" and "T-Hg 2+ -T" structure.

[0046] Then, 4 μL of miRNA-375 and miRNA-148a at different concentrations were added to the above system.

[0047] Subsequently, the two solutions were mixed, 4 μL of Helper 1 (10 μM) and 4 μL of Helper 2 (10 μM) were added thereto, and the mixture was incubated at room temperature in the dark for 90 minutes.

[0048] Finally, 5.5 μL LCDs were added and incubated at room temperature for 10 min to consume Hg 2+ , and then 1.4 μL CdTeQDs were added to recognize Ag + After 10 minutes, the fluorescence intensity was measured at an excitation wavelength of 365 nm. DEPC water was used for sample treatment and preparation of miRNA standard solutions during the analysis.

[0049] 4. Feasibility Verification of miRNAs Analysis

[0050] The inventors first characterized the synthesized CdTeQDs and CDs to verify their feasibility. CdTe QDs are spherical particles with a diameter of about 4nm ( Figure 2 A). When Ag + When interacting with QDs, Ag2Te is generated through cation exchange reaction, resulting in a significant agglomeration effect ( Figure 2 B). CDs are dispersed spherical with a particle size of about 3 nm ( Figure 2 C) When Hg 2+ When introduced, it reacts strongly with CDs and exhibits disordered aggregation ( Figure 2 D). The UV-visible spectrum of CdTeQDs shows an absorption peak at 540 nm ( Figure 2E), the UV-visible spectrum of CDs showed three weak peaks at approximately 280 nm, 410 nm, and 680 nm ( Figure 2 F). Ag + and C-Ag + -C was added to the solution containing CdTeQDs in a concentration gradient to evaluate its selective recognition of Ag + and C-Ag + -C ability.

[0051] The results showed that with the + As the amount of quenched QDs increases ( Figure 2 G) When HP2 is added, C-Ag is generated + -C structure; therefore, the more HP2, the more fluorescence signal QDs recover ( Figure 2 H). Similarly, for CDs, Hg 2+ The more, the more CDs are quenched ( Figure 2 I) T-Hg 2+ -T structure is formed by HP3. As HP3 increases, the fluorescence signal of CDs also increases ( Figure 2 J).

[0052] Based on the above results, a complete nucleic acid amplification-assisted simultaneous analysis of dual miRNAs was performed to illustrate the feasibility of this method ( Figure 2 K). When Ag + and Hg 2+ When both are present, CdTe QDs are added after CDs to ensure specificity. First, HP1 is added to Ag + and Hg 2+ In the process, a dumbbell-shaped hairpin structure "C-Ag + -C" and "T-Hg 2+ -T", at which time the fluorescence signals of CdTeQDs and CDs are the highest ( Figure 2 Ka and Ki).

[0053] After adding 25nM and 100nM miRNA-375 and miRNA-148a, the fluorescence signal decreased significantly, indicating that the selective binding between the dual miRNAs and HP1 can release Ag. + and Hg 2+ , which are recognized by CdTe QDs and CDs, respectively ( Figure 2 Kb to c and 2K-j to k). When HP1, HP2, HP3, Helper1 and Helper2 were all added to the system, the signals of CdTeQDs and CDs did not change significantly, showing good stability ( Figure 2 Kd to e and 2K-l to m).

[0054] Then, when different concentrations of miRNA-375 and miRNA-148a (1, 10, and 1000 fM) were added, the fluorescence of CdTeQDs and CDs was observed to gradually decrease ( Figure 2 Kf to h and 2K-n to p). This demonstrates that signal amplification is achieved, detection sensitivity is improved from nM to fM, and this strategy is feasible for the simultaneous detection of miRNA-375 and miRNA-148a.

[0055] 5. Optimization of miRNAs Analysis Conditions

[0056] After verifying the feasibility of the experiment, the experimental conditions involved in the experiment were investigated. Figure 3 As shown, HP1, HP2 and HP3 can form a stable dumbbell-shaped “C-Ag + -C" and "T-Hg 2+ -T" structure ( Figure 3 A), miRNA can completely bind to HP1 and release Ag within 30 minutes + and Hg 2+ ( Figure 3 B).

[0057] Then, for CHA amplification, Helper1 and Helper2 were added and incubated for 1.5 hours, and the reaction could be fully carried out ( Figure 3 C).

[0058] Finally, 1.4 μL CdTeQDs ( Figure 3 D and E) and 5.5μ LCDs ( Figure 3 F and G) to obtain the best signal-to-noise ratio. CdTeQDs and Ag + After 10 min, the reaction was complete ( Figure 3 H), and CDs can react with Hg in 10 minutes. 2+ Full response ( Figure 3 I).

[0059] 6. Evaluation of miRNAs Analysis Performance

[0060] The inventors investigated the sensitivity and specificity of simultaneous dual miRNA analysis. Figure 4 Under the optimized experimental conditions, this strategy achieved good analytical performance.

[0061] For the simultaneous analysis of miRNA-375 and miRNA-148a, there was significant linearity in the concentration range of 0.1 to 1000 fM ( Figure 4A). The linear equation of miRNA-375 is Y = -893LogC + 6950, and that of miRNA-148a is Y = -97.7LogC + 731 ( Figure 4 B). The detection limits of the dual miRNAs were 30 and 25 aM, respectively (based on a threefold signal-to-noise ratio). Compared to existing miRNA assays, this strategy offers advantages in sensitivity and a simple homogeneous process, requiring room temperature incubation and no enzymes.

[0062] To investigate the specificity of this strategy, single nucleotide polymorphisms (SNPs) were investigated. At the same concentration as miRNA-375 and miRNA-148a, miRNAs with single-base mismatches (mismatch at position 11) and double-base mismatches (mismatch at positions 8 and 16) were measured. For miRNA-375, the signal changes caused by single-base and double-base mismatch miRNAs were approximately 20% and 2%, respectively. Figure 4 C), and for miRNA-148a, approximately 10% and 3%, respectively ( Figure 4 D) The stable CHA system and cascade amplification greatly reduce the theoretical impact of single-base mismatch SNPs, which could be as high as 50%.

[0063] Furthermore, double-base mismatched RNA barely affected the system. However, single-base mismatched RNA significantly quenched QDs and CDs only at concentrations above 100 fM. Consequently, all fluorescence intensity changes caused by miRNA-148a and miRNA-375 were stronger than those caused by mismatches, demonstrating the good specificity of this strategy for miRNA-148a and miRNA-375 detection. This also lays a solid foundation for clinical research and application.

[0064] 7. Conclusion

[0065] This study establishes a sensitive and simple fluorescence-based strategy based on multiplexed signal amplification and the phenomenon of metal ion selective recognition for the simultaneous homogeneous analysis of miRNA-375 and miRNA-148a in urine samples. This technique offers a simple design, high signal amplification efficiency, readily available and stable reagents, high accuracy, and ease of use, establishing a reliable system for noninvasive prostate cancer diagnosis based on dual miRNA detection in urine samples.

[0066] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A system for non-invasive prostate cancer diagnosis by detecting dual miRNAs in urine samples, characterized in that: The system is used to perform the following method, which includes extracting miRNA-375 and miRNA-148a from urine, hybridizing the miRNA-375 and miRNA-148a with the hairpin structure HP, and triggering a catalytic hairpin assembly amplification reaction with an auxiliary chain, while releasing Hg 2+ and Ag + , then selectively recognize Hg based on carbon dots CDs 2+ , and selective recognition of Ag based on CdTe QDs + Finally, the miRNA-375 and the miRNA-148a were quantified based on the fluorescence signals of the carbon dot CDs and the CdTe QDs; The hairpin structure HP includes a hairpin structure HP1, a hairpin structure HP2 and a hairpin structure HP3; The hairpin structure HP1 is C-Ag + -C@T-Hg 2+ -T, the hairpin structure HP2 is a double C-Ag + -C, the hairpin structure HP3 is a double T-Hg 2+ -T; The miRNA-375 and the miRNA-148a hybridize with the hairpin structure HP1 to form an HP1-miRNAs double-stranded structure; The HP1-miRNAs structure binds to the probe Helper 1 and probe Helper 2 to form a Helper 1-HP1-Helper 2 structure. At the same time, the miRNA-375 and the miRNA-148a are released. The free miRNA-375 and the miRNA-148a hybridize with the hairpin structure HP1 again to release Hg 2+ and Ag + ; The hairpin structure HP2 and the hairpin structure HP3 are combined with the Helper 1-HP1-Helper 2 structure at the same time, releasing double Hg 2+ and Ag + , while forming a stable HP2-Helper 1-HP1-Helper 2-HP3 structure; The carbon dots CDs and the CdTe QDs are added one after the other, and the carbon dots CDs consume Hg 2+ , the CdTe QDs consume Ag + .

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