A kit for detecting oligomeric single-stranded nucleic acids, a detection method and applications

Abstract

A kit, detection method, and application for detecting oligomeric single-stranded nucleic acids are disclosed. The kit is constructed based on a signal amplification strategy using a copper ion-responsive cyanine dye probe F-0 and copper oxide nanoparticles. MNPs are coupled with biotin-modified oligo1 to obtain MNPs@oligo1; disulfide-modified oligo2 is coupled with copper oxide nanoparticles to obtain CuO@oligo2. In the presence of the target nucleic acid, complementary hybridization occurs with both to form a three-dimensional sandwich structure of MNPs-target sequence-CuO. After magnetic separation and acid hydrolysis, CuO is released. 2+ The reagent is specifically recognized by the cyanine dye probe F-0 and generates a spectral response, enabling rapid detection of target nucleic acids. Using miRNA as a model, the detection limit can reach 1.5 fM. This kit is easy to use, highly sensitive, and has good specificity, making it suitable for the detection of various oligomeric single-stranded nucleic acids, including miRNAs, nucleic acid aptamers, and antisense oligonucleotides.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a reagent kit, detection method, and application for detecting oligomeric single-stranded nucleic acids. Background Technology

[0002] Oligomeric single-stranded nucleic acids are typically formed by a small number of nucleotides linked by phosphodiester bonds, and are generally several to dozens of nucleotides in length. Compared with long-chain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), their molecular structure is much simpler, but they still play an important role in the regulation of life activities and biotechnology applications.

[0003] MicroRNAs (miRNAs) are important endogenous non-coding RNAs that play a crucial regulatory role in key biological processes such as cell proliferation, differentiation, and apoptosis. Studies have shown that abnormal miRNA expression is closely related to the development and progression of various diseases, especially tumors. Therefore, establishing sensitive, specific, and reliable miRNA detection methods is of great significance for early diagnosis, staging, prognostic assessment, and monitoring of treatment efficacy in diseases.

[0004] Currently, the main nucleic acid detection methods developed include real-time quantitative polymerase chain reaction (qPCR), nucleic acid sequencing, microarray chips, and Northern blotting. However, these methods still have certain limitations in practical applications. For example, while qPCR technology has high sensitivity, probe design is complex and susceptible to sample inhibitors; nucleic acid sequencing technology has high accuracy, but it is expensive, data analysis is complex, and the cycle is long; microarray chips are suitable for high-throughput screening, but their sensitivity is relatively insufficient; Northern blotting, as a classic method, is cumbersome to operate, time-consuming, and requires a large number of samples. In addition, these methods generally require specialized equipment and operators, making it difficult to meet the demand for rapid and convenient detection.

[0005] In complex biological samples, existing fluorescence sensing methods often suffer from limitations such as insufficient detection capability for low-abundance targets, unsatisfactory specificity, and susceptibility to cross-interference. Therefore, developing an oligomeric single-stranded nucleic acid detection technology that combines high sensitivity, high specificity, ease of operation, and strong practicality is of great significance for the detection of tumor-related miRNAs and target analysis using nucleic acid aptamers as recognition elements. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for detecting oligosingle-stranded nucleic acids, its preparation method, and its application. When the kit prepared by this method is applied to the detection of oligosingle-stranded nucleic acids, rapid and highly sensitive detection of targets such as miRNA can be achieved. Moreover, this detection method is simple to operate, highly specific, and low in cost.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A kit for detecting oligomeric single-stranded nucleic acids, the kit comprising a capture probe layer and a signal probe layer; the capture probe layer is obtained by coupling a base sequence in a base sequence layer with nanoparticles in a modification layer; wherein... The base sequence layer includes a biotin-modified base sequence oligo1 and a disulfide-modified base sequence oligo2, oligo1 and oligo2 being partially or completely complementary to the target nucleic acid sequence, respectively. The modified layer includes nano-copper oxide particles and streptavidin-functionalized Fe3O4 magnetic nanoparticles. The capture probe layer comprises MNPs@oligo1 and CuO@oligo2; MNPs@oligo1 is obtained by coupling streptavidin-functionalized Fe3O4 magnetic nanoparticles with a biotin-modified substrate sequence oligo1; CuO@oligo2 is obtained by coupling nano-copper oxide particles with a reduced disulfide bond-modified substrate sequence oligo2. The signal probe layer includes a copper ion detection probe F-0.

[0008] Preferably, the base sequence is selected from the complementary sequence of a nucleic acid aptamer, an antisense oligonucleotide, or a miRNA.

[0009] Preferably, the copper oxide nanoparticles have a particle size of 40 nm; the streptavidin-functionalized Fe3O4 magnetic nanoparticles have a particle size of 200 nm and an oligonucleotide binding capacity ≥400 pmol / mg.

[0010] Preferably, the signal probe layer includes Cu 2+ F-0, a sensitive cyanine dye supramolecular assembly probe.

[0011] To achieve the objective of the invention, the present invention also provides a method for detecting oligomeric single-stranded nucleic acids using the above-mentioned kit, comprising the following steps: (1) Provide the sample to be tested; (2) Mix and incubate the capture probes MNPs@oligo1 and CuO@oligo2 in the kit with the sample to be tested to hybridize the target nucleic acid with the capture probes and form a hybridization complex solution; (3) The hybridization complex solution obtained in step (2) was magnetically separated to obtain the MNPs-target sequence-CuO three-dimensional sandwich structure; (4) Add digestion solution to the MNPs-target sequence-CuO three-dimensional sandwich structure obtained in step (3) for acid hydrolysis to release Cu 2+ And adjust the pH of the digestion solution to neutral; (5) Prepare a detection system by mixing the digestion solution obtained in step (4) with the copper ion detection probe F-0 in the kit, so that the copper ion detection probe F-0 reacts with the Cu in the digestion solution. 2+ It specifically binds and obtains detection results based on the resulting spectral signals.

[0012] Preferably, the incubation conditions in step (2) are incubation at 4°C for 40 minutes.

[0013] Preferably, the detection in step (5) uses fluorescence detection.

[0014] To achieve the purpose of the invention, the present invention also provides the application of the above-mentioned kit in the preparation of detection reagents for detecting miRNA, nucleic acid aptamers or antisense oligonucleotides.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention constructs a specific capture probe and Cu 2+ A responsive signal probe, combined with three-component sandwich hybridization and fluorescence detection, enables rapid and sensitive detection of target oligomeric single-stranded nucleic acids. At the same time, this method simplifies the detection process, improves detection efficiency, and reduces detection costs.

[0016] (2) The present invention uses cyanine dye supramolecular probe F-0 and nano copper oxide particles to construct a dual signal amplification system, which can effectively amplify the detection signal and significantly improve the detection sensitivity (with miRNA as a model, the detection limit can reach 1.5 fM), meeting the needs of low abundance targets (such as trace amounts of miRNA) analysis.

[0017] (3) The present invention effectively reduces non-specific cross-reactions and improves the selectivity and accuracy of detection by optimizing probe design (such as controlling the number of complementary bases) and hybridization conditions (such as incubation at 4°C).

[0018] (4) The base sequence used in this invention is an oligomeric single-stranded nucleic acid sequence, which has the characteristics of low cost, easy synthesis and good specificity, and can provide support for subsequent nanomaterial modification and probe immobilization.

[0019] (5) This invention introduces nanomaterials onto the substrate sequence surface, utilizes the magnetic response characteristics of Fe3O4 nanoparticles to achieve rapid separation of three-dimensional composite structures, and enhances the F-O effect on Cu by leveraging the ion enrichment effect of CuO nanoparticles. 2+ The response signal.

[0020] (6) This kit is easy to operate, has good stability and reproducibility, and provides a reliable tool for the detection of miRNA-related diseases and the target analysis using nucleic acid aptamers as recognition elements. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the base sequence layer, modification layer, and capture probe layer in an embodiment of the present invention; Figure 2 The images show the zeta potential and particle size of the nanoparticles MNPs in Example 1 of this invention; (a) is a comparison of the zeta potentials of different particle types before and after nucleic acid coupling; (b) is a comparison of the particle size of different samples before and after coupling. Figure 3 The calibration curve and visualization of the target nucleic acid miR-221-5p concentration and spectral response in Example 1 of this invention are shown below; (a) is the fluorescence emission spectrum of probe F-0; (b) is the linear calibration curve of fluorescence intensity versus the logarithm of miRNA-221 concentration; (c) is the visual color change of F-0 probe solution under natural light as the miRNA-221 concentration gradient changes. Figure 4 This is a schematic diagram of the miR-221-5p detection principle based on the F-0 and copper nanoparticle-assisted signal amplification strategy in Embodiment 1 of the present invention; Figure 5 The bar chart shows the anti-interference ability of different ion pairs for miR-221-5p detection in Example 1 of this invention; (a) is a graph showing the change in ultraviolet absorbance at 640 nm, and (b) is a graph showing the change in relative fluorescence intensity of probe F-0 at 660 nm. Figure 6 The following are the Zeta potential and particle size test results of nanoparticles MNPs in Example 2 of this invention; (a) is a comparison of the Zeta potentials of different particle types before and after nucleic acid coupling; (b) is a comparison of the particle size of different samples before and after coupling. Figure 2 and Figure 6 In the diagram, 1 represents streptavidin-modified magnetic beads (MNPs), 2 represents magnetic beads after coupling MNPs with oligo1 (MNPs@oligo1), 3 represents nano-copper oxide particles (CuO NPs), 4 represents nano-copper oxide particles after coupling CuO NPs with oligo2 (CuO@oligo2), and 5 represents a three-dimensional sandwich structure.

[0022] Figure 7 (a) is the calibration curve of the target OTA concentration and spectral response in Example 2 of the present invention; (b) is the fluorescence emission spectrum of probe F-0; (c) is the linear calibration curve of fluorescence intensity versus the logarithm of OTA concentration. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] In this invention, microRNAs (miRNAs) are a key class of endogenous non-coding RNA molecules that play an important regulatory role in cell proliferation, differentiation, apoptosis, and other life processes. Their abnormal expression is closely related to the occurrence and development of various diseases and is one of the important biomarkers for tumor diagnosis. The miR-221-5p detected in Example 1 of this invention is closely related to tumor development and can serve as a potential biomarker.

[0025] In this invention, nucleic acid aptamers are a class of oligomeric single-stranded nucleotides that can specifically recognize target molecules. They recognize target events by specifically binding to target molecules and inducing conformational changes, and have important application value in the fields of bioanalysis and biotechnology.

[0026] The capture probes in this invention are nucleic acid probes functionalized with nanomaterials, used for the specific capture of target nucleic acids or target aptamers. Specifically, in Example 1, CuO@oligo2 and MNPs@oligo1 were used to specifically capture miR-221-5p; in Example 2, CuO@oligo2 and MNPs@oligo1 were used to specifically capture the nucleic acid aptamer OBA36.

[0027] The signal probe in this invention is a copper ion-sensitive cyanine dye supramolecularly assembled fluorescent probe F-0, which can react with Cu. 2+ The action generates a detectable optical signal; the F-0, through supramolecular self-assembly behavior, affects Cu. 2+ It generates a response, thereby enabling indirect detection of the target nucleic acid.

[0028] The fluorescence detection used in this invention detects changes in the absorption signal of the sample at a specific wavelength to assess the Cu content in the system. 2+ The analysis was performed, and further quantitative detection of the target nucleic acid was achieved.

[0029] TBS buffer solution is used for hybridization reaction and signal detection in the detection system of the present invention, preferably with a pH value of 7.4-8.0, so as to provide a suitable reaction environment for the hybridization of the specific capture probe and the target sequence.

[0030] Hybridization reaction: The process by which nucleic acid molecules form complex structures through complementary base pairing. This invention utilizes a three-component sandwich hybridization reaction to enable the target nucleic acid to bind to two types of capture probes, thereby achieving specific immobilization of the target molecule.

[0031] The copper ion detection probe used in this invention is a cyanine dye supramolecular assembly fluorescent probe, named F-0, and its structural formula is shown in Formula 1 below.

[0032] Formula 1 The schematic diagram of the synthesis of the cyanine dye supramolecular assembly fluorescent probe F-0 is shown below: The specific preparation process is as follows: a mixture of 3-(2-methylbenzothiazol-3-onthiol-3-yl)propane-1-sulfonate (0.46 g, 1.70 mmol) and squaric acid (0.10 g, 0.88 mmol) was added to a reaction system consisting of 2 mL pyridine and 4 mL 1-butanol, and the mixture was refluxed at 115 °C for 12 h under inert gas protection. After the reaction was completed, the mixture was cooled to room temperature, the precipitate was filtered, and washed with diethyl ether. The crude product was purified using a methanol / dichloromethane mixed solvent (volume ratio 1:5) to obtain 0.26 g of the target product, with a yield of 48%; its melting point was 234–238 °C.

[0033] The characterization data of the fluorescent probe F-0 are as follows: Theoretical value of high-resolution mass spectrometry (ESI) [M] 2- The value was 618.03; the measured values ​​were [M+3H]. + 621.0654, [M+3H+Na] 2+ 643.0476, [M+3H+2Na] 3+ 665.0317, [M] - 619.0463; 1 H NMR (400 MHz, DMSO-d6) δ: 7.82 (t, J = 15.0 Hz, 2H), 7.64 (t, J = 12.9 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 7.25 (t, J = 7.6 Hz, 2H), 5.84 (s, 2H), 4.39 (t, J = 7.4 Hz, 4H), 2.61 (t, J = 7.0 Hz, 4H), 2.07~1.91 (m, 4H); 13 C NMR (101 MHz, DMSO-d6) delta: 175.41, 158.68, 141.58, 128.00, 124.35, 122.85, 112.73, 85.68, 48.34, 45.00, 23.77.

[0034] Example 1

[0035] This embodiment uses miR-221-5p as the detection target, such as Figure 1As shown, a miRNA detection kit based on a cyanine dye supramolecular probe and a signal amplification strategy using nano-copper oxide is provided. The kit includes a capture probe layer and a signal probe layer; the capture probe layer is obtained by coupling a base sequence in a base sequence layer with nanoparticles in a modification layer; wherein, The base sequence layer includes a biotin-modified base sequence oligo1 and a disulfide bond-modified base sequence oligo2, with oligo1 and oligo2 having 11 complementary base pairs with the target nucleic acid, respectively. A capture probe was designed based on the target sequence miR-221-5p. The sequences used are shown in Table 1 below: Table 1. Sequences of miR-221-5p and the capture probe The modified layer comprises copper oxide nanoparticles (CuO NPs, with a particle size of 40 nm) and streptavidin-functionalized Fe3O4 magnetic nanoparticles (MNPs, with a particle size of 200 nm and oligonucleotide binding capacity ≥400 pmol / mg). The capture probe layer comprises MNPs@oligo1 and CuO@oligo2; MNPs@oligo1 is obtained by coupling streptavidin-functionalized Fe3O4 magnetic nanoparticles with a biotin-modified substrate sequence oligo1; CuO@oligo2 is obtained by coupling nano-copper oxide particles with a reduced disulfide bond-modified substrate sequence oligo2. The signal probe layer is for Cu 2+ F-0, a sensitive cyanine dye supramolecular assembly probe.

[0036] The preparation method of the MNPs@oligo1 is as follows: Take 50 μL of MNPs suspension, place it on a magnetic rack and let it stand for 1 min, then discard the supernatant. Add 500 μL of 1×TBS buffer to wash and resuspend the MNPs, place it on a magnetic rack and let it stand for 10 s, then discard the supernatant. Repeat the washing twice. Then wash once with 0.50 mL of 0.10 M NaCl solution. After washing, add 100 μL of 2×Binding and Washing Buffer to resuspend the MNPs.

[0037] Take 100 μL of 50 μM oligo1 solution, add 2.0 μL of 1.25 μM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution, mix well, and then thoroughly mix with the above MNPs suspension. Incubate at 4 °C for 2 h to prepare MNPs@Oligo1. After incubation, collect the supernatant before and after binding and perform nucleic acid quantification analysis. After discarding the supernatant, add 1.0 mL of 1×Binding and Washing Buffer to resuspend the MNPs, place on a magnetic rack and let stand for 1 min, then discard the supernatant. Repeat the washing twice, and finally resuspend the product in 100 μL of ultrapure water for later use.

[0038] The nucleic acid loading of MNPs was determined using a micro-volume nucleic acid / protein analyzer. First, the initial concentration of oligo1 was determined. After adding MNPs, the mixture was incubated at 4°C for 2 h, followed by magnetic separation, and the nucleic acid concentration in the supernatant was quantitatively analyzed. The experiment was repeated three times, and the binding rate of oligo1 on the MNP surface was calculated based on the change in nucleic acid content in the supernatant before and after binding to evaluate its coupling stability. The results are shown in Table 2.

[0039] Table 2. Concentrations of oligo1 and MNPs before and after incubation and their binding rates. Table 2 shows that the binding rates of oligo1 on the surface of MNPs in the three parallel experiments were 80.0%, 81.3%, and 79.5%, respectively, with an average binding rate of 80.3%. These results indicate that MNPs have high coupling efficiency for functionalized nucleic acids, demonstrating that MNPs@Oligo1 has been successfully prepared.

[0040] Further characterization was performed using the zeta potential and particle size measurements of the nanoparticles (see...). Figure 2 As can be seen, the Zeta potential of MNPs is -11.50 mV and the particle size is 660 nm. After coupling with oligo1, the Zeta potential of MNPs@Oligo1 becomes -34.50 mV and the particle size becomes 820 nm. Compared with unmodified MNPs, the surface electronegativity of MNPs@Oligo1 is significantly enhanced and the particle size increases by 160 nm, indicating that nucleic acids have been successfully coupled to the surface of MNPs.

[0041] The preparation method of CuO@oligo2 is as follows: Take 50 μL of 100 μM Oligo2 solution, add 1.5 mL of 0.1 M dithiothreitol (DTT), and react for 30 minutes; remove excess DTT using a Sephadex G-25 NAP-5 desalting column to obtain approximately 50 μL of Oligo2; determine the nucleic acid concentration; mix this solution with 5 μL of CuO NPs solution (1300 ng / μL), and incubate with shaking at 25 °C for 12 hours; finally, add 1 mL of 0.1 M NaCl solution to prepare CuO@Oligo2. Since the resulting mixture is in a homogeneous colloidal state, it is difficult to directly quantify the nucleic acid loading; therefore, further characterization was performed using the zeta potential and particle size assay of the nanoparticles (see...). Figure 2 The results showed that the Zeta potential of CuO NPs was -3.20 mV and the particle size was 960 nm. After coupling with oligo2, the Zeta potential of CuO@Oligo2 changed to -25.80 mV and the particle size changed to 1150 nm. Compared with unmodified CuO NPs, the surface electronegativity of CuO@Oligo2 was significantly enhanced and the particle size increased by 190 nm, indicating that nucleic acids were successfully coupled to the surface of CuO NPs.

[0042] Before using the above kit to detect miR-221-5p in the sample, a linear relationship between miR-221-5p concentration and spectral signal must be established. The specific process is as follows: 1) Dissolve MNPs@Oligo1 and CuO@Oligo2 separately in ultrapure water to prepare probe stock solutions with a concentration of 0.3 nM, and further dilute to 30 pM before use; dissolve miR-221-5p in ultrapure water to prepare a stock solution with a concentration of 0.5 mM, and then dilute stepwise to prepare a series of standard solutions with a concentration range of 0–50 pM; 2) Add different concentrations of miR-221-5p standard solution to the probe solution containing MNPs@Oligo1 and CuO@Oligo2 respectively, and incubate at 4℃ for 40 min to obtain a standard sample solution containing a three-dimensional sandwich structure of MNPs-miR-221-5p-CuO. 3) Perform magnetic separation on the above standard sample solution, and add 5 mL of 10 mM HNO3 solution to the separated complex for digestion for 5 min to completely dissolve the copper oxide nanoparticles in CuO@oligo2 and release Cu. 2+ ; 4) After adjusting the pH of the digestion solution to neutral, evaporate the nitric acid using a hot plate, then add 5 mL of DEPC water to rinse the container, and transfer the resulting mixture to a centrifuge tube. After concentration, bring the volume to 50 μL to obtain the standard sample digestion solution. 5) Add signal probe F-0 to the digestion solution of the standard sample to prepare a detection system with a total volume of 200 μL, and incubate at room temperature in the dark for 60 min; 6) Under 600 nm excitation conditions, fluorescence signals at 630 nm and 700 nm were scanned using a fluorescence microplate reader to measure the fluorescence spectra of standard samples at different concentrations. A linear relationship between miR-221-5p concentration and molecular probe fluorescence signal was established. The results are shown in [Figure number missing]. Figure 3 .

[0043] from Figure 3 As can be seen from b, within the concentration range of 0.05–0.18 pM, the fluorescence intensity has a good linear relationship with the logarithm of the miR-221-5p concentration.

[0044] This embodiment also provides a method for detecting miR-221-5p using the above-mentioned kit, the specific process of which is as follows: (1) Provide a test sample containing the target miR-221-5p; (2) The sample containing the target miR-221-5p was mixed with the capture probes MNPs@Oligo1 and CuO@Oligo2 in a buffer solution and incubated at 4°C for 40 min to form a hybridization complex. During this process, miR-221-5p formed a three-dimensional sandwich structure of MNPs-miR-221-5p-CuO with MNPs@Oligo1 and CuO@Oligo2 through base complementary pairing. (3) After incubation, the resulting hybridization complex was magnetically separated to obtain a three-dimensional sandwich structure of MNPs-miR-221-5p-CuO; (4) Add 5 mL of 10 mM HNO3 solution to the separated MNPs-miR-221-5p-CuO three-dimensional sandwich structure and digest for 5 min to release Cu. 2+ The pH of the digestion solution was then adjusted to neutral, and the nitric acid was evaporated to dryness using a hot plate. 5 mL of DEPC water was added to rinse the container, and the resulting mixture was transferred to a centrifuge tube, concentrated, and brought to a final volume of 50 μL to obtain the sample digestion solution. (5) Add signal probe F-0 to the sample digestion solution to prepare a detection system with a total volume of 200 μL, and incubate at room temperature in the dark for 60 min; then, under 600 nm excitation conditions, use a fluorescence microplate reader to detect the fluorescence signals at 630 nm and 700 nm respectively to obtain the fluorescence spectrum of the sample solution; calculate the content of miR-221-5p in the sample according to the established calibration curve between the concentration of miR-221-5p standard sample and the fluorescence signal. In this embodiment, the detection limit of miR-221-5p can be as low as 1.5 fM, and the linear detection range is 18 fM to 30 nM.

[0045] The detection principle of miR-221-5p in this invention is as follows: Figure 4 As shown: In a probe system containing MNPs@Oligo1 and CuO@Oligo2, when the target miRNA is present, MNPs@Oligo1 and CuO@Oligo2 form a three-dimensional sandwich structure of MNPs-miR-221-5p-CuO with the target nucleic acid through base complementarity pairing; after magnetic separation, the complex containing the three-dimensional sandwich structure is digested and Cu is released. 2+ Then, using the copper ion-responsive probe F-0 to target Cu 2+ This allows for indirect quantitative detection of target miRNAs. When the target nucleic acid is absent, the F-0 probe exhibits a strong fluorescent signal; upon the addition of the target miRNA, a three-dimensional sandwich structure is formed, releasing Cu into the system. 2+ The interaction with F-0 causes a change in its fluorescence signal. Based on the difference in the fluorescence response of F-0 before and after the addition of the target nucleic acid, an "on-off" sensing platform can be constructed. Different concentrations of miR-221-5p can induce different degrees of fluorescence response changes in F-0. Combined with the linear relationship established above, specific and highly sensitive detection of miR-221-5p can be achieved.

[0046] To verify the specificity of the detection system of this invention for the target nucleic acid, fluorescence spectroscopy was used to investigate the effect of different ions on the F-0 fluorescence signal. The tested components were: O-CG (miR-221-5p) and 1-Cd. 2+ 2-K + 3-Ni + 4-Mn 2+ 5-Cr 2+ 6-Na + 7-Zn 2+ 8-Ba 2+ 9-Cd 2+ 10-Sn 2+ 11-Co 2+ .like Figure 5As shown, after adding the above ions to the system, the fluorescence intensity of the system changes little, and both the fluorescence and absorbance are relatively stable, indicating that the detection system constructed in this invention has good anti-interference ability and specificity.

[0047] In summary, this invention constructs a miRNA detection system based on the cyanine dye supramolecular probe F-0 and a signal amplification strategy using nano-copper oxide, enabling qualitative or quantitative analysis of target miRNAs. This detection system offers advantages such as low background interference, ease of operation, no amplification required, rapid response, and high sensitivity. Furthermore, the signal probe possesses good water solubility and excellent optical response characteristics, achieving high selectivity and sensitivity for the detection of target miRNAs, demonstrating promising application prospects. The detection kit described in this invention can be used to detect miRNA expression levels in human serum, exhibiting high sensitivity and selectivity, and can provide technical support for the clinical auxiliary diagnosis of related diseases, demonstrating significant application value.

[0048] Example 2

[0049] This embodiment uses the nucleic acid aptamer OBA36 as the recognition element and ochratoxin A (OTA) as the detection target. Figure 1 As shown, a detection kit based on a cyanine dye supramolecular probe and a signal amplification strategy of nano-copper oxide is provided. The only difference between this kit and Example 1 is the sequence of OBA36 and the capture probe. All other parts are consistent with Example 1.

[0050] The capture probes were designed based on the target sequences, which are shown in Table 3 below: Table 3. Sequences of OBA36 and the capture probe In the preparation method of MNPs@oligo1, only the oligo1 sequence differs from that in Example 1; all other preparation procedures remain consistent with those in Example 1. The procedure for determining the nucleic acid loading of MNPs using a micro-volume nucleic acid / protein analyzer is consistent with that in Example 1. The concentrations of oligo1 before and after incubation with MNPs and their binding rates are shown in Table 4.

[0051] Table 4. Concentrations of oligo1 and MNPs before and after incubation and their binding rates. Table 4 shows that the binding rates of oligo1 on the surface of MNPs in the three parallel experiments were 85.25%, 85.70%, and 84.10%, respectively, with an average binding rate of 85.02%. These results indicate that MNPs have high coupling efficiency for functionalized nucleic acids, demonstrating that MNPs@Oligo1 has been successfully prepared.

[0052] Further characterization was performed using the zeta potential and particle size measurements of the nanoparticles (see...). Figure 6 As can be seen, the Zeta potential of MNPs is -11.50 mV and the particle size is 650 nm. After coupling with oligo1, the Zeta potential of MNPs@Oligo1 becomes -24.00 mV and the particle size becomes 800 nm. Compared with unmodified MNPs, the surface electronegativity of MNPs@Oligo1 is significantly enhanced and the particle size increases by 150 nm, indicating that nucleic acids have been successfully coupled to the surface of MNPs.

[0053] The preparation method of CuO@oligo2 differs from that in Example 1 only in the oligo2 sequence; all other preparation processes remain consistent with those in Example 1. The nanoparticles were characterized using zeta potential and particle size measurements (see [link to example]). Figure 6 The results showed that the Zeta potential of CuO NPs was -3.00 mV and the particle size was 950 nm. After coupling with oligo2, the Zeta potential of CuO@Oligo2 changed to -14.00 mV and the particle size changed to 1320 nm. Compared with unmodified CuO NPs, the surface electronegativity of CuO@Oligo2 was significantly enhanced and the particle size increased by 370 nm, indicating that nucleic acids were successfully coupled to the surface of CuO NPs.

[0054] Before using the above kit to detect OTA in the sample, a linear relationship between OTA concentration and spectral signal must first be established. The specific process is as follows: 1) Dissolve MNPs@Oligo1 and CuO@Oligo2 separately in ultrapure water to prepare probe stock solutions with a concentration of 0.3 nM, and further dilute to 30 pM before use; dissolve OBA36 in ultrapure water to prepare stock solutions with a concentration of 0.3 nM, and further dilute to 30 pM before use; 2) Add an equal concentration of OBA36 solution to the probe solution containing MNPs@Oligo1 and CuO@Oligo2, and incubate at 4℃ for 40 min to obtain a probe solution containing a three-dimensional sandwich structure of MNPs-OBA36-CuO. 3) Subsequently, OTA solutions of different concentrations were added, and the mixture was incubated at 4°C for 40 min. After incubation, the OTA-containing system was magnetically separated, and 5 mL of 10 mM HNO3 solution was added to the separated sample for digestion for 5 min. 4) After adjusting the pH of the digestion solution to neutral, evaporate the nitric acid using a hot plate, then add 5 mL of DEPC water to rinse the container. Transfer the resulting mixture to a centrifuge tube, concentrate it, and bring the volume to 50 μL to obtain the standard sample digestion solution.

[0055] 5) Add signal probe F-0 to the digestion solution of the standard sample to prepare a detection system with a total volume of 200 μL, and incubate at room temperature in the dark for 60 min.

[0056] 6) Under 600 nm excitation conditions, fluorescence signals at 630 nm and 700 nm were detected using a fluorescence microplate reader. Fluorescence spectra of OTA standard samples with different concentrations were measured, and a linear relationship between OTA concentration and molecular probe fluorescence signal was established. Results are shown in [Figure number missing]. Figure 7 .

[0057] from Figure 7 As can be seen from b, within the concentration range of 0.05–0.50 pM, the fluorescence intensity has a good linear relationship with the logarithm of the OTA concentration.

[0058] This embodiment also provides a method for detecting OTA using the above-described kit, the specific process of which is as follows: (1) Provide a test sample containing OTA; (2) The sample containing nucleic acid aptamer OBA36 was mixed with the capture probes MNPs@Oligo1 and CuO@Oligo2 in a buffer solution and incubated at 4°C for 40 min to form a hybridization complex. During this process, OBA36 formed a three-dimensional sandwich structure of MNPs-OBA36-CuO with MNPs@Oligo1 and CuO@Oligo2 through base complementary pairing. (3) After incubation, the resulting hybridization complex was magnetically separated, OTA solution was added, and incubation was continued at 4°C for 40 min. (4) Perform magnetic separation on the reaction system again, and add 5 mL of 10 mM HNO3 solution to the separated sample for 5 min to digest. Adjust the pH of the digestion solution to neutral, and then evaporate the nitric acid using a hot plate. Add 5 mL of DEPC water to rinse the container, transfer the resulting mixture to a centrifuge tube, concentrate it, and bring the volume to 50 μL to obtain the sample digestion solution.

[0059] (5) Add signal probe F-0 to the sample digestion solution to prepare a detection system with a total volume of 200 μL, and incubate at room temperature in the dark for 60 min; then, under 600 nm excitation conditions, use a fluorescence microplate reader to detect the fluorescence signals at 630 nm and 700 nm respectively to obtain the fluorescence spectrum of the sample solution. Calculate the OTA content in the sample based on the established calibration curve between the concentration of the OTA standard sample and the fluorescence signal. In this embodiment, the detection limit of OTA can be as low as 0.8 fM, and the linear detection range is 0.05~0.50 pM.

[0060] The detection kit described in this invention can be used to detect OTA levels in environmental and food samples. It has high sensitivity and selectivity, and can provide an effective technical means for the analysis and detection of OTA in complex matrices. It has good application value in the field of food safety.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principles and essence of the present invention, and all such improvements and modifications should fall within the scope of protection of the present invention.

Claims

1. A reagent kit for detecting oligomeric single-stranded nucleic acids, characterized in that, The kit includes a capture probe layer and a signal probe layer; the capture probe layer is obtained by coupling a substrate sequence in a substrate sequence layer with nanoparticles in a modification layer; wherein... The base sequence layer includes a biotin-modified base sequence oligo1 and a disulfide-modified base sequence oligo2, oligo1 and oligo2 being partially or completely complementary to the target nucleic acid sequence, respectively. The modified layer includes nano-copper oxide particles and streptavidin-functionalized Fe3O4 magnetic nanoparticles. The capture probe layer comprises MNPs@oligo1 and CuO@oligo2; MNPs@oligo1 is obtained by coupling streptavidin-functionalized Fe3O4 magnetic nanoparticles with a biotin-modified substrate sequence oligo1; CuO@oligo2 is obtained by coupling nano-copper oxide particles with a reduced disulfide bond-modified substrate sequence oligo2. The signal probe layer includes a copper ion detection probe F-0.

2. The reagent kit for detecting oligomeric single-stranded nucleic acids according to claim 1, characterized in that, The base sequence is selected from the complementary sequence of nucleic acid aptamers, antisense oligonucleotides, or miRNAs.

3. A reagent kit for detecting oligomeric single-stranded nucleic acids according to claim 1 or 2, characterized in that, The copper oxide nanoparticles have a particle size of 40 nm; the streptavidin-functionalized Fe3O4 magnetic nanoparticles have a particle size of 200 nm and an oligonucleotide binding capacity of ≥400 pmol / mg.

4. A reagent kit for detecting oligomeric single-stranded nucleic acids according to claim 1 or 2, characterized in that, The signal probe layer includes Cu 2+ F-0, a sensitive cyanine dye supramolecular assembly probe.

5. A method for detecting oligomeric single-stranded nucleic acids using the kit according to any one of claims 1 to 4, characterized in that, The steps include: (1) providing the sample to be tested; (2) The capture probes MNPs@oligo1 and CuO@oligo2 in the kit according to any one of claims 1 to 4 are mixed and incubated with the sample to be tested to hybridize the target nucleic acid with the capture probes to form a hybridization complex solution; (3) The hybridization complex solution obtained in step (2) was magnetically separated to obtain the MNPs-target sequence-CuO three-dimensional sandwich structure; (4) Add digestion solution to the MNPs-target sequence-CuO three-dimensional sandwich structure obtained in step (3) for acid hydrolysis to release Cu 2+ And adjust the pH of the digestion solution to neutral; (5) Prepare a detection system by mixing the digestion solution obtained in step (4) with the copper ion detection probe F-0 in the kit according to any one of claims 1 to 4, so that the copper ion detection probe F-0 reacts with the Cu in the digestion solution. 2+ It specifically binds and obtains detection results based on the resulting spectral signals.

6. The method for detecting oligomeric single-stranded nucleic acids according to claim 5, characterized in that, The incubation conditions in step (2) are to incubate at 4°C for 40 minutes.

7. The method for detecting oligomeric single-stranded nucleic acids according to claim 5 or 6, characterized in that, The detection in step (5) uses fluorescence detection.

8. The use of the kit according to any one of claims 1 to 4 in the preparation of a detection reagent for detecting miRNA, nucleic acid aptamers or antisense oligonucleotides.

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