A magnetic dual-fluorescent aptamer sensor and a preparation method and application thereof

Abstract

The application discloses a magnetic dual-fluorescent aptamer sensor and a preparation method and application thereof, and belongs to the technical field of biosensing and medical detection. The sensor comprises a solid phase carrier and a dual-fluorescent switch of aptamer fixed on the solid phase carrier, the switch is composed of two capture arms and CMV and HSV-1 specific fluorescent aptamer capture probes which are hybridized with the two capture arms respectively, when target nucleic acid exists, specific combination of the target nucleic acid and the aptamer can displace the fluorescent probe from the surface of the magnetic beads to the solution; after magnetic separation, the synchronous, high-sensitivity and high-specificity quantitative detection of CMV and HSV-1 nucleic acid can be realized by detecting the intensity of the two kinds of fluorescent signals in the supernatant. The application further relates to a preparation method and an application method of the sensor. The sensor is simple to operate and has low background signal, and has application prospects in clinical rapid diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biosensing and medical detection technology, and more specifically to a magnetic dual-fluorescent aptamer sensor, its preparation method, and its application. Background Technology

[0002] TORCH is a group of pathogens that can cause congenital intrauterine infections and perinatal infections. Cytomegalovirus (CMV) and herpes simplex virus-1 (HSV-1) are the main pathogens causing congenital defects, neurodevelopmental disorders, visual and hearing impairments, and even death in newborns. Simultaneous screening and accurate diagnosis of CMV and HSV-1 in women of childbearing age and newborns are crucial for eugenics, early intervention, and improved prognosis. Therefore, developing a technological platform capable of rapid, simultaneous, and highly sensitive nucleic acid detection of these two pathogens is an urgent need in the field of clinical diagnostics.

[0003] Currently, clinical laboratory diagnosis of CMV and HSV-1 primarily relies on molecular diagnostic and immunological methods. Real-time quantitative polymerase chain reaction (qPCR) is considered the "gold standard" for nucleic acid diagnosis due to its extremely high sensitivity and specificity. However, this method has significant limitations: firstly, it relies on expensive thermal cyclers and specialized operators; secondly, it requires complex nucleic acid extraction and purification steps, making the process cumbersome and time-consuming; and thirdly, it is difficult to achieve simultaneous, quantitative detection of multiple targets in the same reaction tube without multiple primer / probe optimization, typically requiring multiple independent reactions, increasing cost and sample consumption. On the other hand, techniques such as enzyme-linked immunosorbent assay (ELISA) mainly detect pathogen-specific antibodies, cannot distinguish between active and past infections, and have a "window period" in the early stages of infection before antibody production, limiting their application in the early diagnosis of acute infections.

[0004] Nucleic acid aptamers are single-stranded DNA or RNA molecules obtained through in vitro screening techniques. They bind to targets with high affinity and specificity and are often referred to as "chemical antibodies." Compared to protein antibodies, aptamers offer advantages such as high stability, ease of in vitro synthesis and chemical modification, and a broad target range (from small molecules to the entire cell). Aptamer-based biosensors (aptamer sensors) have shown great potential in pathogen detection. However, most existing aptamer sensor designs still focus on single-target detection, failing to meet the throughput demands of simultaneous screening for multiple pathogens in clinical practice. Furthermore, traditional solid-phase sensors (such as those directly fixed to microplates or electrode surfaces) often suffer from strong non-specific adsorption, high background signal, and low mass transfer efficiency, limiting their detection sensitivity and stability. Although magnetic nanoparticles have been introduced into sensing systems to simplify operation due to their excellent separation and enrichment capabilities, combining them with multi-target aptamer recognition and fluorescence signal readout modes to construct an integrated sensor platform that integrates sample separation, simultaneous multi-target recognition, and signal conversion amplification still faces design and integration challenges.

[0005] In summary, clinical practice urgently needs a novel detection method that can overcome the shortcomings of existing technologies and achieve simultaneous, rapid, sensitive, specific, and easy-to-operate detection of CMV and HSV-1 nucleic acids. This is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention develops a magnetic dual-fluorescent aptamer sensor, its preparation method and application, and successfully realizes the simultaneous, highly sensitive and highly specific quantitative detection of CMV and HSV-1.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] The primary objective of this application is to provide: a magnetic dual-fluorescent aptamer sensor, comprising a solid support and an aptamer dual-fluorescent switch fixed on the solid support; the aptamer dual-fluorescent switch comprising a first capture arm, a second capture arm, a first fluorescent aptamer capture probe CMV-1Apt-CP and a second fluorescent aptamer capture probe HSV-1 Apt-CP. The first fluorescent aptamer capture probe includes a first binding region that is partially complementary to the sequence of the first capture arm, and hybridizes with the first capture arm through the first binding region; the second fluorescent aptamer capture probe includes a second binding region that is partially complementary to the sequence of the second capture arm, and hybridizes with the second capture arm through the second binding region. The first fluorescent aptamer capture probe further comprises a first target aptamer sequence and a first fluorescent group, and the second fluorescent aptamer capture probe further comprises a second target aptamer sequence and a second fluorescent group, and the fluorescence spectra of the first fluorescent group and the second fluorescent group are distinguishable.

[0009] As a preferred technical solution, the solid support is a magnetic microsphere with carboxyl groups modified on its surface; the ends of the first and second trapping arms are connected to amino groups through C14 alkane chain spacers; the amino groups form amide bonds with the carboxyl groups on the surface of the magnetic microspheres, thereby covalently fixing the trapping arms to the magnetic beads.

[0010] As a preferred technical solution, the nucleotide sequence of the first fluorescent aptamer capture probe CMV-1Apt-CP is as follows: 5'-tgccgcacattgcCGTCATTAAGCGATT-3', SEQ ID NO.3, with the first fluorescent group FAM labeled at its 5' end; The nucleotide sequence of the second fluorescent aptamer capture probe HSV-1 Apt-CP is as follows: 5'-TCATGACCCTTGTGAaacagtacggcc-3', SEQ ID NO.4, with the second fluorescent group Cy5 labeled at its 3' end; The nucleotide sequence of the first capture arm is as follows: 5'-AAAAAGCGCCTGAGCCGCGACGCCGAGCAATCGCTTAATGACG-3', SEQ ID NO.1, has NH2-C14 connected to its 5' end in the 5' direction to the 3' direction; The nucleotide sequence of the second capture arm is as follows: 5'-AAAAAGTCGCGCACGCGAGCCGCTCTCACAAGGGTCATGA-3', SEQ ID NO.2, has Bio-NH2-C14 connected at its 5' end in the 5' direction to the 3' direction.

[0011] As a preferred technical solution, the diameter of the magnetic microspheres is 1-2 μm.

[0012] Another object of this application is to provide: a method for preparing the above-mentioned magnetic dual-fluorescent aptamer sensor, comprising the following steps: S1. Activate the carboxylated magnetic microspheres to obtain activated magnetic beads; S2. The first and second capture arms are coupled with the activated magnetic beads to fix the capture arms on the surface of the magnetic beads, thus obtaining the capture arm-magnetic bead composite. S3. After the capture arm-magnetic bead complex is sealed, it is hybridized with the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe to fix the probes on the corresponding capture arms through complementary sequences, thus obtaining the sensor.

[0013] As a preferred technical solution, in step S1, EDC and NHS are activated in MES buffer; the final concentration of EDC in the buffer is 5-20 mg / mL; the final concentration of NHS in the buffer is 2-10 mg / mL. As a preferred technical solution, in step S2, the coupling reaction is carried out in PBS buffer with a pH of 5.0-6.0; the molar ratio of the first capture arm to the second capture arm is 1:1, and the dosage of the first capture arm and the second capture arm relative to the carboxylated magnetic microspheres is 0.5-2.0 nmol / mg magnetic beads. As a preferred technical solution, in step S3, the magnetic bead-capture arm coupling product is resuspended in Tris-HCl buffer solution with a pH of 8.0-8.5, and ethanolamine is added to carry out the blocking reaction. The molar ratio of the magnetic bead-capture arm coupling product to ethanolamine is 50-100 mM: 0.1-0.5 M. As a preferred technical solution, in step S3, during the hybridization reaction, the blocked magnetic bead-capture arm coupling product is resuspended in PBS buffer at pH 7.0-7.4; a first fluorescent aptamer capture probe and a second fluorescent aptamer capture probe are added to the suspension, and the mixture is incubated at 35-45℃ under constant temperature, rotation, and in the dark for 30-60 min; wherein, the molar ratio of the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe is 1:1, and the dosage of each of the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe relative to the carboxylated magnetic microspheres is 1.0-4.0 nmol / mg magnetic bead.

[0014] Another object of this application is to provide the application of the above-described magnetic dual fluorescent aptamer sensor or the magnetic dual fluorescent aptamer sensor prepared by the above method in the simultaneous detection of cytomegalovirus and herpes simplex virus-1.

[0015] Another object of this application is to provide: a method for simultaneously detecting at least two target nucleic acids using the above-described magnetic dual-fluorescent aptamer sensor or a magnetic dual-fluorescent aptamer sensor prepared by the above method for non-disease diagnosis and treatment, comprising the following steps: (1) The sensor is mixed with a sample solution containing the nucleic acid to be tested and incubated under conditions that enable the target nucleic acid to specifically bind to the corresponding aptamer and dissociate the fluorescent aptamer capture probe from the sensor; (2) Perform magnetic separation on the incubated system and collect the supernatant containing the displaced fluorescent aptamer capture probe; (3) Detect the fluorescence signals from the first and second fluorophores in the supernatant; (4) Based on the fluorescence signal and the standard curve plotted with known concentrations of nucleic acid standards, perform qualitative or quantitative analysis on the corresponding target nucleic acid in the sample.

[0016] As a preferred technical solution, the at least two target nucleic acids include cytomegalovirus nucleic acid and herpes simplex virus-1 nucleic acid; the incubation conditions in step (1) include: incubation at 85-95℃ for 10-20 minutes, followed by slow cooling to 25℃; the ratio of the magnetic dual-fluorescent aptamer sensor to the nucleic acid sample to be tested during incubation is 5-30 mg / mL: 1×10 2 -1×10 9 copies / µL.

[0017] As a preferred technical solution, in step (3), the first signal is detected by measuring the fluorescence intensity of FAM at an excitation wavelength of 485nm and an emission wavelength of 500-560nm, and the second signal is detected by measuring the fluorescence intensity of Cy5 at an excitation wavelength of 620nm and an emission wavelength of 650-700nm.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Regarding the selection of target genes, this invention uses the UL54 gene of CMV and the UL30 gene of HSV-1 as specific recognition and detection targets, which has the following advantages: 1) As encoding genes for viral DNA polymerase, both are highly conserved among different viral strains. The sequence variation rate of the UL54 gene is less than 5%, and the homology of the core functional region of the UL30 gene exceeds 90%. This selection avoids the risk of missed detection due to gene variation from the source, ensuring the broad applicability of the detection; 2) For highly specific regions in UL54 and UL30, this invention performs matching screening and design of aptamer probe sequences, making the probes In complex samples, it can accurately distinguish between CMV and HSV-1 genomes, fundamentally eliminating the risk of cross-hybridization between probes and non-target viruses, and ensuring the specificity of detection results; 3) Through the synergistic design of probe sequences, this invention enables the aptamer probes of the two viruses to have highly matched hybridization kinetics, ensuring that the two achieve efficient and balanced hybridization signals under the same reaction conditions (such as the same hybridization temperature, ionic strength and closed system), avoiding the problem of one pathogen signal being strong while the other signal is weak or even undetectable due to mismatched reaction conditions, and fundamentally avoiding sensitivity differences caused by mismatched conditions.

[0019] (2) In terms of structural design, this invention has made an innovative design at the fixed interface between the magnetic bead and the capture arm. By introducing a long C14 alkane chain as a spacer arm at the end of the capture arm, the DNA recognition sequence is effectively "pushed away" from the surface of the magnetic bead, greatly reducing the steric hindrance effect. This not only ensures that the fluorescent aptamer probe can fully and quickly hybridize with the capture arm, but more importantly, it provides the necessary spatial freedom for the large-scale conformational change (i.e., displacement reaction) triggered by the target, thereby significantly improving the kinetic speed and sensitivity of the detection. At the same time, after coupling, a high-pH Tris buffer is innovatively used to covalently block the unreacted active sites on the surface of the magnetic bead. This step actively "quenches" all chemical groups that may react with non-target molecules, eliminating the main pathway of non-specific adsorption from the source, and laying a solid foundation for achieving detection with an ultra-high signal-to-noise ratio.

[0020] (3) In terms of detection principle, this invention has developed a unique "target-triggered displacement" signal conversion mechanism. Existing aptamer sensors are mostly based on target-induced fluorescence resonance energy transfer (FRET) or fluorescence quenching / recovery (such as molecular beacons), and their signal changes occur on the solid surface, which is easily interfered with. Unlike traditional detection methods based on conformational changes, this invention breaks through this traditional approach and pioneers a "recognition-detachment" mechanism. By utilizing the thermodynamic advantage that the aptamer-target binding force is much greater than the short-chain hybridization force, the fluorescent reporter group is displaced from the solid magnetic beads into the homogeneous solution.

[0021] (4) In terms of technical performance, this invention effectively eliminates background noise such as light scattering, steric hindrance, and non-specific adsorption caused by the solid support by transferring signal detection from the solid surface to the homogeneous solution, and achieves a high signal-to-noise ratio that is difficult to achieve by traditional methods. At the same time, by integrating two independent recognition units on the functionalized magnetic beads and using dual-color fluorescent labeling, true synchronous single-tube detection of multiple targets is achieved, avoiding the cross-reaction problem that may be caused by simple mixed probes.

[0022] (5) In terms of application expansion, this invention provides not only a specific detection scheme, but also a universal biosensing platform. By changing the sequence of the capture arm and the corresponding aptamer probe, the detection of different targets can be achieved. This design concept has high universality and can be widely applied to the detection of various targets such as pathogens, tumor markers, and small molecule pollutants, with significant technical extensibility and market application potential. At the same time, the entire detection process can be completed at a constant temperature without the need for complex instruments, making it particularly suitable for the application needs of rapid on-site detection scenarios. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Here are: a flowchart (A) of the fabrication process of the magnetic dual fluorescent aptamer sensor described in this invention and a schematic diagram (B) of the detection principle.

[0025] Figure 2 For: Feasibility verification of CMV detection by aptamer sensor in Example 5.

[0026] Figure 3 For: Feasibility verification of HSV-1 detection by aptamer sensor in Example 6.

[0027] Figure 4 Example 7: Feasibility verification of simultaneous detection of CMV and HSV-1 by the aptamer sensor.

[0028] Figure 5 For example: Feasibility study of detecting CMV and HSV-1 using different aptamer sensors in Example 8.

[0029] Figure 6 Here are the fluorescence spectra of different concentrations of CMV nucleic acid detected by the aptamer sensor in Example 9.

[0030] Figure 7 For example: the standard curves of the aptamer sensor detecting different concentrations of CMV nucleic acid in Example 9.

[0031] Figure 8 Here are the fluorescence spectra of HSV-1 nucleic acids detected by the aptamer sensor in Example 9.

[0032] Figure 9 For example: the standard curves of HSV-1 nucleic acid detected by the aptamer sensor in Example 9. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 A magnetic dual-fluorescent aptamer sensor for simultaneous detection of CMV and HSV-1 The invention comprises magnetic microspheres with carboxyl groups on their surface (particle size 1-2 μm, manufactured by Nanjing Rebeads Biotechnology Co., Ltd., product name Rebeads@Mag COOH1400, catalog number NBC315) and an aptamer dual fluorescent switch immobilized on the magnetic microspheres; the aptamer dual fluorescent switch includes a first capture arm, a second capture arm, a first fluorescent aptamer capture probe, and a second fluorescent aptamer capture probe; First capture arm nucleotide sequence: 5'-AAAAAGCGCCTGAGCCGCGACGCCGAGCAATCGCTTAATGACG-3', SEQ ID NO.1, has NH2-C14 connected to its 5' end in the 5' direction to the 3' direction; Second capture arm nucleotide sequence: 5'-AAAAAGTCGCGCACGCGAGCCGCTCTCACAAGGGTCATGA-3', SEQ ID NO.2, has Bio-NH2-C14 connected at its 5' end in the 5' direction to the 3' direction.

[0035] The first fluorescent aptamer captures the nucleotide sequence of the probe (specifically recognizing the cytomegalovirus UL54 gene): 5'-tgccgcacattgcCGTCATTAAGCGATT-3', SEQ ID NO.3, with FAM marked at its 5' end; The second fluorescent aptamer captures the nucleotide sequence of the probe (specifically recognizing the herpes simplex virus-1 UL30 gene): 5'-TCATGACCCTTGTGAaacagtacggcc-3', SEQ ID NO.4, with Cy5 marked at its 3' end; The first fluorescent aptamer capture probe hybridizes with the first capture arm via a complementary sequence, and the second fluorescent aptamer capture probe hybridizes with the second capture arm via a complementary sequence. The first and second capturing arms are covalently fixed by reacting the amino and C14 alkane chain spacer arms at their ends with the carboxyl groups on the surface of the magnetic microspheres to form amide bonds.

[0036] Example 2 A method for preparing a magnetic dual-fluorescent aptamer sensor for simultaneous detection of CMV and HSV-1, as described in Example 1, includes the following steps (preparation process as follows): Figure 1 (As shown in A): a) The magnetic microspheres were resuspended in MES buffer solution with a pH of 5.0-6.0 and a concentration of 10 mM to form a magnetic microsphere suspension with a concentration of 5 mg / mL; EDC and NHS were added to the suspension to make their final concentrations 5 mg / mL and 2 mg / mL, respectively, and the mixture was reacted at room temperature (20°C) on a shaker or rotary mixer for 15 min; after the reaction was completed, the activated magnetic microspheres were washed at least once with MES buffer solution with a pH of 5.0, and the supernatant and excess activator were removed by magnetic separation.

[0037] b) Resuspend the activated magnetic microspheres from step a) in 10 mM PBS buffer at pH 5.0; then add a mixed solution of arm A (first capture arm) and arm B (second capture arm) (final concentration 100 µmol / L) to the suspension, wherein the molar ratio of arm A to arm B is 1:1, and the dosage of arm A and arm B relative to the magnetic microspheres is 0.5 nmol / mg magnetic beads; react at room temperature (20°C) for 10 minutes to allow the amino groups at the ends of the capture arms to form amide bonds with the activated carboxyl groups on the surface of the magnetic beads; after the reaction is complete, wash the coupled magnetic microspheres at least twice with PBS buffer at pH 7.0, and remove uncoupled arms A and arm B by magnetic separation.

[0038] c) Resuspend the magnetic bead-capture arm coupling product obtained in step b) in a 50 mM Tris-HCl buffer at pH 8.0, the buffer containing 0.1-0.5 M ethanolamine; incubate at room temperature (20°C) on a rotary mixer for 30 minutes to block the unreacted activated ester groups on the surface of the magnetic beads; after blocking, wash the magnetic beads at least once with PBS buffer at pH 7.0, and obtain the ready-to-use magnetic beads by magnetic separation.

[0039] d) Resuspend the spare magnetic beads obtained in step c) in PBS buffer at pH 7.0 and a concentration of 10 mM to prepare a suspension; add a mixed solution of CMV fluorescent aptamer probe and HSV-1 fluorescent aptamer probe (final concentration of 100 µmol / L) to the suspension, wherein the molar ratio of CMV fluorescent aptamer probe to HSV-1 fluorescent aptamer probe is 1:1, and the dosage of each fluorescent aptamer probe relative to the magnetic microspheres is 1.0 nmol / mg magnetic bead; incubate at a constant temperature of 35°C in the dark on a rotary mixer for 30 minutes to allow the fluorescent aptamer probe to fully hybridize with the corresponding capture arm on the magnetic bead; after incubation, collect the product by magnetic separation and wash at least twice with preheated hybridization buffer to completely remove unbound fluorescent aptamer probes, thus obtaining the magnetic dual fluorescent aptamer sensor-1.

[0040] Example 3 A method for preparing a magnetic dual-fluorescent aptamer sensor for simultaneous detection of CMV and HSV-1, as described in Example 1, includes the following steps: a) The magnetic microspheres were resuspended in a 50 mM MES buffer solution with a pH of 6.0 to form a magnetic microsphere suspension with a concentration of 20 mg / mL; EDC and NHS were added to the suspension to make their final concentrations 20 mg / mL and 10 mg / mL, respectively, and the mixture was reacted at room temperature (25°C) on a shaker or rotary mixer for 30 min; after the reaction was completed, the activated magnetic microspheres were washed at least once with MES buffer solution, and the supernatant and excess activator were removed by magnetic separation.

[0041] b) Resuspend the activated magnetic microspheres from step a) in 50 mM PBS buffer at pH 6.0; then add a mixed solution of arm A (first capture arm) and arm B (second capture arm) (final concentration 100 µmol / L) to the suspension, wherein the molar ratio of arm A to arm B is 1:1, and the dosage of arm A and arm B relative to the magnetic microspheres is 2.0 nmol / mg magnetic beads; react at room temperature (25°C) for 20 minutes to allow the amino groups at the ends of the capture arms to form amide bonds with the activated carboxyl groups on the surface of the magnetic beads; after the reaction is complete, wash the coupled magnetic microspheres at least twice with PBS buffer at pH 7.4, and remove uncoupled arms A and arm B by magnetic separation.

[0042] c) Resuspend the magnetic bead-capture arm coupling product obtained in step b) in a 100 mM Tris-HCl buffer at pH 8.5 containing 0.5 M ethanolamine; incubate at room temperature (25°C) on a rotary mixer for 60 minutes to block the unreacted activated ester groups on the surface of the magnetic beads; after blocking, wash the magnetic beads at least once with PBS buffer at pH 7.4, and obtain the ready-to-use magnetic beads by magnetic separation.

[0043] d) Resuspend the spare magnetic beads obtained in step c) in PBS buffer at pH 7.4 to prepare a suspension; add a mixed solution (100 µmol / L) of CMV fluorescent aptamer probe and HSV-1 fluorescent aptamer probe to the suspension, wherein the molar ratio of CMV fluorescent aptamer probe to HSV-1 fluorescent aptamer probe is 1:1, and the dosage of each fluorescent aptamer probe relative to the magnetic microspheres is 4.0 nmol / mg magnetic beads; incubate at a constant temperature of 45℃ in the dark on a rotary mixer for 60 minutes to allow the fluorescent aptamer probes to fully hybridize with the corresponding capture arms on the magnetic beads; after incubation, collect the product by magnetic separation and wash at least twice with preheated hybridization buffer to completely remove unbound fluorescent aptamer probes, thus obtaining the magnetic dual fluorescent aptamer sensor-2.

[0044] Example 4 A method for preparing a magnetic dual-fluorescent aptamer sensor for simultaneous detection of CMV and HSV-1, as described in Example 1, includes the following steps: a) The magnetic microspheres were resuspended in a 30 mM MES buffer solution with a pH of 5.5 to form a magnetic microsphere suspension with a concentration of 15 mg / mL; EDC and NHS were added to the suspension to make their final concentrations 15 mg / mL and 8 mg / mL, respectively, and the mixture was reacted at room temperature (22°C) on a shaker or rotary mixer for 20 min; after the reaction was completed, the activated magnetic microspheres were washed at least once with a 5.5 MES buffer solution, and the supernatant and excess activator were removed by magnetic separation.

[0045] b) Resuspend the activated magnetic microspheres from step a) in PBS buffer at pH 5.5 and a concentration of 30 mM. Then, add a mixed solution of arm A (first capture arm) and arm B (second capture arm) (final concentration of 100 µmol / L) to the suspension, wherein the molar ratio of arm A to arm B is 1:1, and the dosage of arm A and arm B relative to the magnetic microspheres is 1.0 nmol / mg of magnetic beads. React at room temperature (22°C) for 15 minutes to allow the amino groups at the ends of the capture arms to form amide bonds with the activated carboxyl groups on the surface of the magnetic beads. After the reaction is complete, wash the coupled magnetic microspheres at least twice with PBS buffer at pH 7.2, and remove uncoupled arms A and arm B by magnetic separation.

[0046] c) Resuspend the magnetic bead-capture arm coupling product obtained in step b) in Tris-HCl buffer (80 mM) at pH 8.2, which contains 0.3 M ethanolamine; incubate at room temperature (22°C) on a rotary mixer for 45 minutes to block the unreacted activated ester groups on the surface of the magnetic beads; after blocking, wash the magnetic beads at least once with PBS buffer at pH 7.2, and obtain the ready-to-use magnetic beads by magnetic separation.

[0047] d) Resuspend the spare magnetic beads obtained in step c) in PBS buffer at pH 7.2 to prepare a suspension; add a mixed solution (100 µmol / L) of CMV fluorescent aptamer probe and HSV-1 fluorescent aptamer probe to the suspension, wherein the molar ratio of CMV fluorescent aptamer probe to HSV-1 fluorescent aptamer probe is 1:1, and the dosage of each fluorescent aptamer probe relative to the magnetic microspheres is 3.0 nmol / mg magnetic beads; incubate at a constant temperature of 40℃ in the dark for 45 minutes on a rotary mixer to allow the fluorescent aptamer probes to fully hybridize with the corresponding capture arms on the magnetic beads; after incubation, collect the product by magnetic separation and wash at least twice with preheated hybridization buffer to completely remove unbound fluorescent aptamer probes, thus obtaining the magnetic dual fluorescent aptamer sensor-3.

[0048] Example 5 Feasibility of aptamer sensor-2 for CMV detection (detection process as follows) Figure 1 (as shown in B) The magnetic dual-fluorescent aptamer sensor-2 (20 mg / mL) was mixed with a 1×10⁻⁶ solution in Tris buffer (pH 7.4, 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂). 6 CMV nucleic acid samples of copies / µL were mixed and incubated at 90℃ for 10 min, then slowly cooled to 25℃ for magnetic separation, and the supernatant was collected. The fluorescence intensity of each fluorophore in the supernatant was detected by fluorescence spectroscopy. For FAM fluorescence spectroscopy, the excitation wavelength was 485 nm and the emission wavelength was 500–560 nm. For Cy5 fluorescence spectroscopy, the excitation wavelength was 620 nm and the emission wavelength was 650–700 nm.

[0049] The fluorescence detection results of Example 5 are shown below. Figure 2In the magnetic dual-fluorescent aptamer sensing system, when only CMV is present, its emission spectrum exhibits a significant FAM characteristic peak near 520 nm, while the characteristic emission band of Cy5 near 670 nm remains at the baseline level. This indicates that the high affinity binding between the CMV target and the FAM-labeled aptamer probe drives the specific dissociation of the probe from the magnetic bead surface, while the Cy5-labeled HSV-1 probe remains unaffected.

[0050] Example 6 Feasibility of Detecting HSV-1 with Aptamer Sensor-2 The magnetic dual-fluorescent aptamer sensor-2 (20 mg / mL) was mixed with a 1×10⁻⁶ solution in Tris buffer (pH 7.5, 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂). 6 HSV-1 nucleic acid samples of copies / µL were mixed and incubated at 85℃ for 10 min, then slowly cooled to 25℃ for magnetic separation, and the supernatant was collected. The fluorescence intensity of each fluorophore in the supernatant was detected by fluorescence spectroscopy. For FAM fluorescence spectroscopy, the excitation wavelength was 485 nm and the emission wavelength was 500–560 nm. For Cy5 fluorescence spectroscopy, the excitation wavelength was 620 nm and the emission wavelength was 650–700 nm.

[0051] The fluorescence detection results of Example 6 are shown below. Figure 3 In the magnetic dual-fluorescent aptamer sensing system, when only HSV-1 is present, its emission spectrum exhibits a significant Cy5 characteristic peak near 670 nm, while the characteristic emission band of CMV near 520 nm remains at the baseline level. This indicates that the high affinity binding between the HSV-1 target and the Cy5-labeled aptamer probe drives the specific dissociation of the probe from the magnetic bead surface, while the FAM-labeled CMV probe remains unaffected.

[0052] Example 7 Feasibility of simultaneous detection of CMV and HSV-1 by aptamer sensor-2 In a Tris buffer solution at pH 7.5 (20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2), the magnetic dual-fluorescent aptamer sensor-2 (20 mg / mL) was mixed with a solution containing 1×10⁻⁶ mg / mL Tris-HCl and 100 mM NaCl, 5 mM MgCl₂. 6CMV and HSV-1 nucleic acid samples (copies / µL) were mixed and incubated at 90℃ for 10 min, then slowly cooled to 25℃ for magnetic separation, and the supernatant was collected. Fluorescence intensity of each fluorophore in the supernatant was detected by fluorescence spectroscopy. For FAM fluorescence spectroscopy, the excitation wavelength was 485 nm and the emission wavelength was 500–560 nm. For Cy5 fluorescence spectroscopy, the excitation wavelength was 620 nm and the emission wavelength was 650–700 nm.

[0053] The fluorescence detection results of Example 7 are shown below. Figure 4 In the magnetic dual-fluorescent aptamer sensing system, when CMV and HSV-1 are present simultaneously, a significant FAM characteristic peak appears near 520 nm, and a significant Cy5 characteristic peak appears near 670 nm. The significant spacing of over 150 nm between the FAM and Cy5 emission peaks ensures the independence and accuracy of dual-channel detection. This indicates that the high affinity binding between the CMV target and the FAM-labeled aptamer probe, and between the HSV-1 target and the Cy5-labeled aptamer probe, drives the specific dissociation of the probes from the magnetic bead surface. At the same target concentration, the fluorescence intensity of CMV and HSV-1 measured in Example 7 is consistent with the detection results of Examples 5 and 6.

[0054] Example 8 Feasibility study of detecting CMV and HSV-1 using different aptamer sensors In a Tris buffer solution at pH 7.5 (20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2), the sensors-1,-2, and-3 (30 mg / mL) prepared in Examples 2, 3, and 4, respectively, were mixed with 1×10... 5 CMV and HSV-1 nucleic acid samples (copies / µL) were mixed and incubated at 90℃ for 10 min, then slowly cooled to 25℃ for magnetic separation, and the supernatant was collected. The fluorescence intensity of each fluorescent group in the supernatant was detected by fluorescence spectroscopy. The fluorescence detection results are shown below. Figure 5 As shown, when CMV and HSV-1 are present in the system, each sensor detects significant FAM and Cy5 characteristic peaks near 520 nm and 670 nm, and the fluorescence intensity is high, indicating that each sensor can effectively detect CMV and HSV-1.

[0055] Example 9 Sensitivity of aptamer sensor-2 for detecting CMV and HSV-1 In a Tris buffer solution at pH 7.5 (30 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2), the magnetic dual-fluorescent aptamer sensor-2 (20 mg / mL) was mixed with a concentration gradient of 1×10⁻⁶ mg / mL. 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 CMV and HSV-1 nucleic acid samples (copies / µL) were mixed and incubated at 90°C for 10 min, then slowly cooled to 25°C for magnetic separation, and the supernatant was collected. The fluorescence intensity of each fluorophore in the supernatant was detected by fluorescence spectroscopy. For FAM fluorescence spectroscopy, the excitation wavelength was 485 nm and the emission wavelength was 500–560 nm. For Cy5 fluorescence spectroscopy, the excitation wavelength was 620 nm and the emission wavelength was 650–700 nm.

[0056] The fluorescence detection results of CMV in Example 9 are shown below. Figure 6 When the magnetic dual-fluorescent aptamer sensing system detects CMV, its emission spectrum shows a significant FAM characteristic peak near 520 nm. The fluorescence intensity increases significantly with increasing target concentration, reaching a peak at 1×10⁻⁶ nm. 2 ~1×10 9 Within the range of copies / µL, CMV concentration showed a linear relationship with the change in fluorescence intensity (F / F0). Figure 7 Its linear equation is y = 1.91947x + 0.65641, R0 2 =0.99031, indicating a relatively wide detection range. According to the 3σ rule, the detection limit for CMV using this method is 32 copies / µL.

[0057] The fluorescence detection results of HSV-1 in Example 9 are shown below. Figure 8 When the magnetic dual-fluorescent aptamer sensing system detects CMV, its emission spectrum shows a significant CY5 characteristic peak near 670 nm. The fluorescence intensity increases significantly with increasing target concentration, reaching a peak at 1×10⁻⁶ nm. 2 ~1×10 9 Within the range of copies / µL, the HSV-1 concentration showed a linear relationship with the change in fluorescence intensity (F / F0). Figure 9 Its linear equation is y = 2.55893x - 2.5346, R0 2 =0.99696. According to the 3σ rule, the detection limit of this method for HSV-1 is 22 copies / µL.

[0058] Example 10 Acromide Sensor-1 for Spiked Recycling Detection of CMV and HSV-1 Non-irritating saliva and urine samples were collected from 10 healthy individuals. pMD19-T-CMV and pMD19-T-HSV-1 standards with known copy numbers were added to these samples to simulate the real-world presence of viral DNA in a complex sample environment, achieving a final concentration of 6 × 10⁻⁶ standards in the samples. 4 Copies / µL were mixed with a magnetic dual fluorescent aptamer sensor (30 mg / mL) and incubated at 90°C for 10 min. After slowly cooling to 25°C, magnetic separation was performed, the supernatant was collected, and spectral detection was performed. The detection results were compared with the standard curves of CMV and HSV-1 in Example 8. The results are shown in Table 1.

[0059] Table 1 Results of Spiked Recovery Tests

[0060] The results showed that the average recovery rates of CMV in saliva and urine samples were 92.2% and 89.7%, respectively, and the average recovery rates of HXV-1 in saliva and urine samples were 92.87% and 88%, respectively, demonstrating that this method has certain guiding significance for actual sample analysis.

[0061] Example 11 Take a concentration of 1×10 2 ~1×10 9 Mixed CMV and HSV-1 samples (copies / µL) were detected using the aptamer sensor-2 and qPCR methods of this invention, respectively. Results showed that the overall concordance rate between this method and qPCR results reached 95.5%. This invention can accurately detect both CMV and HSV-1 positive samples confirmed by qPCR.

[0062] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A magnetic dual-fluorescent aptamer sensor, characterized in that, The invention includes a solid support and an aptamer dual fluorescence switch fixed on the solid support; the aptamer dual fluorescence switch includes a first capture arm, a second capture arm, a first fluorescent aptamer capture probe CMV-1Apt-CP and a second fluorescent aptamer capture probe HSV-1 Apt-CP. The first fluorescent aptamer capture probe includes a first binding region that is partially complementary to the sequence of the first capture arm, and hybridizes with the first capture arm through the first binding region; the second fluorescent aptamer capture probe includes a second binding region that is partially complementary to the sequence of the second capture arm, and hybridizes with the second capture arm through the second binding region. The first fluorescent aptamer capture probe further comprises a first target aptamer sequence and a first fluorescent group, and the second fluorescent aptamer capture probe further comprises a second target aptamer sequence and a second fluorescent group, and the fluorescence spectra of the first fluorescent group and the second fluorescent group are distinguishable.

2. The magnetic dual-fluorescent aptamer sensor according to claim 1, characterized in that, The solid support is a magnetic microsphere with carboxyl groups on its surface; the ends of the first and second trapping arms are connected to amino groups through C14 alkane chain spacers; the amino groups form amide bonds with the carboxyl groups on the surface of the magnetic microspheres, thereby covalently fixing the trapping arms to the magnetic beads.

3. The magnetic dual-fluorescent aptamer sensor according to claim 2, characterized in that, The nucleotide sequence of the first fluorescent aptamer capture probe CMV-1Apt-CP is as follows: 5'-tgccgcacattgcCGTCATTAAGCGATT-3', SEQ ID NO.3, with the first fluorescent group FAM labeled at its 5' end; The nucleotide sequence of the second fluorescent aptamer capture probe HSV-1 Apt-CP is as follows: 5'-TCATGACCCTTGTGAaacagtacggcc-3', SEQ ID NO.4; its 3' end is labeled with a second fluorescent group Cy5; The nucleotide sequence of the first capture arm is as follows: 5'-AAAAAGCGCCTGAGCCGCGACGCCGAGCAATCGCTTAATGACG-3', SEQ ID NO.1, has NH2-C14 connected to its 5' end in the 5' direction to the 3' direction; The nucleotide sequence of the second capture arm is as follows: 5'-AAAAAGTCGCGCACGCGAGCCGCTCTCACAAGGGTCATGA-3', SEQ ID NO.2, has Bio-NH2-C14 connected at its 5' end in the 5' direction to the 3' direction.

4. The magnetic dual-fluorescent aptamer sensor according to claim 2 or 3, characterized in that, The diameter of the magnetic microspheres is 1-2 μm.

5. A method for preparing a magnetic dual-fluorescent aptamer sensor as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Activate the carboxylated magnetic microspheres to obtain activated magnetic beads; S2. The first and second capture arms are coupled with the activated magnetic beads to fix the capture arms on the surface of the magnetic beads, thus obtaining the capture arm-magnetic bead composite. S3. After the capture arm-magnetic bead complex is sealed, it is hybridized with the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe to fix the probes on the corresponding capture arms through complementary sequences, thus obtaining the sensor.

6. The method according to claim 5, characterized in that, In step S1, EDC and NHS are activated in MES buffer; the final concentration of EDC in the buffer is 5-20 mg / mL; the final concentration of NHS in the buffer is 2-10 mg / mL. In step S2, the coupling reaction is carried out in PBS buffer with a pH of 5.0-6.0; the molar ratio of the first capture arm to the second capture arm is 1:1, and the dosage of the first capture arm and the second capture arm relative to the carboxylated magnetic microspheres is 0.5-2.0 nmol / mg magnetic beads. In step S3, the magnetic bead-trapping arm coupling product is resuspended in Tris-HCl buffer at pH 8.0-8.5, and ethanolamine is added to carry out the blocking reaction. The molar ratio of the magnetic bead-trapping arm coupling product to ethanolamine is 50-100 mM: 0.1-0.5 M. In step S3, during the hybridization reaction, the blocked magnetic bead-capture arm coupling product is resuspended in PBS buffer at pH 7.0-7.4; the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe are added to the suspension, and the mixture is incubated at 35-45℃ under constant temperature, rotation, and in the dark for 30-60 min; wherein, the molar ratio of the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe is 1:1, and the dosage of each of the first fluorescent aptamer capture probe and the second fluorescent aptamer capture probe relative to the carboxylated magnetic microspheres is 1.0-4.0 nmol / mg magnetic bead.

7. The application of the magnetic dual-fluorescent aptamer sensor according to any one of claims 1-4 or the magnetic dual-fluorescent aptamer sensor prepared by the method according to any one of claims 5-6 in the simultaneous detection of cytomegalovirus and herpes simplex virus-1.

8. A method for simultaneously detecting at least two target nucleic acids using the magnetic dual-fluorescent aptamer sensor according to any one of claims 1-4 or the magnetic dual-fluorescent aptamer sensor prepared by the method according to any one of claims 5-6, for non-disease diagnosis and treatment, characterized in that, Includes the following steps: (1) The sensor is mixed with a sample solution containing the nucleic acid to be tested and incubated under conditions that enable the target nucleic acid to specifically bind to the corresponding aptamer and dissociate the fluorescent aptamer capture probe from the sensor; (2) Perform magnetic separation on the incubated system and collect the supernatant containing the displaced fluorescent aptamer capture probe; (3) Detect the fluorescence signals from the first and second fluorophores in the supernatant; (4) Based on the fluorescence signal and the standard curve plotted with known concentrations of nucleic acid standards, perform qualitative or quantitative analysis on the corresponding target nucleic acid in the sample.

9. The method according to claim 8, characterized in that, The at least two target nucleic acids include cytomegalovirus nucleic acid and herpes simplex virus-1 nucleic acid; the incubation conditions in step (1) include: incubation at 85-95℃ for 10-20 minutes, followed by slow cooling to 25℃; the ratio of the magnetic dual-fluorescent aptamer sensor to the nucleic acid sample during incubation is 5-30 mg / mL: 1×10 2 -1×10 9 copies / µL.

10. The method according to claim 8 or 9, characterized in that, In step (3), the first signal is detected by measuring the fluorescence intensity of FAM at an excitation wavelength of 485 nm and an emission wavelength of 500-560 nm, and the second signal is detected by measuring the fluorescence intensity of Cy5 at an excitation wavelength of 620 nm and an emission wavelength of 650-700 nm.

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