A spherical nucleic acid based on a double-block DNA probe and its preparation method and application

Abstract

The present invention provides a spherical nucleic acid based on a double-block DNA probe, and a preparation method and application thereof. The spherical nucleic acid uses gold nanoparticles as a carrier, and a double-block DNA probe is modified on its surface. The double-block DNA probe consists of AP, polyA20-TP and FP modified with a fluorescent group. PolyA20 is modified on the surface of the gold nanoparticles, and the spherical nucleic acid is driven to run by the fuel chain FS to achieve room temperature enzyme-free target cyclic amplification. The nucleic acid detection method provided by the present invention utilizes the target and the FP chain with a fluorescent group to be replaced by the fuel chain at the same time. The fluorescent group is away from the surface of the gold nanoparticle, emits light in the solution, and the fluorescence signal is significantly enhanced. As the target concentration increases, the intensity of the fluorescent signal also increases. In a 1×PBS buffer system, the detection limit of this method is 228.7pM. In a complex system of 100% FBS, the detection limit of this method is 6.61nM.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis, detection and medical diagnosis, and in particular relates to a spherical nucleic acid based on a double-block DNA probe and a preparation method and application thereof. Background Art

[0002] Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are among the most essential biomolecules in living organisms. Francis Harry Compton Crick proposed the central dogma in 1958 and published it in Nature in 1970. The mechanism of action of nucleic acids as genetic material has gradually gained acceptance, and genetic research has gradually deepened to the molecular level. DNA stores the genetic information encoding the amino acid sequence of proteins, while RNA plays a crucial role in gene encoding, decoding, regulation, and expression. Therefore, nucleic acids can be used as biomarkers for disease diagnosis and adjunctive therapy. Numerous signal amplification methods have been established for detecting nucleic acids in complex samples, including polymerase chain reaction (PCR), rolling circle amplification (RCA), and signal amplification techniques based on nicking endonucleases and exonuclease III (Exo III). However, PCR requires expensive thermal cycling instruments, which limits its application in fixed-point analysis. RCA and signal amplification technologies based on enzyme cleavage reactions require enzyme assistance, and enzymes are expensive, fragile, and easily inactivated, which restricts their development in nucleic acid detection.

[0003] To overcome these challenges, signal amplification technologies based on DNA strand displacement reactions have gradually developed. DNA strand displacement reactions involve replacing double-stranded DNA with a single-stranded DNA strand, simultaneously generating another strand. This entire process does not require the involvement of enzymes. In recent years, enzyme-free signal amplification systems based on the toehold-mediated DNA strand displacement reaction (TSDR) have garnered widespread attention as a novel signal amplification method for nucleic acid detection. A toehold refers to a single-stranded DNA fragment consisting of 6-10 bases dangling from the end of double-stranded DNA. The DNA strand displacement reaction initiates from the toehold region of the DNA's sticky end—the region where the displaced double-stranded DNA strand is fully complementary to the reactant single-stranded DNA strand. In 2000, Yurke et al. designed the first DNA-driven molecular machine and systematically demonstrated the toehold-mediated strand displacement reaction. This reaction primarily occurs when single-stranded DNA selects the most complementary single-stranded molecule to form a double-stranded DNA strand. The new double-stranded DNA strand is more stable, with a lower Gibbs free energy than the original double-stranded DNA strand.

[0004] Gold nanoparticles (AuNPs), due to their excellent biocompatibility and quenching properties, are excellent scaffold materials for binding nucleic acid probes for fluorescence detection and analysis. Since Chad published a method in 1996 for attaching thiol-modified nucleic acids to the surface of gold nanoparticles to form spherical nucleic acids (SNAs) via gold-sulfur bonds, their application has been widespread. However, thiol-modified nucleic acids are very expensive, and forming DNA-AuNPs via gold-sulfur bonds presents difficulties in precisely controlling the orientation and conformation of the surface-modified oligonucleotides and finely tuning their hybridization capacity. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a spherical nucleic acid based on a double-block DNA probe and a preparation method and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first objective of the present invention is to provide a spherical nucleic acid based on a double-block DNA probe. The spherical nucleic acid uses gold nanoparticles as carriers, and the double-block DNA probe is modified on the surface of the gold nanoparticles. The double-block DNA probe includes AP, polyA20-TP, and FP modified with a fluorescent group. The TP in the polyA20-TP is a first single-stranded DNA, the AP is a second single-stranded DNA, and the FP is a third single-stranded DNA. The first single-stranded DNA includes a first toehold segment, a first segment, a second toehold segment, and a second segment arranged in sequence from 3' to 5'. The first segment is partially complementary to the second single-stranded DNA, and the second segment is completely complementary to the third single-stranded DNA. The first toehold segment is used to recognize a target sequence, and the second toehold segment is used to recognize a burning strand FS sequence. The 3' end of the third single-stranded DNA is modified with a fluorescent group, and the fluorescent group is close to the surface of the gold nanoparticle. The 5' end of the first single-stranded DNA is modified with a polyA20 sequence containing 20 consecutive adenine A's. The polyA20 segment is modified to the surface of the gold nanoparticle to form the spherical nucleic acid.

[0008] Furthermore, the fluorescent group includes FAM, TET, CY3, CY5 or ROX.

[0009] Either one.

[0010] Furthermore, the nucleotide sequence of the polyA20-TP is shown in SEQ ID NO: 1, the nucleotide sequence of the AP is shown in SEQ ID NO: 2, the nucleotide sequence of the FP is shown in SEQ ID NO: 3, the nucleotide sequence of the combustion chain FS is shown in SEQ ID NO: 4, and the nucleotide sequence of the target is shown in SEQ ID NO: 5.

[0011] A second object of the present invention is to provide a method for preparing the above-mentioned spherical nucleic acid, comprising the following specific steps:

[0012] Step S1, preparation of gold nanoparticles

[0013] The aqueous solution of chloroauric acid is heated to boiling, and the sodium citrate solution is added under stirring. After boiling, the heating is continued, the stirring is stopped, and the solution is naturally cooled to room temperature to obtain a gold nanoparticle solution.

[0014] Step S2: polyA20-TP, AP, and three DNA single strands modified with fluorescent groups are mixed in a predetermined ratio, and after uniform mixing, a buffer solution and a magnesium chloride solution are added to obtain a mixed solution. The mixed solution is placed in a PCR amplification instrument to perform an annealing process and hybridization to obtain a diblock DNA probe;

[0015] Step S3, preparation of spherical nucleic acid

[0016] The diblock DNA probe prepared in step S2 was added to the nanogold solution prepared in step S1, shaken overnight, and then SDS was added. Then, salt aging was performed with NaCl solution, centrifuged, the supernatant was discarded, and the precipitate was dispersed with buffer. The precipitate was spherical nucleic acid.

[0017] Furthermore, in step S2, the polyA20-TP, AP and the three modified fluorescent groups are

[0018] The molar concentration ratio of DNA single strands is 1:(1.5 ~ 2):1.

[0019] The third object of the present invention is to provide the use of the above-mentioned spherical nucleic acid in a nucleic acid detection reagent.

[0020] The fourth object of the present invention is to provide a nucleic acid detection system, comprising the above-mentioned spherical nucleic acid and

[0021] The burning chain FS is a fourth single-stranded DNA, and the fourth single-stranded DNA is completely complementary to the first single-stranded DNA.

[0022] Furthermore, the molar concentration ratio of the spherical nucleic acid to the combustion chain FS is 2.5: (400~700).

[0023] It should be noted that the nucleic acid detection system contains spherical nucleic acids and burning chain FS, the molecular weight of the two is

[0024] The molar concentration ratio of 2.5:(400-700) can be understood to include, but is not limited to, 2.5:400, 2.5:450, 2.5:500, 2.5:550, 2.5:600, 2.5:650, and 2.5:700. For example, using a spherical nucleic acid containing 2.5 nM in the nucleic acid detection system, the molar concentration of the fuel chain FS can be 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 700 nM. This also includes, but is not limited to, fuel chain FS containing a specific molar concentration between any two of the aforementioned values, such as, but not limited to, 420 nM and 435 nM between 400 and 450 nM. These are not further detailed here. Excess fuel chain in this nucleic acid detection system does not affect the amount of polyA20-TP or the fluorescent signal generated by the fluorescent group on the FP.

[0025] The fifth object of the present invention is to provide a method for detecting nucleic acid concentration for non-disease diagnosis purposes, comprising the following steps: mixing the nucleic acid sample to be tested with the above-mentioned nucleic acid detection system, and then performing fluorescence signal intensity detection.

[0026] The sixth object of the present invention is to provide a nucleic acid detection kit, comprising the above-mentioned nucleic acid detection system.

[0027] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0028] (1) The present invention provides a spherical nucleic acid based on a diblock DNA probe, which is driven by a fuel chain FS to achieve target cyclic amplification under enzyme-free conditions at room temperature. The diblock DNA probe is composed of three single-stranded DNAs: AP, polyA20-TP, and FP modified with a fluorescent group. The probe is modified onto the surface of a gold nanoparticle through a polyA20 segment to form a spherical nucleic acid. PolyA20 is a polyadenine sequence containing 20 consecutive adenines A. The binding strength of polyadenine (polyA) to AuNPs is equivalent to the binding strength of a gold-sulfur bond, which is much stronger than the binding strength of non-poly A nucleic acid fragments on the surface of AuNPs. The surface of AuNPs is covered with polyadenine, which reduces the possibility of nucleic acid probes adsorbed on the surface of AuNPs, resulting in too little probe binding and probe inactivation. It can also reduce adsorption interference in complex systems, and the additional recognition block adopts an upright conformation that is conducive to DNA hybridization. By using polyadenine to modify the probe onto the gold nanoparticle surface, the probe does not require thiol modification, reducing costs and avoiding the need for capping the AuNP surface with mercaptohexanol. Furthermore, the diblock DNA probe can be tuned by adjusting the length (number of A) of the polyA segment to control the lateral spacing between probes, thereby adjusting the hybridization ability of the probe with the target.

[0029] (2) The present invention provides a method for detecting nucleic acids that utilizes the quenching effect of the fluorescent group on the double-block DNA probe when it approaches the surface of the gold nanoparticles, thereby quenching the fluorescence. After the target nucleic acid is added, the target will hybridize from the first toehold segment of the polyA20-TP and displace the AP chain bound to the polyA20-TP. Subsequently, the fuel chain FS is added, and the FS will hybridize from the first toehold segment of the polyA20-TP and displace the target nucleic acid and the FP chain with the fluorescent group. At this time, the fluorescent group is away from the surface of the gold nanoparticles that produce the quenching effect on it, so that the fluorescence signal of the solution is significantly enhanced; at the same time, the target chain can still participate in the detection cycle in the solution, realizing cyclic amplification of the detection signal in the absence of enzymes and room temperature, thereby achieving high-sensitivity detection of the target.

[0030] (3) The present invention uses gold nanoparticles as fluorescence quenching groups, and FP can be linked to different fluorescent groups for simultaneous detection of multiple nucleic acid samples. The operation is simple, rapid, and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A flow chart for preparing spherical nucleic acids provided by the present invention;

[0032] Figure 2 A diagram showing the principle of detecting a nucleic acid sample to be tested using spherical nucleic acids provided by the present invention;

[0033] Figure 3 This is a gel electrophoresis diagram of the diblock DNA probe prepared in the present invention, wherein 1 is FP-FAM, 2 is AP, 3 is polyA20-TP, 4 is polyA20-TP + AP, 5 is polyA20-TP + FP-FAM, 6 is polyA20-TP:AP:FP-FAM=1:1:1, and 7 is polyA20-TP:AP:FP-FAM=1:2:1;

[0034] Figure 4a TEM image of the gold nanoparticles prepared in Example 1 of the present invention;

[0035] Figure 4b This is a UV spectrum of the gold nanoparticles and spherical nucleic acids prepared in Example 1 of the present invention;

[0036] Figure 4c This is a dynamic light scattering particle size distribution diagram of the gold nanoparticles and spherical nucleic acids prepared in Example 1 of the present invention;

[0037] Figure 4d This is a graph showing the average particle size of gold nanoparticles and spherical nucleic acids prepared in Example 1 of the present invention;

[0038] Figure 5 To draw a standard curve for a single buffer system;

[0039] Figure 6 To draw a standard curve for complex buffer systems. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in further detail below in conjunction with specific examples and accompanying drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0041] The spherical nucleic acid provided herein uses gold nanoparticles as carriers, and is surface-modified with a diblock DNA probe. The diblock DNA probe comprises AP, polyA20-TP, and FP modified with a fluorescent group. The TP in the polyA20-TP is a first single-stranded DNA, the AP is a second single-stranded DNA, and the FP is a third single-stranded DNA. The first single-stranded DNA comprises a first toehold segment, a first segment, a second toehold segment, and a second segment, arranged in 3'-5' order. The first segment is partially complementary to the second single-stranded DNA, and the second segment is fully complementary to the third single-stranded DNA. The first toehold segment is used to recognize a target sequence, and the second toehold segment is used to recognize a fuel chain (FS) sequence. The 3' end of the third single-stranded DNA is modified with a fluorescent group, which is located near the surface of the gold nanoparticle. The 5' end of the first single-stranded DNA is modified with a polyA20 sequence containing 20 consecutive adenines (A). The polyA20 segment is modified onto the surface of the gold nanoparticle to form the spherical nucleic acid. The spherical nucleic acid is driven by the fuel chain (FS), achieving cyclic target amplification at room temperature under enzyme-free conditions. The nucleotide sequences of AP, polyA20-TP, FP modified with a fluorescent group, and burning chain FS can be designed according to the nucleotide sequence of the target nucleic acid to be detected to prepare a spherical nucleic acid for detecting the target nucleic acid to be detected. The fluorescent group can be any one of FAM, TET, CY3, CY5, or ROX.

[0042] The following examples illustrate the concept of the present invention in detail. Using the target DNA, whose nucleotide sequence is shown in SEQ ID NO: 5, the nucleotide sequences of AP, polyA20-TP, FP modified with a FAM fluorescent group, and the combustion chain FS were designed, as detailed in Table 1. The aforementioned biological materials were purchased from Shanghai Bioengineering. The instruments used in this experiment included a PCR amplifier (Thermal Cycler 2720), a fluorescence spectrophotometer (Hitachi F-4700), a gel imaging system (Shanghai Tianneng Tanon 3500), an electrophoresis apparatus (Beijing Liuyi DYY-BC), a transmission electron microscope (Thermo Fisher Scientific Talos F200x), and a particle size analyzer (Malvern Nano zs90).

[0043] Table 1.

[0044]

[0045] Example 1

[0046] The preparation of spherical nucleic acid based on double-block DNA probe is shown in the flowchart Figure 1 As shown,

[0047] Step S1, preparation of gold nanoparticles (AuNPs)

[0048] The magnetic stirrer and a 250 mL two-necked flask used in the experiment were soaked in aqua regia for 12 hours, then rinsed with water for 1 hour and dried. Next, 99 mL of ultrapure water and 1 mL of 24.28 nM chloroauric acid solution were added to the 250 mL two-necked flask. After the apparatus was set up, the magnetic stirrer oil bath temperature was set to 300°C, the magnetic stirrer speed was set to 600 rpm, and the sensor temperature was set to 130°C. When the oil bath temperature reached 120°C, the magnetic stirrer speed was increased to 1100 rpm, and the two-necked flask containing chloroauric acid and ultrapure water was immersed in the oil bath and the oil bath was started. After 10 minutes of oil bathing, the solution in the two-necked flask began to boil, at which point 3.5 mL of freshly prepared 1% sodium citrate solution was added all at once. After boiling for 3 minutes, the solution turned purple and crystal nuclei began to form. After 5 minutes, the entire bottle of solution had a distinct purple color. After 8 minutes, the solution turned purple-red, the crystal nuclei grew, and the solution began to turn red. After 9 minutes, the solution turned wine-red, and the red color deepened with the number of particles. After 12 minutes, the red color deepened. After 20 minutes, the reaction was complete and the solution showed no significant changes. Turn off the heat, maintain the speed at 1100 rpm, and cool at room temperature for 30 minutes. Then, stop stirring, cool to room temperature, and filter through a 0.22 μm filter. Store at 4°C until needed.

[0049] Step S2, preparation of diblock DNA probe

[0050] The three DNA single strands of polyA20-TP, AP and FP-FAM were mixed in a ratio of 1:2:1. Specifically, 8 μL of 100 μM polyA20-TP, 16 μL of 100 μM AP and 8 μL of 100 μM FP-FAM were mixed evenly. 3.6 μL of 10× PBS buffer was added to the mixed solution, and then 0.73 μL of 200 mM magnesium chloride solution was added to make the Mg in the system 2+ The content of 4 mM was mixed evenly and then placed in a PCR amplification instrument to perform the annealing program: 95°C for 10 min, 4°C for 7 min, and 25°C for 5 min to obtain a double-block DNA probe.

[0051] If there is no PCR amplification instrument, you can use a metal bath to keep it at 95℃ for 10 minutes, then take it out, put it in an ice bath for 10 minutes, and then keep it at 25℃ for more than 5 minutes to obtain a double-block DNA probe.

[0052] When the applicant explored the synthesis ratio of polyA20-TP, AP, and FP-FAM, it was found that when the ratio of polyA20-TP:AP:FP-FAM was 1:1:1, a band of a double-stranded structure of polyA20-TP and FP-FAM appeared in the electrophoresis channel, while when the ratio of polyA20-TP:AP:FP-FAM was 1:2:1, no band of a double-stranded structure of polyA20-TP and FP-FAM appeared. This indicates that an excess of AP chain is required when synthesizing the probe, which can prevent polyA20-TP and FP-FAM from hybridizing into a double-stranded structure and effectively increase the number of diblock DNA probes.

[0053] like Figure 3 As shown, 1 is FP-FAM, 2 is AP, 3 is polyA20-TP, 4 is polyA20-TP + AP, 5 is polyA20-TP + FP-FAM, 6 is polyA20-TP: AP: FP-FAM = 1:1:1, and 7 is polyA20-TP: AP: FP-FAM = 1:2:1. It can be clearly seen that when the ratio of polyA20-TP: AP: FP-FAM is 1:2:1, the band moves the slowest, indicating that the double-block DNA probe was successfully prepared.

[0054] Step S3, Preparation of Spherical Nucleic Acids (SNA)

[0055] Add the diblock DNA probe synthesized in step S2 to 100 μL of a 10 nM gold nanoparticle solution and shake overnight at 300-400 rpm in a 25°C shaker. Add SDS to a concentration of 0.1%. Then, perform a 48-hour salt aging process. Add 5 M NaCl solution three times, with 8-hour intervals between each addition, to a final NaCl concentration of 0.3 nM. Centrifuge this solution at 8000-9000 rpm and 4°C for 16-25 minutes. Carefully discard the supernatant, repeat washing twice, and disperse the precipitate with 80-100 μL of 10 mM PB buffer (containing 0.3 M NaCl) to ensure a SNA concentration of 8-10 nM.

[0056] The modification of polyA-modified DNA probes on the gold nanoparticle surface requires a salt aging process of at least 48 hours. In order to allow the conjugation reaction to proceed while maintaining colloidal stability, NaCl needs to be added slowly to reduce charge repulsion. NaCl solution is added three times, with an interval of 8 hours each time, so that the final NaCl concentration of the system is 0.3 nM.

[0057] The concentration of gold nanoparticles can be determined using a UV spectrophotometer, using the absorbance at 520 nm and the extinction coefficient of gold nanoparticles of 5.502x10 8 As the extinction coefficient of SNA, the concentration of SNA was calculated according to the Lambert-Beer law.

[0058] like Figure 4a As shown, the TEM image of gold nanoparticles shows that the prepared gold nanoparticles do not exhibit aggregation;

[0059] like Figure 4b As shown in the figure, it can be seen from the UV spectrum that after the gold nanoparticles bind to the DNA probe, the UV absorption peak red-shifts and the maximum absorption wavelength of SNA appears at 525 nm;

[0060] like Figure 4c As shown in Figure 2, dynamic light scattering detection revealed that the particle size of SNA increased after binding to the diblock DNA probe.

[0061] like Figure 4d As shown in Figure 3, the water and diameter increased, indicating that the diblock DNA probe was successfully modified on the AuNP surface and the polyA-mediated spherical nucleic acid SNA was successfully prepared.

[0062] Example 2

[0063] Nucleic acid detection using the spherical nucleic acid prepared in Example 1

[0064] The principle diagram of nucleic acid detection is as follows Figure 2 As shown, it includes the following specific steps

[0065] Step S1, preparing target solutions of different concentrations;

[0066] Target solutions were prepared at concentrations of 0.001, 0.01, 0.1, 1, 10, 100, and 1000 nM.

[0067] Step S2, draw a standard curve

[0068] The spherical nucleic acid (SNA) prepared in Example 1 and the combustion chain FS were mixed with target solutions of varying concentrations prepared in Step S1 in 10 mM PB buffer (containing 0.3 M NaCl) to a concentration of 2.5 nM SNA and 500 nM FS. A solution with zero target concentration was used as a blank. After 0.5-1 hour of reaction, the fluorescence intensity of the solution was measured using a fluorescence spectrophotometer. A standard curve was plotted using a logistic regression model with target concentration as the horizontal axis and fluorescence signal intensity as the vertical axis.

[0069] Step S3, sample testing

[0070] In a 1× PBS (0.01 M phosphate buffered saline, pH 7.4) buffer system, the sample to be tested was added to the spherical nucleic acid (SNA) and combustion chain FS prepared in Example 1 and mixed in 10 mM PB buffer (containing 0.3 M NaCl) so that the SNA content in the system was 2.5 nM and the FS was 500 nM. The fluorescence intensity after 1 hour of reaction was recorded and the fluorescence intensity was applied to the standard curve drawn in step S2 to calculate the nucleic acid concentration in the sample.

[0071] like Figure 5 As shown, the standard curve was drawn and fitted with the following function: y = (44.39 – 134.26 x ) / (1.37 + x ). The fluorescence intensity also increased with increasing target concentration, indicating that this assay is functional. Using fluorescence intensity to calculate the corresponding target concentration is a viable strategy. The limit of detection for this assay was calculated to be 228.7 pM by adding three times the standard deviation to the blank value and fitting the standard curve.

[0072] Example 3

[0073] The difference from Example 2 is that the buffer solution is a 100% FBS (fetal bovine serum) complex buffer system.

[0074] like Figure 6 As shown, the standard curve was drawn, and the fitted standard curve function was: y = (129 + 47.26 x) / (5.17 + x). In a complex system with 100% FBS, the fluorescence signal increased with increasing target concentration. This detection scheme still works properly in complex environments, and the detected fluorescence intensity can still be used to calculate the target concentration in the solution. The detection limit in this system was calculated by adding three times the standard deviation of the blank value and fitting the standard curve to 6.61 nM.

[0075] Example 4

[0076] Detection of nucleic acid concentration in nucleic acid samples to be tested

[0077] In a 1× PBS buffer system, the synthesized SNA and FS were mixed with various concentrations of target in 10 mM PB buffer (containing 0.3 M NaCl) to achieve a system concentration of 2.5 nM SNA and 500 nM FS. A solution with zero target concentration was used as a blank. After 1 hour of reaction, the fluorescence intensity of the solution was measured using a fluorescence spectrophotometer. A standard curve was plotted using a logistic regression model, with target concentration as the horizontal axis and fluorescence signal intensity as the vertical axis.

[0078] A solution with a 2.5 nM SNA content and a 500 nM FS was prepared in 10 mM PB buffer (containing 0.3 M NaCl) to a total volume of 99 μL. 1 μL of the nucleic acid sample to be tested was added. The fluorescence intensity was measured to be 59.90. Based on the fluorescence signal fit to the standard curve in 1× PBS buffer, the target concentration in this solution was 0.51 nM, and the concentration of the test sample was 51 nM.

[0079] The present invention provides a signal amplification strategy based on enzyme-free, room-temperature target cycling via hybridization chain reaction. A diblock DNA probe is designed, and through the binding of polyadenine to gold nanoparticles, the DNA probe is modified on the surface of gold nanoparticles to form a spherical nucleic acid structure for nucleic acid detection.

[0080] In the design of the DNA molecular probe, three chains, polyA20-TP, FP modified with a fluorescent group, and AP, were selected for annealing to form a diblock structure. Polyadenine was modified on the TP chain to serve as an anchor for binding to gold nanoparticles. In the strategy for forming spherical nucleic acids (SNAs), polyadenine was selected as the anchor. The binding force of polyadenine to gold nanoparticles is comparable to that of gold-sulfur bonds. Furthermore, the use of polyadenine as the anchor allows the number of diblock DNA probes tethered to the gold nanoparticle surface to be controlled by adjusting the length of the polyadenine segment.

[0081] In the operational monitoring of spherical nucleic acid (SNA), the fuel chain serves as the driving force, enabling an enzyme-free, room-temperature target cyclic signal amplification strategy. The target is displaced by the fuel chain and reused as a reaction substrate. Simultaneously, the FP chain carrying the fluorescent group is also displaced by the fuel chain, removing the fluorescent group from the gold nanoparticle surface and emitting light in solution, significantly enhancing the fluorescence signal. As the target concentration increases, the fluorescence signal intensity also increases. In a 1× PBS buffer system, the detection limit (LOD) is 228.7 pM, calculated by adding three times the standard deviation to the blank value. In a complex system with 100% FBS, the fluorescence signal also increases with increasing target concentration, with a detection limit of 6.61 nM. This demonstrates the high sensitivity of the detection method designed in this invention, overcomes the various shortcomings of existing technologies, and has excellent application prospects.

[0082] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

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Claims

1. A spherical nucleic acid based on a diblock DNA probe, characterized in that: The spherical nucleic acid uses gold nanoparticles as carriers, and a double-block DNA probe is modified on the surface of the gold nanoparticles. The double-block DNA probe includes AP, polyA20-TP and FP modified with a fluorescent group. The TP in the polyA20-TP is a first single-stranded DNA, the AP is a second single-stranded DNA, and the FP is a third single-stranded DNA. The first single-stranded DNA includes a first toehold segment, a first segment, a second toehold segment and a second segment arranged in sequence from 3' to 5'. The first segment is partially complementary to the second single-stranded DNA, and the second segment is completely complementary to the third single-stranded DNA. The first toehold segment is used to identify a target sequence, and the second toehold segment is used to identify a burning chain FS sequence. The 3' end of the third single-stranded DNA is modified with a fluorescent group, and the fluorescent group is close to the surface of the gold nanoparticle. The 5' end of the first single-stranded DNA is modified with a polyA20 sequence containing 20 consecutive adenine A's. The polyA20 segment is modified to the surface of the gold nanoparticle to form the spherical nucleic acid.

2. The spherical nucleic acid based on a double-block DNA probe according to claim 1, wherein The fluorescent group includes any one of FAM, TET, CY3, CY5 or ROX.

3. The spherical nucleic acid based on a diblock DNA probe according to claim 2, wherein: The nucleotide sequence of the polyA20-TP is shown in SEQ ID NO: 1, the nucleotide sequence of the AP is shown in SEQ ID NO: 2, the nucleotide sequence of the FP is shown in SEQ ID NO: 3, the nucleotide sequence of the combustion chain FS is shown in SEQ ID NO: 4, and the nucleotide sequence of the target is shown in SEQ ID NO:

5.

4. The method for preparing a spherical nucleic acid according to any one of claims 1 to 3, wherein: The specific steps include: S1. Preparation of gold nanoparticles The aqueous solution of chloroauric acid is heated to boiling, and the sodium citrate solution is added under stirring. After boiling, the heating is continued, the stirring is stopped, and the solution is naturally cooled to room temperature to obtain a gold nanoparticle solution. S2. Preparation of double-block DNA probes The three DNA single strands of polyA20-TP, AP and FP modified with a fluorescent group were mixed in a preset ratio, and after mixing evenly, buffer and magnesium chloride solution were added to obtain a mixed solution. The mixed solution was placed in a PCR amplification instrument to perform annealing and hybridization to obtain a diblock DNA probe; S3. Preparation of spherical nucleic acids The diblock DNA probe prepared in step S2 was added to the nanogold solution prepared in step S1, shaken overnight, and then SDS was added. Then, salt aging was performed with NaCl solution, centrifuged, the supernatant was discarded, and the precipitate was dispersed with buffer. The precipitate was spherical nucleic acid.

5. The preparation method according to claim 4, wherein In step S2, the molar concentration ratio of the three DNA single strands, polyA20-TP, AP, and FP modified with a fluorescent group, is 1:(1.5 to 2):

1.

6. Use of the spherical nucleic acid according to any one of claims 1 to 3 in the preparation of a nucleic acid detection reagent.

7. A nucleic acid detection system, characterized in that: The method comprises the spherical nucleic acid according to any one of claims 1 to 3 and a burning chain FS, wherein the burning chain FS is a fourth single-stranded DNA, and the fourth single-stranded DNA is completely complementary to the first single-stranded DNA.

8. The nucleic acid detection system according to claim 7, wherein: The molar concentration ratio of the spherical nucleic acid to the combustion chain FS is 2.5: (400-700).

9. A method for detecting nucleic acid concentration for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: mixing the nucleic acid sample to be tested with the nucleic acid detection system as described in any one of claims 7 to 8, and then performing fluorescence signal intensity detection.

10. A nucleic acid detection kit, characterized in that: Comprising a nucleic acid detection system as described in any one of claims 7-8.

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