High-stability small-particle-size nucleic acid-gold nanoparticle composite and synthesis method thereof

Abstract

The application discloses a high-stability small-particle-size nucleic acid-gold nanocomposite and a synthesis method thereof, and belongs to the technical field of functional nanomaterial preparation, and the synthesis method comprises the following steps: under the conditions of heating and stirring, a reducing agent solution is added into a boiling chloroauric acid aqueous solution in batches, a solution of a functional nucleic acid molecule with a PolyA sequence at the terminal is added after boiling again, an acetic acid solution is added when the color of the reaction system is changed from light pink or light purple to dark, and the high-stability small-particle-size nucleic acid-gold nanocomposite is prepared after the reaction is completed; the application provides a brand-new "one-pot" synthesis strategy, discards complicated multi-step separation operation, realizes the synthesis of gold nanoparticles and the surface nucleic acid functionalization of the gold nanoparticles in one step in the same reaction system, the preparation process is simple, the product is uniform in particle size, good in dispersity, free from a plasmon resonance absorption peak, and has excellent long-term storage stability without depending on any exogenous polymer protective agent.

Description

Technical Field

[0001] This invention relates to the field of functional nanomaterial preparation technology, specifically to a highly stable small-particle-size nucleic acid-gold nanocomposite, its synthesis method, and its application in biodetection. Background Technology

[0002] Since nucleic acids can be assembled through complementary base pairing and can also be used as probes for complementary sequences and interact with targets such as metal ions, small molecules and proteins, nucleic acid-modified gold nanoparticles are of great significance in a variety of applications, including self-assembled nanoparticles, gene therapy, biosensing, cell targeting, and drug delivery.

[0003] Currently, the synthesis of nucleic acid-gold nanocomposites mostly adopts a two-step method of first synthesizing colloidal gold nanoparticles and then coupling them with nucleic acids. The existing two-step method has the following core problems that urgently need to be solved: (1) The process is complicated and has poor controllability; Specifically, the two-step process involves multiple separation steps, including synthesis, purification, surface modification, coupling, re-purification and stabilization treatment. The multi-step preparation process is extremely time-consuming and labor-intensive (usually requiring 24-72 hours). More importantly, during the transfer and processing of each step, especially for small-diameter gold nanoparticles with high surface energy, irreversible aggregation or Ostwald ripening can easily occur due to environmental disturbances (such as pH changes, ionic strength changes, temperature fluctuations or mechanical shear forces), resulting in large batch-to-batch differences and poor reproducibility; (2) For small-diameter gold particles (<5 The applicability of the synthesis of gold nanoparticles (nm) is poor; specifically, when the size of gold nanoparticles is less than 5nm, their behavior gradually transitions from classical plasmon resonance metal nanoparticles to molecular gold clusters. Particles in this size range have extremely high specific surface area and surface free energy, and are in a thermodynamically unstable state. In the second step of the traditional two-step method, the nucleic acid coupling process, the ionic strength, pH value, and high density negative charge of the nucleic acid molecules themselves in the reaction system are very likely to disrupt the weak electrostatic repulsion balance between particles, causing instantaneous aggregation and precipitation, making the nucleic acid functionalization of small-diameter particles extremely difficult and the success rate low; (3) poor modification efficiency and uniformity; specifically, for the synthesized small-diameter colloidal gold, its surface chemical properties are active but not uniform, with different crystal planes, defect sites and adsorbed ions, and the subsequent passive adsorption of nucleic acids is difficult. Formulas (such as adsorption relying on electrostatic interactions or random grafting relying on terminal thiol groups) are difficult to achieve high-density, directional, uniform and firm modification of nucleic acid molecules on the particle surface. This non-uniform modification directly leads to inconsistent performance of the final complex (such as hybridization efficiency, stability and recognition specificity); (4) unavoidable side effects of exogenous stabilizers; Specifically, in order to avoid aggregation problems, existing technologies often require the introduction of exogenous polymeric stabilizers (such as polyvinylpyrrolidone PVP, polyethylene glycol PEG) or surfactants after synthesis or coupling. Although these additives can temporarily maintain particle dispersion, they will seriously mask the active sites on the particle surface, hinder subsequent biofunctionalization, make it more difficult to achieve high-density, directional coupling of nucleic acids, and may cause background interference, non-specific adsorption or biocompatibility problems in the final application. Therefore, developing a simple method that can directly and stably prepare small-particle-size nucleic acid-gold nanocomposites has become a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0004] To address at least one of the technical challenges in the preparation of small-particle-size (<5 nm) nucleic acid-gold nanocomposites in existing technologies, such as cumbersome process flow, easy particle aggregation and instability, uneven modification efficiency, and dependence on exogenous stabilizers, this invention provides a novel "one-pot" synthesis strategy. This strategy completely eliminates cumbersome multi-step separation operations, enabling the synthesis of gold nanoparticles and the functionalization of their surface nucleic acids to be completed simultaneously and in one step within the same reaction system. Through an in-situ, intrinsic stabilization mechanism, ultra-small-sized nucleic acid-gold nanocomposites with excellent long-term storage stability are prepared without relying on any exogenous polymeric protective agents. This overcomes the aggregation problem of small-particle-size gold nanoparticles during the functionalization process. Utilizing this simultaneous synthesis and functionalization mechanism, the functional nucleic acid molecules (via the PolyA sequence) are ensured to achieve efficient, robust, and uniform surface anchoring in the initial stage of gold core formation, thereby obtaining standardized nanoprobe materials with excellent performance and high reproducibility.

[0005] The objective of this invention is achieved through the following technical solution: A method for synthesizing highly stable small-particle-size nucleic acid-gold nanocomposites includes the following steps: Under heating and stirring conditions, a reducing agent solution was added in portions to a boiling aqueous chloroauric acid solution. After boiling again, a solution of functional nucleic acid molecules with PolyA sequence modified at the end was added. When the color of the reaction system changed from light pink or light purple to darker, an acetic acid solution was added. After the reaction was completed, the highly stable small-particle-size nucleic acid-gold nanocomposite was obtained. The reaction requires heating to maintain the system temperature, which must be kept at a vigorous boiling point (approximately 105°C). This temperature ensures sufficiently fast reaction kinetics, which is conducive to large-scale, simultaneous nucleation. This is a prerequisite for obtaining small-sized, narrow-distribution particles. Too low a temperature will lead to a slow reaction and uneven particle growth. During the reaction, it is necessary to maintain sufficient and uniform stirring, especially at the moment of injection of the mixture, to ensure rapid and uniform mixing and achieve simultaneous nucleation throughout the system. Poor stirring may result in excessively high local concentrations, forming large particles or aggregates.

[0006] The synthesis method described in this invention is a "one-pot" method that simultaneously performs reduction and coordination coupling. Its basic principle is that, within the time window of the classic chemical reduction nucleation and growth kinetics of gold nanoparticles, two key functional components—acetic acid and functional nucleic acid molecules with PolyA-terminal modifications—are introduced simultaneously, so that they deeply participate in and guide the entire nanoparticle formation and surface engineering process, rather than being modified afterward. Among them, acetic acid, as one of the key functional components, has a crucial multiple synergistic mechanism in the technical solution of this invention, rather than simply acting as a pH adjuster. Specifically, it includes: (1) electrostatic shielding and stabilizing precursor; in the initial strong acidity (provided by chloroauric acid itself) and the weak acidity provided by acetic acid in the reaction environment. The ion pairs formed by acetate ions and cations in the solution can effectively shield the instantaneous high charge density generated on the surface of newly formed gold nuclei due to the adsorption of species such as gold chlorate ions, buffer the impact of drastic changes in ionic strength, and provide a relatively "mild" microenvironment for the extremely unstable initial gold atom clusters, preventing them from prematurely and disorderly agglomerating and growing or becoming unstable and precipitating; (2) Morphology and size guiding agent; Acetate ions have a certain coordination ability and can selectively adsorb on specific crystal faces of gold nanocrystals, gently regulating the relative growth rate of different crystal faces. This adsorption effect, together with the regulating effect of reducing agents (such as citrate ions), guides the particles to develop towards smaller and more uniformly distributed spherical shapes, which is the way to achieve precise control of particle size within 5 One of the key aspects below nm; (3) Creating the best interface for PolyA anchoring; The slightly acidic environment (pH3-5) created by acetic acid is conducive to the balance between the protonated and deprotonated states of the nitrogen atoms of the adenine bases in the PolyA sequence, which may optimize the distribution of the coordination electron cloud between it and the gold atoms, thereby enhancing the affinity and binding strength between PolyA and the gold surface. As another key functional component of the "one-pot method" described in this invention, the timing of its introduction and the mode of action are one of the key points of the technical solution of this invention, specifically including: (1) In-situ capture and locking; At the moment when chloroauric acid is reduced and the gold atom clusters begin to nucleate, the PolyA sequence pre-existing in the solution can be rapidly adsorbed and anchored on the surface of these initial nuclei through the strong coordination between its adenine bases and the gold atoms (N-Au coordination); (2) Spatial hindrance and growth restriction; PolyA segments and the functional nucleic acid sequences they are connected to, on the particle surface A molecular layer with moderate steric hindrance is formed. This molecular layer physically restricts the excessive deposition of gold atoms onto the existing nucleus and the mutual approach and fusion between particles, forcing the particle growth to take place in a limited nanospace. This is another key limiting factor for obtaining ultra-small size. (3) Achieve strong covalent-level connection. This coordination anchoring that occurs simultaneously during particle growth is essentially a "co-crystallization" process, which makes the PolyA chain segment and the surface of the gold nucleus form a strong bond with similar covalent bond strength, which is much more stable than the connection method of post-conversion by thiol or electrostatic adsorption in the traditional two-step method.

[0007] The "one-pot method" described in this invention refers to a synthetic method in which all or key reactants are placed in the same container, without intermediate separation steps, and the target product is obtained directly through sequential or simultaneous reactions.

[0008] In some preferred embodiments, the reducing agent is one or more of sodium citrate, ascorbic acid, and sodium borohydride. Since sodium citrate has a moderate reduction rate and its oxidation products (acetone dicarboxylic acid, etc.) and itself have a certain affinity for the gold surface, and can synergistically act with glacial acetic acid and PolyA, sodium citrate is a more preferred embodiment. Regarding the concentration of the reducing agent, its concentration level needs to be sufficient to reduce Au... 3+ For complete reduction, taking sodium citrate as an example, the molar ratio of sodium citrate to chloroauric acid should generally be greater than 5:1. For example, for 20 μM HAuCl4, using sodium citrate with a final concentration of 1-2 mM is safe and effective. Ascorbic acid, sodium borohydride (which should be used with care in an ice bath), etc., can also be used as reducing agents, but because they have different reduction potentials and kinetics, the concentration of glacial acetic acid and reaction conditions may need to be adjusted accordingly. For other non-sodium citrate reducing agents, the concentration of glacial acetic acid is generally slightly higher than that of sodium citrate. However, because the reducing power of ascorbic acid and sodium borohydride is much stronger than that of sodium citrate, it is difficult to control the stable targeted nucleation of gold atoms during the operation.

[0009] In some preferred embodiments, the concentration of acetic acid in the reaction system is 1-50 mmol / L; more preferably, the concentration of acetic acid in the reaction system is 20-50 mmol / L. Within this range, a product with a particle size of 3-5 nm and excellent stability can be stably obtained. When the concentration of acetic acid is too low (e.g., <1 mmol / L), the electrostatic shielding and morphology regulation effects are insufficient, and particles with larger particle sizes (>7 nm) or wider distributions are easily generated, and the stability decreases. When the concentration of acetic acid is too high (e.g., >100 mmol / L), the excessively acidic environment may affect the reduction kinetics of citrate, resulting in slow nucleation, or even acid hydrolysis or denaturation of nucleic acid chains. At the same time, high concentrations of acetate may excessively passivate the gold surface, hindering the effective anchoring of PolyA. The PolyA sequence described in this invention is a polyadenylated acid, a single-stranded nucleic acid sequence composed of multiple adenosine mononucleotides linked by phosphodiester bonds, such as pentaadenylated acid (generally denoted as A5). In some preferred embodiments, the length of the PolyA sequence is 2-10 bases. Experiments show that the length of the PolyA sequence is between A2 and A5. 10 Both are effective, but the anchoring force may be slightly weaker when the length is shorter (e.g., A2), and slightly weaker when the length is longer (e.g., greater than A). 10If the length is too short, the cost will increase and the spatial steric hindrance may be too great, affecting some applications; a moderate length (such as A5) is a better choice that balances anchoring strength, overall cost and spatial steric hindrance effect.

[0010] The functional nucleic acid molecule described in this invention is an oligonucleotide sequence with a specific biochemical function; in some preferred embodiments, the functional nucleic acid molecule is a DNA probe, RNA probe, aptamer, antisense oligonucleotide, or small interfering RNA (siRNA), which can be adjusted by those skilled in the art according to the design goals.

[0011] In some preferred embodiments, the nucleic acid concentration needs to be matched with the gold precursor concentration, expressed as the molar ratio of nucleic acid to gold precursor (denoted as R). When R is too low, the nucleic acid is insufficient to cover all gold nuclei, resulting in the presence of some "naked" nuclei, which can easily lead to aggregation and product instability. When R is too high, excessive free nucleic acid may interfere with the reaction or cause aggregation between particles through nucleic acid bridging, which is also uneconomical. Under optimal nucleic acid sequence length (A5) conditions, the value of R is between 0.2 and 1.0, for example, c[Au] = 300 μmol / L, c[DNA] = 4-20 μmol / L, which usually yields good results. Generally, fine-tuning is required based on the specific nucleic acid sequence length.

[0012] In some preferred embodiments, the reaction time is 20s-10min; from the start of injection of the mixture, 10-20 minutes is usually sufficient to complete the reaction. If the reaction time is too short, the reaction may be incomplete; if the reaction time is too long, it is not beneficial to the stability of small-diameter particles, and may even lead to slow aggregation due to prolonged thermal disturbance. A moderate reaction time (such as 15 minutes) is a robust choice.

[0013] Another objective of this invention is to provide a highly stable small-particle-size nucleic acid-gold nanocomposite, wherein the core of the composite is a gold nanoparticle with a particle size of less than 5 nm, and the surface of the composite is coordinated with the functional nucleic acid molecule whose end is modified with a PolyA sequence through the PolyA sequence. The composite is prepared by the aforementioned synthesis method.

[0014] In some preferred embodiments, the surface of the complex still has hybridizable nucleic acid sequences, which can be further assembled with a second functional module, such as a probe with a different sequence, a fluorescently labeled chain, or a peptide chain for targeting, through DNA hybridization to construct a multifunctional complex system.

[0015] The present invention also aims to provide an intermediate product, which is a newly formed gold nanocrystal nucleus with acetic acid molecules and PolyA sequences adsorbed on its surface. The nucleus can be further grown and stabilized in the reaction system. The intermediate product is formed by the aforementioned synthesis method.

[0016] Another object of the present invention is to provide an application of the aforementioned highly stable small-particle-size nucleic acid-gold nanocomposite, specifically as a biosensing probe, such as a biosensing probe comprising the aforementioned highly stable small-particle-size nucleic acid-gold nanocomposite; a rapid test strip in which the biosensing probe is immobilized or coated on a detection line or conjugate pad; for detecting pathogen nucleic acids, tumor markers or specific proteins, or for preparing diagnostic reagents or kits for detecting pathogen nucleic acids, tumor markers or specific proteins, etc.

[0017] The applications of the highly stable small-particle-size nucleic acid-gold nanocomposite described in this invention include: Ultrasensitive biosensors: The high specific surface area and potential unique catalytic activity resulting from small size can be used to construct next-generation electrochemical, colorimetric, or fluorescence sensors.

[0018] Intracellular imaging and delivery: The ultra-small size facilitates cellular uptake and can be used as a high-performance intracellular mRNA imaging probe or gene therapy vector.

[0019] Nanoelectronics and self-assembly: Uniformly sized ultrasmall particles are ideal building blocks for quantum dot arrays or precision superlattices.

[0020] Rapid diagnostic test strips: can be used directly as labeled probes in lateral chromatography test strips, and their small size may result in faster migration speed and higher label density.

[0021] The biosensing / diagnostic probes described in this invention refer to molecules or nanodevices that can convert biorecognition events (such as DNA hybridization) into detectable signals (such as color changes).

[0022] The beneficial effects of this invention are as follows: To address the problems of poor stability, easy aggregation, and cumbersome process in the traditional two-step method for preparing small-particle nucleic acid-gold nanocomposites, this invention proposes a novel one-pot synthesis strategy that simultaneously introduces glacial acetic acid and nucleic acid molecules with terminal PolyA sequences in the initial stage of the chloroauric acid reduction reaction. This strategy eliminates the need for cumbersome multi-step separation operations, enabling the synthesis of gold nanoparticles and the functionalization of their surface nucleic acids to be completed simultaneously and in one step within the same reaction system. The preparation process is simple. Characterized by transmission electron microscopy, agarose gel electrophoresis, UV-Vis absorption spectroscopy, and long-term stability experiments, and compared with traditional 10nm colloidal gold, the product of this invention exhibits characteristics such as uniform particle size, good dispersibility, no plasmon resonance absorption peak, and unique electrophoretic migration behavior. Furthermore, this composite exhibits excellent long-term storage stability without relying on any exogenous polymeric protective agents. After being stored at 4°C in the dark for 7 days, the solution remained clear, and no aggregation was observed under electron microscopy, demonstrating intrinsic stability. Attached Figure Description

[0023] The present invention will be further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention. For those skilled in the art, other drawings can be obtained based on the following drawings without creative effort.

[0024] Figure 1 This is a schematic diagram illustrating the principle of the synthesis method described in this invention; Figure 2 These are physical appearance diagrams of samples S1 / S2 (right) and C1 (left) as described in Embodiment 1 and Comparative Example 1 of the present invention; Figure 3 This is a transmission electron microscope (TEM) image of sample S1 as described in an embodiment of the present invention; Figure 4 This is a statistical diagram of the particle size distribution of sample S1 as described in the embodiment of the present invention; Figure 5 This is a transmission electron microscope (TEM) image of sample C1 described in Comparative Example 1 of the present invention; Figure 6 This is a statistical diagram of the particle size distribution of sample C1 described in Comparative Example 1 of the present invention; Figure 7 This is a transmission electron microscope (TEM) image of sample D1 described in Comparative Example 2 of the present invention; Figure 8 This is the ultraviolet-visible absorption spectrum of sample S1 described in this embodiment of the invention; Figure 9 This is the ultraviolet-visible absorption spectrum of sample C1 described in Comparative Example 1 of this invention; Figure 10 This is a comparison diagram of the agarose gel electrophoresis behavior of samples S1 and C1 described in Example 1 and Comparative Example 1 of the present invention; Figure 11 These are actual photos of the C1 solution (left) and S1 solution (right) after 7 days of storage. Figure 12 This is a transmission electron microscope (TEM) image of the S1 sample after 7 days of storage; Figure 13 This is a transmission electron microscope (TEM) image of a sample of the S1S2 complex mixture system. Detailed Implementation

[0025] The present invention will be further described in conjunction with the following embodiments.

[0026] Embodiments of the present invention relate to a one-pot synthesis method for highly stable small-particle-size nucleic acid-gold nanocomposites, comprising the following steps: Under heating and stirring conditions, a reducing agent solution is added in portions to a boiling aqueous chloroauric acid solution. After boiling again, a solution of functional nucleic acid molecules with PolyA sequence modified at the ends is added. When the color of the reaction system changes from light pink or pale purple to darker, acetic acid solution is added for reflux reaction. During the reaction, the color of the reaction system changes from pale yellow to colorless (or light gray), and finally to a stable, clear pale yellow or light pink. After the reaction is completed, a dispersion solution containing the highly stable small-particle-size nucleic acid-gold nanocomposite is obtained, which can be used directly or stored for later use after gentle centrifugation purification. Compared to the traditional two-step method, the one-pot method offers advantages in both kinetics and thermodynamics. The traditional two-step method essentially separates thermodynamically unstable "naked" gold nanoparticles as an intermediate product and then attempts to perform a second chemical reaction (nucleic acid modification) on their surface. This poses a significant challenge for small-sized particles with high surface energy. The one-pot method proposed in this invention thermodynamically treats the formation of the gold nucleus and surface modification as a continuous, synergistic process of minimizing free energy. When chloroauric acid is reduced by a reducing agent (such as citrate), gold atoms (Au...)... 0 The system rapidly generates and aggregates into initial atomic clusters (nuclei). If molecules that can strongly interact with the gold surface (such as the PolyA sequence of this invention) are present in the system at this time, these molecules will be instantly adsorbed on the surface of the newly formed nuclei. This adsorption changes the properties of the nucleus-solution interface and reduces the interfacial energy, thereby thermodynamically stabilizing these small nuclei. At the same time, the adsorbed molecules act as a spatial and electrostatic barrier, affecting the diffusion rate and path (growth kinetics) of subsequent gold atoms deposited on the nuclei, effectively inhibiting the excessive growth and aggregation of particles. Glacial acetic acid avoids exposing unstable, highly reactive, "naked" small-particle gold nuclei to environments that could trigger aggregation (such as pH changes, centrifugation, and solvent exchange), thus preventing intermediate instability. The chloroauric acid solution itself is strongly acidic; the added glacial acetic acid, together with components such as sodium citrate in the system, forms a buffer system that stabilizes the pH in the initial reaction stage within a weakly acidic range of 3.5-5.5. At this pH, the reducing power of citrate is moderate, which is conducive to the formation of numerous small nucleation centers, rather than the rapid coarsening of a few nuclei. Most DNA / RNA backbones are stable at this pH, and the coordination properties of adenine bases in PolyA are optimal. The H+ in the solution... + Na + Isocations and acetate (CH3COO) - These ion pairs form ion pairs, which can effectively shield the surface of newly formed gold nuclei from adsorption of [AuCl4]. - [AuCl4] -The transient local high negative charge generated by intermediates and citrate oxidation products prevents nucleus-nucleus aggregation (coalescence) caused by insufficient electrostatic repulsion. In addition to stabilizing intermediates, acetic acid also has a positive guiding effect on the formation of particle morphology. Acetate has a moderate affinity for gold surfaces and may selectively adsorb on certain high-energy crystal faces of gold, inhibiting the growth rate of these faces, thereby promoting the development of particles towards lower energy and more spherical morphology, which helps to obtain particles with narrower size distribution. The weakly acidic environment may affect the protonation state of N atoms in adenine, subtly regulating the availability of its lone pair electrons, thereby optimizing the coordination bond strength and geometry with gold surface atoms. The role of PolyA in synthesis is very obvious. Its "molecular anchoring" and "spatial confinement" mechanisms include: (1) in-situ capture and co-growth; within a few seconds to tens of seconds after the start of the reduction reaction, gold atom clusters begin to form. Free PolyA-nucleic acid molecules in the solution anchor to the surface of these clusters in a polydentate coordination manner through multiple adenine bases in its PolyA segment. This anchoring occurs in the earliest stage of particle growth. Therefore, the modified molecule and the gold core are "co-grown". As more gold atoms are deposited on the core, some of the initially adsorbed adenine may even be "buried" in the near-surface layer, forming an exceptionally strong "organic-inorganic hybrid interface"; (2) kinetically confined growth; the functional nucleic acid sequence (such as a 30-60 nt probe sequence) connected to the other end of PolyA has a considerable hydrodynamic volume in the solution. When these chains are densely anchored in a space with a size of only 2-3 When the tiny gold nuclei are around nm, they form a three-dimensional steric layer. This steric layer physically hinders the free diffusion of gold atom precursors to the surface of the gold nuclei and also prevents direct contact between different gold nuclei. This "spatial confinement" effect forces the particles to grow in a limited volume, which is the decisive factor in achieving a particle size of less than 5 nm; (3) Provide intrinsic stability; After growth, the gold nuclei are tightly wrapped by a layer of nucleic acid molecules that are connected by strong coordination bonds and extend outward. This hydrophilic nucleic acid shell provides strong steric stability (preventing particles from getting close) and hydration. More importantly, the negative charge carried by the nucleic acid backbone provides electrostatic stability for the particles. This "steric + electrostatic" dual stability mechanism is the fundamental reason why the product of this invention can be stored for a long time without external stabilizers.

[0027] Example 1 A small-particle-size nucleic acid-gold nanocomposite probe for colorimetric detection of specific DNA sequences is prepared by the following steps: (1) Solution preparation 10mM chloroauric acid stock solution: Accurately weigh 2.055g HAuCl4·4H2O, dissolve in 500 mL of ultrapure water to obtain a pale yellow transparent stock solution with a concentration of 10mM (calculated as gold). Store in a brown bottle at 4°C and use within two weeks. HAuCl4·4H2O with a purity ≥99.9% was provided by Sinopharm Group; the resistivity of the ultrapure water was 18.2 MΩ·cm, prepared using the Milli-Q system; Reducing agent portion: 2700 μL 34mM trisodium citrate aqueous solution; Trisodium citrate (Na3C6H5O7·2H2O) was of analytical grade and was provided by Sangon Biotech Co., Ltd. Functional nucleic acid molecule portion: 100 μL of reduced and purified probe chain aqueous solution with a concentration of 10 μM (as oligonucleotide). The nucleic acid sequences were synthesized by Sangon Biotech Co., Ltd. and purified by HPLC; Probe DNA A1: 5'-gene-specific sequence (54 bases)-AAAAA-3', with the last (3') being a continuous AAAAA (5 adenines); Probe DNA A2: 5'-gene-specific sequence (54 bases)-AAAAA-3', with the last (3') being a continuous AAAAA (5 adenines); It should be emphasized that the gene-specific sequences described in the embodiments of the present invention are only exemplary sequences and do not represent specific sequences that meet the technical solutions of the present invention. The present invention is applicable to any nucleic acid molecule with a PolyA sequence modified at the 5' or 3' end, including but not limited to: DNA aptamers (for detecting proteins and small molecules), siRNA (for gene silencing), molecular beacon stem-loop structures, sticky end chains for DNA self-assembly, etc. The specific nucleic acid sequences can be adjusted by those skilled in the art according to the design goals; Acetic acid portion: 45 μL pure glacial acetic acid (17.4 M); Glacial acetic acid (CH3COOH) was of analytical grade and had a concentration of ≥99.5%.

[0028] (2) One-pot synthesis reaction Take a clean 150 mL Erlenmeyer flask (standard ground glass joint), place a magnetic stir bar with a diameter of approximately 8 mm inside, and place the flask on a magnetic stirring platform. Accurately pipette 60 mL of ultrapure water into the flask, then add 2.025 mL of 10 mM chloroauric acid stock solution. The total volume in the flask is now 62.025 mL, and the final concentration of chloroauric acid is 326 μM. Turn on the stirrer at a medium speed (approximately 500 rpm) to ensure a clearly visible vortex. Turn on the heating platform and set the temperature to 110°C (this temperature allows the aqueous solution to maintain vigorous reflux). Closely observe the solution. When it begins to boil vigorously and steadily (approximately 105°C) with obvious reflux, immediately start timing. Use a 1 mL pipette (Eppendorf, adjusted to 1...) Quickly and directly inject 2.7 mL of 34 mM trisodium citrate aqueous solution into the center vortex of the boiling solution in three portions. This step is crucial and requires speed and accuracy to ensure complete mixing. After injection, the solution's color and state will undergo a series of characteristic changes, which require careful observation. 0-30 seconds: Boiling may temporarily stop, and the solution color will quickly change from pale yellow to almost colorless or very pale gray. This indicates that gold ions are rapidly reduced to form a large number of tiny atomic clusters / nuclei (size <2 nm). At this time, there is no plasmon resonance effect. Once the solution boils again, quickly add 80 μL (10 μM) of probe chain A1 solution or probe chain A2 solution. 1-3 minutes: The solution color gradually changes from colorless to light pink or pale purple and then quickly deepens, indicating that the nucleus has begun to grow and the size has entered the 2-4 nm range. At this time, add 180 μL of glacial acetic acid (17.4 M) in 4 portions, preferably 45 μL each time. 5-10 minutes: The color continues to evolve, eventually stabilizing into a clear, transparent pale yellow-brown or light wine red (the specific hue depends on the final particle size and nucleic acid concentration). The solution must remain clear at all times; any turbidity or opalescence indicates aggregation failure. Reaction maintenance: Continue vigorous reflux and stirring, and maintain the total reaction time precisely 15 minutes from the start of injection of the mixture. Reaction termination: After 15 minutes, immediately remove the conical flask from the heating platform and place it in a damp towel or cold bath at room temperature to allow it to cool naturally while stirring slowly. After cooling to room temperature (about 25°C), the target product, namely the colloidal solution of small-particle-size nucleic acid-gold nanocomposite, is obtained. The corresponding probe chain A1 solution and probe chain A2 solution are labeled as S1 and S2, respectively.

[0029] For most direct applications, the unpurified S1 or S2 stock solution can be used directly. Alternatively, it can be stored for later use after removing unreacted free nucleic acids, excess citrate, acetic acid, and other small molecules. The purification method is as follows: Aliquot S1 or S2 into 1.5 mL centrifuge tubes and centrifuge at 8000 rpm (approximately 6000 g) for 15 minutes at 4°C. For successfully prepared small-particle complexes, most of the particles should remain in the supernatant at this point, or only a very loose, almost invisible precipitate should form at the bottom of the tube. Discard the supernatant, leaving approximately 100 μL for resuspending. Add 900 μL of pre-chilled 10 mM Tris-HCl buffer (pH 7.4, containing 0.01% Tween-20 to aid resuspending) and gently pipette repeatedly until the precipitate is completely dispersed. This purification step can be repeated once. Store the purified sample at 4°C protected from light.

[0030] Comparative Example 1 The 10 nm thiolated DNA-colloidal gold complex was prepared using a traditional two-step method, including the following steps: (1) Synthesis of 10nm colloidal gold: Take 1.35mL of 0.37% HAuCl4·4H2O (3.7g / L) (about 0.24 mM) and heat to boiling. Quickly add 10mL of 1% (w / v) trisodium citrate solution and continue to reflux for 15 minutes. The solution turns wine red. Cool to obtain about 10nm colloidal gold (C-AuNP). (2) Blocking and stabilization: Add 30 μL of 10% PVP40000 solution (final concentration 0.05%) and continue stirring for 30 minutes; (3) Purification: Add 15 mg of BSPP to approximately 50 mL of colloidal gold solution, rotate and shake for 48 h to ensure the solution is fully mixed and to prevent precipitate aggregation. Add 1 g of NaCl and wait for the solution to change from wine red to light purple or dark red. Centrifuge twice at 4 °C and 17500 rpm for 30 minutes. Discard the supernatant and resuspend the precipitate with 200 μL of BSPP (3 mg / 10 mL). Measure the absorbance at 520 nm to determine the AUNP concentration. (4) Thiolized DNA coupling: Add thiolated DNA single strands and AUNPs at a ratio of 15:1, shake and incubate for 48 h (300 rpm, 25 ℃), then take the backfilled thiolated DNA short single strands and add them to the solution at a ratio of 200:1 (24 h, 300 rpm, 25 ℃) to backfill the surface of AUNPs. The product is recorded as sample C1.

[0031] Comparative Example 2 A two-step method for preparing non-thiol (PolyA) nucleic acid-coupled gold nanoparticles includes the following steps: (1) Prepare a 10 nm colloidal gold purification solution, following the same steps as in Comparative Example 1; (2) PolyA-DNA conjugation: Mix 100 nM BSPP-AuNP with DNA strands of different PolyA lengths at appropriate concentrations (usually 500 nM). After 1 minute, rapidly add citrate-HCl buffer (500 mM, pH 3.1) to the DNA / AuNPs until the final citrate concentration is 10 mM. Incubate the mixture at room temperature for 15 minutes. The product is recorded as sample D1.

[0032] Comparative Example 3 An attempt was made to prepare "naked" colloidal gold with a particle size of <5 nm using traditional methods (without adding nucleic acids and glacial acetic acid, only using strong reducing agents such as ascorbic acid). The sample completely precipitated within hours after synthesis, forming a blackish-purple precipitate, making subsequent operations impossible.

[0033] Characterization experiment (1) Appearance To the naked eye, S1 and S2 are homogeneous, transparent, colored liquids; when observed against white light, they exhibit no or only a very weak Tyndall effect; see also Figure 2 In a transparent glass bottle or plastic tube, the solution appearance of sample C1 is shown in the left image, which is a uniform, bright, and transparent wine red; the solution appearance of samples S1 / S2 is shown in the right image, which is a uniform and clear light pink. This comparison intuitively demonstrates that the two products of different size levels can be distinguished from the macroscopic color alone.

[0034] (2) Shape and size Take 10 μL of S1, S2 and C1, D1 respectively, dilute them 10 times with ultrapure water, and add 5 μL of each to an ultrathin carbon film copper grid. Let them air dry at room temperature without any negative staining, and observe them directly under a transmission electron microscope (Thermo Fisher Scientific, 120 kV).

[0035] See Figure 3-7 , Figure 3-4 The images show the morphology and particle size distribution of sample S1 obtained through transmission electron microscopy. The particles are uniformly distributed within the field of view, and under high magnification, they appear approximately spherical. Using ImageJ software, the diameter of 200 randomly selected particles was measured, yielding an average particle size of 3.8 nm and a standard deviation of 0.5 nm. The particle size distribution histogram shows a unimodal normal distribution, with the vast majority of particles between 2.5 and 4.5 nm. No particles larger than 5 nm were observed, nor were any dimers or oligomers formed by the fusion of 2-3 particles. The TEM results for S2 show no statistically significant difference from S1 in morphology and size distribution, demonstrating that nucleic acid chains containing only A5 can effectively guide and stabilize the formation of small-diameter gold nuclei. Figure 5-6The images show the transmission electron microscope (TEM) morphology and particle size distribution of sample C1. Within the field of view, the particles are typical monodisperse spheres of approximately 10 nm, with an average particle size of 10.5 ± 1.8 nm. Figure 3 The size difference is orders of magnitude. Figure 7 This is a transmission electron microscope image of sample D1, with a field of view similar to that of sample C1.

[0036] (3) Characterization of optical properties S1, S2 and C1 were appropriately diluted with ultrapure water to make the gold concentrations similar (about 5 nM), and the ultraviolet-visible absorption spectra were scanned in a quartz cuvette with a 1 cm optical path (Shimadzu UV-2600).

[0037] See Figure 7-8 , Figure 7 The UV-Vis absorption spectrum of sample S1 shows a smooth decrease in the 400-800 nm range, with no absorption peak near 520 nm. The maximum absorption is located at ~280 nm, which mainly comes from the UV absorption of nucleic acids (DNA has strong absorption at 260 nm). This is a characteristic spectrum of ultra-small (<5 nm) gold nanomaterials. The UV-Vis absorption spectrum of sample S2 is similar to that of sample S2. Figure 8 The UV-Vis absorption spectrum of sample C1 shows a sharp and strong absorption peak at 519 nm (A). max The half-width at half-maximum (WHM) is about 55 nm, which is a typical characteristic of the plasmonic resonance absorption of 10 nm spherical gold nanoparticles; the distinctly different spectra are the most direct physical evidence to prove that S1 / S2 and C1 are fundamentally different in size and electronic structure.

[0038] (4) Characterization of surface charge and uniformity Prepare 1.5% agarose gels and run them in 1×TAE buffer. The agarose gel electrophoresis system was provided by Bio-Rad.

[0039] Sample preparation: Mix samples S1 and C1 with 6× DNA loading buffer (saturated sucrose solution).

[0040] Electrophoresis conditions: constant voltage 90V, electrophoresis for 30 minutes.

[0041] The comparison diagram of the agarose gel electrophoresis behavior of samples S1 and C1 is shown below. Figure 9As can be seen, sample S1 (lane 1) exhibits a fast-migrating, concentrated, and sharp orange-red band near the gel front. The band shows no tailing, indicating high product homogeneity. Sample C1 (lane 2) shows a much slower-migrating, diffuse red band located further back. This slow migration is attributed to the larger particle size and potentially uneven density and conformation of the surface-modified nucleic acids, leading to dispersed electrophoretic migration. This experiment demonstrates the homogeneity of sample S1 and provides a rapid and simple quality control method for determining the success of each batch of products.

[0042] (5) Characterization of long-term storage stability Freshly prepared S1 sample (unpurified) was aliquoted into 2 mL brown liquid chromatography sample vials, filled with nitrogen and sealed (to minimize oxidation), and stored in a 4°C refrigerator protected from light. Meanwhile, C1 was stored under the same conditions as a control.

[0043] During storage, visual observation was conducted once a day. On the third day of storage, C1 showed a small amount of dark flocculent precipitate visible to the naked eye at the bottom of the bottle, and the color of the supernatant became lighter. By the seventh day, the precipitate had increased significantly. S1, on the other hand, remained uniform and transparent throughout the entire 7-day storage period, without any precipitate or color change. Figure 10 These are actual images of C1 solution (left) and S1 solution (right) after 7 days of storage; S1 solution after 7 days of storage is compared with a fresh sample. Figure 2 (Right) Indistinguishable to the naked eye in color and clarity; TEM observation was performed on the S1 sample after 7 days of storage, using the same sample preparation method. See [link / reference]. Figure 11 No large particles or chain-like aggregates formed by the fusion of multiple particles were found in the field of view. The particles still maintained a good monodisperse state. 100 particles were randomly measured and the average particle size was 4.2±0.6 nm. Compared with the fresh sample of 4.0±0.5 nm, there was no significant difference by t test (p>0.05).

[0044] (6) Characterization of synergistic performance of the composite mixture system Samples S1 and S2 were mixed in equal proportions at room temperature. After 15 minutes, 10 μL of the mixture was taken and diluted 10-fold with ultrapure water. 5 μL of the diluted mixture was then dropped onto an ultrathin carbon film copper grid and allowed to air dry at room temperature without any negative staining. The mixture was then observed under a transmission electron microscope (Thermo Fisher Scientific, 120 kV). Figure 12 Transmission electron microscopy revealed that specific hybridization occurred through DNA base pairing. When multiple DNA strands on S1 hybridized with one DNA strand on S2, they brought several gold particles closer together, forming stable, nucleic acid-bridged "DNA-gold nanoparticle polymers," demonstrating that the DNA reacted synergistically and the connection was stable.

[0045] The nucleic acid-gold nanocomposite prepared by the method of this invention has excellent intrinsic long-term stability. This stability comes from the robust core-shell structure formed in situ, rather than relying on an unstable adsorption layer added later. This is the cornerstone for its practical application.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for synthesizing highly stable small-particle-size nucleic acid-gold nanocomposites, characterized in that, Includes the following steps: Under heating and stirring conditions, a reducing agent solution was added in portions to a boiling aqueous solution of chloroauric acid. After boiling again, a solution of functional nucleic acid molecules with PolyA sequences at the ends was added. When the color of the reaction system changed from light pink or pale purple to a deeper color, an acetic acid solution was added. After the reaction was completed, the highly stable small-particle-size nucleic acid-gold nanocomposite was obtained.

2. The method for synthesizing a highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 1, characterized in that, The reducing agent is one or more of sodium citrate, ascorbic acid, and sodium borohydride.

3. The method for synthesizing a highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 1, characterized in that, The concentration of acetic acid in the reaction system is 1-50 mmol / L.

4. The method for synthesizing a highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 1, characterized in that, The length of the PolyA sequence is 2-10 bases.

5. The method for synthesizing a highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 1, characterized in that, The functional nucleic acid molecules are DNA probes, RNA probes, aptamers, antisense oligonucleotides, or small interfering RNAs.

6. The method for synthesizing a highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 1, characterized in that, The reaction time is 5-15 minutes.

7. The highly stable small-particle-size nucleic acid-gold nanocomposite prepared by the synthesis method according to any one of claims 1-6, characterized in that, The core consists of gold nanoparticles with a particle size of less than 5 nm, and the surface of these nanoparticles is coordinated with functional nucleic acid molecules whose ends are modified with PolyA sequences through PolyA sequences.

8. The intermediate product formed by the synthetic method according to any one of claims 1-6, characterized in that, It is a newly formed gold nanocrystal nucleus with acetic acid molecules and PolyA sequences adsorbed on its surface.

9. The application of the highly stable small-particle-size nucleic acid-gold nanocomposite according to claim 7 as a biosensing probe.

10. The application according to claim 9, characterized in that, Applications in the preparation of diagnostic reagents or kits for detecting pathogen nucleic acids, tumor markers, or specific proteins.

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