Preparation method of functional nucleic acid / polymer modified liquid metal core-shell structure microgel

Abstract

The present invention provides a method for preparing a liquid metal core-shell structure microgel modified with a functional nucleic acid / polymer, wherein a monomer, a cross-linking agent, and liquid metal nanoparticles are mixed to form a mixed solution, and then ultrasonically reacted in an ice-water bath to initiate a monomer free radical polymerization reaction on the surface of the liquid metal nanoparticles, thereby obtaining a liquid metal core-shell structure microgel modified with a cross-linked polymer network. The method described in the present invention can obtain a composite microgel with uniform particle size and good dispersibility. According to the proposed synthesis strategy, the present invention further introduces nucleic acid monomers or thermosensitive polymer monomers with functional programmability to successfully construct a composite microgel with multiple properties. These microgels can exhibit specific responsiveness to specific biochemical targets such as small molecules, nucleic acids, proteins, etc., as well as responsiveness to temperature changes and near-infrared light irradiation. The present invention has certain prospects in the field of functional soft material construction and biomedical applications, and is expected to broaden the application of multifunctional liquid metal / polymer composite microstructures in the fields of biosensing, bioimaging, targeted drug release, photothermal and photodynamic synergistic therapy, integrated diagnosis and treatment, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a liquid metal core-shell structure microgel modified with functional nucleic acid / polymer and a preparation method thereof. Background Art

[0002] Compared with traditional metal materials, gallium-based liquid metals have attracted widespread attention due to their unique properties, including thermal conductivity, electrical conductivity, fluidity, deformability, non-toxicity and self-healing. In particular, the combination of metallic properties (thermal conductivity and electrical conductivity) and fluidity has broadened their application range (stretchable electronics, microfluidics, soft robotics, microelectrodes, thermoelectric wearable devices, electronic tattoos). Compared with other liquid metals such as mercury, the low cytotoxicity and high biocompatibility of gallium-based liquid metals make them ideal materials for biomedical applications. Studies have found that exposing liquid metal to ultrasound can strip off the oxides on the surface of liquid metal nanoparticles, which can then induce free radical polymerization reactions on the surface, thereby allowing the local formation of polymer network structures on the liquid metal surface to synthesize liquid metal@polymer composite microgels. Functional nucleic acids are short single chains composed of dozens of nucleotides with unique functions, such as nucleases (DNAzymes) with catalytic ability and nucleic acid aptamers (Aptamers) with the ability to recognize different targets. By combining the characteristics of liquid metal with functional nucleic acids with functional sequence encodeability, composite microgels constructed by integrating the properties of liquid metal and functional nucleic acid / polymer shells can further broaden the application of gallium-based liquid metal in biosensing, drug release, cancer treatment, bioimaging and other fields. Summary of the Invention

[0003] In light of this, the present invention proposes a method for preparing liquid metal core-shell microgels modified with functional nucleic acids and polymers. By combining liquid metal nanoparticles, polymer networks, and functional nucleic acids, a multifunctional composite microgel was successfully constructed. This method has promising applications in the fields of functionalized liquid metals, biosensing, and biomedicine.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A polymer-modified liquid metal core-shell structure microgel comprises a core and a shell layer, wherein the core is liquid metal nanoparticles and the shell layer is a polymer network formed by monomers and a cross-linking agent.

[0006] Preferably, the liquid metal is a gallium-based liquid metal, such as one or more of a gallium-indium alloy, a gallium-indium-tin alloy, a gallium-indium-tin-zinc alloy, and a gallium-indium-tin-zinc-bismuth alloy.

[0007] The monomer is a vinyl monomer, preferably one or more of acrylamide monomer, N-isopropylacrylamide monomer, and acrylic acid monomer.

[0008] The cross-linking agent is methylene bisacrylamide.

[0009] This study, using acrylamide and N-isopropylacrylamide as the primary research targets, explored the feasibility of synthesizing polymer-modified liquid metal microgels under ultrasound. By combining the properties of polymer networks with those of liquid metal nanoparticles, liquid metal@polymer core-shell microgels were prepared.

[0010] Preferably, the microgel shell is further modified with functional nucleic acid, and covalently modified functional nucleic acid is introduced into the shell by introducing functional nucleic acid monomers.

[0011] Preferably, the functional nucleic acid is one or both of DNA and RNA of any length.

[0012] The present invention also provides a method for preparing the polymer-modified liquid metal core-shell structure microgel, which comprises the following steps:

[0013] S1: taking monomers, cross-linking agent, and liquid metal nanoparticles and mixing them to form a mixed solution;

[0014] S2: The mixed solution is sonicated in an ice-water bath and centrifuged to obtain a polymer-modified liquid metal core-shell structure microgel, which is labeled as liquid metal@polymer.

[0015] The present invention also provides another method for preparing the functional nucleic acid / polymer modified liquid metal core-shell structure microgel, which is selected from the following scheme A or scheme B:

[0016] Plan A includes the following steps:

[0017] S1: taking monomers, cross-linking agent, and liquid metal nanoparticles and mixing them to form a mixed solution;

[0018] S2: The mixed solution is sonicated in an ice-water bath, and then the functional nucleic acid is added, sonicated, and the mixed solution is centrifuged to obtain the functional nucleic acid / polymer functionalized liquid metal microgel, which is labeled as liquid metal@polymer / functional nucleic acid.

[0019] Plan B includes the following steps:

[0020] S1: taking liquid metal nanoparticles and functional nucleic acids and mixing them to form a mixed solution;

[0021] S2: The mixed solution is sonicated in an ice-water bath, and then the monomer and cross-linking agent are added, sonicated, and the mixed solution is centrifuged to obtain functional nucleic acid / polymer functionalized liquid metal microgel, which is labeled as liquid metal@polymer / functional nucleic acid.

[0022] Preferably, the liquid metal is a gallium-based liquid metal, specifically one or more of a gallium-indium-tin-zinc alloy and a gallium-indium-tin-zinc-bismuth alloy.

[0023] The monomer is a vinyl monomer, preferably one or more of acrylamide monomer, N-isopropylacrylamide monomer, and acrylic acid monomer.

[0024] The cross-linking agent is methylene bisacrylamide.

[0025] The concentration of the monomer is 10-300 mM, and the concentration of the cross-linking agent is 1-20 mM.

[0026] The final concentration of the liquid metal in the mixed solution is 1-20 mg / mL.

[0027] Preferably, in S1, the ultrasonic time is 30-240 min, and the ultrasonic power is 100-400 W.

[0028] Preferably, in S2, the first ultrasonic time is 30-240 min, the second ultrasonic time is 10-200 min, and the ultrasonic power is 100-400 W.

[0029] Preferably, the functional nucleic acid is one or both of DNA and RNA of any length.

[0030] The functional nucleic acid / polymer modified liquid metal core-shell structure microgel can be applied to the fields of biosensing, biomedicine, etc.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention utilizes the free radical polymerization mechanism. Under ultrasonic conditions, the liquid metal surface can initiate monomer polymerization, polymerizing vinyl monomers on the surface of liquid metal nanoparticles to form polymer-modified liquid metal microgels. This method can obtain liquid metal@polymer microgels with good dispersion and uniform particle size.

[0033] (2) The liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel prepared by the present invention is temperature-sensitive. When the temperature is higher than its critical phase transition temperature, the microgel undergoes a transition from an expanded hydrated state to a contracted dehydrated solid state, exposing the functional nucleic acids in the inner layer of the network, allowing them to more fully bind to the substrate DNA, cleave the substrate DNA, and generate an enhanced fluorescence signal. That is, temperature can regulate the activity of the functional nucleic acids in the microgel.

[0034] (3) The present invention introduces functional nucleic acids to construct nucleic acid-functionalized liquid metal composite materials. This method has certain prospects in the functionalization of liquid metal materials and biosensing and biomedical applications.

[0035] (4) The synthetic strategy proposed in the present invention can modularly synthesize microgels with different functional group distributions by changing the addition time of monomers and functional nucleic acids, thereby achieving the regulation of the microgel's ability to respond to stimuli.

[0036] (5) The liquid metal@polymer / functional nucleic acid microgel synthesized in the present invention is photoresponsive and can undergo directional movement under near-infrared laser irradiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0038] Figure 1 . Schematic diagram of ultrasound-assisted synthesis of polymer-functionalized liquid metal microgels and functional nucleic acid / polymer-functionalized liquid metal microgels.

[0039] Figure 2 Characterization of liquid metal@polyacrylamide microgels: (A) Transmission electron microscopy image of liquid metal@polyacrylamide microgels (scale bar: 200 nm), (B) FTIR spectrum of liquid metal@polyacrylamide microgels, (C) Zeta potential of liquid metal nanoparticles and liquid metal@polyacrylamide microgels.

[0040] Figure 3 (A) Transmission electron microscopy image of liquid metal@poly (N-isopropylacrylamide) microgel, (B) high-angle annular dark-field scanning transmission electron microscopy image of liquid metal@poly (N-isopropylacrylamide) microgel, (C) corresponding X-ray energy spectrum elemental analysis image (red is gallium, green is indium, and yellow is nitrogen) (Scale bar: 200 nm).

[0041] Figure 4 Characterization of liquid metal@poly (N-isopropylacrylamide) microgels: (A) FTIR spectra of liquid metal nanoparticles, poly (N-isopropylacrylamide), and liquid metal@poly (N-isopropylacrylamide) microgels, (B) Zeta potential diagrams of liquid metal nanoparticles and liquid metal@poly (N-isopropylacrylamide) microgels.

[0042] Figure 5Transmission electron microscopy image of liquid metal@polyacrylamide / functional nucleic acid microgel, high-angle annular dark-field scanning transmission electron microscopy image, and corresponding X-ray energy spectrum elemental analysis image (red is gallium, green is indium, yellow is nitrogen, and blue is phosphorus) (Scale bar: 200 nm).

[0043] Figure 6 The functionality of the nucleic acid structure on the surface of catalytically modified liquid metal microgels was demonstrated: (A) Fluorescence intensity generated by the catalytic cleavage of substrate DNA at different concentrations by liquid metal@polyacrylamide / functional nucleic acid microgels: 50nM, 70nM, 100nM, 200nM, 300nM, 400nM, 500nM, 700nM, and 1000nM; (B) Linear equation between substrate DNA concentration and fluorescence intensity.

[0044] Figure 7 Transmission electron microscopy (TEM) images of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels, high-angle annular dark-field scanning TEM images, and corresponding X-ray energy dispersive spectrometry elemental analysis images (red: gallium, green: indium, yellow: nitrogen, blue: phosphorus) (scale bar: 200 nm).

[0045] Figure 8 Characterization of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels: (A) FTIR spectra of poly (N-isopropylacrylamide) and liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels, (B) Zeta potential of liquid metal nanoparticles, liquid metal@poly (N-isopropylacrylamide) and liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels, (C) Changes in particle size of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels at different temperatures.

[0046] Figure 9 The functionality of the nucleic acid structure on the surface of the catalytic nucleic acid-modified thermosensitive liquid metal microgel was verified as follows: (A) Liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel was exposed to 1×10 -6 Fluorescence signals generated by the reaction of M substrate DNA for 1 hour, 40℃ (blue line), 25℃ (red line) and liquid metal@poly (N-isopropylacrylamide) microgel at 25℃ with 1×10 -6 (B) Fluorescence signal generated by the reaction of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel with different concentrations of substrate DNA; (C) Linear equation between substrate DNA concentration and fluorescence signal intensity.

[0047] Figure 10. Laser confocal imaging of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel targeting MCF-7 cells.

[0048] Figure 11 Fluorescence signals generated by free DNA enzyme and microgel at 40°C and 25°C, respectively: (A) NIPAM-DNA (B) DNA-NIPAM; Ratio of fluorescence intensity of free DNA enzyme and microgel at 40°C and 25°C, respectively: (C) NIPAM-DNA; (D) DNA-NIPAM; Fluorescence intensity cycled between 25°C and 40°C: (E) NIPAM-DNA; (F) DNA-NIPAM

[0049] Figure 12 Characterization of the near-infrared laser responsiveness of functional nucleic acid-modified liquid metal microgels: (A) Trajectory distribution of LM@pAM / functional nucleic acid microgels without and (B) with near-infrared laser irradiation (n=10); (C) Dispersion of TRITC-dextran dye (70 kDa) in LM@pAM / functional nucleic acid microgel solutions with the near-infrared laser turned off (I, left) or on (I, right) and in blank solutions with the near-infrared laser turned off (II, left) or on (II, right). DETAILED DESCRIPTION

[0050] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0051] The ingredients and purchasing manufacturers of the main materials involved in the following Examples 1-4 are:

[0052] Liquid metal: Gallium 75wt%, Indium 25wt%, purchased from Shenyang Jiabei Trading Co., Ltd.;

[0053] Acrylamide: 40% (w / v), purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0054] N-isopropylacrylamide: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0055] Methylene bisacrylamide: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0056] Magnesium ion-DNA enzyme: 5′-Acrydite-TTTTTCTCATTCAGCGATCCGGAACGGCACCCATGTTCTGTGA-3′, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0057] Substrate DNA: 5′-ROX-TCACAGAT / rA / GGAATGAG-BHQ2-3′, purchased from Sangon Biotechnology (Shanghai) Co., Ltd.;

[0058] AS1411 aptamer: 5′-Acrydite-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0059] Example 1: Liquid Metal@Polyacrylamide Microgel

[0060] First, 20 mg of liquid metal droplets were dispersed into 1 mL of ultrapure water, and then ultrasonically treated in an ice water bath for 10 minutes (power 230 W, working for 2 seconds, interval 2 seconds) to form a 20 mg / mL liquid metal nanoparticle solution. Then, 50 μL of 20 mg / mL liquid metal nanoparticle solution was mixed with 2.5 μL of 40% (w / v) acrylamide solution and 10 μL of 28 mM methylene bisacrylamide solution, and ultrasonically treated for 2 hours in an ice water bath using a Dingtai Hengsheng silent ultrasonic cleaning machine. The resulting mixture was then centrifuged at 8000 rpm for 10 minutes to redisperse the precipitated part, and then centrifuged at 5000 rpm for 10 minutes. After the precipitated part was redispersed, the supernatant was discarded after centrifugation at 5000 rpm for 10 minutes to obtain liquid metal @ polyacrylamide microgel. The microgel was characterized by transmission electron microscopy, infrared spectrometer and nanoparticle size analyzer. Figure 2 .

[0061] Example 2: Liquid Metal@Poly (N-isopropylacrylamide) Microgel

[0062] The difference from Example 1 is that 2.5 μL of 40% (w / v) acrylamide solution was replaced with 25 μL of 564 mM N-isopropylacrylamide solution. The microgel was characterized by transmission electron microscopy, infrared spectrometer and nanoparticle size analyzer. Figure 3 and Figure 4 .

[0063] Example 3: Liquid Metal@Polyacrylamide / Functional Nucleic Acid Microgel

[0064] First, 20 mg of liquid metal droplets were dispersed into 1 mL of ultrapure water, and then ultrasonically treated in an ice water bath for 10 minutes (power 230 W, working for 2 seconds, interval 2 seconds) to form a 20 mg / mL liquid metal nanoparticle solution. Then, 50 μL of 20 mg / mL liquid metal nanoparticle solution was mixed with 2.5 μL of 40% (w / v) acrylamide solution and 10 μL of 28 mM methylene bisacrylamide solution, and reacted under ultrasonic action for 30 minutes using a Dingtai Hengsheng silent ultrasonic cleaning machine in an ice water bath. Subsequently, magnesium ion-DNA enzyme at a concentration of 50 μM was added and ultrasonic action was continued for 90 minutes. The resulting mixture was then centrifuged at 8000 rpm for 10 minutes, the precipitate was redispersed, and then centrifuged at 5000 rpm for 10 minutes. After the precipitate was redispersed, it was centrifuged again at 5000 rpm for 10 minutes and the supernatant was discarded to obtain liquid metal @ polyacrylamide / functional nucleic acid microgel. The microgel was characterized by transmission electron microscopy, infrared spectrometer and nanoparticle size analyzer. Figure 5 .

[0065] First, the liquid metal@polyacrylamide / functional nucleic acid microgel was used to catalyze the reaction of substrate DNA. The entire catalytic process was carried out in 20 mM (pH 7.0) 4-hydroxyethylpiperazineethanesulfonic acid buffer containing 500 mM NaCl and 20 mM MgCl2. 10 μL of the prepared 10 mg / mL liquid metal@polyacrylamide / functional nucleic acid microgel solution was mixed with 1 μM substrate DNA (DNA sequence: 5'-ROX-TCACAGAT / rA / GGAATGAG-BHQ2-3') and reacted at 25°C for 1 hour. The supernatant was then collected by centrifugation at 14,800 rpm for 10 minutes, and fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength: 580 nm, emission wavelength: 608 nm). Liquid metal@polyacrylamide / functional nucleic acid microgel was used to catalyze substrate DNA solutions of different concentrations to obtain a standard curve. Specifically, 10 μL of the prepared 10 mg / mL liquid metal@polyacrylamide / functional nucleic acid microgel was taken and then mixed with 50 nM, 70 nM, 100 nM, 200 nM, 300 nM, 500 nM, 700 nM, and 1000 nM substrate DNA solutions, respectively, and reacted at 25°C for 1 hour in 20 mM HEPES buffer (pH 7.0 containing 500 mM NaCl and 20 mM MgCl2). The results are shown in Figure 2. Figure 6 .

[0066] Example 4: Liquid Metal@Poly (N-isopropylacrylamide) / Functional Nucleic Acid Microgel

[0067] First, 20 mg of liquid metal droplets were dispersed in 1 mL of ultrapure water and then sonicated in an ice-water bath for 10 minutes (230 W power, 2 seconds on, 2 seconds off) to form a 20 mg / mL LM NPs concentrated solution. Next, 50 μL of the 20 mg / mL liquid metal nanoparticle solution was mixed with 25 μL of 564 mM N-isopropylacrylamide solution and 10 μL of 28 mM methylene bisacrylamide. After sonication for 30 minutes using a Dingtai Hengsheng silent ultrasonic cleaner, 50 μM magnesium ion-DNase was added and sonication continued for another 90 minutes. The mixture was then separated by differential centrifugation to yield composite microgels labeled with NIPAM-DNA. Detection of the substrate DNA was performed in 20 mM (pH 7.0) HEPES buffer containing 500 mM NaCl and 20 mM MgCl₂. Specifically, 10 μL of the prepared 10 mg / mL liquid metal @ poly N-isopropylacrylamide / functional nucleic acid microgel solution, a certain concentration of substrate DNA, and 10 mM phosphate ions were mixed and incubated at 40 ° C for 1 hour. The supernatant of the reaction solution was then collected by centrifugation at 14800 rpm for 10 minutes at 40 ° C, and the fluorescence intensity of the supernatant was measured by a fluorescence spectrophotometer with an excitation wavelength of 580 nm and an emission wavelength of 608 nm. As a control experiment, 10 μL of 10 mg / mL liquid metal @ poly N-isopropylacrylamide microgel solution was mixed with 1 μM substrate DNA solution and reacted at 25 ° C for 1 hour. The results are shown in Figures 7 to 9 .

[0068] Example 5: Liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel targeting cancer cells

[0069] The difference from Example 4 is that the magnesium ion-DNA enzyme was replaced with AS1411 aptamer, and the aptamer / polymer modified liquid metal microgel was synthesized, and the targeting ability of the microgel to cancer cells was investigated. First, MCF-7 cells were cultured in a confocal dish, with 1.5×10 cells per dish. 4 , and kept in a cell culture incubator overnight. The next day, the cells were removed, the culture medium was discarded, and the cells were washed three times with PBS buffer. LM@pNIPAM / AS1411 microgels diluted to 0.5 mg / mL with culture medium were added, cultured in the cell culture incubator for 4 hours, removed, and thoroughly washed three times with PBS buffer. The cells were fixed with 4% paraformaldehyde solution, washed three times with PBS buffer after 15 minutes, and the paraformaldehyde solution was washed off. Subsequently, 1 μg / mL of the dye 4',6-diamidino-2-phenylindole (DAPI) was added to stain the cell nuclei in the cell culture incubator for 15 minutes, and then thoroughly washed three times with PBS buffer. PBS buffer was added to the confocal dish, and the targeting ability of LM@pNIPAM / AS1411 microgels to cells was observed by confocal microscopy. The results are shown in Figure 10 .

[0070] Example 6: Modular synthesis of different liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels

[0071] Due to localized surface polymerization, a modular synthesis process can be achieved by simply adjusting the order of addition of different monomers, thereby controlling the distribution of different functional groups in the microgel layer. The difference from Example 4 is that the order of addition of monomers and functional nucleic acids during the ultrasonic process is adjusted. Specifically, 50 μL of 20 mg / mL liquid metal nanoparticle solution and 50 μM magnesium ion-DNA enzyme are taken, and ultrasonic treatment is performed for 30 minutes using a Dingtai Hengsheng silent ultrasonic cleaning machine. Then, 25 μL of 564 mM N-isopropylacrylamide solution and 10 μL of 28 mM methylene bisacrylamide are added and the mixture is ultrasonically treated for 90 minutes. Finally, the mixture is separated by differential centrifugation to obtain a composite microgel labeled DNA-NIPAM. It exhibits different catalytic activity against substrate DNA than NIPAM-DNA. The specific steps are shown in Example 4, and the results are shown in Figure 11 .

[0072] Example 7: Directed movement of composite microgels driven by near-infrared laser

[0073] Synthesize LM@pAM / functional nucleic acid microgels. The specific steps are shown in Example 3. Use a LM@pAM / functional nucleic acid microgel solution with a near-infrared irradiation concentration of 0.1 mg / mL to record the directional movement of LM@pAM / functional nucleic acid microgels under near-infrared laser irradiation using an inverted fluorescence microscope, and analyze its movement behavior using Image J software. Using TRITC-dextran (70KDa) dye as an indicator, observe the macroscopic movement of LM@pAM / functional nucleic acid microgels under near-infrared laser irradiation. The laser current is 3A, the irradiation time is 1 minute, and the concentration of LM@pAM / functional nucleic acid microgel is 1 mg / mL. The results are shown in Figure 12 .

[0074] Experimental results and discussion

[0075] Figure 2 This is a transmission electron microscope image of the liquid metal@polyacrylamide microgel prepared in Example 1. As can be seen from the figure, the liquid metal@polyacrylamide microgel particles are spherical, with the inner layer being a liquid metal nanoparticle core with a darker contrast, and the outer layer being a polymer layer with a lighter contrast, presenting a uniform core-shell structure as a whole, indicating that the liquid metal@polyacrylamide microgel has been synthesized. In addition, infrared spectroscopy was used to characterize the functional groups of polyacrylamide in the liquid metal@polyacrylamide microgel, such as Figure 2As shown in Figure B, compared with polyacrylamide, the liquid metal@polyacrylamide microgel formed after polymerizing acrylamide on the surface of liquid metal nanoparticles has the same infrared spectrum peak. Specifically, in the infrared spectrum of liquid metal@polyacrylamide microgel, the peak at 1647 cm -1 The carbonyl peak belonging to polyacrylamide was observed at the wave number, indicating that polyacrylamide was polymerized on the surface of the liquid metal nanoparticles. The change of Zeta potential before and after the polymerization of polyacrylamide on the surface of the liquid metal nanoparticles was further characterized by a particle size analyzer. Figure 2 As shown in Figure C, the results indicate that liquid metal nanoparticles are positively charged. Since acrylamide is uncharged, the liquid metal@polyacrylamide microgel maintains its positive charge after surface polymerization with acrylamide. These results demonstrate that under the influence of liquid metal and ultrasound, monomers polymerize on the liquid metal surface via an atom transfer radical polymerization mechanism to form a liquid metal@polyacrylamide microgel with a core-shell structure.

[0076] The feasibility of the proposed strategy was further verified by replacing the acrylamide monomer with N-isopropylacrylamide monomer. The morphology of the liquid metal@poly(N-isopropylacrylamide) microgel synthesized in Example 2 was characterized by transmission electron microscopy. Figure 3 The liquid metal@poly (N-isopropylacrylamide) microgel shown in A is spherical with a size distribution of 118.8 ± 34.6 nm (inset, calculated using ImageJ). The inner layer is a liquid metal nanoparticle core with a darker contrast, and the outer layer is a polymer layer with a lighter contrast, presenting a uniform core-shell structure. As can be seen from the magnified image, the shell thickness is approximately 23.1 nm ( Figure 3 B). In addition, high-angle annular dark-field scanning transmission electron microscopy images also show that the liquid metal@poly (N-isopropylacrylamide) microgel has a core-shell structure. X-ray energy spectrum elemental analysis shows that gallium, indium, and carbon are evenly distributed in the particles ( Figure 3 C).

[0077] In addition, the liquid metal@poly (N-isopropylacrylamide) microgels were characterized by Fourier transform infrared spectroscopy and Zeta potential. Figure 4 Compared with pure poly (N-isopropylacrylamide), the same functional groups were observed in the infrared spectrum of liquid metal@poly (N-isopropylacrylamide) microgel. Specifically, two characteristic peaks of amide group (stretching vibration of carbonyl group ν C＝O : 1634cm -1 and the deformation vibration of the secondary amide NH δ N-H : 1528cm -1 )( Figure 4 A). In addition, if Figure 4As shown in Figure B, similar to pure liquid metal nanoparticles, the zeta potential of the particles remains positive after surface modification with poly (N-isopropylacrylamide) microgels. In summary, the proposed synthetic strategy can still be used to polymerize monomers on the outer layer of liquid metal nanoparticles to synthesize liquid metal@poly (N-isopropylacrylamide) core-shell microgels.

[0078] Based on base diversity and sequence programmability, functional nucleic acids are often used as a universal material. By leveraging the ability of liquid metal to initiate free radical polymerization under ultrasonic conditions, functional nucleic acids are polymerized on the surface of liquid metal nanoparticles to give them the characteristics of functional nucleic acids. For example, target responsiveness, biocompatibility, and precise recognition. In addition, based on the sequence programmability of functional nucleic acids, the functional nucleic acid sequence can be changed to meet the needs of different applications, thereby broadening the application range of liquid metal. Next, using the magnesium ion-responsive DNA enzyme sequence as a model, the feasibility of constructing a functional nucleic acid-modified liquid metal@polymer core-shell structure microgel on the surface of liquid metal nanoparticles by ultrasonically initiating the polymerization of functional nucleic acids and vinyl monomers under the action of liquid metal nanoparticles was explored.

[0079] After 30 minutes of ultrasonic reaction, DNA enzyme was added to synthesize liquid metal@polyacrylamide / functional nucleic acid microgel (see Example 3 for details). The morphology of liquid metal@polyacrylamide / functional nucleic acid microgel was characterized by transmission electron microscopy. Figure 5 As shown, transmission electron microscopy (TEM) images and high-angle annular dark-field scanning TEM images demonstrate that the synthesized microgel has a distinct core-shell structure, with liquid metal nanoparticles as the core and polymer and DNA enzyme as the outer layers. Furthermore, X-ray energy spectrum elemental imaging confirmed the presence of elements in the particles: gallium (red) and indium (green) come from the liquid metal nanoparticles, nitrogen comes from the polymer (yellow), and phosphorus (blue) comes from the phosphate backbone of the DNA enzyme. The results demonstrate that even after the addition of DNA enzyme, the proposed method is still capable of synthesizing liquid metal@polyacrylamide / functional nucleic acid microgels with a core-shell structure. Elemental scanning also verifies that the DNA enzyme has also been successfully modified on the surface of the liquid metal nanoparticles.

[0080] In the presence of magnesium ions, the catalytic activity of DNA enzymes on the surface of liquid metal@polyacrylamide / functional nucleic acid microgels was studied based on the fluorescence signal generated by the catalytic cleavage of substrate DNA by liquid metal@polyacrylamide / functional nucleic acid microgels. Liquid metal@polyacrylamide / functional nucleic acid microgels were reacted with substrate DNA at different concentrations to detect the substrate DNA. A good fluorescence signal response was observed in the range of 50nM-1000nM substrate DNA concentration ( Figure 6 A) If Figure 6As shown in B, the obtained fluorescence signal is linearly related to the substrate DNA concentration, and the linear equation is FL = 0.483c + 9.01 (R 2 =0.996), and the detection limit was 26.1 nM (3σ rule). This indicates that liquid metal@polyacrylamide / functional nucleic acid microgel can be used to detect substrate DNA.

[0081] The acrylamide monomer was replaced with another monomer, N-isopropylacrylamide, and the morphology of the liquid metal@poly(N-isopropylacrylamide) / functional nucleic acid microgel prepared in Example 4 was characterized by transmission electron microscopy. Figure 7 As shown in Figure B, transmission electron microscopy (TEM) and high-angle annular dark-field scanning TEM images demonstrate that the synthesized microgel has a distinct core-shell structure, with the liquid metal nanoparticles as the core and the polymer and DNA enzyme as the outer layers. Furthermore, X-ray energy spectrum elemental imaging confirmed the presence of elements in the particles: gallium (red) and indium (green) originate from the liquid metal nanoparticles, nitrogen from the polymer (yellow), and phosphorus (blue) from the DNA enzyme backbone. These results demonstrate that even after the addition of DNA enzyme, the proposed method is still capable of synthesizing liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels with a core-shell structure, and that DNA enzyme is also successfully modified on the surface of the liquid metal nanoparticles.

[0082] In addition, the difference between liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel and pure poly (N-isopropylacrylamide) was characterized by infrared spectroscopy. Figure 8 As shown in A, the liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgel has the same infrared spectrum as pure poly (N-isopropylacrylamide). For example, at 1636 cm -1 The stretching vibration of carbonyl group can be observed at 1522 cm -1 The NH deformation vibration of the secondary amide was observed, indicating that poly-N-isopropylacrylamide is indeed present in the liquid metal@poly N-isopropylacrylamide / functional nucleic acid core microgel. The charge on the particle surface after each step of the reaction was characterized by a nanoparticle size meter. The results showed that after the negatively charged functional nucleic acid was attached to the surface, the potential of the liquid metal@poly N-isopropylacrylamide / functional nucleic acid decreased but remained positive. This indicates that although the functional nucleic acid has been modified on the microgel surface, its content is low and is not enough to change the overall electrical properties of the particle ( Figure 8 B) Using a nanoparticle size analyzer, we investigated the trend of particle size changes with temperature. As the temperature increased, the particle size decreased. We selected reaction temperatures of 25°C and 40°C as critical points for particle size to investigate the catalytic activity of DNA enzymes before and after the phase transition of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels. Figure 8 C).

[0083] like Figure 9 As shown in Figure A, compared to the reaction temperature at 25°C, when the reaction temperature is 40°C, it is higher than the critical phase transition temperature (LCST), causing the poly (N-isopropylacrylamide) microgel network to shrink. The DNA enzyme is more fully exposed and more effectively used to cleave the substrate DNA, thus generating a stronger fluorescence signal. Different concentrations of substrate DNA were reacted with liquid metal @ poly (N-isopropylacrylamide) / functional nucleic acid microgel. In the range of substrate DNA concentration from 50nM to 1000nM, liquid metal @ poly (N-isopropylacrylamide) / functional nucleic acid microgel showed sensitive signal response ( Figure 9 B). Linear fitting was performed with the concentration of substrate DNA as the horizontal axis and the fluorescence signal intensity as the vertical axis. The obtained linear equation was FL = 0.497c-9.69 (R 2 =0.991), the detection limit was 4.97nM (3σ rule) ( Figure 9 C) To further investigate the temperature-sensitive properties of liquid metal@poly (N-isopropylacrylamide) / functional nucleic acid microgels and their ability to regulate DNA enzyme catalytic activity.

[0084] By changing the base sequence of functional nucleic acids, new functions can be given to composite microgels. For this purpose, a nucleic acid aptamer (AS1411) was selected to achieve specific targeting of composite microgels to tumor cells MCF-7. The 3' end of the AS1411 aptamer was modified with a fluorescent group and observed by laser confocal microscopy. The sample was incubated with cells in a cell culture incubator for 25 minutes, and then the targeting of AS1411-modified liquid metal@poly(N-isopropylacrylamide) microgels to cells was observed under a laser confocal microscope. Figure 10 As shown, bright red fluorescent groups were observed on the cell surface, indicating that the synthesized composite microgels can target cancer cells, which will further broaden the application of liquid metal microgels in the biomedical field.

[0085] In addition, the in situ polymerization reaction on the liquid metal surface allows the performance of the functional nucleic acid in the microgel layer to be regulated by changing the order of adding the monomer and the functional nucleic acid, such as constructing microgels with different catalytic activities towards the substrate (NIPAM-DNA and DNA-NIPAM). Figure 11 As shown in Figure 2, the fluorescence signals generated by the catalytic cleavage of substrate DNA by free DNA enzyme and microgel samples were measured by fluorescence spectrophotometer at 25°C and 40°C, respectively. Figure 11 A and 11B), the fluorescence intensity at 40°C was compared with that at 25°C to obtain Figure 11The data in Figures C and 11D show that when the temperature rises to 40°C, the activity of the free DNA enzyme decreases, and the catalytic ability to the substrate DNA decreases. However, due to the thermoresponsiveness of poly-N-isopropylacrylamide, the microgel shrinks, the DNA enzyme fully binds to the substrate DNA, and the microgel sample produces an enhanced fluorescence signal. Figure 11 As shown in Figure C, when the temperature increased from 25°C to 40°C, the activity of free DNA enzyme decreased to 68%. In contrast, the fluorescence intensity of NIPAM-DNA microgel increased by 1.47 times. The fluorescence intensity of another sample, DNA-NIPAM, at 40°C was 2.26 times that of 25°C. The ratio of the fluorescence intensity of free DNA enzyme at 40°C to 25°C was 0.61 times ( Figure 11 D). By comparing the two different microgels mentioned above, we found that the fluorescence signals generated by NIPAM-DNA microgels at 25°C and 40°C were higher than those of DNA-NIPAM microgels, but the fluorescence intensity ratio at 40°C and 25°C was lower than that of DNA-NIPAM microgels. Therefore, adding DNA enzyme before adding N-isopropylacrylamide monomer will cause more DNA enzyme to be dispersed in the inner layer of the microgel, and a microgel with more sensitive temperature responsiveness can be obtained; while adding it after adding N-isopropylacrylamide monomer will cause more DNA enzyme to be distributed in the outer layer of the microgel, and a microgel with higher enzyme activity will be obtained, thereby confirming the feasibility of regulating the performance of functional nucleic acids in the microgel layer by controlling the order of monomer addition. In addition, the composite microgel also exhibits reversible regulation characteristics. By cyclically increasing and decreasing the solution temperature, the reversible regulation of DNA enzyme catalytic activity is achieved ( Figure 11 E and Figure 11 F).

[0086] Then, the response properties of liquid metal@polyacrylamide / functional nucleic acid microgels to near-infrared light irradiation were studied. Figure 12 As shown, the Brownian motion random displacement of microgels is significantly different from that without near-infrared irradiation ( Figure 12 A), under near-infrared laser irradiation, the microgels undergo directional photoinduced motion, and near-infrared light irradiation significantly enhances the movement speed of the microgels ( Figure 12 B). TRITC-dextran dye (70 kDa) was further used as an indicator to verify the movement of liquid metal@polyacrylamide / functional nucleic acid microgels under near-infrared irradiation. Figure 12 As shown in Figure C, under near-infrared laser irradiation, the liquid metal@polyacrylamide / functional nucleic acid microgel moves, promoting the rapid dispersion of TRITC-dextran dye in the solution, which is in sharp contrast to the sample group without near-infrared laser. This may be beneficial to the biological applications of composite microgels driven by near-infrared.

[0087] In summary, a functional nucleic acid / polymer-modified liquid metal core-shell microgel was synthesized under ultrasound. This microgel boasts uniform particle size, good dispersibility, and a simple preparation method, making it an effective method for functionalizing liquid metal. It also combines the properties of liquid metal with functional nucleic acids with sequence-encodeable functionality. The resulting liquid metal@polymer / functional nucleic acid microgel material has broad application prospects in smart materials, biosensing, biomedicine, and other fields.

[0088] 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.

Claims

1. A polymer-modified liquid metal core-shell microgel, characterized by: The microgel comprises a core and a shell, wherein the core is liquid metal nanoparticles and the shell is a polymer network formed by monomers and cross-linking agents; The liquid metal is one or more of gallium-indium alloy, gallium-indium-tin alloy, gallium-indium-tin-zinc alloy, and gallium-indium-tin-zinc-bismuth alloy; The monomer is one or more of acrylamide monomer, N-isopropylacrylamide monomer, and acrylic acid monomer; Also included is the introduction of covalently modified functional nucleic acids into the shell by introducing functional nucleic acid monomers; The preparation method of the microgel is selected from the following scheme A or scheme B: Plan A includes the following steps: S1: taking monomers, cross-linking agent, and liquid metal nanoparticles and mixing them to form a mixed solution; S2: ultrasonicating the mixed solution in an ice-water bath, then adding the functional nucleic acid monomer, ultrasonicating the mixed solution, and centrifuging the mixed solution to obtain the functional nucleic acid / polymer functionalized liquid metal microgel, which is labeled as liquid metal@polymer / functional nucleic acid; Plan B includes the following steps: S1: taking liquid metal nanoparticles and functional nucleic acid monomers and mixing them to form a mixed solution; S2: The mixed solution is sonicated in an ice-water bath, and then the monomer and cross-linking agent are added, sonicated, and the mixed solution is centrifuged to obtain functional nucleic acid / polymer functionalized liquid metal microgel, which is labeled as liquid metal@functional nucleic acid / polymer.

2. The polymer-modified liquid metal microgel according to claim 1, characterized in that: The cross-linking agent is methylene bisacrylamide.

3. The polymer-modified liquid metal core-shell structure microgel according to claim 1, characterized in that: The functional nucleic acid is one or both of DNA and RNA of any length.

4. The polymer-modified liquid metal core-shell structure microgel according to claim 1, characterized in that: The concentration of the monomer is 10-300 mM, and the concentration of the cross-linking agent is 1-20 mM; The final concentration of the liquid metal in the mixed solution is 1-20 mg / mL.

5. The polymer-modified liquid metal core-shell structure microgel according to claim 1, characterized in that: In S2, the first ultrasonic time is 30-240 min, the second ultrasonic time is 10-200 min, and the ultrasonic power is 100-400W.

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