Two-dimensional high-molecular fluorescent encoding nanosheet, and preparation method and application thereof

Abstract

The application discloses a two-dimensional high-molecular fluorescent coding nanosheet, a preparation method and application thereof. The two-dimensional high-molecular fluorescent coding nanosheet has a nanosheet structure realized by extending and crystallizing in a two-dimensional direction by using a crystalline polymer, and fluorescent groups are introduced into the nanosheet structure in the process of extending and crystallizing of the crystalline polymer to realize a fluorescent coding function. The application increases an assembly driving force by introducing the crystalline polymer, simplifies a preparation method of a nanometer coding material, realizes preparation of the nanometer coding material at room temperature, fast and mild conditions by a crystalline self-assembly mode, realizes fast and accurate signal writing of fluorescence in a two-dimensional nanometer range by a peripheral crystallization mode, and accurate fluorescent information coding capability can be realized by controlling the order, mass and proportion of the added polymer, thereby providing coding signals for fast, efficient, multi-index and high-throughput tracking and marking of multi-target molecules in an in-vitro diagnosis field.

Description

Technical Field

[0001] This invention relates to the field of nano-fluorescent encoded materials, and more specifically, to a method for preparing biodegradable polycaprolactone-based nanosheet materials and a technology for reading and writing fluorescence information. Background Technology

[0002] With the development of biomedicine, cutting-edge scientific fields such as disease diagnosis, drug screening, and gene sequencing have created a significant demand for the development of high-throughput detection technologies. Suspended array microchips represent the most promising high-throughput screening and diagnostic technology. Among these, fluorescence coding technology, as the core design of suspended array microchips, provides a large number of encoded signals for high-throughput analysis by analyzing the arrangement and recombination of fluorescent signal molecules.

[0003] Suspended array microchips using encoded microspheres as carriers are currently the mainstream fluorescence encoding technology, widely used in the analysis of biomolecules such as proteins, nucleic acids, and cytokines. Commercially available fluorescence-encoded microspheres from Luminex and Quanterix dominate applications such as flow cytometry, encapsulating various fluorescent molecules in different proportions within a single microsphere to achieve high-throughput encoding of multiple fluorescence intensities / wavelengths, enabling simultaneous tracking and detection of multiple analytes. However, relying solely on fluorescence intensity / wavelength as the single output information in this design is susceptible to spectral overlap and molecular interference, limiting the number of output encoded signals to 60-200.

[0004] Current research on fluorescence coding technology mainly focuses on two aspects: the development of novel fluorescence readout modes and the design of coding matrix materials. The development of fluorescence readout modes primarily aims to overcome the information capacity limitations caused by fluorescence emission spectra by exploring photophysical parameters of different dimensions as output signals (fluorescence lifetime, fluorescence anisotropy, etc.). However, reading these high-dimensional photophysical parameters typically requires large-scale equipment and is complex to operate, limiting the universality of this coding strategy. Besides the development of readout modes, the arrangement and recombination of fluorescence signals on matrix materials can also achieve high-density information storage.

[0005] The most common design approach currently is to mix fluorescent molecules in zero-dimensional nanomaterials (microspheres) to achieve high-throughput encoding of various fluorescence intensities / wavelengths. This design strategy is widely used in the analysis of biomolecules such as proteins, nucleic acids, and cytokines. However, output signals based on fluorescence intensity / wavelength are limited by spectral overlap and intermolecular interference, making it difficult to achieve higher throughput detection. The introduction of micro / nanoscale spatial information can enable more diverse and flexible encoding modes. By synergistically regulating the recombination and spatial distribution of fluorescent molecules, the barriers of spectral overlap and fluorescence signal interference can be overcome. However, achieving precise distribution of fluorescence signals in a limited space requires highly designable micro / nano matrix materials.

[0006] High-dimensional assembled structures, such as one-dimensional rod-like structures, two-dimensional sheet-like structures, and other complex heterostructures, can provide more flexible and diverse encoding platforms for the spatial distribution of fluorescent molecules. Current research on high-dimensional fluorescent encoding matrices focuses on one-dimensional rod-like assembled structures. Borrowing from the design strategies of macroscopic barcodes, geometric length information is introduced into one-dimensional micro / nano matrices, expanding the fluorescent encoding capacity through the synergistic regulation of fluorescence intensity and spatial information. However, the spatial information carried by one-dimensional structures is relatively simple; changes in spatial distribution such as stacking, bending, and entanglement between materials can affect the accurate reading of signals. Therefore, how to achieve nanoscale fluorescent spatial distribution while ensuring the accuracy of signal reading and writing still requires further in-depth research.

[0007] Two-dimensional structures, compared to one-dimensional structures, possess more complex geometric distributions. They can maintain the spatial geometric information of one-dimensional rod-shaped coding materials while providing patterned signal output, further improving the accuracy and flexibility of signal reading and writing. Although the two-dimensional spatially specific distribution of fluorescence signals can expand information storage capacity, it also poses greater challenges to the design and synthesis of this nanomatrix. Due to limitations in preparation technology, no polymer-based two-dimensional coding materials have been reported to date.

[0008] Therefore, it is essential to develop a two-dimensional fluorescent coding system that combines accurate reading and writing capabilities, flexible modification methods, and controllable costs. Summary of the Invention

[0009] As an important branch of organic macromolecules, polymers can construct assemblies with various structures and functions through monomer design, chain segment arrangement regulation, and assembly method optimization, offering greater design flexibility and lower cost compared to DNA materials. Traditional block polymer nanoassemblies typically utilize solvent interactions between blocks to construct nanoassemblies. Because these assemblies are in a highly dynamic equilibrium and the phase separation driving assembly lacks directionality, it is difficult to construct high-dimensional assembly structures (one-dimensional rod-shaped micelles or two-dimensional sheet-like micelles) or precisely control the spatial distribution of the assembly structure. Developing novel assembly driving forces to construct high-dimensional nanostructures while ensuring the spatially specific distribution of fluorescent molecules is a key issue in the design strategy of nanomatrices in the field of fluorescence encoding. Crystallization-Driven Self-Assembly (CDSA), as a polymer assembly method, achieves polymer self-assembly by introducing crystalline segments into polymer blocks, driven by both phase separation induced by solvent interactions and crystallization induced by the crystalline blocks. The introduction of crystallization can induce polymers to stack in a specific direction, making it easy to construct high-dimensional, multi-level heterogeneous assembly morphology structures and precisely control the spatial distribution of high-dimensional assemblies.

[0010] Based on the above problems and the development ideas of fluorescence coding technology, this invention introduces a crystallization-driven active self-assembly strategy from the perspective of molecular synthesis and material design, and develops a novel fluorescence coding technology that co-encodes spatial geometric information and fluorescence information, so as to expand the storage capacity of fluorescence information.

[0011] This invention is implemented as follows:

[0012] This invention first provides a two-dimensional polymer fluorescently encoded nanosheet, comprising a polymer nucleus and one or more epitaxial polymer rings surrounding the outer edge of the polymer nucleus. The multiple epitaxial polymer rings are arranged sequentially from the inside to the outside. The epitaxial polymer rings are formed by planar crystallization growth of the outer edge of the polymer nucleus through crystallization-driven self-assembly. The polymer nucleus and the epitaxial polymer rings together constitute a nanosheet structure. At least one of the polymer nucleus and all epitaxial polymer rings is a fluorescent polymer layer. The fluorescent polymer layer contains a polymer modified with a fluorescent group, and the fluorescent polymer layer has fluorescent information by introducing the polymer modified with a fluorescent group.

[0013] Furthermore, each fluorescent polymer layer comprises a polymer modified with one color fluorescent group, or a mixture of polymers modified with multiple colors of fluorescent groups; the fluorescent groups include red fluorescent groups, green fluorescent groups, and blue fluorescent groups. The precise fluorescence information encoding capability of the two-dimensional polymer fluorescently encoded nanosheets can be achieved by controlling the order, mass, and ratio of adding different fluorescently modified polycaprolactones; simultaneously, the signal writing of fluorescence in the two-dimensional nanoscale can be achieved quickly and accurately through edge crystallization.

[0014] Furthermore, the materials of the polymer nucleus and the epitaxial polymer layer can be selected from any one or more polymers that have crystallinity and can form nanosheet structures through crystallization-driven self-assembly, such as polycaprolactone (PCL) polymer and polylactic acid polymer (PLA).

[0015] In one specific embodiment, the two-dimensional polymer fluorescently encoded nanosheets are nanosheet structures achieved by extending and crystallizing crystalline polycaprolactone (PCL) in a two-dimensional direction. The introduction of crystalline PCL increases the assembly driving force, thus simplifying the preparation method of the nano-encoded material. The preparation of nano-encoded materials under room temperature, rapid, and mild conditions can be achieved through the self-assembly of PCL crystals. The fluorescent encoding function is achieved by introducing fluorescent groups into the nanosheet structure during the extended crystallization process of PCL modified with fluorescent groups. The polymer nucleus material is a PCL block copolymer, and the epitaxial polymer layer material is a mixture of PCL homopolymer and PCL block copolymer, wherein the PCL block copolymer is a PCL-b-PDMA block copolymer. The PCL homopolymer and PCL block copolymer, as the main building blocks of the two-dimensional sheet-like assembly, determine the stability and functionality of the CDSA sheet-like micelles through their structural and functional design.

[0016] The present invention also provides a method for preparing the above-mentioned two-dimensional polymer fluorescently encoded nanosheets, comprising: preparing a polymer modified with a fluorescent group; obtaining polymer nanorods through crystallization-driven self-assembly; breaking the polymer nanorods to obtain short rod-shaped polymer nuclei; and forming epitaxial polymer layers by extending and crystallizing an epitaxial polymer around the polymer nuclei in a two-dimensional direction through crystallization-driven self-assembly, thereby obtaining crystalline organic polymer nanosheets with a nanosheet structure; wherein at least one of the polymer nuclei and all epitaxial polymer layers is a fluorescent polymer layer, and the polymer used in the preparation of the fluorescent polymer layer includes the polymer modified with the fluorescent group.

[0017] In one specific embodiment, the two-dimensional polymer fluorescently encoded nanosheet is a nanosheet structure achieved by extending and crystallizing polycaprolactone in a two-dimensional direction. The preparation method specifically includes the following steps:

[0018] Step 1: Preparation of polymer substrate: PCL homopolymer was prepared by ε-caprolactone ring-opening polymerization, and PCL block polymer was further synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Specifically, a bifunctional initiator with trithioester and hydroxyl structures was synthesized. By designing the molecular structure of the ε-caprolactone ring-opening polymerization initiator, active modification sites were introduced to the end of the homopolymer. The hydroxyl end of the bifunctional initiator was used to initiate the ring-opening polymerization of cyclocaprolactone, and PCL homopolymer with trithioester end groups was synthesized. This homopolymer can be used as a macromolecular chain transfer agent to further participate in the RAFT polymerization of acrylate monomers such as N,N-dimethylacrylamide (DMA). PCL block copolymer with PCL crystalline segments was prepared from the PCL homopolymer, and a polymer substrate including PCL homopolymer and PCL block copolymer was obtained.

[0019] Step 2: Fluorescent Functionalization of Polymer Substrate: At least one of the prepared PCL homopolymers and PCL block copolymers is fluorescently functionalized by selecting fluorophores with different fluorescence emission. Specifically, a fluorescent dye with carboxyl groups is provided, and the hydroxyl functional groups at the ends of the polymer substrate are coupled to the carboxyl groups of the fluorescent dye via Steglich esterification to modify the polymer substrate with fluorescent groups, obtaining a fluorescently modified polymer. The fluorescently modified polymer includes at least one of fluorescently modified PCL homopolymers and fluorescently modified PCL block copolymers. Fluorescent modification of polycaprolactone is also performed to obtain a fluorescently labeled polymer. For example, three different fluorescently modified PCL homopolymers (denoted as PCL-R, PCL-G, and PCL-B, respectively) are prepared, along with three different fluorescently modified PCL homopolymers (denoted as PCL-R, PCL-G, and PCL-B, respectively). 50 -b-PDMA 200 Block copolymers (referred to as PCL-b-PDMA-R, PCL-b-PDMA-B, and PCL-b-PDMA-G, respectively).

[0020] Step 3, Preparation of polymer nuclei: PCL crystallization-driven seed micelles are prepared according to the self-nucleation method. This process includes two steps: preparing PCL crystallization-driven rod-shaped micelles and ultrasonically manipulating them to prepare seed micelles. First, the PCL block copolymer is driven to self-nucleate and crystallize in an ethanol solution by a thermodynamic method of heating and cooling to form polymer nanorods of rod-shaped micelles. The polymer nanorods synthesized by this method are further subjected to intermittent ultrasonication to obtain polymer nuclei of short rod-shaped seed micelles with a size of less than 50 nm.

[0021] Step 4: Preparation of two-dimensional polymer sheets by seed growth method: An epitaxial polymer solution is added once or multiple times to the polymer nucleus solution prepared in step 3. The epitaxial polymer is a PCL block copolymer or a mixture of PCL block copolymer and PCL homopolymer. Each time the epitaxial polymer is added, it extends and crystallizes along the outer edge of the polymer nucleus to form an epitaxial polymer layer. At least one of the polymer nucleus and all epitaxial polymer layers is a fluorescent polymer layer. The polymer used in the preparation of the fluorescent polymer layer includes the polymer modified with the fluorescent group, thus obtaining two-dimensional polymer fluorescently encoded nanosheets.

[0022] In one specific embodiment, in step 1, the initiator used is 2-cyano-5-pentanol-2-ethyltrithioester (CTA); the PCL homopolymer is subjected to RAFT polymerization with N,N-dimethylacrylamide to obtain a PCL block copolymer. 50 -b-PDMA 200 Block copolymers.

[0023] In one specific implementation, the fluorescent dye used in step 2 is a carboxyl-functionalized BODIPY dye, including BODIPY-630 / 650, BODIPY-R6G, and BODIPY-FITC.

[0024] In a preferred embodiment, in order to enhance the crystallization driving force in the two-dimensional direction, a mixed solution of PCL homopolymer and copolymer is used in step 4 to achieve controllable two-dimensional growth of seed micelles. The added fluorescently labeled polymer solution is a blend solution of fluorescently labeled PCL block copolymer and homopolymer. Furthermore, the ratio of homopolymer / copolymer, the ratio of seed micelles / polymer, and the concentration can be adjusted to achieve changes in the microstructure of the assembly.

[0025] This invention also provides the application of two-dimensional polymer fluorescently coded nanosheets, or two-dimensional polymer fluorescently coded nanosheets prepared according to the above preparation method, in the preparation of anti-counterfeiting materials, including anti-counterfeiting polymer patches, anti-counterfeiting inks, etc.; for example, mixing two-dimensional polymer fluorescently coded nanosheets with PVP solution to prepare polymer patches with different information codes can realize the encoding and encryption of different commodities; the two-dimensional polymer fluorescently coded nanosheets of this invention can also be used to prepare anti-counterfeiting inks for the reading and writing of fingerprint information.

[0026] The beneficial effects of this invention are:

[0027] (1) This invention introduces a crystallization-driven active polymer self-assembly strategy from the perspective of molecular synthesis and material design, and develops a novel fluorescence coding technology that co-encodes two-dimensional spatial geometric information and fluorescence information. It has great application prospects. Through different modification methods, it can realize the specific distribution and functionalization of various molecules on the polymer platform, while avoiding complex preparation methods and high material costs.

[0028] (2) The two-dimensional polymer fluorescent coding nanosheets provided by the present invention can not only use fluorescent emission signals to encode information, but also realize the spatial distribution of fluorescent information to achieve the purpose of ultra-high throughput coding. Compared with the one-dimensional rod-shaped fluorescent coding technology that has been widely reported, the morphology and pattern of the two-dimensional sheet material itself gives it more accurate and more distinguishable signal reading and writing capabilities. The two-dimensional coding material can realize richer coding methods and further expand the information storage capacity. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is the design concept of the two-dimensional polymer fluorescently encoded nanosheets of the present invention;

[0031] Figure 2 This is a schematic diagram of the preparation route of polymer crystal nuclei in the preparation method of two-dimensional polymer fluorescently encoded nanosheets of the present invention;

[0032] Figure 3 This is a schematic diagram of the two-dimensional polymer fluorescently encoded nanosheets preparation method of the present invention, in which fluorescent groups modify polymers and different fluorescent shells are nested to form two-dimensional sheet-like micelles;

[0033] Figure 4 The 1H NMR spectra of different fluorescently modified PCL polymers prepared in the preparation examples of this invention;

[0034] Figure 5 Gel chromatograms of molecular weight of different fluorescently modified PCL polymers prepared in the preparation examples of this invention;

[0035] Figure 6 The assembly morphology of rod-shaped micelles (a), seed micelles (b), and single-layer two-dimensional sheet micelles (c) prepared according to the seed growth method in this invention is shown under TEM, and the assembly morphology of multi-layer nested sheet micelles (df) is shown under AFM.

[0036] Figure 7 This is a figure showing the seed growth size kinetics results of fluorescently modified PCL. Figure 7 Figure a shows the monitoring results of the morphology and size of the two-dimensional assembly under CLSM by increasing the ratio of polymer / seed micelles (1-90); Figure 7 b represents the linear relationship between the polymer / seed micelle ratio and the length of the two-dimensional sheet assembly; Figure 7 c represents the linear relationship between the polymer / seed micelle ratio and the width of the two-dimensional sheet assembly.

[0037] Figure 8 This is a diagram showing the seed growth kinetics of fluorescently modified PCL. Figure 8 Figure a shows the results of monitoring the morphology and size changes of the two-dimensional assembly at different time periods under CLSM to fix the ratio of polymer / seed micelles. Figure 8 bc is a graph showing the relationship between time and the length of the two-dimensional sheet-like assembly;

[0038] Figure 9 The graph shows the monitoring results of dynamic light scattering on two-dimensional PCL nanosheets at different times.

[0039] Figure 10 The image shows the result of writing the encoded information of a CDSA nanosheet onto a single-layer two-dimensional CDSA nanosheet by adjusting the proportion of dye-labeled PCL solution.

[0040] Figure 11 Example diagram of a programmable four-layer CDSA two-dimensional fluorescent coding system with spatial positioning fluorescence information, where G / R / B are PCLs labeled with fluorescent dyes and W represents PCLs without fluorescent dye labels;

[0041] Figure 12 a shows a 3D reconstruction of a z-scan CLSM encrypted PVP film containing different 4-layer codes (BGRB, BGBR). Figure 12 bc is an image showing the results of reading information from BGRB and BGBR barcodes. Figure 12 The image shows the PVP film with (right) and without (left) four layers of barcodes under ultraviolet light.

[0042] Figure 13 A schematic diagram of the design strategy for a double-layer CDSA anti-counterfeiting film with different coded barcodes: Code A (b) and Code B (c). Figure 13 d shows the z-scan confocal 3D reconstruction results of the encrypted PVP membrane. Figure 13 e is a side view of a double-layered CDSA anti-counterfeiting film with spatially localized distribution. Figure 13 f represents a 3D image of the encrypted PVP film in z-scan CLSM with magnified images in the yz and xz directions;

[0043] Figure 14 This image shows the encoding results of CDSA anti-counterfeiting ink on fingerprint information at different magnifications. Detailed Implementation

[0044] To better explain the present invention, detailed descriptions of its embodiments are provided, and the main content of the invention is further clarified in conjunction with specific examples. However, the content of the present invention is not limited to the following embodiments. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0045] Please see Figure 1 The overall design concept of the two-dimensional polymer fluorescently encoded nanosheets of this invention is as follows: Block polymers with biocompatible polymer chain segments such as PCL as crystalline units are synthesized to construct polymer-based two-dimensional sheet-like assemblies. Controllable growth and assembly in the two-dimensional direction are adjusted using homopolymer and copolymer blending assembly methods. To achieve fluorescent encoding, the precise spatial distribution of the two-dimensional nanomaterials is controlled by the epitaxial growth of polymers modified with different fluorescent groups on the crystalline assemblies. Based on this, the preparation method of the two-dimensional polymer fluorescently encoded nanosheets of this invention mainly includes: First, as shown in the figure... Figure 2 The diagram shows the acquisition of short rod-shaped polycaprolactone seed micelles; secondly, as shown... Figure 3 The diagram shows how polycaprolactone modified with fluorescent groups grows layer by layer along the outer edge of seed micelles to form planar nanosheet structures, thereby enabling the writing of different fluorescent signals in different shells.

[0046] The following examples illustrate in more detail the preparation method of the two-dimensional polymer fluorescently encoded nanosheets of the present invention, and the performance of the prepared two-dimensional polymer fluorescently encoded nanosheets is tested. However, the embodiments of the present invention are not limited to the following examples.

[0047] Preparation Example 1

[0048] Synthesis of PCL homopolymers:

[0049] The α-caprolactone was synthesized via ring-opening polymerization (ROP) in an inert nitrogen glove box. Specifically, diphenyl phosphate (DPP, 20 mg, 0.08 mmol) and the bi-terminal initiator ethyl 2-cyano-5-hydroxypentan-2-ylethylcarbotrithioate (20 mg, 0.08 mmol) were dissolved in dry toluene (2 mL) and transferred to a toluene (3 mL) solution of α-caprolactone (543.3 mg, 4.76 mmol, 70 mL). After stirring at room temperature for 6 hours, the solution was removed from the glove box, precipitated in cold diethyl ether, and centrifuged. Chloroform was then added to dissolve the solid, and the polymer precipitated in cold diethyl ether. This process was repeated three times to obtain a light yellow powder, PCL. 50 .

[0050] Preparation Example 2

[0051] The diblock copolymer PCL- was synthesized using the RAFT polymerization method. b -PDMA:

[0052] PCL 50 900 mg (0.14 mmol), DMA (2.88 g, 29 mmol), and AIBN (2.4 mg, 0.014 mmol) were dissolved in 1,4-dioxane (6 mL) and added to a dry ampoule containing a stir bar. The resulting solution was degassed using at least three freeze-pump-thaw cycles, backfilled with nitrogen, sealed, and placed in a preheated oil bath at 70°C. The solution was heated at 70°C for 2 hours and then quenched by immersion of the ampoule in liquid nitrogen. The polymer was precipitated three times in ice-cold diethyl ether, dried under vacuum, and analyzed to obtain PCL. 50 - b -PDMA 200 .

[0053] Preparation Example 3

[0054] For the preparation of fluorescently labeled polymers, three different carboxyl-functionalized BODIPY dyes (BODIPY-630 / 650, BOPIPY-R6G, and BODIPY-FL) were selected to modify PCL:

[0055] 1. PCL 50 50 mg of BODIPY 630 / 650 (3.3 mg), 4-dimethylaminopyridine (DMAP 0.56 mg) and dicyclohexylcarbodiimide (DCC 17.9 mg) were dissolved in 2 mL of dichloromethane and stirred for 24 hours; the reaction mixture was precipitated three times in cold MeOH to obtain pure terminally functionalized fluorescently labeled polymer PCL-R.

[0056] 2. PCL50 (50 mg), BODIPY-R6G (3.3 mg), 4-dimethylaminopyridine (DMAP 0.56 mg), and dicyclohexylcarbodiimide (DCC 17.9 mg) were dissolved in 2 mL of dichloromethane and stirred for 24 hours. The reaction mixture was precipitated three times in cold MeOH to obtain pure terminally functionalized fluorescently labeled polymer PCL-G.

[0057] 3. PCL 50 50 mg of BODIPY-FL (3.3 mg), 4-dimethylaminopyridine (DMAP 0.56 mg), and dicyclohexylcarbodiimide (DCC 17.9 mg) were dissolved in 2 mL of dichloromethane and stirred for 24 hours. The reaction mixture was precipitated three times in cold MeOH to obtain pure terminally functionalized fluorescently labeled polymer PCL-B.

[0058] Preparation Example 4

[0059] Preparation of seed micelles:

[0060] PCL 50 - b -PDMA 200 (5 mg) dissolved in 1 mL of ethanol (5.0 mg / mL) -1 Then, the solution was heated at 70°C for 3 hours. The solution was slowly cooled to room temperature and further aged for 5 days to prepare crystalline polymer nanowires. The obtained crystalline nanowires were ultrasonically treated in an ice bath for 20 minutes (10 cycles, 2 minutes of ultrasonic treatment per cycle, with a 20-minute interval to cool the solution temperature) to obtain seed micelles.

[0061] Preparation Example 5

[0062] Preparation of unfluorescently modified PCL epitaxial polymer solution:

[0063] The prepared unfluorescently labeled PCL 50 and PCL 50 - b -PDMA 200 A PCL blend solution was prepared by mixing the components in THF (10 mg) at a weight ratio of 1:1, and denoted as PCL-W epitaxial polymer solution.

[0064] Preparation Example 6

[0065] Preparation of two-dimensional polymer nanosheets:

[0066] Unfluorescently modified PCL epitaxial polymer solution was gradually added to the seed micelle solution prepared above. After addition, the solution was shaken for 5 seconds. The unmodified PCL... 50The mass ratio of the added material to the seed crystals was 20:1. After shaking for 5 seconds, the addition of the unfluorescently modified PCL epitaxial polymer solution was repeated 2, 3, and 4 times to obtain two-dimensional polymer nanosheets with 2, 3, and 4 epitaxial polymer layers, respectively.

[0067] Preparation Example 7

[0068] Preparation of fluorescently modified PCL epitaxial polymer solutions:

[0069] The prepared red fluorescently labeled polymers PCL-R and PCL 50 - b -PDMA 200 A PCL blend solution was prepared by mixing the PCL in THF (10 mg) at a weight ratio of 1:1, denoted as PCL-R epitaxial polymer solution.

[0070] The prepared green fluorescent labeling polymers PCL-G and PCL 50 - b -PDMA 200 A PCL blend solution was prepared by mixing the PCL in THF (10 mg) at a weight ratio of 1:1, and denoted as PCL-G epitaxial polymer solution.

[0071] The prepared blue fluorescently labeled polymers PCL-G and PCL 50 - b -PDMA 200 A PCL blend solution was prepared by mixing the PCL in THF (10 mg) at a weight ratio of 1:1, and denoted as PCL-B epitaxial polymer solution.

[0072] Performance Testing

[0073] 1. The PCL homopolymers and PCL polymers with different fluorescence modifications prepared in the above preparation examples were characterized by nuclear magnetic resonance (NMR), and the results are as follows: Figure 4 As shown, this result demonstrates the successful preparation of the PCL polymer and the successful modification of it with fluorescent groups.

[0074] 2. The PCL homopolymers and PCL polymers with different fluorescence modifications prepared in the above preparation examples were characterized by gel permeation chromatography (SEC), and the results are as follows: Figure 5 As shown, this result demonstrates the successful preparation of the PCL polymer and the achievement of fluorescent group modification.

[0075] 3. The polymer nanowires and nanocrystal seed precursors prepared in Preparation Example 4 were characterized by transmission electron microscopy (TEM), and the two-dimensional polymer nanosheets prepared in Preparation Example 6 were characterized by TEM and atomic force microscopy (AFM). The results are as follows: Figure 6As shown in the TEM image, rod-shaped crystalline nanowires and short rod-shaped crystalline seeds were successfully prepared. Figure 6 TEM and atomic force microscopy (AFM) images confirmed that the prepared two-dimensional polymer nanosheets were monolayer sheet structures. Figure 6 The atomic force microscopy (AFM) images show that by controlling the number of times the epitaxial polymer solution is added, two-dimensional polymer nanosheets with different numbers of epitaxial polymer layers can be successfully prepared.

[0076] Example 1

[0077] Preparation of two-dimensional polymer fluorescently encoded nanosheets with monolayer epitaxial polymer layers of different sizes:

[0078] Adding a green fluorescent modified PCL epitaxial polymer solution and a PCL-G epitaxial polymer solution to a seed micelle solution alters the PCL content. 50 Two-dimensional polymer fluorescently encoded nanosheets with monolayer epitaxial polymer layers of different sizes were prepared by adding seed crystals (0.01 mg / mL) at a mass ratio of 10-30:1.

[0079] Example 2

[0080] Preparation of two-dimensional polymer fluorescently encoded nanosheets:

[0081] PCL-G epitaxial polymer solution (PCL-G 0.2 mg / mL) was rapidly added to the seed micelle solution (0.01 mg / mL), and after shaking for 5 seconds, two-dimensional polymer fluorescently encoded nanosheets, CDSA polymer sheets, were obtained.

[0082] Example 3

[0083] Preparation of two-dimensional polymer fluorescently encoded nanosheets with monolayer epitaxial polymer layers of different fluorescence colors:

[0084] Fluorescently modified PCL epitaxial polymer solution was added to the seed micelle solution (0.01 mg / mL). PCL epitaxial polymer solutions containing different fluorescently modified PCL or combinations of different fluorescently modified PCL were used. Specifically, the addition ratios of PCL-R, PCL-G, PCL-B and seed crystals were changed according to Table 1 below. The solution was shaken for 5 seconds after each addition to obtain two-dimensional polymer fluorescently encoded nanosheets with monolayer epitaxial polymer rings of different fluorescent colors.

[0085] Table 1. Addition ratio of PCL-R, PCL-G, PCL-B and seed crystals

[0086] serial number PCL-R PCL-G PCL-B 1 0 0 1 2 0 1 0 3 1 0 0 4 1 1 1 5 1 2 1 6 1 1 2 7 2 1 1

[0087] Example 4

[0088] Preparation of multilayer two-dimensional polymer fluorescently encoded nanosheets with different information encoding:

[0089] PCL epitaxial polymer solution was added sequentially to a seed micelle solution (0.01 mg / mL). The order of addition of epitaxial polymer solutions labeled with different dyes was varied, while maintaining a seed-to-PCL ratio of 1:20 for each addition. This yielded multilayer two-dimensional polymer fluorescently encoded nanosheets with different information encodings. The specific order of addition of the epitaxial polymer solution included:

[0090] 1. PCL→PCL-G→PCL-R→PCL-B

[0091] 2. PCL→PCL-G→PCL-B→PCL-R

[0092] 3. PCL-G → PCL-R + PCL-B

[0093] 4. PCL→PCL-G+PCL-R+PCL-B

[0094] 5. PCL-B → PCL-G + PCL-R

[0095] 6. PCL-B→PCL-G→PCL-R→ PCL-R

[0096] Example 5

[0097] Preparation of CDSA anti-counterfeiting film:

[0098] PVP (5000 Da) was dissolved in ethanol and then sonicated to obtain a clear PVP solution. Two-dimensional nanosheets (BGRB, BGBR) (0.1 mg / mL) from Example 4 were added to the PVP solution, and a film was formed on a glass slide and dried overnight to prepare a CDSA anti-counterfeiting film.

[0099] Example 6

[0100] Preparation of double-layer CDSA anti-counterfeiting film:

[0101] PVP (5000 Da) was dissolved in ethanol and then sonicated to obtain a clear PVP solution. Two-dimensional nanosheets (BGRB, BGBR) (0.1 mg / mL) from Example 4 were added to the PVP solution. A film was then formed on a glass slide and dried overnight. Using the same procedure, a double-layer CDSA anti-counterfeiting film was prepared by spin-coating another CDSA two-dimensional encoded PVP solution on top of the formed film.

[0102] Example 7

[0103] Preparation of CDSA-coded anti-counterfeiting ink:

[0104] PVP (5000 Da) was dissolved in ethanol and then sonicated to obtain a clear PVP solution. Two-dimensional nanosheets (BGRB, BGBR) (0.1 mg / mL) from Example 4 were added to the PVP solution to prepare CDSA-coded anti-counterfeiting ink.

[0105] Performance Testing

[0106] 4. Two-dimensional polymer fluorescently encoded nanosheets of different sizes with monolayer epitaxial polymer layers prepared in Example 1 were characterized by confocal laser scanning (CLSM), and the results are as follows: Figure 7 As shown, by Figure 7 It can be seen that gradually increasing the amount of epitaxial polymer added can linearly adjust the thickness of the epitaxial polymer layer and the size of the two-dimensional polymer nanosheets.

[0107] 5. The two-dimensional polymer fluorescently encoded nanosheets prepared in Example 2 were characterized by CLSM at different times, and the results are as follows: Figure 8 As shown, the size and morphology of polymer nanosheets can remain stable over a long period of time.

[0108] 6. Dynamic light scattering test

[0109] Dynamic light scattering monitoring was performed on the two-dimensional polymer fluorescently encoded nanosheets prepared in Example 2 at different times. The results are as follows: Figure 9 As shown, the results indicate that the two-dimensional nanosheets remain stable at different time intervals.

[0110] 7. Laser confocal microscopy (CLSM) testing

[0111] (1) Fluorescence emission coding

[0112] In Example 3 of this invention, PCL (PCL-R, PCL-B, PCL-G) labeled with different fluorescent dyes were premixed in different molar ratios (e.g., GBR: 1:1:1, 2:1:1, 1:2:1, 1:1:2). The premixed solutions were then added to a seed solution to prepare two-dimensional polymer nanosheets with different fluorescence emissions. Decoding was performed using a CLSM to obtain various fluorescent codes, as shown in the results. Figure 10 As shown.

[0113] from Figure 10As can be seen, after treatment with premixed solutions of GBR: 1:1:1, 2:1:1, 1:2:1, and 1:1:2, Example 3 used three primary color (PCL-R, PCL-G, and PCL-B) encoding plates and premixed primary colors in various proportions to write fluorescent information onto nanosheets, resulting in four types of multi-emission fluorescent codes and three types of single-emission fluorescent codes. This demonstrates the feasibility of using solution premixing to achieve multiple fluorescent emission codes.

[0114] (2) Geometric fluorescent coding

[0115] In Example 4 of this invention, PCLs labeled with different fluorescent dyes (PCL-R, PCL-B, PCL-G) were sequentially encoded in different orders and added to a seed solution to prepare two-dimensional polymer nanosheets with various fluorescent encoding modes. Decoding was performed using CLSM to obtain multiple fluorescent codes, as shown in the results. Figure 11 As shown.

[0116] Results Analysis: Epitaxial growth of fluorescent dyes on two-dimensional sheets can be achieved by adding a corresponding PCL mixed solution in the presence of CDSA seed. Figure 11 As can be seen, green / red / blue / white (G / R / B / W) is encoded in different sequences on each layer of the two-dimensional nanosheet. For example, -B / G / R and R / B / G are used as examples of encoding. The color information can be decoded using CLSM, and the diagram clearly shows the color pattern of each layer. Furthermore, this strategy can also utilize both fluorescence emission and geometric encoding for composite encoding to achieve a wider encoding range. For example, mixed color patterns are encoded in a three-layer CDSA sheet, with two patterns as demonstrations: G(R+B), W(G+R+B), and G(R+B)(G+B). By changing the order of polymer dye addition, the encoding pattern can be further extended to a four-layer CDSA polymer nanosheet. In this case, seven different color patterns can generate 343 (7*7*7) different color codes for a three-layer CDSA sheet and 2401 (7*7*7*7) different color codes for a four-layer sheet.

[0117] 8. In-situ analysis of the CDSA anti-counterfeiting film prepared in Example 5 was performed by layer scanning on a CLSM, and the results are as follows: Figure 12 As shown.

[0118] Results analysis: From Figure 12 As can be seen, due to the special two-dimensional pattern, the information of different layers of two-dimensional encoding is clearly identified without affecting the aggregation or overlap between barcodes.

[0119] 9. In-situ analysis of the double-layer CDSA anti-counterfeiting film prepared in Example 6 was performed by layer scanning on a CLSM, and the results are as follows: Figure 13As shown.

[0120] The results show that the design strategy of double-layer CDSA anti-counterfeiting film with different coded barcodes was successfully implemented: coded A and coded B ( Figure 13 The encrypted PVP membrane was scanned using z-scan confocal 3D reconstruction. Figure 13 e indicates that the double-layer coding film has a spatial double-layer local distribution.

[0121] 10. The CDSA-coded anti-counterfeiting ink prepared in Example 7 was pressed onto a glass slide to create fingerprint patterns. The fingerprint information was then decoded using a CLSM at different magnifications on the glass slide. The results are as follows: Figure 14 As shown.

[0122] The results show that: Figure 14 This demonstrates that the 2D image of the encoded fingerprint is magnified by CLSM, and information can be clearly extracted in different channels. The extracted 2D image information is consistent with the written information, and the accuracy of the output is not affected by the aggregation or overlap of barcodes.

Claims

1. A two-dimensional polymer fluorescently encoded nanosheet, characterized in that, The structure includes a polymer nucleus and one or more epitaxial polymer rings surrounding the outer edge of the polymer nucleus. The multiple epitaxial polymer rings are arranged sequentially from the inside to the outside. The epitaxial polymer rings are formed by planar crystallization growth of the polymer nucleus through crystallization-driven self-assembly. The polymer nucleus and the epitaxial polymer rings together constitute a nanosheet structure. At least one of the epitaxial polymer rings is a fluorescent polymer layer. The fluorescent polymer layer contains a polymer modified with a fluorescent group and has fluorescent information by introducing the fluorescent group-modified polymer.

2. The two-dimensional polymer fluorescently encoded nanosheet according to claim 1, characterized in that, Each fluorescent polymer layer comprises a polymer modified with one color fluorescent group, or a mixture of polymers modified with multiple colors fluorescent groups; the fluorescent groups include red fluorescent groups, green fluorescent groups, and blue fluorescent groups.

3. The two-dimensional polymer fluorescently encoded nanosheet according to claim 1, characterized in that, The materials of the polymer nucleus and the epitaxial polymer layer are selected from one or more of polycaprolactone polymers and polylactic acid polymers.

4. The two-dimensional polymer fluorescently encoded nanosheet according to claim 1, characterized in that, The polymer nucleus is made of PCL block copolymer, and the epitaxial polymer layer is made of a mixture of PCL homopolymer and PCL block copolymer.

5. The two-dimensional polymer fluorescently encoded nanosheet according to claim 4, characterized in that, The PCL block copolymer is a PCL-b-PDMA block copolymer.

6. A method for preparing two-dimensional polymer fluorescently encoded nanosheets as described in claim 1, characterized in that, include: Prepare fluorescently modified polymers; obtain polymer nanorods through crystallization-driven self-assembly; The polymer nanorods are broken to obtain short rod-shaped polymer nuclei; through crystallization-driven self-assembly, the epitaxial polymer is extended and crystallized around the polymer nuclei in a two-dimensional direction to form an epitaxial polymer layer, resulting in a crystalline organic polymer nanosheet with a nanosheet structure; at least one of the polymer nuclei and all epitaxial polymer layers is a fluorescent polymer layer, and the polymer used in the preparation of the fluorescent polymer layer includes the polymer modified with the fluorescent group.

7. The method for preparing two-dimensional polymer fluorescently encoded nanosheets according to claim 6, characterized in that, Specifically, the steps include the following: Step 1, Preparation of polymer substrate: A bifunctional initiator with trithioester and hydroxyl structures is synthesized. The hydroxyl end of the bifunctional initiator is used to initiate the ring-opening polymerization of cyclohexanolide to synthesize a PCL homopolymer with trithioester end groups. From the PCL homopolymer, a PCL block copolymer with PCL crystalline segments is prepared to obtain a polymer substrate including PCL homopolymer and PCL block copolymer. Step 2, Fluorescent Functionalization of Polymer Substrate: A fluorescent dye with carboxyl groups is provided, and the fluorescent dye is coupled with at least one of the PCL homopolymer and PCL block copolymer prepared in Step 1 through an esterification reaction to modify the polymer substrate with fluorescent groups, thereby obtaining a fluorescently modified polymer. The fluorescently modified polymer includes at least one of the fluorescently modified PCL homopolymer and fluorescently modified PCL block copolymer. Step 3, Preparation of polymer nuclei: The PCL block copolymer is driven to nucleate and crystallize in an ethanol solution by a thermodynamic method of heating and cooling to form polymer nanorods with rod-shaped micelles. The synthesized polymer nanorods are broken by ultrasonication to obtain polymer nuclei of short rod-shaped seed micelles with a size of less than 50 nm. Step 4: Preparation of two-dimensional polymer sheets by seed growth method: An epitaxial polymer solution is added once or multiple times to the polymer nucleus solution prepared in step 3. The epitaxial polymer is a PCL block copolymer or a mixture of PCL block copolymer and PCL homopolymer. Each time the epitaxial polymer is added, it extends and crystallizes along the outer edge of the polymer nucleus to form an epitaxial polymer layer. At least one of the polymer nucleus and all epitaxial polymer layers is a fluorescent polymer layer. The polymer used in the preparation of the fluorescent polymer layer includes the polymer modified with the fluorescent group, thus obtaining two-dimensional polymer fluorescently encoded nanosheets.

8. The method for preparing two-dimensional polymer fluorescently encoded nanosheets according to claim 7, characterized in that, The initiator used in step 1 is 2-cyano-5-pentanol-2-ethyl trithioester; the PCL homopolymer is subjected to RAFT polymerization with N,N-dimethylacrylamide to obtain a PCL block copolymer, which is a PCL-b-PDMA block copolymer.

9. The method for preparing two-dimensional polymer fluorescently encoded nanosheets according to claim 7, characterized in that, The fluorescent dye used in step 2 is a carboxyl-functionalized BODIPY dye.

10. The method for preparing two-dimensional polymer fluorescently encoded nanosheets according to claim 9, characterized in that, The carboxyl-functionalized BODIPY dye includes at least one of BODIPY-630 / 650, BODIPY-R6G, and BODIPY-FITC.

11. The application of the two-dimensional polymer fluorescent coded nanosheets as described in any one of claims 1-5 or the two-dimensional polymer fluorescent coded nanosheets prepared by the preparation method as described in any one of claims 6-10 in the preparation of anti-counterfeiting materials.

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