DNA-modified π-conjugated fluorescent nanoparticle-based protein detection imaging method capable of signal rapid amplification and high throughput

Abstract

The present disclosure relates to an azide-modified fluorescent π-conjugated polymer and a DNA-modified π-conjugated fluorescent nanoparticle-based protein detection imaging method capable of signal rapid amplification and high throughput. The present disclosure prepares oligonucleotide-modified fluorescent π-conjugated polymers (OCPNs) based on the azide-modified fluorescent π-conjugated polymer. The OCPNs have ultrahigh fluorescence luminance and extremely high detection sensitivity, and can complete detection and imaging of targets in different abundances without the need of an additional fluorescence signal amplification step. In addition, a rapid and specific targeting effect can be achieved with an extremely small dosage and an entire staining and imaging process can be shortened, so that the OCPNs achieve an extremely high imaging efficiency during multi-color imaging and thus exhibit great application prospects.

Description

A method for rapid signal amplification and high-throughput protein detection imaging based on DNA-modified π-conjugated fluorescent nanoparticles

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2024105528370, filed on May 7, 2024, entitled "A method for rapid signal amplification and high-throughput protein detection imaging based on DNA-modified π-conjugated fluorescent nanoparticles", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of biomaterials technology, specifically relating to a method for rapid signal amplification and high-throughput protein detection imaging based on DNA-modified π-conjugated fluorescent nanoparticles. Background Technology

[0004] Proteins, as essential components of cells, tissues, and organs, provide crucial information for the detection, diagnosis, and treatment of many major diseases through their expression levels and systematic distribution. Current bioscience research shows that the onset and exacerbation of specific diseases are often caused by the abnormal expression of multiple proteins. These proteins exhibit varying abundances and systematic distributions within cells, making the development of efficient, convenient, highly specific, and highly sensitive detection methods for multiple biological targets an urgent priority.

[0005] Among the many detection methods currently under development, immunofluorescence imaging (IF) technology, based on the specific binding of fluorescent signals to antibody antigens, has received widespread attention and development. By combining antibodies with multiple fluorescent probes with different fluorescence, the fluorescent probes can be targeted to multiple different antigens to generate corresponding fluorescent signals, thus achieving rapid detection of multiple specific targets. However, as the number of targets to be detected increases, and due to the limitations of the fluorescence spectrum wavelength range that current detection equipment can collect, the optical path overlap caused by using multiple fluorescent probes gradually worsens, limiting the total number of effective detection targets to 3 to 5. Since the abundance of the targets to be detected may vary greatly, the total fluorescence intensity generated by the fluorescent probe at targets with lower abundance is lower, thus being severely interfered with by background signals, leading to the complete neglect of the target signal or the generation of false "positive" signals.

[0006] To address the issue of optical path congestion in fluorescent probes, and assuming no further advancements in detection equipment, an experimental workflow based on "removable fluorescence" can enable the detection of more targets. When several fluorescent probes with non-overlapping optical paths are available, the fluorescence signal in the sample can be "removed" using a specific method. This allows for repeated fluorescence imaging of new targets using several probes of the same type, theoretically enabling the detection of an unlimited number of targets through multiple imaging cycles. Nucleic acid materials (DNA), due to their rich biological functionality, molecular-scale programmability and modifiability, and the specificity resulting from the strict Watson-Crick base pairing principle, are ideal functional materials for this purpose. By controlling the nucleic acid chain length and base sequence, numerous non-interfering orthogonal oligonucleotides (ONs, a general term for short-chain nucleic acids) can be easily designed and synthesized. Fluorescent probes prepared by conjugating ONs with fluorescent materials (ONs fluorescent probes) can recognize antibodies modified with corresponding complementary ONs. The complementary double-stranded ONs can be rapidly eluted by DNA strand substitution technology or by soaking in 75% formamide solution to remove hybridization, thus achieving efficient removal of fluorescence signals in the sample. Then, ONs fluorescent probes with different sequences can be introduced to complete fluorescence imaging of many targets.

[0007] Current fluorescent probes used in multi-target immunofluorescence imaging are limited by the trade-off between fluorescence intensity and imaging efficiency, hindering their application in broader biosensing and detection. Due to the complex, near-porous structure within cells, the sensitivity and specificity of fluorescent probes are severely affected by their size. For example, large-sized fluorescent probes loaded with a large number of fluorescent molecules, as solid-phase micelles, naturally tend to adsorb onto the denatured hydrophobic domains of various proteins within cells. The complex intracellular structure also limits the free diffusion of these probes, leading to severe non-specific binding and affecting imaging specificity and sensitivity.

[0008] When the size of fluorescent probes is limited, the total number of fluorophores contained in a single probe is also limited, resulting in lower brightness of individual probes and affecting the spatiotemporal resolution and sensitivity of fluorescence imaging. Therefore, additional fluorescence amplification steps are required to encourage the accumulation of multiple fluorescent probes on the same target, further increasing the fluorescence brightness from the target. However, such amplification steps often require the post-assembly of complex nanostructures within the cell, leading to reduced imaging efficiency and increased elution difficulty. Only when the fluorescent probe possesses sufficient fluorescence brightness can the complex amplification steps be omitted while ensuring the specificity and sensitivity of the imaging. Therefore, exploring the fabrication of small-sized fluorescent probes with extremely high fluorescence brightness holds great potential for achieving efficient multi-target imaging. π-conjugated polymers (CPs), as organic materials, can be used to prepare organic light-emitting polymers with sufficient photostability and diverse properties by adjusting their molecular structure and aggregation state. Fluorescent nanoparticles prepared using fluorescent π-conjugated polymers (FCPs) exhibit higher fluorescence brightness than quantum dots, providing fundamental optical properties for achieving sensitive fluorescence detection.

[0009] SAKA SK, WANG Y, KISHI JY, et al. Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues[J]. Nature Biotechnology, 2019, 37(9): 1080-1090. This paper presents an immunostaining scheme using a primer exchange reaction (PER) to amplify the signal. This scheme prepares a series of DNA-encoded antibodies by coupling various designed ONs with corresponding antibodies. Orthogonal DNA multiplyes with one end recognizing specific DNA-encoded antibodies are then prepared using PER. Simultaneously, ONs modified with small-molecule fluorescent dyes are prepared. These three components are combined to achieve fluorescent imaging of the target. However, this scheme uses small-molecule fluorescent dyes to modify ONs, which has disadvantages such as poor photostability, the need for different signal amplification methods for targets with different abundances, and high cost. Furthermore, the large amount of small-molecule dye used can easily generate significant background signals, leading to false positives.

[0010] LIU X, MAO D, SONG Y, et al. Computer-aided design of reversible hybridization chain reaction (CAD-HCR) enables multiplexed single-cell spatial proteomics imaging[J]. Science Advances, 2022, 8(2): eabk0133. This paper presents a computer-aided design scheme for reversible hybridization chain reaction (CAD-HCR). The scheme first labels the target by adding multiple DNA-encoded antibodies to the sample, then adds three groups of hairpin-shaped ONs with different fluorescent dyes. After binding with the corresponding initiating chains, chain assemblies are amplified at the target, achieving fluorescence amplification imaging of the target. Currently, this scheme can simultaneously image three targets using three different fluorescently emitting dyes in a single imaging cycle. However, this scheme has disadvantages such as low imaging efficiency, long operation time required for fluorescence removal, and stringent conditions for DNA sequence design and preparation. Furthermore, the imaging process requires the assembly of nanostructures at the target, necessitating a certain self-assembly time, which results in a longer detection time.

[0011] CN112552490B provides a method for detecting target nucleic acids with high sensitivity and specificity using nucleic acid probes, which are obtained by hybridizing nucleic acid assemblies with dye-modified DNA single-stranded S2. However, this method cannot simultaneously detect multiple different target nucleic acids, resulting in low imaging efficiency.

[0012] Therefore, there is an urgent need to find a technical solution that can overcome the shortcomings of existing technologies. Summary of the Invention

[0013] Based on this, this disclosure combines the unique biorecognition and coding properties of ONs with the excellent optical properties of π-conjugated polymers. By designing and controlling the molecular structure of FCP polymer chains to adjust the fluorescence emission wavelength range, various FCP polymers with different fluorescence emission bands and without severe optical path overlap are prepared. Subsequently, ONs are modified onto FCP and self-assembled by solvent substitution to prepare ONs-modified fluorescent π-conjugated polymer nanoparticles (OCPNs) with different fluorescence emission bands. OCPNs have extremely high fluorescence brightness and adjustable particle size (10-100 nm), enabling multi-target immunofluorescence imaging with high specificity, high sensitivity, and high elution efficiency without additional fluorescence amplification steps.

[0014] One object of this disclosure is to provide an azide-modified fluorescent π-conjugated polymer having the following general structural formula:

[0015] Wherein, R4 is independently selected from C1-C30 alkyl groups with straight or branched structures;

[0016] The Ar unit is selected from aromatic rings, aromatic heterocycles, aromatic ring derivatives, and aromatic heterocycle derivatives;

[0017] n is a natural number.

[0018] Furthermore, the Ar unit is selected from any of the following structures:

[0019] R2 and R3 are each independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

[0020] Furthermore, n is selected from 5-15.

[0021] Optionally, n is selected from 7-9.

[0022] Another objective of this disclosure is to provide a method for preparing an azide-modified fluorescent π-conjugated polymer, comprising the following steps:

[0023] S1. Mix monomer M, monomer N, catalyst, base and solvent, and heat and stir the reaction under inert gas protection. After purification, a fluorene precursor containing bromoalkyl is obtained.

[0024] S2. The fluorene precursor containing bromoalkyl groups was mixed with NaN3 and solvent and stirred to react. After purification, the product was obtained.

[0025] The monomer M has the following general structural formula:

[0026] X1 is independently selected from halogen atoms;

[0027] The X2 is independently selected from one of the following: halogen atoms and pinacol borate ester;

[0028] R1 is independently selected from C1-C30 alkyl groups with straight or branched structures;

[0029] The monomer N is selected from one of the following structures:

[0030] R2 and R3 are independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

[0031] Further, in step S1, the heating temperature is 80-100℃ and the reaction time is 1-50h; in step S2, the reaction time is 10-20h.

[0032] Another object of this disclosure is to provide the application of the above-described azide-modified fluorescent π-conjugated polymer in the preparation of oligonucleotide-modified fluorescent π-conjugated polymers.

[0033] Another object of this disclosure is to provide an oligonucleotide-modified fluorescent π-conjugated polymer with the following general structural formula:

[0034] The R4 is independently selected from C1-C30 alkyl groups with straight or branched structures;

[0035] The Ar unit is selected from aromatic rings, aromatic heterocycles, aromatic ring derivatives, and aromatic heterocycle derivatives;

[0036] The P is an oligonucleotide;

[0037] The value of x is selected from 0.1 to 0.6.

[0038] The term "ran" represents random combination, indicating that the entire polymer chain is composed of x units with DNA and 1-x units without ONs in a random order. The grafting of ONs does not change the polymer structure or degree of polymerization; it only involves some units having ONs attached, but the positions are completely random.

[0039] Furthermore, the Ar unit is selected from any of the following structures:

[0040] R2 and R3 are each independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

[0041] Another object of this disclosure is to provide a method for preparing oligonucleotide-modified fluorescent π-conjugated polymers, comprising the following steps:

[0042] L1. Preparation of CPG-modified oligonucleotides via phosphoramide chemical synthesis;

[0043] L2. Using butyn-phosphoramide as the 5' end modification unit, an alkyne group was introduced into the CPG-modified oligonucleotide chain to obtain alkylated CPG-modified oligonucleotides.

[0044] L3. The azide-modified fluorescent π-conjugated polymer was coupled with the alkyne-modified CPG-modified oligonucleotide by click chemistry, and then the CPG was removed by ammonolysis to obtain the oligonucleotide-grafted fluorescent π-conjugated polymer.

[0045] L4. The oligonucleotide-grafted fluorescent π-conjugated polymer was transferred to the aqueous phase by solvent displacement to form an oligonucleotide-modified fluorescent π-conjugated polymer.

[0046] Further, in step L4, the self-assembly is performed by adding the oligonucleotide-grafted fluorescent π-conjugated polymer to a 0.05-2M buffer solution to form an oligonucleotide-modified fluorescent π-conjugated polymer.

[0047] Another objective of this disclosure is to provide the application of the above-mentioned oligonucleotide-modified fluorescent π-conjugated polymer in immunofluorescence imaging detection.

[0048] Another objective of this disclosure is to provide the application of the above-mentioned oligonucleotide-modified fluorescent π-conjugated polymer in imaging multiple molecular spatial distributions within a single cell.

[0049] Furthermore, the application includes: using the above-mentioned oligonucleotide-modified fluorescent π-conjugated polymer to detect the spatial distribution and relative positions of various molecules of different abundances in single cells.

[0050] Another object of this disclosure is to provide a product for single-cell spatial in situ multiplex molecular imaging detection, comprising the above-described oligonucleotide-modified fluorescent π-conjugated polymer.

[0051] Another objective of this disclosure is to provide a method for rapid signal amplification and high-throughput protein detection imaging based on DNA-modified π-conjugated fluorescent nanoparticles, including the aforementioned oligonucleotide-modified fluorescent π-conjugated polymer.

[0052] This disclosure has the following beneficial effects: the OCPNs prepared by this disclosure have ultra-high fluorescence brightness (molar extinction coefficient reaches 107 M). -1 cm -1With a quantum yield of 11-90%, these OCPNs possess extremely high detection sensitivity, enabling the detection and imaging of targets of varying abundances without the need for additional fluorescence signal amplification. Simultaneously, the dense DNA shell of OCPNs provides strong hybridization-driven capabilities, allowing for faster target targeting. Combined with their ultra-high fluorescence brightness, this enables rapid and specific targeting with minimal dosage, generating sufficient fluorescence signals while maintaining a significant signal-to-noise ratio, thus shortening the entire staining and imaging process to as little as 2 minutes. Furthermore, by adding a substitution strand, OCPNs can be replaced in situ for rapid removal of the fluorescence signal, a process that can be completed within 30 seconds. Therefore, a single imaging cycle can be completed in as little as 2.5 minutes. When using three types of OCPNs for multicolor imaging, imaging of nine different proteins can be completed in three cycles, with a total time not exceeding one hour. This imaging efficiency is the fastest known to date.

[0053] This disclosure also optimizes the self-assembly process of OCPNs. The oligonucleotide-grafted fluorescent π-conjugated polymer (ONP) obtained after CPG removal using ammonia is first dried in a slow argon flow, allowing it to exist as a monodisperse brush-like polymer molecule in a solid form before self-assembly. Then, an aqueous solution (10×PBS) with a high ion concentration is added to dissolve it, causing it to self-assemble into nanoparticles. The particle size of the self-assembled nanoparticles is determined solely by the grafting rate of the oligonucleotide-grafted fluorescent π-conjugated polymer (ONP) formed through click chemistry coupling, and is independent of the subsequent solution treatment. Compared to existing technologies, this self-assembly process eliminates the need to control the solution drop rate, add additional solution components, or control the solution volume, while still yielding an aqueous solution of nanoparticles with uniform particle size. Attached Figure Description

[0054] Figure 1 illustrates the synthesis and modification process of ONs;

[0055] in,

[0056] Figure 1(a) shows a schematic diagram of the chemical reaction process for solid-phase synthesis of ONs based on phosphorous amide chemical synthesis method;

[0057] Figure 1(b) shows a schematic diagram of the process of using butyne-phosphoramide as the 5' end modification unit and coupling it to the ONs sequence;

[0058] Figure 2 illustrates the process of self-assembly to form OCPNs;

[0059] in,

[0060] Figure 2(a) illustrates the process of directly coupling azide-modified fluorescent π-conjugated polymers with alkyne-modified CPG-modified oligonucleotides (CPG-ONs) via click chemistry to obtain oligonucleotide-grafted fluorescent π-conjugated polymers (FCP-g-ONs).

[0061] Figure 2(b) illustrates the process of transferring FCP-g-ONs into an aqueous phase via solvent displacement to form OCPNs through self-assembly.

[0062] Figure 3 shows the performance test results of OCPNs;

[0063] in,

[0064] Figure 3(a) shows the UV-Vis absorption spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0065] Figure 3(b) shows the agarose gel electrophoresis results of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0066] Figure 3(c) shows the fluorescence spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs under excitation light of 370 nm, 448 nm and 515 nm, respectively;

[0067] Figure 3(d) shows the particle size distribution of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs as detected by DLS particle size analysis;

[0068] Figure 3(e) shows transmission electron microscopy (TEM) images of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0069] Figure 4 shows the UV-Vis absorption spectra of PFBT-OCPNs with different particle sizes, the hydration particle size results, the fitting curve of ONs grafting rate versus CPG-ONs dosage, and the fitting curve of hydration particle size versus CPG-ONs dosage.

[0070] in,

[0071] Figure 4(a) shows the UV-Vis absorption spectra of PFBT-OCPNs with different particle sizes;

[0072] Figure 4(b) shows the hydration particle size of PFBT-OCPNs with different particle sizes detected by DLS particle size analyzer;

[0073] Figure 4(c) shows the fitting curves of ON grafting rate and CPG-ONs dosage for PFBT-OCPNs with different particle sizes;

[0074] Figure 4(d) shows the fitting curves of the hydration particle size of PFBT-OCPNs with different particle sizes and the amount of CPG-ONs used;

[0075] Figure 5 shows the absolute fluorescence quantum yield detection spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs at 370 nm, 445 nm and 488 nm, respectively, using a photoluminescence spectrophotometer (FLS1000).

[0076] Figure 6 shows the results detected using PAGE gel-paired antibodies, DNA-conjugated antibodies, DNA-conjugated antibodies + complementary DNA, and DNA-conjugated antibodies + OCPNs.

[0077] Figure 7 shows fluorescence images of Lamin A / C, Ki67, α-tubulin, Calnexin, GM130, and mitochondria in HeLa cells after being stained with PFBT-OCPNs grafted with different ONs.

[0078] Figure 8 shows a schematic diagram comparing the photostability of AF488 and PFBT-OCPNs, as well as a comparison of the fluorescence signals of different parts of cells labeled with AF488 and PFBT-OCPNs under continuous laser excitation for 3 minutes.

[0079] in,

[0080] Figure 8(a) shows a schematic diagram comparing the photostability of AF488 and PFBT-OCPNs;

[0081] Figure 8(b) shows a comparison of fluorescence signals of AF488-labeled Lamin A / C and PFBT-OCPNs-labeled α-tubulin under continuous laser excitation for 3 minutes.

[0082] Figure 8(c) shows a comparison of fluorescence signals of AF488-labeled α-tubulin and PFBT-OCPNs-labeled Lamin A / C under continuous laser excitation for 3 min;

[0083] Figure 9 shows fluorescence images of Mitochondria, Lamin A / C, and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody, and AF488-modified ONs, respectively, as well as fluorescence intensity bar charts plotted after quantitative statistical analysis of fluorescence intensity for each cell using ImageJ software.

[0084] in,

[0085] Figure 9(a) shows fluorescence images of Mitochondria, Lamin A / C and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody and AF488-modified ONs, respectively;

[0086] Figure 9(b) shows a fluorescence intensity bar chart plotted after quantitative statistical analysis of the fluorescence intensity of each cell using ImageJ software based on the image in Figure 9(a);

[0087] Figure 10 shows a schematic diagram and operational example of signal removal by in-situ chain replacement;

[0088] Figure 11 shows fluorescence images obtained through rapid imaging and elution using OCPNs;

[0089] in,

[0090] Figure 11(a) shows fluorescence images of Vimentin protein detected using OCPNs;

[0091] Figure 11(b) shows the fluorescence image after fluorescence removal using in-situ chain replacement;

[0092] Figure 11(c) shows the fluorescence patterns after adding OCPNs for detecting Lamin A / C at 10 s, 30 s, 1 min, 1 min, 30 s, 2 min, and after washing;

[0093] Figure 12 shows fluorescence images of six different proteins imaged using six PFBT-OCPNs;

[0094] in,

[0095] Figure 12(a) shows the process of performing six monochromatic six-cycle fluorescence imaging of six different proteins using six PFBT-OCPNs;

[0096] Figure 12(b) shows a collection of fluorescence images of six different proteins from Figure 12(a);

[0097] Figure 13 shows a schematic diagram of multi-target fluorescence imaging using OCPNs and fluorescence images of nine proteins imaged using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs in three cycles.

[0098] in,

[0099] Figure 13(a) shows a schematic diagram of multi-target fluorescence imaging using OCPNs;

[0100] Figure 13(b) shows fluorescence images of α-tubulin, Lamin A / C, mitochoondria, Calnexin, EGFR, Ki67, Lamin B1, Vimentin, and GM130 obtained by imaging with PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs in three cycles.

[0101] Figure 13(c) shows the fluorescence images of nine proteins at the cell scale obtained by merging the fluorescence images in 13(b). Detailed Implementation

[0102] To more clearly illustrate the technical solutions of this disclosure, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well-known to those skilled in the art.

[0103] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0104] The method for synthesizing 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene in this embodiment is as follows:

[0105] 3.24 g of 2,7-dibromofluorene, 16.6 mL of 1,8-dibromooctane, and 322 mg of tetrabutylammonium bromide (TBAB) were added to a 250 mL three-necked flask. Then, 100 mL of 50 wt% NaOH aqueous solution was added under magnetic stirring. The mixture was heated to 75 °C and refluxed for 12 h. After the reaction was complete and the system returned to room temperature, the product was dissolved in 50 mL of DCM and transferred to a 100 mL separatory funnel. Deionized water (50 mL) was added for extraction. The mixture was shaken and allowed to stand to allow for re-separation. The organic phase was collected by opening the valve, and the aqueous phase was removed. This process was repeated three times. The washed organic phase was dried over anhydrous sodium sulfate, filtered, and the DCM was removed by rotary evaporation to obtain a milky yellow oil. The above-mentioned milky yellow oil was subjected to column chromatography (mobile phase: petroleum ether) to remove excess 1,8-dibromooctane and other byproducts, yielding a white solid 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene (3.38 g, yield: 54%).

[0106] The synthesis method of 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacol borate) in this embodiment is as follows:

[0107] 2,7-Dibromo-9,9-bis(8-bromooctyl)fluorene (1 g), pinacol diborate (1.08 g), potassium acetate (KOAc) (836 mg), and 1,1-bis(diphenylphosphine)diferropalladium dichloride (Pd(dppf)Cl2) (62.1 mg) were added to a 100 mL two-necked flask and mixed. The mixture was degassed and re-purged with argon three times. Then, dioxane (20 mL) was added under an argon atmosphere. The mixture was heated to 85 °C and stirred for 12 h. After the reaction was complete and the system was allowed to return to room temperature, dioxane was removed by rotary evaporation to obtain a off-white solid product. The product was dissolved in DCM (50 mL) and transferred to a 100 mL separatory funnel. Deionized water (50 mL) was added for extraction. The mixture was shaken and allowed to stand to allow it to separate into layers again. The valve was opened to collect the organic phase, and then the aqueous phase was removed. The above steps were repeated 3 times. The washed organic phase was dried with anhydrous sodium sulfate, filtered, and DCM was removed by rotary evaporation to obtain a brownish-brown oil. The above brownish-brown oil was subjected to column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:12) to remove byproducts, yielding a white solid 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacol borate) (1.33 g, yield: 77%).

[0108] Example 1

[0109] An azide-modified fluorescent π-conjugated polymer (PF-N3) has the following structural formula:

[0110] The value of n is 8.

[0111] Its preparation method is as follows:

[0112] S1. 2,7-Dibromo-9,9-bis(8-bromooctyl)fluorene (400 mg), 9,9-dioctylfluorene-2,7-bis(pinacol borate) (365 mg) and tetra-triphenylphosphine palladium (Pd(PPh3)4) (11.3 mg) were added to a 50 mL two-necked flask and mixed. The mixture was degassed and re-purged with argon three times. Then, toluene (11.3 mL) and K2CO3 aqueous solution (6.8 mL, 2.0 M) were added under an argon atmosphere. The mixture was heated to 90 °C and stirred for 48 h to obtain the crude product. After the system was cooled to room temperature, the crude product was added dropwise to ice-cold methanol to precipitate. Then, it was extracted with acetone, n-hexane and DCM in sequence. After concentration, it was precipitated again in ice-cold methanol to obtain the yellow solid poly[(9,9-bis(8-bromooctyl)-2,7-diyl)-alt-co-(9,9-dioctylfluorene)](PF-Br).

[0113] S2. Add PF-Br (100 mg) and NaN3 (20 mg) to a 50 mL round-bottom flask and mix. Dissolve the mixture in a mixed solvent of DMF (5 mL) and THF (5 mL) and stir at room temperature for 12 h. Dissolve the product with DCM (50 mL) and transfer to a 100 mL separatory funnel. Add saturated saline (50 mL) for extraction. Shake the mixed solution and let it stand to allow it to separate into layers again. Open the valve to collect the organic phase, then remove the aqueous phase. Repeat the above steps 3 times. Wash the organic phase with deionized water (50 mL) twice. After washing, dry the organic phase with anhydrous sodium sulfate, filter, and remove DCM by rotary evaporation to obtain yellow solid PF-N3.

[0114] Example 2

[0115] An azide-modified fluorescent π-conjugated polymer (PFBT-N3) has the following structural formula:

[0116] The value of n is 9.

[0117] Its preparation method is as follows:

[0118] S1. 2,7-Dibromo-9,9-bis(8-bromooctyl)fluorene (400 mg), 4,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxapentylborane-2-yl)-2,1,3-benzothiadiazole (220 mg) and tetraphenylphosphine palladium (Pd(PPh3)4) (32.8 mg) were added to a 50 mL two-necked flask and mixed. The mixture was degassed and re-purged with argon three times. Then, toluene (11.3 mL) and K2CO3 aqueous solution (6.8 mL, 2.0 M) were added under an argon atmosphere. The mixture was heated to 90 °C and stirred for 48 h to obtain the crude product. After the system was cooled to room temperature, the crude product was added dropwise to ice-cold methanol to precipitate. Then, it was extracted sequentially with acetone, n-hexane, and DCM using a Soxhlet extractor. After concentration, it was precipitated again in ice-cold methanol to obtain an orange-yellow solid poly[(9,9-bis(8-bromooctyl)-2,7-diyl)-alt-co-(1,4-benzo[2,1',3]-thiazole)](PFBT-Br). Then, high-performance liquid chromatography was used to screen for components with molecular weights between 5k and 10k.

[0119] S2. PFBT-Br (75 mg) and NaN3 (17 mg) with a molecular weight of 5k-10k were added to a 50 mL round-bottom flask. The mixture was dissolved in a mixed solvent of DMF (5 mL) and THF (5 mL) and stirred at room temperature for 12 h. The product was dissolved in DCM (50 mL) and transferred to a 100 mL separatory funnel. Saturated saline (50 mL) was added for extraction. The mixture was shaken and allowed to stand to allow it to separate into layers again. The valve was opened to collect the organic phase. The aqueous phase was then removed. The above steps were repeated 3 times. The organic phase was washed with deionized water (50 mL) twice. The washed organic phase was dried with anhydrous sodium sulfate, filtered, and DCM was removed by rotary evaporation to obtain an orange-yellow solid PFBT-N3.

[0120] Example 3

[0121] An azide-modified fluorescent π-conjugated polymer (PFTBT-N3) has the following structural formula:

[0122] The value of n is 7.

[0123] Its preparation method is as follows:

[0124] S1. 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacol borate) ester (80 mg), 4,7-bis(5-bromo-4-hexylthiophene-2-)-2,1,3-benzothiadiazole (62.6 mg), tetra-triphenylphosphine palladium (Pd(PPh3)4) (2.9 mg), TBAB (1.6 mg), and tetrabutylammonium hydroxide aqueous solution (2.2 mL, 40 wt%) were added to a 50 mL two-necked flask and mixed. The mixture was degassed and re-purged with argon three times. Then, toluene (6.7 mL) was added under an argon atmosphere. The mixture was heated to 90 °C and stirred for 2 h to obtain the crude product. After the system was cooled to room temperature, the crude product was added dropwise to ice-cold methanol to precipitate. Then, it was extracted sequentially with acetone, n-hexane, and DCM using a Soxhlet extractor. After concentration, it was precipitated again in ice-cold methanol to obtain a reddish-brown solid poly[2,7-(9,9-bis(8-bromooctyl)fluorene)-alt-(4,7-bis(thiophene-2-yl)benzo-2,1,3-thiazole)](PFTBT-Br). Then, high-performance liquid chromatography was used to screen for components with molecular weights between 5k and 10k.

[0125] S2. Add 48 mg of PFTBT-Br (molecular weight 5k-10k) and 13 mg of NaN3 to a 50 mL round-bottom flask. Dissolve the mixture in a mixed solvent of DMF (5 mL) and THF (5 mL) and stir at room temperature for 12 h. Dissolve the product in DCM (50 mL) and transfer to a 100 mL separatory funnel. Add saturated saline (50 mL) for extraction. Shake the mixture and allow it to stand to recrystallize. Open the valve to collect the organic phase. Remove the aqueous phase. Repeat the above steps 3 times. Wash the organic phase with deionized water (50 mL) twice. Dry the washed organic phase with anhydrous sodium sulfate, filter, and remove DCM by rotary evaporation to obtain a reddish-brown solid, PFTBT-N3.

[0126] Example 4

[0127] An oligonucleotide-modified fluorescent π-conjugated polymer (PF-OCPNs) has the following structural formula:

[0128] The P is an oligonucleotide (ONS) with the sequence: AAAAA ATA AAC ACA ACC GAA;

[0129] The value of x is 0.5.

[0130] Its preparation method is as follows:

[0131] L1. Preparation of CPG-modified oligonucleotides via phosphoramide chemical synthesis:

[0132] Weigh out 50 mg of Universal CPG (which can prepare 1.5 μmol of ONs at 100% yield) and pour it into an ONs synthesis column. Insert sieve plates into the wells at the top and bottom of the column, then insert it into the sample wells of a DNA synthesizer for fixation. Start the DNA synthesizer's self-test program to ensure that all pipelines and gas pressure are normal. Input the ONs sequence to be prepared, and start the preset program to automatically complete the preparation based on phosphorous amide chemistry. The program design is as follows:

[0133] (1) Rinse the synthesis column with anhydrous acetonitrile (1.5 mL), let it stand for 10 seconds, and then pump in argon gas;

[0134] (2) Pour in TCA Deblock deprotecting agent (trichloroacetic acid / dichloromethane) (1.5 mL), let stand for 1 min to react, to remove the 5'-hydroxy protecting group DMT, to obtain free 5'-hydroxy, then rinse with anhydrous acetonitrile (1.5 mL) for washing, repeat 3 times;

[0135] (3) Pour in ETT Activator (5'-ethimercaptotetrazole / acetonitrile) (1.5 mL), let stand for 2 min to react, so that the 3'-end of the phosphorous amide-protected nucleotide monomer is activated to obtain the nucleoside phosphorous acid activation intermediate, which undergoes a condensation reaction with the free 5'-hydroxyl group linked to the base on CPG. Then rinse with anhydrous acetonitrile (1.5 mL) for washing, and repeat twice.

[0136] (4) Pour in a mixture of 10% v / v acetic anhydride (tetrahydrofuran) (1 mL) and 10% v / v N-methylimidazolium (10% v / v pyridine / tetrahydrofuran) (1 mL) and react for 3 min to complete the capping reaction, blocking the 5'-hydroxyl group that did not participate in the previous condensation reaction, so that the short chain fragment containing unreacted hydroxyl group can no longer react, and can be separated and removed during purification. Then rinse with anhydrous acetonitrile (1.5 mL) for washing, and repeat 3 times.

[0137] (5) Pour in iodine solution oxidant (0.05M I2 / pyridine / tetrahydrofuran / water) (1.5mL), let stand for 3min to oxidize phosphorous acyl to a more stable triphosphate, then rinse with anhydrous acetonitrile (1.5mL) and repeat 3 times.

[0138] (6) Repeat the above steps until all sequences are synthesized. Then, anhydrous acetonitrile can be removed by pumping in argon gas to obtain a white powder of CPG-modified ONs with DMT protection at the 5' end, which can be stored at -20°C for a long time.

[0139] L2. Using butyn-phosphoramide as the 5' end modification unit, an alkynyl group was introduced into the CPG-modified oligonucleotide chain to obtain alkynylated CPG-modified oligonucleotides:

[0140] 5'-butyne phosphoramidite was dissolved in anhydrous acetonitrile to prepare a 0.1M solution. A synthesis column containing 50 mg of CPG-modified ONs powder with 5' end DMT protection was inserted into the DNA synthesizer tank. After sealing, the instrument automatically pumped in argon gas to restore the pressure. The DNA synthesizer was then flushed with 3 mL of anhydrous acetonitrile to clean the external reaction glass tube Z. This process was repeated three times. Argon gas was pumped in to clean the tubing and the tube Z was dried. Then, 0.1M butyne phosphoramidite solution was added to the tube Z at a rate of 300 μL / synthesis column. The tube Z was reinserted into the DNA synthesizer, and the pressure was allowed to return to normal. After repeating steps (1), (2), and (3) in L1, mix the liquid in tube Z with anhydrous acetonitrile (800 μL) and flush it into the synthesis column. Let the reaction stand for 8 minutes, then flush with anhydrous acetonitrile (1.5 mL) three times. Continue to complete steps (4) and (5) in L1 to complete the end sealing. After flushing with anhydrous acetonitrile (1.5 mL) three times, continue to pump argon gas until the powder loosens and falls off when the synthesis column is tapped. After confirming that it is dry, take out the synthesis column, seal it, and it can be stored for a long time at -20℃.

[0141] L3. The azide-modified fluorescent π-conjugated polymer was coupled with an alkyne-modified CPG oligonucleotide via click chemistry, and then the CPG was removed by ammonolysis to obtain the oligonucleotide-grafted fluorescent π-conjugated polymer.

[0142] (1) Prepare the storage solution:

[0143] Dissolve PF-N3 polymer in DCM to prepare a 500 μM (monomer concentration, monomer relative molecular mass 858) stock solution. Store the stock solution in a glass bottle with a cap. When storing, seal the gap of the cap with sealant and wrap the entire glass bottle with aluminum foil to protect it from light. Avoid tilting the bottle. Then it can be stored in a -20℃ refrigerator for long-term storage.

[0144] (2) Graft ONs onto PF-N3:

[0145] Weigh out CuI (1.9 mg), acetylated CPG-modified ONs (50 mg), and an A5 magnetic stir bar, and place them together in a Schlenk tube. Seal the bottle opening with a rubber stopper and connect it to a double-row tube. Evacuate for 3 minutes and then introduce argon gas for 3 minutes. Repeat this step 3 times and then maintain a slow argon gas flow. Prepare a DIPEA-DMF solution by dissolving DIPEA (50 μL) in DMF (450 μL), and prepare an OHAc-DMF solution by dissolving glacial acetic acid (50 μL) in DMF (450 μL). Then, take 750 μL of PF-N3 stock solution into an EP tube (2 mL), add 500 μL of DMSO, 500 μL of DMF, 6 μL of HOAc-DMF, and 18 μL of DIPEA-DMF. Slowly pipette the solution with a 100 μL pipette to mix the liquid. Use a syringe to draw out the liquid, gently tap the syringe to expel the gas, insert the rubber cap, and slowly inject the liquid into a Schlenk tube. After removing the syringe, immerse the Schlenk tube in an oil bath and react at 45°C and 900 rpm for 12 h. After the reaction is complete, transfer the liquid to an EP tube (1.5 mL) for temporary storage.

[0146] (3) Remove unreacted PF-N3 and reactive impurities:

[0147] Centrifuge the EP tube containing the temporary reaction product at 12000 rpm for 5 min to collect the CPG-conjugated reaction product at the bottom of the EP tube and remove the liquid using a 1 mL pipette. Then, add 1 mL of DCM and wash the product with the liquid under sonication. Remove the DCM by centrifuging at 12000 rpm for 5 min. Then, add 1 mL of DMSO to the tube, mix under sonication, and remove the DMSO by centrifuging at 12000 rpm for 5 min. Repeat the above washing steps 3 times. Then wash with 1 mL of DCM and remove the liquid. Finally, blow dry the remaining DCM under an argon flow to obtain a white powder deposited at the bottom of the EP tube.

[0148] (4) Disconnect PF-g-ONs from CPG:

[0149] Concentrated ammonia (1 mL, 15-20% w / v) was slowly added to the EP tube containing the dried product. The EP tube was capped and sealed with sealing tape, then the cap was clamped with an explosion-proof clamp. The product and solution were mixed under ultrasonication, and the EP tube was placed in a constant temperature mixer and treated at 55°C and 450 rpm for 2 hours. Afterward, the EP tube was removed and left at room temperature until it returned to room temperature. The explosion-proof clamp was slowly removed to prevent liquid from overflowing, and the remaining ammonia was dried under a slow argon flow to obtain PF-g-ONs powder.

[0150] L4. Oligonucleotide-grafted fluorescent π-conjugated polymers are transferred to an aqueous phase via solvent displacement to self-assemble into oligonucleotide-modified fluorescent π-conjugated polymers:

[0151] 10×PBS (1 mL) was rapidly added to the EP tube, mixed under sonication, and then centrifuged at 12000g for 5 min to complete solvent displacement and allow PF-g-ONs to self-assemble into PF-OCPN nanoparticles. Unreacted alkyne-modified ONs were then removed by ultrafiltration. A new ultrafiltration tube (4 mL, 50 kDa) was used. 1×PBS (4 mL) was added to the filter cartridge, and ultrafiltration was performed at 5000g for 10 min to clean the cartridge, removing any remaining liquid. The nanoparticle aqueous solution was aspirated using a syringe, avoiding contamination with CPG precipitated at the bottom, and transferred to the filter cartridge. 1×PBS was added until the solution volume reached 4 mL, and the ultrafiltration tube was ultrafiltered at 5000g for 10 min. The filtrate in the collection tube was removed. Then, ultrapure water (3.5 mL) was added to the filter cartridge, and ultrafiltration was performed at 5000g for 10 min. The ONs concentration in the filtrate was detected using NanoDrop. This step was repeated until the detected ONs concentration was less than 1 ng / μL. Transfer the product to an ep tube (1.5 mL) using a pipette (100 μL), add ultrapure water or 1×PBS to a solution volume of 500 μL, tighten the cap and seal with sealing film. It can be stored at 4°C for several years.

[0152] Figure 1 illustrates the synthesis and modification process of ONs;

[0153] in,

[0154] Figure 1(a) shows a schematic diagram of the chemical reaction process for solid-phase synthesis of ONs based on phosphoramidite chemical synthesis. A general-purpose CPG (controllable microporous glass microspheres grafted with DMT-protected hydroxyl groups) is used as the solid-phase support for ONs synthesis. Based on phosphoramidite chemistry, DMT-protected phosphoramidite base monomers are sequentially reacted and attached to the ONs chain without catalysis. This reaction is highly efficient and rapid, and can automatically synthesize ONs chains with a predetermined sequence. It should be noted that this reaction is very sensitive to water. The introduction of a small amount of water will significantly reduce the reaction efficiency. Therefore, this reaction is carried out using a DNA synthesizer. When the reaction is started for the first time, argon gas at 0.5 MPa is used to maintain the internal environment of the instrument for more than 1 hour. Then, the ONs synthesis reaction is completed according to the preset program.

[0155] Figure 1(b) shows a schematic diagram of the process of using butyne-phosphorous amide as the 5' end modification unit and coupling it to the ONs sequence; this reaction can be completed by a DNA synthesizer based on the phosphorous amide chemical synthesis method;

[0156] Figure 2 illustrates the process of self-assembly to form OCPNs;

[0157] in,

[0158] Figure 2(a) illustrates the process of directly coupling the azide-modified fluorescent π-conjugated polymer with the alkyne-modified CPG-modified oligonucleotides (CPG-ONs) via click chemistry to obtain oligonucleotide-grafted fluorescent π-conjugated polymers (FCP-g-ONs); using CuI "click" chemistry catalysis, the ONs are grafted onto the FCP by coupling the alkyne group on the ONs with the azide group on the FCP, and then the CPG is removed from the ONs by ammonolysis.

[0159] Figure 2(b) shows the process of transferring FCP-g-ONs to the aqueous phase via solvent displacement to form OCPNs; the product in the filter cartridge is transferred to the EP tube and can be stored at 4°C for more than 3 years.

[0160] Example 5

[0161] An oligonucleotide-modified fluorescent π-conjugated polymer (PFBT-OCPNs) has the following structural formula:

[0162] The P is an oligonucleotide (ONS) with the sequence: AAAAA GAC TCA ACG GAC TAT;

[0163] The value of x is 0.54.

[0164] Its preparation method is as follows:

[0165] The difference between the preparation method in this embodiment and that in Example 4 is that in step L3 (1), the PFBT-N3 polymer is dissolved in DCM to prepare a 500 μM (monomer concentration, monomer relative molecular mass 604) storage solution and applied to subsequent steps. The other preparation methods are the same as in Example 4.

[0166] Example 6

[0167] An oligonucleotide-modified fluorescent π-conjugated polymer (PFTBT-OCPNs) has the following structural formula:

[0168] The P is an oligonucleotide (ONS) with the sequence: AAAAA TAG CTT ATC AGA CTG;

[0169] The value of x is 0.47.

[0170] Its preparation method is as follows:

[0171] The difference between the preparation method in this embodiment and that in Example 4 is that in step L3 (1), the PFTBT-N3 polymer is dissolved in DCM to prepare a 500 μM (monomer concentration, monomer relative molecular mass 936) storage solution and applied to subsequent steps. The other preparation methods are the same as in Example 4.

[0172] Examples 7-8

[0173] The difference between Examples 7-8 and Example 4 is that the sequence of oligonucleotide (ONs)P is replaced with:

[0174] CCAAT ACC TAA AGA CTG AAG;

[0175] TCTCC ACT GAA TAA CAA CAT;

[0176] All other preparation methods are the same as in Example 4.

[0177] Examples 9-14

[0178] The difference between Examples 9-14 and Example 5 is that the sequence of oligonucleotide (ONs)P is replaced with:

[0179] CCAAT ACC TAA AGA CTG AAG;

[0180] AGAAC CAA GGA ACC TAG AAC;

[0181] ACAAC AAA CAA GGA CCC AAC;

[0182] GCAAC TAT CCA CAG AAA CAC;

[0183] AACAA AGG AAA TGG AAC TAA;

[0184] AAAAA ATA AAC ACA ACC GAA;

[0185] All other preparation methods are the same as in Example 5.

[0186] Examples 15-16

[0187] The difference between Examples 15-16 and Example 6 is that the sequence of oligonucleotide (ONs)P is replaced with:

[0188] AACAA AGG AAA TGG AAC TAA;

[0189] ACAAC AAA CAA GGA CCC AAC;

[0190] All other preparation methods are the same as in Example 6.

[0191] Test Example 1

[0192] Test methods: The PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs prepared in Examples 4-6 were subjected to ultraviolet-visible absorption spectroscopy, agarose gel electrophoresis, fluorescence spectra under different wavelengths of excitation light, particle size distribution and transmission electron microscopy (TEM) images.

[0193] Figure 3 shows the performance test results of OCPNs;

[0194] in,

[0195] Figure 3(a) shows the UV-Vis absorption spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0196] Figure 3(b) shows the agarose gel electrophoresis results of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0197] Figure 3(c) shows the fluorescence spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs under excitation light of 370 nm, 448 nm and 515 nm, respectively;

[0198] Figure 3(d) shows the particle size distribution of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs as detected by DLS particle size analysis;

[0199] Figure 3(e) shows transmission electron microscopy (TEM) images of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs, with a scale bar of 100 nm.

[0200] Test results show that OCPNs with a particle size of about 10 nm can be prepared for all three different FCPs, and the fluorescence spectra can be separated for simultaneous detection.

[0201] Test Example 2

[0202] Test method: The particle size of PFBT-OCPNs was controlled by changing the amount of CPG-ONs (adjusting the molar ratio of azide functional groups on CPG-ONs and PFBT-N3 to 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, and 8:1).

[0203] Figure 4 shows the UV-Vis absorption spectra of PFBT-OCPNs with different particle sizes, the hydration particle size results, the fitting curve of ONs grafting rate versus CPG-ONs dosage, and the fitting curve of hydration particle size versus CPG-ONs dosage.

[0204] in,

[0205] Figure 4(a) shows the UV-Vis absorption spectra of PFBT-OCPNs with different particle sizes; Figure 4(b) shows the results of DLS particle size analyzer detection of the hydrated particle size of PFBT-OCPNs with different particle sizes; Figure 4(c) shows the fitting curves of ONs grafting rate and CPG-ONs dosage for PFBT-OCPNs with different particle sizes; Figure 4(d) shows the fitting curves of hydrated particle size and CPG-ONs dosage for PFBT-OCPNs with different particle sizes; the fitted data represent the mean ± standard deviation (n = 3) of ONs grafting rate and average hydrated particle size obtained under the same conditions.

[0206] Test results show that as the amount of CPG-ONs increases, the particle size of PFBT-OCPNs decreases. The minimum particle size of PFBT-OCPNs prepared using the current method is about 10 nm, and the maximum grafting rate of the obtained ONs is about 50%.

[0207] Test Example 3

[0208] Test method: The absolute fluorescence quantum yields of PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs prepared in Examples 4-6 were measured at 370 nm, 445 nm, and 488 nm using a photoluminescence spectrophotometer (FLS1000). The quantum yield (QY) was automatically calculated by the test software, and the results are shown in Table 1.

[0209] Table 1 Optical information of three OCPNs and fluorescent probes with similar excitation and emission spectra.

[0210] Figure 5 shows the absolute fluorescence quantum yield detection spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs detected at 370 nm, 445 nm and 488 nm using a photoluminescence spectrophotometer (FLS1000).

[0211] Test Example 4

[0212] Test method: PAGE gel was used to detect antibodies, DNA-conjugated antibodies, DNA-conjugated antibodies + complementary DNA, and DNA-conjugated antibodies + OCPNs. The OCPNs used were PFBT-OCPNs from Example 5.

[0213] Preparation method of DNA-conjugated antibodies:

[0214] Take one ultrafiltration tube with a molecular weight cutoff of 50 kDa and a core volume of 500 μL, add 500 μL of 1×PBS solution containing Tween-20 (0.1% v / v), shake, and ultrafilter at 12000 g and 4 °C for 2 min. Use a pipette to remove the core solution and the solution in the filter tube. Add 10×PBS (400 μL) to the filter core, then add 30 μg of antibody, and then ultrafilter at 12000 g and 4 °C for 8 min. Remove the liquid in the filter tube. Prepare a 7.5 mM TCEP solution using EDTA (2 μL, 0.5 M), TCEP (6 μL, 0.5 M), and 10×PBS (392 μL). Add 360 μL of the TCEP solution (7.5 mM) to a filter tube, gently pipette to mix the liquid, centrifuge at 1000 g for 10 s, and let stand at room temperature for 30 min to complete the reduction of the antibody.

[0215] After ultrafiltration at 8000g and 4℃ for 8 min, remove the liquid from the filter tube. Add 1×PBS (450 μL), mix well, and ultrafilter at 8000g and 4℃ for 8 min. Remove the liquid from the filter tube. Add 1×PBS (450 μL), mix well, and ultrafilter at 12000g and 4℃ for 8 min. Weigh out 60 μg of maleimide-modified ONs (twice the antibody mass) and dissolve it in 10×PBS (100 μL). Add the solution to the ultrafiltration tube, gently pipette to mix the liquid, centrifuge at 1000g for 10 s, and incubate at room temperature for 2 h to complete the coupling.

[0216] Add 1×PBS (350 μL) and ultrafilter at 12000 g and 4 °C for 8 min. Remove the filter solution, add 1×PBS (450 μL) again, and ultrafilter at 12000 g and 4 °C for 8 min. Repeat three times. Add 1×PBS (100 μL), gently aspirate and mix the liquid using a pipette, remove the filter cartridge, invert it and place it in a new filter tube, ultrafilter at 3000 g and 4 °C for 2 min. Transfer the collected liquid to a 200 μL EP tube and store at 4 °C.

[0217] Figure 6 shows the results detected using PAGE gel-paired antibodies, DNA-conjugated antibodies, DNA-conjugated antibodies + complementary DNA, and DNA-conjugated antibodies + OCPNs.

[0218] Lanes 1-4 represent the original antibody (antibodies typically have a molecular weight of 150 kDa; those closer to 110 kDa are antibodies after removing two light chains), DNA-conjugated antibody, the result of incubation of DNA-conjugated antibody with complementary DNA, and the result of incubation of DNA-conjugated antibody with OCPNs, respectively. The results show that lanes 1-4 did not contain BSA bands, indicating that they were BSA-free. The DNA-conjugated antibody band was positioned higher than the original antibody, and the band after incubation with complementary DNA was even higher, proving that the DNA-conjugated antibody was successfully prepared and could perform its corresponding hybridization function. Simple mixing of DNA-conjugated antibody and OCPNs resulted in one OCPNs hybridizing with multiple DNA-conjugated antibodies, forming a larger structure that could not enter the gel through the channels, further demonstrating that the DNA-conjugated antibody could successfully hybridize with OCPNs.

[0219] The results of this test case show that using the OCPNs disclosed herein to detect DNA-coupled antibodies can greatly reduce the amount of antibody used in a single experiment (the amount of antibody used in the literature is generally greater than 100 μg), and the efficiency of DNA coupling on the antibody can reach almost 100%.

[0220] Test Example 5

[0221] Test Method: The detection effect of OCPNs was detected using HeLa cells. HeLa cells were seeded at a density of 10,000 cells / well in 96-well plates and incubated in DMEM medium containing fetal bovine serum (10% v / v) and 1% penicillin-dextrose antibody at 37°C and CO2 (5% v / v) for 24 h. Cells were then washed three times with 1×PBS preheated to 37°C for 3 min each time. Cells were then fixed with paraformaldehyde (4% w / v) at room temperature for 20 min. The fixation process was quenched by incubation with NH4Cl solution (100 mM) dissolved in 1×PBS for 20 min, followed by washing three times with 1×PBS for 5 min each time. Wells were punched with Triton X-100 (0.1% v / v) dissolved in 1×PBS for 10 min, followed by washing three times with 1×PBS for 5 min each time. Cells were blocked at room temperature for 1 hour with blocking buffer containing cleaved salmon extract (0.2 mg / mL), dextran sulfate (0.05% w / v), EDTA (4 mM), and BSA (5% w / v). Then, an appropriate concentration of DNA-conjugated antibody was dissolved in the blocking buffer and added to the cell samples, which were incubated overnight at 4°C. The next day, the cell samples were brought to room temperature and washed three times with 1×PBS for 5 min each time. Then, the PFBT-OCPNs prepared in Examples 5 and 9-13 were dissolved in 2×SSC hybridization buffer containing BSA (5% w / v), dextran sulfate (0.05 w / v), and glycine (0.3 M), and the cell samples were added. The cells were incubated for 5-10 min and washed three times with 1×PBS for 3 min each time. The cells were then examined using a confocal microscope.

[0222] Figure 7 shows fluorescence images of Lamin A / C, Ki67, α-tubulin, Calnexin, GM130, and mitochondria in HeLa cells after staining with PFBT-OCPNs prepared in Examples 5 and 9-13, respectively. Scale bar: 40 μm.

[0223] The results of this test case show that OCPNs have sufficient detection sensitivity for proteins of different abundances.

[0224] Test Example 6

[0225] Test Method: In the same cell sample, two different proteins, Lamin A / C and α-tubulin, were targeted by AF488-labeled secondary antibody and PFBT-OCPNs, respectively. In the first group, PFBT-OCPNs prepared in Example 14 were used to image α-tubulin, and AF488 was used to image Lamin A / C. In the second group, PFBT-OCPNs prepared in Example 10 were used to image Lamin A / C, and AF488 was used to image α-tubulin. Under 488 nm excitation light, the fluorescence signals of AF488 and PFBT-OCPNs were detected simultaneously. The photostability of the two was compared under continuous laser irradiation.

[0226] Figure 8 shows a schematic diagram comparing the photostability of AF488 and PFBT-OCPNs, as well as a comparison of the fluorescence signals of different parts of cells labeled with AF488 and PFBT-OCPNs under continuous laser excitation for 3 minutes.

[0227] in,

[0228] Figure 8(a) shows a schematic diagram comparing the photostability of AF488 and PFBT-OCPNs;

[0229] Figure 8(b) shows a comparison of fluorescence signals of AF488-labeled Lamin A / C and PFBT-OCPNs-labeled α-tubulin under continuous laser excitation for 3 minutes.

[0230] Figure 8(c) shows a comparison of fluorescence signals of α-tubulin labeled with AF488 and Lamin A / C labeled with PFBT-OCPNs under continuous laser excitation for 3 min.

[0231] The results of this test case show that: (1) Under continuous laser irradiation, the fluorescence of AF488 gradually quenched, while the fluorescence signal of PFBT-OCPNs remained. (2) Under continuous excitation for 3 minutes, the fluorescence signal at the target location labeled by AF488 decreased significantly. In Figure 8(b), the Lamin A / C labeled by AF488 secondary antibody is the cell nucleus region. The fluorescence curves before and after excitation along the yellow line show that the fluorescence at the cell nucleus decreased significantly, while the decrease at other locations was very small, proving that the microtubules labeled by PFBT-OCPNs have excellent photostability. In Figure 8(c), the microtubules were labeled with AF488 secondary antibody. After continuous excitation, the signal at the microtubule location decreased significantly, while the Lamin A / C signal labeled by PFBT-OCPNs in the cell nucleus region did not decrease significantly. Both of these results indicate that PFBT-OCPNs have excellent photostability.

[0232] Test Example 7

[0233] Test method: The PFBT-OCPNs, AF488 secondary antibody and AF488 modified ONs prepared in Example 5 were used to detect Mitochondria, Lamin A / C and α-tubulin proteins, respectively. The fluorescence amplification effect of PFBT-OCPNs, AF488 secondary antibody and AF488 modified ONs were compared.

[0234] Figure 9 shows fluorescence images of Mitochondria, Lamin A / C, and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody, and AF488-modified ONs, respectively, as well as fluorescence intensity bar charts plotted after quantitative statistical analysis of fluorescence intensity for each cell using ImageJ software.

[0235] in,

[0236] Figure 9(a) shows fluorescence images of Mitochondria, Lamin A / C and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody and AF488-modified ONs, respectively;

[0237] Figure 9(b) shows a fluorescence intensity bar chart plotted using ImageJ software to quantify the fluorescence intensity of each cell based on the image in Figure 9(a). Data were collected from different locations of the same sample, and the mean ± standard deviation (n = 10) was calculated. The scale bar is 50 μm. Statistically, the fluorescence intensity produced by PFBT-OCPNs is 3-11 times that of the AF488 secondary antibody.

[0238] The results of this test show that the fluorescence intensity of OCPNs is 3-11 times that of traditional dye molecules that amplify fluorescence based on secondary antibodies.

[0239] Test Example 8

[0240] Signal removal is achieved by in situ strand replacement of DNA-encoded antibodies.

[0241] Test method: The PFBT-OCPNs prepared in Example 9 were used to remove the signal by binding to the DNA-encoded antibody with a replacement strand that has more complementary sequences to the ONs on the antibody.

[0242] Figure 10 shows a schematic diagram and operational example of signal removal by in-situ chain replacement. Scale bar: 40 μm.

[0243] After the replacement strand is introduced, the fluorescence signal is completely removed. This is because the replacement strand has more complementary sequences to the ONs on the antibody, and its binding affinity to the DNA-encoded antibody is greater than that of the OCPNs. Therefore, after the replacement strand is introduced, it can replace the OCPNs and hybridize to the antibody, causing the OCPNs that have already hybridized with the DNA-encoded antibody to dissociate into the solution and be washed away, thus causing the fluorescence signal to disappear. At this point, the ONs on the antibody are completely blocked by hybridization of the replacement strand, and the reintroduction of OCPNs will not be able to hybridize with the DNA-coupled antibody again, so the signal cannot reappear.

[0244] The results of this test case show that OCPNs can avoid residual fluorescence signals in multi-target imaging, which can affect the imaging results.

[0245] Test Example 9

[0246] Rapid imaging and elution using OCPNs.

[0247] Test method: First, Vimentin protein was detected using PFBT-OCPNs prepared in Example 12, and fluorescence signal was detected. Then, in situ strand replacement was used to remove fluorescence. Subsequently, Lamin A / C was detected by adding PFBT-OCPNs prepared in Example 10 at 10s, 30s, 1min, 1min, 30s, and 2min after adding OCPNs.

[0248] Figure 11 shows fluorescence images obtained using rapid imaging and elution with OCPNs;

[0249] in,

[0250] Figure 11(a) shows fluorescence images of Vimentin protein detected using OCPNs;

[0251] Figure 11(b) shows the fluorescence image after fluorescence removal using in-situ chain replacement;

[0252] Figure 11(c) shows the fluorescence images at 10 s, 30 s, 1 min, 1 min, 30 s, 2 min after adding OCPNs for detecting Lamin A / C, and after washing. The test results show that the fluorescence signal is completely removed within 15 s after in-situ chain replacement. After adding OCPNs for detecting Lamin A / C, the morphology of Lamin A / C appears rapidly. Direct washing after 2 min yields a fluorescence image with a higher signal-to-noise ratio, indicating that using OCPNs for fluorescence imaging has high detection efficiency and high elution efficiency.

[0253] The results of this test case show that fluorescence imaging using OCPNs takes less than 2 minutes and can obtain fluorescence images with a good signal-to-noise ratio without the need for washing. The removal of fluorescence signals (i.e., the elution step) only takes less than 30 seconds, which is more efficient than known imaging techniques.

[0254] Test Case 10

[0255] Test method: Six different proteins were fluorescently imaged using six monochromatic cycles of six PFBT-OCPNs prepared in Examples 5 and 9-13.

[0256] Figure 12 shows fluorescence images of six different proteins imaged using six PFBT-OCPNs;

[0257] in,

[0258] Figure 12(a) shows the process of performing six monochromatic six-cycle fluorescence imaging of six different proteins using six PFBT-OCPNs;

[0259] Figure 12(b) shows a collection of fluorescence images of the six different proteins in Figure 12(a).

[0260] The results of this test case show that, for proteins with different abundances in cells, no additional adjustment of the OCPNs concentration is required to image these six proteins separately, and the cell morphology is not damaged during the elution process.

[0261] Test Example 11

[0262] Simultaneously, different PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs were used to perform fluorescence imaging on nine different proteins—α-tubulin, Lamin A / C, mitochondria; Calnexin, EGFR, Ki67; Lamin B1, Vimentin, and GM130—in three cycles. The nine proteins were divided into three groups for sequential imaging. The first group consisted of PF-OCPNs from Example 4 for α-tubulin imaging, PFBT-OCPNs from Example 10 for Lamin A / C imaging, and PFTBT-OCPNs from Example 6 for mitochondria imaging. The second group consisted of PF-OCPNs from Example 7 for Calnexin imaging, PFBT-OCPNs from Example 5 for Ki67 imaging, and PFTBT-OCPNs from Example 15 for EGFR imaging. The third group consisted of PF-OCPNs from Example 8 for Lamin B1 imaging. B1 imaging, Vimentin imaging with PFBT-OCPNs prepared in Example 12, and GM130 imaging with PFTBT-OCPNs prepared in Example 16.

[0263] Test method: First, nine DNA-encoded antibodies were incubated in cells. In each cycle, PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs were added to detect three different proteins simultaneously. Then, in situ strand replacement was used to remove the fluorescence signal before adding the next batch of PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs. Fluorescence imaging detection of the nine target proteins was completed in three cycles.

[0264] Figure 13 shows a schematic diagram of multi-target fluorescence imaging using OCPNs and fluorescence images of nine proteins imaged using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs in three cycles.

[0265] in,

[0266] Figure 13(a) shows a schematic diagram of multi-target fluorescence imaging using OCPNs;

[0267] Figure 13(b) shows fluorescence images of α-tubulin, Lamin A / C, mitochoondria, Calnexin, EGFR, Ki67, Lamin B1, Vimentin, and GM130 obtained by imaging with PF-OCPNs, PFBT-OCPNs, and PFTBT-OCPNs in three cycles.

[0268] Figure 13(c) shows the fluorescence images of nine proteins at the cell scale obtained by merging the fluorescence images in 13(b).

[0269] The results of this test case show that the total imaging time for the nine proteins is less than 1 hour, which exceeds all currently known multi-target fluorescence imaging techniques.

[0270] In summary, the OCPNs prepared in this disclosure possess extremely high fluorescence brightness and detection sensitivity, enabling the detection and imaging of targets with varying abundances without the need for additional fluorescence signal amplification steps. Furthermore, they achieve rapid and specific targeting with minimal dosage, reducing the entire staining and imaging process to as little as 2 minutes. On the other hand, in-situ displacement enables rapid removal of the fluorescence signal, reducing a single imaging cycle to as little as 2.5 minutes. When using three OCPNs for multicolor imaging, imaging of nine different proteins can be completed in three cycles, with a total time not exceeding 1 hour. This imaging efficiency is the fastest known and has excellent application prospects.

[0271] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0272] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. Industrial applicability

[0273] This application prepares oligonucleotide-modified fluorescent π-conjugated polymers (OCPNs) based on azide-modified fluorescent π-conjugated polymers. These OCPNs exhibit ultra-high fluorescence brightness and extremely high detection sensitivity, enabling the detection and imaging of targets with varying abundances without the need for additional fluorescence signal amplification. Furthermore, they achieve rapid and specific targeting with minimal dosage, shortening the entire staining and imaging process, and demonstrating extremely high imaging efficiency in multicolor imaging. These OCPNs resolve the contradiction between bioconjugation modification and the stability of small-sized nanoparticle micelle structures, thereby enabling the application of π-conjugated polymers in imaging multiple molecular spatial distributions within single cells. Therefore, the OCPNs presented in this application possess excellent practical performance and broad market application prospects.

Claims

1. An azide-modified fluorescent π-conjugated polymer, characterized in that, The general structural formula of the azide-modified fluorescent π-conjugated polymer is shown below: Wherein, R4 is independently selected from C1-C30 alkyl groups with straight or branched structures; The Ar unit is selected from aromatic rings, aromatic heterocycles, aromatic ring derivatives, and aromatic heterocycle derivatives; n is a natural number.

2. The azide-modified fluorescent π-conjugated polymer according to claim 1, characterized in that, The Ar unit is selected from any of the following structures: R2 and R3 are each independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

3. The azide-modified fluorescent π-conjugated polymer according to claim 1, characterized in that, The number n is selected from 5 to 15.

4. The method for preparing the azide-modified fluorescent π-conjugated polymer according to any one of claims 1-3, characterized in that, The preparation method of the azide-modified fluorescent π-conjugated polymer includes the following steps: S1. Mix monomer M, monomer N, catalyst, base and solvent, and heat and stir the reaction under inert gas protection. After purification, a fluorene precursor containing bromoalkyl is obtained. S2. The fluorene precursor containing bromoalkyl groups was mixed with NaN3 and solvent and stirred to react. After purification, the product was obtained. The monomer M has the following general structural formula: X1 is independently selected from halogen atoms; The X2 is independently selected from one of the following: halogen atoms and pinacol borate ester; R1 is independently selected from C1-C30 alkyl groups with straight or branched structures; The monomer N is selected from one of the following structures: R2 and R3 are independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

5. The method for preparing the azide-modified fluorescent π-conjugated polymer according to claim 4, characterized in that, In step S1, the heating temperature is 80-100℃ and the reaction time is 1-50h; in step S2, the reaction time is 10-20h.

6. The use of the azide-modified fluorescent π-conjugated polymer as described in any one of claims 1-3 or the azide-modified fluorescent π-conjugated polymer obtained by the preparation method described in claim 4 or 5 in the preparation of oligonucleotide-modified fluorescent π-conjugated polymers.

7. An oligonucleotide-modified fluorescent π-conjugated polymer based on the azide-modified fluorescent π-conjugated polymer according to any one of claims 1-3, characterized in that, The general structural formula of the oligonucleotide-modified fluorescent π-conjugated polymer is shown below: The R4 is independently selected from C1-C30 alkyl groups with straight or branched structures; The Ar unit is selected from aromatic rings, aromatic heterocycles, aromatic ring derivatives, and aromatic heterocycle derivatives; The P is an oligonucleotide; The value of x is selected from 0.1 to 0.

6.

8. The oligonucleotide-modified fluorescent π-conjugated polymer according to claim 7, characterized in that, The Ar unit is selected from any of the following structures: R2 and R3 are each independently selected from one or more of the following: straight-chain or branched C1-C30 alkyl, straight-chain or branched C1-C30 alkoxy, straight-chain or branched C1-C30 alkylthio, and straight-chain or branched C1-C30 silyl.

9. A method for preparing the oligonucleotide-modified fluorescent π-conjugated polymer according to any one of claims 7-8, characterized in that, Includes the following steps: L1. Preparation of CPG-modified oligonucleotides via phosphoramide chemical synthesis; L2. Using butyn-phosphoramide as the 5' end modification unit, an alkyne group was introduced into the CPG-modified oligonucleotide chain to obtain alkylated CPG-modified oligonucleotides. L3. The azide-modified fluorescent π-conjugated polymer was coupled with the alkyne-modified CPG-modified oligonucleotide by click chemistry, and then the CPG was removed by ammonolysis to obtain the oligonucleotide-grafted fluorescent π-conjugated polymer. L4. The oligonucleotide-grafted fluorescent π-conjugated polymer was transferred to the aqueous phase by solvent displacement to form an oligonucleotide-modified fluorescent π-conjugated polymer.

10. The method for preparing the oligonucleotide-modified fluorescent π-conjugated polymer according to claim 9, characterized in that, In step L4, the self-assembly involves adding the oligonucleotide-grafted fluorescent π-conjugated polymer to a 0.05-2M buffer solution to self-assemble into an oligonucleotide-modified fluorescent π-conjugated polymer.

11. The application of the oligonucleotide-modified fluorescent π-conjugated polymer according to any one of claims 7-8 or the oligonucleotide-modified fluorescent π-conjugated polymer obtained by the preparation method according to any one of claims 9-10 in immunofluorescence imaging detection.

12. The application of the oligonucleotide-modified fluorescent π-conjugated polymer according to any one of claims 7-8 or the oligonucleotide-modified fluorescent π-conjugated polymer obtained by the preparation method according to any one of claims 9-10 in single-cell multiple molecular spatial distribution imaging.

13. The application according to claim 12, characterized in that, The applications include: using the oligonucleotide-modified fluorescent π-conjugated polymer to detect the spatial distribution and relative positions of various molecules with different abundances in single cells.

14. A product for single-cell in-situ spatial multiplex molecular imaging detection, characterized in that, The fluorescent π-conjugated polymer modified with oligonucleotides as described in any one of claims 7-8.

15. A method for rapid signal amplification and high-throughput protein detection imaging based on DNA-modified π-conjugated fluorescent nanoparticles, characterized in that, The fluorescent π-conjugated polymer modified with oligonucleotides as described in any one of claims 7-8.

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