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

Abstract

The present application relates to a kind of azide modified fluorescent pi conjugated polymer and the signal rapid amplification and high throughput protein detection imaging method based on DNA modified pi conjugated fluorescent nanoparticles.The present application prepares the oligonucleotide modified fluorescent pi conjugated polymer (OCPNs) based on azide modified fluorescent pi conjugated polymer, the OCPNs has super high fluorescent brightness and extremely high detection sensitivity, without additional fluorescent signal amplification step, different abundance target detection and imaging can be completed;At the same time, in the case of little use, fast, specific targeting effect can be completed, shorten the whole dyeing imaging process, when carrying out multicolor imaging, imaging efficiency is extremely high, has excellent application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a signal rapid amplification and high-throughput protein detection imaging method based on DNA modified pi conjugated fluorescent nanoparticles. BACKGROUND

[0002] Proteins are important components of cells, tissues and organs in the body, and their expression levels and systemic distribution in life activities are important references for the detection, diagnosis and treatment of many major diseases. Current biological research shows that the occurrence and deterioration of certain diseases are often caused by abnormal expression of multiple proteins. These proteins have different content abundance and systemic distribution in cells, making it urgent to develop a detection method that is efficient, convenient, highly specific and sensitive for multiple biological targets.

[0003] Among the many detection methods currently developed, the immunofluorescence imaging (IF) technology based on fluorescence signal and antibody-antigen specific binding has received extensive attention and development. By combining antibodies with multiple fluorescent probes with different fluorescence, the fluorescent probes can be targeted to multiple different antigens and generate corresponding fluorescence signals, i.e., the rapid detection of multiple specific targets is completed. However, as the number of targets to be detected increases, and due to the limitation of the wavelength range of fluorescence spectrum that can be collected by the current detection equipment, the light path overlap phenomenon caused by the use of multiple fluorescent probes gradually increases, limiting the total number of effective detection objects to 3 to 5. Since the abundance of the targets to be detected may differ greatly, the total fluorescence intensity generated by the fluorescent probes at the detection targets with lower abundance is lower, and thus is severely disturbed by the background signal, resulting in complete neglect of the target signal or the generation of false "positive" signals.

[0004] To solve the problem of light path congestion of fluorescent probes, based on the experimental process of "removable fluorescence", more targets can be detected without further development of detection equipment. When there are several fluorescent probes without light path overlap, the fluorescence signal in the sample can be "removed" by a certain method, and the same type of several fluorescent probes can be used to image new targets again, and theoretically unlimited targets can be detected in multiple cycles of imaging. Among them, nucleic acid materials (DNA) are ideal functional materials based on this idea due to their rich biological functions, programmability and modification at the molecular scale, and the specificity generated by the strict Waston-Crick base pairing rule. By controlling the length and base sequence of the nucleic acid, a large number of orthogonal oligonucleotides (ONs, a general term for short-chain nucleic acids) that do not interfere with each other can be easily designed and synthesized. The fluorescent probes (ONs fluorescent probes) prepared by coupling ONs with fluorescent materials can recognize antibodies modified with corresponding complementary ONs. The double-stranded ONs formed by complementation can be quickly washed out based on the DNA strand displacement technology or by adding 75% formamide solution to soak and remove the hybridization to achieve efficient removal of the fluorescence signal in the sample, and then introduce ONs fluorescent probes with different sequences to complete the fluorescence imaging of a large number of targets.

[0005] The current fluorescent probes used for multi-target immunofluorescence imaging are limited by the incompatibility of fluorescence intensity and imaging efficiency, which hinders their application in a wider range of biological sensing and detection. Due to the complex porous structure in cells, the sensitivity and specificity of fluorescent probes are severely affected by their size. For example, large-size fluorescent probes loaded with a large number of fluorescent molecules, as a kind of solid-phase micelle, naturally have the tendency to adsorb to the denatured hydrophobic domain of various proteins in cells. The complex structure in cells also limits the degree of free diffusion of the probe, causing serious non-specific binding phenomenon, affecting the specificity and sensitivity of imaging.

[0006] When the size of the fluorescent probe is limited in a small range, the total number of fluorophores contained in a single fluorescent probe is also limited, making the brightness of a single fluorescent probe lower, which affects the spatial and temporal resolution and sensitivity of fluorescence imaging. Therefore, additional fluorescence amplification steps are needed to accumulate multiple fluorescent probes on the same target to further increase the fluorescence brightness from the targeted target. However, such fluorescence amplification steps often require the post-assembly of complex nanostructures in cells, which reduces the imaging efficiency and increases the elution difficulty. Only when the fluorescent probe has sufficient fluorescence brightness, the complex amplification step can be omitted, and the specificity and sensitivity of the imaging can be ensured. Therefore, exploring the preparation of small-size fluorescent probes with extremely high fluorescence brightness has great potential for efficient multi-target imaging. As an organic material, π-conjugated polymer (CP) can be prepared into organic luminescent polymers with sufficient optical stability and diverse properties by adjusting its molecular structure and aggregation state. The fluorescent nanoparticles prepared by fluorescent π-conjugated polymer (FCP) have higher fluorescence brightness than quantum dots, providing the basic optical properties for sensitive fluorescence detection.

[0007] SAKA S K, WANG Y, KISHI J Y, et al. Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues [J]. Nature Biotechnology, 2019, 37(9): 1080-1090 provides a scheme of signal amplification by exchange reaction for immuno staining (Immuno-SABER), which prepares a series of DNA coded antibodies by coupling a plurality of designed ONs with corresponding antibodies respectively, uses primer exchange reaction (PER) to prepare a positive DNA concatemer that can recognize a specific DNA coded antibody, and prepares ONs modified with small molecule fluorescent dyes at the same time, combines the three to realize fluorescence imaging of the target. However, this scheme uses small molecule fluorescent dye modified ONs, which has the disadvantages of poor light stability, different signal amplification methods need to be taken for different abundance targets, expensive, etc., at the same time, the amount of small molecule dye is large, which is easy to produce significant background signal and cause "false positive".

[0008] 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. A computer-aided design of reversible hybridization chain reaction (CAD-HCR) scheme is provided, which first puts multiple DNA coded antibodies into the sample to label the target, and then adds 3 groups of hairpin ONs with different fluorescent dyes, which are combined with the corresponding priming chains to amplify the chain assembly at the target to realize fluorescent amplification imaging of the target. The scheme can currently use 3 different fluorescent dyes to image 3 targets simultaneously in a single imaging cycle. However, this scheme has the disadvantages of low imaging efficiency, long operation time required for fluorescent removal, and harsh conditions for DNA sequence design and preparation. At the same time, the imaging process of this scheme requires the assembly of nanostructures at the target, which requires a certain self-assembly time, which makes the detection time of this scheme longer.

[0009] CN112552490B provides a method for high-sensitivity and high-specificity detection of target nucleic acids using nucleic acid probes obtained by hybridizing nucleic acid assemblies with dye-modified DNA single-stranded S2. However, this method cannot detect multiple different target nucleic acids at the same time, and the imaging efficiency is low.

[0010] Therefore, it is urgent to find a technical solution to solve the defects existing in the prior art. SUMMARY

[0011] Based on this, the present application combines the unique biological recognition and codeability of ONs and the good optical properties of pi-conjugated polymers, and by designing and regulating the molecular structure of FCP polymer chains to adjust the fluorescence emission wavelength range, a plurality of FCP polymers with different fluorescence emission wavelength bands and without serious optical overlap are prepared. Then, ONs are modified on FCP to prepare ONs modified fluorescent pi-conjugated polymer nanoparticles (OCPNs) with different fluorescence emission wavelength bands by solvent displacement self-assembly. OCPNs have extremely high fluorescence brightness and adjustable particle size (10-100 nm), and can realize multi-target immunofluorescence imaging with high specificity, high sensitivity and high elution efficiency without additional fluorescence amplification steps.

[0012] One object of the present application is to provide an azide-modified fluorescent pi-conjugated polymer, the structure of which is as follows:

[0013]

[0014] R4 is independently selected from C1-C30 alkyl of linear or branched structure;

[0015] Ar unit is selected from aromatic ring, aromatic heterocycle, aromatic ring derivative, aromatic heterocycle derivative;

[0016] n is a natural number.

[0017] Further, Ar unit is selected from any one of the following structures:

[0018]

[0019] R2 and R3 are independently selected from one or more of C1-C30 alkyl of linear or branched structure, C1-C30 alkoxy of linear or branched structure, C1-C30 alkylthio of linear or branched structure, and C1-C30 silyl of linear or branched structure.

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

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

[0022] Another object of the present application is to provide a preparation method of azide-modified fluorescent π-conjugated polymer, comprising the following steps:

[0023] S1, mixing monomer M, monomer N, catalyst, base and solvent, and stirring and reacting under heating with inert gas protection, to obtain a fluorene precursor containing bromoalkyl after purification;

[0024] S2, blending and stirring the fluorene precursor containing bromoalkyl with NaN3 and solvent, and reacting, to obtain a product after purification;

[0025] The monomer M has the following general structure:

[0026]

[0027] X1 is independently selected from halogen atom;

[0028] X2 is independently selected from halogen atom and pinacol borate;

[0029] R1 is independently selected from C1-C30 alkyl of linear or branched structure;

[0030] The monomer N is selected from one of the following structures:

[0031]

[0032] R2, R3 are independently selected from one or more of C1-C30 alkyl of linear or branched structure, C1-C30 alkoxy of linear or branched structure, C1-C30 alkylthio of linear or branched structure, C1-C30 silyl of linear or branched structure.

[0033] 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.

[0034] Another object of the present application is to provide an oligonucleotide-modified fluorescent pi-conjugated polymer, whose structural general formula is as follows:

[0035]

[0036] R4 is independently selected from C1-C30 alkyl of linear or branched structure;

[0037] The Ar unit is selected from aromatic ring, aromatic heterocycle, aromatic ring derivative, aromatic heterocycle derivative;

[0038] P is an oligonucleotide;

[0039] x is selected from 0.1-0.6.

[0040] "ran" represents random combination, indicating that the entire polymer chain is combined by x units with DNA and 1-x units without ONs in random order, and the grafting of ONs does not change the polymer structure and polymerization degree, only part of the units are grafted with ONs, but the position is completely random.

[0041] Further, the Ar unit is selected from any one of the following structures:

[0042]

[0043] R2, R3 are independently selected from one or more of C1-C30 alkyl of linear or branched structure, C1-C30 alkoxy of linear or branched structure, C1-C30 alkylthio of linear or branched structure, C1-C30 silyl of linear or branched structure.

[0044] Another object of the present application is to provide a preparation method of an oligonucleotide-modified fluorescent pi-conjugated polymer, comprising the following steps:

[0045] L1, CPG-modified oligonucleotide is prepared by phosphoramidite chemistry synthesis method;

[0046] L2, introducing the alkyne group to the CPG modified oligonucleotide by using butynyl-phosphoramidite as the 5' end modification unit to obtain the alkyne modified CPG modified oligonucleotide;

[0047] L3, coupling the azide modified fluorescent pi-conjugated polymer with the alkyne modified CPG modified oligonucleotide by click chemistry, and then removing the CPG by aminolysis to obtain the oligonucleotide grafted fluorescent pi-conjugated polymer;

[0048] L4, transferring the oligonucleotide grafted fluorescent pi-conjugated polymer to the aqueous phase by a solvent displacement method to self-assemble to form the oligonucleotide modified fluorescent pi-conjugated polymer.

[0049] Further, in step L4, the self-assembly is that the oligonucleotide grafted fluorescent pi-conjugated polymer is added into 0.05-2M buffer solution to self-assemble to form the oligonucleotide modified fluorescent pi-conjugated polymer.

[0050] Another object of the present application is to provide a signal rapid amplification and high-throughput protein detection imaging method based on DNA modified pi-conjugated fluorescent nanoparticles, comprising the above oligonucleotide modified fluorescent pi-conjugated polymer.

[0051] The present application has the following beneficial effects: the OCPNs prepared by the present application have super-high fluorescence brightness (molar extinction coefficient reaches 107M -1 cm -1 , and quantum yield is 11-90%), which makes it have extremely high detection sensitivity, and can complete the detection and imaging of different abundance targets without additional fluorescence signal amplification steps. At the same time, the dense DNA shell of OCPNs makes it have extremely strong hybridization driving force, which can target the target more quickly, and combined with its super-high fluorescence brightness, it can complete rapid and specific targeting effect with extremely small amount, produce enough fluorescence signal and have significant signal-to-noise ratio, so that the entire staining and imaging process can be shortened to 2min. On the other hand, by adding a displacement chain, the OCPNs can be replaced in the original position to quickly remove the fluorescence signal, and this process can be completed within 30s, so the shortest single imaging cycle can be shortened to 2.5min. When using three kinds of OCPNs for multicolor imaging, nine different proteins can be imaged in three cycles, and the total time consumption is not more than 1h. The imaging efficiency is the fastest among known inventions.

[0052] The self-assembly process of the OCPNs is also optimized. The oligonucleotide grafted fluorescent pi conjugated polymer obtained after cleaving the CPG using ammonia is first blown dry in a slow argon stream, so that it exists in the form of a solid monodisperse brush polymer molecule before self-assembly occurs. Then, the oligonucleotide grafted fluorescent pi conjugated polymer ONs is dissolved in a solution to form nanoparticles by adding an aqueous solution containing a high ion concentration (10×PBS). The particle size of the nanoparticles formed by self-assembly is only related to the grafting rate of the oligonucleotide grafted fluorescent pi conjugated polymer ONs formed by coupling through click chemistry, and is not related to the subsequent solution treatment method. Compared with the existing technology, the self-assembly process does not need to control the solution drop speed, does not need to add additional solution components, and does not need to control the solution volume, so that a nanoparticle aqueous solution with uniform particle size can be obtained. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The synthesis and modification process of ONs is shown;

[0054] wherein,

[0055] Figure 1 (a) shows a chemical reaction process schematic diagram for realizing solid-phase synthesis of ONs based on phosphoramidite chemistry;

[0056] Figure 1 (b) shows a process schematic diagram for using butyne-phosphoramidite as a 5' end modification unit and coupling it to the ONs sequence;

[0057] Figure 2 The process of self-assembly to form OCPNs is shown;

[0058] wherein,

[0059] Figure 2 (a) shows the process of directly coupling the azide-modified fluorescent pi conjugated polymer and the alkyne-modified CPG-modified oligonucleotide (CPG-ONs) through click chemistry to obtain the oligonucleotide grafted fluorescent pi conjugated polymer (FCP-g-ONs);

[0060] Figure 2 (b) shows the process of transferring the FCP-g-ONs into the aqueous phase by solvent replacement to form OCPNs by self-assembly;

[0061] Figure 3 The performance detection results of OCPNs are shown;

[0062] wherein,

[0063] Figure 3 (a) shows the ultraviolet-visible light absorption spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0064] Figure 3 (b) shows the agarose gel electrophoresis detection results of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0065] Figure 3 (c) shows the fluorescence spectra of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs under the action of excitation light at 370 nm, 448 nm and 515 nm, respectively;

[0066] Figure 3 (d) shows the particle size distribution of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs detected by DLS particle size analysis;

[0067] Figure 3 (e) shows the transmission electron microscope (TEM) detection images of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0068] Figure 4 shows the fitting curves of the UV-visible absorption spectra, the hydrated particle size results, the ONs grafting rate and the CPG-ONs dosage of PFBT-OCPNs with different particle sizes, and the fitting curves of the hydrated particle size and the CPG-ONs dosage;

[0069] wherein,

[0070] Figure 4 (a) shows the UV-visible absorption spectra of PFBT-OCPNs with different particle sizes;

[0071] Figure 4 (b) shows the hydrated particle size results of PFBT-OCPNs with different particle sizes detected by DLS particle size analyzer;

[0072] Figure 4 (c) shows the fitting curves of the ONs grafting rate and the CPG-ONs dosage of PFBT-OCPNs with different particle sizes;

[0073] Figure 4 (d) shows the fitting curves of the hydrated particle size and the CPG-ONs dosage of PFBT-OCPNs with different particle sizes;

[0074] Figure 5 shows the absolute fluorescence quantum yield detection map of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs detected by photoluminescence spectrophotometer (FLS1000) at 370 nm, 445 nm and 488 nm, respectively;

[0075] Figure 6 Results of detection of antibodies, DNA conjugated antibodies, DNA conjugated antibodies + complementary DNA, and DNA conjugated antibodies + OCPNs using PAGE gel are shown;

[0076] Figure 7 Fluorescent imaging images of Lamin A / C, Ki67, a-tubulin, Calnexin, GM130, and mitochondria in HeLa cells after being stained by different ONs grafted PFBT-OCPNs are shown;

[0077] Figure 8 A schematic diagram of the light stability comparison of AF488 and PFBT-OCPNs, and a comparison diagram of fluorescent signals of different parts of cells labeled by AF488 and PFBT-OCPNs respectively under the continuous excitation of laser for 3 min are shown;

[0078] wherein,

[0079] Figure 8 (a) a schematic diagram of the light stability comparison of AF488 and PFBT-OCPNs is shown;

[0080] Figure 8 (b) a comparison diagram of fluorescent signals of Lamin A / C labeled by AF488 and a-tubulin labeled by PFBT-OCPNs under the continuous excitation of laser for 3 min is shown;

[0081] Figure 8 (c) a comparison diagram of fluorescent signals of a-tubulin labeled by AF488 and Lamin A / C labeled by PFBT-OCPNs under the continuous excitation of laser for 3 min is shown;

[0082] Figure 9 Fluorescent images of detection of mitochondria, Lamin A / C, and a-tubulin proteins by PFBT-OCPNs, AF488 secondary antibody, and AF488 modified ONs respectively, and a fluorescent intensity bar graph drawn after quantitatively counting the fluorescent intensity of each cell using ImageJ software are shown;

[0083] wherein,

[0084] Figure 9 (a) fluorescent images of detection of mitochondria, Lamin A / C, and a-tubulin proteins by PFBT-OCPNs, AF488 secondary antibody, and AF488 modified ONs respectively are shown;

[0085] Figure 9 (b) a fluorescent intensity bar graph drawn according toFigure 9 (a) The fluorescence intensity bar graph was plotted after quantifying the fluorescence intensity of each cell using ImageJ software in (a);

[0086] Figure 10 The schematic diagram and operation example of in situ chain displacement for signal removal are shown;

[0087] Figure 11 The fluorescence images of fast imaging and elution using OCPNs are shown;

[0088] wherein,

[0089] Figure 11 (a) The fluorescence images of detecting Vimentin protein using OCPNs are shown;

[0090] Figure 11 (b) The fluorescence images after removing fluorescence using in situ chain displacement are shown;

[0091] Figure 11 (c) The fluorescence images after adding OCPNs for detecting Lamin A / C for 10 s, 30 s, 1 min, 1 min 30 s, 2 min, and after washing are shown;

[0092] Figure 12 The fluorescence images of using 6 PFBT-OCPNs to image 6 different proteins are shown;

[0093] wherein,

[0094] Figure 12 (a) The process of using 6 PFBT-OCPNs to image 6 different proteins by monochrome 6 cycles is shown;

[0095] Figure 12 (b) The fluorescence images of using 6 PFBT-OCPNs to image 6 different proteins by monochrome 6 cycles are shown; Figure 12 (a) The collection of fluorescence images of 6 different proteins;

[0096] Figure 13 The schematic diagram of using OCPNs for multi-target fluorescence imaging and the fluorescence images of using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs to image 9 proteins in three cycles are shown;

[0097] wherein,

[0098] Figure 13 (a) The schematic diagram of using OCPNs for multi-target fluorescence imaging is shown;

[0099] Figure 13(b) shows fluorescence images of the imaging of a-tubulin, Lamin A / C, mitochondria; Calnexin, EGFR, Ki67; Lamin B1, Vimentin, GM130 using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs in three cycles;

[0100] Figure 13 (c) shows fluorescence images of the cell-scale nine proteins obtained by merging the fluorescence images in 13(b). DETAILED DESCRIPTION

[0101] In order to more clearly illustrate the technical solutions of the present application, the following examples are listed. The raw materials, reactions and post-processing methods appearing in the examples are all common raw materials on the market and technical means familiar to those skilled in the art, unless otherwise stated.

[0102] The experimental methods used in the following examples are conventional methods unless otherwise specified. The reagents, materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0103] The synthesis method of 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene in the embodiment of the present application is as follows:

[0104] 2,7-dibromofluorene (3.24 g), 1,8-dibromo octane (16.6 mL) and tetrabutylammonium bromide (TBAB) (322 mg) were added to a 250 mL three-necked flask, then NaOH aqueous solution (100 mL, 50 wt%) was added under magnetic stirring to mix, the mixture was heated to 75°C and condensed to reflux for 12 h. After the reaction was completed, the system was allowed to return to room temperature, 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 mixed solution was shaken and allowed to stand to re-layer, the valve was opened to collect the organic phase, then the water phase was removed, the above steps were repeated 3 times, the organic phase after washing was dried with anhydrous sodium sulfate, filtered, and DCM was removed by rotary evaporation to obtain a milky yellow oil. The milky yellow oil was removed by column chromatography (mobile phase: petroleum ether) to remove excess 1,8-dibromo octane and the remaining by-products to obtain white solid 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene (3.38 g, yield: 54%).

[0105] The synthesis method of 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacol borate) in the embodiment of the present application is as follows:

[0106] To a 100 mL two-necked flask, 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene (1 g), bis(pinacolato)diboron (1.08 g), potassium acetate (KOAc) (836 mg), palladium (Pd(dppf)Cl2) (62.1 mg) were added and mixed, the mixture was degassed and refilled with argon for 3 times, then toluene (20 mL) and aqueous K2CO3 solution (6.8 mL, 2.0 M) were added under argon atmosphere, the mixture was heated to 85 °C and stirred for 12 h. After the reaction was completed, the system was allowed to cool to room temperature, and the toluene was removed by rotary evaporation to obtain a 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 mixed solution was shaken and allowed to reseparate, the valve was opened to collect the organic phase, then the water phase was removed, the above steps were repeated 3 times, the organic phase after washing was dried with anhydrous sodium sulfate, filtered, and the DCM was removed by rotary evaporation to obtain a brownish oil. The brownish oil was removed by column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:12) to remove the byproduct to obtain a white solid 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacolato borate) (1.33 g, yield: 77%).

[0107] Example 1

[0108] A kind of azide modified fluorescent π conjugated polymer (PF-N3), its structural formula is shown as follows:

[0109]

[0110] The 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(pinacolato borate) (365 mg) and palladium tetraphenylphosphine (Pd(PPh3)4) (11.3 mg) were added to a 50 mL two-necked flask and mixed, the mixture was degassed and refilled with argon for 3 times, then toluene (11.3 mL) and aqueous K2CO3 solution (6.8 mL, 2.0 M) were added under argon atmosphere, the mixture was heated to 90 °C and stirred for 48 h to obtain a crude product. After the system cooled to room temperature, the crude product was added dropwise into ice methanol for precipitation, then Soxhlet extraction was performed with acetone, n-hexane and DCM in sequence, after concentration, re-precipitation was performed in ice methanol to obtain a yellow solid poly[(9,9-bis(8-bromooctyl)-2,7-diyl)-alt-co-(9,9-dioctylfluorene)] (PF-Br).

[0113] S2, PF-Br (100 mg) and NaN3(20 mg) were added into a 50 mL round bottom flask and mixed, the mixture was dissolved in a mixed solvent of DMF (5 mL) and THF (5 mL), and stirred at room temperature for 12 h. After the product was dissolved in DCM (50 mL) and transferred to a 100 mL separatory funnel, saturated brine (50 mL) was added for extraction, the mixed solution was shaken and then allowed to re-layer after standing, the valve was opened to collect the organic phase, and then the aqueous phase was removed, the above steps were repeated 3 times, and then the organic phase was washed with deionized water (50 mL) for 2 times, and the organic phase after washing was dried with anhydrous sodium sulfate, filtered, and rotary evaporated to remove DCM to obtain a yellow solid PF-N3.

[0114] Example 2

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

[0116]

[0117] The n is 9.

[0118] The preparation method is as follows:

[0119] S1, 2,7-dibromo-9,9-bis(8-bromooctyl)fluorene (400 mg), 4,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,1,3-benzothiazole (220 mg), and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (32.8 mg) were added into a 50 mL two-neck flask and mixed, the mixture was degassed and recharged with argon for 3 times, then toulene (11.3 mL) and aqueous K2CO3 solution (6.8 mL, 2.0 M) were added under argon atmosphere, the mixture was heated to 90°C and stirred for 48 h to obtain a crude product. After the system was cooled to room temperature, the crude product was added dropwise into ice methanol for precipitation, and then sequentially extracted with acetone, n-hexane, and DCM, concentrated and precipitated in ice methanol again 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), and then a component with a molecular weight of 5k-10k was screened by high performance liquid chromatography.

[0120] S2, PFBT-Br (75 mg) and NaN3(17 mg) with molecular weight of 5k-10k were added into a 50 mL round-bottom flask, the mixture was dissolved in a mixed solvent of DMF (5 mL) and THF (5 mL), and the reaction was stirred at room temperature for 12 h. After the product was dissolved in DCM (50 mL) and transferred to a 100 mL separatory funnel, saturated brine (50 mL) was added for extraction, the mixed solution was shaken and then allowed to re-layer after standing, the valve was opened to collect the organic phase, then the aqueous phase was removed, the above steps were repeated 3 times, and then the organic phase was washed with deionized water (50 mL) for 2 times. After washing, the organic phase was dried over anhydrous sodium sulfate, filtered, and DCM was removed by rotary evaporation to obtain an orange-yellow solid PFBT-N3.

[0121] Example 3

[0122] An azide-modified fluorescent π-conjugated polymer (PFTBT-N3) has the following structural formula:

[0123]

[0124] The n is 7.

[0125] The preparation method is as follows:

[0126] S1, 9,9-bis(8-bromooctyl)fluorene-2,7-bis(pinacolborate) (80 mg), 4,7-di(5-bromo-4-hexylthiophenyl-2-)-2,1,3-benzothiadiazole (62.6 mg), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (2.9 mg), TBAB (1.6 mg), and aqueous tetrabutylammonium hydroxide (2.2 mL, 40 wt%) were added into a 50 mL two-neck flask for mixing, the mixture was degassed and recharged with argon for 3 times, then toluene (6.7 mL) was added under argon atmosphere, the mixture was heated to 90°C and stirred for 2 h to obtain a crude product. After the system was cooled to room temperature, the crude product was added dropwise into ice methanol for precipitation, then sequentially extracted with acetone, n-hexane, and DCM, concentrated and re-precipitated in ice methanol to obtain a red-brown solid poly[2,7-(9,9-bis(8-bromooctyl)fluorene)-alt-(4,7-bis(thiophen-2-yl)benzo-2,1,3-thiazole)] (PFTBT-Br), and then a component with a molecular weight of 5k-10k was screened by high performance liquid chromatography.

[0127] S2, PFTBT-Br (48 mg) and NaN3(13 mg) with molecular weight of 5k-10k were added into a 50 mL round bottom flask, the mixture was dissolved in a mixed solvent of DMF (5 mL) and THF (5 mL), and the reaction was stirred at room temperature for 12 h. After dissolving the product in DCM (50 mL) and transferring it into a 100 mL separatory funnel, saturated brine (50 mL) was added for extraction. After shaking the mixed solution and allowing it to re-layer, the valve was opened to collect the organic phase, and then the aqueous phase was removed. The above steps were repeated 3 times, and then the organic phase was washed with deionized water (50 mL) twice. After washing, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to remove DCM to obtain a red-brown solid PFTBT-N3.

[0128] Example 4

[0129] A fluorescent pi-conjugated polymer modified by oligonucleotides (PF-OCPNs) has the following structure:

[0130]

[0131] The P is an oligonucleotide (ONs) with the sequence: AAAAA ATA AAC ACA ACC GAA;

[0132] The x is 0.5.

[0133] The preparation method is as follows:

[0134] L1, preparation of CPG modified oligonucleotides by phosphoramidite chemistry:

[0135] Universal CPG (50 mg, 1.5 pmol ONs can be prepared under 100% yield) was weighed into an ONs synthesis column, a sieve plate was inserted into the upper and lower holes of the synthesis column, and then inserted into the sample hole of the DNA synthesizer for fixation. The self-checking program of the DNA synthesizer was started to ensure that the pipeline and air pressure were normal. The sequence of the ONs to be prepared was input, and the preset program was started to automatically complete the preparation according to phosphoramidite chemistry. The program design is as follows:

[0136] (1) Wash the synthesis column with anhydrous acetonitrile (1.5 mL), and pump in argon after standing for 10 s;

[0137] (2) Wash with TCA Deblock deprotection agent (trichloroacetic acid / dichloromethane) (1.5 mL), stand for 1 min, and then wash with anhydrous acetonitrile (1.5 mL) to remove the 5'-hydroxyl protecting group DMT to obtain a free 5'-hydroxyl group. Repeat 3 times;

[0138] (3) flush in ETT Activator activator (5'-ethylmercaptotetrazole / acetonitrile) (1.5 mL) and let stand for 2 min to react, to activate the 3'-end of the phosphoramidite-protected nucleotide monomer to obtain a nucleoside phosphorous acid activation intermediate, which condenses with the free 5'-hydroxyl group of the base linked to the CPG, after which flush in anhydrous acetonitrile (1.5 mL) to clean, repeat 2 times;

[0139] (4) flush in 10% v / v acetic anhydride (tetrahydrofuran) (1 mL) and 10% v / v N-methylimidazole (10% v / v pyridine / tetrahydrofuran) (1 mL) mixed solution to react for 3 min to complete the capping reaction, to close the 5'-hydroxyl group not involved in the reaction in the previous condensation reaction, so that the short chain fragment containing the unreacted hydroxyl group cannot continue to react, and at the same time can be separated and removed during purification, after which flush in anhydrous acetonitrile (1.5 mL) to clean, repeat 3 times;

[0140] (5) flush in iodine liquid oxidant (0.05M I2 / pyridine / tetrahydrofuran / water) (1.5 mL), let stand for 3 min to react, to oxidize the phosphorous to a more stable phosphoric acid triester, after which flush in anhydrous acetonitrile (1.5 mL) to clean, repeat 3 times;

[0141] (6) repeat the above steps until all sequence synthesis is completed, after which the anhydrous acetonitrile can be removed by pumping in argon, to obtain a 5'-end DMT-protected CPG modified ONs white powder, which can be stored for a long time at -20°C.

[0142] L2, use butyne-phosphoramidite as a 5'-end modification unit to introduce an alkyne group to the CPG modified oligonucleotide chain to obtain an alkyne-modified CPG modified oligonucleotide:

[0143] Dissolve butyne-phosphoramidite in anhydrous acetonitrile to make a 0.1M solution, insert the synthesis column containing 50mg of CPG modified ONs powder with 5' end protected by DMT into the synthesis slot of the DNA synthesizer, after capping, the instrument checks the pump to restore the argon pressure. Control the DNA synthesizer to flush the Z number of external reaction glass tube with anhydrous acetonitrile (3mL), repeat 3 times, pump in argon to clean the pipeline and dry the Z number tube, then add butyne-phosphoramidite solution (0.1M) in the Z number tube at 300μL / synthesis column, reinsert the Z number tube into the DNA synthesizer and wait for the pressure to recover. Set the program to repeat steps (1) (2) (3) in L1, after flushing the liquid mixture in the Z number tube with anhydrous acetonitrile (800μL) into the synthesis column, react for 8min, then flush with anhydrous acetonitrile (1.5mL) for 3 times, continue to complete steps (4) (5) in L1 to complete the end capping, after flushing with anhydrous acetonitrile (1.5mL) for 3 times, continue to pump in argon until the powder is loose and falls off when tapping the synthesis column, determine that it is dry after taking out the synthesis column, seal and store at -20℃ for a long time.

[0144] L3, coupling of azide-modified fluorescent π-conjugated polymer with alkyne-modified CPG modified oligonucleotide through click chemistry, and then removing CPG through aminolysis to obtain oligonucleotide grafted fluorescent π-conjugated polymer:

[0145] (1) Prepare the storage solution:

[0146] Dissolve PF-N3 polymer in DCM to prepare a 500μM (monomer concentration, monomer relative molecular mass 858) storage solution, store the storage solution in a glass bottle with a bottle cap, seal the gap of the bottle cap with sealing glue when storing, wrap the entire glass bottle with aluminum foil to avoid light, avoid tilting the bottle, and then store in a -20℃ refrigerator for a long time;

[0147] (2) Graft ONs onto PF-N3:

[0148] CuI (1.9 mg), alkynylated CPG modified ONs (50 mg) and a magnetic stir bar were placed in a Schlenk tube, which was capped with a rubber septum and connected to a double manifold. The tube was evacuated for 3 min, backfilled with argon for 3 min, and this cycle was repeated three times. A slow stream of argon was maintained. DIPEA (50 μί) was dissolved in DMF (450 μί) to make a DIPEA-DMF solution, and HOAc (50 μί) was dissolved in DMF (450 μί) to make an OHAc-DMF solution. PF-N3 stock solution (750 μί) was removed from an ep tube (2 mL) and added to DMSO (500 μί), DMF (500 μί), OHAc-DMF (6 μί), and DIPEA-DMF (18 μί) in an ep tube (1.5 mL). The solution was mixed by slowly pipetting up and down with a 100 μί pipette tip. The solution was removed from the tip by flicking the tip to remove the air and then slowly injected into the Schlenk tube through the rubber septum. The Schlenk tube was immersed in an oil bath at 45 °C and stirred at 900 rpm for 12 h. After the reaction was complete, the solution was transferred to an ep tube (1.5 mL) for temporary storage;

[0149] (3) Removal of unreacted PF-N3 and reaction impurities:

[0150] The ep tube containing the reaction product was centrifuged at 12,000 rpm for 5 min, and the CPG-bound reaction product was collected at the bottom of the ep tube. The liquid was removed using a 1 mL pipette. DCM (1 mL) was added, and the product was mixed in the liquid under sonication. The DCM was removed by centrifugation at 12,000 rpm for 5 min. DMSO (1 mL) was added, mixed under sonication, and removed by centrifugation at 12,000 rpm for 5 min. This washing step was repeated three times. The product was washed with DCM (1 mL) and the liquid was removed. The residual DCM was blown off under a stream of argon to obtain a white powder deposited at the bottom of the ep tube;

[0151] (4) Cleavage of PF-g-ONs from CPG:

[0152] Concentrated aqueous ammonia (1 mL, 15-20% w / v) was slowly added to the ep tube containing the dried product. The ep tube was capped with a cap and sealed with Parafilm. The tube was clamped with a bomb clamp. The product was mixed with the solution under sonication. The ep tube was placed in a constant temperature shaker at 55 °C and 450 rpm for 2 h. The ep tube was removed and left to stand at room temperature until it reached room temperature. The bomb clamp was slowly removed, and the liquid was allowed to flow out. The remaining ammonia was blown off under a slow stream of argon to obtain the PF-g-ONs powder.

[0153] L4. Transferring the oligonucleotide-grafted fluorescent π-conjugated polymer to an aqueous phase by solvent exchange to self-assemble the oligonucleotide-modified fluorescent π-conjugated polymer:

[0154] In the ep tube, quickly add 10x PBS (1 mL), mix under ultrasonic, and centrifuge at 12000 g for 5 min to complete solvent replacement to allow PF-g-ONs to self-assemble into nanoparticles PF-OCPNs. Then remove the unreacted alkyne-modified ONs by ultrafiltration. Select a new ultrafiltration tube (4 mL, 50 kDa), first add 1x PBS (4 mL) in the filter core, ultrafiltrate at 5000 g for 10 min to complete washing, and remove the remaining liquid in the filter core. Use a syringe to suck the nanoparticle aqueous solution, avoid mixing the CPG precipitated at the bottom, and transfer it to the filter core. First add 1x PBS until the solution volume reaches 4 mL, ultrafiltrate the tube at 5000 g for 10 min, remove the filtrate in the collection tube. Then, add ultrapure water (3.5 mL) in the filter core, ultrafiltrate at 5000 g for 10 min, and detect the ONs concentration in the filtrate by NanoDrop, repeat the step until the detected ONs concentration is less than 1 ng / μL. Use a pipette (100 μL) to transfer the product to an ep tube (1.5 mL), add ultrapure water or 1x PBS to a solution volume of 500 μL, tightly cap the tube and seal it with sealing film, and it can be stored at 4°C for several years.

[0155] Figure 1 The synthesis and modification process of ONs is shown;

[0156] Among them,

[0157] Figure 1 (a) shows a chemical reaction process diagram for realizing solid-phase synthesis of ONs based on phosphoramidite chemistry; universal CPG (controllable microporous glass beads grafted with DMT-protected hydroxyl groups) is used as a solid-phase carrier for ONs synthesis, and DMT-protected phosphoramidite base monomers are sequentially reacted and coupled to the ONs chain based on phosphoramidite chemistry without catalysis. The reaction is efficient and rapid, and can automatically synthesize ONs chains with predetermined sequences. It needs to be noted that the reaction is very sensitive to water, and the introduction of a small amount of water will greatly reduce the reaction efficiency, so the reaction is carried out by a DNA synthesizer, and 0.5 MPa argon gas is used to maintain the instrument environment for more than 1 h for the first time, and then the ONs synthesis reaction is completed according to the preset program;

[0158] Figure 1 (b) shows a flowchart of using butyne-phosphoramidite as a 5' end modification unit and coupling it to the ONs sequence; the reaction can be completed by a DNA synthesizer based on phosphoramidite chemistry synthesis method;

[0159] Figure 2 The process of self-assembly to form OCPNs is shown;

[0160] Among them,

[0161] Figure 2 (a) shows the process of direct coupling of azide-modified fluorescent π-conjugated polymer with alkyne-modified CPG-modified oligonucleotide (CPG-ONs) by click chemistry to obtain oligonucleotide-grafted fluorescent π-conjugated polymer (FCP-g-ONs); using CuI "click" chemistry catalysis, the grafting of ONs to FCP is realized by coupling the alkyne on ONs with the azide on FCP, and then the CPG is removed from ONs by aminolysis method;

[0162] Figure 2 (b) shows the process of transferring FCP-g-ONs into water phase by solvent displacement method to self-assemble into OCPNs; the product in the filter core is transferred into an EP tube, which can be stored at 4℃ for more than 3 years.

[0163] Example 5

[0164] A fluorescent π-conjugated polymer modified by oligonucleotide (PFBT-OCPNs) has the following structural formula:

[0165]

[0166] The P is an oligonucleotide (ONs) with the sequence of: AAAAA GAC TCA ACG GAC TAT.

[0167] The x is 0.54.

[0168] The preparation method is as follows:

[0169] The difference between the preparation method of this example and that of 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) stock solution and applied in the subsequent steps, and the other preparation methods are the same as those of Example 4.

[0170] Example 6

[0171] A fluorescent π-conjugated polymer modified by oligonucleotide (PFTBT-OCPNs) has the following structural formula:

[0172]

[0173] The P is an oligonucleotide (ONs) with the sequence of: AAAAA TAG CTT ATC AGA CTG.

[0174] The x is 0.47.

[0175] The preparation method is as follows:

[0176] The preparation method of this example is different from that of example 4 in that in step L3(1), the PFTBT-N3 polymer is dissolved in DCM to prepare a 500 μΜ (monomer concentration, monomer relative molecular mass 936) stock solution and used in the subsequent steps, and the other preparation methods are the same as those of example 4.

[0177] Examples 7-8

[0178] Examples 7-8 are different from example 4 in that the sequence of oligonucleotide (ONs) P is replaced by, respectively:

[0179] CCAAT ACC TAA AGA CTG AAG;

[0180] TCTCC ACT GAA TAA CAA CAT;

[0181] The other preparation methods are the same as those of example 4.

[0182] Examples 9-14

[0183] Examples 9-14 are different from example 5 in that the sequence of oligonucleotide (ONs) P is replaced by, respectively:

[0184] CCAAT ACC TAA AGA CTG AAG;

[0185] AGAAC CAA GGA ACC TAG AAC;

[0186] ACAAC AAA CAA GGA CCC AAC;

[0187] GCAAC TAT CCA CAG AAA CAC;

[0188] AACAA AGG AAA TGG AAC TAA;

[0189] AAAAA ATA AAC ACA ACC GAA;

[0190] The other preparation methods are the same as those of example 5.

[0191] Examples 15-16

[0192] Examples 15-16 are different from example 6 in that the sequence of oligonucleotide (ONs) P is replaced by, respectively:

[0193] AACAA AGG AAA TGG AAC TAA;

[0194] ACAAC AAA CAA GGA CCC AAC;

[0195] Other preparation methods are the same as example 6.

[0196] Test example 1

[0197] Test method: UV-Vis absorption spectrum, agarose gel electrophoresis, fluorescence spectrum under different wavelength excitation light, particle size distribution and transmission electron microscopy (TEM) image detection of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs prepared in examples 4-6.

[0198] Figure 3 The performance detection results of OCPNs are shown;

[0199] Among them,

[0200] Figure 3 (a) shows the UV-Vis absorption spectrum of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0201] Figure 3 (b) shows the agarose gel electrophoresis detection results of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs;

[0202] Figure 3 (c) shows the fluorescence spectrum of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs under the action of 370nm, 448nm and 515nm excitation light respectively;

[0203] Figure 3 (d) shows the particle size distribution of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs detected by DLS particle size analysis;

[0204] Figure 3 (e) shows the transmission electron microscopy (TEM) detection image of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs, the scale is 100nm.

[0205] The test results show that OCPNs with a particle size of about 10nm can be prepared for three different FCPs, and the fluorescence spectrum is separated, which can be used for simultaneous detection.

[0206] Test example 2

[0207] Test method: By changing the amount of CPG-ONs (adjusting the molar ratio of CPG-ONs and azide functional groups on PFBT-N3 to 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, 8:1), the particle size of PFBT-OCPNs is regulated.

[0208] Figure 4 UV-Vis absorption spectra, hydrodynamic size results, fitting curves of ONs grafting ratio vs. CPG-ONs dosage and fitting curves of hydrodynamic size vs. CPG-ONs dosage of PFBT-OCPNs with different particle sizes are shown;

[0209] wherein,

[0210] Figure 4 (a) UV-Vis absorption spectra of PFBT-OCPNs with different particle sizes are shown; Figure 4 (b) Hydrodynamic size results of PFBT-OCPNs with different particle sizes detected by DLS particle size analyzer are shown; Figure 4 (c) Fitting curves of ONs grafting ratio vs. CPG-ONs dosage of PFBT-OCPNs with different particle sizes are shown; Figure 4 (d) Fitting curves of hydrodynamic size vs. CPG-ONs dosage of PFBT-OCPNs with different particle sizes are shown; the data of fitting represent the mean ± standard deviation (n = 3) of ONs grafting ratio and average hydrodynamic size statistically obtained under the same conditions.

[0211] The test results show that as the amount of CPG-ONs increases, the particle size of PFBT-OCPNs decreases, and the minimum particle size of PFBT-OCPNs prepared by the current method is about 10 nm, and the maximum grafting rate of ONs obtained is about 50%.

[0212] Test Example 3

[0213] Test method: The absolute fluorescence quantum yield of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs prepared in Examples 4-6 was detected at 370 nm, 445 nm and 488 nm respectively by photoluminescence spectrophotometer (FLS1000), and the quantum yield (QY) was automatically calculated by the test software, and the results are shown in Table 1:

[0214] Table 1 Optical information of three kinds of OCPNs and fluorescent probes with similar excitation and emission spectra

[0215]

[0216] Figure 5 The detection spectrum of the absolute fluorescence quantum yield of PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs detected by photoluminescence spectrophotometer (FLS1000) at 370 nm, 445 nm and 488 nm respectively is shown.

[0217] Test Example 4

[0218] Test method: PAGE gel was used to detect the antibody, DNA conjugated antibody, DNA conjugated antibody + complementary DNA, and DNA conjugated antibody + OCPNs. The OCPNs used were PFBT-OCPNs of Example 5.

[0219] Method for preparing DNA conjugated antibody:

[0220] Take one ultrafiltration tube with an inner core volume of 500 μL and a molecular weight cut-off of 50 kDa, add 1xPBS solution (500 μL) containing Tween-20 (0.1% v / v), shake, and ultrafiltrate at 12000 g, 4°C for 2 min. Remove the inner core solution using a pipette and remove the solution in the filter tube. Add 10xPBS (400 μL) to the filter core, then add 30 μg of antibody, and then ultrafiltrate at 12000 g, 4°C for 8 min, and remove the liquid in the filter tube. Use EDTA (2 μL, 0.5 M), TCEP (6 μL, 0.5 M) and 10xPBS (392 μL) to prepare a 7.5 mM TCEP solution, add the TCEP solution (360 μL, 7.5 mM) to the filter tube, gently mix the liquid using a pipette, then centrifuge at 1000 g for 10 s, and stand at room temperature for 30 min to complete the reduction of the antibody.

[0221] After ultrafiltration at 8000 g, 4°C for 8 min, remove the liquid in the filter tube. Add 1xPBS (450 μL), mix, and ultrafiltrate at 8000 g, 4°C for 8 min, remove the liquid in the filter tube. Add 1xPBS (450 μL), mix, and ultrafiltrate at 12000 g, 4°C for 8 min. Weigh the maleimide-modified ONs (60 μg, 2 times the mass of the antibody) and dissolve them in 10xPBS (100 μL), add them to the ultrafiltration tube, gently mix the liquid using a pipette, then centrifuge at 1000 g for 10 s, and stand at room temperature for 2 h to complete the conjugation.

[0222] Add 1xPBS (350 μL) and ultrafiltrate at 12000 g, 4°C for 8 min, remove the solution in the filter tube, then add 1xPBS (450 μL) and ultrafiltrate at 12000 g, 4°C for 8 min, repeat three times. Add 1xPBS (100 μL), gently mix the liquid using a pipette, then remove the filter core and place it in a new filter tube upside down, ultrafiltrate at 3000 g, 4°C for 2 min, and transfer the collected liquid to a 200 μL EP tube, and store it at 4°C.

[0223] Figure 6 The results of the detection of antibody, DNA conjugated antibody, DNA conjugated antibody + complementary DNA, and DNA conjugated antibody + OCPNs using PAGE gel are shown.

[0224] Lane 1-4 are the original antibody (the molecular weight of the antibody is generally 150 kDa, close to 110 kDa is the antibody after two light chain), DNA conjugated antibody, DNA conjugated antibody and complementary DNA incubation results, DNA conjugated antibody and OCPNs incubation results. From the results, lane 1-4 are no BSA band, which proves that there is no BSA; DNA conjugated antibody band position is higher than the original antibody, and the band position is higher after incubation with complementary DNA, which proves that the DNA conjugated antibody is successfully prepared, and can complete the corresponding hybridization function; DNA conjugated antibody and OCPNs simple mixing can lead to a OCPNs can hybridize multiple DNA conjugated antibody, form a structure with large size, so it can not enter the hole into the glue, also proves that the DNA conjugated antibody can complete the hybridization with OCPNs normally.

[0225] The test example results show that using the OCPNs of the application to detect DNA conjugated antibody can greatly reduce the amount of antibody in a single experiment (the amount of antibody in the literature is generally greater than 100 μg), and the efficiency of DNA conjugated on the antibody can almost reach 100%.

[0226] Test Example 5

[0227] Test method: The detection effect of OCPNs was tested by using HeLa cells. HeLa cells were seeded in 96-well plates at a density of 10000 cells per well in DMEM medium containing fetal bovine serum (10% v / v), 1% double-antibiotic, and incubated at 37°C in a CO2 (5% v / v) environment for 24 h. After that, the cells were washed with 1x PBS preheated to 37°C for 3 times, each for 3 min, and then fixed with polyformaldehyde (4% w / v) at room temperature for 20 min. Subsequently, the fixation process was quenched by incubating the cells with NH4Cl solution (100 mM) dissolved in 1x PBS for 20 min, and then the cells were washed with 1x PBS for 3 times, each for 5 min. Permeabilization was performed by using Triton X-100 (0.1% v / v) dissolved in 1x PBS for 10 min, and then the cells were washed with 1x PBS for 3 times, each for 5 min. Blocking was performed by adding blocking solution containing sheared salmon sperm (0.2 mg / mL), dextran sulfate (0.05% w / v), EDTA (4 mM) and BSA (5% w / v) to the cells at room temperature for 1 h, and then the DNA conjugated antibody was dissolved in the blocking solution and added to the cell sample for incubation at 4°C overnight. The next day, the cell sample was restored to room temperature, and the cells were washed with 1x PBS for 3 times, each for 5 min. Subsequently, the PFBT-OCPNs prepared in Example 5 and Examples 9-13 were dissolved in 2x SSC hybridization solution containing BSA (5% w / v), dextran sulfate (0.05 w / v) and glycine (0.3 M), and then added to the cell sample for incubation for 5-10 min. The cells were washed with 1x PBS for 3 times, each for 3 min, and then confocal microscopy was used for detection.

[0228] Figure 7 Fluorescence imaging graphs of Lamin A / C, Ki67, a-tubulin, Calnexin, GM130 and mitochondria in HeLa cells after being stained by PFBT-OCPNs prepared in Example 5 and Examples 9-13 are shown, and the scale is 40 μm.

[0229] The results of this test example show that OCPNs have sufficient detection sensitivity for different abundance of proteins.

[0230] Test Example 6

[0231] Test method: In the same cell sample, two different proteins Lamin A / C, α-tubulin were respectively labeled by AF488 secondary antibody and PFBT-OCPNs. 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 the action of excitation light at 488 nm, the fluorescence signals of AF488 and PFBT-OCPNs were detected at the same time. Under the action of continuous laser irradiation, the light stability of the two was compared.

[0232] Figure 8 The light stability comparison diagram of AF488 and PFBT-OCPNs and the fluorescence signal comparison diagram of using AF488 and PFBT-OCPNs to label different parts of the cell under the action of continuous excitation of laser for 3 min are shown.

[0233] wherein,

[0234] Figure 8 (a) shows the light stability comparison diagram of AF488 and PFBT-OCPNs;

[0235] Figure 8 (b) shows the fluorescence signal comparison diagram of using AF488 to label Lamin A / C and PFBT-OCPNs to label α-tubulin under the action of continuous excitation of laser for 3 min.

[0236] Figure 8 (c) shows the fluorescence signal comparison diagram of using AF488 to label α-tubulin and PFBT-OCPNs to label Lamin A / C under the action of continuous excitation of laser for 3 min.

[0237] The test results show that: (1) Under the action of continuous laser irradiation, the fluorescence of AF488 gradually quenches, and the fluorescence signal of PFBT-OCPNs still exists. (2) Under the action of continuous excitation for 3 min, it can be seen that the fluorescence signal of the target position labeled by AF488 decreases significantly. Figure 8 In (b), Lamin A / C labeled by AF488 secondary antibody is in the nuclear region. As can be seen from the fluorescence curve diagram detected before and after excitation along the yellow line, the fluorescence of the nuclear position decreases significantly, while the decrease of the rest is very small, which proves that the microtubule part labeled by PFBT-OCPNs has excellent light stability. Figure 8(c) The microtubules were labeled with AF488 secondary antibody. After continuous excitation, the microtubule position signal decreased significantly, while the LaminA / C signal labeled with PFBT-OCPNs in the cell nucleus did not decrease significantly. Both of these results indicate that PFBT-OCPNs have excellent photostability.

[0238] Test Example 7

[0239] Test method: The PFBT-OCPNs, AF488 secondary antibody and AF488 modified ONs prepared in Example 5 were used to detect Mitochondria, LaminA / C and α-tubulin proteins, respectively. The fluorescence amplification effect of PFBT-OCPNs, AF488 secondary antibody and AF488 modified ONs were compared.

[0240] Figure 9 The images show fluorescence images of Mitochondria, LaminA / C, and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody, and AF488-modified ONs, respectively, as well as fluorescence intensity bar graphs plotted after quantitative statistical analysis of fluorescence intensity for each cell using ImageJ software.

[0241] in,

[0242] Figure 9 (a) shows fluorescence images of Mitochondria, LaminA / C and α-tubulin proteins detected using PFBT-OCPNs, AF488 secondary antibody and AF488-modified ONs, respectively;

[0243] Figure 9 (b) shows the results according to Figure 9 The image in (a) is a fluorescence intensity bar chart plotted using ImageJ software after quantitative statistical analysis of the fluorescence intensity of each cell. 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. Statistical results show that the fluorescence intensity produced by PFBT-OCPNs is 3-11 times that of the AF488 secondary antibody.

[0244] 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.

[0245] Test Example 8

[0246] Signal removal is achieved by in situ strand replacement of DNA-encoded antibodies.

[0247] Test method: PFBT-OCPNs prepared in Example 9 were used to remove the signal by using displacement strands with more complementary sequences to the ONs on the antibody to bind to the DNA-encoded antibody.

[0248] Figure 10 A schematic diagram and operation example of in situ strand displacement for signal removal are shown. Scale bar: 40 pm.

[0249] After the displacement strands were added, the fluorescence signal was completely removed, because the displacement strands had more complementary sequences to the ONs on the antibody, and their binding force to the DNA-encoded antibody was stronger than that of the OCPNs. Therefore, after the displacement strands were added, the OCPNs could replace the OCPNs hybridized to the antibody, so that the OCPNs that had hybridized to the DNA-encoded antibody were dissociated into the solution and were washed away, thereby causing the fluorescence signal to disappear. At this time, the ONs on the antibody were completely hybridized and blocked by the displacement strands, and the re-addition of OCPNs could not hybridize to the DNA-conjugated antibody again, so the signal could not reappear.

[0250] The results of this test example show that OCPNs can avoid the residual fluorescence signal in multi-target imaging, which affects the imaging results.

[0251] Test Example 9

[0252] Fast imaging and elution using OCPNs.

[0253] Test method: First, PFBT-OCPNs prepared in Example 12 were used to detect Vimentin protein, and fluorescence signal detection was performed, and then in situ strand displacement was used to remove the fluorescence. Subsequently, PFBT-OCPNs prepared in Example 10 were added to detect Lamin A / C, and detection was performed at 10 s, 30 s, 1 min, 1 min 30 s, and 2 min after the addition of OCPNs, respectively.

[0254] Figure 11 Fluorescence images showing fast imaging and elution using OCPNs are shown.

[0255] Among them,

[0256] Figure 11 (a) shows fluorescence images of Vimentin protein detection using OCPNs;

[0257] Figure 11 (b) shows fluorescence images after removing fluorescence using in situ strand displacement;

[0258] Figure 11(c) shows the fluorescence pattern after 10s, 30s, 1 min, 1 min 30s, 2 min and washing after adding OCPNs for detecting Lamin A / C. The test results show that the fluorescence signal is completely removed within 15s after using in situ strand displacement, and the morphology of Lamin A / C can be seen after adding OCPNs for detecting Lamin A / C. The fluorescence image with higher signal-to-noise ratio can be obtained after washing directly after 2 min, which shows that the use of OCPNs for fluorescence imaging has high detection efficiency and high elution efficiency.

[0259] The test example results show that the time consumption of fluorescence imaging using OCPNs is within 2 min, and a fluorescence image with good signal-to-noise ratio can be obtained without washing. The removal of fluorescence signal (i.e. elution step) only needs time within 30s, which is more efficient than known imaging techniques.

[0260] Test Example 10

[0261] Test method: Six different proteins were imaged by single-color 6-cycle fluorescence imaging using six PFBT-OCPNs prepared in Examples 5 and 9-13.

[0262] Figure 12 shows the fluorescence images of six different proteins imaged by six PFBT-OCPNs respectively;

[0263] wherein,

[0264] Figure 12 (a) shows the process of fluorescence imaging of six different proteins by single-color 6-cycle using six PFBT-OCPNs;

[0265] Figure 12 (b) shows Figure 12 (a) a collection of fluorescence images of six different proteins.

[0266] The test example results show that for proteins with different abundances in cells, the six proteins can be imaged respectively without additional adjustment of OCPNs concentration, and the cell morphology is not damaged during the elution process.

[0267] Test Example 11

[0268] Meanwhile, different PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs were used to image nine different proteins, including a-tubulin, Lamin A / C, mitochondria; Calnexin, EGFR, Ki67; Lamin B1, Vimentin, GM130 in three cycles. The nine proteins were divided into three groups and imaged in turn. The first group was PF-OCPNs of Example 4 for a-tubulin imaging, PFBT-OCPNs prepared in Example 10 for Lamin A / C imaging, and PFTBT-OCPNs of Example 6 for mitochondria imaging. The second group was PF-OCPNs prepared in Example 7 for Calnexin imaging, PFBT-OCPNs of Example 5 for Ki67 imaging, and PFTBT-OCPNs prepared in Example 15 for EGFR imaging. The third group was PF-OCPNs prepared in Example 8 for Lamin B1 imaging, PFBT-OCPNs prepared in Example 12 for Vimentin imaging, and PFTBT-OCPNs prepared in Example 16 for GM130 imaging.

[0269] Test method: First, incubate the nine DNA-encoded antibodies in cells. Add PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs to detect three different proteins in each cycle. Then remove the fluorescence signal using in situ strand displacement. Next, add PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs to complete the fluorescence imaging detection of nine target proteins in three cycles.

[0270] Figure 13 The schematic diagram of multi-target fluorescence imaging using OCPNs and the fluorescence images of using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs to image nine proteins in three cycles are shown.

[0271] wherein,

[0272] Figure 13 (a) shows the schematic diagram of multi-target fluorescence imaging using OCPNs;

[0273] Figure 13 (b) shows the fluorescence images of using PF-OCPNs, PFBT-OCPNs and PFTBT-OCPNs to image a-tubulin, Lamin A / C, mitochondria; Calnexin, EGFR, Ki67; Lamin B1, Vimentin, GM130 in three cycles.

[0274] Figure 13 (c) shows the merged fluorescence images of the 9 proteins at the cellular scale in 13(b).

[0275] The results of the test example show that the total time for imaging the 9 proteins is less than 1 h, which exceeds all known multi-target fluorescence imaging techniques.

[0276] In summary, the OCPNs prepared by the present application have ultra-high fluorescence brightness and extremely high detection sensitivity, and can complete the detection and imaging of different abundance targets without additional fluorescence signal amplification steps; at the same time, the rapid and specific targeting effect can be achieved with very small amount of use, and the entire staining and imaging process can be shortened to 2 min. On the other hand, the fluorescence signal can be quickly removed by in situ replacement, so that the shortest single imaging cycle can be shortened to 2.5 min, and when 3 kinds of OCPNs are used for multi-color imaging, the imaging of 9 different proteins can be completed in 3 cycles, and the total time is not more than 1 h. This imaging efficiency is the fastest among known inventions, and has excellent application prospects.

[0277] It is apparent for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all aspects as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and all changes falling within the meaning and range of the equivalent elements of the claims are intended to be included in the present application.

[0278] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not contain only one independent technical solution, and the description manner of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be properly combined to form other embodiments that those skilled in the art can understand.

Claims

1. An oligonucleotide-modified fluorescent π-conjugated polymer, characterized in that, The structural formula of the oligonucleotide-modified fluorescent π-conjugated polymer is shown below: ； in, The value of x is 0.5; The P is an oligonucleotide with the following sequence: AAAAA ATA AAC ACA ACC GAA; or CCAAT ACC TAA AGA CTG AAG; or TCTCC ACT GAA TAA CAA CAT; or The structural formula of the oligonucleotide-modified fluorescent π-conjugated polymer is shown below: ； in, The value of x is 0.54; The P is an oligonucleotide with the following sequence: AAAAA GAC TCA ACG GAC TAT; or CCAAT ACC TAA AGA CTG AAG; or AGAAC CAA GGA ACC TAG AAC; or ACAAC AAA CAA GGA CCC AAC; or GCAAC TAT CCA CAG AAA CAC; or AACAA AGG AAA TGG AAC TAA; or AAAAA ATA AAC ACA ACC GAA; Alternatively, the structural formula of the oligonucleotide-modified fluorescent π-conjugated polymer is shown below: ； in, The value of x is 0.47; The P is an oligonucleotide with the following sequence: AAAAA TAG CTT ATC AGA CTG; or AACAA AGG AAA TGG AAC TAA; or ACAAC AAA CAA GGA CCC AAC; The "ran" represents random combination, indicating that the entire polymer chain is composed of x DNA-bearing units and 1-x units without oligonucleotides in a random order.

2. The method for preparing the oligonucleotide-modified fluorescent π-conjugated polymer according to claim 1, 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.

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

4. The application of the complex formed by the oligonucleotide-modified fluorescent π-conjugated polymer and DNA-coupled antibody as described in claim 1 in the preparation of a signal amplification and high-throughput protein detection imaging reagent based on DNA-modified π-conjugated fluorescent nanoparticles.

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