Aggregation Behavior Of Peptide-Conjugated Aggregation-Induced Luminescent Probes In Confined Space For Sensing To Tumor Markers

Tumor biomarkers play an important role in the diagnosis of tumors,and their qualitative and quantitative analysis can predict the natural course of tumors in a comprehensive and detailed manner,providing a basis for formulating the best treatment plan.Fluorescent bioprobes have the advantages of high sensitivity and specificity in the detection of tumor biomarkers so that attracted extensive attention from researchers.However,due to the complex tumor microenvironment and the low abundance of some biomarkers in the early stage of the tumor,the detection performance of fluorescent bioprobes in the clinic still needs to be continuously improved to achieve accurate tumor detection.With the rapid development of nanotechnology and interface analytical chemistry,new strategies for regulating the properties of fluorescent molecules have been developed at the micro-nano interface,which can improve the detection sensitivity and specificity of fluorescent bioprobes through fluorescence signal amplification.Given the special structure and optical properties at the micro-nano/biological structure interface,based on the summary of previous research results,this paper designed and prepared three kinds of peptide/AIEgens-conjugated biological probes for the detection of tumor biomarkers.The micro-nano/biological confinement space interface is used to regulate the aggregation behavior of AIEgens so that the fluorescence signal of AIEgens molecules can be output and amplified stably.Thus,it can be achieving high sensitivity and specificity of tumor markers in vitro and in vivo detection.The main contents of the thesis are as follows:（1）To achieve highly sensitive and specific detection of tumor biomarker matrix metalloproteinase-2（MMP-2）,a novel method based on peptide/AIEgens covalent probe MP and nano-confined space interface to modulate the molecular properties of AIEgens were designed（MP/NPs-SLIPS sensing system）.The hydrophilic probe MP was synthesized by covalently linking a positively charged hydrophilic polypeptide（RRRRRR-GG-PLGLAG-Pra-NH₂）and AIEgens Py TPA through a"click reaction".MP is cleaved under the catalysis of MMP-2 to generate hydrophobic Py TPA residues,which emit a fluorescent response signal of MMP-2 after aggregation.However,the random aggregation of Py TPA residues in an aqueous solution can cause weak and unstable fluorescence response signals.Nano-confined space interface,using electrostatic adsorption and liquid repulsion on the interface to control the aggregation behavior of Py TPA residues,effectively amplifying the fluorescence response signal and improving the stability of the fluorescence output signal,thereby improving the performance of the MP/NPs-SLIPS sensing system,such as sensitivity and specificity.The MP/NPs-SLIPS sensing system can be directly applied to the detection of MMP-2secreted by tumor cells,and can also be prepared into a high-throughput detection device to achieve high-efficiency detection of MMP-2.（2）To explore the effect of AIEgens molecular aggregates on fluorescence signal amplification,based on the molecular design and confinement space interface regulation in Chapter 2,this chapter designed a highly sensitive and specific TP/NPs-SLIPS sensing system for detection of tumor biomarker furin.The trimeric form of the peptide/AIEgens conjugated probe TP was synthesized from a positively charged hydrophilic trimeric peptide（3x RRRRRR-GG-RVRRCK-Pra-NH₂）and AIEgens Py TPE through a"click reaction".Hydrophilic TP is cleaved under the catalysis of Furin to generate hydrophobic Py TPE trimer residues,which emit a fluorescent response signal after aggregation.Aiming at the problem that the aggregation behavior of Py TPE monomers may be regulated by the weak force of the NPs and SLIPS confined space interface,resulting in poor fluorescence response signal enhancement.we introduced Py TPE trimer residues from the molecular structure design of the probe,due to increasing the hydrophobic aggregation and the positive charge of its molecules on the surface,which further enhance the electrostatic effect of the confined space interface regulating AIEgens so that effectively improving the aggregation behavior of AIEgens and its fluorescence response signal amplification effect.Since the Py TPE trimer residues show faster aggregation speed and higher aggregation degree than Py TPE monomers,this greatly improves the amplification effect of the fluorescence response signal and improves the sensitivity and specificity of TP/NPs-SLIPS sensing system.The TP/NPs-SLIPS sensing system can be directly applied to the ultrasensitive detection of Furin in various tumor cells,and can also be prepared into high-throughput detection devices and glass biochips to achieve high-efficiency and small-volume trace detection of Furin.（3）To explore the effect of biological confinement space interface regulating the molecular properties of AIEgens,based on the molecular design and confinement space interface regulation in the previous two chapters,this chapter designs a D-PA+PA+TP-K sensing system with a peptide/AIEgens conjugated probe TP-K and biological confined space interface regulating molecular action for highly sensitive and specific detection of tumor biomarker programmed death ligand 1（PDL1）.TP-K was synthesized from a self-assembling peptide（Pra-FFVLK）and AIEgens TPESPY through a"click reaction".After targeting PDL1 on the cell membrane,the D-PA aptamer undergoes a"bioorthogonal reaction"with the self-assembling short peptide PA and induces the aggregation of TP-K near PDL1 through the self-assembling peptide to generate a fluorescent response signal.To solve the problems of weak fluorescence turn-on signal and low efficiency caused by the low aggregation degree of AIEgens probes in targeting targets,we introduced positive charges and hydrophobic alkyl chains from the molecular structure design of the probes and utilized the confined space of the cell membrane.The charge on the interface and the hydrophobic region of the liposome generate electrostatic adsorption and hydrophobic interaction with TP-K,which enhances the aggregation of TP-K near PDL1 on the cell membrane,and effectively amplifies the fluorescence response signal of PDL1,thereby realizing the highly sensitive and specific detection of PDL1 on cell membranes.The D-PA+PA+TP-K sensing system can be used to quantitatively detect the expression levels of up-regulated,normal,and down-regulated PDL1 on the tumor He La cell membrane,thereby establishing an accurate evaluation platform for PDL1 on the cell membrane of different tumor cell lines.Then,it is hoped that we will continue to explore and build an integrated platform for the diagnosis and treatment of PDL1.