Design Of Biosensors Using Dual-functional Organic Molecules-based Assembles

Organic molecules can assemble into different nanostructures with photo-or electroactivity functions through metal coordination bond,hydrophobic interaction,π-πstacking interaction,electrostatic interaction,hydrogen bond,or van der Waals force.Materials formed by the assembly of organic molecules have received wide widespread attention in various fields,such as material science,chemistry,and biomedicine.Among them,metal organic frameworks（MOFs）constructed by metal-ligand coordination bond,covalent organic frameworks（COFs）constructed by covalent bond,hydrogen-bonded organic frameworks（HOFs）constructed by hydrogen bond,and organic molecular assemblies constructed by multiple noncovalent forces are several types of the most representative assembly materials.Various nanostructures with specific functions can be synthesized through the rational design of building blocks based on the close relationship of organic molecules and the functions of nanostructures.In this thesis,under the guidance of the properties of molecular assembly materials and the structural characteristics of analytes,several organic assemblies with dual functions were prepared and applied for the sensitive detection of biomolecules.Briefly,MOFs formed by metal-ligand coordination interactions and molecular assemblies constructed by multiple noncovalent forces with dual functions were prepared and utilized for the development of electrochemical and fluorescence biosensors.The detailed research contents are as follows:1.Based on the molecular structure characteristics of glycoproteins and boronate affinity interaction,dual-funtional MOFs were synthesized for the recognition and signal output of glycoprotein.First,Cu-MOFs were prepared using 2-aminoterephthalic acid as the organic linker and Cu²⁺as the metal ion through the hydrothermal method,and further modified with4-formylphenylboric acid（FPBA-Cu-MOFs）via the Shiff base reaction.Then,the formed FPBA-Cu-MOFs were used as electroactive labels for the sensitive detection of glycoprotein prostate specific antigen（PSA）.In this work,PSA was captured by the aptamer-modified electrode and further labeled by electroactive FPBA-Cu-MOFs throught the boronate interaction between boronic acid and cis-diol in glycan.The concentration of PSA was quantified by directly measuring the reduction peak of large numbers of Cu²⁺ions in FPBA-Cu-MOFs captured on the electrode surface.This electrochemical biosensor for the detection of PSA exhibited a linear range from 0.01 to 25 ng/m L,and the minimum detectable concentration was lower than that of PSA in male serum samples.2.Based on the characteristic that commercially available proteins contain the oligohistidine（His₆）tag and glycosyl chain simultaneously,a biorecognition element-free electrochemical method was developed for the detection of recombinant glycoprotein using NTA-Ni²⁺complex modified-magnetic beads(MBs-NTA-Ni²⁺)and FPBA-Cu-MOFs.MBs-NTA-Ni²⁺were employed to capture and enrich target recombinant glycoprotein human erythropoietin（rhu EPO）via the metal chelation interaction between NTA-Ni²⁺complex and His₆ tag in rhu EPO.Then,glycans in captured rhu EPO were labeled by electroactive FPBA-Cu-MOFs for signal output.This electrochemical method with a linear range from 0.01to 50 ng/m L did not require the use of any biorecognition elements（e.g.aptamer and antibody）,providing important information for the design and application of novel biosensors.3.Most of proteins in commercial immunoassay kits are recombinat and engineered with His₆ tags on the N-or C-terminal.Based on this fact,this paper proposed a strategy for the immobilization of recombinant proteins based on the metal chelation interaction and then developed competitive biosensors,in which the His₆ tag in recombinant protein could interact with the coordinatively-unsaturated Cu²⁺ions present on the surface of Cu-MOFs.The resulting recombinant proteins-modified Cu-MOFs serving as the specific signal labels could be captured by antibodies on the sensing electrode,thereby generating a strong electrochemical signal.Once antibodies bound to the target in samples,the immobilization of recombinant protein-modified Cu-MOFs on the electrode was limited,leading to a decrease in electrochemical signal.With nucleocapsid protein（N-protein）as the model analyte,this method achieved the linear range from 1 pg/m L to 1 ng/m L.Based on the structural characteristics of recombinant proteins in the commercial kits,this method is expected to be used for the detection of other protein biomarkers.4.This work investigated the existence states of aggregation-induced emission（AIE）molecules with a single charged or reactive active group under different organic solvent contents.It was found that AIE molecules existed in the form of small aggregates or nanoclusters in aqueous solution with low organic solvent content,but the fluorescence signal was weak.The small aggregates with AIE property and target-responsive ability can be used as dual-functional signal probe.Upon the introduction of external stimuli,they could further assemble into larger aggregates through electrostatic interactions or covalent bonds,accompanied with an obvious increase in the fluorescence signal.Using tetraphenylethene-substituted pyridinium salt（TPE-Py）as an example,the effect of organic solvent content on the stimuli-induced assembly behavior of TPE-Py were studied.Pyrophosphate ion（PPi）were selected as the external stimuli and the enzymatic substrate,and the system of TPE-Py/PPi was applied to the determination of alkaline phosphatase（ALP）activity.Furthermore,a“signal-on”competitive immunoassay was developed for the detection of N-protein by coupling the TPE-Py/PPi system with the Cu-MOFs/ALP-labeled immunoassay.5.Phenylalanine-containing organic molecules can self-assemble into different nanostructures with specific functions through hydrophobic andπ-πstacking interaction.This work designed and synthesized phenylalanine derivatives labeled with biotin and pyrene at both ends（denoted as bio-Py F）.Bio-Py F can self-assemble into nanoparticles（bio-Py FNPs）with the spefici recognition and signal output ability.A dual-signal immunosensor was constructed for the detection of alpha fetoprotein（AFP）based on the SA-biotin interaction and the streptavidin（SA）-assisted in-situ self-assembly of bio-Py FNPs on the sensing surface.In the presence of target AFP,the sandwich-type immunocomplexs of capture antibodies,AFP and biotinylated detection antibodies were formed on the sensing interface.Biotin molecules conjugated with detection antibodies could sequentially capture the tetramer protein SA and bio-Py FNPs in the solution.The assembly process occurred repeatedly,and（SA-bio-Py FNPs）_n networks were in-situ formed on the sensing surface.Bio-Py FNPs in the network could release a large number of assembly units after the treatment with methanol.Then,fluorescence and electrochemical techniques were used to quantify those monomers,achieving the dual-signal detection of AFP.The linear ranges of fluorescence and electrochemical methods were found to be 0.001～2.5 ng/m L and 0.01～2 ng/m L,respectively.This work can provide a new idea for the design,synthesis,and analytical application of molecular self-assembly materials.