Signal Amplification Strategy Based On Controlled Radical Polymerization For Sensitive Detection Of Tumor Markers

The sensitive,rapid and accurate detection of tumor-related bio-molecules in complex biological samples is of great significance for the study of cancer occurrence,development,diagnosis and treatment.Signal amplification technology plays an important role in improving the detection sensitivity of biosensors.Polymer signal amplification is based on the fact that the polymer chain is composed of thousands of monomer units,which contains many active functional groups or active units.Through polymer labeling,a large number of signal probes or functional groups that can react with signal probes can be introduced into the sensing interface,enabling the sensor to output strong signals when detecting trace target analytes,thereby improving detection sensitivity.Polymer-based signal amplification strategies have been widely used for sensitive detection of biomolecules.Focusing on the requirements of signal amplification methods for tumor marker detection,this paper has carried out the following four aspects of work based on controlled free radical polymerization:1.Highly sensitive detection ofMMP-2 based on electrochemically mediated ATRP signal amplification strategy.A peptide-based electrochemical biosensor was developed for sensitive and selective analysis of matrix metalloproteinase 2（MMP-2）by using the signal amplification strategy of electrochemically mediated atomic transfer radical polymerization（eATRP）.In this study,by linking anα-bromophenylacetic acid（BPAA）initiator to the end ofMMP-2 specific polypeptides,using Cu（Ⅱ）as catalyst and ferrocene methyl methacrylate（FMMA）as monomer,eATRP reaction was carried out under constant potential control,and ferrocene（Fc）was de novo polymerized on the electrode surface.The targetMMP-2 can specifically recognize and cleave peptide,resulting in a decrease in the electrochemical signal of Fc on the electrode surface.The experimental results show that there is a linear relationship between Fc oxidation peak current andMMP-2 concentration（8 fM～80 pM）.The detection limit was as low as 0.53 fM.At the same time,the peptide-based biosensor showed good performance in the determination ofMMP-2 in human serum samples.2.The electrochemical sensitive detection of miRNA-141 based on AGET ATRP signal amplification.An electrochemical sensing platform based on activators（Re）generated by electron transfer for ATRP（AGET ATRP）signal amplification technology was prepared for sensitive determination of miRNA-141,a marker of breast cancer and prostate cancer.In this experiment,hairpin DNA was immobilized on the benzoic acid modified electrode to capture the target miRNA-141,the sulfhydryl group was released from the open end of the hairpin DNA probe,and then theATRP initiator-modified gold nanoparticles were combined with the sulfhydryl group at the end of the hairpin DNA.By adding ascorbic acid reductant and copper（Ⅱ）catalyst,AGET ATRP reaction was initiated on the electrode surface,and a large number of monomers with Fc signal molecules were introduced into the sensor interface.The linear range of the biosensor is 10 aM to 10 pM,and the detection limit is 3.23 aM.At the same time,the sensor also shows good selectivity and stability.3.Sensitive electrochemiluminescence analysis of lung cancer marker miRNA-21 based on thermo-initiated RAFT signal amplification.Combining RAFT signal amplification technology with electrochemiluminescence technology,a thermo-initiated RAFT signal amplification electrochemiluminescence sensor was proposed to detect lung cancer tumor marker miRNA-21.In this experiment,the target miRNA-21 was recognized by sulfhydryl-functionalized peptide nucleic acid（PNA）capture probe.Through the coordination of Zr⁴⁺with the phosphate group and the carboxyl group,the chain transfer agent CPAD of RAFT was introduced into the phosphate group site of the side chain of miRNA-21.The initiator AIBN is decomposed to generate free radicals at 55℃,and reacts with the monomer N-acryloyloxysuccinimide（NAS）.After polymerization,the sensor is placed in luminol solution with amino groups for further reaction,thus the signal molecule with electrochemiluminescence performance is indirectly grafted on the polymer chain.Under the optimal experimental conditions,the electrochemiluminescence detection of miRNA-21 was carried out.The ECL intensity increased with the increase of target concentration.In the range of 10 aM to 1 pM,there was a good linear relationship between the electrochemiluminescence intensity and the logarithm of miRNA-21 concentration and the detection limit is 0.21 aM.4.Sensitive electrochemical sensor for miRNA-21 detection based on photo-induced charge/energy transfer reversible addition-fragmentation chain transfer（PET-RAFT）signal amplification.A greener signal amplification strategy based on Zn（TCPP）catalyzed PET-RAFT polymerization was proposed for the electrochemical detection of tumor marker miRNA-21.Based on the construction of the sensor in chapter four,after the target miRNA-21 was captured by PNA,a trithiocarbonate（CDTPA）was introduced at the phosphate group site of the RNA side chain through Zr⁴⁺.Since Zn（TCPP）can efficiently and selectively activate trithiocarbonate through electron transfer under blue light irradiation,the polymerization process of monomer FMMA can be controlled by adjusting the light source.The advantage of this reaction system is that PET-RAFT polymerization can be carried out under fully open conditions,Under the optimum experimental conditions,the detection range of miRNA-21 by the sensor is 10 aM to 0.1 nM and the detection limit is 4.48 aM.