Construction Of Anti-Fouling ECL Biosensor And Its Application In Tumor-Related Nucleic Acid Detection

Cancer has become one of the most dangerous factors that threaten the safety of human life and is of great significance to the early detection and diagnosis of cancer patients.Electrochemiluminescence(ECL)biosensors are widely used in the detection of tumor-related nucleic acids due to their advantages such as no external light source,simple operation,high sensitivity,and practical applications in many fields.Due to the low abundance of many tumor-related nucleic acids,and in order to achieve early diagnosis of tumors,it is very important to amplify the signal of the target.In this paper,two amplification programs were designed to achieve effective signal amplification for the target nucleic acid.In addition,because it is inevitable to face complex actual samples in the analysis and detection process,the non-specific adsorption of impurities therein will seriously affect the accurate response of the biosensor.For this reason,the anti-fouling peptide provide anti-fouling performance for the constructed biosensor.In this article,two electrochemiluminescence biosensors based on signal amplification and anti-fouling peptide have been constructed to realize the sensitive analysis and detection of p53 and miRNA nucleic acids.The specific schemes are as follows:(1)A sensitive anti-fouling ECL biosensor based on the nucleic acid cycle signal amplification program mediated by anti-fouling peptide and exo? was proposed.The peptide exhibits excellent anti-fouling performance,but due to its poor conductivity,polyaniline(PANI)was formed on the electrode surface through electropolymerization,which significantly enhanced the conductivity of the modified electrode,and made the sensor more sensitive to response electrochemical signal.The designed cyclic amplification program based on exo? could effectively amplify the target p53.This anti-fouling biosensor realized sensitive and accurate analysis and detection of DNAp53,with a linear range of 0.5 fM to 50 nM,and a detection limit of 0.06 fM.And it has realized the detection of the target in the actual complex sample,which is expected to play an important value in the fields of clinical diagnosis and biopathological analysis and research.(2)An anti-fouling electrochemiluminescence biosensor based on dual signal amplification design was proposed.A nucleic acid cycle amplification strategy based on DNAzyme was designed,and the first amplification process of the target was realized through the magnetic separation of anti-fouling magnetic beads.The output DNA generated by the nucleic acid cycle amplification program stimulated the chain hybridization reaction(HCR)process on the surface of the modified electrode to generate long double-stranded DNA.The ECL signal molecule Ru(bpy)₂(cpaphen)²⁺was inserted into the double-stranded DNA by electrostatic adsorption and generated ECL signal response.Titanium dioxide nanoneedles(TiO₂ NNs)are used as co-reaction promoters of Ru(bpy)₂(cpaphen)²⁺and tripropylamine(TPrA)systems,which can enhance the ECL luminescence intensity of Ru(bpy)₂(cpaphen)²⁺.However,due to the poor electrical conductivity of titanium dioxide,this article used conductive polymer PANI to wrap TiO₂ NNs to generate TiO₂ NNs/PANI composite nanomaterials.The introduction of PANI significantly compensated for the extremely poor conductivity of TiO₂ NNs and introduced functional groups for modification on the surface of the nanoneedles,enabling DNA to be modified to the surface of the composite material through amide bonds.The significant enhancement of Ru(bpy)₂(cpaphen)²⁺ECL luminescence realized the second signal amplification.The surface of the magnetic beads was wrapped with a dendritic peptide anti-fouling layer with a newly designed sequence.The anti-fouling peptide made the magnetic beads resistant to non-specific adsorption of impurities such as proteins in complex samples,thereby achieving ultra-sensitive and accurate target identification and signal response.The design of the dual signal amplification process made this biosensor exhibit ultra-sensitive analysis and detection performance for miRNA-21,with a linear range of 1 fM to 1 nM and a detection limit of 0.13 fM.Anti-fouling dendritic peptide made the sensor capable of analyzing complex real samples.This ultra-sensitive ECL biosensor based on dual signal amplification strategies and anti-fouling peptide was expected to realize clinical analysis and detection of actual samples in the future.