The Sensing Analysis And Research Of The Novel Nano-Electrochemistry For P16^INK4a Gene And Its Expression Protein

Malignant tumor is one of the main reasons that threaten human health.Tumor biomarkers are quantifiable indicators of specific cancer status,including DNA,RNA,proteins,lipids,metabolites and other biological active substances and factors.The analysis and evaluation of tumor biomarkers can be used to study the occurrence and development of cancer at cellular and molecular level,which provides the basis for the formulation of relevant measures to prevent cancer,and has profound significance for the comprehensive prevention and treatment of cancer.Traditional analysis methods of tumor biomarker have great limitations in sensitivity,speed and detection cost.Biosensor is a new technology which uses bioactive materials as identification elements to transform biochemical reactions into quantifiable physical/chemical signals,and can be used to detect and monitor the bioactive and chemical substances.At present,according to the different physical and chemical transducers,the common biosensors can be classified into optical,piezoelectric and electrochemical types.The electrochemical biosensor is the most potential method for detecting tumor biomarkers,which has advantages of simple and rapid,high sensitivity,good selectivity,low cost,unaffected by the color and turbidity of the sample,and simple instrument and equipment.In recent ten years,electrochemical biosensor of tumor biomarkers has developed rapidly.However,it still needs to be further strengthened and improved in terms of sensitivity and selectivity in the face of complex biological samples with very low concentration of the target.In view of the key problems of tumor biomarker analysis and detection,which are low sensitivity,complex detection and poor specificity,we start from the two aspects of the biomolecular fixed carrier material and electrochemical detection signal amplification strategy to establish a novel nano-electrochemical sensing system in the paper.The functional nanocomposite modified electrodes with large specific surface area,fast electron transfer rate and good biocompatibility are used to construct a new type of biomolecular immobilization interface.Rapid and simple continuous amplification of electrochemical detection signals can be obtained through the codoped alloy core-shell optical composite nanoparticles/enzyme-elec-tronic medium polymer catalytic immune system.The novel nano-electrochemical sensing system with high sensitivity for detection of the tumor biomarkers was established by combining new type of biomolecular immobilization interface,multiple signal amplification,and high specificity and sensitivity of base complementary hybridization/double site sandwich method.It provides a new idea for the analysis and detection of tumor biomarkers.The thesis was consisted of five chapters and the specific contents were as follows:Chapter 1 IntroductionThe present situation of the traditional and the new type detection technologies of p16^INK4aNK4a gene and its product protein at home and abroad were summarized and compared in the chapter.Specifically,the latest applications and shortages of the electrochemical biosensors of p16^INK4aNK4a gene and its product protein were mainly described.The research purpose,the innovative features and significance of this paper were given.Chapter 2 An electrochemiluminescence biosensor for detection of the p16^INK4a gene based on the NPTh modified electrodeIn this work,a nano polythionine?NPTh?-based universal sandwich-type electrochemi-luminescence?ECL?biosensor for sensitive and specific detection of the p16^INK4a gene using core-shell luminescent nanocomposites as excellent DNA labels was constructed.The NPTh electrode was prepared by in situ electropolymerization technique,which was served as effective biosensing substrates for ssDNA1 immobilization by charge desorption.Meanwhile,tri?2,2'-bipyridyl?ruthenium?II?(Ru?bpy?₃²⁺)/silver nanoparticles?AgNPs?doped gold?Au?core-shell nanocomposites?RuAg@AuNPs?were utilized as excellent ssDNA2 labels and exhibited excellent ECL behaviors on the NPTh electrode.The construction of dsDNA was fabricated through a hybridization reaction of ssDNA1,p16^INK4a gene and RuAg@AuNPs labled ssDNA2?RuAg@Au-ssDNA2?.The change of ECL intensity on the NPTh electrode was found to have a linear relationship in respect to the logarithm of the p16^INK4a gene concentrations in the wide range of 1.0?10^-131.0?10^-10 mol/L,with a detection limit of 5.0?10^-1414 mol/L?S/N=3?.The presented biosensor can discriminate the mismatch with single bases sequences.Chapter 3 An electrochemiluminescence biosensor for detection of p16^INK4a gene based on the PG/GR/CS/PPy-modified SPCEIn this work,we offered a novel and sensitive ECL biosensing system for detection of p16^INK4a gene using the functional paste-like nanofibers composites-modified screen-printed carbon electrode?SPCE?.The paste-like nanofibers composites?PG/GR/CS?which were comprised of the electrospun nanofibers?PG,the graphene?GR?doped polycaprolactam 6?PA6?were prepared via one-step electrospinning?,graphene?GR?and chitosan?CS?were served as the nanosized backbones for pyrrole?Py?electropolymerization.The functional paste-like nanofibers composites?PG/GR/CS/PPy?used as a substrate for dsDNA(hybridization reaction of ssDNA1,p16^INK4a gene and RuAg@AuNPs-ssDNA2)immobilization.Under optimal conditions,a linear relationship between ECL intensity and p16^INK4a gene concentration was found in a range of 1.0?10^-131.0?10^-9 nmol/L with the detection limit of 5.0?10^-1414 mol/L?S/N=3?.The presented biosensor with wide linear range can be regenerated and has great potential for practical application.Chapter 4 An electrochemiluminescence biosensor for detection of p16^INK4a gene based on the PA6-MWCNTs-SiO2 modified electrodeAn ECL biosensor based on functional electrospun nanofibers for hybridization detection of specific p16^INK4a gene at trace level via binding luminescent composite nanoparticles for signal amplification has been developed.The carboxylated multiwalled carbon nanotubes?MWCNTs?doped PA6 electrospun nanofibers?PA6-MWCNTs?was prepared via electrospinning,which served as the nanosized backbones for silica nanoparticles?SiO₂?electrodeposition.The electrochemical surface areas of PA6-MWCNTs-SiO₂ modified electrode is about 4 times higher than that of SiO₂ modified electrode.The PA6-MWCNTs-SiO₂ modified electrode used as supporting scaffolds for single-stranded DNA1?ssDNA1?immobilization can dramatically increase the amount of DNA attachment and the sensitivity of hybridization.It was observed that high amount of doped Ru?bpy?₃²⁺in RuAg@AuNPs successfully amplify the recognition signal by adding tripropylamine?TPrA?.The change of ECL intensity was found to have a linear relationship in respect to the logarithm of the p16^INK4aNK4a gene concentrations in the wide range of 1.0?10^-151.0?10^-1212 mol/L,with a detection limit of5.0?10^-1616 mol/L?S/N=3?which is reduced by two orders of magnitude.Chapter 5 An electrochemical immunosensor for detection of AG_p16INK4a protein based on the PG/GR/CS/PMB-modified electrodeIn this work,we offered a novel and sensitive electrochemical immunosensor for detection of AG_p16INK4a protein based on the functional paste-like nanofibers composites?PG/GR/CS/PMB?.The paste-like nanofibers composites?PG/GR/CS?were served as the nanosized backbones for carboxylation MWCNTs doped methylene blue?MB?electropolymerization.The functional paste-like nanofibers composites?PG/GR/CS/PMB?used as a substrate for Ab₁.After a sandwich immunoreaction,AG_p16INK4a and horseradish peroxidase?HRP?-labeled detection antibody?HRP@Ab₂?captured onto the electrode surface produced an amplified electrocatalytic response by the PMB?poly?methylene blue??-HRP-hydrogen peroxide?H₂O₂?.The increase of response current was proportional to the AG_p16INK4a concentration in the range of1 pg/mL to 1 ng/mL and the detection limit was down to 0.5 pg/mL?S/N=3?.