Gold Nanoparticle Based Chemiluminescent Sequence-Specific DNA Detection

Medical reports indicate that most of the cancers and genetic diseases are associated with genetic mutation.Therefore,it is very important to develop sequence-specific DNA biosensors detection techniques due to their significant advantages in various fields such as genetic screening,forensic analysis,and the early-stage identification of genetic disorders.DNA analysis has recently attracted much attention,and numerous assays such as electrochemical,optical,fluorescent,radiochemical,chemiluminescent(CL) have been developed due to its short assay time,low sample consumption and reduced overall cost.Each has its own pros and cons.Amount these techniques,electrochemistry, enzymic method,and CL method have been developing quickly in recent years.Chemiluminescence(CL) has been exploited within a wide range of applications in various fields,due to their extremely high sensitivity along with their extra advantages such as simple instrumentation,wide calibration ranges,and suitability for miniaturization in analytical chemistry.Gold nanoparticle is one of the ideal biological tags,due to the advantage of simple protocol,cheap price,stable character of the prepared nanoparticle tags,and retention of the biochemical activity of the labeled biomolecules.Hence,a sequence-specific DNA assay based on gold nanoparticle as label has been developed.Description of research in my thesis is presented as follows: (1) This thesis first addresses the need to develop sequence-specific DNA detection techniques,and then presents current state of knowledge on different classes of labels including nanoparticle,DNAzyme,and enzyme based DNA detection assay. (2) Sequence-specific DNA CL detection based on magnetic beads and gold nanoparticles:Virus hepatitis type B,as a world-wide disease,has attracted great attention of public and government agencies.It is important to develop a practical and effective method to diagnose,treat Virus hepatitis type B,and control its infections in public.In this chapter,we develop a novel DNA CL detection protocol based on magnetic beads and gold nanoparticles,by using a 35 based HBV-DNA as target DNA.First,the amine-modified capture DNA are immobilzed on carboxyl terminated magnetic beads. Then,the target DNA are hybridized with magnetic beads-linked capture probes, followed by the hybridization of the biotin-terminated reporter probe and the binding of streptavidin-coated gold nanoparticles.The amounts of target DNA were determined by the CL reaction with Luminol-AgNO3 system.The results showed a good linear correlation in the concentration range of 0.1-100 fmol(R²=0.9819),and the detection limit was estimated to be 50 amol. (3) Sequence-specific DNA CL detection based on 96-well plates and gold nanoparticles: Traditional methods for gold nanoparticles detection are comparatively time-consuming and complicated,and often required specialized and cumbersome technical processing.Thus an ultrasensitive,simple and user-friendly technique for the detection of DNA hybridization is in urgent need.In this chapter,we develop a novel DNA CL detection method and its amplification technique based on 96-well plates and gold nanoparticles.First,the amine-modified capture DNA is immobilzed on the surface of 96-well plates.Then,the target DNA is hybridized with capture probes,followed by the hybridization of the biotin-terminated reporter probe and the binding of streptavidin-coated gold nanoparticles.Gold nanoparticles that assembled on the surface of the 96-well plates were catalytically enlarged in the presence of NH₂OH and HAuCl₄ at room temperature for 20 min.The amounts of target DNA were determined by the CL reaction with Luminol-AgNO₃ system.The results showed a good linear correlation in the concentration range of 0.0025-1 fmol(R²=0.9874),and the detection limit was estimated to be 0.8 amol.