Gold Nanoparticle-based Chemiluminescent Detection Of Platelet-derived Growth Factor B-chain Homodimer And Adenosine Triphosphate

Chemiluminescence (CL) is a trace analysis method, which used optical radiation(chemiluminescence) generated by a chemical reaction to determine the mattercontent. This method has been growing in popularity because of its advantages suchas high sensitivity, rapid assay speed and simple instrumentation. Gold nanoparticleswere one of the most widely used nanomaterials because of rapid and simple chemicalsynthesis, easy preparation in a wide range of sizes and high activity of the labeledbiomolecules, which led to a better label molecular. This chemiluminescent assaydeveloped platelet-derived growth factor and adenosine triphosphate detectionmethods based on the colloidal gold as label. The research in my assay consists ofthree parts:Chapter1: Review-research progress of gold nanoparticles.Au NPs are particularly attractive in bioassay by virtue of their facile synthesisand biocompatibility. This chapter descripted the properties, synthetic method anddetection method of Au NPs.Chapter2: Hydroxylamine amplified gold nanoparticle-based aptameric systemfor the highly selective and sensitive detection of platelet-derived growth factor.A sensitive and selective aptamer-based chemiluminescent (CL) method for thedetermination of platelet-derived growth factor (PDGF)-BB using hydroxylamineenlarged gold nanoparticles (Au NPs) was developed. Rabbit anti-human PDGF-BBpolyclonal antibody was covalently coupled on the96-well plate that offers reactiveN-oxysuccinimide ester (referred to as NOS group) surface. In the presence of targetprotein, the biotinylated aptamer was captured on the96-well plate forming anantibody/PDGF-BB/biotinylated aptamer sandwiched complex, which was followedby the assembly of streptavidin coated Au NPs (streptavidin–gold). Au NPsassembled on the surface of96-well plate reacted with HAuCl4and NH2OH, whichenabled the catalytic deposition of gold metal onto the Au NPs surfaces. A hugenumber of Au3+ions were released from the hydroxylamine enlarged Au NPs afteroxidative gold metal dissolution, which was determined by a simple and sensitiveluminol CL reaction. The results showed that the detection limit of the assay is60pMof PDGF-BB (corresponding to6fmol in a100μL volume), which comparesfavorably with those of other PDGF-BB detection techniques. In addition, this aptameric CL biosensor demonstrated extraordinary specificity. And PDGF-BB hasbeen determined in diluted serum indicating the applicability of this assay.Chapter3: A cascade amplification strategy based on rolling circle amplificationand hydroxylamine amplified gold nanoparticles enables chemiluminescent detectionof adenosine triphosphate.A highly sensitive and selective chemiluminescent biosensor for adenosinetriphosphate (ATP) was developed by taking advantage of the ATP-dependentenzymatic reaction (ATP-DER), the powerful signal amplification capability ofrolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles(Au NPs). The strategy relies on the ability of ATP, a cofactor of T4DNA ligase, totrigger the ligation-RCA reaction. In the presence of ATP, the T4DNA ligasecatalyzes the ligation reaction between the two ends of the padlock probe, producing aclosed circular DNA template that initiates the RCA reaction with phi29DNApolymerase and dNTP. Therein many complementary copies of the circular templatecan be generated. The ATP-DER is eventually converted into detectable CL signalafter a series of processes, including gold probe hybridization, hydroxylamineamplification, and oxidative gold metal dissolution coupled with a simple andsensitive luminol CL reaction. The CL signal is directly proportional to the ATP level.The results showed that the detection limit of the assay is100pM of ATP, whichcompares favorably with those of other ATP detection techniques. In addition, bytaking advantage of ATP-DER, the proposed CL sensing system exhibitedextraordinary specificity towards ATP and could distinguish the target molecule ATPfrom its analogues. The proposed method provides a new and versatile platform forthe design of novel DNA ligation reaction-based CL sensing systems for othercofactors. This novel ATP-DER based CL sensing system may find wide applicationsin clinical diagnosis as well as environmental and biomedical fields.