Rolling Circle Amplification Based Chemiluminescent Detection Of Platelet-derived Growth Factor And Adenosine Triphosphate

Chemiluminescence(CL)is a trace analysis method,which has been widely used in detecting drug,heavy metal ion,protein and nucleic acid,because of its advantages such as simple instrumentation,rapid assay speed,high sensitivity and wide linear range.Rolling circle amplification(RCA)is an isothermal amplification method.Owing to its high selectivity,high throughput,wide range of application and simple operation,RCA has been growing in popularity.This chemiluminescent assay developed platelet-derived growth factor and adenosine triphosphate detection methods based on the RCA.The research in this assay consists of three parts:Chapter 1: Review-research progress of RCA.This chapter described the principle,advantages and classification of RCA,and the application of RCA in biosensors and nanomaterials.Chapter 2: An aptamer-based chemiluminescence method for ultrasensitive detection of platelet-derived growth factor by cascade amplification combining rolling circle amplification with hydroxylamine-enlarged gold nanoparticles.A cascade amplification strategy for ultrasensitive CL detection of platelet-derived growth factor(PDGF)-BB(PDGF-BB)was developed based on RCA and hydroxylamine-amplified gold nanoparticles(Au NPs).The strategy takes advantage of using aptamers as recognition elements,and combining inherent signal amplification of the RCA reaction with HAuCl4–NH2OH redox reaction-based catalytic enlargement of assembled Au NPs.The enlarged Au NPs were then dissolved by an acid dissolution and the released gold ions were detected by a sensitive luminol CL reaction.The proposed CL detection system exhibits extraordinary sensitivity and high specificity.The proposed CL method exhibited a broad linear range over 3 orders of magnitude and high sensitivity with a detection limit of 0.06 pM.In addition,the proposed method demonstrated extraordinary specificity towards PDGF-BB and could be applied to detecting PDGF-BB in diluted human serum samples.Chapter 3: A label-free chemiluminescent strategy for highly selective and sensitive detection of adenosine triphosphate by cofactor-dependent enzymatic ligation-triggered rolling circle amplification.A simple and sensitive sensing system for label-free CL detection of adenosine triphosphate(ATP)was developed by taking advantage of graphene oxide(GO)nano-platform,instantaneous derivatization of guanine bases and powerful signal amplification capability of RCA.In the presence of ATP,the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP.Therein,many complementary copies of the circular template can be generated.The formed long single-stranded DNA which contained an amount of guanine bases could adsorb on the surface of GO forming DNA-GO complexes.And the CL signal of DNA-GO complexes can be obtained via the instantaneous derivatization reaction between phenylglyoxal(PGO)and guanine bases.The CL signal increased linearly with the concentration of ATP from 0.1 to 2.5 nM with a detection limit of 0.03 nM.In addition,the proposed method demonstrated extraordinary specificity towards ATP and could be applied to detecting ATP in diluted human serum samples.