Novel Electrochemiluminescence Approaches For DNA Analysis

Electrochemiluminescence(ECL)technique,offering a lot of advantages such as low cost,wide range of analytes,low background signal and high sensitivity,has attracted growing attention in bioanalytical field.Recently,semiconductor nanocrystals(SNCs)have been exploited as a new kind of ECL emitters.Compared with conventional molecule emitters,SNCs own several distinctive merits like size/surface-trap controlled luminescence and good stability against photobleaching.The SNCs-based ECL has accordingly been applied in biosensing and bioanalysis.This doctoral dissertation focuses on developing highly sensitive analytical methods by coupling with the DNA hybridization,nanoparticles and enzymatic cycling amplification techniques on SNCs-DNA sensing interfaces.1.Ultrasensitive DNA Detection Based on Au Nanoparticles and Isothermal Circular Double-Assisted ECL Signal AmplificationWe have developed a novel ultrasensitive ECL DNA sensing interfaces which combines a powerful isothermal amplification method with a "DNA machine",and distance-dependent quenching or enhancing of ECL from CdS nanocrystals(NCs)films by Au NPs.Such energy transfer in ECL systems opens a new way for transduction of biological recognition events and the isothermal DNA amplification can be utilized under normal temperature condition,thus eliminating the requirement for thermal cycling.The results of this study not only offer a new approach to detect DNA at an ultralow level but also broaden the perspective for future development of DNA-based biosensors for the detection of other analytes.2.Highly Sensitive ECL Detection of Single-Nucleotide Polymorphisms Based on Isothermal Cycle-Assisted Triple-Stem Probe with Dual-Nanoparticle LabelsWe report here a new ECL approach for detection of single nucleotide polymorphisms(SNPs)based on isothermal cycle-assisted triple-stem probe labeled with Au nanoparticles(NPs)and CdTe NPs.The system is composed of a CdS nanocrystals(NCs)film on glassy carbon electrode as ECL emitter attached a double-stem DNA probe labeled with Au NPs.Then,the third stem labeled with CdTe NPs hybridizes with the double-stem DNA to form a triple-stem probe with the two labels near the CdS NCs film.A dual-quenched ECL of CdS NCs film is achieved due to energy transfer(ET)from CdS NCs to Au NPs and CdTe NPs,which makes the sensor exhibit relatively low background.Once the one base mutant DNA(m-DNA)sequence as target of SNPs analysis displaces the third stem and hybridizes with the double-stem probe,forcing Au NPs away from the CdS NCs film,an ECL enhancement by the ECL-induced surface plasmon resonance of Au NPs is observed.Furthermore after isothermal cycle induced by primer,polymerase and nicking endonuclease(NEase),a further enhancement of ECL is obtained.Taking advantages of the triple-stem probe architecture and "DNA machine",the reported biosensor shows excellent discrimination capabilities of SNPs with high selectivity and low detection limit(35 aM).In conclusion,this strategy realized SNP discovery and analysis,and hold great promise for clinical research and diagnostics of genetic disease and cancer.3.ECL-Resonance Energy Transfer Between CdS:Eu Nancrystals and Au Nanorods for Sensitive DNA DetectionThis work provides a novel ECL-resonance energy transfer(ECL-RET)system using CdS:Eu nanoscrytals as an ECL donor and Au nanorods(Au NRs)as an ECL acceptor.The absorption spectrum of Au NRs with high extinction coefficient could be easily tuned to match well with the ECL emission spectrum of the CdS:Eu NCs film by adjusting the aspect ratio of the nanorods to get the highly effective ECL-RET.Here,we studied the spectrum-,distance-and shape-dependence of the efficiency of ECL-RET between the NCs-ECL and different Au nanoparticles based on the stem-loop structure DNA with a 6-base-pair stem and a 12,30 or 45 nucleotides loop.At the optimized conditions,the system could be used to ultrasensitively and specifically detect target DNA,providing significant potential application in clinical analysis.4.The ECL Biosensor Based on the Target Recycling-Assisted Amplification and Efficient Quenching of DNAzymewe prepared a novel K-doped graphene-NCs(K-GR-NCs)composites via electrostatic interactions between negatively charged 3-mercaptopropionic acid(MPA)modified CdS:Eu NCs and positively charged graphene,which was noncovalently functionalized with poly(diallyldimethylammonium chloride)(PDDA)via electrostatic interactions.Here,the K-doped graphene nanomaterials can improve the electron transfer at the electrode interface.So the layer-by-layer assembled K-GR-NCs composites film on the glassy carbon electrode can effectually increase the surface area and meanwhile improve the ECL intensity.Based on the K-doped graphene-CdS:Eu NCs composites and nicking endonuclease(NEase)assisted strand-scission cycle,we have developed a simple,label-free,ultrasensitive and selective ECL DNA biosensor using the G-quadruplex/hemin-based DNAzyme which acts as an electrocatalyst for the reduction of H2O2,the coreactant of CdS:Eu NCs ECL.