Study And Applications Of Electrochemiluminescence Sensors Based On Signal Amplification Technologies

Electrochemiluminescence(ECL)detection has the advantages of fast response,high sensitivity,strong controllability,convenient operation and simple equipment,while signal anplification techniques can significantly improve the sensitivity of biosensors.In this thesis,combining the merits of some signal amplification techniques and ECL detection,three different biosensors with high sensitivity and selectivity have been developed and applied to detect target analytes in complicated samples.This thesis is composed of four chapters.In chapter 1,the concept,the classification of ECL and analytical applications of ECL biosensors were firstly reviewed.Then,the basic categories of signal amplification techniques and their applications in the biosensing field were introduced.In the end,the purpose and the content of this thesis were described.In chapter 2,an ultrasensitive ECL biosensor based on hyperbranched rolling circle amplification(HRCA)was developed for the determination of ochratoxin A(OTA).The thiolated capture probe(CDNA)was firstly immobilized on the gold electrode surface via Au-S covalent bonds.Then the OTA aptamer hybridized with CDNA on the electrode surface.Since OTA can competitively bind with the aptamer due to their high affinity,which would induce the releasing of aptamer from the electrode surface.Subsequently,the free CDNA on the electrode surface can hybridize with the padlock probe,and the padlock probe was ligated and the HRCA reaction proceeded following.Thus,the HRCA products which contained large amount of double-stranded DNA(dsDNA)fragments were accumulated on the electrode surface.Since Ru(phen)32+ could intercalate into the dsDNA grooves to act as ECL probe,leading to a remarkable enhancement of the ECL signal.The limit of detection of the proposed sensor is 0.02 pg/mL and the sensor has been applied to detect OTA in the corn samples with satisfied results.In chapter 3,combining the merits of cascade signal amplification of nicking endonuclease signal amplification(MESA)coupled with HRCA and ECL detection,an ultrasensitive biosensor has been designed for p53 DNA sequence determination.A tiny amount of the target DNA could trigger NESA to produce multple complementary sequences for the padlock probes and then the HRCA reaction was initiated.Afterwards,Ru(phen)32+ could act as ECL signal reporter for the following ECL measurements.Using this cascade anplificatibn strategy,an ultrasensitive p53 DNA sequence detection method was developed with a low detection limit of 0.02 fM.Moreover,this cascade amplified ECL biosensor exhibited high distinction ability to single-base mismatch.In chapter 4,a label-free and ultrasensitive electrochemiluminescence(ECL)biosensor based on telomeric repeat amplication protocol(TRAP)has been fabricated to detect the activity of telomerase extracted from HeLa cells.The thiolated telomerase substrate(TS)primer was firstly immobilized on the gold electrode surface via Au-S covalent bonds.Then TS primer could be discriminated specifically and elongated by telomerase and TRAP process was initiated,generating a much longer single stranded DNA with telomeric repeat units.Subsequently,the conplementary DNA was added to hybridize with the extended TS telomeric repeat strand and then dsDNA segments were formed on the electrode surface.Finally,Ru(phen)32+ was added to act as ECL indicator for the ECL measurements.The proposed biosensor could determine the number of HeLa cells by detecting the activity of telomerase,and the limit of detection of the biosensor is 2 HeLa cells.