Research On A New Method For Biosensing Of Electrochemiluminescence Arrays

With the continuous damage to nature,the living environment is deteriorated and the incidence of various diseases in the world is rising.Therefore,it is very important to simultaneously detect of multiple biomarkers with high sensitivity.Electrogenerated chemiluminescence(ECL)is the process whereby species generated at electrodes undergo high-energy electron-transfer reactions to form excited states that emit light.Due to the advantages of ECL,such as high sensitivity,low background,simplified optical setup,and good temporal and spatial control,ECL method has been widely used in clinical diagnosis,pharmaceutical analysis,environmental analysis,and bioanalysis.Compared with other molecular recognition,peptide as a molecular recognition substrate,has many advantages,such as cost-effective,small molecular weight,high biological affinity and high biological activity.Therefore,peptide as a molecular recognition substrate has becoming increasingly attention in biological analysis.The aim of this work is to develop simple and sensitive peptide based multiplexed ECL biosensors to simultaneously detect multiple biomarkers.Peptide was used as molecular recognition elements while Ru(phen)32+/Ru(bpy)32+ was used as ECL signals.Two simple and sensitive ECL methods were established to simultaneously detect PKA and CK2,cTnI and ATP using peptide phosphorylation and super sandwich technique.The main contents of this paper are as follows:First part,General introduction,that briefly introduces the concept,characteristics and devices of the ECL and reviews the progress of ECL multiple-component and current research of ECL imaging.Finally,the purpose and contents of this research work was presented.The second part is the research content.First chapter,electrogenerated chemiluminescence bioassay of two protein kinases incorporating peptide phosphorylation and versatile probe.A sensitive electrogenerated chemiluminescence(ECL)bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe.Casein kinase ?(CK2)and cyclic adenosine monophosphate-dependent protein kinase(PKA)were used as proof-of concept targets while a CK2-specific peptide and a PKA-specific peptide were used as the recognition substrates.Taking advantage of the ability of protein A binding with the fragment crystallizable region of a variety of antibodies with high affinity,a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases.A specific peptide was first selfassembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5 '-triphosphate.After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody,the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface.The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.05U/mL and 0.04U/mL,respectively.In addition,the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates.Moreover,an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL,respectively.This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitors.Second chapter,highly sensitive electrochemiluminescence assay for cardiac troponin I and adenosine triphosphate using supersandwich amplification and bifunctional aptamer.A highly sensitive electrochemiluminescence(ECL)assay was developed for sequential detection of cardiac troponin I(cTnI)and adenosine triphosphate(ATP)via supersandwich amplification and bifunctional aptamer.The bifunctional aptamer(S1)contained ATP binding aptamer and cTnl binding aptamer.A gold electrode was modified with specific peptide of cTnI via self-assembly technique.A sandwich-type conjugate(peptide<cTnI>S1)was formed when the peptide modified electrode was successively reacted with cTnI and S1.Then,hybridizations between S1 and two ss-DNA auxiliary probes,in which one is complementary with ATP binding aptamer and the other is ATP binding aptamer,lead to the formation of long-range ds-DNA cm the electrode surface.In the presence of ECL indicator Ru(phen)32+,a great amount of Ru(phen)32+ was intercalated into ds-DNA grooves,resulting in amplification of ECL signals.After detecting cTnI,ATP was detected based on switching structures of aptamers from ds-DNA to ss-DNA/target complex.Low detection limit is 3×10-13 g/mL and 10 nM for cTnI and ATP,respectively.The employment of bifunctional aptamer probe and supersandwich signal amplification is promising for the sensitive detection of multiple targets.This paper constructed two different kinds of ECL biosensors array,combining the high sensitive of ECL technology,high selective of bio-recognition and simultaneous determination of multi-component,which could be applied to the detection of inhibitors and cell lysate.The method provided a new idea for the detection of biomarkers,which is of great significance to the biosensors research,clinical detection and the life science development.