| Biosensors are defined as analytical devices incorporating a biological material(e.g.tissue, microorganisms,organelles,cell receptors,enzymes,antibodies,nucleic acids,natural products etc.), a biologically derived material(e.g.recombinant antibodies,engineered proteins) or a biomimic(e.g. synthetic catalysts,combinatorial ligands,imprinted polymers) intimately associated with or integrated within a physicochemical transducer or transducing microsystem,which may be optical, electrochemical,thermometric,piezoelectric,magnetic or micromechanical.The research of biosensors has already become new frontier of modern analytical chemistry.Aptamers have several advantages over traditional antibody-based reagents.Unlike antibodies,aptamers can be synthesized chemically and selected by SELEX(Systematic Evolution of Ligands by Exponential Enrichment) process,undergo ligand-dependent conformational changes,and offer long-term stability,target versatility,and convenient regeneration.Because of their specific binding abilities,aptamers would be extremely useful to make aptamer-based biosensing for the determination of small molecular and protein substances.Electrogenerated chemiluminescence(also called electrochemiluminescence,abbreviated as ECL) is the process where species generated at electrodes undergo electron-transfer reactions to form excited states that emit light.ECL method has many distinct advantages over fluorescence method because it does not involve a light source and avoids the attendant problems of scattered light and impurities luminescent.Moreover,the specificity of the ECL reaction associated with the ECL label and the coreactant species decreases problems with side reactions and is characterized by good spatial and temporal resolution.It has also widely been used in pharmaceutical analysis, bioanalysis,environmental analysis and clinical analysis.In recent years,highly selective and sensitive analytical methods such as DNA hybridization assay and biosensor have been received much attention.The development of a simple,rapid,sensitive and selective ECL biosensor and the fabrication of a simple,cheap and stable analytical detector in ECL analysis have been a long-term goal.The aim of this thesis is to design and fabricate ECL DNA hybridization and aptamer-based biosensors for the determination of biological molecule with high sensitivity,selectivity and simplify. In this thesis,taking advantages of the unique properties of nanoparticles and the specificity of biological molecular recognition substances,such as DNA,aptamer,we have designed a series of ECL DNA and aptamer-based biosensors for the determination of DNA,cocaine,thrombin and lysozyme.Research work in this thesis is financially supported by the National Natural Science Foundation of China(Grant No.20775046,No.90607016,No.20375025).The thesis includes two parts.First part,chapter 1,is general introduction while second part consisting of four chapters,is a research report.In Chapter 1,general introduction to ECL,DNA biosensor and aptamer biosensor including their principles and research development,and the purpose of this research work were presented.In chapter 2,an ultrasensitive ECL detection method of DNA hybridization based on single-walled carbon-nanotubes(SWNT) carrying a large number of ruthenium complex tags was developed.The probe single strand DNA(ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe.When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode,and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate,a strong ECL response was electrochemically generated.The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4×10-14 to 1.7×10-12mol/L with a detection limit of 9×10-15 mol/L.The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA.This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization.In chapter 3,a novel ECL aptamer-based biosensor for the determination of a small molecule drug is designed employing cocaine-binding aptamer as molecular recognition element for cocaine as a model analyte and ruthenium complex served as an ECL label.A 5'-terminal cocaine-binding aptarner with the ECL label at 3'-terminal of the aptamer was utilized as an ECL probe.The ECL-AB biosensors were fabricated by immobilizing the ECL probe onto a gold electrode surface via thiol-Au interactions.An enhanced ECL signal is generated upon recognition of the target cocaine, attributed to a change in the conformation of the ECL probe from random coil-like configuration on the probe-modified film to three-way junction structure,in close proximity to the sensor interface. The integrated ECL intensity versus the concentration of cocaine was linear in the range from 5.0×10-9 to 3.0×10-7 mol/L.The detection limit was 1×10-9 mol/L.This work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design ECL aptamer-based biosensor is a great promising approach for the determination of small molecule drugs.In chapter 4,three aptamer-based ECL biosensors for the determination of thrombin were designed.As a model system,thrombin-binding aptamer was utilized as molecular recognition element and thrombin as a target analyte and tris(2,2'-bipyridyl) ruthenium derivatives as aptamer ECL label.First one was designed on basis of a structure-switching ECL-dequenching mechanism.The thiolated DNA capture probe,composed of a DNA sequence to adopt two distinct structures-a DNA duplex with a complementary DNA sequence tagged with tris(2,2'-bipyridyl) ruthenium derivatives (RuDNA)-binding motif and a DNA duplex with a complementary DNA sequence tagged with ferrocene(Fc)-DNA probe(FcDNA) with the target for which the aptamer is created,was self-assembled onto surface of a gold electrode.In the presence of thrombin,the aptamer prefers to form the aptamer-target complex and the switch of the binding partners for the aptamer occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA.The integrated ECL intensity versus the concentration of thrombin was linear in the range from 2.0×10-10 to 2.0×10-7 mol/L.The detection limit was 6×10-11 mol/L thrombin.Second one was designed with a "signal off" model.Gold nanoparticles were assembled onto a gold electrode through 1,6-hexanedithiol and then thiolated ss-DNA capture probe was assembled onto the surface of the gold nanoparticles.The ECL aptamer probe was designed to hybridize with capture DNA sequence and specifically recognize thrombin.The introduction of thrombin triggers structure switching of the aptamer and thus the ECL probe is forced to dissociate from the sensing interface,resulting in a decrease in ECL intensity.The decrement of integrated ECL intensity is proportional to the concentration of thrombin in the range from 2.7×10-12 to 2.7×10-9 mol/L.The detection limit was 8×10-13 mol/L.Third one was designed in sandwich model using the two aptamers(aptamerâ… and aptamerâ…¡), which recognize different positions of thrombin.Aptamerâ… immobilized onto a gold electrode can captured the analyte thrombin and then the captured thrombin binds to an ECL probe composed of aptamerâ…¡and ruthenium complex loaded at SWNT,resulting ECL generation.The detection limit as low as 3×10-15 mol/L thrombin was achieved.In chapter 5,a novel ECL aptamer-based biosensors for label-free determination of lysozyme was developed using aptamer-self-assembly electrodes.Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly,for which the surface concentration of aptamers was determined by ECL studies of Ru(bpy)32+ bound to the surface via electrostatic interaction with the DNA phosphate backbone.Upon incubation of the electrode with a solution containing lysozyme,the ECL response of surface-bound Ru(bpy)32+ changed substantially,and the relative decrease in the integrated the ECL intensity can be tabulated as a quantitative measure of the protein concentration 6.4×10-10 mol/L to 6.4×10-7 mol/L.It is significant that the on-electrode protein/aptamer binding constant and the optimized surface concentration of lysozyme aptamer to achieve the lower detection limit can be evaluated.This biosensor is label-free and offers a sensitive and versatile method for protein detection.In conclusion,one ECL DNA hybridization biosensor and five ECL aptamer-based biosensors were fabricated for the determination of biological molecule in this thesis.This thesis work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design ECL aptamer-based biosensor is a great promising approach for the determination of small molecule drugs and proteins.The sensitivity of ECL DNA hybridization and aptamer-based biosensors can be much improved using the nanoparticles modified electrode and nanotubes carrying multiple ECL probes.
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