Study On Electrochemiluminescence Biosensor Based On Peptide And Aptamer

Electrochemiluminescence (electrogenerated chemiluminescence, ECL) involves the generation of species at electrode surfaces that then undergo electron-transfer reactions to form excited states that emit light. ECL biosensors combine the advantages of both electrochemical and chemiluminescent biosensors, such as high sensitivity, ease of control and using of simple equipment and specific selectivity offered by the biological recognition elements, which has widely been used in pharmaceutical analysis, environmental analysis, bioanalysis, and clinical analysis. The aim of this paper is to design and fabricate three ECL biosensors for determination of bacteria, protein and adenosine with high sensitivity, selectivity and regeneration.This paper includes two parts. First part, chapter1, is general introduction while second part containing four chapters, is a research report.In Chapter1, general introduction to ECL, ECL biosensor and aptamer biosensor including their principles and research development, and the purpose of this research work were presented.In Chapter2, two electrogenerated chemiluminescence (ECL) biosensors for selective and sensitive detection of Escherichia coli (E. coli) O157:H7as a model target of pathogenic bacteria were first designed by employing antimicrobial peptide Magainin I as a biological recognition element and ruthenium complex (Rul) as ECL label. In a direct format, the biosensor (Ⅰ) was fabricated by self-assembling Rul-labelled peptide taken as capture and signal probe onto the surface of gold electrode, and showed that a decreased ECL intensity was logarithmically direct proportion to the concentration of E. coli O157:H7in the range from1.O×103CFU/mL to5.0×105CFU/mL with a detection limit of226CFU/mL. In a sandwich format, the biosensor (Ⅱ) was fabricated by self-assembling the modified Magainin I taken as capture probe onto the surface of gold electrode. After this biosensor captured E. coli O157:H7and then bound the Rul-labelled peptide as signal probe, the increased ECL intensity was logarithmically direct proportion to the concentration of E. coli from5.0×102CFU/mL to5.O×105. CFU/mL with a detection limit of116CFU/mL. Importantly, the ECL biosensors designed showed a satisfactory selectivity in discriminating Gram-negative E. coli O157:H7from Gram-positive bacteria, and pathogenic E. coli from and nonpathogenic E. coli. The strategy of using Rul-labelled peptide as a capture/signal probe is a promising approach to the design of ECL biosensors for highly sensitive and rapid detection of E. coli O157:H7and could be extended to the design of ECL biosensors for other desired bacteria. In Chapter3, a novel electrogenerated chemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin incorporating an auxiliary probethe was designed by employing specific anti-thrombin aptamer as an capture probe and ruthenium(II) complex-tagged the relevant complementary ssDNA as an ECL probe and an auxiliary probe to assist the ECL probe close to the surface of the electrode. The ECL aptasensor was fabricated by self-assembling a thiolated capture probe on the surface of gold electrode and then hybridizing the ECL probe with the capture probe, and further self-assembling the auxiliary probe. When analyte thrombin was bound with the capture probe, the part of the dehybridized ECL probe was hybridized with the neighboring auxiliary probe, led to tagged ruthenium(Ⅱ) complex close to the electrode surface, resulted in greatly increasing the ECL intensity. The results showed that the increased ECL intensity was directly related to the logarithm of thrombin concentrations in the range from1.0×10-15M to5.0×10-12M with a detection limit of5.0×10-16M. This work demonstrates that employing an auxiliary probe which exists nearby the capture probe can enhance the sensitivity of the ECL aptasensor. This promising strategy will be extend to design the other biosensors for the detecting of other proteins and genes.In Chapter4, a highly sensitive electrogenerated chemiluminescence (ECL) aptasensor for the determination of adenosine was designed by simply adsorbing ruthenium complex-tagged aptamer onto the surface of single-walled carbon nanotubes (SWNTs) as a biosensing platform. A specific anti-adenosine binding aptamer was used as recognition molecular element and ruthenium(II) complex (Rul) was done as ECL signal compound. Rul-tagged aptamer was utilized as an ECL probe and the ECL probe was non-covalently assembled on the surface of SWNTs to form ECL probe/SWNTs composite. The ECL aptasensors were fabricated by simply adsorbing ECL probe/SWNTs composite onto the surface of a glassy carbon electrode. Analyte adenosine was bound with the aptamer of the ECL probe on the SWNTs so that the ECL probe was dropped from the SWNTs, resulted in the decrease of ECL signal. The results showed that the decreased ECL intensity was directly related to the logarithm of adenosine concentration in the range from1.0×10-10M to5.0×10.-7M with a detection limit of5.0×10.-11M. This work demonstrates that the strategy of simply adsorbing ECL probe/SWNTs composite as a biosensing platform is a promising approach to design ECL aptasensors with high sensitivity and selectivity.In Chapter5, the conclusions was presented. This work establishes "direct format" and "sandwich format" ECL biosensor for the detection of bacteria and two ECL biosensors for the determination of protein and adenosine with high sensitivity. This thesis work demonstrates that the combination of antimicrobial peptide Magainin I and aptamer to analyte with a highly sensitive ECL technique to design ECL biosensor is a great promising approach for the determination of bacteria, proteins and small molecule.