Electrochimical/Electrochemiluminescent Aptasensors Based On Tripropylamine Oxidation And Their Application In Protein Analysis

This dissertation is composed of three chapters, including the introduction, electrochemiluminescent aptasensor via a low potential approach at DNA-modified gold electrodes, electrochemical aptasensor using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array.In chapter 1, the definition, properties and current status of the aptamer are summarized. The development, basic theories and application of the aptasensors are introduced in detail. The mechanism and current status of electrochemical aptasensor are discussed deeply.Chapter 2 presented an electrochemiluminescent aptasensor via a low potential approach at DNA-modified gold electrodes. Electrochemiluminescence (ECL)-based biosensors are often used in the field of DNA-and protein-assay. Although ruthenium complex-based ECL is sensitive, its high exciting potential may lead to oxidation damage to biomolecules. A non-damaging, low potential ECL aptasensor was constructed for bioassay with lysozyme as a model in this chapter. After a single-stranded anti-lysozyme aptamer was attached to a gold electrode, a double stranded (ds)-DNA formed with its complementary strand. Ru(phen)32+, as an ECL probe, was intercalated into the ds-DNA. The hybridization of lysozyme with its aptamer led to the dissociation of ds-DNA because of the high stability of the aptamer-lysozyme and therefore the Ru(phen)32+ intercalated into ds-DNA was released. A low potential ECL was observed at the ds-DNA-modified electrode because ds-DNA was able to preconcentrate tripropylamine (TPA) and acted as the acceptor of the protons released from protonated TPAH+. While the DNA sequence (anti-lysozyme aptamer) was used as the special recognition element for lysozyme, the formed ds-DNA also provided a micro-environment for low potential ECL. The low potential ECL aptasensor achieved the determination of lysozyme with a detection limit of 0.45 pM. The day-to-day precision (RSDs, n=5) for the determination of lysozyme was lower than 5%, showing the reliability of the aptasensor. The regeneration of the aptasensor confirmed that the low potential for ECL could decrease oxidation damage to biomolecules. Further, the proposed method was successfully used to analyze diluted egg white sample directly. The protocol exhibited a promising platform for sensitive bioassay and could be further applied for the development of other low potential ECL sensing systems.In Chapter 3, an electrochemical aptasensor was constructed using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array. Tripropylamine (TPA) has different oxidation efficiency at double stranded (ds)-and single stranded (ss)-DNA-modified electrode. Using this property, a simple but sensitive biosensor using TPA oxidation to probe the intramolecular displacement was constructed with the analysis of lysozyme as model. After the complementary ss-DNA strand of anti-lysozyme aptamer was immobilized onto gold electrode via gold-thiol bond, the incubation with the aptamer resulted in the formation of ds-DNA. Lysozyme (in 10μL sample) binding with aptamer displaced the complementary strand because of the high affinity of lysozyme and its aptamer, corresponding to the dissociation of the ds-DNA. The modified electrode was swept in 20 mM TPA solution from 0.2 to 0.95 V. The difference in oxidation current was used to quantify the content of lysozyme with a linear range from 1.0 pM to 1.1 nM. That means 10 amol or 6.0×106 lysozyme molecules can be detected. Because the signal is produced from the preconcentrated TPA at the electrode surface, the high sensitivity is achieved over the single site labeling strategy. The proposed method is simple, stable, specific, and time-saving while the complicated sample pre-treatment and the labelling to the DNA strand are avoided. The biosensor was validated by the analysis of the diluted egg white sample directly. The recovery and reproducibility were 93.3-100% and 1.4-4.2%, respectively.