Studies Of Functional Nucleic Acid-based Electrochemical Biosensors

Aptamers, nucleic acid enzymes, and aptazymes are collectively called functional nucleic acids (FNAs), whose functions are beyond the conventional genetic roles of nucleic acids. FNAs, which have superior properties as excellent sensors, including high stability, low cost, and ease of synthesis and modification, are generally believed to be able to target any analyte of choice. Hence, FNAs have taken an increasingly significant role in biosensing events. The topics presented in this paper focus on three signaling methods developed to construct electrochemical FNA sensors. Aptamer-based sensors are described first, followed by DNAzyme and aptazyme sensors. The details are summarized as follows:In Chapter 2, a label-free electrochemical sensor based on target-induced displacement was reported with adenosine as the model analyte. The sensing substrate was prepared using a gold electrode modified with a gold nanoparticle film. An aptamer for adenosine was applied to hybridizing with the capture probe. The interaction of adenosine with the aptamer displaced the aptamer sequence and caused it to dissociate from the interface. This resulted in a decrease in the amount of aptamer/capture probe duplex form, and accordingly, the desorption of methylene blue from the electrode. Then, the redox current of the indicator could reflect the concentration of the analyte. The fabricated sensor was shown to exhibit high sensitivity, desirable selectivity and a three-decade wide linear range.In Chapter 3, a novel electrochemical DNAzyme sensor was reported for the detection of nucleic acids based on proximity-dependent DNA ligation assays. When the target DNA was introduced into the system, part of it was complementary to 5'-end of the recognition probe, resulting in the ligation of a stable duplex. This duplex containing a complete 10-23 DNAzyme structure could cleave the purine-pyrimidine cleavage site of the hairpin substrate, which resulted in the fragmentation of the hairpin structure and the release of two single-stranded nucleic acids, one of which was biotinylated and acted as the signal probe. An immobilized thiolated capture probe could bind with the signal probe, using biotin as a tracer in the signal probe, and streptavidin-alkaline phosphatase (SA-ALP) as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 5 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The results revealed that the sensor showed a sensitive response to complementary target sequences of H. pylori. In addition, the sensing system could discriminate the complementary sequence from mismatched sequences.In Chapter 4, an aptazyme-based electrochemical biosensor for the detection of adenosine was reported. Aptazyme activity was modulated by appending an "inhibitor" oligonucleiotide strand containing a 32-base adenosine aptamer to the 8-17 DNAzyme. In the absence of adenosine, the DNAzyme could not form appropriate catalytic structure due to the binding with the inhibitor strand. Upon adenosine binding to the aptamer, the inhibitor strand was dissociated from the DNAzyme sequence. This allowed the DNAzyme to open and bind with the hairpin substrate, cleaving the substrate at its ribonucleotide site in the presence of Pb2+. Cleavage of the substrate yields two single-stranded products, one of which was ferrocene-tagged and acted as the signal probe. The thiolated probe modified on the gold electrode could capture the signal probe. As a result, the ferrocene moiety was brought in close proximity to the electrode surface and the Faradaic current was observed. The fabricated sensor is shown to exhibit high sensitivity and desirable selectivity, which might be promising for the rational construction of aptazyme-based biosensors and the determination of adenosine in clinical examination.