Studies On New Methods And Applications Of Aptameric Sensing Analysis Based On Conformational Switch And Polymerase Reaction

With a combinatorial method called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX), short synthetic DNA and RNA sequences known as aptamers have been selected as ligands to bind analytes, ranging from small molecules to large proteins and even cells. The ability of aptamers to fold into numerous tertiary conformations to interact with their target is related to their diversity in sequences making aptamers ideal elements to generate capture molecules of high affinity and specificity. Aptamers possess unprecedented advantages as recognition elements in biosensing when compared to traditional antibodies. Aptamers with high specificity and affinity can in principle be selected in vitro for any given target. They can be synthesized and modified with high reproducibility and purity from commercial sources. They are small in size, chemically stable and cost effective. They have fast response time, long shelf life, and good generality for detecting a broad range of analytes with the same class of recognition element. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures.In the field of analytical chemistry, aptamer can be used as molecular recognition element in biosensor design instead of antibodies and enzymes. The principle of aptameric sensors is that the conformational transition of the aptamer upon binding to its target leads to the change of optical or electrochemical signaling properties. Summing up the previous work, we developed some conformational switch based aptameric sensors with high sensitivity and selectivity for protein or small molecule detection. However, the use of conformational switch based aptameric sensing methods has been limited in further sensitivity improvement. Nevertheless, some success has been obtained by signal amplification or background reduction for ultrasensitive target detection. Strand displacement amplification based aptamer sensor is an ultrasensitive sensing approach based on polymerase reaction. After the aptamer-target binding, the aptamer conformational change initiates polymerase reaction. The further change of aptamer conformation resulted from polymerization leads to optical or electrochemical signal amplification. Thus the sensitivity has been improved by our design.The details are summarized as follows:(1) In chapter 2, a new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement. Here we describe new probes, the hairpin-probe and the single strand-probe, possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double-stranded form, the single strand-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed-tube, offering a convenient approach for homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective and economical.(2) In chapter 3, an electrochemical aptameric sensor based on polymerase extension reaction was produced for sensitive cocaine detection. An unmodified hairpin strand in solution contains the cocaine aptamer sequence, while a thiol-short strand immobilized on the gold electrode surface contains the sequence complementary to stem part of the hairpin strand. When cocaine is induced, the ternary complex is formed and the hairpin strand is opened. The hybridization of hairpin strand and the short strand initiates polymerase extension with dATP, dGTP, dCTP and ferrocene-dUTP. Electrochemical signal amplification could be observed due to the extended thiol-strand labeled with several ferrocene molecules.(3) In chapter 4, we developed a label free aptamer fluorescence probe using intercalating dye (SYBR Green I) to improve the sensitivity of adenosine sensing and reduce the cost of experiments. The binding site of adenosine aptamer has been split into two half sites. In the absence of the adenosine, little hybridization occur between the two DNA probes, each containing half of the DNA sequence corresponding to a binding site. In the presence of the adenosine, its high affinity for aptamer of the adenosine binding site drives to form a double strand structure that preferably binds to SYBR Green I. As a result, the fluorescence of a mixture of two DNA probes and SYBR Green I increased in the presence of adenosine. it was demonstrated that the presented aptamer sensing method was highly sensitive and specific with a detection range of 3 orders of magnitude. (4) In chapter 5, we demonstrate a fluorescence immunoglobulin E (IgE) assay probe based on DNA aptamer. A Texas red labeled short DNA strand (T-DNA) complementary with part of the IgE aptamer sequence was used to produce the fluorescence enhancement affected upon the binding of IgE to the aptamer. Another short DNA strand labeled with Dabcyl quencher (Q-DNA) complementary with part of aptamer sequence nearby the T-DNA location was used to lower the background fluorescence. The IgE can be detected in the concentration range from 9.2×10⁽-11 to 3.7×10^-8 molL^-1 with a detection limit of 5.7×10^-11 mol·L^-1.(5) In chapter 6, we report a fluorescence aptamer probe for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand complementary to part of the aptamer sequence or the loop of molecular beacon. The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensing method can detect as little as 0.34 nM of HNE, and the response is linear in the tested concentration range of 0.34 nM 68 nM with the detection limit of 47 pM.(6) In chapter 7, we describe the biocatalytic growth of high-density gold agglomerates on gold electrode surface to form a carrier for aptamer probe immobilization. The present approach provides a simple strategy to promote the seed-mediated deposition of Au from AuCl4·onto surface-attached 12 nm diameter Au nanoparticles (AuNPs) in the presence of reductive coenzyme and surfactant. The growth process was studied by electrochemical impedance spectroscopy and scanning electron microscopy. This nanostructured platform is effective and prospective toward the aptamer probe immobilization. For the nice performance of enhanced substrate, the aptamer sensing interface showed excellent applicability under the investigations such as alternating current voltammetry and surface-enhanced Resonance Raman scattering spectra.