| Aptamers are synthetic oligonucleotides with high binding affinity for a broad range of targets, including small molecules, proteins, or even whole cells. They are isolated from random-sequence nucleic acid libraries by"in vitro selection". Some aptamers with guanine (G)-rich segments, biologically active RNA and DNA sequences including anti-proliferative, anti-HIV and anti-coagulation aptamers[1], can assemble into G-quartets, planar structures of four H-bonded Gs, which stack on top of each other to form G-quadruplexes that are essential for nucleotide fuctions. Some species that bind to G-quadruplexes can find significant applications as therapeutics or as probes for gene function[2]. In biochemical analysis, aptamers, including G-rich oligonucleotides, rival antibodies as highly promising tools due to the specificity, the easy storage, the fast tissue penetration, the high binding affinity and simplicity of in vitro selection. Aptamers have been used for the preparation of numerous sensing atrageies for the detection of various proteins and provide a interesting alternative to immunoassays and immunosensors[3]. Nevertheless, aptamer research is still in its infancy compared with the bellwether antibody technology[4]. The key in the development of aptamer-based sensing systems is to transducer successfully aptamer recognition events to detectable signals.Single-nucleotide polymorphisms (SNPs) are point mutations that occur at specific positions in a genome and constitute the most common form of genetic variation. SNP may affect gene function resulting from amino acid substitution, modification of gene expression or alteration of gene splicing and are closely associated with various common diseases and individual differences in drug metabolism[5]. Although numerous technologies for the point mutation detection have been reported to date, most of these approaches require target amplification, typically with PCR. Additional efforts are thus needed to explore more broadly applicable methods for the sensitive, accurate, rapid, and low-cost SNP identification[6]. In this doctoral thesis, several bioassay systems have been developed based on aptamers with the common sequences and those with with G-rich segments that can form the intramolecular or intermolecular G-quadruplex structures; the aggregation behavior of G-rich DNA-modified nanoparticles has been inveastigatedd; electrochemical sensing interfaces as well as optical methods for the detection of DNA hybridization and the screening of SNPs. The details are summarized as follows:1. Aptamer–based bioassay systemsA) The aggregation behavior of gold nanoparticles modified with G-rich DNA sequences and electrochemical biosensors based on G-quadruplex structures.(1) In chapter 2, a reusable electrochemical sensing platform based on structure-switching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is firstly modified with polytyramine (Pty) and gold nanoparticles (GNPs). Then, thiolated-capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for adenosine assay.(2) In chapter 3, the aggregation behavior of the nanoparticles, which are functionalized with four-guanine-terminated 27-base sequences, is investigated. To some extent, the guanine-quadruplex structures between gold nanoparticles (GNPs) promote the nanoparticle aggregation. However, the coordination site of the metal ion on the nanoparticle surfaces is partly passivated: the stability of guanine-rich DNA-GNPs is slightly lower than that of the common DNA-GNPs, and the metal-ion specificity of nanoparticle assembly is substantially decreased. Accordingly, a mechanism for the aggregation of guanine-rich sequence-modified GNPs is proposed.In chapter 4, a novel on/off electronic nanoswitch is described based on the conformational change of DNA sequence possessing a single guanine (G)-rich stretch. A thiolated, amine-containing G-rich DNA sequence is immobilized on the surface of gold electrode and is then labeled with redox-active ferrocene molecules. The surface-confined DNA sequence is able to change its configuration between rigid tetramolecular G-quadruplex and flexible single-stranded structures. The large conformational change enables the probes to perform an inchworm like extending-shrinking motion, which is reflected by the fluctuation in current intensity. Since potassium ion can specifically bind to G-quadruplex, using this reagentless reusable electrochemical sensing platform, the simple, rapid and selective detection of potassium ion can be accomplished without the use of exogenous reagents.In chapter 5, a highly sensitive electrochemical DNA biosensor without any signal amplifier has been reported by employing an intermolecular tetrameric guanine (G)-quadruplex structure. To fabricate a sensing interface, thiolated DNA sequence (capture DNA) is assembled onto the surface of a gold electrode through the covalent thiol-gold binding. Redox-active ferrocene (Fc)-conjugated DNAs with single guanine (G)-rich stretches are used as signaling probes, which can form intermolecular G-quadruplexes and are complementary sequences of capture DNAs. The introduction of signaling probes onto the sensing interface can lead to a redox current. A marked current response is observed even if small amounts of target DNA are prehybridized with the surface-confined capture DNA. Target sequences can be detected in a competition setup in the concentration range from 9.92×10-14 to 9.92×10-10 M with a detection limit of 2.48×10?19 mol, indicating a substantial improvement in the sensitivity compared with a common signaling probe-based scheme. The present strategy exhibits other advantages of simplicity, rapidity, low cost and circumvents various problems associated with the additional signal amplifiers. B) To demonstrate that aptamers can provide several advantages over the corresponding antibodies, we have developed biosensing systems using IgE and aptamers as the model analyte and probe molecules, respectively, and investigated their analytical characteristic by comparison.In chapter 6, an aptamer-based electrochemical sensing platform for the direct protein detection has been developed using immunoglobulin E (IgE) and a specifically designed oligonucleotide strand with hairpin structure that has the standard aptamer segment as the model analyte and probe sequence, respectively. In the absence of IgE, the aptamer immobilized on an electrode surface forms a large hairpin due to the hybridization of the two complementary arm sequences, and peak currents of redox species dissolved in solution can be achieved. However, the target protein-binding can not only cause the increase of the dielectric layer but also trigger the significant conformational switching of the aptamer due to the opening of the designed hairpin structure that pushes the biomolecule layer/electrolyte interface away from the electrode surface, suppressing substantially the electron transfer (eT) and resulting in a strong detection signal.In chapter 7, we demonstrate a fluorescence immunoglobulin E (IgE) assay sensor 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 enhanced fluorescence 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 mol·L-1 with a detection limit of 5.7×10-11 mol? L-1.2. The methods for the detection of DNA hybridization and the identification of SNPs.In chapter 8, a novel strategy is described for highly sensitive DNA detection and point mutation identification based on the combination of reverse molecular beacon with DNA ligase. A 5'-phosphorylated and 3'-ferrocene terminated DNA sequence is used as detection probe, which may be ligated to capture DNA immobilized on an electrode surface in the presence of a target DNA strand that is complementary to the ends of each DNA since this allows formation of a nicked, double-stranded DNA. A redox current is observed. The ligation product may form a hairpin structure after the removal of target DNA, resulting in an enhanced electrochemical signal. By this method, target DNA can be determined in the range from 3.4×10-12 to 1.4×10-7 M with a detection limit of 1.0×10-12 M.In chapter 9, a novel system for the detection of DNA hybridization in a homogenous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nano-scaffolds for the immobilization of capture sequences and nano-quenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base olignucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and olignucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine(TAMRA)-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with olignucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM.In chapter 10, an exciting discovery that the adsorption of negatively charged molecules onto nanoparticle surfaces can induce the intensive aggregation of citrate-stabilized gold nanoparticles has been reported. Along this line, a homogeneous, colorimetric system for DNA detection is described based on the aggregation of gold nanoparticles induced by the products of DNA cleavage. Excellent response characteristics (for example, sensitivity, selectivity and linear response range) are achieved using either UV/vis spectrophotometer or the naked eye.
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