| Various diseases, such as cancers and genetic ones, were associated with gene mutation and many infectious diseases were caused by the virus in the environment. Thus, sequence-specific and sensitive detection of sequence-specific DNA is of great importance in a variety of applications, including gene screening, life science, medical diagnosis, pathogen identification, and environmental and food safety monitoring. Generally, according to the use of tags or not, DNA biosensors can be divided into two types, i.e., the labeled and label-free ones. Among them, the latter ones were well developed and applied in molecular biology, forensic testing and clinical diagnosis, taking the advantages of simplicity, high-speed and cost-efficiencyChemiluminescence(CL) is considered one of the most suited optical detection techniques for developing miniaturized and highly sensitive analytical devices, by taking advantages of high sensitivity, the wide dynamic range of the CL measurements (up to6orders of magnitude), without the need of sample dilution and an excitation light source. In2003, based on an instantaneous derivatization reaction between a specific CL reagent3,4,5-trimethoxyphenylglyoxal (TMPG) and guanine nucleotides consisted in telomeres, our group firstly reported on the detection of telomeres by coupling of the label-free guanine CL detection route with an efficient magnetic isolation of the hybrid, which was published in Chemical Communications (2003,2888-2889). In2008, we described the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. One hybridization occured between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel, and then CL detection proceeded via the reaction between TMPG and the guanine nucleotide-rich regions within the target DNA, which was published in Analytical Chemistry (2008,1606-1613). In2011, we reported on a highly sensitive aptameric assay system for the determination of IgE. A "sandwich-type" detection strategy was employed, where magnetic beads functionalized with a capture antibody were reacted with the target protein IgE, and then sandwiched with the aptamer-barcodes which were prepared by assembling polystyrene beads with IgE. After that, TMPG reacts with G bases of the aptamer-barcodes on the MB surface to form an unstable CL siginals. Further signal amplification is achieved by extending the G nucleotide-rich domain on the aptamer backbone for second amplification.Nucleic acid amplification technologies (NAATs) are used in the field of molecular biology and recombinant DNA technologies. NAATs have been developed which can be broadly divided into those that require thermal cycling (such as Polymerase Chain Reaction, PCR) and isothermal amplification, which has emerged as an alternative amplification technique that proceeds at constant temperature, such as Rolling Circle Amplification (RCA) and Hybridization Chain Reaction (HCR). For which, no specialized instrumentation is needed and the time required for DNA amplification is less than that for thermal cycling techniques. Recently, NAATs has widely used in the detection of the sequence-specific DNA, proteins, small molecules and other biomolecules.Based on our previous researchs, herein we developed two highly sensitive CL technologies for the detection of DNA based on CL instantaneous derivatization reaction between TMPG and guanine bases, including the following parts:Chapter1:This thesis first introduces the importance to develop the techniques for sequence-specific DNA and single nucleotide polymorphism detection, and then presents current state of knowledge on label-free DNA analysis, including electrochemical, colorimetry, SPR and QCM techniques, etc. The second phase of this chapter reviews the principle of the CL systems, and their recent applications in various fields, especially in the DNA assay. Following that, objectives and significance of this research are summarized.Chapter2:We propose here a new amplifying strategy based on HCR to detect sequences-specific of DNA, where one of two HCR monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, CL detection proceeds via an instantaneous derivatization reaction between a specific CL reagent TMPG and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe this simple technique is promising for improving medical diagnosis and treatment.Chapter3:The streptavidin-modified polystyrene microspheres (PS-SA) coupling of HCR were employed as an amplification platform to build a specific DNA sequence detection technology. Briefly, a "sandwich-type" detection strategy was employed in our design, which involved capture probe DNA immobilized on the surface of carboxyl terminated magnetic beads and the reporter DNA and the HCR trigger immobilized on the surface of PS-SA based on streptavidin-biotin reaction. After that, two hairpin-type monomers were added in the reaction system, HCR could be carried out in the reaction platform. The CL signal was obtained via the instantaneous derivatization reaction between TMPG and guanines in the target and the HCR product binding on the magnetic beads. The results showed that it was a simple, stable, rapid and sensitive method. An excellent linearity was found within the range of0.01finol-10pmol (0.1pM-100nM)(R2=0.991) with the lowest detection concentration of5amol (50fM), which was2×104-fold improvement on direct detection method. This novel approach provided great promise for DNA assay with good differentiation ability for various single-base mismatches. |
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