Novel Aptamer-based Techniques For The Chemiluminescent Biosensors

DNA aptamers are synthetic single-strand nucleic acids discriminated by screening in vitro, which are of high affinity and specificity to many given targets ranging from small molecules to large proteins and even cells. New methods for efficient and fast detection in biochemical and biomedical fields have been developed based on the molecular recognition of aptamers. Aptamers have been demonstrated to have advantages over antibodies with regard to chemical stability, readily availability, simple modifiability, and high flexibility in biosensor design for analyte detection. As a novel functional molecule, aptamer have gained considerable interest in bio-analytical application in many fields such as clinical diagnosis, environmental supervision, pharmaceutical study, etc.Chemiluminescence (CL) has been exploited within a wide range of applications in various fields, due to their extremely high sensitivity along with their extra advantages such as needless special light, simple instrument, wide calibration ranges, and suitability for automatical operation in analytical chemistry. Nowadays, based on cross-research with other disciplines, CL has been successfully served for different applications. Our study developed an aptasensor on the strength of the high specifically binding between aptamers and ligand in the thesis. Description of research in the thesis is presented as follows:Chapter 1:Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.Chapter 2:We reported on a highly sensitive aptameric assay system for the determination of IgE, where a special CL reagent,3,4,5-trimethoxylphenylglyoxal (TMPG), acted as the signaling molecule and polystyrene beads as the amplification platform. Briefly, a "sandwich-type" detection strategy is employed in our design, 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 aptamer. The target immunoreaction event could be sensitively detected via an instantaneous derivatization reaction between TMPG and the guanine (G) nucleotides within the aptamer-barcodes to form an unstable CL intermediate for the generation of light. Further signal amplification was achieved by extending the G nucleotide-rich domain on the aptamer backbone for second amplification. Such simple amplified CL transduction allows the detection of IgE down to the 4.60 pM level, which is better than most previous aptameric methods for IgE detection.Chapter 3:A novel and high sensitive method for the detection of platelet-derived growth factor BB(PDGF-BB) based on antibody-antigen-aptamer sandwich assay by using rolling circle amplification(RCA) has been reported. The primer of the RCA was hybridized with the 3'-terminal of the aptamer. With phi29 DNA polymemse,deoxyribonucleic acid and circle template, rolling circle amplification can perform. The reaction form a long single chain DNA which repetitive sequence is complementary with circle template DNA sequence. The product of RCA hybridized with biotinylated oligonucleotide probe. Following with streptavidin-HRP and CL detection was performed. The results showed that RCA can improve remarkably the sensitivity. The linear ranges for the PDGF-BB is 10 fM-1 nM and the lower limit is 10 fM. It will provide wide application of detecting of protein and diagnosing disease.Chapter 4:Herein, we wish to introduce a new CL method for the determination of the adenosine by the coupling of catalytic nucleic acid label (DNAzyme) based aptamer. The assay relied on the affinity between aptamer and ligand is stronger than the binding of DNA. When adenosine closed to aptamer, the double-stranded DNA is unstable, and then the hemin DNA is free. The hemin DNA and hemin can form a "cage", named DNAzyme with HRP-mimicking property, and then the CL is detected. The linear ranges for the adenosine is 7.81×10-6-5.00×10-4M and the lower limit is 7.81×10-6 M. Overall, the protocol described here may have value in a variety of clinical, pharmacal and environmental applications for which the simple, fast and accurate quantitative analysis of biomolecular adenosine is desired.