Studies On Competitive Mechanism Triggered Signal Amplification Based Aptasensors

Biosensors have valuable applications in chemistry, biomedicine, environmental protection, food industry, medicine and military affairs because of its good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system. Aptamers have become highly promising recognition probes due to the high specificity, high binding affinity, the stable bioactivity, and the easy systhesis and storage. Aptamers have provided an interesting alternative to biochemical analysis and biosensors. In this paper, in order to solve the focus and difficult problems in building interfaces of sensors for current detection of proteins, we combined with the advantages of our laboratory and made use of the competition reaction alone target proteins complement DNA and aptamer ,which can trigger signal amplification(eg RCA). We developed several high specifity and sensitivity biosensors construction methods for the detection of proteins and small molecules. Main works are as follows:In chapter two, a reusable aptameric recognition system was described for the electrochemical detection of protein PDGF-BB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe and primer probe were utilized to introduce a RCA process into the aptamer-target binding event while a newly designed aptamer probe was used to recognize the target protein. Towards this goal, this adapted aptamer probe was designed via lengthening an original aptamer sequence by the complement to CDNA and extending the outer helix of three-way junction. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: the unique electrochemical technique, attractive RCA process, reversible DNA hybridization on an electrode surface and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits the excellent performance,but also overcomes the limitations associated with conventional aptameric biosensors (e.g. dependence of signaling target binding on specific aptamer sequence and requirement of sandwich assays for two or more binding sites per target molecule). The recovery test demonstrated the feasibility of the designed sensing scheme for target protein assay. Given attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research. Based on chapter two, we explore a new method for protein detection in chapter three, which combined signal amplification of the L-RCA system and background elimination of molecular beacon. The strategy is depended on the competition reaction between target protein, aptamer probe and a complementary single-strand DNA (the CDNA) perfectly matched with the aptamer probe. The presences of target protein in the assay system made the aptamer probe bind specifically with the target protein to form aptamer probe/analyte complex and triggered the end of aptamer strand displace with itself, that made the CDNA released from aptamer and allowed the CDNA to hybridize with the padlock probe. With the assistance of DNA ligase, the padlock probe could be circularised and subsequently be amplified by RCA reaction with Phi 29 DNA polymerase. The RCA products contained thousands of repeated sequences which could hybridize with the molecular beacon (the detection probe). By contraries, In the absence of target protein, the aptamer probe hybridized with the CDNA to form aptamer probe/CDNA duplex, which hindered the binding of CDNA and the padlock probe and resulted in the failure of L-RCA reaction. Using platelet-derived growth factor BB (PDGF-BB) as the model analyte, as low as 1.36 amol of protein molecules could be detected. The proposed strategy is universal since the sequences of aptamer probe, CDNA, and the padlock probe could be easily designed to adapt for the aptamer-based detection of other proteins without other strict conditions.In chapter four, a new working mechanism, hairpin sticky-end pairing-induced GNP assembly, is introduced on the basis of the unique instability of aptamer-modified nanoparticles. The salt-induced aggregation of oligonucleotide-modified GNPs can readily occur while addition of target molecules favors the formation of hairpin structure and inhibits substantially the nanoparticle assembly. Along this line, we developed a proof-of-concept colorimetric homogeneous assay using immunoglobulin E (IgE) as analyte model via transforming commonly designed"light-down"colorimetric biosensor into"light-up"one. From the points of view of both conformational transition of aptamer and steric bulk, oligonucleotide-GNPs display an additional stability upon binding to target molecules. The assay showed an excellent sensitivity with both the naked eye and absorbance measurements. Compared with almost all existing IgE sensing strategies, the proposed colorimetric system possesses a substantially improved analytical performance. Success in this biosensing protocol indicates that investigating the assembly behavior of hairpin aptamer-modified GNPs would offer new insight into the dependence of GNP property on the structure switching and open a new way to design signaling probes and develop colorimetric assay schemes.