| With the continuous development of the analytical science, various analysis and testing process in life sciences are increasingly needed the help of biosensing technology to obtain the required information. Due to many advantages such as good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system, biosensors have valuable applications in chemistry, biomedicine, environmental protection, food industry, medicine and military affairs. Ideally, biosensors for the target detection should have high detection sensitivity (low detection limit), high specificity (low interference), wide dynamic range, fast response time, and universal characteristics.Functional nucleic acids are short synthetic DNA and RNA sequences which have been isolated via a combinatorial biology technique known as in vitro selection, or a process also known as systematic evolution of ligands by exponential enrichment (SELEX). The functional nucleic acids can specifically bind a broad of analytes including small biomolecules, proteins, cancer cells as well as metal ions with high affinity. Typically, functional nucleic acids include two types of nucleic acid molecules, one type of them have been shown to perform catalytic reactions (called DNAzymes, or deoxyribozymes) like protein enzymes, and the other can bind to a specific target molecule (called aptamers) like antibodies. In the traditional understanding, the nucleic acids are only the storage and transfer carrier of genetic information. However, the discovery of functional nucleic acids has resulted in a breakthrough in such traditional sense, and provided an interesting alternative to biosensing system.One of the most important performances of biosensor is sensitivity, and it is very limited in further improving the sensitivity by simply using the conformational changes of the functional nucleic acid before and after bind to the target. In recent years, the design of new signal amplification method for ultrasensitive detection of target has attracted attentions of researchers. Two different strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection. Recently, signal amplification technology has attracted more concerns due to the significant effect on improving sensitivity.Based on the above considerations and the reported literatures, in this doctoral thesis, several bioassay systems have been developed focused on new methods for enhancing the detection sensitivity of biosensors by using signal amplification technology. The details are summarized as follows:(1) Study on the biosensors based on gold nanoparticle signal amplification technology. Due to the large specific surface area, good biocompatibility and surface characteristics of nucleic acids of high load, a novel electrochemical label-free biosensor for Hg2+has been developed in chapter2based on the "T-Hg2+-T" specific interaction and the gold nanoparticle signal amplification technology. In the presence of Hg2+, the linker DNA hybridized with the electrode surface-tethered capture probe via the "T-Hg2+-T" specific interaction, and the gold nanoparticles functionalized reporter DNA subsequently hybridized with the linker. Methylene blue was selected as electrically active substances, which can specifically bind with guanine bases to form methylene blue-guanine complexes, resulting in a significant increase in the electrochemical signal. The proposed biosensor exhibits high sensitivity, high specificity, and a detection limit of0.5nM could be achieved for Hg2+, which makes the biosensor favorable for Hg2+assays in practical samples of very low concentration. Based on the distance dependent exponentially decreased enhancement of the EM (electromagnetic) field, in chapter3, the use of a nanoscale DNA-Au dendrimer as a signal amplifier was proposed for the design of functional DNA-based ultra-sensitive SERS biosensors. As a proof-of-concept, a Pb2+-dependent DNAzyme and the anti-adenosine aptamer was chosen to develop novel SERS biosensors to verify the feasibility of our DNA-Au dendrimer amplification strategy. For the detection of Pb2+the DNAzyme strand was immobilized on the electrode surface first, in the presence of Pb2+, the DNAzyme was activated and it cleaved the substrate section into two parts. The remaining oligonucleotide moiety on the electrode surface hybridized with the reporter DNA1and DNA2to form nanoscale junctions through layer by layer assembling. The DNA-Au dendrimer could then be formed to give a strong SERS signal, and a detection limit of0.1nM could be achieved for Pb2+. For the detection of adenosine, the SERS aptamer sensor was designed based on target-induced strand displacement. A thiolated DNA sequence was first immobilized on gold electrode surface as a capture probe, and then hybridized with the anti-adenosine aptamer. The introduction of target adenosine could trigger the release of the aptamer from the duplex. The free capture probe could then hybridize with the reporter DNA2and DNA1, thereby forming the DNA-Au dendrimer to trigger a remarkable SERS signal enhancement. A low detection limit of50nM was obtained with good selectivity. (2) Study on the biosensors based on enzymatic recycling signal amplification technology. Based on ssDNA-cleaved property of nicking endonuclease, and take advantage of the unique features of molecular beacons (MBs) such as high sensitivity and low fluorescent background, in chapter4, a new MB-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs was developed. In the presence of a target, the assistant probe, together with the target, can hybridize with the MB and open its hairpin structure to form a "Y" junction structure, as well as form the doublestranded recognition sequence for nicking endonuclease Nt.BbvCI. Once the MB is cleaved, it is dissociated from the sensing system, and fluorescence is restored. The released hybrid of the assistant probe with the target can then hybridize with another MB and trigger the second cycle of cleavage. Eventually, each hybrid of the assistant probe with the target can undergo many cycles to trigger the cleavage of many MBs, providing a true and efficient target-triggered enzymatic recycling amplification signal. Moreover, compared with the first-generation junction sensing system, our proposed sensing sytem also afforded a faster and more sensitive response, and a detection limit of5pM was obtained. In chapter5, by separating the molecular recognition module from the signal reporter to avoid DNAzyme modifications, and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification, we constructed a new fluorescent cascadic catalytic beacon sensing platform. An L-histidine-dependent DNAzyme was chosen as a model to construct the cascadic catalytic beacon. The unmodified unimolecular DNAzyme was linked by a polyT sequence which serves as a molecular recognition module for L-histidine, and a molecular beacon was selected as the signal reporter. The introduction of target resulted in the cleavage of DNAzyme, and the cleaved partial substrate is released from the DNAzyme and hybridized with the MB, the further using of nicking endonuclease resulted in signal amplification detection for the target. Under the optimized experimental conditions, the proposed sensing strategy provides a low detection limit of200nM with high selectivity. Due to the enzymatic recycling cleavage property, the DNAzyme also can be used to develop signal amplification technology. In chapter6, we proposed a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based signal amplification to develop a ligation-triggered DNAzyme cascade for small molecules detection. ATP and NAD+were chosen as analytical models to verify the feasibility of the CAMB sensing system. As two kinds of DNA ligases which specifically employ ATP and NAD+as cofactors, respectively, we employed the ligation to trigger DNAzyme cascade and realize zero-background signal. Then the activated DNAzyme could hybridize with substrate sequence which was designed as MB to form the CAMB, which was employed for amplified signal detection. The cycling and regenerating of DNAzyme could lead to a true enzymatic multiple turnovers of catalytic beacons. This combination of and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of100pM and50pM for ATP and NAD+, respectively, much lower than those of previously reported biosensors. Moreover, the developed DNAzyme cascades show significantly high selectivity towards the target cofactor (ATP or NAD+), and the target biological small molecule can be distinguished from its analogues.(3) Study on the biosensors based on dual signal amplification strategy that combines the gold nanoparticle with enzymatic recycling signal amplification technologies. In chapter7, a label-free electrochemical DNA biosensor with high sensitivity and selectivity has been reported by employing both gold nanoparticle and enzymatic recycling signal amplification technology. In the design, take advantage of the improved mismatch distinguishing ability of MB, the hairpin probe first hybridizes with target DNA to trigger the enzymatic recycling reaction in the presence of exonuclease Ⅲ, which could avoid the low enzymatic reaction efficiency, effectively. Then, the cleaved sequences hybridize with the hairpin capture probe, which can further improve the mismatch distinguishing ability of the sensing system. Finally, the gold nanoparticles functionalized reporter DNA hybridized with the opened capture probe to amplify the electrochemical signal. Methylene blue was selected as electrically active substances as the sensor in chapter2, and the results demonstrated that the presented dual signal amplification strategy was highly sensitive and specific with a detection limit of0.6pM for target DNA. |
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