The Construction Of Fluorescence Aptasensor Based On Signal Amplification Strategy For Ultrasensitive Detection Of Adenosine

Biosensor is a rapid detection and monitoring system which could transform the recognition of targets into a physically detectable signal. They have broad application prospects in food and drug analysis, biotechnology, clinical diagnosis and other fields. The biosensors based on aptamer as recognition element have been a hot and key research because of its high specificity and affinity. Up to now, many aptasensors based on signal amplification have been established, especially fluorescence aptasensors due to its simplicity, speediness and good selectivity. Thus, this paper mainly focuses on how to build novel fluorescence aptasensor based on existing biological and chemical analysis techniques.Adenosine is a medicinal molecules with crucial physiological and pharmacological activities, such as extension of the blood vessels and regulation of the smooth muscle contraction. It is a major regulator of the human life activities. Adenosine performs extremely important functions in the peripheral/central nervous system and immune system. Moreover, studies have shown that adenosine can also be used as a potential biomarker for tumor. So the detection of adenosine is of great value in the diagnosis and treatment of cardiovascular disease, neurological disease, malignant tumor and the disease of immune system, etc.Recently, a large amount of fluorescence aptasensors has been used to detect adenosine. However, most of the aptasensors have unsatisfactory sensitivities, which cannot meet the requirement of the low level adenosine detection in complex biological samples, such as human serum and urine samples. Meanwhile, the realization of adenosine detection in complex biological samples is of great importance in clinical research and disease diagnosis and prognosis. Therefore, the sensitivity of adenosine detection needs to be further enhanced. So in this paper, using the effective strategy of signal amplification, we developed new and general fluorescence aptasensors, which improved the sensitivity of the assay and realized the quantitative analysis of the adenosine detection in complex biological samples.The first chapter is the introduction section, and this article firstly summarizes the biosensors, especially the characteristics, advantages and application prospects of the fluorescence aptasensors. Secondly, we summarize the commonly used signal amplification strategy. Then, we introduce the significance of detection and traditional detection methods of adenosine. Finally, we discussed the development of adenosine aptasensors and the current problems we are facing.In the second chapter, a label-free and dual-amplified fluorescence aptasensor for sensitive analysis of adenosine is developed based on exonuclease Ⅲ-assisted DNA cycling and hybridization chain reaction. Firstly, we fabricated a trifunctional probe that consists of the catalytic strand, the aptamer sequence and a streptavidin-magnetic nanobead (streptavidin-MNB). The streptavidin-MNB played a role of enrichment and separation to achieve a low background. The aptamer sequence was employed as a recognition element to bind the target adenosine, leading to the release of the catalytic stand. Then, the catalytic strand induced the Exo Ⅲ-assisted DNA cycling reaction and produced a large amount of DNA fragments, which got a primary amplification. Subsequently, the DNA fragments acted as trigger strands to initiate HCR, forming nicked double helices with multiple G-quadruplex structures, which achieved a secondary amplification. Finally, the G-quadruplex structures bonded with the N-nethyl mesopor-phyrinIX (NMM) and yielded an enhanced fluorescence signal, realizing the label-free detection. In the optimum conditions, the detection range of this method was from 1.0×10-6mol L-1 to 1.0× 10-4 mol L-1, and the detection limit of this assay is 4.2×10-7 mol L-1, which was about 10 times lower than that of the reported label-free strategies. Moreover, this assay can significantly distinguish the content of adenosine in urine samples of cancer patients and normal human, indicating that our method will offer a new strategy for reliable quantification of adenosine.Although the above aptasensor could detect adenosine successfully and sensitively, the existence of MNB increased the complexity and the cost of the assay. So we developed our next aptasensor. In the third chapter, we develop a label-free aptasensor for adenosine based on nicking-assisted strand-displacement amplification and catalytic hairpin assembly. In this assay, an ingenious hairpin probe is designed, which contains the aptamer sequence of adenosine, the recognition site for nicking enzyme, and the complementary sequence of the CHA trigger. When the target exists, the aptamer sequence would recognize adenosine and result in the conformational change of the hairpin probe. Once adding the polymerase, nicking enzyme, and dNTPs, it would initiate polymerase and nicking cycling and release of numerous ssDNA fragment. The ssDNA fragment can be used as the triggers of CHA, inducing the hybridization of H1 and H2, leading to forming a large amount of dsDNA with multiple G-quadruplex structures. Finally, the G-quadruplex structures bond with NMM and yield an enhanced fluorescence signal, which realizes the label-free and homogeneous detection of adenosine. The detection of adenosine is 1.3×10-7 mol L-1, which is two order lower than other aptasensors for adenosine[171,175] and about 50 times lower than our group’s last work.This method is sensitive, label-free, and separation-free. In addition, it can also distinguish the content of adenosine in urine samples of cancer patients and normal human. Moreover, this system can be extend to detect other target through changing the sequence of hairpin probe, providing a universal platform.