Study On Detection Of Proteins And Small Molecules Based On Label-free Fluorescence Biosensor

Biosensor technology has many unique advantages, such as simple equipment, low cost, fast response, high sensitivity and so on. Base on its inherent advantages, biosensor technology has aroused great concern in the field of analytical chemistry, biochemistry, biomedicine and biotechnology analysis. Among them, fluorescence biosensor, the use of the fluorescent signal as a mean of detecting, has attracted more and more attention in the field of bioengineering, environmental monitoring and medicine. Analysis of small molecules and proteins and other substances in human has been a very important modern in analytical chemistry research. The detection of small molecules and proteins has a very important significance for diagnosis of disease, nutrition and health research and clinical analysis. How to use fluorescent biosensor technology for realizing the fast, sensitive and accurate detection of small molecules and proteins is still pursuing for researches in biomedical and analytical area. Based on the current research situation, we combines signal amplification technology and use of fluorescent dye as marker to development a number of new fluorescent biosensor technology for sensitively detecting small molecule and protein. The specific contents in our paper are as follows:Part 1 Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplificationIn this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease â…¢ (Exo â…¢)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo â…¢ to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specificaffinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules.Part 2 Terminal protection of small molecule-linked ssDNA for label-free and sensitive fluorescent detection of folate receptorIn this work, based on terminal protection of folate-linked ssDNA (FA-ssDNA) and the SYBR Gold fluorescent dye, we describe the development of a label-free fluorescent strategy for the detection of folate receptors (FRs). The binding between the target FR and the FA moiety of the FA-ssDNA protects the FR bound FA-ssDNA from digesting by Exo I. The binding of SYBR Gold to the terminal protected, un-digested FA-ssDNA leads to enhanced fluorescent emission for the monitoring of FR with a detection limit of 30 pM. Besides, the developed method also shows high selectivity toward FR against other control proteins. Moreover, our approach avoids the labeling of the probes with fluorescent tags and achieves label-free detection of FR. With these advantages, the proposed method thus holds promising potential for the development of simple and convenient strategies for the detection of other proteins by using different small molecule receptor/protein ligand pairs.Part 3 Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombinBased on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs.