| Research on the biomolecules has been a vital part for people to study the mystery of human bodies and has aroused much interest since last century, of which the detection for oligonucleotides, proteins, aptamers, adenosine, etc is of great importance for common disease diagnosis and clinical medicine. For instance, adenine triphosphate (ATP), generally acknowledged as "energy currency" in most animate begins, plays an essential role in most enzymatic activities and it is also a vital component of many biological cofactors. In addition, ATP has also been used as an indicator for cell vitability and injury. The lack of ATP would lead to some disease like Anemia, Hypoglycemia and Parkinson. Lysozyme, acted as the necessary protein for mammal species, can dissolve bacteria into the solution overflew from the cell walls broken by it. Lysozyme is anti-inflammation since it can combine with negative viral proteins directly and inactivate them. These proteins and small molecules can bind with aptamers to form the corresponding compounds, which provide the theoretical basis for many research works.The interaction of metal ions or complexes with cyclodextrins has been of extensive interest to bring diverse properties to the receptor, hence the definition of metallocyclodextrins. The design of sensors and molecular wires based on metallocyclodextrins is attractive due to the inherent redox, photochemical, conductivity properties introduced by metal centres. Usually, when cyclodextrin cavity encapsulates a guest molecule, the intermolecular energy and electron transfer process will happen between guest molecule and metal centre, which leads to the changes for fluorescence signal. These unique properties make metallocyclodextrins special in building blocks for the development of sophisticated supramolecular devices and arouse much attention.Nowadays, a lot of works about the fluorescent biosensing based on bipyridine-ruthenium have been reported, which are quite valuable in biology, clinical medicine and food security. Herein, to improve a higher fluorescent efficiency and extend its application, much effort has been made on synthesizing new material modified with bipyridine-ruthenium to realized sensitive detection on biomolecules while maintained their specific biological activities.A new series of metallocyclodextrins, named (bipyridine)ruthenium(II)-β-cyclodextrin, has been synthesized by our team, including two groups: multi-(bipyridine)ruthenium(Ⅱ) and multi-β-cyclodextrins. multi-(bipyridine)ruthenium (Ⅱ) (multi-Ru-β-cyclodextrin) exhibites excellent electrochemiluminescence and host-guest recognition properties. We have fabricated many sensitive sensors to realize the determination on DNA, lysozyme, thrombin and ATP; While in this work, (bipyridine)ruthenium(Ⅱ)-multi-β-cyclodextrin is utilized to develop aptasensing on biomolecules based on its brilliant fluorescent properties, which can provide new research direction for the exploration on human bodies. Therefore, this work offers great potential.Chapter 1:IntroductionIn Chapter 1, the cyclodextrin, as one of the most common receptors, has been introduced generally about its structure and features. The excellent performance of metallocyclodextrins and their applications in biosensing have been illustrated systematically. Then, we have studied the principle of the fluorescence biosensor since fluorescence based detection method exhibits excellent benefits in sensitivity, simplicity and convenience. The aptamer based and cyclodextrin modified biosensing based on fluorescence are highlighted. In the end, we summarized the background, significance, objectives and main content of this thesis. Chapter 2:Sensitive fluorescence detection of lysozyme using a tris(bipyridine)ruthenium(Ⅱ) complex containing multiple cyclodextrins.A new series of metallocyclodextrins comprising a ruthenium core and multiple cyclodextrins were synthesized and the photophysical studies indicates their higher emission efficiency than the parent compound tris(bipyridine)ruthenium(II). ssDNA/aptamer could further increase the fluorescence intensity of metallocyclodextrins through the host-guest recognition between bases on ssDNA/aptamer and cyclodextrin cavities, which can enhance the shielding effect of the ruthenium cores from quenching by oxygen in the solution and lead to the increased fluorescence signal. On this basis, we propose a sensitive fluorescence method for protein detection. First, ssDNA/aptamers (lysozyme-aptamer in this case) bind with metallocyclodextrin and increase its fluorescence intensity. While the addition of lysozyme lead to the formation of aptamer/lysozyme complex and thus weaken the interaction between aptamers and CD cavities. This process ultimately results in a reduction of fluorescence intensity which can be used to determine lysozyme quantitatively. An excellent linear relationship between the fluorescence decrease and the logarithm of lysozyme concentrations ranging from 5×10-7 M to 5×10-10 M was obtained with a detection limit of 48 pM (S/N=3). The proposed fluorescence aptasensing method shows simplicity, high sensitivity and selectivity without time-consuming signal-labeling process, which can be potential in detecting other aptamer-specific biomolecules. Chapter 3:A fluorescence aptasensing strategy for ATP detection using tris(bipyridine)ruthenium(Ⅱ) complex containing six cyclodextrin units.A sensitive label-free fluorescent aptasening strategy for the adenosine triphosphate (ATP) detection has been developed with a novel metallocyclodextrin, tris(bipyridine)ruthenium (Ⅱ) complex containing six cyclodextrin units (6CD-Ru). The photophysical properties of 6CD-Ru was investigated, exhibiting much stronger emission signal compared to the parent compound Ru(bpy)3Cl2. Furthermore, the emission spectrum showed that aptamer could increase the fluorescence intensity of 6CD-Ru dramatically, attributed to the interaction between aptamer and cyclodextrin, which could provide protection to the ruthenium core from the quenching of emission by oxygen in the solution. With the addition of ATP to the mixture system, the interaction between aptamer and cyclodextrins on the 6CD-Ru was diminished, since the ATP/aptamer complex had the priority to be formed, leading to the corresponding reduction of fluorescence intensity, which could be utilized to detect ATP quantitatively. In this work, the assay was linear between the fluorescence decrease and the logarithm of ATP concentrations in the ranges from 1 nM to 0.5 μM with the detection limit of 0.5 nM (S/N=3). The proposed fluorescent aptasensing strategy exhibited high sensitivity and specificity, without any labeling or amplification procedures, and it could also be applied for the detection of many other aptamer-specific targets. |
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