Study On Sensitivity Detection Of Adenosine Deaminase Based On Conjugated Polymer

Water-soluble conjugated polymers (WSCPs) with unique structure have attracted much attention because of their good light-harvesting ability and signal amplification effect, which exhibits the broad applications in biological sensing, cell imaging, drug screening and delivery, disease diagnosis and treatment. In this thesis, to sensitively detect the adenosine deaminase (ADA), a label-free aptasensor was set up firstly. Then, by taking advantage of excellent optical properties of conjugated polymers, two sensitive biosensors for ADA sensing was designed based on conjugated polymers poly (9,9-bis (6’-N,N,N-trimethylammonium)hexyl)fluorine pHenylene(PFP). The results are as follows:1. Label-free aptasensor for adenosine deaminase sensing based on fluorescence turn-onA label-free and fluorescence turn-on aptamer biosensor has been developed for the detection of adenosine deaminase (ADA) activity with simplicity and selectivity. Adenosine aptamer will form a tight stem-loop structure upon binding with adenosine. In the absence of ADA, only a small quantity of picagreen intercalates into the stem section of aptamer, resulting in a low fluorescence of picagreen when excited at 490 nm. Interestingly, after the addition of ADA, adenosine is hydrolyzed to inosine, and the released aptamer forms double-stranded DNA (dsDNA) with its complementary single-stranded DNAc, followed by the intercalation of picagreen to dsDNA. When the solution is excited, picagreen emits strong green fluorescence. The increased fluorescence intensity of picagreen is dependent on the concentration of ADA. The detection limit of the ADA is determined to be 2 U/L. Furthermore, compared to other previous ADA sensors, the assay is not only label-free but also a turn-on signal, and possesses properties of lower cost and simpler detection system.2. Water-soluble conjugated polymer as a platform for adenosine deaminase sensing based on fluorescence resonance energy transfer techniqueTo improve the detection sensitivity, we further develop a new biosensor for ADA sensing based on water-soluble conjugated PFP and fluorescence resonance energy transfer technique. In this biosensor, PFP, DNAc-FI labeled with fluorescein (FAM), and ethidium bromide (EB) were used as the fluorescence energy donor, resonance gate, and the final fluorescence energy acceptor, respectively. In the absence of ADA, the adenosine aptamer forms a hairpin-like conformation with adenosine, which is far from its complementary single-stranded DNA (DNAc-FI).When PFP is excited at 380 nm, fluorescein emits strong green fluorescence via one-step fluorescence resonance energy transfer (FRET) while EB has no fluorescence. After addition of ADA, adenosine is hydrolyzed to inosine and then double-stranded DNA (dsDNA) is formed between the aptamer and DNAc-FI, followed by EB intercalating into dsDNA. Once PFP is excited, EB will emit strong yellow fluorescence after two-step FRET from PFP to fluorescein and from fluorescein to EB. The sensitive ADA detection then is realized with a low detection limit of 0.5 U/L by measuring the FRET ratio of EB to fluorescein. Hence, this method demonstrates the sensitive, cost-effective, and rapid detection of ADA activity. The ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), was also studied based on this assay, and the detection limit of EHNA is 50 pM.3. Adenosine deaminase biosensor combining cationic conjugated polymer-based FRET with deoxyguanosine-based pHotoinduced electron transferIn order to improve the detection sensitivity and simplify the detection system, we further demonstrated a ADA detection by modulating the FRET between cationic conjugated PFP and the deoxyguanosine-tailored hairpin aptamer. The hairpin aptamer was labeled with a fluorophore FAM at one end and three deoxyguanosines (Gs) at the other end as a quencher. In the absence of ADA, aptamer forms hairpin-like conformation with adenosines making close affinity of Gs and FAM, which results in the weak FRET from PFP to FAM because of FAM fluorescence being quenched by Gs via photoinduced electron transfer (PET). After addition of ADA, adenosine was hydrolyzed by ADA, followed by the release of free aptamer. In this case, FAM being far away from Gs, the strong FRET thus was obtained due to the quenching process being blocked. Therefore, the new strategy based on the FRET ratio enhancement is reasonably used to detect the ADA sensitively, combining the fluorescence signal amplification of conjugated polymers with the initiative signal decreasing by Gs. The detection limit of the ADA assay is 0.3 U/L in both buffer solution and human serum, which is more sensitive than most of those previously documented methods. The ADA inhibitor EHNA was also studied based on this assay, and the detection limit of EHNA is 10 pM.In conclusion, we have designed three different sensing systems for the sensitive detection of ADA enzyme. Comparing these three methods, we found that the first method is simple, cheap, but relatively insensitive. The second method has good sensitivity, but more complex than the first one. The third method is the most sensitive, and the sensing system is relatively simple. From the following experimental research, it is not hard to know that the systems are more sensitive in the presence of conjugated polymers, which further confirmed that the conjugated polymers applied in biological sensing system can greatly improve the detection sensitivity. In addition, the assay is rapid, homogeneous, and simple, without any excess operation, such as separation or washing steps. These three methods also provide new platforms for the detection of other enzymes and the screening of their inhibitors.