Study On The Construction Of Electrochemiluminescence Aptasensor Based On Nanocomposites And Multiple Amplifying Techniques For Signal Amplification

The electrochemiluminescence(ECL) aptasensor is one kind of novel biosensor, which combines the recognition of aptamer and the ECL analytical technology. It plays a vital role in detection of target protein and judgment of early diagnosis due to their advantages of high sensitivity,good selectivity, fast response, simple operation, lowcost, and so on. Seeking an effective method to amplify the ECL signal for improving the sensitivity of aptasensor in the detection of target protein is one of the most important things in the ECL aptasensors construction. In recent years, a variety of amplification technology, such as nanomaterials, co-reactant and enzymatic reaction to in situ generate co-reactant, are widely employed to fabricate ECL aptasensor. Therefore, it is meaningful to work on reliable,effective and high sensitive ECL aptasensor by the combination of amplifying technique and ECL biosensors. In our paper, research work is carried out from the aspects as follows:Part 1 Amplified electrochemiluminescent aptasensor using mimicking bi-enzyme nanocomplexes as signal enhancementTo in situ generated coreactant can effectively enhance the ECL intensity and thus improve the sensitivity of aptasensor. In this work, hollow Au nanoparticles(HAu NPs) with large surface area were first used as carriers for loading glucose oxidase nanoparticles(GOx NPs), Pt nanoparticles(Pt NPs) and detection aptamer of thrombin(TBA 2) with the aim of increasing the loading amount of the nanomaterails and improving the bio-compatibility of the nanocomposites. Afterwards, hemin was intercalated into the TBA 2 to obtain the hemin/G-quadruplex structure. The hemin/G-quadruplex and Pt NPs were then synergied as the mimiced horseradish peroxidase(HRP)to catalyze H2O2 to generate O2. Meanwhile, C60 nanoparticles(nano-C60) with excellent stability and large surface area were utilized to construct a novel and effective sensitive interface for immobilizing of thiol-terminated thrombin capture aptamer(TBA 1). With the sandwich-type format,GOx NPs, hemin/G-quadruplex and Pt NPs formed mimicking bi-enzyme cascade catalysis system.When proper amounts of glucose were added in the peroxydisulfate(S2O82-) solution, GOx NPs could catalyze the glucose to generate H2O2, which could be further catalyzed by hemin/G-quadruplex and Pt NPs to in situ generate dissolved O2 of high concentration, resulting in a considerably enhancement of ECL signal. This method had successfully overcome the disadvantage of difficulty to label the dissolved O2 and realized the ECL signal amplification. The experiment proved that the aptasensor had high sensitivity, wide linear range, good reproducibility and acceptable precision for TB detection. The linear range was 1×10-6-10 n M, with a detection limit of0.3 f M.Part 2 An amplified electrochemiluminescent aptasensor using Au nanoparticles capped by3,4,9,10-perylene tetracarboxylic acid-thiosemicarbazide functionalized C60 nanocomposites as a signal enhancement tagC60, with zero-dimensional and conjugate π electron structure, can serve as structural platforms for the construction of multifunctional materials. More notably, C60 was reported that it could improve the ECL response of the S2O82-/O2 system. However, due to the poor solubility of C60 in water,we prepared a kind of multi-functionalized water-soluble C60nanoparticles(C60NPs) to achieve ECL signal amplification of the S2O82-/O2 system in this work. Firstly, 3,4,9,10-perylene tetracarboxylic acid(PTCA) with a flat π system was used to functionalize C60 NPs via π–π stacking interactions for increasing the ECL intensity of the S2O82-/O2 system. More importantly,thiosemicarbazide(TSC), with the active groups of –NH2, was selected and introduced into the ECL S2O82-/O2 system for the first time, which could not only absorb Au nanoparticles(Au NPs) for TBA2 anchoring but also formed the TSC-PTC/C60 NPs nanocomposites. Herein, TSC-PTC could act as the coreactant of S2O82- to remarkably enhance the ECL signal of the S2O82-/O2 system and thus improve the sensitivity of aptasensor. Finally, with the sandwich-type format, we constructed a highly sensitive ECL aptasensor for TB detection based on the employment of Au NPs/TSC-PTC/C60 NPs nanocomposites as a ECL signal tag. This aptasensor exhibited high sensitivity, a wide linear range, a low detection limit and high selectivity for TB detection in the range of 1×10-5–10 n M with the detection limit of 3.3 f M, and the S2O82-/O2 ECL system has provided a new perspective in the application in bioanalysis.Part 3 New Signal Amplification Strategy Using Semicarbazide as Coreaction Accelerator for Highly Sensitive Electrochemiluminescent Aptasensor ConstructionThe co-reaction accelerator is a species that when it is introduced into the ECL system containing luminophore and co-reactant, it can interact with co-reactant rather than luminophore to promote the ECL reaction rate of luminophore and co-reactant; thus the ECL signal is significantly amplified in comparison with that in which only luminophore and co-reactant are present. In thiswork, we first proposed a new signal amplification strategy based on the employment of Sem as co-reaction accelerator to promote the ECL reaction rate of Cd Te quantum dots(Cd Te QDs) and the co-reactant of S2O82-for boosting signal amplification. Initially, hollow Au nanocages(Au NCs) with the large specific surface area were used to alternately immobilize the large amount of Sem and Au NPs to obtain the multilayered nanomaterials of(Au NPs-Sem)n-Au NCs for anchoring TBA 2 as TBA 2 signal probes. Notably, since abundant Sem as co-reaction accelerator was introduced into the TBA2 signal probes, the ECL reaction rate of Cd Te QDs and S2O82- was promoted to enhance the ECL response for signal amplification. Then, we utilized TB as the experimental model to construct a novel ECL aptasensor. Utimately, with the sandwich-type format, when we tested in S2O82-solution,the Sem in the TBA2 signal probes could accelerate the reduction of S2O82- to produce the more oxidant mediators of SO4?-, which further boosted the production of excited states of Cd Te QDs to emit light. With the employment of the novel co-reaction accelerator Sem, the proposed ECL biosensor exhibited ultrahigh sensitivity to quantify the concentration of TB from 1× 10-7 to 1 n M with a detection limit of 0.03 f M, which demonstrated that the co-reaction accelerator could provide a simple, efficient, and low-cost approach for signal amplification and hold great potential for other ECL biosensors construction.