A Highly Sensitive Immunoassay Based On Controllable Hierarchical Self-assembled Enzymatic Nano-composite

With the improvement of living standards, accelerated pace of life and diet changes, the morbidity and mortality of cardiovascular disease (CVD) has shown an increasing trend in China, which poses a serious threat to the life and health of our residents, while it is also a big burden on our healthy national economy. As one of the acute CVD, the acute myocardial infarction (AMI) is increasing year by year which became a main cause of death. High sensitive detection of an early stage AMI biochemical marker:cardiac troponin (cTn) which is extremely low in concentration facilitates timely intervention, thus significantly reducing its mortality. But conventional immunoassay methods cannot meet the demands for sensitivity in early diagnosis of AMI, the current clinical methods used basically achieving the requirements of sensitivity, however, it requires large specialized equipment and additional staff training, which causes cTn analysis expensive, hindering the development of low-cost-healthcare. Even so, it was noted that the existing analytical methods still cannot meet the clinical needs for assay precision and sensitivity by the European Society of Cardiology.Aiming to the above problems, we established a size-controlled hierarchical self-assembled enzyme nanocomposite (ENC) design method. By adjusting the number of self-assembled interaction sites of building block, the control of the number of structural unit in the nanocomposite can be achieved. Based on this design, human recombinant heavy chain ferritin (rHF), capable of self-assembly to form the cage structure, was fused with biotin acceptor peptide (BAP) to construct fusion protein BAP-rHF by genetic modification. Utilizing the biotin ligase (BirA), the fusion protein could be biotinylated when it was expressed in E.coli. By manipulating the process of self-assembly of biotinylated ferritin and horseradish peroxidase labeled streptavidin (SA-HRP) in vitm, a multifunctional nanocomposite was constructed, whose number of enzyme molecules could be controlled. Applied it in the cardiac troponin I (cTnI) immunoassay, the detection sensitivity was significantly enhanced more than10,000folds compared to traditional enzyme linked immunosorbent assay (ELISA). This study demonstrated a new design to construct a size-controlled hierarchical self-assembled enzyme nanocomposite, and successfully constructs a controlled hierarchical self-assembled enzyme nanocomposite by using ferritin as the backbone. This verified the feasibility of the design. By fixing certain quantity of enzyme molecules with a high density, the nanocomposite integrated two functions of binding and catalytic ability together. Under the situation of no extra professional equipment and technology, the enzyme nanocomposites could greatly increase the detection sensitivity when applied in immunoassay. It is verified that the design and preparation of enzyme nanocomposite is feasible, and it provides a new technical routine for the development of fabrication of multifunctional nanocomposite.