# These two authors contributed equally to this paper.
## 2 Results and Discussion
### Application in the detection of free hCG\(\beta\)  
Magnetic beads came into use in enzyme chemiluminescence commence immunoassay (ECLIA) just in recent years. In this method magnetic separation, immunological protocols and chemiluminescence detection technique were combined ingeniously, which left out the complicated package of antibodies in microcells, thus the time and procedures were cut down. [PERSON] et al. [19] thought that the high selectivity displayed between the antibodies and their antigens had motivated the development of immunoassay analytical techniques, while magnetic beads provided immunosensing systems with renewable surfaces for the sake of regeneration. **Scheme 2. ** SEM and TEM pictures of the SPMP microbeads synthesized by the modified suspension microemulsion polymerization method (a. left picture; b. right picture). In our point, the advantage of superparamagnetic microbeads in ECLIA was mainly in their superparamagnetism. Namely, their magnetism was proportional to the strength of outside eternal magnetic field without any remnant magnetism. When the field was removed, the good dispersion of these SPMP microbeads could be ensured after repeated uses of magnetic field. Otherwise, the specific biomolecules bound to these beads would not has much possibility to bump and react with their corresponding biomolecules in liquid, or nonspecifically reacted molecules could not be diluted effectively due to the aggregation among particles. But superparamagnetism was usually demonstrated when the size of the particles was less than 10 nm. Thus some obstacles prevented the very tiny superparamagnetic nanoparticles to realize their application. First, some biological molecules are larger than 10 nm, some (E.G., cells) are even several tens microns. Significant reduction in particle size must be achieved to provide the surface area required, but too small a particle may not carry enough magnetite and, in practice, would cease to be magnetic [12(b)] due to their little magnetophoresisability [6]. Second, as a result of anisotropic dipolar attraction, pristine nanoparticles tend to aggregate into large clusters and thus lose the specific properties associated with single-domain, magnetic nanostructures [24]. Immobilizing these very tiny pristine nanoparticles in the framework of polymers successfully solved both of the two obstacles. **Scheme 3. ** the magnetic hysteris loop by vibrating samples magneticity  
Whatever advantages of magnetic beads in assays people focused on, the magnetic ECLIA has by and by come into use in the detection of other tumor markers such as carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP)**,** carbohydrate antigen (CA) 125**, CA199, and [PERSON] et al [25] detected the hCG in saliva samples in this method. Scheme 4 demonstrated the proposed mechanism of the newly developed assay. In the assay, the molecules for determination were successively reacted with the two kinds of monoclonal antibodies, which were separately labeled by alkaline phosphatase (ALP) and fluoreszeinthiocyanat (FITC) in their ferrocenemonocarboxylic acid (Fc) fragments, to form a sandwich double antibodies' complex, ALP-labeled-antibody-antigen-FITC-labeled-antibody. The SPMP microbeads, which were coupled with anti-FITC antibody, then specifically reacted with the sandwich complex, and deposited to the bottom of the microcells in the attraction of the eternal magnetic field. The unreacted molecules were removed by repeated cleansing and magnetic separation. The new chemiluminescence substrate, 3(2'spiroadamantane)-4- methoxy-4(3' phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), was used to produce stable photo signals with the aid of alkaline phosphatase (ALP) without any background.