With the help of iron oxide nanoparticles, electron spin resonance spectroscopy (ESR) was applied to immunoassay. Iron oxide nanoparticles were used as the ESR probe in order to achieve an amplification of the signal resulting from the large amount of Fe 3+ ion enclosed in each nanoparticle. Rabbit IgG was used as antigen to test this method. Polyclonal antibody of rabbit IgG was used as antibody to detect the antigen. Iron oxide nanoparticle with a diameter of either 10 or 30 nm was labeled to the antibody, and Fe 3+ in the nanoparticle was probed for ESR signal. The sepharose beads were used as solid phase to which rabbit IgG was conjugated. The nanoparticle-labeled antibody was first added in the sample containing antigen, and the antigen-conjugated sepharose beads were then added into the sample. The nanoparticle-labeled antibody bound to the antigen on sepharose beads was separated from the sample by centrifugation and measured. We found that the detection ranges of the antigen obtained with nanoparticles of different sizes were different because the amount of antibody on nanoparticles of 10 nm was about one order of magnitude higher than that on nanoparticles of 30 nm. When 10 nm nanoparticle was used as probe, the upper limit of detection was 40.00 μg mL −1 , and the analytical sensitivity was 1.81 μg mL −1 . When 30 nm nanoparticle was used, the upper limit of detection was 3.00 μg mL −1 , and the sensitivity was 0.014 and 0.13 μg mL −1 depending on the ratio of nanoparticle to antibody.
Schematic diagram of procedure and ESR spectra
Water soluble copper sulfide (CuS) nanoparticles were synthesized using l -cysteine as the ligand. Multiple biotins were conjugated to the antibody of rabbit IgG, and the streptavidin was attached to the CuS nanoparticles. The Cu 2+ ions enclosed in the nanoparticles were used as the electron spin resonance (ESR) probes and detected with ESR spectrometer. The immunoassay reaction was resulted in the formation of the coating antibody attached to the microplate well, the detecting antibody labeled with the biotins, and the streptavidin attached to the CuS nanoparticles. After the immunoassay reaction was performed, large amount of Cu 2+ /Cu + ions inside the nanoparticles were released with the help of diethyldithiocarbamate (DDC) and the Cu 2+ -DDC complex formed. The Cu 2+ -DDC complex was extracted into n -butanol, which was used as the analytical sample. Both ESR and UV–vis signals were collected for the analytical sample. The double logarithm standard curve was well simulated with a linear regression equation. The limits of detection of the rabbit IgG were 1.76 pg/mL and 2.36 pg/mL by ESR and UV–vis method, respectively. The detection range using ESR as the detector was from 8.8 pg/mL to 500 ng/mL, covering almost 5 magnitude orders of the rabbit IgG concentrations. The rabbit serum was analyzed and the rabbit IgG concentration was found to be 7.76 mg/mL. The reproducibility of the present method was good enough with the intra-assay error within 3.4 % and the inter-assay error within 11.2 %. The spiked serum samples were analyzed and the experimental results indicated that the recoveries were from 108.2 to 113.7%.
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