Ultrasensitive, selective and simultaneous detection of cytochrome c and insulin based on immunoassay and aptamer-based bioassay in combination with Au/Ag nanoparticle tagging and ICP-MS detection

An ultrasensitive method for simultaneous determination of cytochrome c (cyt-c) and insulin was developed by combining aptamer-based bioassay and immunoassay, multielement-tagging and inductively coupled plasma mass spectrometry (ICP-MS). Aptamer-modified gold nanoparticles (apt-AuNPs) and antibody-modified silver nanoparticles (ab-AgNPs) were employed as specific element tags for cyt-c and insulin, respectively. The prepared surface-functionalized magnetic microparticles (MMPs) were used for efficient and fast magnetic separation. The bioassay conditions were carefully optimized, including the amount of MMPs, the concentration of AuNPs and AgNPs, and the reaction time. Under optimal conditions, the developed method gave a linear range of 0.1-20 nM for cyt-c and 0.2-40 nM for insulin, a detection limit (3s) of 1.5 fmol (30 pM in 50 mu L) for cyt-c and 5.5 fmol (110 pM in 50 mu L) for insulin. The precision (relative standard deviation) for six replicate determinations of cyt-c (0.6 nM) and insulin (2.0 nM) was 6.6%, and 6.0%, respectively. The present method exhibits good specificity with recoveries from 87% to 98% for spiked cyt-c and insulin in human serum samples. The methodology demonstrated here provides a new possibility for bioassays and clinical diagnoses, which has potential for simultaneous determination of two or more low-abundance biomarkers of interest via multi-element tags.