Demonstration of sandwich and competitive modulated supraparticle fluoroimmunoassay applied to cardiac protein biomarker myoglobin

Modulated supraparticle structures are used to improve sandwich and competitive fluoroimmunoassays. The improved methods are demonstrated on myoglobin, a key diagnostic protein for detection of heart damage. The resulting method uses microliter volumes with bovine serum samples doped with varying concentrations of equine myoglobin. These immunoassays use micron-diameter iron oxide particles as a solid phase for antibody anchoring. Introduction of a magnetic field creates dipole moments on the particles, which attracts them to each other to form rod-like supraparticle structures. These structures can rotate within an alternating magnetic field generating convective flow and a periodic signal that can be analyzed with lock-in amplification enabling more sensitive detection. The system is demonstrated on a target associated with acute myocardial infarction (AMI). This disease causes decreased oxygen delivery to the heart resulting in tissue death and the release of cardiac myoglobin into the bloodstream. Studies have shown that the assessment and monitoring of serum myoglobin concentrations is important when making an early diagnosis of AMI. Early diagnosis is crucial since treatment is most effective when done within the first two hours of symptoms. The modulated assay is rapid, accurate, and sensitive for myoglobin assessment of small-volume serum samples. Using a cut-off value of 5.0 nM (85 ng/mL) for AMI induced myoglobin, the modulated competitive assay was able to diagnose AMI-like conditions in serum doped with myoglobin after an incubation time of only 10 min. The standard curve developed for the modulated sandwich assay was linear over a range of zero to 1 nM (17 ng/mL) with a lower limit of detection at 50 pM (0.85 ng/mL).