Improved detectability and signal strength for rotating phase fluorescence immunoassays through image processing.

Fluorescence immunoassays based on rotating solid phase have shown promise of lowered detection limits, among other advantages. However, intrinsic background distortion effects have limited their utility. Here, novel image processing strategies are used to minimize these effects and improve the estimate of concentration and lower the detection limit. This initial demonstration of a new processing capability is performed on data for a protein, myoglobin, which is a biomarker for acute myocardial infarction. For these data, compared with published results, the detection limit is improved by a factor of approximately one hundred (to 700 fM), which is competitive with or better than other immunoassay strategies (ELISA, for example) that are fully developed. This work suggests that image and video processing technologies can provide a valuable alternative approach to biochemical detection and concentration estimation.

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).

Detection of FITC-cortisol via modulated supraparticle lighthouses.

Hormones are important bioactive compounds in blood and tissue that vary in concentration in response to stress and certain disease states. Establishing the changes in physiological hormone concentrations over time can lead to more effective diagnoses and perhaps a better understanding of the evolution of stress and disease. To monitor concentration over time, the sampling must be rapid and noninvasive; specimens such as saliva that require little effort to collect are preferred. However, more sensitive assay techniques are needed when compared to blood analysis since free hormone concentration in saliva is only a small fraction of the concentration in circulating blood. In this work, magnetic field-induced structures of paramagnetic particles are used as a solid substrate to demonstrate improved detection limits for a separation-free assay of cortisol. Once formed, the structures are subjected to a rotating magnetic field and this leads to two important features. First is the ability to utilize frequency and phase filtering (lock-in amplification) for the signal generated from surface-bound labeled species. Second is the improved mass transport of the antigen to the surface of the rotating structures. These two unique capabilities result in a quantifiable signal at a relatively low target antigen concentration. This method has been demonstrated with the detection of fluorescein isothiocyanate-labeled cortisol (FITC-cortisol) at a concentration of 300 pM.