Demonstration of sandwich and competitive modulated supraparticle fluoroimmunoassay applied to cardiac proteinbiomarkermyoglobin

Abstract

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).

Diagnostics on acute myocardial infarction: Cardiac troponin biomarkers

“…Compared to the conventional technique such as ECG, biosensors possess high sensitivity, high selectivity, fast analysis, reliable pretreatment and simple instrumentation (Burcu Bahadır and Kemal Sezgintürk, 2015). Different methods have been developed for cardiac troponin detection and quantification which include enzyme-linked immunosorbent assay (De Antonio et al, 2013), chemiluminescent immunoassay (Cho et al, 2009), fluoro-immunoassays (Hayes et al, 2009), electrical detections (Tuteja et al, 2014), surface plasmon resonance (SPR)-based detection (Liu et al, 2011), colorimetric protein array , point-of-care (POC) assays (Dittmer et al, 2010), and aptamer-based biosensor (Shu-hai et al, 2014). This review focus on the advancement, sensitivity and limitations of these methods towards detection of cardiac troponin biomarkers.…”

Sensitive detection of cardiac biomarkers using a magnetic microbead immunoassay

“…30 In terms of data acquisition, previous work noted differences in signal strength depending on their location in the field of view, increasing variation in both signal and noise. The changes to optics and acquisition conditions eliminated this issue, producing rotors with similar signal intensities independent of their location.…”

“…The optimized collection of the video sets allowed for improvement in detection over several orders of magnitude compared to previously collected myoglobin data, from 50 pM to 36 aM (Table 1). 30 In addition to approaching these fundamental limits of quantification, the linear range of this method may be easily scaled for the detection of higher concentration samples through the addition of more magnetic microparticles or through sample dilution. The limits of quantitation observed in the present work compare favorably to the metrics of a fully optimized immunoassay, achieving detection on the same order of magnitude as fundamental limitations.…”

Rapid electrical immunoassay of the cardiac biomarker troponin I through dielectrophoretic concentration using imbedded electrodes

“…The methods used for the diagnosis of AMI include conventional enzyme-linked immunosorbent assays (Wang et al, 2001), fluoroimmunoassays (Hayes et al, 2009), immunochromatographic tests (Penttil et al, 1999), spectrophotometric methods (Shiomi et al, 2005), electrochemiluminescence assays (Shen et al, 2011), and radioimmunoassays (Apple et al, 1997;Carraro et al, 1994). However, these approaches require multistep processing of samples, long diagnostic times, high costs, and trained personnel.…”