Liquid-Phase Immunoassays Using Brownian Relaxation of Magnetic Markers

We have developed a liquid-phase immunoassay technique using Brownian relaxation of magnetic markers. In this method, biological targets are fixed on the surface of large polymer beads whose size is typically a few μm. When the markers are bound to the targets, their Brownian relaxation time is dominated by that of the polymer bead, becoming much longer than that of unbound (free) markers. The resulting difference between the magnetic properties of the bound and free markers was detected by relaxation measurements. Therefore, we can magnetically distinguish between the bound and free markers, i.e., we can omit a time consuming washing process called bound/free separation. We developed a detection system using a magneto-resistive (MR) sensor and showed that we can detect 1.4 × 10⁷ bound markers in 60 μl of solution. If we assume that a single marker is bound to a single target, this sensitivity can be expressed as 3.8 × 10^-16 mol/ml (or 0.38 fmol/ml) in terms of the molecular-number concentration. We also demonstrated the detection of biological targets called biotins, which were conjugated on the surface of the polystyrene beads with a diameter of 3.3 μm. A strong relationship was obtained between the number of bound markers and the number of biotin-conjugated polymer beads, which confirmed the validity of the present method.