We demonstrate the wash-free detection of biological targets based on the Brownian relaxation of magnetic markers. The Brownian relaxation time of bound markers was prolonged by using a cluster (or agglomerate) of bound markers that was formed via the binding reaction between markers and targets. Thus, bound and free markers were differentiated magnetically by using the difference in their relaxation times. Two types of magnetic properties, i.e., third-harmonic signal and magnetic relaxation, were used for measurement. The concentration of target was detected from the decrease in the third-harmonic signal and the increase in the magnetic-relaxation signal. We demonstrated the detection of C-reactive proteins using these two properties. In both cases, a good correlation was obtained between the measured signal and the concentration of target. It was also shown that the two methods had different concentration dependences, which will be caused mainly by the distribution of cluster size of the bound markers.
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