Common labels include enzymes, fluorescent dyes, radioisotopes, or magnetic particles. to the targets of interest and provide a means of detection. Common labels include enzymes, fluorescent dyes, radioisotopes, or magnetic particles. This general technique has various applications. In an immunoassay, the goal is to detect and quantify specific targets. Tagged antibodies can also be used to separate target antigens selectively or to measure the affinity between antibody and antigen. In this article, we present a sensitive method for detecting magnetically labeled bacteria by using a superconducting quantum interference device (SQUID), a highly sensitive detector of magnetic flux. This assay can be used to monitor bacteria in a liquid sample and to determine the rate of binding between antibody-linked particles and bacteria. Magnetic particles have several advantages as labels. They are stable and nontoxic and can be manipulated with a magnetic field, making it possible to individual target antigens magnetically (3). Methods have been developed to detect small numbers of such particles by using Hall probes (4), giant magnetoresistance arrays (5), atomic force microscopy (6), force-amplified biological sensors (7), and SQUIDs (8C10). Weitschies, K?titz, and colleagues pioneered the use of SQUIDs for magnetic immunoassays (8, 11C16). They developed a magnetic relaxation immunoassay in which PF-00562271 magnetic particles bound to targets are distinguished from unbound particles by their different relaxation times. By using a low-critical-temperature ((17) used a high-and add them to a suspension of that organism. After allowing time for PF-00562271 the particles to bind to the targets, we place the sample 130 m above a high- B after the field is usually turned off, Esam unbound particles have randomized PF-00562271 direction by Brownian rotation, whereas particles bound to bacteria are still aligned. The magnetic moments of the bound particles reorient slowly by means of Nel relaxation. This straightforward assay format does not require immobilization of the targets or washing away of the unbound particles. It has the potential for improved accuracy over conventional immunoassays because no materials are lost. We demonstrate that this technique can successfully differentiate between bound and unbound particles and present results from titration experiments in which the concentration of either bacteria or particles is usually varied. We show how the relaxation signal depends on the applied magnetic field and present time-resolved data illustrating that this technique can measure binding reaction rates. Finally, we discuss improvements to the technique and potential applications. Theory We differentiate between bound and unbound particles by the different mechanisms by which they relax after the removal of a magnetic field. Brownian relaxation (18) is usually a physical rotation of the particles, with a relaxation time for a sphere [1] where is the viscosity of the medium, is the temperature. Taking = 293 K and = 10C3 kgmC1sC1, we find B is usually 50 s for particles with a hydrodynamic diameter of 50 nm. Nel relaxation (19) originates from the anisotropy of the crystalline lattice. Many magnetic materials have an easy axis of magnetization; when the crystal is usually magnetized along that axis, PF-00562271 the energy is usually minimized. If an external field rotates the magnetization away from the easy axis, the magnetization eventually returns to its preferred direction on removal of the field. The Nel relaxation time for a single domain particle is usually [2] where 0 is usually 10C9 s, is the magnetic anisotropy constant, and depend on the shape of the particle, and values of 0 vary by up to four orders of magnitude (20). The particles used here were composed of -Fe2O3, for which the bulk anisotropy constant.