Various analytical procedures to detect an analyte in a test sample or sample volume are known in the prior art.
For example, immunoassays use the mechanisms of the immune system, wherein antibodies and the respective antigens are capable of binding to each other. This specific reaction mechanism is used to determine the presence or quantity of the antigen in a test sample. In particular, the antibody or the antigen (analyte of interest) is labeled to quantify the interactions between antibody and antigen. Common labels are, for example, fluorescent and chemiluminescent molecules, colored particles (beads) or radioisotopes. In general all these are referred to here as particles. A certain application is the optical detection of magnetic particles bound to the antigen to be detected in various manners. Thereby, by detecting the magnetic particles, the amount of antigen or analyte can be concluded.
Recently, magnetic labels have been used in microfluidic assays to detect the presence or quantity of an analyte. The use of magnetic labels as, for example, magnetic particles, also denominated as magnetic beads or beads, has several advantages. The magnetic particles can be actuated by applying a magnetic field such that the analytical procedure can be accelerated. Further, there is no magnetic background signal in the biological test sample influencing the detection of the magnetic particles.
The performance of known systems in the sub-pM regime is limited by optical baseline drift and non-specifically bound beads. A non-specifically bound bead normally is not wanted for detection, it is an aim to receive a detection signal based only on bound beads or particles. Thus, increasing the optical resolution would be beneficial in terms of higher stability and detailed information on single particles or beads.
High numerical apertures, required for single bead identification will result in a high degree of optical aberrations, limiting the resolution of the imaging optics of known systems to several microns.
A recently known magnetic bio sensor system makes use of Frustrated Total Internal Reflection (FTIR) to detect the presence of magnetic beads near a surface of an assay. The signal is more or less linearly dependent on the concentration of beads on the surface (the surface density ñ). The signal (i.e. the decrease of the totally internal reflected signal) can be expressed as:S=β·ñ
where S is the measured signal change in % and β is a conversion factor from surface density to signal change. The limit of detection of this technique is mainly determined by drift in the background signal which contributes to about 0.1% of signal change. This is equivalent to a surface density of 1 bead per 200 um2 which can be detected with this FTIR platform.
For a channel height of 480 μm, the minimum detectable target concentration would be about 20 fM. However, in practice the assay efficiency is an order of magnitude lower, bringing the detection limit to 200 fM. Besides the drift in the background signal, also the signal of nonspecifically bound beads limits the minimum detectable concentration. These beads are bound to the surface via a non-specific bond, e.g. an antibody-antibody bond instead of a specific bond like antibody-target-antibody.