*2.3. Detection of Zearalenone by Planar Waveguide Immunoosensor*

The detection of ZON was carried out using the experimental PW set-up described in Section 2.2, which has an RIS of approximately 5100 rad/RI unit. Experiments of detecting ZON were performed in a direct immunosensor format with specific antibodies immobilised electrostatically on the sensor surface via the layers poly-allylamine hydrochloride (PAH) and protein A (ProtA) in a following sequence: (i) deposition of PAH polycations carrying positive charge; (ii) deposition of ProtA being negatively charged at pH = 7, (iii) deposition of polyclonal antibodies to ZON via a biding site at the second constant domain to ProtA. In the above experiments, a very thin (≈1 nm) layer of PAH [24] deposited on the waveguide surface yields a phase shift of about a <sup>1</sup> 2 period. The absorption of larger molecules of ProtA (42 kDa) causes a larger phase shift of about two periods, while much larger molecules of polyclonal antibodies to ZON (150–900 kDa) gave about 3.5 periods of phase change.

The detection of ZON was undertaken by sequential injections of ZON standards in increasing order of concentrations, e.g., 0.01, 0.1, 1, 10, 100, and 1000 ng/mL. Typical responses to injections of different concentrations of ZON are shown in Figure 4. As one can see, the number of periods of signal oscillation increases with the increase in ZON concentration. The accuracy of phase shift calculation was about 0.1 of a period or about 0.6 rad. However, the increase in concentration of ZON cannot lead to a limitless increase in the number oscillation periods of the output signal. The sequential injections of ZON at increasing concentration cause a gradual saturation of the ZON-specific antibodies. The saturation and even slight decrease of the individual phase shifts is due to the beginning saturation of the binding sites of the antibodies. A complete saturation of binding sites resulted in a very small phase shift due to non-specific binding. This corresponds to the exhaustion of the immobilised antibodies on the sensor surface. It has also to be noted that the washing out of non-specifically bound ZON molecules during purging with pure buffer solution through the cell caused a phase shift of about 1/4 of a period, which was the baseline (background) level of the experiments.

**Figure 4.** Typical sensor responses to injection of zearalenone (ZON) of different concentrations: 0.01 ng/mL (**a**), 0.1 ng/mL (**b**), 1 ng/mL (**c**), 10 ng/mL (**d**), 100 ng/mL (**e**), and 1000 (ng/mL) (**f**).

As seen in Figure 4, the minimal detection concentration of ZON (limit of detection, LOD) was 0.01 ng/mL, which is an order of magnitude lower than the results obtained earlier using TIRE in a similar direct immunoassay format and have the same LOD as in TIRE measurements in more sensitive competitive assay format [3]. Cross-reactivities (CRs) of the immobilised antibodies, which were carried out with aflatoxin B1 (AFB1) and ochratoxin A (OTA), did not show any signal oscillations and thus no phase shifts, even at a concentration of 1000 ng/mL. This demonstrated the high specificity of the antibodies towards ZON.
