Aspergillus is known as one of the most frequent toxigenic fungi in Europe. It produces aflatoxin B1 transformed into aflatoxin M1 (AFM1) present in milk. The International Agency for Research on Cancer (IARC) has included AFM1 in the group I human carcinogens. Acceptable maximum level of AFM1 in milk according to EU regulation is 50 ppt equivalent to 15.2×10-2 nM (the molecular weight of AFM1 is 328.27 g/mol). Up to now, the most used techniques for AFM1 detection in milk are time consuming Enzyme-Linked Immuno-Sorbent Assay (ELISA) and high-performance liquid chromatography associated to fluorescence (HPLC). Silicon photonics based biosensors such as Mach-Zehnder Interferometers and Microring Resonators thanks to their ability to be miniaturize and integrated with electronics and microfluidics in lab-on-a-chip devices, are new candidates to become faster, cheaper and more accurate tools for AFM1 detection in milk. Here, we validate photonic AFM1 biosensors based on an array of four Si3N4 asymmetric Mach-Zehnder Interferometers (aMZI) functionalized by F(ab’)2 fragments and passivated with casein.
Analyses of AFM1 binding by Fab’ in MES buffer and in real milk samples, using different concentrations of AFM1, are performed. The dependence of the phase shifts to the AFM1 concentration allows to calculate the association and dissociation rate constants. The proposed biosensor is capable to detect AFM1 concentration down to 50 ppt. For the average dissociation constant (KD) of AFM1-Fab’ interaction, values of (1.8÷5)×10-8 M in MES buffer and 0.8×10-9 M in milk samples are measured. These are in the same order of magnitude as published results. The difference of one order of magnitude between KD in MES buffer and in milk might be caused by the fact that during the preparation of milk samples, an additional concentration of salt is added to the solution which yields a stronger ionic interaction to occur. Finally, the specificity of the interaction is confirmed by using blank solutions that are free from AFM1.
Integrated optical biosensors based on Mach-Zehnder Interferometers and Microring Resonators are widely used for food/drug monitoring and protein studies thank to their high intrinsic sensitivity, easy integration and miniaturization, and low cost.<sup>1, 2</sup> In this study, we present a system to perform antibody interaction analysis using a photonic chip made of an array of six microring resonators (MRRs) based on the TriPleX platform. A compact system is presented where the input light is provided by a Vertical Cavity Surface Emitting Laser (VCSEL) pigtailed to a single mode fiber and operating at a ≈ 850nm wavelength. The output signal is detected by PIN photodetectors placed in the optical signal read-out module (the so-called OSROM) and processed by an easy-to-use Fourier Transform algorithm. Bulk sensitivity (S<sub>b</sub>=98±2.1 nm/RIU) and Limit of Detection (LOD=(7.5± 0.5) x10<sup>-6</sup> RIU) are measured and appeared to be very similar for the six MRRs on the same chip,<sup>3</sup> which is an important property for multianalyte detection. An analysis of the anti-biotin interaction with immobilized biotin is performed by using different concentrations of anti-biotin antibody. The dependence of the resonance wavelength shift from the antibody concentration, as well as the association and the dissociation rate constants are calculated. For the average dissociation constant (K<sub>D</sub>) of anti-biotin antibody toward immobilized biotin, a value of (1.9±0.5) x10<sup>-7</sup>M is estimated, which is of the same order of magnitude of other published data.<sup>4</sup> Furthermore, the specificity of the interaction is confirmed by using negative control antibodies and by performing competition with free, i.e., dissolved, biotin. In addition, the functional surface of the sensors could be regenerated for repeated measurements up to eight times by using 10 mM glycine/HCl pH 1.5.
In this work, we present a study on photonic biosensors based on Si<sub>3</sub>N<sub>4</sub> asymmetric Mach-Zehnder Interferometers (aMZI) for Aﬂatoxin M1 (AFM1) detection. AFM1 is an hepatotoxic and a carcinogenic toxin present in milk. The biosensor is based on an array of four Si<sub>3</sub>N<sub>4</sub> aMZI that are optimized for 850nm wavelength. We measure the bulk Sensitivity (S) and the Limit of Detection (LOD) of our devices. In the array, three devices are exposed and have very similar sensitivities. The fourth aMZI, which is covered by SiO<sub>2</sub>, is used as an internal reference for laser (a VCSEL) and temperature ﬂuctuations. We measured a phase sensitivity of 14300±400 rad/RIU. To characterize the LOD of the sensors, we measure the uncertainty of the experimental readout system. From the measurements on three aMZI, we observe the same value of LOD, which is ≈ 4.5×10<sup>−7</sup> RIU. After the sensor characterization on homogeneous sensing, we test the surface sensing performances by ﬂowing speciﬁc Aﬂatoxin M1 and non-speciﬁc Ochratoxin in 50 mM MES pH 6.6 buﬀer on the top of the sensors functionalized with Antigen-Recognising Fragments (Fab’). The diﬀerence between speciﬁc and non-speciﬁc signals shows the speciﬁcity of our sensors. A moderate regeneration of the sensors is obtained by using glycine solution.