*4.2. Instrumentation*

RFUs were determined by the prototype of a novel instrumentation developed in project Aquafluosense and realized in a modular setup (Figure 6). The instrument was developed partly (motor, optics, sample holder) at Optimal Optik Ltd. (Budapest, Hungary) and partly (detector electronics) at the Budapest University of Technology and Economics (Budapest, Hungary), and was designed to fit the 96-well ELISA microplate format using a self-designed, 3D-printed holder for ELFIA (Figure 6). The samples were illuminated in a dual head configuration with a high-power LED (Cree XPEBGR-L1-0000-00F01 with 520 to 535 nm minimum to maximum dominant wavelength range) in each head. The emitted fluorescence is measured in a dichroic beam path with silicon photodiodes (PIN-25D, OSI Optoelectronics) having large active area (d = 27.9 mm). The necessary high-spectral blocking and contrast was achieved by a combination of dichroic (Semrock FF562-Di03, edge: 562 nm) and bandpass optical filters on both the excitation (Semrock FF01-531/40-25, peak: 531 nm, width: 40 nm) and emission (Semrock FF01-593/40-25, peak: 593 nm, width: 40 nm) paths. The photodetector signal was coupled to a 2-stage amplifier unit (1st stage: OPA129 electrometer preamplifier, Texas Instruments; 2nd stage: AD620 instrumentation amplifier, Analog Devices) and then fed to a 12-bit analog-to-digital converter (Analog Devices AD7864-2 with 0 to 5 V unipolar input range) yielding 4095 resolvable RFUs. Gain and offset of the 2nd stage and the LED optical power were controlled by 256-stage (8-bit) digital potentiometers. As the LED power adjustment was nonequidistant, the optical power-control number curve was calibrated by a FieldMaxII-TO (Coherent) power meter with an OP-2 VIS sensor head set to a nominal wavelength of 532 nm. The instrument was equipped with stepping motors to move the detector heads over the 96-well microplates which provides fast and effective determination of individual RFUs in each microplate well. The instrument development is currently in the experimental phase; further decision about possible commercialization will be made by the project consortium.

ject consortium.

**Figure 6.** The immunofluorescence module developed in project Aquafluosense and appropriate for zearalenone determination (top left). The optical path in the detector head (top right). The modular instrumental setup during on site operation in a laboratory motor vehicle (bottom). **Figure 6.** The immunofluorescence module developed in project Aquafluosense and appropriate for zearalenone determination (**top left**). The optical path in the detector head (**top right**). The modular instrumental setup during on site operation in a laboratory motor vehicle (**bottom**).

by 256-stage (8-bit) digital potentiometers. As the LED power adjustment was nonequidistant, the optical power-control number curve was calibrated by a FieldMaxII-TO (Coherent) power meter with an OP-2 VIS sensor head set to a nominal wavelength of 532 nm. The instrument was equipped with stepping motors to move the detector heads over the 96-well microplates which provides fast and effective determination of individual RFUs in each microplate well. The instrument development is currently in the experimental phase; further decision about possible commercialization will be made by the pro-

## *4.3. Determination of Zearalenone by Autofluorescence 4.3. Determination of Zearalenone by Autofluorescence*

Fluorescence spectra of ZON were recorded on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) by scanning the excitation wavelengths between 250 and 830 nm and emission wavelengths between 270 and 850 nm, with step sizes of 10 nm, where the emission wavelength must be a minimum of 20 nm greater than excitation. A spectrum map was established by RFUs measured for a solution of ZON in PBS at a concentration of 2000 ng/mL with a corresponding RFU for PBS at each point as a background. An optimized peak on the basis of the fluorescence spectral map obtained was applied to establish a calibration curve (in the concentration range of 0.6–2000 ng/mL) and a LOD of ZON based on autofluorescence. Fluorescence spectra of ZON were recorded on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) by scanning the excitation wavelengths between 250 and 830 nm and emission wavelengths between 270 and 850 nm, with step sizes of 10 nm, where the emission wavelength must be a minimum of 20 nm greater than excitation. A spectrum map was established by RFUs measured for a solution of ZON in PBS at a concentration of 2000 ng/mL with a corresponding RFU for PBS at each point as a background. An optimized peak on the basis of the fluorescence spectral map obtained was applied to establish a calibration curve (in the concentration range of 0.6–2000 ng/mL) and a LOD of ZON based on autofluorescence.
