16 th International Conference on Environmental Science and Technology Rhodes, Greece, 4 to 7 September 2019 CEST2019_00586 On-chip Mach-Zehnder Interferometers for rapid detection of bacteria in drinking water Angelopoulou Μ. 1,* , Petrou P.S. 1 , Misiakos K. 2 , Raptis I. 2 , Kakabakos S.E. 1 1 IMMUNOASSAYS-IMMUNOSENSORS LAB, INRASTES, NCSR “DEMOKRITOS”, ATHENS, GREECE 2 OPTICAL SENSORS LAB, INSTITUTE OF NANOSCIENCE & NANOTECHNOLOGY, NCSR “DEMOKRITOS”, ATHENS, GREECE *corresponding author: Michailia Angelopoulou: e-mail: mikangel@ipta.demokritos.gr Abstract A miniaturized optical immunosensor for the simultaneous label-free determination of bacteria in drinking water is presented. The sensor consists of an array of ten Mach-Zehnder interferometers (MZIs) integrated on silicon chip along with their corresponding broad-band light sources. The transmitted spectra of the MZIs were continuously recorded through a spectrometer. The spectral shifts caused by changes of the effective refractive index on the sensor surface due to bioreaction were converted to phase shifts through discrete Fourier transform. For the analysis, the different MZIs of the chip were biofunctionalized with the respective S. typhimiurim and E. coli membrane antigens. Then, mixtures of bacteria solutions with anti-bacteria antibodies were pumped over the chip followed by reaction with biotinylated anti-species specific antibody and streptavidin. The assays were fast (10 min), sensitive (LODs <2X10 2 CFU/mL), accurate (recovery 86-115%), repeatable with intra- and inter-assay CVs <5% and 8%, respectively, and the chip could be regenerated/reused for at least 20 times. Considering the low detection limits achieved in combination with the short analysis time and the small chip size, the proposed immunosensor could find wide application for bacteria detection in drinking water at the point-of-need. Keywords: immunosensor, bacteria, drinking water, Mach-Zehnder Interferometers 1. Introduction The contamination of drinking water by pathogenic bacteria, such as E. coli, Vibrio cholera, Salmonella spp. and Shigella is a global health alarm. According to WHO, 5 million deaths are associated to water related diseases annually (Cabral et al., 2010). For this reason, the assessment of drinking water contamination by bacteria is of crucial importance to protect consumer’s health. The conventional methods for bacteria detection are based on culturing and plating, which are reliable but rather time- consuming. In order to shorten the analysis time, ELISA- and DNA-based methods have been employed for bacteria identification (Shabani et al., 2015). However, the above methods require skilled personnel and cannot be performed at the point-of-need. Recently, biosensors based on electrochemical, piezoelectric or optical transducers are gaining ground in waterborne bacteria detection (Li et al., 2014; Zhang et al., 2016; Guo et al., 2012). Although these sensors claim inexpensive analysis and potential for miniaturization, they lack in sensitivity and often require labels for signal enhancement to improve detection limits. In this report, for the first time, we investigated the potential of an optical biosensor based on an array of ten Mach-Zehnder interferometers (MZI) integrated along with their corresponding light sources on silicon chips (Angelopoulou et al., 2015; Angelopoulou et al., 2018) for the simultaneous and sensitive detection of S. typhimiurim and E. coli in drinking water. 2. Experimental 2.1. Reagents, chip fabrication and signal processing The rabbit polyclonal anti-S. typhimiurim LPS antibody was from Bio-Rad (UK) and the mouse monoclonal anti- E. coli LPS antibody was from Origene (Germany). E. coli LPS was from Creative Diagnostics (USA). APTES, bovine serum albumin (BSA) and S. typhimiurim LPS were from Sigma-Aldrich (Germany). The chips are fabricated following mainstream silicon technology as described previously (Angelopoulou et al., 2015; Angelopoulou et al., 2018). The ten MZIs converge at the edge of the chip where an external a miniaturized spectrometer is positioned (QE65000, Ocean Optics). During the assay, the spectra of the MZIs were continuously recorded and the spectral shifts caused due to bioreaction were converted to phase shifts (analytical signal) through discrete Fourier transform. 2.2. Chemical and biological activation of the chip The chips were cleaned/hydrophylized through O 2 plasma treatment for 30 s and immersed for 2 min in a 0.5% (v/v) APTES solution. Then, the chips were washed, dried, and heated at 120 o C for 20 min. After that, 3 MZIs per chip were spotted with 200 μg/mL of S. typhimurium LPS solution, 4 MZIs with 200 μg/mL of E. coli LPS solution, and the remaining 3 with 100 μg/mL BSA solution (non- specific binding) using the BioOdyssey Calligrapher Mini Arrayer. After spotting completion, the chips were washed, blocked for 2 h with 1% (w/v) BSA in 0.1 M