Milk can be contaminated with pathogenic bacteria such as Salmonella typhimurium and Escherichia coli, which can cause acute foodborne illnesses. In this study, we present an optical immunosensor designed for the simultaneous detection of these two bacteria in milk samples. The sensor is based on a silicon chip that incorporates two U-shaped silicon nitride waveguides configured as Mach-Zehnder Interferometers (MZIs). The sensing windows of the MZIs, located at one end of the chip, differ in length and allow for sequential immersion into reagent solutions during assay procedures. At the opposite end, the chip is optically coupled to a broadband white LED and a spectrophotometer via a bifurcated optical fiber and a dedicated coupler. To enable selective detection, the sensing windows of the MZIs are functionalized with lipopolysaccharides from the outer membranes of S. typhimurium and E. coli, respectively. A competitive immunoassay, completed in just 15 min, enabled detection limits of 45 cfu/mL for S. typhimurium and 125 cfu/mL for E. coli in milk. With a pre-enrichment step of approximately 8 h, single-cell detection became possible for both bacteria. The sensor excellent performance, quantitative determinations capability, and compact design, achieved by eliminating the need for microfluidics or pumps, make it a powerful and practical tool for bacterial detection in milk.

Aflatoxin M1 (AFM1) appears in the milk of animals that have consumed feed contaminated with aflatoxin B1. AFM1 presence in milk is regulated by the European Commission, which has set the maximum allowable limits for adult and infant consumption to 50 and 25 pg/mL, respectively. Here, a rapid and sensitive method for detecting AFM1 in milk based on an immersible silicon photonic chip is presented. The chip features two U-shaped silicon nitride waveguides formed as Mach–Zehnder interferometers. One interferometer is functionalized with AFM1–bovine serum albumin conjugate and the other with BSA to serve as a blank. The chip is connected to a broad-band white LED and a spectrophotometer by a bifurcated optical fiber and an assay is performed by immersing the chip in a mixture of milk with the anti-AFM1 antibody. Then, the chip is sequentially immersed in biotinylated anti-rabbit IgG antibody and streptavidin solutions for signal enhancement. The assay is completed in 20 min and the detection limit for AFM1 in undiluted milk is 20 pg/mL. Given its analytical performance and the absence of pumps and fluidics that lead to a compact instrument design, the proposed immunosensor is ideal for the on-site detection of AFM1 in milk samples.

Rapid and sensitive methods to detect allergenic proteins in foods at the point-of-need could help to protect allergic consumers from life-threatening accidental exposure. In this context, the development of a label-free optical immunosensor for detecting hazelnut proteins in cookies is presented. The sensor is based on silicon photonic chips containing two integrated Mach-Zehnder interferometers (MZI) with light input and output on one side of the chip, and sensing window openings on the other side. Coupling with a white-light LED and a spectrometer for signal recording is achieved via a bifurcated fiber. The sensing window of one of the MZIs is modified with a mouse monoclonal antibody against hazelnut proteins, while the other sensing window is modified with bovine serum albumin to serve as blank. The assay follows a two-step sandwich immunoassay format, involving a 10-min reaction with the hazelnut proteins calibrator or cookie sample, followed by a 2-min reaction with a mixture of monoclonal antibodies. All reactions were performed by immersing the chip side where the MZIs windows are located into the solutions. The recorded spectrum is processed in real-time to transform the spectrum shifts due to immunoreactions taking place on the sensing windows of the two MZIs into phase shifts. The developed assay detects hazelnut proteins at concentrations as low as 25 ng/mL in calibrators prepared in buffer or in aqueous extract of hazelnut-free cookies. Additionally, cookies containing hazelnuts were analyzed demonstrating the sensor ability for fast and sensitive detection of the hazelnut proteins in commercial products.

Aflatoxin M1 (AFM1) is the hydroxylated form of Aflatoxin B1 (AFB1) and is expelled in the milk of both humans and animals following the consumption of AFB1-contaminated food. AFM1 has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer. Consequently, the European Commission has established a maximum allowable concentration of 50 pg/mL for AFM1 in dairy products and milk. Here, a rapid and sensitive approach for detecting AFM1 in bovine milk is presented. The analytical setup comprises a broad-band white LED, a spectrophotometer, and a silicon photonic probe, all interconnected by a bifurcated optical fiber [1]. Additionally, a laptop powers the system and facilitates signal monitoring through specialized software. The silicon photonic probe is equipped with two Mach–Zehnder interferometers: one functionalized with AFM1-bovine serum albumin conjugate, and the other with bovine serum albumin to serve as a blank. The analysis involves immersing the probe directly into a mixture of anti-AFM1 antibodies and the sample, followed by sequential immersion into biotinylated anti-rabbit IgG antibody and streptavidin solutions. The entire assay process takes 12 min, and the limit of detection in undiluted milk is 20 pg/mL, below the EU maximum allowable limit of 50 pg/mL. The assay demonstrates accuracy, with %recovery values ranging from 87.5 to 112%, and repeatability, with intra/inter-assay coefficients of variation below 7.6%. Given its analytical performance and compact instrumentation, the proposed immunosensor proves to be an ideal solution for precise on-site determination of AFM1 in milk samples.

Silicon chips that monolithically integrate ten Mach-Zehnder interferometers (MZIs), their respective broad-band optical sources, and spectral analyzers, as well as photodiode arrays that record the spectrally-resolved output signals of the ten MZIs, are exploited for the multiplexed immunochemical determination of allergens and mycotoxins. The monolithically integrated light sources emit light in the visible/infrared spectrum (530-950 nm), and thus the detection based on broad-band Mach-Zehnder interferometry provided information about changes in the refractive index on the transducer surface due to binding reactions across the entire spectrum, surpassing the limitations of traditional monochromatic interferometry. The assays were run using a portable automated reader incorporating the chip fluidic and electronic interfacing, a micropump for continuous fluid delivery, and control electronics combined with a software for real-time signal monitoring. The photonic chips and portable reader were applied for allergen detection in dairy industry rinsing waters and mycotoxin detection in beer samples. All analytes were determined through competitive immunoassays. For the multiplexed detection of three allergens (bovine kappa-casein, peanut protein, and gliadin), the respective proteins were immobilized onto the sensing windows of different MZIs on a single chip. For the detection of mycotoxins (fumonisin B1 and deoxynivalenol), the respective mycotoxin-protein conjugates were employed. In all cases, the reaction with mixtures of calibrators/samples and analyte-specific antibodies was followed by a reaction with appropriate secondary antibodies to enhance the signal and reduce the assay duration. The allergen assays were completed in 10 min with detection limits of 0.01, 0.25, and 0.05 mu g/mL for kappa-casein, peanut protein, and gliadin, respectively. The mycotoxin assays took 15 min with detection limits of 2.0 and 10 ng/mL for fumonisin B1 and deoxynivalenol, respectively, in beer samples. The results demonstrate the potential of the developed solution for the rapid and sensitive on-site multiplexed detection of targeted analytes.

The quality and authenticity of milk are of paramount importance. Cow milk is more allergenic and less nutritious than ewe, goat, or donkey milk, which are often adulterated with cow milk due to their seasonal availability and higher prices. In this work, a silicon photonic dipstick sensor accommodating two U-shaped Mach–Zehnder Interferometers (MZIs) was employed for the label-free detection of the adulteration of ewe, goat, and donkey milk with cow milk. One of the two MZIs of the chip was modified with bovine κ-casein, while the other was modified with bovine serum albumin to serve as a blank. All assay steps were performed by immersion of the chip side where the MZIs are positioned into the reagent solutions, leading to a photonic dipstick immunosensor. Thus, the chip was first immersed in a mixture of milk with anti-bovine κ-casein antibody and then in a secondary antibody solution for signal enhancement. A limit of detection of 0.05% v/v cow milk in ewe, goat, or donkey milk was achieved in 12 min using a 50-times diluted sample. This fast, sensitive, and simple assay, without the need for sample pre-processing, microfluidics, or pumps, makes the developed sensor ideal for the detection of milk adulteration at the point of need.

Cow milk is more allergenic than milk from other species, and therefore the adulteration of ewe or goat milk with cow milk can pose a serious threat to consumers. In this work, a silicon-based photonic immunosensor, which includes two U-shaped Mach–Zehnder Interferometers (MZIs), was employed for the detection of ewe and goat milk adulteration with cow milk through the immunochemical determination of the milk. The method was fast and sensitive with a detection limit of 0.04 μg/mL bovine k-casein (which corresponds to approximately 0.06% cow milk) in ewe or goat milk, respectively, and with a total assay time of 12 min.

Aflatoxin M1 (AFM1) is detected in the milk of animals after ingestion of aflatoxin B1-contaminated food; since 2002, it has been categorized as a group I carcinogen. In this work, a silicon-based optoelectronic immunosensor for the detection of AFM1 in milk, chocolate milk, and yogurt has been developed. The immunosensor consists of ten Mach-Zehnder silicon nitride waveguide interferometers (MZIs) integrated on the same chip with the respective light sources, and an external spectrophotometer for transmission spectra collection. The sensing arm windows of MZIs are bio-functionalized after chip activation with aminosilane by spotting an AFM1 conjugate with bovine serum albumin. For AFM1 detection, a three-step competitive immunoassay is employed, including the primary reaction with a rabbit polyclonal anti-AFM1 antibody, followed by biotinylated donkey polyclonal anti-rabbit IgG antibody and streptavidin. The assay duration was 15 min with limits of detection of 0.005 ng/mL in both full-fat and chocolate milk, and 0.01 ng/mL in yogurt, which are lower than the maximum allowable concentration of 0.05 ng/mL set by the European Union. The assay is accurate (% recovery values 86.7-115) and repeatable (inter- and intra-assay variation coefficients <8%). The excellent analytical performance of the proposed immunosensor paves the way for accurate on-site AFM1 determination in milk.

Microgreens have gained attention for their exceptional culinary characteristics and high nutritional value. The present study focused on a novel approach for investigating the easy extraction of plant samples and the utilization of immersible silicon photonic sensors to determine, on the spot, the nutrient content of microgreens and their optimum time of harvest. For the first time, it was examined how these novel sensors can capture time-shifting spectra caused by the molecules' dynamic adhesion onto the sensor surface. The experiment involved four types of microgreens (three types of basil and broccoli) grown in a do-it-yourself hydroponic installation. The sensors successfully distinguished between different plant types, showcasing their discriminative capabilities. To determine the optimum harvest time, this study compared the sensor data with results obtained through standard analytical methods. Specifically, the total phenolic content and antioxidant activity of two basil varieties were juxtaposed with the sensor data, and this study concluded that the ideal harvest time for basil microgreens was 14 days after planting. This finding highlights the potential of the immersible silicon photonic sensors for potentially replacing time-consuming analytical techniques. By concentrating on obtaining plant extracts, capturing time-shifting spectra, and assessing sensor reusability, this research paves the way for future advancements in urban farming.

The consumption of water and milk contaminated with bacteria can lead to foodborne disease outbreaks. For this reason, the development of rapid and sensitive analytical methods for bacteria detection is of primary importance for public health protection. Here, a miniaturized immunosensor based on broadband Mach–Zehnder Interferometry for the simultaneous determination of S. typhimurium and E. coli O157:H7 in drinking water and milk is presented. For the assay, mixtures of bacteria solutions with anti-bacteria-specific antibodies were run over the chip, followed by solutions of biotinylated anti-species-specific antibody and streptavidin. The assay was fast (10 min for water, 15 min for milk), accurate, sensitive (LOD: 40 cfu/mL for S. typhimurium; 110 cfu/mL for E. coli) and reproducible. The analytical characteristics achieved combined with the small chip size make the proposed biosensor suitable for on-site bacteria determination in drinking water and milk samples.

: Amongst label-free optical sensors, those relying on silicon photonics are especially promising for the development of small-sized devices appropriate for applications at the point-of-need. In this context, our work over the last 10 years has focused on the development of silicon photonic chips that combine all optical components, both active and passive, onto the same substrate. The approach followed for this monolithic integration, as well as the application of the different silicon photonic chip versions as immunosensors for the determination of single or panels of analytes, related to biodiagnostics or the food safety sector, will be presented.

Drinking water contamination by pathogenic bacteria poses a great danger for public health, since according to WHO 5 million deaths are associated to water related diseases annually. To safeguard drinking water quality, several techniques for bacteria detection, such as culturing and plating, ELISA and DNA-based methods have been developed; which, however, are laborious and time consuming. In this work, we present a miniaturized immunosensor for the rapid, simultaneous label-free determination of bacteria in drinking water. The sensor consists of ten broad-band Mach-Zehnder interferometers (MZIs) integrated on silicon chip along with their corresponding light sources. For the analysis, the MZIs sensing areas were biofunctionalized with bacteria membrane antigens. Then, bacteria mixtures with anti-bacteria specific antibodies were pumped over the chip followed by biotinylated anti-species specific antibody and streptavidin solutions. Binding of antibodies onto the MZI-immobilized antigen, changes the effective refractive index over the MZI area causing an interference spectrum shift which is monitored continuously by an external spectrometer. Due to the short analysis time (12 min), the low detection limits achieved (<5X102 CFU/mL) for S. typhimurium and E. coli, and the small chip size, the proposed immunosensor could find wide application for on-site bacteria detection in drinking water samples.

Silicon photonic probes based on broad-band Mach-Zehnder interferometry are explored for the first time as directly immersible immunosensors alleviating the need for microfluidics and pumps. Each probe includes two U- shaped waveguides allowing light in- and out-coupling from the same chip side through a bifurcated fiber and a mechanical coupler. At the opposite chip side, two Mach-Zehnder interferometers (MZI) are located enabling real-time monitoring of binding reactions by immersion of this chip side into a sample. The sensing arm windows of the two MZIs have different length resulting in two distinct peaks in the Fourier domain, the phase shift of which can be monitored independently through Fast Fourier Transform of the output spectrum. The photonic probes analytical potential was demonstrated through detection of antibodies against SARS-CoV-2 in human serum samples. For this, one MZI was functionalized with the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike 1 protein, and the other with bovine serum albumin to serve as reference. The biofunctionalized probes were immersed for 10 min in human serum sample and then for 5 min in goat anti-human IgG Fc specific antibody solution. Using a humanized rat antibody against SARS-CoV-2 RBD, a detection limit of 20 ng/mL was determined. Analysis of human serum samples indicated that the proposed sensor discriminated completely non- infected/non-vaccinated from vaccinated individuals, and the antibodies levels determined correlated well with those determined in the same samples by ELISA. These results demonstrated the potential of the proposed sensor to serve as an efficient tool for expeditious point-of-care testing

The increasing demand for miniaturized, portable and low-cost imaging systems has led to the development of compact, optical components that integrate multiple functions and have short optical paths. We describe a novel opto-electronic chip for imaging opaque or semi -transparent samples in epi-illumination mode. The chip integrates of an array of ball lenses - 1 mm in diameter - and ring-shape Light Emitting Diodes (LEDs). A sample, when placed at closed proximity to the chip (<300 mu m), is illuminated by the LEDs, and the reflected light is collected by the lenses and focused onto a detector. The proposed architecture separates the excitation and emission optical paths and enables epi-illumination imaging without the need of using bulky and complex optical components. With an optical resolution of 8.8 mu m and a total optical path of less than 2.5 mm, we believe that the developed chip is an ideal module for compact portable, reflection-based imaging systems. (C) 2020 Elsevier B.V. All rights reserved.

Immunochemical detection of Mozzarella di Bufala Campana and Feta cheese adulteration with cow milk using integrated silicon Mach–Zehnder interferometers.

An optical immunosensor based on White Light Reflectance Spectroscopy is described for the determination of the herbicide glyphosate in drinking water samples. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. Glyphosate determination is accomplished by functionalizing the chip with a protein conjugate of the herbicide followed by a competitive immunoassay format. Prior to the assay, glyphosate derivatization in the calibrators and/or the samples was performed through reaction with succinic anhydride. Under the optimized assay protocol, a detection limit of 10 pg mL(-1) was achieved. Recovery values ranging from 90.0 to 110% were determined in spiked bottled and tap water samples, demonstrating the accuracy of the method. In addition, the sensor could be regenerated and re-used for at least 14 times without statistically significant effect on the assay sensitivity and accuracy. The excellent analytical performance and short analysis time (approx. 25 min), combined with the small sensor size, should be helpful for the fast on-site determination of glyphosate in drinking water samples.

Time-of-flight secondary ion mass spectrometry has been employed to characterize micropatterning of aminosilane layer by photolithography and oxygen plasma treatment to achieve spatially selective biofunctionalization of Si3N4 waveguides surface corresponding to the sensing arm area of Mach-Zehnder interferometric biosensors integrated on silicon-chip. Si3N4 surface with (3-aminopropyl)triethoxysilane (APTES) layer was examined after photolithography, plasma treatment, photoresist removal, and after robotic spotting with biotinylated bovine serum albumin (BSA), blocking with BSA and specific binding of streptavidin. TOF-SIMS chemical imaging and microanalysis provided an inside view regarding the resolution and selectivity of surface modification after each step of both the APTES layer patterning and biofunctionalization procedures. More particular, the effective APTES removal and surface oxidization to create 20-mu m wide APTES stripes through photolithography and oxygen plasma treatment was demonstrated. Exclusive adsorption of biotinylated-BSA on the APTES stripes through spotting of the patterned surface is then revealed, followed by a preferential but not exclusive BSA adsorption during the blocking step. The pattern was clearly developed through exclusive streptavidin binding to biotinylated-BSA only onto the APTES regions. The proposed spatially-selective biofunctionalization, performed with biotinylated-BSA, was demonstrated for the Si3N4 waveguide surface of an integrated on chip interferometric biosensor sensing arm for the detection of streptavidin.

Despite the tremendous advances in micro- and nanoelectronics and the fast-pacing advances in photonic circuit designs, seamless monolithic integration of electronic and photonic components on single chips still remains elusive. In this work, a radically designed silicon-based chip that monolithically integrates in a 37 mm(2) footprint 10 interferometric optical sensors along with their respective optical sources, spectral analyzers, and photodetector arrays is presented. The chip is fabricated with mainstream CMOS-compatible fabrication techniques and employs optical devices operating in the visible/infrared spectrum and waveguides with a critical dimension of 1.0 mu m. In addition, it exploits the newly introduced detection principle of broad-band Mach Zehnder interferometry that surpasses the stringent requirement for external monochromatic sources and inherent limitations of traditional interferometry and introduces alternative designs of on-chip spectral analyzers and mode-filtering components, aspiring thus to become a novel lab-on-a-chip that can address the needs of next-generation analytical systems. Apart from the conceptual design, novel photonic features, fabrication steps, and out-of-the-box system development that circumvents the need for fluidic interfacing and employs only electrical interconnects, the present work tests the potential of the fully spectroscopic chip for analytical applications through real-time monitoring of immunochemical reactions and demonstrates limits of detection for antimouse IgG antibody and CRP of 60 and 8 pM, respectively.

A miniaturized optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the rapid and label-free detection of aflatoxin M1 (AFM(1)) in milk samples has been developed. WLRS sensing system consists of the measurement set-up and the biochip. The first encompasses the reflection probe, the light source and the spectrometer, while the latter is a Si chip with a SiO2 layer on top where an AFM(1)-bovine serum albumin conjugate has been immobilized. The assay was performed by running mixtures of rabbit polyclonal anti-AFM(1) antibody with the calibrators or the samples, followed by reaction with biotinylated anti-rabbit IgG antibody and streptavidin. The assay cycle was completed in 25 min, the limit of detection was 6 pg/mL, and the linear working range extended from 0.012 to 2.0 ng/mL. The assay was repeatable (intra-and inter-assay coefficients of variation ranged from 2.1 to 6.3% and 3.5 to 8.2%, respectively) and accurate (percent recovery values ranged from 92.5 to 110%). AFM(1) could be detected with the immunosensor developed in both processed and unprocessed milk of different animal species without any dilution. The excellent analytical characteristics and the small instrument size make the proposed sensor suitable for accurate low-cost AFM(1) determination in milk samples at the point-of-need.

A silicon microphotonic chip with ten monolithically integrated interferometers and an equal number of light sources is employed for the label-free and multiplexed detection of two proteins of the cytokine family. The chip implements broad-band Mach-Zehnder interferometry and is used for the detection of the IL-6 and IL-8 interleukins. The silicon transducer is made through mainstream silicon technology and integrates avalanche diode based white light LEDs and monomodal waveguides forming Mach-Zehnder interferometers. The light spectrum is modulated through the interferometer and the spectrally resolved interference fringes are monitored by a digital spectrometer. Biomolecular built-up on the sensing arm causes pattern shifts that are evaluated through Fourier Transform. The microsystem level set-up includes microfluidic components, direct contacting of the chip contact pads through spring loaded pins and alignment guides and a spectrometer. The dual polarization optics allows the simultaneous derivation of the TE and TM sensitivity plots and the calculation of the refractive indices of either protein. (C) 2017 Published by Elsevier B.V.