Simultaneous Detection of SARS-CoV-2 Nucleoprotein and Receptor Binding Domain by a Multi-Area Reflectance Spectroscopy Sensor.

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications.

Mach-Zehnder Interferometric Immunosensor for Detection of Aflatoxin M1 in Milk, Chocolate Milk, and Yogurt.

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.

Simultaneous Detection of Salmonella typhimurium and Escherichia coli O157:H7 in Drinking Water and Milk with Mach-Zehnder Interferometers Monolithically Integrated on Silicon Chips.

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.

Directly immersible silicon photonic probes: Application to rapid SARS-CoV-2 serological testing.

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.

Rapid Detection of Salmonella typhimurium in Drinking Water by a White Light Reflectance Spectroscopy Immunosensor.

Biosensors represent an attractive approach for fast bacteria detection. Here, we present an optical biosensor for the detection of Salmonella typhimurium lipopolysaccharide (LPS) and Salmonella bacteria in drinking water, based on white light reflectance spectroscopy. The sensor chip consisted of a Si die with a thin SiO2 layer on top that was transformed into a biosensor through the immobilization of Salmonella LPS. The optical setup included a reflection probe with seven 200 µm fibers, a visible and near-infrared light source, and a spectrometer. The six fibers at the reflection probe circumference were coupled with the light source and illuminated the biosensor chip vertically, whereas the central fiber collected the reflected light and guided it to the spectrometer. A competitive immunoassay configuration was adopted for the analysis. Accordingly, a mixture of LPS or bacteria solution, pre-incubated for 15 min, with an anti-Salmonella LPS antibody was pumped over the chip followed by biotinylated secondary antibody and streptavidin for signal enhancement. The binding of the free anti-Salmonella antibody to chip-immobilized LPS led to a shift of the reflectance spectrum that was inversely related to the analyte concentration (LPS or bacteria) in the calibrators or samples. The total assay duration was 15 min, and the detection limits achieved were 4 ng/mL for LPS and 320 CFU/mL for bacteria. Taking into account the low detection limits, the short analysis time, and the small size of the chip and instrumentation employed, the proposed immunosensor could find wide application for bacteria detection in drinking water.

Rapid detection of mozzarella and feta cheese adulteration with cow milk through a silicon photonic immunosensor.

Mozzarella di Bufala Campana and Feta are two cheeses with Protected Designation of Origin the fraudulent adulteration of which with bovine milk must be routinely checked to ensure that consumers actually buy these high-end products and avoid health issues related to bovine milk allergy. Here, we employed, for the first time, a silicon-based photonic immunosensor for the detection of mozzarella and feta adulteration with bovine milk. The photonic immunosensor used relies on Mach-Zehnder interferometers monolithically integrated along with their respective light sources on a silicon chip. A rabbit polyclonal antiserum raised against bovine κ-casein was used for the development of a competitive immunoassay realized in three steps, including a reaction with the antiserum, a biotinylated anti-rabbit IgG antibody, and streptavidin. The implementation of this assay configuration significantly reduced the non-specific signal due to the cheese matrix, and allowed completion of the assay in ∼9 min. After optimization of all assay conditions, bovine cheese could be quantified in mozzarella or feta at concentrations as low as 0.5 and 0.25% (w/w), respectively; both quantification limits were below the maximum allowable content of bovine milk in mozzarella and feta (1% w/w) according to the EU regulations. Equally important, the assays were reproducible with intra- and inter-assay coefficients of variation <10%, and exhibited a wide linear dynamic range that extended up to 50 and 25% (w/w) for mozzarella and feta, respectively. Taking into account its performance, the proposed immunosensor may be transformed to a new tool against fraudulent activities in the dairy industry.

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips.

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach-Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 µg/mL for bovine k-casein, 1.0 µg/mL for peanut protein, 0.80 µg/mL for soy protein, and 0.10 µg/mL for gliadin. The assays were accurate (recoveries 88-118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations.

Lab-on-a-Membrane Foldable Devices for Duplex Drop-Volume Electrochemical Biosensing Using Quantum Dot Tags.

This work describes a new type of integrated lab-on-a-membrane foldable device suitable for on-site duplex electrochemical biosensing using drop-size sample volumes. The devices are fabricated entirely by screen-printing on a nylon membrane and feature two assay zones which are located symmetrically on either side of a three-electrode voltammetric cell with a bismuth citrate-loaded graphite working electrode. After the completion of two spatially separated drop-volume competitive immunoassays on the assay zones using biotinylated antibodies labeled with streptavidin-conjugated Pb- and Cd-based quantum dots (QDs), respectively, the QD labels are dissolved releasing Pb(II) and Cd(II) in the assay zones. Then, the two assay zones are folded over, and they are brought in contact with the voltammetric cell for simultaneous anodic stripping voltammetric (ASV) determination of Pb(II) and Cd(II) at the bismuth nanostructured layer formed on the working electrode by reduction of the bismuth citrate during the preconcentration step. The fabrication of the devices is discussed in detail, and their operational characteristics are exhaustively studied. In order to demonstrate their applicability to the analysis in complex matrices, duplex ASV-QDs-based determination of bovine casein and bovine immunoglobulin G is carried out in milk samples yielding limits of detection of 0.04 µg mL(-1) and 0.02 µg mL(-1), respectively. The potential of the devices to detect milk adulteration is further demonstrated. These new membrane devices enable duplex biosensing with distinct advantages over existing approaches in terms of cost, fabrication, and operational simplicity and rapidity, portability, sample size, disposability, sensitivity, and suitability for field analysis.