Cancer Marker Immunosensing through Surface-Enhanced Photoluminescence on Nanostructured Silver Substrates.

Nanostructured noble metal surfaces enhance the photoluminescence emitted by fluorescent molecules, permitting the development of highly sensitive fluorescence immunoassays. To this end, surfaces with silicon nanowires decorated with silver nanoparticles in the form of dendrites or aggregates were evaluated as substrates for the immunochemical detection of two ovarian cancer indicators, carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4). The substrates were prepared by metal-enhanced chemical etching of silicon wafers to create, in one step, silicon nanowires and silver nanoparticles on top of them. For both analytes, non-competitive immunoassays were developed using pairs of highly specific monoclonal antibodies, one for analyte capture on the substrate and the other for detection. In order to facilitate the identification of the immunocomplexes through a reaction with streptavidin labeled with Rhodamine Red-X, the detection antibodies were biotinylated. An in-house-developed optical set-up was used for photoluminescence signal measurements after assay completion. The detection limits achieved were 2.5 U/mL and 3.12 pM for CA125 and HE4, respectively, with linear dynamic ranges extending up to 500 U/mL for CA125 and up to 500 pM for HE4, covering the concentration ranges of both healthy and ovarian cancer patients. Thus, the proposed method could be implemented for the early diagnosis and/or prognosis and monitoring of ovarian cancer.

Rational Design, Synthesis and Binding Affinity Studies of Anthraquinone Derivatives Conjugated to Gonadotropin-Releasing Hormone (GnRH) Analogues towards Selective Immunosuppression of Hormone-Dependent Cancer.

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone-GnRH conjugates (con1-con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2-con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies.

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.

Covalent and Non-covalent In-Flow Biofunctionalization for Capture Assays on Silicon Chips: White Light Reflectance Spectroscopy Immunosensor Combined with TOF-SIMS Resolves Immobilization Stability and Binding Stoichiometry.

Immunosensors that combine planar transducers with microfluidics to achieve in-flow biofunctionalization and assay were analyzed here regarding surface binding capacity, immobilization stability, binding stoichiometry, and amount and orientation of surface-bound IgG antibodies. Two IgG immobilization schemes, by physical adsorption [3-aminopropyltriethoxysilane (APTES)] and glutaraldehyde covalent coupling (APTES/GA), followed by blocking with bovine serum albumin (BSA) and streptavidin (STR) capture, are monitored with white light reflectance spectroscopy (WLRS) sensors as thickness dΓ of the adlayer formed on top of aminosilanized silicon chips. Multi-protein surface composition (IgG, BSA, and STR) is determined by time of flight secondary ion mass spectrometry (TOF-SIMS) combined with principal component analysis (applying barycentric coordinates to the score plot). In-flow immobilization shows at least 1.7 times higher surface binding capacity than static adsorption. In contrast to physical immobilization, which is unstable during blocking with BSA, chemisorbed antibodies desorb (reducing dΓ) only when the bilayer is formed. Also, TOF-SIMS data show that IgG molecules are partially exchanged with BSA on APTES but not on APTES/GA modified chips. This is confirmed by the WLRS data that show different binding stoichiometry between the two immobilization schemes for the direct binding IgG/anti-IgG assay. The identical binding stoichiometry for STR capture results from partial replacement with BSA of vertically aligned antibodies on APTES, with fraction of exposed Fab domains higher than on APTES/GA.

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.

SERS Immunosensors for Cancer Markers Detection.

Early diagnosis and monitoring are essential for the effective treatment and survival of patients with different types of malignancy. To this end, the accurate and sensitive determination of substances in human biological fluids related to cancer diagnosis and/or prognosis, i.e., cancer biomarkers, is of ultimate importance. Advancements in the field of immunodetection and nanomaterials have enabled the application of new transduction approaches for the sensitive detection of single or multiple cancer biomarkers in biological fluids. Immunosensors based on surface-enhanced Raman spectroscopy (SERS) are examples where the special properties of nanostructured materials and immunoreagents are combined to develop analytical tools that hold promise for point-of-care applications. In this frame, the subject of this review article is to present the advancements made so far regarding the immunochemical determination of cancer biomarkers by SERS. Thus, after a short introduction about the principles of both immunoassays and SERS, an extended presentation of up-to-date works regarding both single and multi-analyte determination of cancer biomarkers is presented. Finally, future perspectives on the field of SERS immunosensors for cancer markers detection are briefly discussed.

Simultaneous determination of procalcitonin and interleukin-6 in human serum samples with a point-of-care biosensing device.

The simultaneous determination of two inflammatory diseases biomarkers, namely procalcitonin (PCT) and interleukin-6 (IL-6), in human serum samples employing a Point-of-Care device based on Multi Area Reflectance Spectroscopy is presented. Dual-analyte detection was achieved using silicon chips with two silicon dioxide areas of different thickness, one functionalized with an antibody specific for PCT and the other with an antibody specific for IL-6. The assay included reaction of immobilized capture antibodies with mixtures of PCT and IL-6 calibrators with the biotinylated detection antibodies, streptavidin and biotinylated-BSA. The reader provided for the automated execution of the assay procedure, as well as for the collection and processing of the reflected light spectrum, the shift of which is correlated to analytes concentration in the sample. The assay was completed in 35 min and the detection limits for PCT and IL-6 were 2.0 and 0.01 ng/mL respectively. The dual-analyte assay was characterized by high reproducibility (the intra- and inter-assay coefficients of variation were less than 10% for both analytes) and accuracy (the percent recovery values ranged from 80 to 113% for both analytes). Moreover, the values determined for the two analytes in human serum samples with the assay developed were in good agreement with the values determined for the same samples by clinical laboratory methods. These results support the potential of the proposed biosensing device application for inflammatory biomarkers determination at the Point-of-Need.

SERS Determination of Oxidative Stress Markers in Saliva Using Substrates with Silver Nanoparticle-Decorated Silicon Nanowires.

Glutathione and malondialdehyde are two compounds commonly used to evaluate the oxidative stress status of an organism. Although their determination is usually performed in blood serum, saliva is gaining ground as the biological fluid of choice for oxidative stress determination at the point of need. For this purpose, surface-enhanced Raman spectroscopy (SERS), which is a highly sensitive method for the detection of biomolecules, could offer additional advantages regarding the analysis of biological fluids at the point of need. In this work, silicon nanowires decorated with silver nanoparticles made by metal-assisted chemical etching were evaluated as substrates for the SERS determination of glutathione and malondialdehyde in water and saliva. In particular, glutathione was determined by monitoring the reduction in the Raman signal obtained from substrates modified with crystal violet upon incubation with aqueous glutathione solutions. On the other hand, malondialdehyde was detected after a reaction with thiobarbituric acid to produce a derivative with a strong Raman signal. The detection limits achieved after optimization of several assay parameters were 50 and 3.2 nM for aqueous solutions of glutathione and malondialdehyde, respectively. In artificial saliva, however, the detection limits were 2.0 and 0.32 µM for glutathione and malondialdehyde, respectively, which are, nonetheless, adequate for the determination of these two markers in saliva.

Cortisol Immunosensors: A Literature Review.

Cortisol is a steroid hormone that is involved in a broad range of physiological processes in human/animal organisms. Cortisol levels in biological samples are a valuable biomarker, e.g., of stress and stress-related diseases; thus, cortisol determination in biological fluids, such as serum, saliva and urine, is of great clinical value. Although cortisol analysis can be performed with chromatography-based analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), conventional immunoassays (radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), etc.) are considered the "gold standard" analytical methodology for cortisol, due to their high sensitivity along with a series of practical advantages, such as low-cost instrumentation, an assay protocol that is fast and easy to perform, and high sample throughput. Especially in recent decades, research efforts have focused on the replacement of conventional immunoassays by cortisol immunosensors, which may offer further improvements in the field, such as real-time analysis at the point of care (e.g., continuous cortisol monitoring in sweat through wearable electrochemical sensors). In this review, most of the reported cortisol immunosensors, mainly electrochemical and also optical ones, are presented, focusing on their immunosensing/detection principles. Future prospects are also briefly discussed.

Post-Polymerization Modification of Fluoropolymers via UV Irradiation in the Presence of a Photoacid Generator.

Fluorinated polymers have unique wettability and protein adsorption properties. The site-specific alteration of these properties could expand their application to different research areas. In this work, a fluorinated homopolymer and two of its copolymers with 4-vinylbenzyl glycidyl ether (VBGE) are synthesized by free radical polymerization. The produced polymers are then used to develop resist formulations by the addition of a photoacid generator. Films of these formulations are exposed to ultraviolet radiation through a binary mask and heated to create the pattern. It is found that the water contact angle values of the exposed films areas are reduced compared to those of the unexposed ones, with the exception of pentafluorophenyl methacrylate (PFMA) homopolymer film. This is attributed to the reaction of the epoxy groups creating x-links and producing hydroxyl groups and the cleavage of the pentafluorophenyl group from the ester group leading to carboxylic acid groups. Both modifications on the exposed areas are verified by FTIR spectroscopy and ToF-SIMS analysis. In addition, the biomolecules adsorption ability of the exposed area is increasing 10-15 times compared to the unexposed one for the PFMA homopolymer and the PFMA/VBGE 1:1 copolymer. Thus, the proposed polymers and patterning procedure could find application to spatially directed immobilization of biomolecules and/or cells onto a surface for both biosensing and tissue engineering purposes.

Photolithographically Patterned Cell-Repellent PEG-b-PTHPMA Diblock Copolymer for Guided Cell Adhesion and Growth.

Surfaces for guided cell adhesion and growth are indispensable in several diagnostic and therapeutic applications. Towards this direction, four diblock copolymers comprising polyethylene glycol (PEG) and poly(2-tetrahydropyranyl methacrylate) (PTHPMA) are synthesized employing PEG macroinitiators of different chain lengths. The copolymer with a 5000 Da PEG block and a PEG-PTHPMA comonomers weight ratio of 43-57 provides a film with the highest stability in the culture medium and the strongest cell repellent properties. This copolymer is used to develop a positive photolithographic material and create stripe patterns onto silicon substrates. The highest selectivity regarding smooth muscle cell adhesion and growth and the highest fidelity of adhered cells for up to 3 days in culture is achieved for stripe patterns with widths between 25 and 27.5 µm. Smooth muscle cells cultured on such patterned substrates exhibit a decrease in their proliferation rate and nucleus area and an increase in their major axis length, compared to the cells cultured onto non-patterned substrates. These alterations are indicative of the adoption of a contractile rather than a synthetic phenotype of the smooth muscle cells grown onto the patterned substrates and demonstrate the potential of the novel photolithographic material and patterning method for guided cell adhesion and growth.

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform.

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need.

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.

Recent Developments in the Field of Optical Immunosensors Focusing on a Label-Free, White Light Reflectance Spectroscopy-Based Immunosensing Platform.

Optical immunosensors represent a research field of continuously increasing interest due to their unique features, which can mainly be attributed to the high-affinity and specific antibodies they use as biorecognition elements, combined with the advantageous characteristics of the optical transducing systems these sensors employ. The present work describes new developments in the field, focusing on recent bioanalytical applications (2021-2022) of labeled and label-free optical immunosensors. Special attention is paid to a specific immunosensing platform based on White Light Reflectance Spectroscopy, in which our labs have gained specific expertise; this platform is presented in detail so as to include developments, improvements, and bioanalytical applications since the mid-2000s. Perspectives on the field are been briefly discussed as well, highlighting the potential of optical immunosensors to eventually reach the state of a reliable, highly versatile, and widely applicable analytical tool suitable for use at the Point-of-Care.

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.

Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon.

The orientation of antibodies, employed as capture molecules on biosensors, determines biorecognition efficiency and bioassay performance. In a previous publication we demonstrated for antibodies attached covalently to silicon that an increase in their surface amount Γ, evaluated with ellipsometry, induces changes in their orientation, which is traced directly using Time-of-Flight Secondary Ion Mass Spectroscopy combined with Principal Component Analysis. Here, we extend the above studies to antibodies adsorbed physically on a 3-aminopropyltriethoxysilane (APTES) monolayer. Antibodies physisorbed on APTES (0 ≤ Γ ≤ 3.5 mg/m2) reveal the Γ ranges for flat-on, side-on, and vertical orientation consistent with random molecular packing. The relation between orientation and Γ is juxtaposed for silicon functionalized with APTES, APTES modified with glutaraldehyde (APTES/GA) and N-hydroxysuccinimide-silane (NHS-silane). Antibody reorientation occurs at lower Γ values when physisorption (APTES) is involved rather than chemisorption (APTES/GA, NHS-silane). At high Γ values, comparable proportions of molecules adapting head-on and tail-on vertical alignment are concluded for APTES and the NHS-silane monolayer, and they are related to intermolecular dipole-dipole interactions. Intermolecular forces seem to be less decisive than covalent binding for antibodies on the APTES/GA surface, with dominant head-on orientation. Independently, the impact of glutaraldehyde activation of APTES on vertical orientation is confirmed by separate TOF-SIMS measurements.

Development of a Point-of-Care System Based on White Light Reflectance Spectroscopy: Application in CRP Determination.

The development of methods and miniaturized systems for fast and reliable quantitative determinations at the Point-of-Care is a top challenge and priority in diagnostics. In this work, a compact bench-top system, based on White Light Reflectance Spectroscopy, is introduced and evaluated in an application with high clinical interest, namely the determination of C-Reactive protein (CRP) in human blood samples. The system encompassed all the necessary electronic and optical components for the performance of the assay, while the dedicated software provided the sequence and duration of assay steps, the reagents flow rate, the real-time monitoring of sensor response, and data processing to deliver in short time and accurately the CPR concentration in the sample. The CRP assay included two steps, the first comprising the binding of sample CRP onto the chip immobilized capture antibody and the second the reaction of the surface immunosorbed CRP molecules with the detection antibody. The assay duration was 12 min and the dynamic range was from 0.05 to 200 µg/mL, covering both normal values and acute inflammation incidents. There was an excellent agreement between CRP values determined in human plasma samples using the developed device with those received for the same samples by a standard diagnostic laboratory method.

Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy.

Carbendazim is a systemic benzimidazole-type fungicide with broad-spectrum activity against fungi that undermine food products safety and quality. Despite its effectiveness, carbendazim constitutes a major environmental pollutant, being hazardous to both humans and animals. Therefore, fast and reliable determination of carbendazim levels in water, soil, and food samples is of high importance for both food industry and public health. Herein, an optical biosensor based on white light reflectance spectroscopy (WLRS) for fast and sensitive determination of carbendazim in fruit juices is presented. The transducer is a Si/SiO2 chip functionalized with a benzimidazole conjugate, and determination is based on a competitive immunoassay format. Thus, for the assay, a mixture of an in-house developed rabbit polyclonal anti-carbendazim antibody with the standards or samples is pumped over the chip, followed by biotinylated secondary antibody and streptavidin. The WLRS platform allows for real-time monitoring of biomolecular interactions carried out onto the Si/SiO2 chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. The sensor is able to detect 20 ng/mL of carbendazim in fruit juices with high accuracy and precision (intra- and inter-assay CVs ≤ 6.9% and ≤9.4%, respectively) in less than 30 min, applying a simple sample treatment that alleviates any "matrix-effect" on the assay results and a 60 min preincubation step for improving assay sensitivity. Excellent analytical characteristics and short analysis time along with its small size render the proposed WLRS immunosensor ideal for future on-the-spot determination of carbendazim in food and environmental samples.

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.