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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 (

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

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

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.

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 mm2 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 μm. 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 (AFM1) 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 AFM1-bovine serum albumin conjugate has been immobilized. The assay was performed by running mixtures of rabbit polyclonal anti-AFM1 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%). AFM1 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 AFM1 determination in milk samples at the point-of-need.

An optical immunosensor based on White Light Reflectance Spectroscopy for the simultaneous determination of the herbicides atrazine and paraquat in drinking water samples is demonstrated. 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 due to reaction to effective biomolecular layer thickness. Dual-analyte determination is accomplished by functionalizing spatially distinct areas of the chip with protein conjugates of the two herbicides and scanning the surface with an optical reflection probe. A competitive immunoassay format was adopted, followed by reaction with secondary antibodies for signal enhancement. The sensor was highly sensitive with detection limits of 40 and 50 pg/mL for paraquat and atrazine, respectively, and the assay duration was 12 min. Recovery values ranging from 90.0 to 110% were determined for the two pesticides in spiked bottled and tap water samples, demonstrating the sensor accuracy. In addition, the sensor could be regenerated and re-used at least 20 times without significant effect on the assay characteristics. Its excellent analytical performance and short analysis time combined with the small sensor size should be helpful for fast on-site determinations of these analytes.

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.

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

An immunosensor for fast and accurate determination of C-reactive protein (CRP) in human serum samples based on an array of all-silicon broad-band Mach-Zehnder interferometers (BB-MZIs) is demonstrated. The detection was based on monitoring the spectral shifts during the binding of CRP on the antibody molecules that have been immobilized on the sensing arms of the BB-MZIs. By employing the reaction rate as the analytical signal the assay time was compressed to few minutes. The detection limit was 2.1ng/mL, the quantification limit was 4.2ng/mL and the linear dynamic range extended up to 100ng/mL. The measurements performed in human serum samples with the developed immunosensor were characterized by high repeatability and accuracy as it was demonstrated by dilution linearity and recovery experiments. In addition, the concentration values determined were in excellent agreement with those determined for the same samples by a standard clinical laboratory method. The compact size of the chip makes the proposed immunosensor attractive for incorporation into miniaturized devices for the determination of clinical analytes at the point-of-need.

The simultaneous determination of pesticides chlorpyrifos, imazalil and thiabendazole in drinking water and wine samples with a biosensor based on White Light Reflectance Spectroscopy is presented. A competitive immunoassay format was implemented followed by reaction of immunoadsorbed anti-analyte specific antibody molecules with a secondary antibody in order to enhance the signal and reduce the analysis time. The whole analysis was completed in less than 10 min and the limits of quantification achieved with calibrators prepared in buffer were 60 pg/mL for chlorpyrifos and imazalil, and 80 pg/mL for thiabendazole. Bottled water samples were analysed without any treatment, whereas white and red wine samples were analysed after 10-times dilution with assay buffer. This way pesticide concentrations as low as 0.6 ng/mL for chlorpyrifos and imazalil, and 0.8 ng/mL for thiabendazole could be detected in wines. The measurements were repeatable with intra- and inter-assay CVs values ranging from 4.9 to 8.2% and from 7.3 to 9.7%, respectively, for all assays. The accuracy of the measurements was evaluated through recovery experiments using spiked water and wine samples. Percent recovery values ranged from 86 to 116%, demonstrating the accuracy of the measurements performed with the developed biosensor. In addition, 5 wines spiked with different concentrations of the three pesticides were analysed both with the developed biosensor and a validated LC–MS/MS method. The good agreement of the results obtained with the two methods is a further testimony of the developed method accuracy and a good starting point to demonstrate its Point-of-Need application capabilities in the food analysis field.

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.

A dual-analyte assay for the simultaneous determination of C-reactive protein (CRP) and D-dimer in human blood plasma based on a white light interference spectroscopy sensing platform is presented. Measurement is accomplished in real-time by scanning the sensing surface, on which distinct antibody areas have been created, with a reflection probe used both for illumination of the surface and collection of the reflected interference spectrum. The composition of the transducer, the sensing surface chemical activation and biofunctionalization procedures were optimized with respect to signal magnitude and repeatability. The assay format involved direct detection of CRP whereas for D-dimer a two-site immunoassay employing a biotinylated reporter antibody and reaction with streptavidin was selected. The assays were sensitive with detection limits of 25 ng/mL for both analytes, precise with intra- and inter-assay CV values ranging from 3.6% to 7.7%, and from 4.8% to 9.5%, respectively, for both assays, and accurate with recovery values ranging from 88.5% to 108% for both analytes. Moreover, the values determined for the two analytes in 35 human plasma samples were in excellent agreement with those received for the same samples by standard diagnostic laboratory instrumentation employing commercial kits. The excellent agreement of the results supported the validity of the proposed system for clinical application for the detection of multiple analytes since it was demonstrated that up to seven antibody areas can be created on the sensing surface and successfully interrogated with the developed optical set-up.

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 (

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-µm 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.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B

This chapter pertains to a succinct but comprehensive presentation of the research on interferometric transducers, which have emerged as extremely promising candidates for viable, truly marketable solutions for point-of-need/point-of-care applications due to their attested analytical performance reaching down to 10−8 in terms of effective refractive index changes. The chapter, on one hand, explores the principles of operation of the various interferometric architectures, while on the other hand, the issues of biosensor functionalization and immobilization of receptors are addressed. In the final section, interferometric sensors employed for immunosensing applications are described along with their bioanalytical performance.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1) and deoxynivalenol (DON), in beer samples is presented. The biosensor is based on an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources onto a single chip. Multi-analyte determination is accomplished by functionalizing the sensing arms of individual MZIs with mycotoxin-protein conjugates. Assay is performed by pumping over the chip mixtures of calibrators or samples with a mixture of specific monoclonal antibodies, followed by reaction with a secondary anti-mouse IgG antibody. Reactions are monitored in real-time by continuously recording the MZI output spectra, which are then subjected to Discrete Fourier Transform to convert spectrum shifts to phase shifts. The detection limits achieved for AFB1, FB1 and DON were 0.8, 5.6 and 24 ng/ml, respectively, while the assay duration was 12 min. Recovery values ranging from 85 to 115% were determined in beer samples spiked with known concentrations of the three mycotoxins. In addition, beers of different types and origin were analysed with the biosensor developed and the results were compared with those provided by established laboratory methods, further supporting the accuracy of the proposed device.

β- and γ-Cyclodextrins connected on their narrow side via 6 bond-linkers with monosaccharides (Man, GlcNAc) suitable for bacterial lectin recogni tion, along with reference mono-analogues were prepared. The resulting compounds: (a) posses a functional cavity forming inclusion complexes with a model guest molecule and with the antibiotic ampicillin; (b) inhibit by more than 30% the hydrolysis of ampicillin in the presen ce of the enzyme β-lactamase; (c) interact stronger with surface immobilized lectins than the reference mono-analogues. The present work suggests that combining the lectin recognition prop erty of the compounds with their ability to encapsulate antibiotics could be used to target and kill bacteria.

A label-free immunosensor based on white-light reflectance spectroscopy for the determination of total- and free-PSA in human serum is presented. The sensor consists of a silicon die with an overlayer of transparent material. Thermally grown silicon dioxide layers with thicknesses ranging between 700 and 3000 nm proved to be the most appropriate transparent materials for monitoring bioreactions compared to silicon dioxide layers of lower thickness as well as to silicon nitride layers. Highly specific antibodies against either total-PSA or free-PSA were selected and used to develop the respective immunosensors. To achieve the required analytical detection sensitivity required for the determination of both analytes in human serum samples, a two site immunoassay format was selected employing a biotinylated antibody as reporter molecule in combination with streptavidin. Due to the employment of this detection scheme, the effect of serum on the signal of the calibrators and the assay sensitivity was negligible. The assays developed with the proposed sensor were sensitive with detection limits of 0.2 and 0.15 ng/mL for total- and free-PSA, respectively, accurate with recovery values ranging from 92 to 108% for both analytes, and reproducible (within-sensor CVs less than 5.6%, between-sensors CVs less than 6.7%). The working ranges of the assays extended from 0.5 to 100 ng/mL, and 0.5 to 20 ng/mL for total- and free-PSA, respectively, and cover the concentration ranges of the analytes in the serum of normal male individuals as well as of those suffering from benign hyperplasia and prostate cancer. The particular sensor due to its small size and low cost could be the heart of a miniaturized device suitable for point-of-care determinations.

A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2-5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection.

In this chapter, the main categories of integrated optical sensors that is grating-couplers, interferometers, photonic crystals, microring resonators, slot waveguides or silicon wires will be discussed. Emphasis will be given to the analytical performance of these sensors upon their application in clinical diagnostics, food analysis schemes or environmental monitoring. Finally, the attempts to build up lab-on-chips or portable devices for application of these sensors at the point-of-need will be mentioned. The comparison of the various types of sensors will be performed using the detection sensitivity and in particular the limit of detection (LOD) as figures of merit. The LOD is referred to the lowest amount of analyte that the sensor can detect as a signal distinguishable from the measurement noise. Usually, the LOD is expressed as refractive index units (RIU) or as surface mass density (g mm-2). It can be also expressed as analyte concentration, although this value could complicate the comparison between different sensors which have been evaluated using different analytes or recognition molecules with different affinity constants. Therefore, whenever applicable, the first two expressions of LOD are preferable to be employed as a more objective performance criterion.

The arrangement, composition and orientation of immunoreagents employed in an indirect immunoassay for determination of mycotoxin OchraToxin A (OTA) are specified for Si3N4 substrate, aiming to imitate biosensor transducers made of the same material. Si3N4 surfaces are examined after modification with (3-aminopropyl)triethoxysilane, spotting with OTA-ovalbumin conjugate (probe), blocking with bovine serum albumin, reaction with a mouse monoclonal antibody against OTA and, finally, reaction with a goat anti-mouse secondary antibody. Atomic force micrographs, their autocorrelation and height histogram parameters, show the stepwise development of a multi-component monolayer covered by groups of secondary antibody molecules. Time-Of-Flight Secondary Ion Mass Spectrometry reveals the composition of probe and blocking protein, as well as their partial desorption during the primary immunoreaction. Ellipsometry provides surface amount of all proteins, increasing step-by-step from 0.7 to 6.9mg/m2. In addition, ellipsometry combined with TOF-SIMS reveals the mass loadings of different molecules in the intermediate and the final overlayer. Based on this, some orientations of the immobilized molecules are proposed and a molar ratio of ∼2.5 for secondary to primary antibody is calculated. The orientations of the primary and secondary antibody are further clarified by Principal Component Analysis of TOF-SIMS data, through which a side-on and a head-on orientation is deduced for the primary and the secondary antibody, respectively. These findings demonstrate how the combination of multiple surface analysis techniques can provide insight on the arrangement, composition and orientation of biomolecules in the course of multi-step procedures employed in biosensors.

Mass fabrication of integrated biosensors on silicon chips is facilitated by contact pin-printing, applied for biofunctionalization of individual Si 3 N 4 -based transducers at wafer-scale. To optimize the biofunctionalization for immunochemical (competitive) detection of fungicide thiabendazole (TBZ), Si 3 N 4 surfaces are modified with (3-aminopropyl)triethoxysilane and examined after: immobilization of BSA-TBZ conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin (BSA), and immunoreaction with a mouse monoclonal antibody against TBZ. Nanostructure, surface density, probe composition and coverage uniformity of protein layers are evaluated with Atomic Force Microscopy, Spectroscopic Ellipsometry, Time-of-Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy. Contact pin-printing of overlapping probe spots is compared with hand spotted areas. Contact pin-printing resulted in two-fold increase of immobilized probe surface density as compared to hand spotting. Regarding BSA-TBZ immobilization, an incomplete monolayer develops into a bilayer as the concentration of BSA-TBZ molecules in the printing solution increases from 25 to 100 μg/mL. Upon blocking, however, a complete protein monolayer is formed for all the BSA-TBZ concentrations used. Free surface sites are filled with BSA for low surface coverage with BSA-TBZ, whereas loosely bound BSA-TBZ molecules are removed from the BSA-TBZ bilayer. As a consequence immunoreaction efficiency increases with the printing probe concentration.

In this work, interferometric silicon chips with monolithically-integrated light-emitting devices coupled to co-integrated monomodal waveguides shaped as Young interferometers through mainstream silicon technology, are presented. Although the light sources are broad-band emitters, Young interferometry is possible through filtering. Chips with arrays of ten multiplexed interferometers have been employed for the label-free determination of pesticides in drinking water currently achieving detection limits in the ng/ml range.

An optical biosensor for label-free detection of ochratoxin A (OTA) in beer samples is presented. The biosensor consists of an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources on the same Si chip (37mm2). The chip was transformed to biosensor by functionalizing the MZIs sensing arms with an OTA-ovalbumin conjugate. OTA determination was performed by pumping over the chip mixtures of calibrators or samples with anti-OTA antibody following a competitive immunoassay format. An external miniaturized spectrometer was employed to continuously record the transmission spectra of each interferometer. Spectral shifts obtained due to immunoreaction were transformed to phase shifts through Discrete Fourier Transform. The assay had a detection limit of 2.0ng/ml and a dynamic range 4.0-100ng/ml in beer samples, recoveries ranging from 90.6 to 116%, and intra- and inter-assay coefficients of variation of 9% and 14%, respectively. The results obtained with the sensor using OTA-spiked beer samples spiked were in good agreement with those obtained by an ELISA developed using the same antibody. The good analytical performance of the biosensor and the small size of the proposed chip provide for the development of a portable instrument for point-of-need determinations.

Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

Protein detection and characterization based on Broad-band Mach-Zehnder Interferometry is analytically outlined and demonstrated through a monolithic silicon microphotonic transducer. Arrays of silicon light emitting diodes and monomodal silicon nitride waveguides forming Mach-Zehnder interferometers were integrated on a silicon chip. Broad-band light enters the interferometers and exits sinusoidally modulated with two distinct spectral frequencies characteristic of the two polarizations. Deconvolution in the Fourier transform domain makes possible the separation of the two polarizations and the simultaneous monitoring of the TE and the TM signals. The dual polarization analysis over a broad spectral band makes possible the refractive index calculation of the binding adlayers as well as the distinction of effective medium changes into cover medium or adlayer ones. At the same time, multi-analyte detection at concentrations in the pM range is demonstrated.

A White Light Reflectance Spectroscopy (WLRS) methodology is applied in the evaluation of the effectiveness of biomolecules immobilization onto solid surfaces as well as their subsequent reaction with counterpart biomolecules via measuring the respective layers thickness. In particular we investigated the adsorption of rabbit and mouse gamma-globulins as well as their reaction with complementary antibodies. The results obtained with the proposed methodology were compared with those received by atomic force microscopy analysis of the same samples. It was found that the developed method provides a simple, very fast and accurate approach for thin biomolecular layers thickness determination. (C) 2009 Elsevier B.V. All rights reserved. Microelectronic Engineering

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible–near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150 pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin-horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction.

We present a label-free, real-time sensor based on white light reflectance spectroscopy for quantitating the complement activation product C3b and its metabolites as a biomarker in human serum. Our novel sensor allows real-time monitoring of biomolecular reactions (in this case, antigen–antibody reactions) taking place on a reflective surface within a flow cell. Detection was based on monitoring the increase in film thickness caused by its immunoreaction with a specific antibody; this reaction was seen as a shift in the wavelength at which constructive interference was observed . Quantitation of C3b was achieved by immobilizing a specific mouse monoclonal antibody onto the refractive surface and monitoring the rate of the signal changes occurring during the first 60 s of the immunoreaction between the antibody and known concentrations of purified C3b or dilutions of complement-activated human serum. The lowest detectable concentration of purified C3b was 20 ng/mL, and complement activation products in human serum samples could be detected at dilutions as high as 6000-fold. The advantages of the method include its relatively low cost, short analysis time, and high assay sensitivity and reliability. Thus, this novel assay method can be used to monitor serum C3b produced as a result of complement activation in a variety of normal and pathologic conditions.

Methods for homogeneous coverage of surfaces with biomolecules are critical for the development of bioanalytical microsystems. Here we present a simple method for coating glass capillaries with an ultra-thin poly(dimethylsiloxane) (PDMS) film in order to be used as optical waveguide fluoroimmunosensors. A model rabbit @c-globulin binding assay was employed for the evaluation of modified capillaries in terms of protein binding capacity and protein coating homogeneity. It was found that the PDMS-modified capillaries provided excellent protein coating homogeneity inside each one capillary (CVs 2.9-6.6%) and between-capillaries repeatability (CVs 2.1-5.0%) compared to aminosilanized ones. Furthermore, the waveguiding properties of the glass were not affected by the proposed surface modification.

A simple approach that employs black drawing ink (BDI) as bulk fluorescence light blocker and improves considerably the homogeneous signal detection in capillary-waveguide fluoroimmunosensors is presented. The concept was proved using a capillary sensor configuration. Fluorescent molecules in the capillary were excited by a laser beam vertically to its axis and the emitted photons that were trapped and waveguided through the capillary wall were then collected. Two competitive fluoroimmunoassays, for rabbit gamma-globulins in buffer and thyroxine in human serum, respectively, were set-up to evaluate this approach. It was found that the presence of ink improved the specific to bulk fluorescence signal ratio by approximately 60-times without affecting the analyte-antibody binding reaction thus, facilitating homogeneous detection. The analytical characteristics of the two assays developed with the sensor operating in homogeneous detection mode were similar to those determined following the heterogeneous detection mode (i.e. after removal/washing of the immunoreaction mixture).

The monolithic silicon optocoupler presented here offers a platform for a new generation of fully integrated devices fabricated through the mature and inexpensive silicon technology. Using the developed optocoupler, real-time detection of the most common mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was accomplished. For this purpose, oligonucleotides corresponding to wild- and mutant-type sequences were immobilized onto different optocouplers and the hybridization with fluorescently labeled complementary or non-complementary sequences was monitored in real time. Hybridization of fluorescently labeled oligonucleotides to the immobilized ones modulated the coupling efficiency between the light emitting diode and the detector in a concentration-dependent manner. Using as label the AlexaFluor® 647 dye (whose absorption maximum fits the emission maximum of the light source) a detection limit of 0.9 nM (9 fmol) was achieved. Real-time signal monitoring, especially during dehybridization, improved considerably the discrimination between wild-type and mutant sequences due to the ability to calculate dissociation kinetics upon washing independently for each one mutation. The bioanalytical capabilities of the transducer along with the fact that dense transducer arrays can be fabricated on a single chip open new frontiers in the manufacturing of microsystems appropriate for point-of-care analysis.