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Bioprocess development and fermentation control would benefit from a timely knowledge of metabolic parameters, e.g., glucose, glutamine, and lactate concentration etc., enabling adequate intervention, maximizing product yield, and providing process analytical tools for reliable and traceable bioprocessing in the context of process analytical technology. A polymer laminate technology based microsytem comprising integrated pre-calibrated electrochemical biosensors for the simultaneous monitoring of lactate, glucose, glutamine, and glutamate in a 0.15-μL flow-through cell for process monitoring and control is presented. Flexible manufacturing technologies used for these devices allow a cost effective customization of them. [ABSTRACT FROM AUTHOR]

For simultaneous measurement of glucose, lactate, glutamine, and glutamate a biosensor array is implemented in a micro flow-system thus giving a microsystem. The microsystem consists of a glass chip with the integrated biosensor array and a bottom part, which comprises a gold counter electrode, a 300 μm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 μl when integrated with a mixer on chip. The biosensors with no crosstalking and high long term stability were produced by modifying the electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producable devices for in vivo and ex vivo applications in whole blood and fermentation broth. Due to a novel glutaminase with an activity optimum in the neutral pH range direct and simultaneous monitoring of glutamine together with glucose, lactate, and glutamate could be performed. [Copyright &y& Elsevier]

Describes the development of a miniaturized device for simultaneous measurement of glucose and lactate levels. Photopatterning of enzyme-containing photosensitive membrane precursors; Linear response ranges for glucose and lactate; Potential for mass production of devices by means of thin-film technology on flexible carriers; Results of ex vivo experiments performed with human volunteers.

[Display omitted] • Ten MZIs, optical sources, spectral analyzers, and photodiode arrays on a single chip. • Portable reader based on all-in-one monolithic photonic chips. • Multiplexed determination of 3 allergens in industry rinsing water samples in 10 min. • Dual mycotoxins determination in beer samples within 15 min. 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 κ-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 μg/mL for κ-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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fabrication, and evaluation of an integrated Lab-on-Chip platform. • Bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification on chip. • Pathogen screening in milk samples for cell lysates corresponding to <500 cells. Abstract Strict regulations govern food safety as the latter is of extremely high importance and a major public issue, while it is demanding in respect of analytical effort. The duration of common food analysis practices is typically 2–5 days. Microfluidics can reduce labor and analysis time to a few hours, proving its beneficial use and rendering the response of food industry as well as public safety agents faster. The aim of the present work is the fabrication and evaluation of a modular integrated Lab-on-Chip platform for sample preparation, accommodating bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification, addressing pathogen screening in milk samples for cell lysates corresponding to <500 cells. We note that individual modules have been tested to work down to 10 cells. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Optical immunosensor for label-free detection of AFB1, FB1and DON in beer. • The three mycotoxins are determined following a competitive immunoassay format. • The multiplexed assay duration is 12 min. • The sensor can determine the three mycotoxins in different beer types. Abstract A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B 1 (AFB 1), fumonisin B 1 (FB 1) 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 AFB 1 , FB 1 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. [ABSTRACT FROM AUTHOR]

The fast and efficient detection of foodborne pathogens is a societal priority, given the large number of food-poisoning outbreaks, and a scientific and technological challenge, given the need to detect as little as 1 viable cell in 25 gr of food. Here, we present the first approach that achieves the above goal, thanks to the use of a micro/nano-technology and the detection capability of acoustic wave sensors. Starting from 1 Salmonella cell in 25 ml of milk, we employ immuno-magnetic beads to capture cells after only 3 h of pre-enrichment and subsequently demonstrate efficient DNA amplification using the Loop Mediated Isothermal Amplification method (LAMP) and acoustic detection in an integrated platform, within an additional ½ h. The demonstrated 4 h sample-to-analysis time comes as a huge improvement to the current need of few days to obtain the same result. In addition, the work presents the first reported Lab-on-Chip platform that comprises an acoustic device as the sensing element, exhibiting impressive analytical features, namely, an acoustic limit of detection of 2 cells/μl or 3 aM of the DNA target and ability to detect in a label-free manner dsDNA amplicons in impure samples. The use of food samples together with the incorporation of the necessary pre-enrichment step and ability for multiple analysis with an internal control, make the proposed methodology highly relevant to real-world applications. Moreover, the work suggests that acoustic wave devices can be used as an attractive alternative to electrochemical sensors in integrated platforms for applications in food safety and the point-of-care diagnostics. [ABSTRACT FROM AUTHOR]

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 2.1 ng/mL, the quantification limit was 4.2 ng/mL and the linear dynamic range extended up to 100 ng/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. [ABSTRACT FROM AUTHOR]

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 (37 mm 2 ). 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 2.0 ng/ml and a dynamic range 4.0–100 ng/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The label-free detection of bovine milk in goat milk through a miniaturized optical biosensor is presented. The biosensor consists of ten planar silicon nitride waveguide Broad-Band Mach–Zehnder interferometers (BB-MZIs) monolithically integrated and self-aligned with their respective silicon LEDs on the same Si chip. The BB-MZIs were transformed to biosensing transducers by functionalizing their sensing arm with bovine k-casein. Measurements were performed by continuously recording the transmission spectra of each interferometer through an external spectrometer. The amount of bovine milk in goat milk was determined through a competitive immunoassay by passing over the sensor mixtures of anti-k-casein antibodies with the calibrators or the samples. The output spectra of each BB-MZI recorded during the reaction were subjected to Discrete Fourier Transform in order to convert the observed spectral shifts to phase shifts in the wavenumber domain. The method had a detection limit of 0.04 % (v/v) bovine milk in goat milk, dynamic range 0.1–1.0 % (v/v), recoveries 93–110 %, and intra- and inter-assay coefficients of variation less than 12 and 15 %, respectively. The proposed biosensor compared well in terms of analytical performance with a competitive ELISA developed using the same monoclonal antibodies. Nevertheless, the duration of the biosensor assay was 10 min whereas the ELISA required 2 h. Thus, the fast and sensitive determinations along with the small size of the sensor make it ideal for incorporation into portable devices for assessment of goat or ewe’s milk adulteration with bovine milk at the point-of-need. [ABSTRACT FROM AUTHOR]

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min−1) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium. [ABSTRACT FROM AUTHOR]

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles' heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004-2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations. [ABSTRACT FROM AUTHOR]