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

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

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Unlocking the power of polyphenols: A promising biomarker of improved metabolic health and anti-inflammatory diet in adolescents.

BACKGROUND: Metabolic syndrome (MetS) in adolescence is a risk factor for future cardiovascular disease. The chronic inflammation associated with MetS can be attenuated by the anti-inflammatory effect of polyphenols. We aimed to evaluate total urinary polyphenols as a biomarker of anti-inflammatory diets and their effect on MetS in adolescents. METHODS: In this retrospective analysis of a longitudinal cohort study, the relationship between total polyphenol excretion (TPE) in urine, the inflammatory potential of the diet measured through the Children's Dietary Inflammatory Index (C-DII), and the presence of metabolic syndrome was evaluated. The study population consisted of adolescents enrolled in the SI! Program for Secondary Schools trial, who had completed all the study forms and provided urine samples at baseline and at the two-year follow-up. Multivariate linear regression and multinominal logistic regression models were generated to evaluate the relationship of changes in TPE with changes in the C-DII score and changes in MetS status, respectively. An analysis of the ROC curve was performed to assess the potential of TPE as a biomarker of an anti-inflammatory diet. RESULTS: This study included 662 adolescents, 51.2% were males, and 48.8% were females, with a mean age of 12 (0.38) years at baseline. The relationship between changes in TPE and changes in the C-DII score was stratified by sex with a p-value <0.001 for the interaction. TPE and C-DII were inversely associated in males (-0.13 mg GAE/g creatinine [-0.26; -0.01] per 1-SD increase, p-value = 0.037). In addition, an increase in changes in TPE levels were associated with a reversal in MetS status in all adolescents (1.30 [1.27; 1.34] per 1-SD increase, p-value<0.001). The ROC curve showed that urinary TPE levels can predict dietary inflammatory potential with an AUC = 0.793 (0.725; 0.863) in males. CONCLUSION: Polyphenols excreted in urine are a potential biomarker of anti-inflammatory diets in males and are associated with a reversal of MetS status in adolescents. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, Identifier: NCT03504059, https://clinicaltrials.gov/study/NCT03504059.

Microbial Phenolic Metabolites Are Associated with Improved Cognitive Health.

SCOPE: Diets rich in polyphenols has been associated with better cognitive performance. The aim of this study is to assess the relationship between microbial phenolic metabolites (MPM) in urine and cognition in the context of an older population at high cardiovascular risk. METHODS AND RESULTS: A cross-sectional analysis is conducted in 400 individuals of the PREDIMED-Plus study. Liquid chromatography coupled to mass spectrometry is used to identify urinary MPM. Mediterranean diet (MedDiet) adherence is estimated with a 17-item questionnaire and cognitive function is evaluated with a battery of neuropsychological tests. Multivariable-adjusted linear regression models are fitted to assess the relationship of urinary MPM with the MedDiet and cognitive tests. Protocatechuic acid and enterolactone glucuronide are associated with higher adherence to the MedDiet. Regarding cognitive function, protocatechuic acid, vanillic acid glucuronide, 3-hydroxybenzoic acid, enterodiol glucuronide, and enterolactone glucuronide are directly associated with a global composite score of all the cognitive tests. Furthermore, protocatechuic acid and enterolactone glucuronide are associated with higher scores in the Mini-Mental State Examination, whereas enterodiol glucuronide is associated with improved Clock Drawing Test scores. CONCLUSIONS: These results suggest that the MedDiet is linked to MPM associated with better cognitive performance in an older population.

Urinary Phenolic Metabolites Associated with Peanut Consumption May Have a Beneficial Impact on Vascular Health Biomarkers.

Phenolic compounds in peanuts may moderate inflammation and endothelial function. Thus, the aim of this study was to evaluate the association of urinary phenolic metabolites (UPMs) with vascular biomarkers after peanut product consumption. A three-arm parallel-group randomized controlled trial was conducted in 63 healthy young adults who consumed 25 g/day of skin roasted peanuts (SRP), 32 g/day of peanut butter (PB), or 32 g/day of a control butter for six months. UPMs were analyzed by liquid chromatography coupled to mass spectrometry. Additionally, urinary eicosanoids, prostacyclin I2 (PGI2), and thromboxane A2 (TXA2) were determined using two competitive enzyme-linked immunosorbent assay kits. Consumers of SRP and PB presented significantly higher excretion of UPMs (enterodiol glucuronide (p = 0.018 and p = 0.031), 3-hydroxybenzoic acid (p = 0.002 and p < 0.001), vanillic acid sulfate (p = 0.048 and p = 0.006), p-coumaric acid (p = 0.046 and p = 0.016), coumaric acid glucuronide I (p = 0.001 and p = 0.030) and II (p = 0.003 and p = 0.036), and isoferulic acid (p = 0.013 and p = 0.015) in comparison with the control group. An improvement in PGI2 (p = 0.037) levels and the TXA2:PGI2 ratio (p = 0.008) was also observed after the peanut interventions compared to the control. Interestingly, UPMs with significantly higher post-intervention levels were correlated with an improvement in vascular biomarkers, lower TXA2 (r from -0.25 to -0.48, p < 0.050) and TXA2:PGI2 ratio (r from -0.25 to -0.43, p < 0.050) and higher PGI2 (r from 0.24 to 0.36, p < 0.050). These findings suggest that the UPMs with higher excretion after peanut product consumption could have a positive impact on vascular health.

Effect of Peanut Consumption on Cardiovascular Risk Factors: A Randomized Clinical Trial and Meta-Analysis.

Although numerous studies have reported the protective effect of nut consumption on cardiovascular risk, evidence for the role of peanuts in maintaining cardiometabolic health is inconclusive. Presented here are the results from the ARISTOTLE study, a parallel randomized controlled trial evaluating the impact of regular peanut intake on anthropometric, biochemical, and clinical measurements. The 63 healthy subjects that completed the study consumed their habitual diet plus either: a) 25 g/day of skin roasted peanuts (SRP, n = 21), b) two tablespoons (32 g)/day of peanut butter (PB, n = 23) or c) two tablespoons (32 g)/day of a control butter based on peanut oil (CB, n = 19) for 6 months. In addition, a meta-analysis of clinical trials, including data from the ARISTOTLE study, was carried out to update the evidence for the effects of consuming peanuts, including high-oleic peanuts, and peanut butter on healthy subjects and those at high cardiometabolic risk. After a systematic search on PubMed, Web of Science, Cochrane Library and Scopus databases up to July 2021, 11 studies were found to meet the eligibility criteria. In the ARISTOTLE study, lower total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratios were found in the SRP group compared to the CB group (p = 0.019 and p = 0.008). The meta-analysis of clinical trials revealed that peanut consumption is associated with a decrease in triglycerides (MD: -0.13; 95% CI, -0.20 to -0.07; p < 0.0001) and that healthy consumers had lower total cholesterol and LDL-cholesterol/HDL-cholesterol ratios compared to the control groups (MD: -0.40; 95% CI, -0.71 to -0.09; p = 0.01 and MD: -0.19; 95% CI, -0.36 to -0.01; p = 0.03, respectively). However, individuals at high cardiometabolic risk experienced an increase in body weight after the peanut interventions (MD: 0.97; 95% CI, 0.54 to 1.41; p < 0.0001), although not in body fat or body mass index. According to the dose-response analyses, body weight increased slightly with higher doses of peanuts. In conclusion, a regular consumption of peanuts seems to modulate lipid metabolism, reducing triglyceride blood levels. Systematic Review Registration: https://osf.io/jx34y/, identifier: 10.17605/OSF.IO/MK35Y.

Effect of Crushing Peanuts on Fatty Acid and Phenolic Bioaccessibility: A Long-Term Study.

BACKGROUND: Peanuts are consumed worldwide and have been linked to multiple health benefits. Processing may affect the bioavailability of peanut bioactive compounds. Therefore, we aim to evaluate the effects of crushing peanuts on the bioavailability of fatty acids and phenolic compounds in healthy adults. METHODS: 44 participants from the ARISTOTLE study consumed 25 g/day of whole peanuts (WP) or 32 g/day of peanut butter (PB) for 6 months. Fatty acids and phenolic compounds in peanut products and biological samples were assessed by gas chromatography coupled to flame ionization detection and liquid chromatography coupled to high resolution mass spectrometry, respectively. RESULTS: Plasma concentrations of very long chain saturated fatty acids (VLCSFAs) increased significantly after 6 months of WP or PB intake (p < 0.001 in both cases). Participants in the WP group excreted twice as many VLCSFAs in feces as those in the PB group (p = 0.012). The most abundant polyphenols found in WP and PB were p-coumaric and isoferulic acids. Urinary excretion of isoferulic acid increased after the intake of WP and PB (p = 0.032 and p = 0.048, respectively), with no significant difference observed between interventions. CONCLUSION: The crushing step in peanut butter production seems to enhance the bioavailability of bioactive compounds.

One-Year Changes in Urinary Microbial Phenolic Metabolites and the Risk of Type 2 Diabetes-A Case-Control Study.

The intake of polyphenols has been associated with a risk reduction of type 2 diabetes. Nevertheless, to the best of our knowledge, the molecules that might be metabolically active after ingestion are only starting to be investigated regarding this metabolic disease. To investigate the association between one-year changes in urinary microbial phenolic metabolites (MPM) and the incidence of type 2 diabetes, we performed a case-control study using data and samples of the PREDIMED trial including 46 incident type 2 diabetes cases of 172 randomly selected participants. Eight urinary MPMs were quantified in urine by liquid chromatography coupled to mass spectrometry and used to assess their associations with type 2 diabetes risk by multivariable logistic regression models. Compared to participants in the lowest tertile of one-year changes in hydroxybenzoic acid glucuronide, those in the highest tertile had a significantly lowered probability of developing type 2 diabetes (OR [95% CI], 0.39 [0.23−0.64]; p < 0.001 for trend). However, when additionally adjusting for fasting plasma glucose, the statistical significance was lost. Changes in the dietary pattern can increase the concentrations of this compound, derived from many (poly)phenol-rich foods, and might be changing the gut microbial population as well, promoting the production of the metabolite.

Total urinary polyphenols and ideal cardiovascular health metrics in Spanish adolescents enrolled in the SI Program: a cross-sectional study.

To study the relationship between urinary total polyphenol excretion (TPE) in adolescents and ideal cardiovascular (CVH) metrics. 1151 adolescents aged 12.04 (0.46) years participating in the SI! Program for Secondary Schools were selected based on the availability of urine samples and information required to assess CVH metrics. Data on health behaviours (smoking status, body mass index, physical activity, and healthy diet) and health factors (blood pressure, total cholesterol, and blood glucose) were used to calculate the CVH metrics. TPE in urine was analysed by a Folin-Ciocalteu method after solid-phase extraction. Associations between TPE (categorized into tertiles) and CVH metrics (total and separate scores) were assessed using multilevel mixed-effect regression models. Higher TPE levels were associated with higher (healthier) CVH scores and ideal smoking status (OR 1.54, 95% CI 1.10; 1.87, p value = 0.007), physical activity (OR 1.12, 95% CI 1.02; 1.23, p value = 0.022) and total cholesterol (OR 1.78, 95% CI 1.16; 2.73, p value = 0.009) after multivariate adjustment. An association between TPE and total CVH scores was observed only in boys. Girls with higher TPE had higher rates of ideal total cholesterol and blood pressure. According to our findings, higher urinary TPE is related to better CVH scores, with relevant differences in this association by gender.

Consumption of peanut products improves memory and stress response in healthy adults from the ARISTOTLE study: A 6-month randomized controlled trial.

BACKGROUND: Peanuts are rich in bioactive compounds that may have a positive impact on memory and stress response. OBJECTIVE: To evaluate the effect of regular consumption of peanut products on cognitive functions and stress response in healthy young adults. DESIGN: A three-arm parallel-group randomized controlled trial was conducted in 63 healthy young adults that consumed 25 g/day of skin roasted peanuts (SRP, n = 21), 32 g/d of peanut butter (PB, n = 23) or 32 g/d of a control butter made from peanut oil (free of phenolic compounds and fiber) (CB, n = 19) for six months. Polyphenol intake, cognitive functions, and anxiety and depression scores were evaluated using validated tests. Fecal short-chain fatty acids (SCFAs) and plasma and fecal fatty acids were assessed by chromatographic methods. Urinary cortisol was quantified by an enzymatic method. RESULTS: Comparing the two interventions with the control, a significant reduction in anxiety scores was observed in the SRP compared to the CB group. After the intervention, consumers of SRP and PB had an improved immediate memory (p = 0.046 and p = 0.011). Lower anxiety scores were associated with SRP and PB (p < 0.001 and p = 0.002, respectively) and lower depression scores with SRP, PB and CB (p = 0.007, p = 0.003 and p = 0.032, respectively). Memory functions and stress response were significantly correlated with polyphenol intake, fecal SCFAs, plasma and fecal very long chain saturated fatty acids (VLCSFAs). CONCLUSIONS: Regular peanut and peanut butter consumption may enhance memory function and stress response in a healthy young population. These effects seem to be associated with the intake of peanut polyphenols, increased levels of fecal SCFAs, and unexpectedly, VLCSFAs, which were also present in the control product.

Urinary Tartaric Acid, a Biomarker of Wine Intake, Correlates with Lower Total and LDL Cholesterol.

Postmenopausal women are at higher risk of developing cardiovascular diseases due to changes in lipid profile and body fat, among others. The aim of this study was to evaluate the association of urinary tartaric acid, a biomarker of wine consumption, with anthropometric (weight, waist circumference, body mass index (BMI), and waist-to-height ratio), blood pressure, and biochemical variables (blood glucose and lipid profile) that may be affected during the menopausal transition. This sub-study of the PREDIMED (Prevención con Dieta Mediterránea) trial included a sample of 230 women aged 60-80 years with high cardiovascular risk at baseline. Urine samples were diluted and filtered, and tartaric acid was analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Correlations between tartaric acid and the study variables were adjusted for age, education level, smoking status, physical activity, BMI, cholesterol-lowering, antihypertensive, and insulin treatment, total energy intake, and consumption of fruits, vegetables, and raisins. A strong association was observed between wine consumption and urinary tartaric acid (0.01 µg/mg (95% confidence interval (CI): 0.01, 0.01), p-value < 0.001). Total and low-density lipoprotein (LDL) cholesterol were inversely correlated with urinary tartaric acid (-3.13 µg/mg (-5.54, -0.71), p-value = 0.016 and -3.03 µg/mg (-5.62, -0.42), p-value = 0.027, respectively), whereas other biochemical and anthropometric variables were unrelated. The results suggest that wine consumption may have a positive effect on cardiovascular health in postmenopausal women, underpinning its nutraceutical properties.

Polyphenols in Urine and Cardiovascular Risk Factors: A Cross-Sectional Analysis Reveals Gender Differences in Spanish Adolescents from the SI! Program.

(1) Background: Epidemiological studies have shown an inverse association between polyphenol intake and cardiovascular risk factors (CVRFs) in adults, but few have provided information about adolescents. The aim of this study was to evaluate the relationship between urinary total polyphenol excretion (TPE) and CVRFs in adolescents. (2) Methods: A cross-sectional study was performed in 1194 Spanish adolescents from the SI! (Salud Integral) program. TPE in urine samples was determined by the Folin-Ciocalteu method, after solid-phase extraction, and categorized into quartiles. The association between TPE and CVRFs was estimated using mixed-effect linear regression and a structural equation model (SEM). (3) Results: Linear regression showed negative associations among the highest quartile of TPE and body fat percentage (B = -1.75, p-value = <0.001), triglycerides (TG) (B = -17.68, p-value = <0.001), total cholesterol (TC) (B = -8.66, p-value = 0.002), and low-density lipoprotein (LDL)-cholesterol (LDL-C) (B = -4.09, p-value = 0.008) in boys, after adjusting for all confounder variables. Negative associations between TPE quartiles and systolic blood pressure (SBP), diastolic blood pressure (DBP), and TC were also found in girls. Moreover, a structural equation model revealed that TPE was directly associated with body composition and blood glucose and indirectly associated with blood pressure, TG, LDL-C, and high-density lipoprotein-cholesterol (HDL-C) in boys. (4) Conclusions: Higher concentrations of TPE were associated with a better profile of cardiovascular health, especially in boys, while in girls, the association was not as strong.

Interstitial Glucose and Lactate Levels Are Inversely Correlated With the Body Mass Index: Need for In Vivo Calibration of Glucose Sensor Results With Blood Values in Obese Patients.

BACKGROUND: Continuously measured glucose and lactate levels in interstitial fluid (ISF) may markedly differ from their respective blood levels. METHODS: Combining microdialysis with a bioanalytical microsystem, the interstitial glucose and lactate concentrations of eight male volunteers with different body mass index (BMI) were monitored during a 2-fold glucose tolerance test over the period of three hours. RESULTS: Significant correlations were found between abdominally measured sensor results and reference measurements ( R2 = .967 for glucose and R2 = .936 for lactate, P < .05). The physiological delay of the abdominally observed glucose appearance in the ISF correlated positively with the BMI ( R2 = .787, P < .05). The relative in vivo recovery of glucose and lactate was inversely proportional to the BMI of the volunteers ( R2 = .540 for glucose, R2 = .609 for lactate, P < .05). One subject with a BMI of > 34 kg/m2 showed abdominally as well as the antebrachially significantly reduced tissue glucose values compared to blood glucose values ( P < .001). CONCLUSIONS: A very good correlation between abdominally measured sensor results and the results of the reference method verified the reliability of the BioMEMS. The abdominally measured glucose level in ISF decreased significantly with increasing BMI. Therefore, an in vivo calibration of glucose levels in ISF with blood levels seems to be necessary especially in markedly obese subjects.

Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label-free monolithically integrated optoelectronic biosensor.

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.

Simultaneous determination of CRP and D-dimer in human blood plasma samples with White Light Reflectance Spectroscopy.

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 25ng/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.

Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.

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 microm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 microl 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.

Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

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.