Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique.

Here, we present a novel approach to increase the degree of miniaturization as well as the sensitivity of biosensor platforms by the optimization of microfluidic stop-flow techniques independent of the applied detection technique (e.g. electrochemical or optical). The readout of the labeled bioassays, immobilized in a microfluidic channel, under stop-flow conditions leads to a rectangular shaped peak signal. Data evaluation using the peak height allows for a high level miniaturization of the channel geometries. To study the main advantages and limitations of this method by numerical simulations, a universally applicable model system is introduced for the first time. Consequently, proof-of-principle experiments were successfully performed with standard and miniaturized versions of an electrochemical biosensor platform utilizing a repressor protein-based assay for tetracycline antibiotics. Herein, the measured current peak heights are the same despite the sextuple reduction of the channel dimensions. Thus, this results in a 22-fold signal amplification compared to the constant flow measurements in the case of the miniaturized version.

Electrochemical characteristics of nanostructured platinum electrodes--a cyclic voltammetry study.

Platinum surfaces play a decisive role in catalysis in sensors, fuel cells, solar cells and other applications like neuronal stimulation and recording. Technical advances in nanotechnology contributed tremendously to the progress in these fields. A fundamental understanding of the chemical and physical interactions between the nanostructured surfaces and electrolytes is essential, but was barely investigated up to now. In this article, we present a wet-chemical process for the deposition of nanostructures on polycrystalline platinum surfaces. The electrochemically active surface area was increased by a factor of over 1000 times with respect to the geometrical surface. The influence of the nanostructures was examined in different acidic, alkaline, and neutral electrolytes. Comparing cyclic voltammograms of nanostructured and planar polycrystalline platinum revealed new insights into the microenvironment at the electrode-electrolyte interface. The characteristic features of the cyclic voltammograms were altered in their shape and strongly shifted with respect to the applied potential. In neutral buffered and unbuffered electrolytes the water window was expanded from 1.4 V to more than 2 V. The shifts were interpreted as local pH-changes and exhausted buffer capacity in direct proximity of the electrode surface due to the strong release and binding of protons, respectively. These polarized electrodes induce significant changes in the electrochemical potential of the electrolyte due to the high roughness of their surface. The electrochemical phenomena and the observed voltage shifts are crucial for the understanding of the basic mechanism at nanostructured electrodes and mandatory for designing fuel cells, sensors and many other devices.

Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

Prolongation of electrode lifetime in biofuel cells by periodic enzyme renewal.

Enzymatically catalyzed biofuel cells show unique specificity and promise high power densities, but suffer from a limited lifetime due to enzyme deactivation. In the present work, we demonstrate a novel concept to extend the lifetime of a laccase-catalyzed oxygen reduction cathode in which we decouple the electrode lifetime from the limited enzyme lifetime by a regular resupply of fresh enzymes. Thereto, the adsorption behavior of laccase from Trametes versicolor to buckypaper electrode material, as well as its time-dependent deactivation characteristics, has been investigated. Laccase shows a Langmuir-type adsorption to the carbon nanotube-based buckypaper electrodes, with a mean residence time of 2 days per molecule. In a citrate buffer of pH 5, laccase does not show any deactivation at room temperature for 2 days and exhibits a half-life of 9 days. In a long-term experiment, the laccase electrodes were operated at a constant galvanostatic load. The laccase-containing catholyte was periodically exchanged against a freshly prepared one every second day to provide sufficient active enzymes in the catholyte for the replacement of desorbed inactive enzymes. Compared to a corresponding control experiment without catholyte exchange, this procedure resulted in a 2.5 times longer cathode lifetime of 19 ± 9 days in which the electrode showed a potential above 0.744 V vs. normal hydrogen electrode at 110 µA cm(-2). This clearly indicates the successful exchange of molecules by desorption and re-adsorption and is a first step toward the realization of a self-regenerating enzymatic biofuel cell in which enzyme-producing microorganisms are integrated into the electrode to continuously resupply fresh enzymes.

Multichannel Cell Detection in Microcompartments by Means of True Parallel Measurements using the Solartron S-1260.

Designing proper frontend electronics is critical in the development of highly sophisticated electrode systems. Multielectrode arrays for measuring electrical signals or impedance require multichannel readout systems. Even more challenging is the differential or ratiometric configuration with simultaneous assessment of measurement and reference channels. In this work, an eight-channel frontend was developed for contacting a 2×8 electrode array (8 measurement and 8 reference electrodes) with a large common electrode to the impedance gain-phase analyzer Solartron 1260 (S-1260). Using the three independent and truly parallel monitor channels of the S-1260, impedance of trapped cells and reference material was measured at the same time, thereby considerably increasing the performance of the device. The frontend electronics buffers the generator output and applies a potentiostatic signal to the common electrode of the chip. The applied voltage is monitored using the current monitor of the S-1260 via voltage/current conversion. The frontend monitors the current through the electrodes and converts it to a voltage fed into the voltage monitors of the S-1260. For assessment of the 8 electrode pairs featured by the chip, a relay-based multiplexer was implemented. Extensive characterization and calibration of the frontend were carried out in a frequency range between 100 Hz and 1 MHz. Investigating the influence of the multiplexer and the frontend electronics, direct measurement with and without frontend was compared. Although differences were evident, they have been negligible below one per cent. The significance of measurement using the complex S-1260-frontend-electrode was tested using Kohlrausch's law. The impedance of an electrolytic dilution series was measured and compared to the theoretical values. The coincidence of measured values and theoretical prediction serves as an indicator for electrode sensitivity to cell behavior. Monitoring of cell behavior on the microelectrode surface will be shown as an example.

Percolation limit and stability conditions for the spin glass state in the spinel families based on the two matrices CuCr(2)S(4) and CuCr(2)Se(4) doped by Sb ions.

The percolation limit of the appearance of the spin glass state in the compounds under study has been obtained experimentally. The conditions of stability of the spin glass state have been analyzed here on the basis of the de Almeida-Thouless theory for two spinels differing in the magnetic coupling constants. It turned out that for the higher value of the coupling constant the magnetic field influences the freezing temperature more strongly. Moreover, the greater the coupling constant the broader the range of the possible values of freezing temperatures, in other words the greater the temperature range of the appearance of the spin glass states. It was proved that for the stability of the spin glass state the existence of a small magnetic field is necessary. In our case the value of this field is equal to 3.46 × 10(-23)T(G). For the compounds under study the value of the magnetic coupling constant J cannot exceed 130 K for the spin glass state to appear.

Electrochemical Stability of Thin-Film Platinum as Suitable Material for Neural Stimulation Electrodes.

Only thin-film technology can satisfy the requirements of high spatial selectivity at high-channel-count electrode array designs by simultaneously good conformability to the targeted tissue through mechanical flexibility enriching future applications of functional neural stimulation. However, caused by the high impact of the microstructure on the mechanical and electrochemical film properties, varying fabrication processes of the same thin-film makes the difference between acute and chronic long-term stable electrodes. The influence of standard clinical electrical pulsing on flexible polyimide-based thin-film platinum electrodes for neuroprostheses, either sputter deposited or evaporated, and different diameters was assessed and compared. The electrochemical and morphological analysis showed a higher corrosion susceptibility and electrochemical degradation for the sputter deposited platinum electrodes with even total failures of smaller diameters. In contrast, the evaporated thin-films provided itself as more stable and reliable metallization with also smaller electrodes keeping their film integrity intact over the experimental period, -appearing to be the preferable material for improving thin-film electrodes' longevity.

A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction.

To improve detection sensitivity, molecular diagnostics require preconcentration of low concentrated samples followed by rapid nucleic acid extraction. This is usually achieved by multiple centrifugation, lysis and purification steps, for instance, using chemical reagents, spin columns or magnetic beads. These require extensive infrastructure as well as time consuming manual handling steps and are thus not suitable for point of care testing (POCT). To overcome these challenges, we developed a microfluidic chip combining free-flow electrophoretic (FFE) preconcentration (1 ml down to 5 µl) and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis. The integration of these techniques in a single chip is unique and enables fast, easy and space-saving sample pretreatment without the need for laboratory facilities, making it ideal for the integration into small POCT devices. A preconcentration efficiency of nearly 100% and a lysis/gel-electrophoresis efficiency of about 65% were achieved for the detection of E. coli. The genetic material was analyzed by RT-qPCR targeting the superfolder Green Fluorescent Protein (sfGFP) transcripts to quantify mRNA recovery and qPCR to determine DNA background.

Correction: A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction.

[This corrects the article DOI: 10.1039/C8RA02177E.].

Platinum nanowires anchored on graphene-supported platinum nanoparticles as a highly active electrocatalyst towards glucose oxidation for fuel cell applications.

The limited performance of platinum-based electrocatalysts for glucose electrooxidation is a major concern for glucose fuel cells, since glucose electrooxidation is characterized by slow reaction kinetics and low diffusion coefficient. Here, the presented graphene-supported platinum-based hierarchical nanostructures attain highly enhanced electrocatalytic activity towards glucose oxidation. Platinum nanoparticles electrodeposited on graphene support retain mechanical stability and act as junctions allowing a reliable, smooth and dense growth of platinum nanowires with extremely small diameters (>10 nm) on graphene. The electrode's surface roughness was increased by factors up to 4000 to the geometrical surface area enabling maximized exploitation of the electrocatalytic activity of platinum and efficient electron transfer between nanowires and the substrate. The unique three-dimensional geometry of these hierarchical nanostructures has a significant impact on their catalytic performance offering short diffusional paths for slow glucose species, thus, mass transport limitations are optimized leading to lower polarization losses. This was examined by galvanostatic measurements of the operation as anodes in glucose half-cells under conditions corresponding to implantable glucose fuel cells. The presented hierarchical nanostructures show remarkably enhanced catalytic performance for glucose electrooxidation, i.e. a negatively shifted open circuit potential of -580 mV vs. Ag/AgCl, hence, representing appropriate electrocatalysts for use as anodes in glucose fuel cells. In combination with a non-metal N-doped graphene cathode, a cell potential of 0.65 V was achieved at a galvanostatic load of 17.5 µA cm-2 which noticeably surpasses the performance of state of the art catalysts for the aforementioned operation conditions.

Clinical on-site monitoring of ß-lactam antibiotics for a personalized antibiotherapy.

An appropriate antibiotherapy is crucial for the safety and recovery of patients. Depending on the clinical conditions of patients, the required dose to effectively eradicate an infection may vary. An inadequate dosing not only reduces the efficacy of the antibiotic, but also promotes the emergence of antimicrobial resistances. Therefore, a personalized therapy is of great interest for improved patients' outcome and will reduce in long-term the prevalence of multidrug-resistances. In this context, on-site monitoring of the antibiotic blood concentration is fundamental to facilitate an individual adjustment of the antibiotherapy. Herein, we present a bioinspired approach for the bedside monitoring of free accessible ß-lactam antibiotics, including penicillins (piperacillin) and cephalosporins (cefuroxime and cefazolin) in untreated plasma samples. The introduced system combines a disposable microfluidic chip with a naturally occurring penicillin-binding protein, resulting in a high-performance platform, capable of gauging very low antibiotic concentrations (less than 6 ng ml-1) from only 1 µl of serum. The system's applicability to a personalized antibiotherapy was successfully demonstrated by monitoring the pharmacokinetics of patients, treated with ß-lactam antibiotics, undergoing surgery.

Nanomolar detection of methylparaben by a cost-effective hemoglobin-based biosensor.

This work describes the development of a new biosensor for methylparaben determination using electrocatalytic properties of hemoglobin in the presence of hydrogen peroxide. The voltammetric oxidation of methylparaben by the proposed biosensor in phosphate buffer (pH=7.0), a physiological pH, was studied and it was confirmed that methylparaben undergoes a one electron-one proton reaction in a diffusion-controlled process. The biosensor was fabricated by carbon paste electrode modified with hemoglobin and multiwalled carbon nanotube. Based on the excellent electrochemical properties of the modified electrode, a sensitive voltammetric method was used for determination of methylparaben within a linear range from 0.1 to 13µmolL(-1) and detection limit of 25nmolL(-1). The developed biosensor possessed accurate and rapid response to methylparaben and showed good sensitivity, stability, and repeatability. Finally, the applicability of the proposed biosensor was verified by methylparaben evaluation in various real samples.

Antibody response to collagen after functional implantation of different polyester vascular prostheses in pigs.

Besides inflammation, specific immune responses are seen also after implantation of biomaterials. The aim was to investigate the humoral response to bovine collagen type I following implantation of various polyester (Dacron) prostheses into pigs. In 24 randomized pigs, the infrarenal aorta was replaced with a segment of collagen-impregnated, woven polyester prosthesis of low, medium, or high porosity. IgG antibodies were detected by immunoassay using native and denatured collagen type I as a target for blood samples taken on day 1 (implantation), 10, 17, 24, 62, and 116. As generally observed, antibodies to native and denatured collagen are of low titer and were significantly correlated with enhanced binding to the denatured form (p < 0.001). The highest overall antibody prevalence to native and denatured collagen was obtained on day 116 with 68% and on day 62 with 59%, respectively. Prostheses with high porosity induced an early immune response on day 10; those with low and medium porosity induced the highest antibody levels later after 2 months. Collagen antibodies neither correlated with serum IgG contents nor with antibodies to the prosthesis polyester matrix. Thus, humoral immune response against implant components may provide a further parameter in describing biocompatibility but also a potential marker that may facilitate monitoring of individual perigraft reaction.

Superoxide microsensor integrated into a Sensing Cell Culture Flask microsystem using direct oxidation for cell culture application.

A new electrochemical sensor system for reliable and continuous detection of superoxide radical release from cell culture was developed utilizing direct oxidation of superoxide on polymer covered gold microelectrodes. Direct superoxide oxidation was demonstrated to provide robust measurement principle for sensitive and selective reactive oxygen species (ROS) quantification without the need for biocomponent supported conversion. Sensor performance was investigated by using artificial enzymatic superoxide production revealing a sensitivity of 2235AM(-1)m(-2). An electrode protection layer with molecular weight cut-off property from adsorbed linear branched polyethylenimine was successfully introduced for long term and selectivity improvement. Thin-film based sensor chip fabrication with implemented three-electrode setup and full integration into the technological platform Sensing Cell Culture Flask was described. Cell culturing directly on-chip and free radical release by phorbol-12-myristate-13-acetate (PMA) stimulation was demonstrated using T-47D human breast cancer carcinoma cell model. Transient extracellular superoxide production upon stimulation was successfully observed from amperometric monitoring. Signal inhibition from scavenging of extracellular superoxide by specific superoxide dismutase (SOD) showed the applicability for selective in vitro ROS determination. The results confirm the possibility of direct superoxide oxidation, with exclusion of the main interfering substances uric acid and hydrogen peroxide. This offers new insights into the development of reliable and robust ROS sensors.

Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms.

In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 µm spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2.

Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.

Serine/threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling.

BACKGROUND: In most cells glucocorticoid receptors (GR) reside predominantly in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR. RESULTS: To further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP. CONCLUSION: These studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

Effects of aerobic exercise and weight loss on riboflavin requirements of moderately obese, marginally deficient young women.

In a previous study, exercise was shown to increase riboflavin requirements of active, normal weight young women. The present study examined the effect of exercise and weight loss on riboflavin status of moderately overweight women. The experiment was designed as a two-period cross-over with an initial base-line period and two 5-wk metabolic periods. The basic diet contained 1200 kcal with a riboflavin concentration of 0.8 mg/1000 kcal. Exercise consisted of a program of dance exercise. Riboflavin depletion, as measured by increased erythrocyte glutathione reductase activity coefficients and decreased urinary excretion of riboflavin, occurred during both nonexercise and exercise periods. Erythrocyte glutathione reductase activity coefficients increased from a base-line mean of 1.28 +/- 0.11 to 1.40 +/- 0.12 during nonexercise and to 1.49 +/- 0.16 during exercise. Urinary excretion of riboflavin fell from 48 +/- 12% of intake during base-line to 30 +/- 13% during nonexercise and to 19 +/- 6% during exercise. Riboflavin depletion was not related to the rate or composition of weight loss or to change in aerobic capacity.

Serotonin syndrome induced by transitioning from phenelzine to venlafaxine: four patient reports.

With the use of the newer antidepressants beyond the traditional tricyclics and monoamine oxidase inhibitors, newer options in headache prophylaxis are provided as well as the potential for undesirable and even potentially life-threatening interactions between medications. In this article, four patient reports of a specific interaction--the serotonin syndrome--are presented. These events resulted from transitioning headache patients from an older antidepressant (phenelzine) to a newer antidepressant (venlafaxine).

Biosensors with modified electrodes for in vivo and ex vivo applications.

Integrated and miniaturized biosensor arrays were developed exhibiting outstanding performance. Biosensors with negligible sensitivity to interferences and high long-term stability were produced by modifying electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producible devices for in vivo and ex vivo applications.