Advanced multichannel submersible probe for autonomous high-resolution in situ monitoring of the cycling of the potentially bioavailable fraction of a range of trace metals.

We report here on the development and application of a submersible, compact, low power consumption, integrated multichannel trace metal sensing probe (TracMetal). This probe is unique in that it allows high-resolution, simultaneous in-situ measurements of the potentially bioavailable (so-called dynamic) fraction of Hg(II), As(III), Cd(II), Pb(II), Cu(II), Zn(II). The TracMetal incorporates nanostructured Au-plated and Hg-plated gel-integrated microelectrode arrays. In addition to be selective to the fraction of metal potentially bioavailable, they offer protection against fouling and ill-controlled convective interferences. Sensitivities in the low pM for Hg(II) and sub-nM for the other target trace metals is achieved with precision ≤ 12%. The TracMetal is capable of autonomous operation during deployment, with routines for repetitive measurements (1-2 h-1), data storage and management, data computer visualization, and wireless data transfer. The system was successfully applied in the Arcachon Bay, to study the temporal variation of the dynamic fraction of the trace metals targeted. The in situ autonomous TracMetal measurements were combined with in situ measurements of the master bio-physicochemical parameters and sample collection for complementary measurements of the dissolved metal concentrations, organic matter concentrations and proxy for biological activities. The integration of all data revealed that various biotic and abiotic processes control the temporal variation of the dynamic fractions of the target metals (Medyn). The difference in the percentage of the dynamic forms of the metals studied and the short-term processes influencing their variation highlight the TracMetal potentiality as metal bioavailability-assessment sentinel to achieve comprehensive environmental monitoring of dynamic aquatic systems.

In Situ Voltammetric Sensor of Potentially Bioavailable Inorganic Mercury in Marine Aquatic Systems Based on Gel-Integrated Nanostructured Gold-Based Microelectrode Arrays.

The development and field validation of newly designed nanostructured gold-plated gel-integrated microelectrode (Au-GIME) arrays applied to the direct in situ square wave anodic stripping voltammetry (SWASV) quantification of the potentially bioavailable inorganic mercury (Hg(II)) species in the coastal area are presented. The Au-GIME consists of arrays of 100-500 interconnected iridium (Ir)-based microdisks that are electroplated with renewable Au nanoparticles (AuNPs) or Au nanofilaments (AuNFs) and covered with an agarose gel. The gel protects the sensor surface from fouling and ensures that mass transport of analytes toward the sensor surface is by pure diffusion only and therefore independent of the ill-controlled convective conditions of the media. The responses of these sensors to direct SWASV measurements of inorganic Hg(II) at near-neutral pH were investigated first in synthetic media and then in UV-irradiated marine samples. The analytical responses were found to be correlated to the number of interconnected microelectrodes and the morphology of the nanostructured Au deposits and independent of the media composition for chloride concentration ≥0.2 M (salinity S ≥ 13) and pH ranging from 7 to 8.5. The AuNF-GIMEs have detection and quantification limits at a low pM level, fulfilling the requirement of sentinel tools for real-time monitoring of the dynamic fraction of Hg(II) in coastal area. The AuNF-GIMEs were incorporated in an in-house advanced multichannel sensing probe for remote in situ high-resolution trace metal monitoring. Field evaluation and validation were successfully performed as a part of a field study in Arcachon Bay (France), from which environmental data are presented. This work marks the first time that an autonomous electrochemical sensing probe successfully measures Hg(II) and its hourly temporal variation in situ without chemical modification of the sample.

Multisite monitoring of choline using biosensor microprobe arrays in combination with CMOS circuitry.

A miniature device enabling parallel in vivo detection of the neurotransmitter choline in multiple brain regions of freely behaving rodents is presented. This is achieved by combining a biosensor microprobe array with a custom-developed CMOS chip. Each silicon microprobe comprises multiple platinum electrodes that are coated with an enzymatic membrane and a permselective layer for selective detection of choline. The biosensors, based on the principle of amperometric detection, exhibit a sensitivity of 157±35 µA mM(-1) cm(-2), a limit of detection of below 1 µM, and a response time in the range of 1 s. With on-chip digitalization and multiplexing, parallel recordings can be performed at a high signal-to-noise ratio with minimal space requirements and with substantial reduction of external signal interference. The layout of the integrated circuitry allows for versatile configuration of the current range and can, therefore, also be used for functionalization of the electrodes before use. The result is a compact, highly integrated system, very convenient for on-site measurements.

Real-time amyloid aggregation monitoring with a photonic crystal-based approach.

We propose the application of a new label-free optical technique based on photonic nanostructures to real-time monitor the amyloid-beta 1-42 (Aß(1-42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer's Disease (AD). The aggregation of Aß peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aß peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real-time monitoring of the refractive index variation of the solution, wherein Aß(1-42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages.

Cocaine detection by a mid-infrared waveguide integrated with a microfluidic chip.

A germanium (Ge) strip waveguide on a silicon (Si) substrate is integrated with a microfluidic chip to detect cocaine in tetrachloroethylene (PCE) solutions. In the evanescent field of the waveguide, cocaine absorbs the light near 5.8 µm, which is emitted from a quantum cascade laser. This device is ideal for (bio-)chemical sensing applications.

Development and validation of a low cost blood filtration element separating plasma from undiluted whole blood.

Clinical point of care testing often needs plasma instead of whole blood. As centrifugation is labor intensive and not always accessible, filtration is a more appropriate separation technique. The complexity of whole blood is such that there is still no commercially available filtration system capable of separating small sample volumes (10-100 µl) at the point of care. The microfluidics research in blood filtration is very active but to date nobody has validated a low cost device that simultaneously filtrates small samples of whole blood and reproducibly recovers clinically relevant biomarkers, and all this in a limited amount of time with undiluted raw samples. In this paper, we show first that plasma filtration from undiluted whole blood is feasible and reproducible in a low-cost microfluidic device. This novel microfluidic blood filtration element (BFE) extracts 12 µl of plasma from 100 µl of whole blood in less than 10 min. Then, we demonstrate that our device is valid for clinical studies by measuring the adsorption of interleukins through our system. This adsorption is reproducible for interleukins IL6, IL8, and IL10 but not for TNFα. Hence, our BFE is valid for clinical diagnostics with simple calibration prior to performing any measurement.

Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell-granule cell loops.

Despite a wealth of in vitro and modelling studies it remains unclear how neuronal populations in the cerebellum interact in vivo. We address the issue of how the cerebellar input layer processes sensory information, with particular focus on the granule cells (input relays) and their counterpart inhibitory interneurones, Golgi cells. Based on the textbook view, granule cells excite Golgi cells via glutamate forming a negative feedback loop. However, Golgi cells express inhibitory mGluR2 receptors suggesting an inhibitory role for glutamate. We set out to test this glutamatergic paradox in Golgi cells. Here we show that granule cells and Golgi cells interact through extra-synaptic signalling mechanisms during sensory information processing, as well as synaptic mechanisms. We demonstrate that such interactions depend on granule cell-derived glutamate acting via inhibitory mGluR2 receptors leading causally to the suppression of Golgi cell activity for several hundreds of milliseconds. We further show that granule cell-derived inhibition of Golgi cell activity is regulated by GABA-dependent extra-synaptic Golgi cell inhibition of granule cells, identifying a regulatory loop in which glutamate and GABA may be critical regulators of Golgi cell­granule cell functional activity. Thus, granule cells may promote their own prolonged activity via paradoxical feed-forward inhibition of Golgi cells, thereby enabling information processing over long timescales.

A novel enzyme entrapment in SU-8 microfabricated films for glucose micro-biosensors.

The present work investigates the utilisation of the widely used SU-8 photoresist as an immobilisation matrix for glucose oxidase (GOx) for the development of glucose micro-biosensors. The strong advantage of the proposed approach is the simultaneous enzyme entrapment during the microfabrication process within a single step, which is of high importance for the simplification of the BioMEMS procedures. Successful encapsulation of the enzyme GOx in "customised" SU-8 microfabricated structures was achieved through optimisation of the one-step microfabrication process. Although the process involved contact with organic solvents, UV-light exposure, heating for pre- and post-bake and enzyme entrapment in a hard and rigid epoxy resin matrix, the enzyme retained its activity after encapsulation in SU-8. Measurements of the immobilised enzyme's activity inside the SU-8 matrix were carried out using amperometric detection of hydrogen peroxide in a 3-electrode setup. Films without enzyme showed negligible variation in current upon the addition of glucose, as opposed to films with encapsulated enzyme which showed a very clear increase in current. Experiments using films of increased thickness or enzyme concentration, showed a higher response, thus proving that the enzyme remained active not only on the film's surface, but also inside the matrix as well. The proposed enzyme immobilisation in SU-8 films opens up new possibilities for combining BioMEMS with biosensors and organic electronics.

Continuous-flow multi-analyte biosensor cartridge with controllable linear response range.

This article presents the design and fabrication of a microfluidic biosensor cartridge for the continuous and simultaneous measurement of biologically relevant analytes in a sample solution. The biosensor principle is based on the amperometric detection of hydrogen peroxide using enzyme-modified electrodes. The low-integrated and disposable cartridge is fabricated in PDMS and SU-8 by rapid prototyping. The device is designed in such a way that it addresses two major challenges of biosensors using microfluidics approaches. Firstly, the enzymatic membrane is deposited on top of the platinum electrodes via a microfluidic deposition channel from outside the cartridge. This decouples the membrane deposition from the cartridge fabrication and enables the user to decide when and with what mixture he wants to modify the electrode. Secondly, by using laminar sheath-flow of the sample and a buffer solution, a dynamic diffusion layer is created. The analyte has to diffuse through the buffer solution layer before it can reach the immobilized enzyme membrane on the electrode. Controlling of the thickness of the diffusion layer by variation of the flow-rate of the two layers enables the user to adjust the sensitivity and the linear region of the sensor. The point where the buffer and sample stream join proved critical in creating the laminar sheath-flow. Results of computational simulations considering fluid dynamics and diffusion are presented. The consistency of the device was investigated through detection of glucose and lactate and are in accordance with the CFD simulations. A sensitivity of 157+/-28 nA/mM for the glucose sensor and 79+/-12 nA/mM for the lactate sensor was obtained. The linear response range of these biosensors could be increased from initially 2 mM up to 15 mM with a limit of detection of 0.2 mM.

Computer simulation and theory of the diffusion- and flow-induced concentration dispersion in microfluidic devices and HPLC systems based on rectangular microchannels.

The dynamics of formation of solute peaks in microfluidic systems are investigated by computer simulation. A finite-element numerical procedure is applied to analyze the diffusion- and flow-controlled concentration dispersion in a 40 microm-high rectangular flow-through channel. Two-dimensional concentration profiles are shown for channels with cross sections of large aspect ratio. The final shapes of the peaks are formed during a very short time period, ranging from a few milliseconds to about 1s for low and high flow velocities, respectively. The observed standard half-width sigma of the peaks is found to strictly follow a linear function of t(1/2) over the whole time range. The extrapolated long-term peak characteristics are in perfect agreement with theoretical predictions. For comparison, theoretical results on the concentration dispersion for solute peaks in open-channel liquid-chromatography (HPLC) are re-examined and applied.

Development and characterization of choline and L-glutamate biosensor integrated on silicon microprobes for in-vivo monitoring.

A silicon microprobe to measure in real-time choline and L-glutamate in brain tissue is described. The biosensors are prepared by coating integrated platinum thin-film electrodes with an enzymatic membrane by electrochemically aided deposition. This method allows a very local and reproducible immobilization of the enzymes and the achievement of high sensitivities as required for in vivo recordings. Moreover, several closely-spaced electrode sites on the same microprobe can be coated with different enzyme membranes without any cross-contamination.