in vivo Monitoring with micro-implantable hypoxia sensor based on tissue acidosis.

Hypoxia is a common medical problem, sometimes difficult to detect and caused by different situations. Control of hypoxia is of great medical importance and early detection is essential to prevent life threatening complications. However, the few current methods are invasive, expensive, and risky. Thus, the development of reliable and accurate sensors for the continuous monitoring of hypoxia is of vital importance for clinical monitoring. Herein, we report an implantable sensor to address these needs. The developed device is a low-cost, miniaturised implantable electrochemical sensor for monitoring hypoxia in tissue by means of pH detection. This technology is based on protonation/deprotonation of polypyrrole conductive polymer. The sensor was optimized in vitro and tested in vivo intramuscularly and ex vivo in blood in adult rabbits with respiration-induced hypoxia and correlated with the standard device ePOCTM. The sensor demonstrated excellent sensitivity and reproducibility; 46.4 ± 0.4 mV/pH in the pH range of 4-9 and the selectivity coefficient exhibited low interference activity in vitro. The device was linear (R2 = 0.925) with a low dispersion of the values (n = 11) with a cut-off of 7.1 for hypoxia in vivo and ex vivo. Statistics with one-way ANOVA (α = 0.05), shows statistical differences between hypoxia and normoxia states and the good performance of the pH sensor, which demonstrated good agreement with the standard device. The sensor was stable and functional after 18 months. The excellent results demonstrated the feasibility of the sensors in real-time monitoring of intramuscular tissue and blood for medical applications.

Non-invasive monitoring of pH and oxygen using miniaturized electrochemical sensors in an animal model of acute hypoxia.

BACKGROUND: One of the most prevalent causes of fetal hypoxia leading to stillbirth is placental insufficiency. Hemodynamic changes evaluated with Doppler ultrasound have been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. As a first step, the present work aimed to evaluate the performance of miniaturized electrochemical sensors in the continuous monitoring of oxygen and pH changes in a model of acute hypoxia-acidosis. METHODS: pH and oxygen electrochemical sensors were evaluated in a ventilatory hypoxia rabbit model. The ventilator hypoxia protocol included 3 differential phases: basal (100% FiO2), the hypoxia-acidosis period (10% FiO2) and recovery (100% FiO2). Sensors were tested in blood tissue (ex vivo sensing) and in muscular tissue (in vivo sensing). pH electrochemical and oxygen sensors were evaluated on the day of insertion (short-term evaluation) and pH electrochemical sensors were also tested after 5 days of insertion (long-term evaluation). pH and oxygen sensing were registered throughout the ventilatory hypoxia protocol (basal, hypoxia-acidosis, and recovery) and were compared with blood gas metabolites results from carotid artery catheterization (obtained with the EPOC blood analyzer). Finally, histological assessment was performed on the sensor insertion site. One-way ANOVA was used for the analysis of the evolution of acid-based metabolites and electrochemical sensor signaling results; a t-test was used for pre- and post-calibration analyses; and chi-square analyses for categorical variables. RESULTS: At the short-term evaluation, both the pH and oxygen electrochemical sensors distinguished the basal and hypoxia-acidosis periods in both the in vivo and ex vivo sensing. However, only the ex vivo sensing detected the recovery period. In the long-term evaluation, the pH electrochemical sensor signal seemed to lose sensibility. Finally, histological assessment revealed no signs of alteration on the day of evaluation (short-term), whereas in the long-term evaluation a sub-acute inflammatory reaction adjacent to the implantation site was detected. CONCLUSIONS: Miniaturized electrochemical sensors represent a new generation of tools for the continuous monitoring of hypoxia-acidosis, which is especially indicated in high-risk pregnancies. Further studies including more tissue-compatible material would be required in order to improve long-term electrochemical sensing.

Micro-needle implantable electrochemical oxygen sensor: ex-vivo and in-vivo studies.

Oxygen is vital for energy metabolism in mammals and the variability of the concentration is considered a clinical alert for a wide range of metabolic malfunctions in medicine. In this article, we describe the development and application of a micro-needle implantable platinum-based electrochemical sensor for measuring partial pressure of oxygen in intramuscular tissue (in-vivo) and vascular blood (ex-vivo). The Pt-Nafion® sensor was characterized morphological and electrochemically showing a higher sensitivity of -2.496 nA/mmHg (-1.495 nA/µM) when comparing with its bare counterpart. Our sensor was able to discriminate states with different oxygen partial pressures (pO2) for ex-vivo (blood) following the same trend of the commercial gas analyzer used as standard. For in-vivo (intramuscular) experiments, since there is not a gold standard for measuring pO2 in tissue, it was not possible to correlate the obtained currents with the pO2 in tissue. However, our sensor was able to detect clear statistical differences of O2 between hyperoxia and hypoxia states in tissue.

Enhanced detection of quantum dots labeled protein by simultaneous bismuth electrodeposition into microfluidic channel.

In this study, we propose an electrochemical immunoassay into a disposable microfluidic platform, using quantum dots (QDs) as labels and their enhanced detection using bismuth as an alternative to mercury electrodes. CdSe@ZnS QDs were used to tag human IgG as a model protein and detected through highly sensitive stripping voltammetry of the dissolved metallic component (cadmium in our case). The modification of the screen printed carbon electrodes (SPCEs) was done by a simple electrodeposition of bismuth that was previously mixed with the sample containing QDs. A magneto-immunosandwich assay was performed using a micromixer. A magnet placed at its outlet in order to capture the magnetic beads used as solid support for the immunoassay. SPCEs were integrated at the end of the channel as detector. Different parameters such as bismuth concentration, flow rate, and incubation times, were optimized. The LOD for HIgG in presence of bismuth was 3.5 ng/mL with a RSD of 13.2%. This LOD was about 3.3-fold lower than the one obtained without bismuth. Furthermore, the sensitivity of the system was increased 100-fold respect to experiments carried out with classical screen-printed electrodes, both in presence of bismuth.

Simple on-plastic/paper inkjet-printed solid-state Ag/AgCl pseudoreference electrode.

A miniaturized, disposable, and low cost Ag/AgCl pseudoreference electrode based on inkjet printing has been developed. Silver ink was printed and chlorinated with bleach solution. The reference electrodes obtained in this work showed good reproducibility and stability during at least 30 min continuous measurement and even after 30 days storage without special care. Moreover, the strategy used in this work can be useful for large scale production of a solid-state Ag/AgCl pseudoreference electrode with different designs and sizes, facilitating the coupling with different electrical/electrochemical microsensors and biosensors.

An integrated phenol 'sensoremoval' microfluidic nanostructured platform.

Phenol is a widely used chemical that for several reasons may be released into the environment and, consequently, its detection and subsequent destruction into the ground and surface waters are of special importance. Herein, a simple lab-on-a-chip (LOC) device based on biocompatible and biodegradable CaCO3- poly(ethyleneimine) (PEI) nanostructured microparticles (MPs) to detect and remove phenolic wastes is proposed. The detection of phenol using a hybrid polydimethylsiloxane (PDMS)/glass chronoimpedimetric microchip and its removal in the same LOC system through the use of an extra CaCO3-PEI MPs microcolumn is achieved. For the first time, the chronoimpedance technique is applied in a LOC system for phenol sensing in a range of 0.01-10 µM achieving the limit of detection (LOD) of 4.64 nM. Moreover, this device shows a high repeatability with a relative standard deviation of 3% which is almost 4 times lower than that for the chronoamperometry technique. This LOC system represents an integrated platform for phenol sensing and removal (sensoremoval) that can be easily fabricated and is of a low cost, disposable and amenable to mass production.

On-chip magneto-immunoassay for Alzheimer's biomarker electrochemical detection by using quantum dots as labels.

Electrochemical detection of cadmium-selenide/zinc-sulfide (CdSe@ZnS) quantum dots (QDs) as labeling carriers in an assay for apolipoprotein E (ApoE) detection has been evaluated. The immunocomplex was performed by using tosylactivated magnetic beads as preconcentration platform into a flexible hybrid polydimethylsiloxane (PDMS)-polycarbonate (PC) microfluidic chip with integrated screen printed electrodes (SPE). All the immunoassay was performed in chip and in flow mode. The sensitive electrochemical detection was obtained by square wave anodic stripping voltammetry. ApoE was evaluated for its potential as biomarker for Alzheimer's disease detection, achieving a limit of detection (LOD) of ~12.5 ng mL(-1) with a linear range from 10 to 200 ng mL(-1) and high accuracy for diluted human plasma.

Nanostructured CaCO3-poly(ethyleneimine) microparticles for phenol sensing in fluidic microsystem.

A new and simple strategy based on nanostructured CaCO3-poly(ethyleneimine) (PEI) microparticles (MPs) for phenol sensing using PDMS/glass fluidic microchip is developed. This fluidic microsystem including integrated screen-printed electrodes modified with CaCO3-PEI MPs and tyrosinase (Tyr) through cross-linking with glutaraldehyde, represents a low-cost platform for phenol detection. The designed fluidic microsystem improves the sensitivity of the biosensor allowing the detection of very low concentrations of phenol (up to 10 nM). This device shows high repeatability and low detection limit, is easy to be fabricated, inexpensive, disposable, and amenable to mass production.

On-chip electrochemical detection of CdS quantum dots using normal and multiple recycling flow through modes.

A flexible hybrid polydimethylsiloxane (PDMS)-polycarbonate (PC) microfluidic chip with integrated screen printed electrodes (SPE) was fabricated and applied for electrochemical quantum dots (QDs) detection. The developed device combines the advantages of flexible microfluidic chips, such as their low cost, the possibility to be disposable and amenable to mass production, with the advantages of electrochemistry for its facility of integration and the possibility to miniaturize the analytical device. Due to the interest in biosensing applications in general and particularly the great demand for labelling alternatives in affinity biosensors, the electrochemistry of cadmium sulfide quantum dots (CdS QDs) is evaluated. Square wave anodic stripping voltammetry (SWASV) is the technique used due to its sensitivity and low detection limits that can be achieved. The electrochemical as well as the microfluidic parameters of the developed system are optimized. The detection of CdS QDs in the range between 50 to 8000 ng mL(-1) with a sensitivity of 0.0009 µA/(ng mL(-1)) has been achieved. In addition to the single in-chip flow through measurements, the design of a recirculation system with the aim of achieving lower detection limits using reduced volumes (25 µL) of sample was proposed as a proof-of-concept.

Fabrication of thermoplastics chips through lamination based techniques.

In this work, we propose a novel strategy for the fabrication of flexible thermoplastic microdevices entirely based on lamination processes. The same low-cost laminator apparatus can be used from master fabrication to microchannel sealing. This process is appropriate for rapid prototyping at laboratory scale, but it can also be easily upscaled to industrial manufacturing. For demonstration, we used here Cycloolefin Copolymer (COC), a thermoplastic polymer that is extensively used for microfluidic applications. COC is a thermoplastic polymer with good chemical resistance to common chemicals used in microfluidics such as acids, bases and most polar solvents. Its optical quality and mechanical resistance make this material suitable for a large range of applications in chemistry or biology. As an example, the electrokinetic separation of pollutants is proposed in the present study.

Nanomaterials and lab-on-a-chip technologies.

Lab-on-a-chip (LOC) platforms have become important tools for sample analysis and treatment with interest for DNA, protein and cells studies or diagnostics due to benefits such as the reduced sample volume, low cost, portability and the possibility to build new analytical devices or be integrated into conventional ones. These platforms have advantages of a wide set of nanomaterials (NM) (i.e. nanoparticles, quantum dots, nanowires, graphene etc.) and offer excellent improvement in properties for many applications (i.e. detectors sensitivity enhancement, biolabelling capability along with other in-chip applications related to the specificities of the variety of nanomaterials with optical, electrical and/or mechanical properties). This review covers the last trends in the use of nanomaterials in microfluidic systems and the related advantages in analytical and bioanalytical applications. In addition to the applications of nanomaterials in LOCs, we also discuss the employment of such devices for the production and characterization of nanomaterials. Both framed platforms, NMs based LOCs and LOCs for NMs production and characterization, represent promising alternatives to generate new nanotechnology tools for point-of-care diagnostics, drug delivery and nanotoxicology applications.

Evaluation of microchip material and surface treatment options for IEF of allergenic milk proteins on microchips.

The use of glass and PDMS microchips has been investigated to perform rapid and efficient separation of allergenic whey proteins by IEF. To decrease EOF and to limit protein adsorption, two coating procedures have been compared. The first one consists in immobilizing hydroxypropyl cellulose (HPC) and the second one poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE). EOF limitation has been evaluated using frontal electrophoresis of a fluorescent marker of known effective mobility. EOF velocity was decreased by a factor about 100 and 30, respectively. pH gradient formation has been evaluated for each microchip using fluorescent pI markers. It was demonstrated that as expected a coating was essential to avoid pH gradient drift. Both coatings were efficient on glass microchips, but only PDMA-AGE allowed satisfying focusing of pI markers on PDMS microchips. Fluorescent covalent and noncovalent labelings of milk proteins have been compared by IEF on slab-gels. IEF separation of three major allergenic whey proteins [beta-lactoglobulin A (pI 5.25) and B (pI 5.35) and alpha-lactalbumin (pI 4.2-4.5)] was performed in both microchips. Milk proteins were separated with better resolution and shorter analysis time than by classical CIEF. Finally, better resolutions for milk allergens separation were obtained on glass microchips.

High-throughput simultaneous detection of point mutations and large-scale rearrangements by CE.

The detection of unknown mutations is important both in population genetics research and in diagnosis. At present, two different methods must be used to detect either point mutations or large-scale genetic rearrangements, which is costly and time-consuming. We describe here a new method for the simultaneous detection of these two types of mutations. It is based on electrophoretic heteroduplex analysis (HDA) using enhanced mismatch mutation analysis (EMMA) and semiquantitative multiplexed PCR conditions. The use of such conditions allows the simultaneous search of any kind of mutation in up to five different fragments per capillary, in a single or multi-CE system. The method was validated on patient samples with mutations in the breast predisposition gene BRCA1. It leads to highly reliable and high-throughput mutation detection at low cost, as compared with classical methods.

Lamination-based rapid prototyping of microfluidic devices using flexible thermoplastic substrates.

Transposing highly sensitive DNA separation methods (such as DNA sequencing with high read length or the detection of point mutations) to microchip format without loss of resolution requires fabrication of relatively long (approx. 10 cm) microchannels along with sharp injection bands. Conventional soft lithography methods, such as mold casting or hot-embossing in a press, are not convenient for fabricating long channels. We have developed a lamination-based replication technique for rapid fabrication of sealed microfluidic devices with a 10 cm long, linear separation channel. These devices are fabricated in thin cyclo-olefin copolymer (COC) plastic substrates, thus making the device flexible and capable of assuming a range of 3-D configurations. Due to the good optical properties of COC, this new family of devices combines multiple advantages of planar microfluidics and fused-silica capillaries.