PoCOsteo: generic novel platform for bone turnover marker measurement & monitoring.

Despite the increasing efforts in improving bone health assessments, current diagnostics suffer from critical shortcomings. The present article therefore describes a multiplex label-free immunosensor designed and validated for the assessment of two bone turnover markers (BTMs), namely beta isomerized C-terminal telopeptide of type I collagen (CTx) and Procollagen I Intact N-Terminal (PINP), the combination of which is needed to illustrate an accurate overview of bone health. The immunosensor was then tested outside and inside of a microsystem, with the aim of becoming compatible with a point of care system fabricated for automated assessment of these biomarkers later-on at patient side. Custom-made monoclonal antibodies were specifically designed for this purpose in order to guarantee the selectivity of the immunosensor. In the final platform, a finger prick blood sample is introduced into the microfluidic manifolds without any need for sample preparation step, making the tool suitable for near patient and outside of the central laboratory applications. The platform was exploited in 30 real blood samples with the results validated using electrochemiluminescence immunoassay. The results revealed the platform was capable of measuring the target analyte with high sensitivity and beyond the recommended clinical reference range for each biomarker (CTx: 104-1028 ng L-1 and PINP: 16-96 µg L-1, correspondingly). They also showed the platform to have a limit of detection of 15 (ng L-1) and 0.66 (µg L-1), a limit of quantification of 49 (ng L-1) and 2.21 (µg L-1), and an inter- and intra-assay coefficient of variance of 5.39-6.97% and 6.81-5.37%, for CTx and PINP respectively, which is comparable with the gold standard. The main advantage of the platform over the state-of-the art was the capability of providing the results for two markers recommended for assessing bone health within 15 minutes and without the need for skilled personnel or costly infrastructure.

Multifunctional Poly(2-ethyl-2-oxazoline) Copolymers Containing Dithiolane and Pentafluorophenyl Esters as Effective Reactive Linkers for Gold Surface Coatings.

Surface functionalization with biological macromolecules is an important task for the development of sensor materials, whereby the interaction with other biological materials should be suppressed. In this work, we developed a novel multifunctional poly(2-ethyl-2-oxazoline)-dithiolane conjugate as a versatile linker for gold surface immobilization of amine-containing biomolecules, containing poly(2-ethyl-2-oxazoline) as antifouling polymer, dithiolane for surface immobilization, and activated esters for protein conjugation. First, a well-defined carboxylic acid containing copoly(2-ethyl-2-oxazoline) was synthesized by cationic ring-opening copolymerization of 2-ethyl-2-oxazoline with a methyl ester-containing 2-oxazoline monomer, followed by postpolymerization modifications. The side-chain carboxylic groups were then converted to amine-reactive pentafluorophenyl (PFP) ester groups. Part of the PFP groups was used for the attachment of the dithiolane moiety, which can efficiently bind to gold surfaces. The final copolymer contained 1.4 mol% of dithiolane groups and 4.5 mol% of PFP groups. The copolymer structure was confirmed by several analytical techniques, including NMR spectroscopy and size-exclusion chromatography. The kinetics of the PFP ester aminolysis and hydrolysis demonstrated significantly faster amidation compared to hydrolysis, which is essential for subsequent protein conjugation. Successful coating of gold surfaces with the polymer was confirmed by spectroscopic ellipsometry, showing a polymer brush thickness of 4.77 nm. Subsequent modification of the coated surfaces was achieved using bovine serum albumin as a model protein. This study introduces a novel reactive polymer linker for gold surface functionalization and offers a versatile polymer platform for various applications including biosensing and surface functionalization.

Methods to Improve Accuracy of Electronic Component Positioning in Thermoformed Electronics.

Three new methods for accurate electronic component positioning for thermoformed electronics are presented in this paper. To maintain the mechanical and electrical properties of printed-ink tracks, prevent deformation and stretching during thermoforming, and ensure reproducibility, the component positioning principle for all three proposed methods is based on keeping the temperature of some regions in the thermoplastic substrate less than the glass transition temperature of the thermoplastic carrier, to keep those regions resistant to plastic deformation. We have verified the accuracy of the different approaches by implementing these methods in a semi-sphere mold for positioning seven LEDs and one printed capacitive touch sensor. We compared the result of our fabrication processes with the typical fabrication process of in-mold electronics (direct printing on a thermoplastic foil and followed by a thermoforming step) and noticed that the sample produced by the typical process had tracks that were randomly stretched, tracks were not in a straight path after thermoforming and they were not electrically conductive. Furthermore, the final 3D position of the components was not reproducible sample by sample. However, with our proposed fabrication methods, the tracks and pads do not deform or expand during thermoforming and are electrically conductive after. Moreover, the round shape of the touch sensor remains the same as in the 2D design. Based on the results of the experiments, it appears that the proposed methods are capable of positioning electronic components with high precision in thermoformed electronics.

Measuring the Flex Life of Conductive Yarns in Narrow Fabric.

Due to constant advancements in materials research, conductive textile-based materials have been used increasingly in textile-based wearables. However, due to the rigidity of electronics or the need for their encapsulation, conductive textile materials, such as conductive yarns, tend to break faster around transition areas than other parts of e-textile systems. Thus, the current work aims to find the limits of two conductive yarns woven into a narrow fabric at the electronics encapsulation transition point. The tests consisted of repeated bending and mechanical stress, and were conducted using a testing machine built from off-the-shelf components. The electronics were encapsulated with an injection-moulded potting compound. In addition to identifying the most reliable conductive yarn and soft-rigid transition materials, the results examined the failure process during the bending tests, including continuous electrical measurements.

Parameter Study on Force Curves of Assembled Electronic Components on Foils during Injection Overmolding Using Simulation.

The integration of assembled foils in injection-molded parts is a challenging step. Such assembled foils typically comprise a plastic foil on which a circuit board is printed and electronic components are mounted. Those components can detach during overmolding when high pressures and shear stresses prevail due to the injected viscous thermoplastic melt. Hence, the molding settings significantly impact such parts' successful, damage-free manufacturing. In this paper, a virtual parameter study was performed using injection molding software in which 1206-sized components were overmolded in a plate mold using polycarbonate (PC). In addition, experimental injection molding tests of that design and shear and peel tests were made. The simulated forces increased with decreasing mold thickness and melt temperature and increasing injection speed. The calculated tangential forces in the initial stage of overmolding ranged from 1.3 N to 7.3 N, depending on the setting used. However, the experimental at room temperature-obtained shear forces at break were at least 22 N. Yet, detached components were present in most of the experimentally overmolded foils. Hence, the shear tests performed at room temperature can only provide limited information. In addition, there might be a peel-like load case during overmolding where the flexible foil might bend during overmolding.

Development of a Microfluidic Chip Powered by EWOD for In Vitro Manipulation of Bovine Embryos.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.

Process Optimization of Injection Overmolding Structural Electronics with Regard to Film Distortion.

The integration of structural electronics in injection-molded parts is a challenging step. The films-comprising of laminated stacks with electronics-are exposed to shear stresses and elevated temperatures by the molten thermoplastic. Hence, molding settings have a significant impact on the successful, damage-free manufacturing of such parts. In this paper, test films with polycarbonate (PC) sheets as outer and two different thermoplastic polyurethanes (TPUs) as middle layers incorporating conductive tracks on a flexible printed circuit board (flexPCB) are manufactured and overmolded with PC. Parameter studies investigating the influence of the melt temperature, mold temperature, injection speed and used TPU layer were performed. The molded parts were inspected visually and compared with a numerical simulation using injection molding software. A shear distortion factor for the TPU layer was derived based on the simulations that linked the shear stresses with the injection time and the softening (melting) of the TPU. The distortion of the films was found to reduce with higher melt temperature, lower mold temperature and faster injection speed. Films using the TPU with the higher melting temperature yielded significantly better results. Moreover, distortion on the films reduced with the increasing distance to the gate and a larger cavity thickness was found to be beneficial. All those relations could be correlated with the shear distortion factor.

A Study on Over-Molded Copper-Based Flexible Electronic Circuits.

Over-molding has been proposed in recent years as an integrated functional flexible circuit board in a plastic part. This method uses the conventional process for film insert technology. Over-molding has attracted significant attention across many industries due to its potential to deliver different electrical functions in a variety of different part geometries, especially in automotive interiors and home appliances. While it has great application potential, manufacturing challenges continue throughout foil fabrication and injection molding. This raises challenges for designers and researchers responsible for maintaining the reliability of such electronic flexible circuits. Therefore, the purpose of this research paper is to improve some of the over-molding process parameters. On 0805 and 1206 over-molded zero-ohm resistors, electrical, mechanical, and failure characterization was performed. Those components were mounted in parallel, perpendicular, and 45° angled arrangements on two different polymer substrates, polyimide (PI) and polyethylene terephthalate (PET) using lead-free solder, low-melt solder, and conductive adhesive paste. Moreover, as an over-molding material, polycarbonate (PC) with medium viscosity was used. The effect of using different mold shapes (corner mold, 2 mm flat mold, and 3 mm flat mold) and injection molding process parameters (injection speeds and melt temperature) was studied.

Electrically-driven handling of gametes and embryos: taking a step towards the future of ARTs.

Electrical stimulation of gametes and embryos and on-chip manipulation of microdroplets of culture medium serve as promising tools for assisted reproductive technologies (ARTs). Thus far, dielectrophoresis (DEP), electrorotation (ER) and electrowetting on dielectric (EWOD) proved compatible with most laboratory procedures offered by ARTs. Positioning, entrapment and selection of reproductive cells can be achieved with DEP and ER, while EWOD provides the dynamic microenvironment of a developing embryo to better mimic the functions of the oviduct. Furthermore, these techniques are applicable for the assessment of the developmental competence of a mammalian embryo in vitro. Such research paves the way towards the amelioration and full automation of the assisted reproduction methods. This article aims to provide a summary on the recent developments regarding electrically stimulated lab-on-chip devices and their application for the manipulation of gametes and embryos in vitro.

In vivoblue light illumination for optogenetic inhibition: effect on local temperature and excitability of the rat hippocampus.

Objective.The blue light-activated inhibitory opsin, stGtACR2, is gaining prominence as a neuromodulatory tool due its ability to shunt-inhibit neurons and is being frequently used inin vivoexperimentation. However, experiments involving stGtACR2 use longer durations of blue light pulses, which inadvertently heat up the local brain tissue and confound experimental results. Therefore, the heating effects of illumination parameters used forin vivooptogenetic inhibition must be evaluated.Approach.To assess blue light (473 nm)-induced heating of the brain, we used a computational model as well as direct temperature measurements using a fiber Bragg grating (FBG). The effects of different light power densities (LPDs) and pulse durations on evoked potentials (EP) recorded from dentate gyrus were assessed. For opsin-negative rats, LPDs between 127 and 636 mW mm-2and pulse durations between 20 and 5120 ms were tested while for stGtACR2 expressing rats, LPD of 127 mW mm-2and pulse durations between 20 and 640 ms were tested.Main results.Increasing LPDs and pulse durations logarithmically increased the peak temperature and significantly decreased the population spike (PS) amplitude and latencies of EPs. For a pulse duration of 5120 ms, the tissue temperature increased by 0.6 °C-3.4 °C. All tested LPDs decreased the PS amplitude in opsin-negative rats, but 127 mW mm-2had comparatively minimal effects and a significant effect of increasing light pulse duration was seen from 320 ms and beyond. This corresponded with an average temperature increase of 0.2 °C-1.1 °C at the recorded site. Compared to opsin-negative rats, illumination in stGtACR2-expressing rats resulted in much greater inhibition of EPs.Significance.Our study demonstrates that light-induced heating of the brain can be accurately measuredin vivousing FBG sensors. Such light-induced heating alone can affect neuronal excitability. Useful neuromodulation by the activation of stGtACR2 is still possible while minimizing thermal effects.

Ex Vivo Generation and Characterization of Human Hyaline and Elastic Cartilaginous Microtissues for Tissue Engineering Applications.

Considering the high prevalence of cartilage-associated pathologies, low self-repair capacity and limitations of current repair techniques, tissue engineering (TE) strategies have emerged as a promising alternative in this field. Three-dimensional culture techniques have gained attention in recent years, showing their ability to provide the most biomimetic environment for the cells under culture conditions, enabling the cells to fabricate natural, 3D functional microtissues (MTs). In this sense, the aim of this study was to generate, characterize and compare scaffold-free human hyaline and elastic cartilage-derived MTs (HC-MTs and EC-MTs, respectively) under expansion (EM) and chondrogenic media (CM). MTs were generated by using agarose microchips and evaluated ex vivo for 28 days. The MTs generated were subjected to morphometric assessment and cell viability, metabolic activity and histological analyses. Results suggest that the use of CM improves the biomimicry of the MTs obtained in terms of morphology, viability and extracellular matrix (ECM) synthesis with respect to the use of EM. Moreover, the overall results indicate a faster and more sensitive response of the EC-derived cells to the use of CM as compared to HC chondrocytes. Finally, future preclinical in vivo studies are still needed to determine the potential clinical usefulness of these novel advanced therapy products.

Testing for Wearability and Reliability of TPU Lamination Method in E-Textiles.

Electronic textiles (e-textiles) and wearable computing have been emerging increasingly during the last decade. Since the market interest and predictions have grown, the research into increasing reliability and durability of wearables and e-textiles is developing rapidly. The washability of different integration methods and resistance to mechanical stress are the main obstacles being tackled. However, the freedom of movement and overall comfort is still often overlooked during the development phase. It is essential to see the e-textile product as a whole and consider several aspects of user experience. This work will focus on developing and improving the thermoplastic polyurethane (TPU) lamination integration method for e-textiles. In the work, a stretchable copper-polyimide based circuit was laminated onto knit fabric using various TPU films and stacks. The study shares measurable characteristics to determine which material assembly and design would ensure the highest durability for the electronics part without losing its original textile softness, flexibility and stretchability.

Technological Challenges in the Development of Optogenetic Closed-Loop Therapy Approaches in Epilepsy and Related Network Disorders of the Brain.

Epilepsy is a chronic, neurological disorder affecting millions of people every year. The current available pharmacological and surgical treatments are lacking in overall efficacy and cause side-effects like cognitive impairment, depression, tremor, abnormal liver and kidney function. In recent years, the application of optogenetic implants have shown promise to target aberrant neuronal circuits in epilepsy with the advantage of both high spatial and temporal resolution and high cell-specificity, a feature that could tackle both the efficacy and side-effect problems in epilepsy treatment. Optrodes consist of electrodes to record local field potentials and an optical component to modulate neurons via activation of opsin expressed by these neurons. The goal of optogenetics in epilepsy is to interrupt seizure activity in its earliest state, providing a so-called closed-loop therapeutic intervention. The chronic implantation in vivo poses specific demands for the engineering of therapeutic optrodes. Enzymatic degradation and glial encapsulation of implants may compromise long-term recording and sufficient illumination of the opsin-expressing neural tissue. Engineering efforts for optimal optrode design have to be directed towards limitation of the foreign body reaction by reducing the implant's elastic modulus and overall size, while still providing stable long-term recording and large-area illumination, and guaranteeing successful intracerebral implantation. This paper presents an overview of the challenges and recent advances in the field of electrode design, neural-tissue illumination, and neural-probe implantation, with the goal of identifying a suitable candidate to be incorporated in a therapeutic approach for long-term treatment of epilepsy patients.

Numerical simulation of a multi-inlet microfluidic device for biosensing purposes in osteoporosis management.

OBJECTIVES: In this paper, the effect of the position of the inlet and outlet microchannels on the flow profile and the geometry of the recognition chamber for sample pre-treatment in an electrochemical biosensor to be used in osteoporosis management were investigated. METHODS: All numerical computation presented in this work were performed using COMSOL Multiphysics and Fluent. Simulation was performed for a three-dimensional, incompressible Navier-Stokes flow and so explicit biphasic volume of fluid (VOF) equations were used. RESULTS: In the designed microfluidic system, a pressure-driven laminar flow with no-slip boundary condition was responsible for fluid actuation through microchannels in a reproducible approach. Based on the simulation results, the number of outlets was increased and the angel through which the inlets and outlets were attached to the microchamber was changed so that the dead volume would be eliminated and the fluid flow trajectory, the velocity field and pressure were evenly distributed across the chamber. The Re number in the inlets was equal to 4.41, suggesting a laminar flow at this site. CONCLUSION: The simulation results along with the fact that the design change was tested using laser ablated tape and a color dye at different steps provided the researchers with the opportunity to study the changes in a fast and accurate but cheap method. The absence of backflow helps with the cross-talk concern in the channels and the lack of bubbles and complete coverage of the chamber helps with a better surface modification and thus better sensing performance.

Stretchability-The Metric for Stretchable Electrical Interconnects.

Stretchable circuit technology, as the name implies, allows an electronic circuit to adapt to its surroundings by elongating when an external force is applied. Based on this, early authors proposed a straightforward metric: stretchability-the percentage length increase the circuit can survive while remaining functional. However, when comparing technologies, this metric is often unreliable as it is heavily design dependent. This paper aims to demonstrate this shortcoming and proposes a series of alternate methods to evaluate the performance of a stretchable interconnect. These methods consider circuit volume, material usage, and the reliability of the technology. This analysis is then expanded to the direct current (DC) resistance measurement performed on these stretchable interconnects. A simple dead reckoning approach is demonstrated to estimate the magnitude of these measurement errors on the final measurement.

Design and Integration of Flexible Sensor Matrix for in Situ Monitoring of Polymer Composites.

Sensory polymer composites are highly desirable for applications such as in situ and real-time production processes and structural health monitoring, and for technologies that include human-machine interfaces for the next generation of Internet of Things. However, the development of these materials is still in its infancy: these materials have been reported, but the large-scale fabrication of polymer composites with versatile and customizable sensing capabilities has yet to be demonstrated. Here, we report on a scalable fabrication strategy that enables such materials by designing and integrating PCB technology-inspired large-area flexible sensor matrices into polymer composites. The integrated sensor matrices successfully monitored in situ the production processes and structural health of an industrial polymer composite: from the application of vacuum, resin flow and polymerization, production defects, and temperature distribution. Our results demonstrate that the proposed strategy is a simple and effective solution as a distributed monitoring platform for polymer composites and shows the potential toward next generation of sensory polymer composites.

Microphysiological flux balance platform unravels the dynamics of drug induced steatosis.

Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15-20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of ß-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.

From Fibrils to Toughness: Multi-Scale Mechanics of Fibrillating Interfaces in Stretchable Electronics.

Metal-elastomer interfacial systems, often encountered in stretchable electronics, demonstrate remarkably high interface fracture toughness values. Evidently, a large gap exists between the rather small adhesion energy levels at the microscopic scale ('intrinsic adhesion') and the large measured macroscopic work-of-separation. This energy gap is closed here by unravelling the underlying dissipative mechanisms through a systematic numerical/experimental multi-scale approach. This self-containing contribution collects and reviews previously published results and addresses the remaining open questions by providing new and independent results obtained from an alternative experimental set-up. In particular, the experimental studies on Cu-PDMS (Poly(dimethylsiloxane)) samples conclusively reveal the essential role of fibrillation mechanisms at the micro-meter scale during the metal-elastomer delamination process. The micro-scale numerical analyses on single and multiple fibrils show that the dynamic release of the stored elastic energy by multiple fibril fracture, including the interaction with the adjacent deforming bulk PDMS and its highly nonlinear behaviour, provide a mechanistic understanding of the high work-of-separation. An experimentally validated quantitative relation between the macroscopic work-of-separation and peel front height is established from the simulation results. Finally, it is shown that a micro-mechanically motivated shape of the traction-separation law in cohesive zone models is essential to describe the delamination process in fibrillating metal-elastomer systems in a physically meaningful way.

Fabrication and Verification of Conjugated AuNP-Antibody Nanoprobe for Sensitivity Improvement in Electrochemical Biosensors.

This study was designed to obtain covalently coupled conjugates as means for achieving higher stability and better coverage of the AuNPs by antibodies on the particle surface suitable for sensor performance enhancement. Starting by using a modified protocol, colloid gold solution, with mean AuNP core size of ~6 nm was synthesized. The protocol used for conjugation of AuNPs to osteocalcin antibody in this study relies on covalent and electrostatic attractions between constituents. Varieties of conjugates with varying combinations of crosslinkers and different concentrations were successfully synthesized. The obtained products were characterized and their properties were studied to determine the best candidate in sense of antibody - antigen reactivity. Using AuNP-GSH-NHS-Ab combination (1:1:1), the tertiary structure of the protein was maintained and thus the antibody remained functional in the future steps. This one-pot method provided a simple method for covalently coupling antibodies on the particle surface while keeping their functionality intact. The AuNP content of the solution also accelerated electron transfer rate and thus amplifies the detection signal. With the developed and discussed technique herein, a simple solution is modeled to be used for measuring serum levels of biomarkers in single and/or multiplexed sensor systems.

A highly sensitive electrochemical biosensor based on AuNP-modified gold electrodes for selective determination of serum levels of crosslaps.

This article explains a step-wise protocol to develop an electrochemical sensor to quantify serum levels of C-telopeptide (CTX) crosslinks also known as crosslaps in a matter of minutes and with high level of accuracy. The technique needs only one-step (incubation) and can thus be used for point of care screening. Due to the excellent electrical properties of the as-prepared immunosensor, CTX levels were successfully measured from 1 to 1000 pg/mL. This is while the normal reference of the marker is 50-450 pg/mL, suggesting that the sensor can acceptably detect CTX. The results also showed a good correlation with ECLIA in measuring serum levels of CTX.