This PDF file contains the front matter associated with SPIE Proceedings Volume 9518, including the Title Page, Copyright information, Table of Contents, Introduction, Authors, and Conference Committee listing.

The first industrial revolution focused on machines, the second one was data-centric - a third revolution combining the power of devices and information has just started and transforms our understanding of life itself. Thereby novel sensors and networks from wearable biometric devices to lab-on-a-chip platforms for exploratory fundamental research on single-biomolecule characterization and design occupy a key role. In combination with recent advances in big data analytics for life sciences, healthcare and genomics such sensors are essential tools for moving from fast and cheap personalized DNA-sequencing via smart genomics towards one-off prevention and treatment plans. Replacing state-of-the-art, one-fits-all approaches, this paradigm shifting individual "assess & response" scheme commonly referred to as precision medicine merges biomedical engineering, systems biology, systems genomics, and information technology. Integrated sensors for isolating, investigating and eventually manipulating single biomolecules are important experimental tools for developing next-generation DNA-sequencing platforms and for conducting ‘omics research which is a defining part of systems biology. In that context resistive pulse sensing has emerged as a powerful technology at the intersection of biotechnology and nanotechnology allowing electrical, label-free screening of biological compounds such as proteins or DNA with single-molecule, single-nucleotide and even single binding site resolution. Resistive pulse sensing technology has been at the center of recent commercial $100Ms investments in the next-generation DNA-sequencing sector. While next-generation sequencing platforms based on resistive pulse sensing techniques will mature further, the technology is also increasingly used for screening other biomolecules such as for example proteins. This allows for developing novel diagnostics and ultra-high throughput pre-clinical drug screening systems which might help to transform the pharma pipeline similarly to how the $1000-genome has revolutionized DNA-sequencing.

A 16-electrode flexible microprobe presenting an array of holes in its sensing area has been developed using SU-8 negative photoresist as a substrate material. The design of the microprobe consisted on six groups of 2-3 electrodes (50 μm in diameter and separated by 200 μm) placed in-between the array of holes. Distances between each group were appropriated for recording from a ferret slice, allowing us to evaluate the close versus the distant network activity. The holes were designed to allow the flow of oxygenated solution through the probe when measuring the neural activity of cortical slides maintained in vitro, while the gold electrodes have been modified with platinum black to decrease its impedance value and, therefore, improve the measurements.

In particular, with this SU-8 microprobe it has been possible to register spontaneous slow oscillations and also induced activity in all 16 channels. The holes were useful to position puffers that allowed us to locally delivered drugs (glutamate) and to register its response. In addition, they were also used for sticking electrodes to simultaneously record single units that allowed us the synchronous recording of population activity (local field potential) with the electrodes of the probe and single unit activity (with glass or tungsten electrodes), demonstrating the feasibility of using SU-8 for the fabrication of neural microprobes as it can be customized to the required design.

This paper presents a low power fully integrated class-E power amplifier and its integration with remotely powered sensor system. The class-E power amplifier is suitable solution for low-power applications due to its high power efficiency. However, the required high inductance values which make the on-chip integration of the power amplifier difficult. The designed power amplifier is fully integrated in the remotely powered sensor system and fabricated in 0.18 μm CMOS process. The power is transferred to the implantable sensor system at 13.56 MHz by using an inductively coupled remote powering link. The induced AC voltage on the implant coil is converted into a DC voltage by a passive full-wave rectifier. A voltage regulator is used to suppress the ripples and create a clean and stable 1.8 V supply voltage for the sensor and communication blocks. The data collected from the sensors is transmitted by on-off keying modulated low-power transmitter at 1.2 GHz frequency. The transmitter is composed of a LC tank oscillator and a fully on-chip class-E power amplifier. An additional output network is used for the power amplifier which makes the integration of the power amplifier fully on-chip. The integrated power amplifier with 0.2 V supply voltage has a drain efficiency of 31.5% at -10 dBm output power for 50 Ω load. The measurement results verify the functionality of the power amplifier and the remotely powered implantable sensor system. The data communication is also verified by using a commercial 50 Ω chip antenna and has 600 kbps data rate at 1 m communication distance.

This paper reports an integrated 64-channel neural recording sensor. Neural signals are acquired, filtered, digitized and compressed in the channels. Additionally, each channel implements an auto-calibration mechanism which configures the transfer characteristics of the recording site. The system has two transmission modes; in one case the information captured by the channels is sent as uncompressed raw data; in the other, feature vectors extracted from the detected neural spikes are released. Data streams coming from the channels are serialized by an embedded digital processor. Experimental results, including in vivo measurements, show that the power consumption of the complete system is lower than 330μW.

Current microfabrication methods are often restricted to two-dimensional (2D) or two and a half dimensional (2.5D) structures. Those fabrication issues can be potentially addressed by emerging additive manufacturing technologies. Despite rapid growth of additive manufacturing technologies in tissue engineering, microfluidics has seen relatively little developments with regards to adopting 3D printing for rapid fabrication of complex chip-based devices. This has been due to two major factors: lack of sufficient resolution of current rapid-prototyping methods (usually >100 μm ) and optical transparency of polymers to allow in vitro imaging of specimens. We postulate that adopting innovative fabrication processes can provide effective solutions for prototyping and manufacturing of chip-based devices with high-aspect ratios (i.e. above ration of 20:1). This work provides a comprehensive investigation of commercially available additive manufacturing technologies as an alternative for rapid prototyping of complex monolithic Lab-on-a-Chip devices for biological applications. We explored both multi-jet modelling (MJM) and several stereolithography (SLA) processes with five different 3D printing resins. Compared with other rapid prototyping technologies such as PDMS soft lithography and infrared laser micromachining, we demonstrated that selected SLA technologies had superior resolution and feature quality. We also for the first time optimised the post-processing protocols and demonstrated polymer features under scanning electronic microscope (SEM). Finally we demonstrate that selected SLA polymers have optical properties enabling high-resolution biological imaging. A caution should be, however, exercised as more work is needed to develop fully bio-compatible and non-toxic polymer chemistries.

This paper presents the design and realization of a compact, portable and cost effective microfluidic system for isolation and detection of rare circulating tumor cells (CTCs) in suspension. The innovative aspect of the proposed isolation method is that it utilizes superparamagnetic particles (SMPs) to label CTCs and then isolate those using microtraps with integrated current carrying microconductors. The magnetically labeled and trapped CTCs can then be detected by integrated magnetic microsensors e.g. giant magnetoresistive (GMR) or giant magnetoimpedance (GMI) sensors. The channel and trap dimensions are optimized to protect the cells from shear stress and achieve high trapping efficiency. These intact single CTCs can then be used for additional analysis, testing and patient specific drug screening. Being able to analyze the CTCs metastasis-driving capabilities on the single cell level is considered of great importance for developing patient specific therapies. Experiments showed that it is possible to capture single labeled cells in multiple microtraps and hold them there without permanent electric current and magnetic field.

We present an infrared biopsymeter to assist pathologists in the diagnosis of melanoma presence in skin biopsies. The designed and realized system combines the features of visual inspection and physical sensing to reduce false positives and false negatives occurring during standard histopathological analyses. The biopsymeter determines the CH₂-stretch ratio by infrared absorbance measurements of skin biopsies. Investigations conducted with the biopsymeter shows that malignant melanomas and melanoma metastases have higher CH₂-stretch ratio values compared to healthy skin tissues.

A new process for implementing vertical metal nanotubes on a multi-electrode array biosensor is described here. The nanotubes can be fabricated on bulk quartz in precise arrangements and with a high degree of freedom in regards of size, aspect ratio and pitch; furthermore they can be aligned with the electrodes. The nanotubes show good biocompatibility with cultured primary neurons and allow to record extracellular neuronal activity. Moreover, the metal nanotubes present plasmonic features such as high electromagnetic field enhancement and localization; these properties are used to combine spectroscopic analysis to electro-physiological recordings.

We present a method to graft a layer of poly-ethylene-glycol (PEG) to the surface of stereo-lithography fabricated or 3D-printed microfluidic devices rendering it hydrophilic and repellent to the adhesion of proteins. The PEG forms a rigid bond with the surface that is more stable than many coatings or surface treatments. This makes stereolithography much more attractive as a prototyping platform for microfluidics. The method has been proven with two different resins by different manufacturers, showing the universality of said treatment.

Enumerating and analyzing circulating tumor cells (CTCs)—cells that have been shed from primary solid tumors—can potentially be used to determine patient prognosis and track the progression of disease. There is a great challenge to create an effective platform that can isolate these cells, as they are extremely rare: only 1-10 CTCs are present in a 7.5mL of a cancer patient’s peripheral blood. We have developed a novel microfluidic system that can isolate CTC populations label free. Our system consists of a multistage separator that employs inertial migration to sort cells based on size. We demonstrate the feasibility of our device by sorting colloids that are comparable in size to red blood cells (RBCs) and CTCs.

Changes in behavioral traits exhibited by small aquatic invertebrates are increasingly postulated as ethically acceptable and more sensitive endpoints for detection of water-born ecotoxicity than conventional mortality assays. Despite importance of such behavioral biotests, their implementation is profoundly limited by the lack of appropriate biocompatible automation, integrated optoelectronic sensors, and the associated electronics and analysis algorithms. This work outlines development of a proof-of-concept miniaturized Lab-on-a-Chip (LOC) platform for rapid water toxicity tests based on changes in swimming patterns exhibited by Artemia franciscana (Artoxkit M™) nauplii. In contrast to conventionally performed end-point analysis based on counting numbers of dead/immobile specimens we performed a time-resolved video data analysis to dynamically assess impact of a reference toxicant on swimming pattern of A. franciscana. Our system design combined: (i) innovative microfluidic device keeping free swimming Artemia sp. nauplii under continuous microperfusion as a mean of toxin delivery; (ii) mechatronic interface for user-friendly fluidic actuation of the chip; and (iii) miniaturized video acquisition for movement analysis of test specimens. The system was capable of performing fully programmable time-lapse and video-microscopy of multiple samples for rapid ecotoxicity analysis. It enabled development of a user-friendly and inexpensive test protocol to dynamically detect sub-lethal behavioral end-points such as changes in speed of movement or distance traveled by each animal.

Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. LOC systems integrate and scale down one or several laboratory functions on a single chip of a few mm² to cm² in size, and account for many advantages on biochemical analyses, such as low sample and reagent consumption, low cost, reduced analysis time, portability and point-of-need compatibility. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. Here, we present a comparison between static chamber and continuous flow miniaturized PCR devices, in terms of energy consumption for devices fabricated on the same material stack, with identical sample volume and channel dimensions. The comparison is implemented by a computational study coupling heat transfer in both solid and fluid, mass conservation of species, and joule heating. Based on the conclusions of this study, we develop low-cost and fast DNA amplification devices for both PCR and isothermal amplification, and we implement them in the detection of mutations related to breast cancer. The devices are fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zones necessary for PCR or isothermal amplification methods.

Advances in diagnostic technologies enabled scientists to link a large number of diseases with structural changes of the intracellular organisation. This intrinsic biophysical characteristic opened up the possibility to perform clinical assessments based on the measurement of single-cell mechanical properties. In this work, we combine microfluidics, high speed imaging and computational automatic tracking to measure the single-cell deformability of large samples of prostate cancer cells at a rate of ~ 10⁴cells/s. Such a high throughput accounts for the inherent heterogeneity of biological samples and enabled us to extract statistically meaningful signatures from each cell population. In addition, using our technique we investigate the effect of Latrunculin A to the cellular stiffness.

The increasing demand of enhanced sensitivity in the detection of various biochemical analytes paves the way for the development of a new generation of biosensors. Label free multianalyte immunosensing methods utilizing photonic probing have proven to result in better sensitivity and reliability than other types of biosensing methods. Here we described a monolithic silicon optoelectronic transducer capable of label-free and multianalyte determinations. The transducer includes ten Mach-Zehnder interferometers each of which is coupled to its own broad band light emitting device. The adlayers on the sensing arm cause spectral shifts detected at the output of the interferometers by coupling a portable spectrometer through an external fiber. Fourier transform techniques are employed to determine with a high degree of accuracy the shifts of the sinusoidal spectral outputs. The microphotonic chip was integrated with a microfluidic module and a model binding assay (mouse-antimouse) was run to demonstrate the operation.

We present a microfluidic system suitable for parallel label-free detection of several biomarkers utilizing a compact imaging measurement system. The microfluidic system contains a filter unit to separate the plasma from human blood and a functionalized, photonic crystal slab sensor chip. The nanostructure of the photonic crystal slab sensor chip is fabricated by nanoimprint lithography of a period grating surface into a photoresist and subsequent deposition of a TiO₂ layer. Photonic crystal slabs are slab waveguides supporting quasi-guided modes coupling to far-field radiation, which are sensitive to refractive index changes due to biomarker binding on the functionalized surface. In our imaging read-out system the resulting resonance shift of the quasi-guided mode in the transmission spectrum is converted into an intensity change detectable with a simple camera. By continuously taking photographs of the sensor surface local intensity changes are observed revealing the binding kinetics of the biomarker to its specific target. Data from two distinct measurement fields are used for evaluation. For testing the sensor chip, 1 μM biotin as well as 1 μM recombinant human CD40 ligand were immobilized in spotsvia amin coupling to the sensor surface. Each binding experiment was performed with 250 nM streptavidin and 90 nM CD40 ligand antibody dissolved in phosphate buffered saline. In the next test series, a functionalized sensor chip was bonded onto a 15 mm x 15 mm opening of the 75 mm x 25 mm x 2 mm microfluidic system. We demonstrate the functionality of the microfluidic system for filtering human blood such that only blood plasma was transported to the sensor chip. The results of first binding experiments in buffer with this test chip will be presented.

Microneedles are newly developed biomedical devices, whose advantages are mainly in the non-invasiveness, discretion and versatility of use both as diagnostics and as therapeutics tool. In fact, they can be used both for drugs delivery in the interstitial fluids and for the analysis of the interstitial fluid. In this work we present the preliminary results for two devices based on micro needles in PolyEthylene (Glycol). The first for the drugs delivery includes a membrane whose optical reflected wavelength is related to the concentration of drug. Here, we present our preliminary result in diffusion of drugs between the membrane and the microneedles. The second device is gold coated and it works as electrode for the electrochemical detection of species in the interstitial fluid. A preliminary result in detection of glucose will be shown.

The LiPhos project (EU FP7 Grant Agreement No.: 317916, www.liphos.eu) aims to develop three different biophotonic diagnosis tools (BDTs), based on living photonics, namely: single layer living photonics (SLLP), single cell analysis (SCA); and multi-layer living photonics (MLLP). By Measuring of what we term the Photonic Fingerprint (or PIN), of the cells in such BDTs, should make it is possible to differentiate between healthy and non-healthy cell or tissue states. Moreover, the effect of specific drugs and pro-inflammatory agents could be assessed. This concept is currently being applied to the diagnosis of cardiovascular diseases (CVD).

In this work, we present the use of interdigitated electrodes (IDEs) for performing electrical impedance spectroscopy (EIS) measurements to monitor a microfluidic blood brain barrier model. In particular, an electrode configuration which would not impair the optical visualization of the cell culture is proposed. Numerical studies have been performed to evaluate the electrical impedance sensitivity of the proposed tetrapolar configuration along the cell barrier in a given microfluidic chamber geometry. The system has been validated using a home-made cyclo olefin polymer (COP) bioreactor and perforated poly (methyl methacrylate) (PMMA) sheets with different pore densities in order to simulate different cell barrier impedances.

This article details the process layout required for realizing a three-dimensional arrangement of electrodes in a microfluidic device for field flow fractionation based on dielectrophoresis. The metal electrodes are placed horizontally, in a stair-case arrangement, and pass through the bulk of the fluid. Several standard microfabrication processes are employed, in realizing this microdevice, including multi-layer photolithography, casting and plasma bonding. Thus the process layout is repeatable and reproducible. The feasibility of this process layout is demonstrated using three electrodes arranged in aforementioned manner; nevertheless, this process can be extended to as many electrodes as desired in the horizontal direction. This process layout can will make applications possible that were not possible till date due to the inability in microfabricating three-dimensional horizontal metal electrodes that run through the entire width of the microchannel.

The artificial cochlear implant is the only way how to get lost hearing back in some cases. Existing artificial cochlear devices use two separated parts for this purpose: a signal processing unit with transmitter and an implantable receiver with electrodes. This approach is applicable but not fully implantable. A new complex approach to design of a fully implantable artificial cochlea is described in this article.

The proposed artificial cochlea consists of many subcircuits which have to be designed in close context to reach optimal performance and the lowest power consumption. Power consumption should be decreased to a value which allows using cochlear implant as a zero-powered system. A combination of micro-mechanized diaphragm filter bank, possible energy harvesting power source and especially ultra-low power processing electronics is presented in this article. A unique technique for nerve stimulatory output signal generation is discussed. This new technique named charge push-through electronics should use the major part of energy generated by energy harvesting subcircuit for output useful signal generation with minimal undesirable current.

Mechanical parts of the subcircuits were simulated as complex electro-mechanical simulation models in ANSYS, CoventorWare, Matlab and SPICE environment. First, the real energy harvesting power source (human motion and temperature) behavior was measured. The model of this behavior was created in simulation environment and then the whole electronics simulation model for energy harvesting circuits was estimated. Next, signal processing circuits powered from energy harvesting power source were designed and simulated. The new signal processing circuits were simulated in relation to the results of complex electro mechanical diaphragm and SPICE energy harvesting power source simulation.

The future of research in biology and medicine depends on modular tridimensional cell culture platforms with suitable on-demand geometries that can imitate any cell-specific environment with micron-size features. A high level of control of physico-chemical properties of the substrates is critical, as mechanotransduction signals are passed to the cells that sense their environment. The possibility to pattern nanoscale geometries on chip are also leading to better culture results. All these biomimicry parameters influence the cells phenotype, structure and behavior and are now opening new perspectives in 3D cell culturing for basic biology, medicine and drug testing applications. However, this growing need for on-demand 3D platforms is currently limited by two factors: the specificity of the commercial biochips is not suitable for many cell types and the high cost of technology used to design and fabricate custom-made substrates.

In this work, we present the application of a simple, low-cost alternative technique enabling the rapid fabrication of on-demand, custom-made biochips for cell culture with micron-scale resolution. We developed a process that enables the use of low-power, low-cost lasers to etch on-demand micropatterns in transparent biopolymers, circumventing the need for high power lasers or photolithography. We also report the integration of embedded electronics for in situ monitoring or actuation and microchannels on chip. We also succeeded in producing localized carbon nanodomains that are enhancing cell cultures and allowing regionalization in 3D cell culture platforms.

In this work, we present a new miniaturized culture medium based sensor system where we apply an optical reference in an impedance measurement approach for the detection of mold in archives. The designed sensor comprises a chamber with pre-loaded culture medium which promotes the growth of archive mold species. Growth of mold is detected by measuring changes in the impedance of the culture medium caused due to increase in the pH (from 5.5 to 8) with integrated electrodes. Integration of the reference measurement helps in determining the sensitivity of the sensor. The colorimetric principle serves as a reference measurement that indicates a pH change after which further pH shifts can be determined using impedance measurement. In this context, some of the major archive mold species Eurotium amstelodami, Aspergillus penicillioides and Aspergillus restrictus have been successfully analyzed on-chip. Growth of Eurotium amstelodami shows a proportional impedance change of 10 % (12 chips tested) per day, with a sensitivity of 0.6 kΩ/pH unit.

In this work we describe the use of a micro-scale array of polysilicon doubly clamped beams, based on the approach of embedding microfluidic channels inside the resonators, as an innovative platform for multiplexed biosensors. Finite element methods in COMSOL were employed to simulate the structural mechanical behavior and to know the conditions to determine the frequency response in order to achieve optimal sensitivities and quality factors. Particularly, we studied the effect of microchannel cross-section area, length and sidewall thickness respect to the microchannel dimensions with the objective of injecting solutions of different densities. By integrating additional multiphysics models we analyzed the governing microfluidics, and we estimated that a maximum pressure difference of 7 MPa along the microchannels is required to establish an optimum water flow rate of 0.1 μl/min, which is adequate for biosensor applications. To validate the simulations we compared the thermal noise response of a fabricated array of microbridges in air, and we obtained resonant frequencies between 700 KHz and 1 MHz, in good agreement with our simulated results but with downward frequency shifts due to the undercut effect after fabrication.

A label-free compact method for performing photonic characterization of "healthy" versus "diseased" arteries has been developed. It permits the detection of atherosclerotic lesion in living mouse arteries. Using this prototype, we observed that the spectral response (photonic fingerprint, PIN) obtained from aortas of wild-type mice differs from the response of ApoE-KO mice fed with high-fat diet (an atheroprone mouse model). Benchmark of the results against gold standard was performed by staining the aortas with Oil-Red-O to visualize atherosclerotic plaques.

This paper presents a novel microfluidic biosensor for in-vitro detection of biomolecules labeled by magnetic biomarkers (Nanomag-D beads) suspended in a static fluid in combination with giant magnetoresistance (GMR) sensors. While previous studies were focused mainly on exploring the MR change for biosensing of bacteria labeled with magnetic microparticles, we show that our biosensor can be used for the detection of much smaller pathogens in the range of a few hundred nanometers e.g., viruses labeled with Nanomag-D beads (MNPs). For the measurements we also used a novel method for signal acquisition and demodulation. Expensive function generators, data acquisition devices and lock-in amplifiers are substituted by a generic PC sound card and an algorithm combining the Fast Fourier Transform (FFT) of the signal with a peak detection routine. This way, costs are drastically reduced, portability is enabled, detection hands-on time is reduced, and sample throughput can be increased using automation and efficient data evaluation with the appropriate software.

This article deals with the development of a two-dimensional dynamic model for tracking the path of cells subjected to dielectrophoresis, in a continuous flow microfluidic device, for purposes of field-flow fractionation. The nonuniform electric field exists between the top and bottom surface of the microchannel; the top electrode runs over the entire length of the microchannel while the bottom surface of the same holds multiple finite sized electrodes of opposite polarity. The model consists of two governing equations with each describing the movement of the cell in one of the two dimensions of interest. The equations governing of the cell trajectories as well as that of the electric potential inside the microchannel are solved using finite difference method. The model is subsequently used for parametric study; the parameters considered include cell radii, actuation voltage, microchannel height and volumetric flow rate. The model is particularly useful in the design of microfluidic device employing dielectrophoresis for field flow fractionation.

We present a microfluidic chip where droplets of different liquids and sizes can be generated and merged independent of the droplets size and inter-droplet separation. The designed chip consists of two T-junctions located at each side of the long channel and a droplet merging area located at the center of the channel. Our system uses an extra outlet placed before the merging area, which allow the control of the travel time, hitting speed, and merging location of the droplets. From one side of the channel droplets can be generated and stored for several hours. At the other side of the channel droplets containing reagents can be made. Individual droplets generated at each side of the channel can be selected, transferred to the centre location of the channel, and merged with each other. Due to simplicity and efficiency of this droplet merging method, the system can be easily used for on-chip cell analysis.

The development of a low-cost multiparametric platform for enzymatic electrochemical biosensing that can be integrated in a disposable, energy autonomous analytical device is the target of the current work. We propose a technology to fabricate nano-electrodes and ultimately biosensors on flexible polymeric-based substrates (cyclo olefin polymer, and polyimide) using standard microfabrication (step and repeat lithography and lift-off) and rapid prototyping techniques (blade cutting). Our target is towards the fabrication of a miniaturized prototype that can work with small sample volumes in the range of 5-10μL without the need for external pumps for sample loading and handling. This device can be used for the simultaneous detection of metabolites such as glucose, cholesterol and triglycerides for the early diagnosis of diabetes.

A method for characterization of small particles in downstream regime of water suspension is presented. Lowcoherence interferometric technique, based on fiber-optic Mach–Zehnder interferometer (MZI) integrated into the optofluidic platform, is applied for measuring refraction index and size, i.e. diameter of glass particles. Water suspension of glass balls and cylinders of different size (from 50-230μm in diameter) has been involved into the microchannels of the optofluidic platform under laminar flow. Two complementary algorithms have been applied for calculation of index of refraction and diameter of spherical glass parts out of raw interference signals. The accuracy of index of refraction measurement is about 1% that is predominantly determined by the accuracy of reading the position of mechanical scanner.

In-situ integration of microfluidic channels into the microfabrication process flow of implantable microsystems is desirable, for example to enable efficient drug delivery. We propose a fabrication method for such microfluidic channels using parylene C, a biocompatible material whose inert nature favours water flow. A single deposition of parylene C enabled monolithical integration of fully-sealed micro-channels in a silicon substrate. The channel geometry was predefined by etching 100 μm-deep grooves into a silicon substrate. A PVC foil was fixed manually on the wafer and served as a top-cover for the grooves. The wafers were coated with the adhesion promoter AdPro Poly® and a 15 μm-thick parylene C film was deposited conformally into the grooves-foil enclosed space. The outgasing nature of the PVC foil hindered the adhesion of parylene C, allowing the foil to be peeled off easily from the parylene surface. The functionality of the fully-sealed parylene channels, embedded in the silicon wafer, was verified by injecting DI water with dispersed polystyrene microbeads (diameter 6 μm): the polystyrene beads were successfully transported along the channel. Further, a fully-sealed parylene chamber remained leak-tight throughout a stepwise application of hydrostatic pressures from 0.2 to 3.0 bar (15 s step-interval). In short, our parylene channels are: (1) suitable for microsystem drug-delivery; (2) in-situ enclosed hollow spaces embedded in the silicon substrate, realized with a single parylene deposition; (3) intact at hydrostatic pressures up to 3 bar.

Traditional marine ecotoxicity testing is inherently labor intensive, requiring extensive manual procedures both to set up the tests and more importantly to collect experimental readouts. Moreover, static test procedures offer poor control of water parameters such as toxicant concentration and dissolved oxygen, which are important considerations in evaluating environmental impacts of aquatic pollution. So far only minimal levels of automation have been adopted in ecotoxicology. Our current work attempts to address the current limitations by capitalizing on latest advances in microfluidics, 3D printing and laser micromachining technologies to develop highly customized, low cost and high-throughput devices.

Here we for the first time introduce a proof-of-concept laboratory automation system to perform ecotoxicity tests on the marine amphipod Allorchestes compressa in a microfluidic environment. Our innovative system incorporated a microperfusion Lab-on-a-Chip device that enabled the biotests to be run in both closed- or open-loop regimens. Miniaturized video cameras were utilized to monitor the amphipods movement patterns during the experiments. Furthermore innovative video analysis algorithms was applied for detection of sub-lethal endpoints such as changes in swimming activity that would otherwise go unnoticed. A key advantage of this flow-through system as compared to conventional approach is the automation of analysis and emphasis on sub-lethal behavioral parameters.

We present preliminary data validating the technology and compared to a gold standard method for testing organisms from the order Amphipoda This work provides a foundation to enable automation of ecotoxicity biotests performed on marine test organisms.

Non-invasive and real-time visualisation of metabolic activities in living small organisms such as zebrafish embryo and larvae has not yet been attempted due to profound analytical limitations of existing technologies. Significant progress in the development of physico-optical oxygen sensors using luminescence quenching by molecular oxygen has recently been made. Sensing using such microsensors is, however, still performed in small glass chambers that hold single specimens and thus not amenable for high-throughput data acquisition.

In this work, we present a proof-of-concept approach by using microfluidic Lab-on-a-Chip (LOC) technologies combined with sophisticated optoelectronic sensors. The LOC device is capable of immobilising live zebrafish embryos with continuous flow perfusion, while the sensor uses innovative Fluorescence Ratiometric Imaging (FRIM) technology that can kinetically quantify the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. By embedding the sensor foil onto the microfluidic living embryo array system, we demonstrated in situ FRIM on developing zebrafish embryos. Future integration of microfluidic chip-based technologies with FRIM technology represents a noteworthy direction to miniaturise and revolutionise research on metabolism and physiology in vivo.

At present, the major hurdle to widespread deployment of zebrafish embryo and larvae in large-scale drug development projects is lack of enabling high-throughput analytical platforms. In order to spearhead drug discovery with the use of zebrafish as a model, platforms need to integrate automated pre-test sorting of organisms (to ensure quality control and standardization) and their in-test positioning (suitable for high-content imaging) with modules for flexible drug delivery. The major obstacle hampering sorting of millimetre sized particles such as zebrafish embryos on chip-based devices is their substantial diameter (above one millimetre), mass (above one milligram), which both lead to rapid gravitational-induced sedimentation and high inertial forces. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator’s fatigue. In this work, we present an innovative design of a micromechanical large particle in-flow sorter (MILPIS) capable of analysing, sorting and dispensing living zebrafish embryos for drug discovery applications. The system consisted of a microfluidic network, revolving micromechanical receptacle actuated by robotic servomotor and opto-electronic sensing module. The prototypes were fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining. Elements of MILPIS were also fabricated in an optically transparent VisiJet resin using 3D stereolithography (SLA) processes (ProJet 7000HD, 3D Systems). The device operation was based on a rapidly revolving miniaturized mechanical receptacle. The latter function was to hold and position individual fish embryos for (i) interrogation, (ii) sorting decision-making and (iii) physical sorting..The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers embedded in the system. Digital oscilloscope were used to distinguish the diffraction signals from IR sensors when both LIVE and DEAD embryos were flow through in the chip. Image process analysis were also used as detection module to track DEAD embryos as it flowed in the channel.