Validation of HepG2/C3A Cell Cultures in Cyclic Olefin Copolymer Based Microfluidic Bioreactors.

Organ-on-chip (OoC) technology is one of the most promising in vitro tools to replace the traditional animal experiment-based paradigms of risk assessment. However, the use of OoC in drug discovery and toxicity studies remain still limited by the low capacity for high-throughput production and the incompatibility with standard laboratory equipment. Moreover, polydimethylsiloxanes, the material of choice for OoC, has several drawbacks, particularly the high absorption of drugs and chemicals. In this work, we report the development of a microfluidic device, using a process adapted for mass production, to culture liver cell line in dynamic conditions. The device, made of cyclic olefin copolymers, was manufactured by injection moulding and integrates Luer lock connectors compatible with standard medical and laboratory instruments. Then, the COC device was used for culturing HepG2/C3a cells. The functionality and behaviour of cultures were assessed by albumin secretion, cell proliferation, viability and actin cytoskeleton development. The cells in COC device proliferated well and remained functional for 9 days of culture. Furthermore, HepG2/C3a cells in the COC biochips showed similar behaviour to cells in PDMS biochips. The present study provides a proof-of-concept for the use of COC biochip in liver cells culture and illustrate their potential to develop OoC.

Critical Study on the Tube-to-Chip Luer Slip Connectors.

Luer slip is one of the gold standards for chip-to-world interface in microfluidics. They have outstanding mechanical and operational robustness in a broad range of applications using water and solvent-based liquids. Still, their main drawbacks are related to their size: they have relatively large dead volumes and require a significant footprint to assure a leak-free performance. Such aspects make their integration in systems with high microchannel density challenging. To date, there has been no geometrical optimization of the Luer slips to provide a solution to the mentioned drawbacks. This work aims to provide the rules toward downscaling the Luer slips. To this effect, seven variations of the Luer slip male connectors and five variations of Luer slip female connectors have been designed and manufactured focusing on the reduction of the size of connectors and minimization of the dead volumes. In all cases, female connectors have been developed to pair with the corresponding male connector. Characterization has been performed with a tailor-made test bench in which the closure force between male and female connectors has been varied between 7.9 and 55 N. For each applied closure force, the test bench allows liquid pressures to be tested between 0.5 and 2.0 bar. Finally, the analysis of a useful life determines the number of cycles that the connectors can withstand before leakage.

Optofluidic systems enabling detection in real samples: A review.

Optofluidics, understood as the synergistic combination between microfluidics and photonics, has been at the forefront of the scientific research due to its outmatching properties: on the one hand, microfluidics allows the handling of minute amounts of liquid samples at the microscale. On the other hand, photonics has proved to outmatch other detection methods (e.g. electrochemistry) in terms of sensitivity and selectivity. From the initial single analyte or spiked samples, currently the technology is mature enough for selective detection of a variety of analytes in raw, complex liquid samples. This will pave the way for the applicability of optofluidic devices for applications in the field or at the point of care. Here, we will revisit the current state of the art of optofluidic and photonic lab-on-a-chip systems for the analysis of real and biologically relevant samples: body fluids and water.

Disease-Relevant Single Cell Photonic Signatures Identify S100ß Stem Cells and their Myogenic Progeny in Vascular Lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100ß+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- ß1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100ß+ vSCs and identified the presence of S100ß+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.

Ultrasensitive Photonic Microsystem Enabling Sub-micrometric Monitoring of Arterial Oscillations for Advanced Cardiovascular Studies.

Cardiovascular diseases are the first cause of death globally. Their early diagnosis requires ultrasensitive tools enabling the detection of minor structural and functional alterations in small arteries. Such analyses have been traditionally performed with video imaging-based myographs, which helped to investigate the pathophysiology of the microvessels. Since new vascular questions have emerged, substantial modifications are necessary to improve the performance of imaging and tracking software, reducing the cost and minimizing the microvessel cleaning and manipulation. To address these limitations, we present a photonic microsystem fabricated in polydimethylsiloxane and integrating micro-optical elements and a lightguide-cantilever for sub-micrometric analysis of small arteries (between 125 and 400 µm of basal diameter). This technology enables simultaneous measurement of arterial distension, stiffness, vasomotion, and heartbeat and without the need for advanced imaging system. The microsystem has a limit of detection of 2 µm, five times lower than video imaging-based myographs, is two times more sensitive than them (0.5 µm/mmHg), reduces variability to half and doubles the linear range reported in these myographs. More importantly, it allows the analysis of intact arteries preserving the integrity and function of surrounding tissues. Assays can be conducted in three configurations according to the surrounding tissue: (i) isolated arteries (in vitro) where the surrounding tissue is partially removed, (ii) non-isolated arteries (in vivo) with surrounding tissue partially removed, and (iii) intact arteries in vivo preserving surrounding tissue as well as function and integrity. This technology represents a step forward in the prediction of cardiovascular risk.

Microfluidic-controlled optical router for lab on a chip.

In multiplexed analysis, lab on a chip (LoC) devices are advantageous due to the low sample and reagent volumes required. Although optical detection is preferred for providing high sensitivity in a contactless configuration, multiplexed optical LoCs are limited by the technological complexity for integrating multiple light sources and detectors in a single device. To address this issue, we present a microfluidic-controlled optical router that enables measurement in four individual optical channels using a single light source and detector, and without movable parts. The optofluidic device is entirely fabricated in polydimethylsiloxane (PDMS) by soft-lithography, compatible with standard microfabrication technologies, enabling monolithic integration in LoCs. In the device, in-coupled light from an optical fiber is collimated by a polymeric micro-lens and guided through a set of four sequentially connected micro-chambers. When a micro-chamber is filled with water, light is transmitted to the next one. If it is empty of liquid, however, total internal reflection (TIR) occurs at the PDMS-air interface, re-directing the light to the output optical fiber. The router presents high performance, with low cross-talk (<2%) and high switching frequencies (up to 0.343 ± 0.006 Hz), and provides a stable signal for up to 91% of the switching time. With this miniaturized, low-cost, simple and robust design, we expect the current technology to be integrated in the new generation of multiplexed photonic LoCs for biomarker analysis, even at the point of care.

Label-Free Multi Parameter Optical Interrogation of Endothelial Activation in Single Cells using a Lab on a Disc Platform.

Cellular activation and inflammation leading to endothelial dysfunction is associated with cardiovascular disease (CVD). We investigated whether a single cell label-free multi parameter optical interrogation system can detect endothelial cell and endothelial progenitor cell (EPC) activation in vitro and ex vivo, respectively. Cultured human endothelial cells were exposed to increasing concentrations of tumour necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS) before endothelial activation was validated using fluorescence-activated cell sorting (FACS) analysis of inflammatory marker expression (PECAM-1, E-selectin and ICAM-1). A centrifugal microfluidic system and V-cup array was used to capture individual cells before optical measurement of light scattering, immunocytofluorescence, auto-fluorescence (AF) and cell morphology was determined. In vitro, TNF-α promoted specific changes to the refractive index and cell morphology of individual cells concomitant with enhanced photon activity of fluorescently labelled inflammatory markers and increased auto-fluorescence (AF) intensity at three different wavelengths, an effect blocked by inhibition of downstream signalling with Iκß. Ex vivo, there was a significant increase in EPC number and AF intensity of individual EPCs from CVD patients concomitant with enhanced PECAM-1 expression when compared to normal controls. This novel label-free 'lab on a disc' (LoaD) platform can successfully detect endothelial activation in response to inflammatory stimuli in vitro and ex vivo.

Recent trends in capillary electrophoresis for complex samples analysis: A review.

CE has been a continuously evolving analytical methodology since its first introduction in the 1980s of the last century. The development of new CE separation procedures, the coupling of these systems to more sensitive and versatile detection systems, and the advances in miniaturization technology have allowed the application of CE to the resolution of new and complex analytical problems, overcoming the traditional disadvantages associated with this method. In the present work, different recent trends in CE and their application to the determination of high complexity samples (as biological fluids, individual cells, etc.) will be reviewed: capillary modification by different types of coatings, microfluidic CE, and online microextraction CE. The main advantages and disadvantages of the different proposed approaches will be discussed with examples of most recent applications.

Broadcasting photonic lab on a chip concept through a low cost manufacturing approach.

A low cost fabrication process for photonic lab on a chip systems is here proposed. For the implementation of the masters suitable for cast molding fabrication, an inexpensive dry film photoresist, patternable using standard laboratory equipment, is benchmarked against standardized SU-8 masters obtained using UV lithography and systems manufacture in clean room facilities. Results show adequate system fabrication and a comparable performance of the photonic structures for absorbance/extinction measurements.

A simple and fast Double-Flow microfluidic device based liquid-phase microextraction (DF-µLPME) for the determination of parabens in water samples.

A fast double-flow microfluidic based liquid phase microextraction (DF-µLPME) combined with a HPLC-UV procedure using diode array detection has been developed for the determination of the four most widely used parabens: Ethyl 4-hydroxybenzoate (EtP), Propyl 4-hydroxybenzoate (PrP), Butyl 4-hydroxybenzoate (BuP) and IsoButyl 4-hydroxybenzoate (iBuP) in water samples. Parabens have successfully been determined in environmental (lake and river water) samples with excellent clean up, high extraction efficiency and good enrichment factor using double-flow conditions. The microfluidic device consists of two micro-channels, which contain the acceptor and sample solution separated by a flat membrane (support liquid membrane). The sample (0.32mM HCl) and acceptor phase (5.6mM NaOH) are delivered to the µLPME at 10µLmin-1 and 1µLmin-1 flow rate, respectively. The extraction efficiencies are over 84% for all compounds in water samples with enrichment factors within the range of 9-11 and recoveries over 80%. The procedure provides very low detection limits between 1.6 and 3.5µgL-1. The extraction time and the volume required for the extraction are 5min and 50µL, respectively; which are greatly lower compared to any previous extraction procedure for parabens analysis. In addition, this miniaturized DF- µLPME procedure significantly reduces costs compared to not only the existing methods for paraben detection, but also to the existing analytical techniques for sample preparation.

An effective microfluidic based liquid-phase microextraction device (µLPME) for extraction of non-steroidal anti-inflammatory drugs from biological and environmental samples.

In this work, the traditional liquid phase microextraction (LPME) has been miniaturized into a microfluidic device (µLPME) where liquid phase microextraction is combined with an HPLC procedure. This integration enables extraction and determination of acid drugs by µLPME and HPLC, respectively. The analytes selected for the test are five widely used non-steroidal anti-inflammatory drugs (NSAIDs): salicylic acid (SAC), ketoprofen (KTP), naproxen (NAX), diclofenac (DIC) and ibuprofen (IBU). They have successfully been detected in biological (urine and saliva) and environmental (lake and river water) samples with excellent clean up, high extraction efficiency and good enrichment factor under stopped-flow conditions. The µLPME consists of two small channels (acceptor and donor channel) separated by a support liquid membrane and has been implemented to allow a simple membrane replacement an arbitrary number of times. The sample (pH 12) and acceptor phase (pH 1.5) are delivered to the µLPME at 1 µL min-1 flow rate and the extraction is completed after 6 min. Under these conditions, the recoveries obtained in urine samples are over 87% for all compounds. For environmental water analysis, different types of water samples have been analyzed obtaining recoveries over 75% for all compounds. The sample consumption is dramatically decreased (<7 µL) as compared to traditional LPME. This confirms the advantages of the here proposed µLPME when using small volume/high cost samples. Finally, when the acceptor flow is turned off during the extraction time, high enrichment factor significantly increases with the extraction time for all compounds. As an example, the IBU is enriched by a factor of 75 after 25 min extraction consuming only 500 µL of sample.

Continuous Sensing Photonic Lab-on-a-Chip Platform Based on Cross-Linked Enzyme Crystals.

Microfluidics or lab-on-a-chip technology offer clear advantages over conventional systems such as a dramatic reduction of reagent consumption or a shorter analysis time, which are translated into cost-effective systems. In this work, we present a photonic enzymatic lab-on-a-chip reactor based on cross-linked enzyme crystals (CLECs), able to work in continuous flow, as a highly sensitive, robust, reusable, and stable platform for continuous sensing with superior performance as compared to the state of the art. The microreactor is designed to facilitate the in situ crystallization and crystal cross-linking generating enzymatically active material that can be stored for months/years. Thus, and by means of monolithically integrated micro-optics elements, continuous enzymatic reactions can be spectrophotometrically monitored. Lipase, an enzyme with industrial significance for catalyzed transesterification, hydrolysis, and esterification reactions, is used to demonstrate the potential of the microplatforms as both a continuous biosensor and a microreactor for the synthesis of high value compounds.

Plug and measure - a chip-to-world interface for photonic lab-on-a-chip applications.

The integration of detection mechanisms with microfluidics may be one of the most promising routes towards widespread application of Lab-on-a-Chip (LoC) devices. Photonic detection methods like in the so-called Photonic Lab-on-a-Chip (PhLoC) have advantages such as being non-invasive, easy to sterilize and highly sensitive even with short integration times and thus allow in situ monitoring and quantification of biological and chemical processes. The readout of such detection methods usually requires special training of potential users, as in most cases they are confronted with the need of establishing fiber-optics connections to and from the PhLoC and/or rely on the use of complex laboratory equipment. Here, we present a low-cost and robust chip-to-world interface (CWI), fabricated by CO2-laser machining, facilitating the non-expert use of PhLoCs. Fiber-optics with standard SMA-connectors (non-pigtailed) and PhLoCs can be plugged into the CWI without the need for further adjustments. This standardization bestows great versatility on the interface, providing a direct link between PhLoCs and a wide range of light sources and photo-detectors. The ease-of-use of the proposed simple plug mechanism represents a step forward in terms of user-friendliness and may lead PhLoC devices to practical applications.

Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems.

The integration of micro-optical elements with microfluidics leads to the highly promising photonic lab-on-a-chip analytical systems (PhLoCs). In this work, we re-examine the main principles which are underneath the on-chip spectrophotometric detection, approaching the PhLoC concept to a nonexpert audience.

Integrated Photonic Nanofences: Combining Subwavelength Waveguides with an Enhanced Evanescent Field for Sensing Applications.

Photonic nanofences consisting of high aspect ratio polymeric optical subwavelength waveguides have been developed for their application into photonic sensing devices. They are up to millimeter long arrays of 250 nm wide and 6 µm high ridges produced by an advanced lithography process on a silicon substrate enabling their straightforward integration into complex photonic circuits. Both simulations and experimental results show that the overlap of the evanescent fields propagating from each photonic nanofence allows for the formation of an effective waveguide that confines the overall evanescent field within its limits. This permits a high interaction with the surrounding medium which can be larger than 90% of the total guided light intensity (approximately 20000 times larger than the evanescent field of a standard waveguide with equivalent dimensions). In this work, we not only investigate the photonic properties of these structures but also demonstrate their successful integration into a photonic sensor. An absorbance-based sensor for the determination of lead in water samples is therefore achieved by the combination of the photonic nanofences with an ion-sensitive optical membrane. The experimental results for lead detection in water show a sensitivity of 0.102 AU/decade, and a linear range between 10(-6) M and 10(-2) M Pb(II). A detection limit as low as 7.3 nM has been calculated according to IUPAC for a signal-to-noise ratio of 3.

McCLEC, a robust and stable enzymatic based microreactor platform.

A microfluidic chip for cross-linked enzyme crystals (McCLEC) is presented and demonstrated to be a stable, reusable and robust biocatalyst-based device with very promising biotechnological applications. The cost-effective microfluidic platform allows in situ crystallization, cross-linking and enzymatic reaction assays on a single device. A large number of enzymatic reuses of the McCLEC platform were achieved and a comparative analysis is shown illustrating the efficiency of the process and its storage stability for more than one year.

A multiple path photonic lab on a chip for parallel protein concentration measurements.

We propose a PDMS-based photonic system for the accurate measurement of protein concentration with minute amounts of the sample. As opposed to the state of the art approach, in the multiple path photonic lab on a chip (MPHIL), analyte concentration or molar absorptivity is obtained with a single injection step, by performing simultaneous parallel optical measurements varying the optical path length. Also, as opposed to the standard calibration protocol, the MPHIL approach does not require a series of measurements at different concentrations. MPHIL has three main advantages: firstly the possibility of dynamically selecting the path length, always working in the absorbance vs. concentration linear range for each target analyte. Secondly, a dramatic reduction of the total volume of the sample required to obtain statistically reliable results. Thirdly, since only one injection is required, the measurement time is minimized, reducing both contamination and signal drifts. These characteristics are clearly advantageous when compared to commercial micro-spectrophotometers. The MPHIL concept was validated by testing three commercial proteins, lysozyme (HEWL), glucose isomerase (d-xylose-ketol-isomerase, GI) and Aspergillus sp. lipase L (BLL), as well as two proteins expressed and purified for this study, B. cereus formamidase (FASE) and dihydropyrimidinase from S. meliloti CECT41 (DHP). The use of MPHIL is also proposed for any spectrophotometric measurement in the UV-VIS range, as well as for its integration as a concentration measurement platform in more advanced photonic lab on a chip systems.

Characterization of oxygen transfer in vertical microbubble columns for aerobic biotechnological processes.

This paper presents the applicability of a microtechnologically fabricated microbubble column as a screening tool for submerged aerobic cultivation. Bubbles in the range of a few hundred micrometers in diameter were generated at the bottom of an upright-positioned microdevice. The rising bubbles induced the circulation of the liquid and thus enhanced mixing by reducing the diffusion distances and preventing cells from sedimentation. Two differently sized nozzles (21 × 40 µm(2) and 53 × 40 µm(2) in cross-section) were tested. The gas flow rates were adjustable, and the resulting bubble sizes and gas holdups were investigated by image analysis. The microdevice features sensor elements for the real-time online monitoring of optical density and dissolved oxygen. The active aeration of the microdevice allowed for a flexible oxygen supply with mass transfer rates of up to 0.14 s(-1). Slightly higher oxygen mass transfer rates and a better degassing were found for the microbubble column equipped with the smaller nozzle. To validate the applicability of the microbubble column for aerobic submerged cultivation processes, batch cultivations of the model organism Saccharomyces cerevisiae were performed, and the specific growth rate, oxygen uptake rate, and yield coefficient were investigated.

Optofluidic router based on tunable liquid-liquid mirrors.

We present an electrically tunable 1 × 5 optofluidic router for on-chip light routing. The device can redirect light from an optical input channel into five output channels by exploiting total internal reflection (TIR) at a liquid-liquid interface. The liquid-liquid mirrors, demonstrated for the first time, are tuned using integrated electrowetting-on-dielectrics (EWOD) actuators. The router is assembled from two chips fabricated by standard MEMS techniques. Through a combination of microfluidic with micro-optical components on chip, reliable light routing is achieved with switching times of [1.5-3.3] s, efficiencies of coupling into channels of up to 12%, optical cross-talk as low as -24 dB, a required drive voltage of 50 V, and a low power consumption of <5 mW, using a device 12 × 13 × 2 mm(3) in size. The optofluidic approach enables addressing of multiple channels over a broad wavelength range. Such optical routing capabilities are important for lab-on-chip devices focusing on optical spectroscopy, optical detection, or even optical manipulation. When integrated with external light sources and a low-cost disposable photonic lab-on-a-chip, the router could thus lead to novel laboratory measurement systems.

Analysis of the structural integrity of SU-8-based optofluidic systems for small-molecule crystallization studies.

The use of SU-8-based optofluidic systems (OFS) is validated as an affordable and easy alternative to expensive glass device manufacturing for small-molecule crystallization studies and, in comparison with other polymers, able to withstand most organic solvents. A comparison between two identical OFS (using SU-8 and poly(dimethylsiloxane), PDMS) against the 36 most commonly used organic solvents for small-molecule crystallization studies have confirmed both the structural and optical stability of the SU-8, whereas PDMS suffered from unsealing or tearing in most cases. In order to test its compatibility, measurements before and after 24 h of continued exposure against solvents have been pursued. Here, three aspects have been considered: in the macroscale, swelling has been determined by analyzing the variations in the optical path in the OFS. For determining compatibility at microscale, fabricated SU-8 micropatterns were solvent-etched and subsequently characterized by scanning electron microscopy (SEM). Roughness of the polymer has also been studied through atomic force microscopy (AFM) measurements at the nanoscale. Experimental measurements of PDMS swelling were in accordance with previously reported observations, while SU-8 displayed a great stability against all the tested solvents. Through this experimental procedure we also show that the OFS are suitable for real-time, on-chip, UV-vis spectroscopy. Micro- and nanoscale observations did not show apparent corrosion on SU-8 surface. Also, two commonly used carrier fluids for microdroplet generation (FC-70 Fluorinert oil and silicone oil) were also tested against the different solvents with the aim of providing useful information for later microbatch experiments.