In-line Raman imaging of mixing by herringbone grooves in microfluidic channels.

The control over fluid flow achievable in microfluidic devices creates opportunities for applications in many fields. In simple microchannels, flow is purely laminar when one solvent is used, and hence, achieving reliable mixing is an important design consideration. Integration of structures, such as grooves, into the channels to act as static mixers is a commonly used approach. The mixing induced by these structures can be validated by determining concentration profiles in microfluidic channels following convergence of solvent streams from separate inlets. Spatially resolved characterisation is therefore necessary and requires in-line analysis methods. Here we report a line-focused illumination approach to provide operando, spatially resolved Raman spectra across the width of channels in the analysis of single- and multi-phase liquid systems and chemical reactions. A scientific complementary metal oxide semiconductor (sCMOS) sensor is used to overcome smearing encountered during spectral readout of images with CCD sensors. Isotopically labelled probes, in otherwise identical flow streams, show that z-confocality limits the spatial resolution and certainty as to the extent of mixing that can be achieved. These limitations are overcome using fast chemical reactions between reagents entering a microchannel in separate solvent streams. We show here that the progression of a chemical reaction, for which only the product is observable, is a powerful approach to determine the extent of mixing in a microchannel. Specifically resonance enhancement of Raman scattering from a product formed allows for determination of the true efficiency of mixing over the length and width of microchannels. Raman spectral images obtained by line-focused illumination show onset of mixing by observing the product of reagents entering from the separate inlets. Mixing is initially off-centre and immediately before the apex of the first groove of the static mixer, and then evolves along the entire width of the channel after a full cycle of grooves.

Efficient Electrochemiluminescence Sensing in Microfluidic Biosensors: A Review.

Microfluidic devices are capable of handling 10-9 L to 10-18 L of fluids by incorporating tiny channels with dimensions of ten to hundreds of micrometers, and they can be fabricated using a wide range of materials including glass, silicon, polymers, paper, and cloth for tailored sensing applications. Microfluidic biosensors integrated with detection methods such as electrochemiluminescence (ECL) can be used for the diagnosis and prognosis of diseases. Coupled with ECL, these tandem devices are capable of sensing biomarkers at nanomolar to picomolar concentrations, reproducibly. Measurement at this low level of concentration makes microfluidic electrochemiluminescence (MF-ECL) devices ideal for biomarker detection in the context of early warning systems for diseases such as myocardial infarction, cancer, and others. However, the technology relies on the nature and inherent characteristics of an efficient luminophore. The luminophore typically undergoes a redox process to generate excited species which emit energy in the form of light upon relaxation to lower energy states. Therefore, in biosensor design the efficiency of the luminophore is critical. This review is focused on the integration of microfluidic devices with biosensors and using electrochemiluminescence as a detection method. We highlight the dual role of carbon quantum dots as a luminophore and co-reactant in electrochemiluminescence analysis, drawing on their unique properties that include large specific surface area, easy functionalization, and unique luminescent properties.

Rapid prototyping of PMMA-based microfluidic spheroid-on-a-chip models using micromilling and vapour-assisted thermal bonding.

The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.

Mitochondrial transplantation rescues neuronal cells from ferroptosis.

Ferroptosis is a type of oxidative cell death that can occur in neurodegenerative diseases and involves damage to mitochondria. Previous studies demonstrated that preventing mitochondrial dysfunction can rescue cells from ferroptotic cell death. However, the complexity of mitochondrial dysfunction and the timing of therapeutic interventions make it difficult to develop an effective treatment strategy against ferroptosis in neurodegeneration conditions. In this study, we explored the use of mitochondrial transplantation as a novel therapeutic approach for preventing ferroptotic neuronal cell death. Our data showed that isolated exogenous mitochondria were incorporated into both healthy and ferroptotic immortalized hippocampal HT-22 cells and primary cortical neurons (PCN). The mitochondrial incorporation was accompanied by increased metabolic activity and cell survival through attenuating lipid peroxidation and mitochondrial superoxide production. Further, the function of mitochondrial complexes I, III and V activities contributed to the neuroprotective activity of exogenous mitochondria. Similarly, we have also showed the internalization of exogenous mitochondria in mouse PCN; these internalized mitochondria were found to effectively preserve the neuronal networks when challenged with ferroptotic stimuli. The administration of exogenous mitochondria into the axonal compartment of a two-compartment microfluidic device induced mitochondrial transportation to the cell body, which prevented fragmentation of the neuronal network in ferroptotic PCN. These findings suggest that mitochondria transplantation may be a promising therapeutic approach for protecting neuronal cells from ferroptotic cell death.

Enhanced passive mixing for paper microfluidics.

Imprecise control of fluid flows in paper-based devices is a major challenge in pushing the innovations in this area towards societal implementation. Assays on paper tend to have low reaction yield and reproducibility issues that lead to poor sensitivity and detection limits. Understanding and addressing these issues is key to improving the performance of paper-based devices. In this work, we use colorimetric analysis to observe the mixing behaviour of molecules from two parallel flow streams in unobstructed (on unpatterned paper) and constricted flow (through the gap of a patterned hourglass structure). The model system used for characterization of mixing involved the reaction of Fe3+ with SCN- to form the coloured, soluble complex Fe(SCN)2+. At all tested concentrations (equal concentrations of 50.0 mM, 25.0 mM or 12.5 mM for KSCN and FeCl3 in each experiment), the reaction yield increases (higher colorimetric signal) and better mixing is obtained (lower relative standard deviation) as the gap of the flow constriction becomes smaller (4.69-0.32 mm). This indicates enhanced passive mixing of reagents. A transition window of gap widths exhibiting no mixing enhancement (about 2 mm) to gap widths exhibiting complete mixing (0.5 mm) is defined. The implementation of gap sizes that are smaller than 0.5 mm (below the transition window) for passive mixing is suggested as a good strategy to obtain complete mixing and reproducible reaction yields on paper. In addition, the hourglass structure was used to define the ratio of reagents to be mixed (2 : 1, 1 : 1 and 1 : 2 HCl-NaOH) by simply varying the width ratio of the input channels of the paper. This allows easy adaptation of the device to reaction stoichiometry.

A versatile, compartmentalised gut-on-a-chip system for pharmacological and toxicological analyses.

A novel, integrated, in vitro gastrointestinal (GI) system is presented to study oral bioavailability parameters of small molecules. Three compartments were combined into one hyphenated, flow-through set-up. In the first compartment, a compound was exposed dynamically to enzymatic digestion in three consecutive microreactors, mimicking the processes of the mouth, stomach, and intestine. The resulting solution (chyme) continued to the second compartment, a flow-through barrier model of the intestinal epithelium allowing absorption of the compound and metabolites thereof. The composition of the effluents from the barrier model were analysed either offline by electrospray-ionisation-mass spectrometry (ESI-MS), or online in the final compartment using chip-based ESI-MS. Two model drugs, omeprazole and verapamil, were used to test the integrated model. Omeprazole was shown to be broken down upon treatment with gastric acid, but reached the cell barrier unharmed when introduced to the system in a manner emulating an enteric-coated formulation. In contrast, verapamil was unaffected by digestion. Finally, a reduced uptake of verapamil was observed when verapamil was introduced to the system dissolved in apple juice, a simple food matrix. It is envisaged that this integrated, compartmentalised GI system has potential for enabling future research in the fields of pharmacology, toxicology, and nutrition.

Visualization of Bacterial Colonization and Cellular Layers in a Gut-on-a-Chip System Using Optical Coherence Tomography.

Imaging of cellular layers in a gut-on-a-chip system has been confined to two-dimensional (2D)-imaging through conventional light microscopy and confocal laser scanning microscopy (CLSM) yielding three-dimensional- and 2D-cross-sectional reconstructions. However, CLSM requires staining and is unsuitable for longitudinal visualization. Here, we compare merits of optical coherence tomography (OCT) with those of CLSM and light microscopy for visualization of intestinal epithelial layers during protection by a probiotic Bifidobacterium breve strain and a simultaneous pathogen challenge by an Escherichia coli strain. OCT cross-sectional images yielded film thicknesses that coincided with end-point thicknesses derived from cross-sectional CLSM images. Light microscopy on histological sections of epithelial layers at the end-point yielded smaller layer thicknesses than OCT and CLSM. Protective effects of B. breve adhering to an epithelial layer against an E. coli challenge included the preservation of layer thickness and membrane surface coverage by epithelial cells. OCT does not require staining or sectioning, making OCT suitable for longitudinal visualization of biological films, but as a drawback, OCT does not allow an epithelial layer to be distinguished from bacterial biofilms adhering to it. Thus, OCT is ideal to longitudinally evaluate epithelial layers under probiotic protection and pathogen challenges, but proper image interpretation requires the application of a second method at the end-point to distinguish bacterial and epithelial films.

Digestion-on-a-chip: a continuous-flow modular microsystem recreating enzymatic digestion in the gastrointestinal tract.

In vitro digestions are essential for determining the bioavailability of compounds, such as nutrients. We have developed a cell-free, miniaturized enzymatic digestive system, employing three micromixers connected in series to mimic the digestive functions of the mouth, stomach and small intestine. This system continuously processes samples, e.g. containing nutrients, to provide a constant flow of digested materials which may be presented to a subsequent gut-on-a-chip absorption module, containing living human intestinal cells. Our system incorporates three-compartment enzymatic digestion, one of the key functions of the gastrointestinal tract. In each of these compartments, we modify the chemical environment, including pH, buffer, and mineral composition, to closely mimic the local physiological environment and create optimal conditions for digestive processes to take place. It will therefore provide an excellent addition to existing gut-on-a-chip systems, providing the next step in determining the bio-availability of orally administered compounds in a fast and continuous-flow ex vivo system. In this paper, we demonstrate enzymatic digestion in each separate compartment using compounds, starch and casein, as model nutrients. The use of transparent, microfluidic micromixers based on chaotic advection, which can be probed directly with a microscope, enabled enzyme kinetics to be monitored from the very start of a reaction. Furthermore, we have digested lactoferrin in our system, demonstrating complete digestion of this milk protein in much shorter times than achievable with standard in vitro digestions using batch reactors.

Electrochemical sensing with single nanoskived gold nanowires bisecting a microchannel.

We suspended a single nanoskived gold nanowire in a microfluidic channel. In this preliminary report, a 200 nm-diameter nanowire was used as an electrode to perform hydrodynamic voltammetry in the center of solution flow. Suspended nanowires exhibit superior current response due to highly efficient mass transport in the area of fastest flow.

Water-based alkyl ketene dimer ink for user-friendly patterning in paper microfluidics.

We propose the use of water-based alkyl ketene dimer (AKD) ink for fast and user-friendly patterning of paper microfluidic devices either manually or using an inexpensive XY-plotter. The ink was produced by dissolving hydrophobic AKD in chloroform and emulsifying the solution in water. The emulsification was performed in a warm water bath, which led to an increased rate of the evaporation of chloroform. Subsequent cooling led to the final product, an aqueous suspension of fine AKD particles. The effects of surfactant and AKD concentrations, emulsification procedure, and cooling approach on final ink properties are presented, along with an optimized protocol for its formulation. This hydrophobic agent was applied onto paper using a plotter pen, after which the paper was heated to allow spreading of AKD molecules and chemical bonding with cellulose. A paper surface patterned with the ink (10 g L-1 AKD) yielded a contact angle of 135.6° for water. Unlike organic solvent-based solutions of AKD, this AKD ink does not require a fume hood for its use. Moreover, it is compatible with plastic patterning tools, due to the effective removal of chloroform in the production process to less than 2% of the total volume. Furthermore, this water-based ink is easy to prepare and use. Finally, the AKD ink can also be used for the fabrication of so-called selectively permeable barriers for use in paper microfluidic networks. These are barriers that stop the flow of water through paper, but are permeable to solvents with lower surface energies. We applied the AKD ink to confine and preconcentrate sample on paper, and demonstrated the use of this approach to achieve higher detection sensitivities in paper spray ionization-mass spectrometry (PSI-MS). Our patterning approach can be employed outside of the analytical lab or machine workshop for fast prototyping and small-scale production of paper-based analytical tools, for use in limited-resource labs or in the field.

Countercurrent liquid-liquid extraction on paper.

Proof-of-concept is shown for two-phase countercurrent flow on paper. The device consists of two paper layers, one of which has been modified with a sizing agent to be hydrophobic. The layers exhibit different wetting behavior for water and octanol. Both phases dominate wetting in one of the layers and can be made to move in different directions along the interface to achieve liquid-liquid extraction.

Fused Deposition Modeling 3D Printing for (Bio)analytical Device Fabrication: Procedures, Materials, and Applications.

In this work, the use of fused deposition modeling (FDM) in a (bio)analytical/lab-on-a-chip research laboratory is described. First, the specifications of this 3D printing method that are important for the fabrication of (micro)devices were characterized for a benchtop FDM 3D printer. These include resolution, surface roughness, leakage, transparency, material deformation, and the possibilities for integration of other materials. Next, the autofluorescence, solvent compatibility, and biocompatibility of 12 representative FDM materials were tested and evaluated. Finally, we demonstrate the feasibility of FDM in a number of important applications. In particular, we consider the fabrication of fluidic channels, masters for polymer replication, and tools for the production of paper microfluidic devices. This work thus provides a guideline for (i) the use of FDM technology by addressing its possibilities and current limitations, (ii) material selection for FDM, based on solvent compatibility and biocompatibility, and (iii) application of FDM technology to (bio)analytical research by demonstrating a broad range of illustrative examples.

Implementing oxygen control in chip-based cell and tissue culture systems.

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

Bisecting Microfluidic Channels with Metallic Nanowires Fabricated by Nanoskiving.

This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage (<5 V). Further, passive functionality is demonstrated by visualizing individual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.

3D-printed paper spray ionization cartridge with fast wetting and continuous solvent supply features.

We report the development of a 3D-printed cartridge for paper spray ionization (PSI) that can be used almost immediately after solvent introduction in a dedicated reservoir and allows prolonged spray generation from a paper tip. The fast wetting feature described in this work is based on capillary action through paper and movement of fluid between paper and the cartridge material (polylactic acid, PLA). The influence of solvent composition, PLA conditioning of the cartridge with isopropanol, and solvent volume introduced into the reservoir have been investigated with relation to wetting time and the amount of solvent consumed for wetting. Spray has been demonstrated with this cartridge for tens of minutes, without any external pumping. It is shown that fast wetting and spray generation can easily be achieved using a number of solvent mixtures commonly used for PSI. The PSI cartridge was applied to the analysis of lidocaine from a paper tip using different solvent mixtures, and to the analysis of lidocaine from a serum sample. Finally, a demonstration of online paper chromatography-mass spectrometry is given.

Microdialysis-coupled enzymatic microreactor for in vivo glucose monitoring in rats.

Continuous glucose monitoring (CGM) is an important aid for diabetic patients to optimize glycemic control and to prevent long-term complications. However, current CGM devices need further miniaturization and improved functional performance. We have coupled a previously described microfluidic chip with enzymatic microreactor (EMR) to a microdialysis probe and evaluated the performance of this system for monitoring subcutaneous glucose concentration in rats. Nanoliter volumes of microdialysis sample are efficiently reacted with continuously supplied glucose oxidase (GOx) solution in the EMR. The hydrogen peroxide produced is amperometrically detected at a (polypyrrole (PPy)-protected) thin-film Pt electrode. Subcutaneous glucose concentration was continuously monitored in anesthetized rats in response to intravenous injections of 20% glucose (w/v), 5 U/kg insulin, or saline as a control. In vitro evaluation showed a linear range of 2.1-20.6 mM and a sensitivity of 7.8 ± 1.0 nA/mM (n = 6). The physical lag time between microdialysis and the analytical signal was approximately 18 min. The baseline concentration of blood glucose was 10.2 ± 2.3 mM. After administering glucose to the rats, glucose levels increased by about 2 mM to 12.1 ± 2.3 mM in blood and 11.9 ± 1.5 mM in subcutaneous interstitial fluid (ISF). After insulin administration, glucose levels decreased by about 8 mM relative to baseline to 2.1 ± 0.6 mM in blood and 2.1 ± 0.9 mM in ISF. A microfluidic device with integrated chaotic mixer and EMR has been successfully combined with subcutaneous microdialysis to continuously monitor glucose in rats. This proof-of-principle demonstrates the feasibility of improved miniaturization in CGM based on microfluidics.

Innovations in studying in vivo cell behavior and pharmacology in complex tissues--microvascular endothelial cells in the spotlight.

Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs. We focus on microvascular endothelial cells that actively engage in a wide range of physiological and pathological processes. We propose a new strategy in which in vivo molecular control of cells is studied directly in the diseased endothelium instead of at a (far) distance from the site where drugs have to act, thereby accounting for tissue-controlled cell responses. The strategy uses laser microdissection-based enrichment of microvascular endothelium which, when combined with transcriptome and (phospho)proteome analyses, provides a factual view on their status in their complex microenvironment. Combining this with miniaturized sample handling using microfluidic devices enables handling the minute sample input that results from this strategy. The multidisciplinary approach proposed will enable compartmentalized analysis of cell behavior and drug effects in complex tissue to become widely implemented in daily biomedical research and drug development practice.

Monolithically integrated biophotonic lab-on-a-chip for cell culture and simultaneous pH monitoring.

A poly(dimethylsiloxane) biophotonic lab-on-a-chip (bioPhLoC) containing two chambers, an incubation chamber and a monitoring chamber for cell retention/proliferation and pH monitoring, respectively, is presented. The bioPhLoC monolithically integrates a filter with 3 µm high size-exclusion microchannels, capable of efficiently trapping cells in the incubation chamber, as well as optical elements for real-time interrogation of both chambers. The integrated optical elements made possible both absorption and dispersion measurements, which were comparable to those made in a commercially available cuvette. The size-exclusion filter also showed good and stable trapping capacity when using yeast cells of variable size (between 5 and 8 µm diameter). For cell culture applications, vascular smooth muscle cells (VSMC), with sizes between 8 and 10 µm diameter, were used as a mammalian cell model. These cells were efficiently trapped in the incubation chamber, where they proliferated with a classical spindle-shaped morphology and a traditional hill-and-valley phenotype. During cell proliferation, pH changes in the culture medium due to cell metabolism were monitored in real time and with high precision in the monitoring chamber without interference of the measurement by cells and other (cell) debris.

Comparison of biocompatibility and adsorption properties of different plastics for advanced microfluidic cell and tissue culture models.

Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision-cut liver slices, that are not possible with conventional systems. However, PDMS, a silicone rubber material, is very hydrophobic and tends to exhibit significant adsorption and absorption of hydrophobic drugs and their metabolites. Although glass could be used as an alternative, thermoplastics are better from a cost and fabrication perspective. Thermoplastic polymers (plastics) allow easy surface treatment and are generally transparent and biocompatible. This study focuses on the fabrication of biocompatible microfluidic devices with low adsorption properties from the thermoplastics poly(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC) as alternatives for PDMS devices. Thermoplastic surfaces were oxidized using UV-generated ozone or oxygen plasma to reduce adsorption of hydrophobic compounds. Surface hydrophilicity was assessed over 4 weeks by measuring the contact angle of water on the surface. The adsorption of 7-ethoxycoumarin, testosterone, and their metabolites was also determined after UV-ozone treatment. Biocompatibility was assessed by culturing human hepatoma (HepG2) cells on treated surfaces. Comparison of the adsorption properties and biocompatibility of devices in different plastics revealed that only UV-ozone-treated PC and COC devices satisfied both criteria. This paper lays an important foundation that will help researchers make informed decisions with respect to the materials they select for microfluidic cell-based culture experiments.

Microfluidics enables small-scale tissue-based drug metabolism studies with scarce human tissue.

Early information on the metabolism and toxicity properties of new drug candidates is crucial for selecting the right candidates for further development. Preclinical trials rely on cell-based in vitro tests and animal studies to characterize the in vivo behavior of drug candidates, although neither are ideal predictors of drug behavior in humans. Improving in vitro systems for preclinical studies both from a technological and biological model standpoint thus remains a major challenge. This article describes how microfluidics can be exploited to come closer to this goal in combination with precision-cut liver slices (PCLS) as an improved organomimetic system. Recently, we developed a novel microfluidic-based system incorporating a microchamber for slice perifusion to perform drug metabolism studies with mammalian PCLS under continuous flow. In the present study, the viability and metabolism of human PCLS were assessed by the measurement of the leakage of liver-specific enzymes and metabolism of four different substrates: lidocaine, 7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone. All experiments were verified with well plates, an excellent benchmark for these experiments. Clearly, however, human tissue is not readily available, and it is worth considering how to perform a maximum number of informative experiments with small amounts of material. In one approach, the microfluidic system was coupled to an HPLC system to allow on-line monitoring and immediate detection of unstable metabolites, something that is generally not possible with conventional well-plate systems. This novel microfluidic system also enables the in vitro measurement of interorgan interactions by connecting microchambers containing different organ slices in series for sequential perfusion. This versatile experimental system has the potential to yield more information about the metabolic profiles of new drug candidates in human and animal tissues in an early stage of development compared with well plates alone.