Interfacial instabilities affect microfluidic extraction of small molecules from non-Newtonian fluids

As part of a project to develop an integrated microfluidic biosensor for the detection of small molecules in saliva, practical issues of extraction of analytes from non-Newtonian samples using an H-filter were explored. The H-filter can be used to rapidly and efficiently extract small molecules from a complex sample into a simpler buffer. The location of the interface between the sample and buffer streams is a critical parameter in the function of the H-filter, so fluorescence microscopy was employed to monitor the interface position; this revealed apparently anomalous fluorophore diffusion from the samples into the buffer solutions. Using confocal microscopy to understand the three-dimensional distribution of the fluorophore, it was found that the interface between the non-Newtonian sample and Newtonian buffer was both curved and unstable. The core of the non-Newtonian sample extended into the Newtonian buffer and its position was unstable, producing a fluorescence intensity profile that gave rise to the apparently anomalously fast fluorophore transport. These instabilities resulted from the pairing of rheologically dissimilar fluid streams and were flowrate dependent. We conclude that use of non-Newtonian fluids, such as saliva, in the H-filter necessitates pretreatment to reduce viscoelasticity. The interfacial variation in position, stability and shape caused by the non-Newtonian samples has substantial implications for the use of biological samples for quantitative analysis and analyte extraction in concurrent flow extraction devices.     

Optically trapped microsensors for microfluidic temperature measurement by fluorescence lifetime imaging microscopy

The novel combination of optical tweezers and fluorescence lifetime imaging microscopy (FLIM) has been used, in conjunction with specially developed temperature-sensitive fluorescent microprobes, for the non-invasive measurement of temperature in a microfluidic device. This approach retains the capability of FLIM to deliver quantitative mapping of microfluidic temperature without the disadvantageous need to introduce a fluorescent dye that pervades the entire micofluidic system. This is achieved by encapsulating the temperature-sensitive Rhodamine B fluorophore within a microdroplet which can be held and manipulated in the microfluidic flow using optical tweezers. The microdroplet is a double bubble in which an aqueous droplet of the fluorescent dye is surrounded by an oil shell which serves both to contain the fluorophore and to provide the refractive index differential required for optical trapping of the droplet in an external aqueous medium.     

Integrated wavelength-selective optical waveguides for microfluidic-based laser-induced fluorescence detection

We demonstrate the fabrication and characterization of a novel, inexpensive microchip capable of laser induced fluorescence (LIF) detection using integrated waveguides with built-in optical filters. Integrated wavelength-selective optical waveguides are fabricated by doping poly(dimethysiloxane) (PDMS) with dye molecules. Liquid-core waveguides are created within dye-doped PDMS microfluidic chips by filling channels with high refractive index liquids. Dye molecules are allowed to diffuse into the liquid core from the surrounding dye-doped PDMS. The amount of diffusion is controlled by choosing either polar (low diffusion) or apolar (high diffusion) liquid waveguide cores. The doping dye is chosen to absorb excitation light and to transmit fluorescence emitted by the sample under test. After 24 h, apolar waveguides demonstrate propagation losses of 120 dB cm(-1) (532 nm) and 4.4 dB cm(-1) (633 nm) while polar waveguides experience losses of 8.2 dB cm(-1) (532 nm) and 1.1 dB cm(-1) (633 nm) where 532 and 633 nm light represent the excitation and fluorescence wavelengths, respectively. We demonstrate the separation and detection of end-labelled DNA fragments using polar waveguides for excitation light delivery and apolar waveguides for fluorescence collection. We demonstrate that the dye-doped waveguides can provide performance comparable to a commercial dielectric filter; however, for the present choice of dye, their ultimate performance is limited by autofluorescence from the dye. Through the detection of a BK virus polymerase chain reaction (PCR) product, we demonstrate that the dye-doped PDMS system is an order of magnitude more sensitive than a similar undoped system (SNR: 138 vs. 9) without the use of any external optical filters at the detector.     

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera‐based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR‐FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR‐FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR‐FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR‐FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross‐correlation functions (ΔCCF). With the determination of the PSF by ITIR‐FCS and the optimization of measurement conditions ITIR‐FCS becomes a calibration‐free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.     

Fluorescence correlation spectroscopy for flow rate imaging and monitoring--optimization, limitations and artifacts

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.     

Highly sensitive fluorescence detection system for microfluidic lab-on-a-chip

We demonstrate a compact, low cost and practical fluorescence detection system for lab-on-a-chip applications. The system comprises a commercially available InGaN light emitting diode (501 nm) as light source, an organic or silicon photodiode detector, absorptive dye coated colour filters and linear and reflective polarisers. An injection moulded polystyrene microfluidic chip is used as the platform for fluorescence immunoassays for cardiac markers myoglobin and CK-MB. The optical limit of detection (LOD) is measured using a TransFluoSphere? suspension at 5.6 × 10(4) beads μl(-1) which can be equated to ～3 nM fluorescein equivalent concentration. The LOD for the human plasma immunoassays is measured as 1.5 ng ml(-1) for both myoglobin and CK-MB.     

Visualizing transport processes at liquid-liquid interfaces--the application of laser-induced fluorescence

Modeling of liquid-liquid extraction processes involves the concentration of the extracted component directly at the interface. Currently, only very few and specialized methods are available for the direct measurement of these concentrations. Therefore a new, fluorescence-based measurement system with a high spatial resolution and a broad application spectrum was developed and tested. The detection principle is based on the use of fluorescent dyes excited by an argon ion laser. The intensity of the emitted light is dependent on the concentration of the extracted component in the very near surroundings of the dye. This intensity distribution is reproduced by an optical, microscope-based system onto a highly sensitive camera with a spatial resolution of 1 mum. This distribution is converted into a concentration profile at the interface using a calibration function and digital image processing routines. Measurements were performed in a commonly used stirred two-phase reactor modified to meet the requirements of an optical measurement system. It was shown that the concentration profiles at moving and nonmoving interfaces could be visualized with a resolution of 1 mum. The profiles formed at the interface differ significantly according to the kinetic of the used extraction system and the flow profiles in the reactor and can be used for further modeling of the extraction processes.     

Assay for molecular transport across gap junction channels in one-dimensional cell arrays

Transport across gap junction channels (GJCs) between neighboring cells is critical to synchronizing cell's electrical and metabolic activities and maintaining cell homeostasis. Here we present a non-invasive microfluidic method to measure molecular diffusion across GJCs in multiple 1D cell arrays in real time. Using the chip, selective loading of a membrane permeant fluorescence dye (carboxyfluorescein) in Normal Rat Kidney (NRK) cells shows that the dye was able to diffuse through three cells along single cell chains in ～35 minutes. Application of 100 μM 2-aminoethoxydiphenyl borate (2-APB) reversibly inhibits connexin-43 gap junctions in NRK cells; 0.8 mM 1-heptanol inhibits the diffusion partially. The method offers rapid exchange of reagents, enabling sequential screening of multiple gap junction specific drugs with only one preparation of cells. It is capable of measuring gap junction mediated diffusion between single cells.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

Optimisation of a microfluidic analysis chamber for the placement of microelectrodes

The behaviour of droplets entering a microfluidic chamber designed to house microelectrode detectors for real time analysis of clinical microdialysate is described. We have designed an analysis chamber to collect the droplets produced by multiphase flows of oil and artificial cerebral spinal fluid. The coalescence chamber creates a constant aqueous environment ideal for the placement of microelectrodes avoiding the contamination of the microelectrode surface by oil. A stream of alternating light and dark coloured droplets were filmed as they passed through the chamber using a high speed camera. Image analysis of these videos shows the colour change evolution at each point along the chamber length. The flow in the chamber was simulated using the general solution for Poiseuille flow in a rectangular chamber. It is shown that on the centre line the velocity profile is very close to parabolic, and an expression is presented for the ratio between this centre line velocity and the mean flow velocity as a function of channel aspect ratio. If this aspect ratio of width/height is 2, the ratio of flow velocities closely matches that of Poiseuille flow in a circular tube, with implications for connections between microfluidic channels and connection tubing. The droplets are well mixed as the surface tension at the interface with the oil dominates the viscous forces. However once the droplet coalesces with the solution held in the chamber, the no-slip condition at the walls allows Poiseuille flow to take over. The meniscus at the back of the droplet continues to mix the droplet and acts as a piston until the meniscus stops moving. We have found that the no-slip conditions at the walls of the chamber, create a banding effect which records the history of previous drops. The optimal position for sensors is to be placed at the plane of droplet coalescence ideally at the centre of the channel, where there is an abrupt concentration change leading to a response time ?16 ms, the compressed frame rate of the video. Further away from this point the response time and sensitivity decrease due to convective dispersion.     

Characterization of electrokinetic gating valve in microfluidic channels

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.     

A facile LED backlight in situ imaging technique to investigate sub-micron level processing

Many laboratory experimental techniques used for investigating fine fluid structure, such as fiber spinning, microfluidic flow, and electrospinning, require high quality images with good contrast. Common processes of observation and image recording rely heavily on highly technical light and camera setups which can be difficult to operate in some processing conditions and expensive as well. Here, we report a facile technique using LED backlight imaging to investigate ultrathin fluid profile in two different processes, melt electrospinning and tubeless siphoning. The setup comprises of a simple LED light source facing toward the camera, directly shining into the camera lens. The object under investigation was placed between the camera and the light source. The high-quality captured images and video recordings enable the precise analysis of the cone diameter and jet solidification in case of melt electrospinning, and extensional behavior profiles for tubeless siphoning. The LED backlight setup with high resolution camera is a useful tool to investigate sub-micron scale dimensions in fiber spinning, microfluidic flow, solution electrospinning, contact angle measurement for surface properties analysis, etc.     

Use of directly molded poly(methyl methacrylate) channels for microfluidic applications

A direct molding method for creating a homogeneous, polymer microfluidic channel is presented. By utilizing capillary rise and subsequent absorption of poly(methyl methacrylate) (PMMA) solution into a solvent-permeable poly(dimethyl siloxane) (PDMS) mold, various circular or elliptic polymer microchannels were fabricated without channel bonding and additional surface modification processes. In addition, the channel diameter was tunable from several micrometres to several hundreds of micrometres by controlling concentration and initial amount of polymer solution for a given PDMS mold geometry. The molded PMMA channels were used for two applications: blocking absorption of Rhodamine B dye and constructing artificial endothelial cell-cultured capillaries. It was observed that the molded PMMA channels effectively prevented absorption and diffusion of Rhodamine molecules over 5 h time span, demonstrating approximately 40 times higher blocking efficiency as compared to porous PDMS channels. Also, calf pulmonary artery endothelial cells (CPAEs) adhered, spread, and proliferated uniformly within the molded microchannels to form near confluency within 3 days and remained viable at day 6 without notable cell death, suggesting high biocompatibility and possibility for emulating in vivo-like three-dimensional architecture of blood vessels.     

Non-emissive colour filters for fluorescence detection

We describe a simple technique for fabricating non-emissive colour filters based on the sensitisation of a highly porous nanostructured metal-oxide film with a monolayer of dye molecules. Ultrafast electron transfer at the oxide/dye interface induces efficient quenching of photogenerated excitons in the dye, reducing the photoluminescence quantum yield by many orders of magnitude. The resultant filters exhibit much less autofluorescence than conventional colour filters (where the chromophore is dispersed in a glass or polymer host), and are a viable low cost alternative to interference filters for microfluidic devices and other applications requiring non-emissive filtering.     

Temperature measurements in microfluidic systems: heat dissipation of negative dielectrophoresis barriers

The manipulation of living biological cells in microfluidic channels by a combination of negative dielectrophoretic barriers and pressure-driven flows is widely employed in lab-on-a-chip systems. However, electric fields in conducting media induce Joule heating. This study investigates if the local temperatures reached under typical experimental conditions in miniaturized systems cause a potential risk for hyperthermic stress or cell damage. Two methods of optical in situ temperature detection have been tested and compared: (i) the exposure of the thermo-dependent fluorescent dye Rhodamine B to heat sources situated in microfluidic channels, and (ii) the use of thermoprecipitating N-alkyl-substituted acrylamide polymers as temperature threshold probes. Two-dimensional images of temperature distributions in the vicinity of active negative dielectrophoresis (nDEP)-barriers have been obtained and local temperature variations of more than 20 degrees C have been observed at the electrode edges. Heat propagation via both buffer and channel walls lead to significant temperature increases within a perimeter of 100 microm and more. These data indicate that power dissipation has to be taken into account when experiments at physiological temperatures are planned.     

Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices

This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.     

Multiplexed pressure sensing with elastomer membranes

We demonstrate a novel optical pressure measurement platform for microfluidics. The pressure sensors operate as pneumatically-tunable microlenses whose focal lengths vary with pressure. We show that pneumatic lens arrays can be used to perform sensitive multiplexed pressure measurements in microfluidic channels.     

A microfluidic diffusion chamber for reversible environmental changes around flaccid lipid vesicles

The reversible environmental changes around flaccid lipid vesicles represent a considerable experimental challenge, particularly because of remarkable softness of flaccid membranes, which can warp irreversibly under the slightest hydrodynamic flow. As a result, we have developed a microfluidic device for the controlled analysis of individual flaccid, giant lipid vesicles in a changing chemical environment. The setup combines the advantages of a flow-free microfluidic diffusion chamber and optical tweezers, which are used to load the sample vesicles into the chamber. After a vesicle is loaded into the diffusion chamber, its chemical environment is controllably and reversibly changed solely by means of diffusion. The chamber is designed as a 250 micrometres-long and 100 micrometres-wide dead-end microchannel, which extends from a T-junction of the main microchannels. Measurements of the flow-velocity profile in the chamber show that the flow rate decreases exponentially and scales linearly with the flow rate in the main channel. The characteristic length of the exponential decrease is 15 (1 ± 0.13) micrometres, meaning that a large part of the diffusion chamber is effectively flow-free. The diffusion properties are assessed by monitoring the diffusion of a dye into the chamber. It was found that a simple 1D diffusion model fits well to the experimental data. The time needed for the exchange of solutes in the chamber is of the order of minutes, depending on the solute's molecular weight. Here, we demonstrate how the diffusion chamber can be used for reversible environmental changes around flaccid, giant lipid vesicles and membrane tethers (nanotubes).     

Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation

A highly effective, reagentless, mechanical cell lysis device integrated in microfluidic channels is reported. Sample preparation, specifically cell lysis, is a critical element in 'lab-on-chip' applications. However, traditional methods of cell lysis require purification steps or complicated fabrication steps that a simple mechanical method of lysis may avoid. A simple and effective mechanical cell lysis system is designed, microfabricated, and characterized to quantify the efficiency of cell lysis and biomolecule accessibility. The device functionality is based on a microfluidic filter region with nanostructured barbs created using a modified deep reactive ion etching process. Mechanical lysis is characterized by using a membrane impermeable dye. Three main mechanisms of micro-mechanical lysis are described. Quantitative measurements of accessible protein as compared to a chemically lysed sample are acquired with optical absorption measurements at 280 and 414 nm. At a flow rate of 300 microL min(-1) within the filter region total protein and hemoglobin accessibilities of 4.8% and 7.5% are observed respectively as compared to 1.9% and 3.2% for a filter without nanostructured barbs.