Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.     

Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.     

A study of the mechanism of response of liquid ion exchanger calcium selective electrodes: Part III. Membrane behaviour under non-zero current conditions

The I-E response of the liquid membrane of the calcium selective electrode is studied under constant or linearly varying current and voltage. An increase in the membrane resistance, recorded when an electrical current crosses the membrane, is due to the outflow of Cl^? ions initially present in the membrane. When calcium ions are replaced by alkaline ions inside the membrane at constant current, the decrease of the membrane resistance due to an ion exchange is in agreement with the conductivity measurements (Part II). When the applied voltage is imposed besides the ion exchange one must take into account the interfacial overpotential to explain the important rectification effect observed. The interfacial transfer constant rate of alkaline ions seems greater than that of Ca²⁺ ion.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

Smart portable electrophoresis instrument based on multipurpose microfluidic chips with electrochemical detection

A second generation of a battery-powered portable electrophoresis instrument for the use of ME with electrochemical detection was developed. As the first-generation, the main unit of the instrument (150 mm × 165 mm × 95 mm) consists of four-outputs high-voltage power supply (HVPS) with maximum voltage of 3 KV and acquisition system (bipotentiostat) containing 2-channels for dual electrochemical detection. A new reusable microfluidic platform was designed in order to incorporate the microchips with the portable instrument. In this case, the platform is integrated to the main unit of the instrument so that it is not necessary to have any external cable for the interconnection of both parts, making the use of the complete system easier. The new platform contains all the electrical connections for the HVPS and bipotentiostat, as well as fluidic ports for driving the solutions. The microfluidic electrophoresis instrument is controlled by means of a user-friendly interface from a computer. The possibility of wireless connection (Bluetooth?) allows the use of the instrument without any external cable improving the portability. Therefore, the second generation brings a more compact and integrated electrophoresis instrument for "in situ" applications using microfluidic chips in an easy way. The performance of the electrophoresis system was initially evaluated using single- and dual-channel SU-8/Pyrex microchips with different models of integrated electrodes including microelectrodes and interdigitated arrays. The method was tested in different analytical applications such as separation of neurotransmitters, chlorophenols, purine derivatives, vitamins, polyphenolic acids, and flavones.     

Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.     

Microfluidic flow transducer based on the measurement of electrical admittance

A new flow transducer for measuring the flow rate of a conducting fluid in a microchannel is reported. In this paper, the measure of flow of such fluid under laminar flow conditions based on the change of electrical admittance is established with the aid of a pair of electrodes parallel to the line of flow in a glass-PDMS microfluidic device. This flow sensor is simple in design and can be integrated to most of the microfluidic platforms. The effect of flow rate of the electrolyte, the frequency of the applied ac voltage, the voltage applied across the detector electrodes, and the conductivity of the electrolyte are varied to optimize for high sensitivity. The optimized values are then used to demonstrate the measurements of very low flow rates (<1 nL s(-1)). This flow sensor can be extended towards the measurement of chemical and biochemical buffers and reagents.     

Electro-optical BLM chips enabling dynamic imaging of ordered lipid domains

Studies of lipid rafts, ordered microdomains of sphingolipids and cholesterol within cell membranes, are essential in probing the relationships between membrane organization and cellular function. While in vitro studies of lipid phase separation are commonly performed using spherical vesicles as model membranes, the utility of these models is limited by a number of factors. Here we present a microfluidic device that supports simultaneous electrical measurements and confocal imaging of on-chip bilayer lipid membranes (BLMs), enabling real-time multi-domain imaging of membrane organization. The chips further support closed microfluidic access to both sides of the membrane, allowing the membrane boundary conditions to be rapidly changed and providing a mechanism for dynamically adjusting membrane curvature through application of a transmembrane pressure gradient. Here we demonstrate the platform through the study of dynamic generation and dissolution of ordered lipid domains as membrane components are transported to and from the supporting annulus containing solvated lipids and cholesterol.     

Measurement of microchannel fluidic resistance with a standard voltage meter

A simplified method for measuring the fluidic resistance (R_fluidic) of microfluidic channels is presented, in which the electrical resistance (R_elec) of a channel filled with a conductivity standard solution can be measured and directly correlated to R_fluidic using a simple equation. Although a slight correction factor could be applied in this system to improve accuracy, results showed that a standard voltage meter could be used without calibration to determine R_fluidic to within 12% error. Results accurate to within 2% were obtained when a geometric correction factor was applied using these particular channels. When compared to standard flow rate measurements, such as meniscus tracking in outlet tubing, this approach provided a more straightforward alternative and resulted in lower measurement error. The method was validated using 9 different fluidic resistance values (from ∼40 to 600 kPa s mm⁻³) and over 30 separately fabricated microfluidic devices. Furthermore, since the method is analogous to resistance measurements with a voltage meter in electrical circuits, dynamic R_fluidic measurements were possible in more complex microfluidic designs. Microchannel R_elec was shown to dynamically mimic pressure waveforms applied to a membrane in a variable microfluidic resistor. The variable resistor was then used to dynamically control aqueous-in-oil droplet sizes and spacing, providing a unique and convenient control system for droplet-generating devices. This conductivity-based method for fluidic resistance measurement is thus a useful tool for static or real-time characterization of microfluidic systems.     

499 bis—The surface compartment model—voltage clamp

Solutions of the SCM equations under voltage clamp show that a transient inward sodium flux can occur at certain settings of the parameters. If there is a nonspecific increase in cation permeability upon depolarization, the positive sodium flux arises because the ion concentration changes in the electrical double layer compensate for the decrease in the electrical driving force, while parallell changes in the potassium ion concentrations lead to a decreased (but still outward) potassium flux. The sodium flux is quite sensitive to the magnitudes of the ionic permeabilities and mobilities, as well as the ion binding equilibrium and release rate constants. The values of the parameters determined here suggest magnitudes for the properties of the membrane components associated with the permeability change.     

Whole cell immobilised biosensors for toxicity assessment of a wastewater treatment plant treating phenolics-containing waste

Wastewater treatment plants dealing with industrial wastes are often susceptible to overload of toxic influent that can partially or completely destroy treatment for extended periods. An obvious candidate for monitoring toxicity in such wastewater systems is bioluminescent bacteria. However, the natural bioluminescent bacteria can be particularly sensitive to some industrial wastes and therefore their response to normal operational conditions does not reflect the status of the microbial community responsible for treatment. Moreover, the salt dependence of the marine bioluminescent bacteria, and the temperature sensitivity of some strains, further complicate their use. Here we describe the construction of whole cell genetically modified bioluminescent biosensors and their immobilisation for use in monitoring the toxicity of a complex industrial wastewater containing phenolic materials. A hand-held luminometer was designed for laboratory or field use, and the immobilisation system designed with several things in mind: the geometry of the instrument; the need for containment of GM bacteria; the maximisation of the bioavailability of the wastewater to the biosensor. The performance of a candidate GM sensor was compared with Vibrio fischeri in liquid culture and after immobilisation in thin films of poly(vinyl alcohol) (PVA) cryogels. The biosensors were tested against pure phenol and 3-chlorophenol as a reference toxic chemical known to be much more toxic to bacteria than phenol. The biosensors were then tested with the phenolics-containing industrial wastewater. The immobilisation system proved to operate predictably with pure toxicants, and was able to discriminate toxicity of various zones within the wastewater treatment plant.     

Fuel cell-powered microfluidic platform for lab-on-a-chip applications

The achievement of a higher degree of integration of components--especially micropumps and power sources--is a challenge currently being pursued to obtain portable and totally autonomous microfluidic devices. This paper presents the integration of a micro direct methanol fuel cell (μDMFC) in a microfluidic platform as a smart solution to provide both electrical and pumping power to a Lab-on-a-Chip system. In this system the electric power produced by the fuel cell is available to enable most of the functionalites required by the microfluidic chip, while the generated CO(2) from the electrochemical reaction produces a pressure capable of pumping a liquid volume through a microchannel. The control of the fuel cell operating conditions allows regulation of the flow rate of a liquid sample through a microfluidic network. The relation between sample flow rate and the current generated by the fuel cell is practically linear, achieving values in the range of 4-18 μL min(-1) while having an available power between 1-4 mW. This permits adjusting the desired flow rate for a given application by controlling the fuel cell output conditions and foresees a fully autonomous analytical Lab-on-a-Chip in which the same device would provide the electrical power to a detection module and at the same time use the CO(2) pumping action to flow the required analytes through a particular microfluidic design.     

Field-effect flow control in a polydimethylsiloxane-based microfluidic system.

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.     

The influence of membrane ion-permselectivity on electrokinetic concentration enrichment in membrane-based preconcentration units

The performance of nanoporous hydrogel microplugs with varying surface charge density is described in concentrating charged analytes electrokinetically in a microfluidic device. A neutral hydrogel plug with a mean pore size smaller than the size of charged analytes acts as a simple size-exclusion membrane. The presence of fixed charges on the backbone of a nanoporous hydrogel creates ion-permselectivity which results in charge-selective transport through the hydrogel. This leads to the development of concentration polarization (CP) in the adjoining bulk electrolyte solutions under the influence of an applied electrical field. CP strongly affects the distribution of the local electrical field strength, in particular, in the vicinity of the hydrogel plug which can significantly reduce the concentration enrichment factors compared to the neutral hydrogel. A theoretical model and simulations are presented, together with experimental data, to explain the interplay of hydrogel or membrane cation-selectivity, electrical field-induced CP, and the distribution of the local electrical field strength with respect to concentration enrichment of negatively charged analytes at the cathodic membrane-solution interface.     

Immobilisation of Chirasil-Val on glass capillaries

Summary Immobilisation methods of the chiral silicone stationary phase Chirasil-L-Val have been studied. In addition to the standard techniques of radical-induced immobilisation with dicumyl peroxide and azo tert-butane, immobilisation was solely achieved by thermal treatment: conditioning at low flow-rates or under stop-flow conditions led to unexpectedly high immobilisation despite mild temperatures. The presence of water vapor in the carrier gas accelerated the immobilisation process. While up to 75% immobilisation was attainable with both ATB and thermal methods, immobilisation with DCUP did not exceed 50%, despite relatively high DCUP concentrations.Whilst all immobilisation methods were accompanied by varying degrees of racemisation at the chiral centre, it was possible to establish conditions that yielded a high immobilisation (in excess of 70%) with a negligible loss in enantiomeric selctivity. The immobilisation reactions also slightly affected the polarity of the stationary phase, reflected in a shift of the Kovats-Indices of amino acid derivatives and in the accompanying Squalane-Effect. With respect to immobilisation, racemisation and polarity shift, thermal immobilisation with water-saturated carrier gas proved to be the optimum method.     

499—The surface compartment model (SCM) during transients

The surface compartment model (SCM) has been extended to transient states by including kinetic expressions for ion binding equilibria, surface capacitances in the electrical double layer regions and a mechanism for the rapid flow of charge within the membrane, such as gating currents. As a result of these changes the model can be applied to transient states, such as the voltage clamp.     

Integrated micro/nano-fluidic system for mixing and preconcentration of dissolved proteins

An integrated micro/nano-fluidic system is presented for protein analysis. It is comprised of an integrated micromixer (IMM) and a preconcentrator with a separation column. The passive and planar type of IMM is based on an unbalanced split and the cross collision of the fluidic streams. The IMM can be easily fabricated and integrated to the microfluidic system. The preconcentrator has nanochannels formed by the electrical breakdown of polydimethylsiloxane (PDMS) membrane by applying a high electrical shock, but without any nano-lithography. The integrated microdevice was used for sample preparation (mixing with tagging molecules) and subsequent concentration of proteins. Proteins were electrokinetically trapped near the junction of the micro/nanochannels. We show a conceptual design and a simple microfluidic system for purposes of mixing and preconcentration.

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.     

A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami

The lab-on-a-chip concept has led to several point-of-care (POC) diagnostic microfluidic platforms. However, few of these can process raw samples for molecular diagnosis and fewer yet are suited for use in a resource-limited setting without permanent electrical infrastructure. We present here a very low cost paper microfluidic device for POC extraction of bacterial DNA from raw viscous samples--a challenge for conventional microfluidic platforms. This is an example of "microfluidic origami" in that the system is activated by folding; demonstrated here is room temperature cell lysis and DNA extraction from pig mucin (simulating sputum) spiked with E. coli without the use of external power. The microfluidic origami device features dry reagent storage and rehydration of the lysis buffer. We demonstrate DNA extraction from samples with a bacterial load as low as 33 CFU ml(-1). Extraction times, starting from the raw sample, have been optimized to about 1.5 h without the use of external power, or to within 1 h using an oven or a heater block. The fabrication of this paper microfluidic device can be translated into high volume production in the developing world without the need for a semiconductor clean room or a microfabrication facility. The sample preparation can be performed with the addition of just the sample, water, ethanol and elute buffer to the device, thus reducing chemical hazards during transport and handling.