Michele Zagnoni - Academia.edu (original) (raw)
Papers by Michele Zagnoni
Methods in Cell Biology, 2011
Droplet microfluidics (DM) is an area of research which combines lab-on-a-chip (LOC) techniques w... more Droplet microfluidics (DM) is an area of research which combines lab-on-a-chip (LOC) techniques with emulsion compartmentalization to perform high-throughput, chemical and biological assays. The key issue of this approach lies in the generation, over tens of milliseconds, of thousands of liquid vessels which can be used either as a carrier, to transport encapsulated particles and cells, or as microreactors, to perform parallel analysis of a vast number of samples. Each compartment comprises a liquid droplet containing the sample, surrounded by an immiscible fluid. This microfluidic technique is capable of generating subnanoliter and highly monodispersed liquid droplets, which offer many opportunities for developing novel single-cell and single-molecule studies, as well as high-throughput methodologies for the detection and sorting of encapsulated species in droplets. The aim of this chapter is to give an overview of the features of DM in a broad microfluidic context, as well as to show the advantages and limitations of the technology in the field of LOC analytical research. Examples are reported and discussed to show how DM can provide novel systems with applications in high-throughput, quantitative cell and particle analysis.
Physical Review E, 2009
Two phase microfluidic systems creating size-controlled microdroplets have recently emerged as po... more Two phase microfluidic systems creating size-controlled microdroplets have recently emerged as powerful tools to achieve liquid compartmentalization for high throughput chemical and biological assays. Emulsion electrocoalescence is a destabilization process that can be used in droplet-based platforms for water phase separation to enable lab-on-a-chip applications in biotechnology, including particle or cell recovery. In this paper, we report upon a series of phenomena associated with electrocoalescence of water microdroplet-in-oil populations in microfluidics. In our experiments, we formed microdroplets whose size and dispersion in the channel were varied according to the ratio of the flow rates of the two phases. Different types of electrocoalescence between droplets were obtained. For low applied voltages, drops merged in pairs over the electrode region; for higher values of the applied voltage, a cascade of droplet coalescence was produced against the flow direction, for a range of droplet sizes, lateral distributions of droplets in the channel and localized electric fields.
Langmuir, 2010
Multiphase microfluidics offer a wide range of functionalities in the fields of fluid dynamics, b... more Multiphase microfluidics offer a wide range of functionalities in the fields of fluid dynamics, biology, particle synthesis, and, more recently, also in logical computation. In this article, we describe the hysteresis of immiscible, multiphase flow obtained in hydrophilic, microfluidic systems at a T-junction. Stable and unstable state behaviors, in the form of segmented and parallel flow patterns of oil and water, were reliably produced, depending upon the history of the flow rates applied to the phases. The transition mechanisms between the two states were analyzed both experimentally and using numerical simulations, describing how the physical and fluid dynamic parameters influenced the hysteretic behavior of the flow. The characteristics of these multiphase systems render them suitable to be used as pressure comparators and also for the implementation of microfluidic logic operations.
Langmuir, 2010
Electrocoalescence of water-in-oil microdroplets in microfluidic channels is an active technique ... more Electrocoalescence of water-in-oil microdroplets in microfluidic channels is an active technique that enables droplet-based mixing functionalities to be achieved in lab-on-a-chip applications. In this work, a characterization of the electrocoalescence mechanisms of water microdroplets in oil is presented, using localized electric field systems. We report a theoretical and experimental description of the electrocoalscence behavior of droplet pairs by varying the physical and fluid dynamic conditions of the phases. Our results demonstrate that localized electric field systems can be reliably used to merge droplets in pairs, regardless of the distance between the drops. The coalescence behavior was dependent upon the viscosity of the continuous phase for water droplets that were separated by a thick layer of oil and upon interfacial tension for droplets that were in close proximity. We showed that these systems have the potential to be used for high-throughput applications and that, unlike other examples of active systems in the literature, the need of droplet synchronization and the application of high voltages is considerably reduced.
Langmuir, 2007
To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be e... more To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.
Lab on a Chip, 2008
We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electri... more We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.
Lab on a Chip, 2009
Electric fields have previously been used in microfluidic devices for the manipulation, sorting a... more Electric fields have previously been used in microfluidic devices for the manipulation, sorting and mixing of microemulsions. Here, an active system for on-demand electrocoalescence of water droplets in oil is presented. The platform does not require precise electrode alignment nor droplet-droplet or droplet-electric field synchronisation. Droplets can be reliably merged in pairs at a rate up to 50 fusion events per second. The fusion mechanism is based on the balance between viscous, electric and interfacial stresses at the droplet interface and depends upon the flow behaviour in the microchannel. Experimental results show that, under different conditions of frequency, applied potential and size of the droplets with respect to the channel geometry, diverse types of droplet coalescence occur. The fusion mechanism and general trends which enabled different merging results are proposed. This system has potential for being applied and multiplexed for high throughput, emulsion-based applications in the field of combinatorial reactions and screening bioassays.
Lab on a Chip, 2007
The study and the exploitation of membrane proteins for drug screening applications requires a co... more The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.
Journal of Micromechanics and Microengineering, 2007
The use of spark assisted chemical engraving (SACE) to produce glass apertures that are suitable ... more The use of spark assisted chemical engraving (SACE) to produce glass apertures that are suitable for the formation of artificial bilayer lipid membranes is described. Prior to use, the glass apertures were rendered hydrophobic by a silanization process and were then incorporated into a simple microfluidic device. Successful bilayer lipid membrane (BLM) formation and the subsequent acquisition of single-channel recordings
IEE Proceedings - Nanobiotechnology, 2006
The study of ion channels and other membrane proteins and their potential use as biosensors and d... more The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render proteincarrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatinergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the b-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.
Biosensors and Bioelectronics, 2009
In recent years, protein array technologies have found widespread applications in proteomics. How... more In recent years, protein array technologies have found widespread applications in proteomics. However, new methods for high-throughput analysis of protein-protein and protein-compound interactions are still required. In this paper, an array of lipid bilayer membranes formed within a microfluidic system with integrated electrodes is presented. The system is comprised of three layers that are clamped together, thus rendering the device cleanable and reusable. The device microfluidics enable the simultaneous formation of an array of lipid bilayers using a previously developed air-exposure technique, thereby avoiding the need to manually form individual bilayers. The Ag/AgCl electrodes allow for ion channel measurements, each of the sites being independently addressable. Typically, a 50% yield in simultaneous lipid bilayer formation over 12 sites was obtained and ion channel recordings have been acquired over multiple sites. This system has great potential for the development of an automatable platform of suspended lipid bilayer arrays.
Biophysical Journal, 2008
In addition to the annular or boundary lipids that surround the transmembrane surface of the pota... more In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each proteinprotein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 6 0.06 in units of mol fraction, implying that the nonannular sites will only be ;70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from ;2.5% in the presence of 25 mol % phosphatidylglycerol to ;62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K 1 close to the membrane surface.
Analytical Chemistry, 2009
Limitations imposed by conventional analytical technologies for cell biology, such as flow cytome... more Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (∼300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15 000-30 000 cells.
Analytical and Bioanalytical Chemistry, 2009
We describe a system that provides a rapid and simple way of forming suspended lipid bilayers wit... more We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid-liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid-alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.
Human molecular genetics, 2015
The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human... more The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human CNS. Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC) derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analysed the expression, and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10+16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from F...
Methods in Cell Biology, 2011
Droplet microfluidics (DM) is an area of research which combines lab-on-a-chip (LOC) techniques w... more Droplet microfluidics (DM) is an area of research which combines lab-on-a-chip (LOC) techniques with emulsion compartmentalization to perform high-throughput, chemical and biological assays. The key issue of this approach lies in the generation, over tens of milliseconds, of thousands of liquid vessels which can be used either as a carrier, to transport encapsulated particles and cells, or as microreactors, to perform parallel analysis of a vast number of samples. Each compartment comprises a liquid droplet containing the sample, surrounded by an immiscible fluid. This microfluidic technique is capable of generating subnanoliter and highly monodispersed liquid droplets, which offer many opportunities for developing novel single-cell and single-molecule studies, as well as high-throughput methodologies for the detection and sorting of encapsulated species in droplets. The aim of this chapter is to give an overview of the features of DM in a broad microfluidic context, as well as to show the advantages and limitations of the technology in the field of LOC analytical research. Examples are reported and discussed to show how DM can provide novel systems with applications in high-throughput, quantitative cell and particle analysis.
Physical Review E, 2009
Two phase microfluidic systems creating size-controlled microdroplets have recently emerged as po... more Two phase microfluidic systems creating size-controlled microdroplets have recently emerged as powerful tools to achieve liquid compartmentalization for high throughput chemical and biological assays. Emulsion electrocoalescence is a destabilization process that can be used in droplet-based platforms for water phase separation to enable lab-on-a-chip applications in biotechnology, including particle or cell recovery. In this paper, we report upon a series of phenomena associated with electrocoalescence of water microdroplet-in-oil populations in microfluidics. In our experiments, we formed microdroplets whose size and dispersion in the channel were varied according to the ratio of the flow rates of the two phases. Different types of electrocoalescence between droplets were obtained. For low applied voltages, drops merged in pairs over the electrode region; for higher values of the applied voltage, a cascade of droplet coalescence was produced against the flow direction, for a range of droplet sizes, lateral distributions of droplets in the channel and localized electric fields.
Langmuir, 2010
Multiphase microfluidics offer a wide range of functionalities in the fields of fluid dynamics, b... more Multiphase microfluidics offer a wide range of functionalities in the fields of fluid dynamics, biology, particle synthesis, and, more recently, also in logical computation. In this article, we describe the hysteresis of immiscible, multiphase flow obtained in hydrophilic, microfluidic systems at a T-junction. Stable and unstable state behaviors, in the form of segmented and parallel flow patterns of oil and water, were reliably produced, depending upon the history of the flow rates applied to the phases. The transition mechanisms between the two states were analyzed both experimentally and using numerical simulations, describing how the physical and fluid dynamic parameters influenced the hysteretic behavior of the flow. The characteristics of these multiphase systems render them suitable to be used as pressure comparators and also for the implementation of microfluidic logic operations.
Langmuir, 2010
Electrocoalescence of water-in-oil microdroplets in microfluidic channels is an active technique ... more Electrocoalescence of water-in-oil microdroplets in microfluidic channels is an active technique that enables droplet-based mixing functionalities to be achieved in lab-on-a-chip applications. In this work, a characterization of the electrocoalescence mechanisms of water microdroplets in oil is presented, using localized electric field systems. We report a theoretical and experimental description of the electrocoalscence behavior of droplet pairs by varying the physical and fluid dynamic conditions of the phases. Our results demonstrate that localized electric field systems can be reliably used to merge droplets in pairs, regardless of the distance between the drops. The coalescence behavior was dependent upon the viscosity of the continuous phase for water droplets that were separated by a thick layer of oil and upon interfacial tension for droplets that were in close proximity. We showed that these systems have the potential to be used for high-throughput applications and that, unlike other examples of active systems in the literature, the need of droplet synchronization and the application of high voltages is considerably reduced.
Langmuir, 2007
To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be e... more To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.
Lab on a Chip, 2008
We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electri... more We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.
Lab on a Chip, 2009
Electric fields have previously been used in microfluidic devices for the manipulation, sorting a... more Electric fields have previously been used in microfluidic devices for the manipulation, sorting and mixing of microemulsions. Here, an active system for on-demand electrocoalescence of water droplets in oil is presented. The platform does not require precise electrode alignment nor droplet-droplet or droplet-electric field synchronisation. Droplets can be reliably merged in pairs at a rate up to 50 fusion events per second. The fusion mechanism is based on the balance between viscous, electric and interfacial stresses at the droplet interface and depends upon the flow behaviour in the microchannel. Experimental results show that, under different conditions of frequency, applied potential and size of the droplets with respect to the channel geometry, diverse types of droplet coalescence occur. The fusion mechanism and general trends which enabled different merging results are proposed. This system has potential for being applied and multiplexed for high throughput, emulsion-based applications in the field of combinatorial reactions and screening bioassays.
Lab on a Chip, 2007
The study and the exploitation of membrane proteins for drug screening applications requires a co... more The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.
Journal of Micromechanics and Microengineering, 2007
The use of spark assisted chemical engraving (SACE) to produce glass apertures that are suitable ... more The use of spark assisted chemical engraving (SACE) to produce glass apertures that are suitable for the formation of artificial bilayer lipid membranes is described. Prior to use, the glass apertures were rendered hydrophobic by a silanization process and were then incorporated into a simple microfluidic device. Successful bilayer lipid membrane (BLM) formation and the subsequent acquisition of single-channel recordings
IEE Proceedings - Nanobiotechnology, 2006
The study of ion channels and other membrane proteins and their potential use as biosensors and d... more The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render proteincarrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatinergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the b-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.
Biosensors and Bioelectronics, 2009
In recent years, protein array technologies have found widespread applications in proteomics. How... more In recent years, protein array technologies have found widespread applications in proteomics. However, new methods for high-throughput analysis of protein-protein and protein-compound interactions are still required. In this paper, an array of lipid bilayer membranes formed within a microfluidic system with integrated electrodes is presented. The system is comprised of three layers that are clamped together, thus rendering the device cleanable and reusable. The device microfluidics enable the simultaneous formation of an array of lipid bilayers using a previously developed air-exposure technique, thereby avoiding the need to manually form individual bilayers. The Ag/AgCl electrodes allow for ion channel measurements, each of the sites being independently addressable. Typically, a 50% yield in simultaneous lipid bilayer formation over 12 sites was obtained and ion channel recordings have been acquired over multiple sites. This system has great potential for the development of an automatable platform of suspended lipid bilayer arrays.
Biophysical Journal, 2008
In addition to the annular or boundary lipids that surround the transmembrane surface of the pota... more In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each proteinprotein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 6 0.06 in units of mol fraction, implying that the nonannular sites will only be ;70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from ;2.5% in the presence of 25 mol % phosphatidylglycerol to ;62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K 1 close to the membrane surface.
Analytical Chemistry, 2009
Limitations imposed by conventional analytical technologies for cell biology, such as flow cytome... more Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (∼300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15 000-30 000 cells.
Analytical and Bioanalytical Chemistry, 2009
We describe a system that provides a rapid and simple way of forming suspended lipid bilayers wit... more We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid-liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid-alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.
Human molecular genetics, 2015
The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human... more The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human CNS. Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC) derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analysed the expression, and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10+16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from F...