• L-DEP based rapid dispensing of supported lipid bilayer (SLB) and bilayer lipid vesicle (BLV). ... more • L-DEP based rapid dispensing of supported lipid bilayer (SLB) and bilayer lipid vesicle (BLV). • Functionalization of dispensed SLBs and BLVs with quantum dots labeled nucleic acid bio-probes/markers. • Application of functionalized SLBs and BLVs for on-chip, FRET based nucleic acid isolation and detection assays. g r a p h i c a l a b s t r a c t This work demonstrates liquid dielectrophoresis (L-DEP) based droplet microfluidic (DMF) methodology to dispense multitude of precisely positioned supported lipid bilayers (SLB) and bilayer lipid vesicles (BLV). It furthermore demonstrate their utility for on-chip nucleic acid detection. Keywords: Supported lipid bilayer (SLB) Bilayer lipid vesicle (BLV) Liquid-dielectrophoresis (L-DEP) Droplet-dielectrophoresis (D-DEP) Nucleic acid assays a b s t r a c t Lipid bilayer membranes in biological cells are an essential component of cellular infrastructure and furthermore responsible for various transductional afunctionalities. Synthetic lipid membranes can be deposited as supported lipid bilayer (SLB) on functionalized solid surfaces or, as large bilayer lipid vesicles (BLV). Such artificial lipid membranes can serve as fundamental building blocks and models for mimicking biological membrane interfaces, which maybe leveraged for chip based bio-sensor applications. In this paper, we have utilized liquid dielectrophoresis (L-DEP) based droplet microfluidic (DMF) methodology to dispense, multitude of precisely positioned SLB and BLV membranes, over the size range of: 2.8–100 m, in an on-chip rapid, multiplexed dispensing process, not readily achieved by conventional dispensing methods. The deposited SLB and BLV assemblies have been utilized to demonstrate nucleic acid isolation and hybridization assays, using a Q-Dot ® nano-particles FRET analysis.
Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by levera... more Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples OPEN ACCESS Micromachines 2015, 6 64 were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.
Nano-textured super hydrophobic (SH) surfaces can achieve very high droplet contact angles (typic... more Nano-textured super hydrophobic (SH) surfaces can achieve very high droplet contact angles (typically >150 •) and are furthermore associated with reduced surface adsorption of enzymes and other macro-molecules. The advantages of such SH surfaces have already been explored for electrostatic based digital microfluidic (DMF) devices where the high CA and the reduced surface adsorption largely benefits the handling and manipulation of complex biochemical samples/reagents. Liquid dielectrophoresis (LDEP) based digital microfluidic (DMF) technology has demonstrated its capability for achieving combinato-rial assays on standard hydrophobic surfaces. However, such devices suffer from similar issues as DMF devices when it comes to handling bio-reagents/samples where off-chip sample handling steps (wash-ing/buffer exchange) are often required prior to the on-chip bio-assay. In this work, we have optimized a comprehensive fabrication protocol to develop a nano-textured SH surface topology for LDEP device applications. The static and transient behavior of LDEP actuation over the SH surface has been investigated using an improved lumped model and furthermore experimentally verified using specifically tailored liquid samples. The developed SH surface has also been analyzed for liquid actuation of TAQ DNA polymerase enzyme which is a key component of most nucleic acid amplification assays. The performance of LDEP actuation of tailored liquid samples is analyzed and compared for both hydrophobic and SH surfaces. The reported work demonstrates that specific textured SH surfaces can successfully achieve reliable LDEP actuation and furthermore dispense/manipulate TAQ DNA polymerase enzyme and various other aqueous samples.
• L-DEP based rapid dispensing of supported lipid bilayer (SLB) and bilayer lipid vesicle (BLV). ... more • L-DEP based rapid dispensing of supported lipid bilayer (SLB) and bilayer lipid vesicle (BLV). • Functionalization of dispensed SLBs and BLVs with quantum dots labeled nucleic acid bio-probes/markers. • Application of functionalized SLBs and BLVs for on-chip, FRET based nucleic acid isolation and detection assays. g r a p h i c a l a b s t r a c t This work demonstrates liquid dielectrophoresis (L-DEP) based droplet microfluidic (DMF) methodology to dispense multitude of precisely positioned supported lipid bilayers (SLB) and bilayer lipid vesicles (BLV). It furthermore demonstrate their utility for on-chip nucleic acid detection. Keywords: Supported lipid bilayer (SLB) Bilayer lipid vesicle (BLV) Liquid-dielectrophoresis (L-DEP) Droplet-dielectrophoresis (D-DEP) Nucleic acid assays a b s t r a c t Lipid bilayer membranes in biological cells are an essential component of cellular infrastructure and furthermore responsible for various transductional afunctionalities. Synthetic lipid membranes can be deposited as supported lipid bilayer (SLB) on functionalized solid surfaces or, as large bilayer lipid vesicles (BLV). Such artificial lipid membranes can serve as fundamental building blocks and models for mimicking biological membrane interfaces, which maybe leveraged for chip based bio-sensor applications. In this paper, we have utilized liquid dielectrophoresis (L-DEP) based droplet microfluidic (DMF) methodology to dispense, multitude of precisely positioned SLB and BLV membranes, over the size range of: 2.8–100 m, in an on-chip rapid, multiplexed dispensing process, not readily achieved by conventional dispensing methods. The deposited SLB and BLV assemblies have been utilized to demonstrate nucleic acid isolation and hybridization assays, using a Q-Dot ® nano-particles FRET analysis.
Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by levera... more Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples OPEN ACCESS Micromachines 2015, 6 64 were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.
Nano-textured super hydrophobic (SH) surfaces can achieve very high droplet contact angles (typic... more Nano-textured super hydrophobic (SH) surfaces can achieve very high droplet contact angles (typically >150 •) and are furthermore associated with reduced surface adsorption of enzymes and other macro-molecules. The advantages of such SH surfaces have already been explored for electrostatic based digital microfluidic (DMF) devices where the high CA and the reduced surface adsorption largely benefits the handling and manipulation of complex biochemical samples/reagents. Liquid dielectrophoresis (LDEP) based digital microfluidic (DMF) technology has demonstrated its capability for achieving combinato-rial assays on standard hydrophobic surfaces. However, such devices suffer from similar issues as DMF devices when it comes to handling bio-reagents/samples where off-chip sample handling steps (wash-ing/buffer exchange) are often required prior to the on-chip bio-assay. In this work, we have optimized a comprehensive fabrication protocol to develop a nano-textured SH surface topology for LDEP device applications. The static and transient behavior of LDEP actuation over the SH surface has been investigated using an improved lumped model and furthermore experimentally verified using specifically tailored liquid samples. The developed SH surface has also been analyzed for liquid actuation of TAQ DNA polymerase enzyme which is a key component of most nucleic acid amplification assays. The performance of LDEP actuation of tailored liquid samples is analyzed and compared for both hydrophobic and SH surfaces. The reported work demonstrates that specific textured SH surfaces can successfully achieve reliable LDEP actuation and furthermore dispense/manipulate TAQ DNA polymerase enzyme and various other aqueous samples.
Uploads
Papers by Ravi Prakash