Numerical Simulations were performed to see the effect of geometrical misalignment on the progres... more Numerical Simulations were performed to see the effect of geometrical misalignment on the progression of a sample plug in electrokinetic flows. The effect of material mismatch along with combined material and geometrical mismatch has also been investigated. A sample plug with low diffusion coefficient was introduced after the flow was fully developed. The effect of material and geometrical mismatch on the cross-sectional average of the plug concentrations were evaluated from the point of view of an optical detector. Various degrees of geometrical mismatch ranging from 0% to 75% were introduced and its effect on sample plug was evaluated. Comparison has been made with pressure driven flows to quantify the effect of geometrical mismatch.
The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point muta... more The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). A microscale Ligase Detection Reaction (LDR) device was designed and manufactured in polycarbonate. There are at least two mixing stages involved in the LDR identification process. Various micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries were selected for fabrication. Passive diffusional micromixers were made with aspect ratios from 7 to 20. The mixers were made by SU-8 lithography, LIGA, laser ablation and micromilling to characterize each fabrication method. It was found that LIGA was best for making the micromixers, but was the longest process. The micromixers were fabricated and are being tested using fluorescent dyes. For a successful reaction temperatures of 0°C, 95°C and 65°C were needed. A stationary chamber method was used with thermal cycling in which the sample held while the temperature is cycled. Finit...
In microarray studies alterations in gene expression in circulating leukocytes have shown utility... more In microarray studies alterations in gene expression in circulating leukocytes have shown utility for ischemic stroke diagnosis. We studied forty candidate markers identified in three gene expression profiles to (1) quantitate individual transcript expression, (2) identify transcript clusters and (3) assess the clinical diagnostic utility of the clusters identified for ischemic stroke detection. Using high throughput next generation qPCR 16 of the 40 transcripts were significantly up-regulated in stroke patients relative to control subjects (p<0.05). Six clusters of between 5 and 7 transcripts were identified that discriminated between stroke and control (p values between 1.01e-9 and 0.03). A 7 transcript cluster containing PLBD1, PYGL, BST1, DUSP1, FOS, VCAN and FCGR1A showed high accuracy for stroke classification (AUC=0.854). These results validate and improve upon the diagnostic value of transcripts identified in microarray studies for ischemic stroke. The clusters identified...
Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) assoc... more Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 → 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (μSPE) consisting of a plastic chip that is activated with UV/...
... Nanolayered microposts were tested for their thermal expansion behavior on a thermomechanical... more ... Nanolayered microposts were tested for their thermal expansion behavior on a thermomechanical analyzer (TMA) (model 2940, TA Instruments, New Castle, DE) at Stork Technimet, Inc. (New Berlin, WI). ... JDErvin, JDBrei, and D.Brei, IEEE/ASME Trans. ...
Simulations and experiments to assess the predictability of dimensional and locational tolerances... more Simulations and experiments to assess the predictability of dimensional and locational tolerances of passive alignment structures on injection molded microfluidic components were performed. A center-gated disk with microscale assembly features, to aid metrology, was reproduced using injection molding. The feature dimensions were 100, 200, 300, and 400 μ. Dimensions of the features were measured using optical profilometery and optical microscopy. Simulations using a commercial package overestimated replication fidelity. Mold surface temperatures and injection speeds significantly affected the replication fidelity as the ratio of surface area to volume increased. The location of better replication fidelity, at each mold surface temperature, moved from the edge of the mold cavity to the injection point as the mold surface temperature increased from 100°C to 150°C. Therefore, process parameters and the design of a mold have to be considered for successful replication of the features.
A polymer-based, optical waveguide with an integrated coupling prism and microlenses was designed... more A polymer-based, optical waveguide with an integrated coupling prism and microlenses was designed and fabricated to improve overall optical performances. The use of flycutting allowed accurate control of the polymer waveguide thickness. A large prism facilitated better coupling of the light to the waveguide. The highest intensity evanescent excitation of the waveguide was obtained at the critical angle of the waveguide. Fluorescent radiation was highly focused by the array of microlenses. The microfabricated waveguide will allow the rapid, low-cost detection of the fluorescent samples in biomedical applications.
Fluidic interconnects provide the passages for the transport of liquid analytes, containing mass ... more Fluidic interconnects provide the passages for the transport of liquid analytes, containing mass and information, from one component to another in a microfluidic system. The pressure capacity of a novel, modular, gasketless, chip-to-chip microfluidic interconnect that forms a seal with a liquid bridge suspended between concentric through-holes was evaluated experimentally (see Figure 1). [5] The maximum rupture pressure measured for the gasketless interconnect was 21.4 kPa (3.1 psig), which corresponded to a measured assembly gap distance of 3.4 m. The gasketless interconnect withstood maximum pressures seen in microfluidic systems and is realizable within manufacturing variation.
Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of li... more Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of liquids between component chips and to/from microfluidic systems. Unfortunately, most interconnect designs add additional physical constraints to chips with each additional interconnect leading to over-constrained microfluidic systems. The competing constraints provided by multiple interconnects induce strain in the chips, creating indeterminate dead volumes and misalignment between chips that comprise the microfluidic system. A novel, gasketless superhydrophobic fluidic interconnect (GSFI) that uses capillary forces to form a liquid bridge suspended between concentric through-holes and acting as a fluid passage was investigated. The GSFI decouples the alignment between component chips from the interconnect function and the attachment of the meniscus of the liquid bridge to the edges of the holes produces negligible dead volume. This passive seal was created by patterning parallel superhyd...
A method for the design, construction, and assembly of modular, polymer-based, microfluidic devic... more A method for the design, construction, and assembly of modular, polymer-based, microfluidic devices using simple micro-assembly technology was demonstrated to build an integrated fluidic system consisting of vertically stacked modules for carrying out multi-step molecular assays. As an example of the utility of the modular system, point mutation detection using the ligase detection reaction (LDR) following amplification by the polymerase chain reaction (PCR) was carried out. Fluid interconnects and standoffs ensured that temperatures in the vertically stacked reactors were within ± 0.2 C° at the center of the temperature zones and ± 1.1 C° overall. The vertical spacing between modules was confirmed using finite element models (ANSYS, Inc., Canonsburg, PA) to simulate the steady-state temperature distribution for the assembly. Passive alignment structures, including a hemispherical pin-in-hole, a hemispherical pin-in-slot, and a plate-plate lap joint, were developed using screw theory to enable accurate exactly constrained assembly of the microfluidic reactors, cover sheets, and fluid interconnects to facilitate the modular approach. The mean mismatch between the centers of adjacent through holes was 64 ± 7.7 μm, significantly reducing the dead volume necessary to accommodate manufacturing variation. The microfluidic components were easily assembled by hand and the assembly of several different configurations of microfluidic modules for executing the assay was evaluated. Temperatures were measured in the desired range in each reactor. The biochemical performance was comparable to that obtained with benchtop instruments, but took less than 45 min to execute, half the time.
Low-cost modular, polymer microfluidic platforms, integrating several different functional units,... more Low-cost modular, polymer microfluidic platforms, integrating several different functional units, may potentially reduce the cost of molecular and environmental analyses and enable broader applications. Proper function of such systems depends on well-characterized assembly of the instruments. Passive alignment is one approach to obtaining such assemblies. Model modular devices, containing passive alignment features, hemispherical pins in v-grooves, and integrated alignment standards for characterizing the accuracy of the assemblies, were replicated in polycarbonate using doubled-sided injection molding. The dimensions and locations of the assembly features and alignment standards were measured. The assemblies had mismatches from 16±4 µmt o2 0 ±6 µm along the X-axis and from 103±7 µm to 118±11 µm along the Y-axis. The vertical variation from the nominal value of 287 µm ranged from −10±4 µmt o 34±7 µm. An assembly tolerance model was used to estimate the accuracy of the assemblies based on the manufacturing variations of the alignment structures. Variation of the alignment structure features were propagated through the assembly using Monte Carlo methods. The estimated distributions matched the measured experimental results well, with differences of 2∼13% due to unmodeled aspects of the variations. Accurate assembly of advanced polymer microsystems is feasible and predictable in the design phase.
A fluidic control chip was incorporated to control delivery of PCR cocktail to a multi-well (MW) ... more A fluidic control chip was incorporated to control delivery of PCR cocktail to a multi-well (MW) continuous flow PCR (CFPCR) chip. Passive micro-assembly was implemented for modular integration of the fluidic control chip and the CFPCR chip using three sets of passive alignment structures to obtain exact kinematic constraint. The fluidic control chip was assembled with the CFPCR chip by thermal bonding and used for parallel amplification of DNA fragments. Successful, simultaneous amplification of DNA fragments, with lengths of 99bp, 125bp, 150bp, 200bp, 500bp, 997bp, was demonstrated on the whole, high throughput CFPCR platform with a distributed thermal system.
ABSTRACTThis work is a part of an on-going research effort to develop an array of micro thermoele... more ABSTRACTThis work is a part of an on-going research effort to develop an array of micro thermoelectric coolers (TECs) for highly localized control of temperature at the cellular level. Prefabrication experimentation and modeling were carried out to understand the behavior of the proposed device. Mathematical models were used to identify important device parameters and optimal device dimensions. Preliminary experiments have shown that it is feasible to produce the TECs through electrodeposition of bismuth and telluride on modules produced using a modified multistep LIGA (Lithographie, Galvanoformung and Abformung) technique. The development and characterization of the proposed TECs would enable the bioengineer highly localized control of temperature in a native or artificial tissue system. Thus enabling further usage of low temperatures in biological systems for both destructive (cryosurgical) and beneficial (cryopreservation) procedures.
This paper present a method of rapid replication of polymeric high aspect ratio microstructures (... more This paper present a method of rapid replication of polymeric high aspect ratio microstructures (HARMs) and a method of rapid reproduction of metallic micromold inserts for HARMs using polydimethylsiloxane (PDMS) casting and standard LIGA processes. A high aspect ratio (HAR) metallic micromold insert, featuring a variety of test microstructures made of electroplated nickel with 15:1 height-to-width ratio for 300 lm microstructures, was fabricated by the standard LIGA process using deep X-ray lithography (DXRL). A 10:1 mixture of pre-polymer PDMS and a curing agent were cast onto the HAR metallic micromold insert, cured and peeled off to create reverse images of the HAR metallic micromold insert in PDMS. In addition to the replication of polymeric HARMs, replicated PDMS HARMS were coated with a metallic sacrificial layer and electroplated in nickel to reproduce another metallic micromold insert. This method can be used to rapidly and massively reproduce HAR metallic micromold inserts in low cost mass production manner without further using DXRL.
Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for micro... more Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC-68 uC) zones; above 6 mm s 21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s 21 to 20 mm s 21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle 21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle 21). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).
A novel microfluidic device that can selectively and specifically isolate exceedingly small numbe... more A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 μm width × 150 μm depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.
Technologies and methods of prototyping microfluidic devices are widely used in solving many biol... more Technologies and methods of prototyping microfluidic devices are widely used in solving many biological problems and testing of operability of new microanalytic systems. This study is devoted to analyzing the features of the formation of microstructures in SU-8 photoresist and the preparation of replicas in polydimethyl siloxane by the soft lithography method. It has been shown that the aspect ratio of the resultant microstructures is determined by their shape, size, and the force of resist adhesion to the silicon substrate and the efficiency of the circulation of the developer around microstructures. In the replication of complex microstructures, an aspect ratio of ~25 is attained. The technology considered here is used to prepare microfluidic chips with mechanical traps for fixation and the in vitro analysis of living cells.
High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-l... more High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-long 50-m-wide high aspect ratio columns will be the separation component of a handheld gas chromatograph device for detecting semivolatile and volatile compounds. As a first step, 600-m-deep electrodeposited nickel columns were fabricated. The serpentine columns were sealed and pressure-flow rate characteristics compared with the theoretical values. The response of the sealed columns was studied by running methane gas plugs through uncoated columns with a flame ionization detector at the exit. Negligible flow-induced dispersion was observed in the sealed metal columns. Unretained peak widths of 15 ms were measured, and the experimental pressure and flow rate distributions matched those predicted by established analytical models within 2.5%. Columns were coated with OV-1 stationary phase using static coating methods. A mixture of four hydrocarbons C 6 , C 8 , C 10 , and C 12 was separated in a coated 50 m by 600 m by 0.5 m column in less than 2 s at 70 C.
High flow rate capture of circulating tumor cells (CTCs) was performed with a high recovery rate.... more High flow rate capture of circulating tumor cells (CTCs) was performed with a high recovery rate. A new concept for rare target cell capture using a micro device was investigated parametrically using numerical simulations. High flow rate device (HFRD) prototypes were fabricated based on the simulation results. The devices were coated with antibodies (anti-EpCAM) after UV modification and amine functionalization. The simulated CTCs (MCF-7 cells) were collected and spiked in ~40% hematocrit solutions of human red blood cells. The polymer high flow rate device captured the CTCs with an 80% average recovery rate at a flow rate of 750 μL/min.
A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate ... more A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate functional titer plate-based microfluidic platforms. Nickel, large area mold inserts were fabricated using an SU-8 based, UV-LIGA technique on 150 mm diameter silicon substrates. Prior to UV exposure, the prebaked SU-8 resist was flycut to reduce the total thickness variation to less than 5 μm. Excellent UV lithography results, with highly vertical sidewalls, were obtained in the SU-8 by using an UV filter to remove high absorbance wavelengths below 350 nm. Overplating of nickel in the SU-8 patterns produced high quality, high precision, metal mold inserts, which were used to replicate titer plate-based SPRI reactors using hot embossing of polycarbonate (PC). Optimized molding conditions yielded good feature replication fidelity and feature location integrity over the entire surface area. Thermal fusion bonding of the molded PC chips at 150°C resulted in leak-free sealing, which was verified in leakage tests using a fluorescent dye. The assembled SPRI reactor was used for simple, fast purification of genomic DNA from whole cell lysates of several bacterial species, which was verified by PCR amplification of the purified genomic DNA.
Numerical Simulations were performed to see the effect of geometrical misalignment on the progres... more Numerical Simulations were performed to see the effect of geometrical misalignment on the progression of a sample plug in electrokinetic flows. The effect of material mismatch along with combined material and geometrical mismatch has also been investigated. A sample plug with low diffusion coefficient was introduced after the flow was fully developed. The effect of material and geometrical mismatch on the cross-sectional average of the plug concentrations were evaluated from the point of view of an optical detector. Various degrees of geometrical mismatch ranging from 0% to 75% were introduced and its effect on sample plug was evaluated. Comparison has been made with pressure driven flows to quantify the effect of geometrical mismatch.
The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point muta... more The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). A microscale Ligase Detection Reaction (LDR) device was designed and manufactured in polycarbonate. There are at least two mixing stages involved in the LDR identification process. Various micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries were selected for fabrication. Passive diffusional micromixers were made with aspect ratios from 7 to 20. The mixers were made by SU-8 lithography, LIGA, laser ablation and micromilling to characterize each fabrication method. It was found that LIGA was best for making the micromixers, but was the longest process. The micromixers were fabricated and are being tested using fluorescent dyes. For a successful reaction temperatures of 0°C, 95°C and 65°C were needed. A stationary chamber method was used with thermal cycling in which the sample held while the temperature is cycled. Finit...
In microarray studies alterations in gene expression in circulating leukocytes have shown utility... more In microarray studies alterations in gene expression in circulating leukocytes have shown utility for ischemic stroke diagnosis. We studied forty candidate markers identified in three gene expression profiles to (1) quantitate individual transcript expression, (2) identify transcript clusters and (3) assess the clinical diagnostic utility of the clusters identified for ischemic stroke detection. Using high throughput next generation qPCR 16 of the 40 transcripts were significantly up-regulated in stroke patients relative to control subjects (p<0.05). Six clusters of between 5 and 7 transcripts were identified that discriminated between stroke and control (p values between 1.01e-9 and 0.03). A 7 transcript cluster containing PLBD1, PYGL, BST1, DUSP1, FOS, VCAN and FCGR1A showed high accuracy for stroke classification (AUC=0.854). These results validate and improve upon the diagnostic value of transcripts identified in microarray studies for ischemic stroke. The clusters identified...
Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) assoc... more Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 → 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (μSPE) consisting of a plastic chip that is activated with UV/...
... Nanolayered microposts were tested for their thermal expansion behavior on a thermomechanical... more ... Nanolayered microposts were tested for their thermal expansion behavior on a thermomechanical analyzer (TMA) (model 2940, TA Instruments, New Castle, DE) at Stork Technimet, Inc. (New Berlin, WI). ... JDErvin, JDBrei, and D.Brei, IEEE/ASME Trans. ...
Simulations and experiments to assess the predictability of dimensional and locational tolerances... more Simulations and experiments to assess the predictability of dimensional and locational tolerances of passive alignment structures on injection molded microfluidic components were performed. A center-gated disk with microscale assembly features, to aid metrology, was reproduced using injection molding. The feature dimensions were 100, 200, 300, and 400 μ. Dimensions of the features were measured using optical profilometery and optical microscopy. Simulations using a commercial package overestimated replication fidelity. Mold surface temperatures and injection speeds significantly affected the replication fidelity as the ratio of surface area to volume increased. The location of better replication fidelity, at each mold surface temperature, moved from the edge of the mold cavity to the injection point as the mold surface temperature increased from 100°C to 150°C. Therefore, process parameters and the design of a mold have to be considered for successful replication of the features.
A polymer-based, optical waveguide with an integrated coupling prism and microlenses was designed... more A polymer-based, optical waveguide with an integrated coupling prism and microlenses was designed and fabricated to improve overall optical performances. The use of flycutting allowed accurate control of the polymer waveguide thickness. A large prism facilitated better coupling of the light to the waveguide. The highest intensity evanescent excitation of the waveguide was obtained at the critical angle of the waveguide. Fluorescent radiation was highly focused by the array of microlenses. The microfabricated waveguide will allow the rapid, low-cost detection of the fluorescent samples in biomedical applications.
Fluidic interconnects provide the passages for the transport of liquid analytes, containing mass ... more Fluidic interconnects provide the passages for the transport of liquid analytes, containing mass and information, from one component to another in a microfluidic system. The pressure capacity of a novel, modular, gasketless, chip-to-chip microfluidic interconnect that forms a seal with a liquid bridge suspended between concentric through-holes was evaluated experimentally (see Figure 1). [5] The maximum rupture pressure measured for the gasketless interconnect was 21.4 kPa (3.1 psig), which corresponded to a measured assembly gap distance of 3.4 m. The gasketless interconnect withstood maximum pressures seen in microfluidic systems and is realizable within manufacturing variation.
Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of li... more Chip-to-chip and world-to-chip fluidic interconnections are paramount to enable the passage of liquids between component chips and to/from microfluidic systems. Unfortunately, most interconnect designs add additional physical constraints to chips with each additional interconnect leading to over-constrained microfluidic systems. The competing constraints provided by multiple interconnects induce strain in the chips, creating indeterminate dead volumes and misalignment between chips that comprise the microfluidic system. A novel, gasketless superhydrophobic fluidic interconnect (GSFI) that uses capillary forces to form a liquid bridge suspended between concentric through-holes and acting as a fluid passage was investigated. The GSFI decouples the alignment between component chips from the interconnect function and the attachment of the meniscus of the liquid bridge to the edges of the holes produces negligible dead volume. This passive seal was created by patterning parallel superhyd...
A method for the design, construction, and assembly of modular, polymer-based, microfluidic devic... more A method for the design, construction, and assembly of modular, polymer-based, microfluidic devices using simple micro-assembly technology was demonstrated to build an integrated fluidic system consisting of vertically stacked modules for carrying out multi-step molecular assays. As an example of the utility of the modular system, point mutation detection using the ligase detection reaction (LDR) following amplification by the polymerase chain reaction (PCR) was carried out. Fluid interconnects and standoffs ensured that temperatures in the vertically stacked reactors were within ± 0.2 C° at the center of the temperature zones and ± 1.1 C° overall. The vertical spacing between modules was confirmed using finite element models (ANSYS, Inc., Canonsburg, PA) to simulate the steady-state temperature distribution for the assembly. Passive alignment structures, including a hemispherical pin-in-hole, a hemispherical pin-in-slot, and a plate-plate lap joint, were developed using screw theory to enable accurate exactly constrained assembly of the microfluidic reactors, cover sheets, and fluid interconnects to facilitate the modular approach. The mean mismatch between the centers of adjacent through holes was 64 ± 7.7 μm, significantly reducing the dead volume necessary to accommodate manufacturing variation. The microfluidic components were easily assembled by hand and the assembly of several different configurations of microfluidic modules for executing the assay was evaluated. Temperatures were measured in the desired range in each reactor. The biochemical performance was comparable to that obtained with benchtop instruments, but took less than 45 min to execute, half the time.
Low-cost modular, polymer microfluidic platforms, integrating several different functional units,... more Low-cost modular, polymer microfluidic platforms, integrating several different functional units, may potentially reduce the cost of molecular and environmental analyses and enable broader applications. Proper function of such systems depends on well-characterized assembly of the instruments. Passive alignment is one approach to obtaining such assemblies. Model modular devices, containing passive alignment features, hemispherical pins in v-grooves, and integrated alignment standards for characterizing the accuracy of the assemblies, were replicated in polycarbonate using doubled-sided injection molding. The dimensions and locations of the assembly features and alignment standards were measured. The assemblies had mismatches from 16±4 µmt o2 0 ±6 µm along the X-axis and from 103±7 µm to 118±11 µm along the Y-axis. The vertical variation from the nominal value of 287 µm ranged from −10±4 µmt o 34±7 µm. An assembly tolerance model was used to estimate the accuracy of the assemblies based on the manufacturing variations of the alignment structures. Variation of the alignment structure features were propagated through the assembly using Monte Carlo methods. The estimated distributions matched the measured experimental results well, with differences of 2∼13% due to unmodeled aspects of the variations. Accurate assembly of advanced polymer microsystems is feasible and predictable in the design phase.
A fluidic control chip was incorporated to control delivery of PCR cocktail to a multi-well (MW) ... more A fluidic control chip was incorporated to control delivery of PCR cocktail to a multi-well (MW) continuous flow PCR (CFPCR) chip. Passive micro-assembly was implemented for modular integration of the fluidic control chip and the CFPCR chip using three sets of passive alignment structures to obtain exact kinematic constraint. The fluidic control chip was assembled with the CFPCR chip by thermal bonding and used for parallel amplification of DNA fragments. Successful, simultaneous amplification of DNA fragments, with lengths of 99bp, 125bp, 150bp, 200bp, 500bp, 997bp, was demonstrated on the whole, high throughput CFPCR platform with a distributed thermal system.
ABSTRACTThis work is a part of an on-going research effort to develop an array of micro thermoele... more ABSTRACTThis work is a part of an on-going research effort to develop an array of micro thermoelectric coolers (TECs) for highly localized control of temperature at the cellular level. Prefabrication experimentation and modeling were carried out to understand the behavior of the proposed device. Mathematical models were used to identify important device parameters and optimal device dimensions. Preliminary experiments have shown that it is feasible to produce the TECs through electrodeposition of bismuth and telluride on modules produced using a modified multistep LIGA (Lithographie, Galvanoformung and Abformung) technique. The development and characterization of the proposed TECs would enable the bioengineer highly localized control of temperature in a native or artificial tissue system. Thus enabling further usage of low temperatures in biological systems for both destructive (cryosurgical) and beneficial (cryopreservation) procedures.
This paper present a method of rapid replication of polymeric high aspect ratio microstructures (... more This paper present a method of rapid replication of polymeric high aspect ratio microstructures (HARMs) and a method of rapid reproduction of metallic micromold inserts for HARMs using polydimethylsiloxane (PDMS) casting and standard LIGA processes. A high aspect ratio (HAR) metallic micromold insert, featuring a variety of test microstructures made of electroplated nickel with 15:1 height-to-width ratio for 300 lm microstructures, was fabricated by the standard LIGA process using deep X-ray lithography (DXRL). A 10:1 mixture of pre-polymer PDMS and a curing agent were cast onto the HAR metallic micromold insert, cured and peeled off to create reverse images of the HAR metallic micromold insert in PDMS. In addition to the replication of polymeric HARMs, replicated PDMS HARMS were coated with a metallic sacrificial layer and electroplated in nickel to reproduce another metallic micromold insert. This method can be used to rapidly and massively reproduce HAR metallic micromold inserts in low cost mass production manner without further using DXRL.
Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for micro... more Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC-68 uC) zones; above 6 mm s 21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s 21 to 20 mm s 21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle 21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle 21). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).
A novel microfluidic device that can selectively and specifically isolate exceedingly small numbe... more A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 μm width × 150 μm depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.
Technologies and methods of prototyping microfluidic devices are widely used in solving many biol... more Technologies and methods of prototyping microfluidic devices are widely used in solving many biological problems and testing of operability of new microanalytic systems. This study is devoted to analyzing the features of the formation of microstructures in SU-8 photoresist and the preparation of replicas in polydimethyl siloxane by the soft lithography method. It has been shown that the aspect ratio of the resultant microstructures is determined by their shape, size, and the force of resist adhesion to the silicon substrate and the efficiency of the circulation of the developer around microstructures. In the replication of complex microstructures, an aspect ratio of ~25 is attained. The technology considered here is used to prepare microfluidic chips with mechanical traps for fixation and the in vitro analysis of living cells.
High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-l... more High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-long 50-m-wide high aspect ratio columns will be the separation component of a handheld gas chromatograph device for detecting semivolatile and volatile compounds. As a first step, 600-m-deep electrodeposited nickel columns were fabricated. The serpentine columns were sealed and pressure-flow rate characteristics compared with the theoretical values. The response of the sealed columns was studied by running methane gas plugs through uncoated columns with a flame ionization detector at the exit. Negligible flow-induced dispersion was observed in the sealed metal columns. Unretained peak widths of 15 ms were measured, and the experimental pressure and flow rate distributions matched those predicted by established analytical models within 2.5%. Columns were coated with OV-1 stationary phase using static coating methods. A mixture of four hydrocarbons C 6 , C 8 , C 10 , and C 12 was separated in a coated 50 m by 600 m by 0.5 m column in less than 2 s at 70 C.
High flow rate capture of circulating tumor cells (CTCs) was performed with a high recovery rate.... more High flow rate capture of circulating tumor cells (CTCs) was performed with a high recovery rate. A new concept for rare target cell capture using a micro device was investigated parametrically using numerical simulations. High flow rate device (HFRD) prototypes were fabricated based on the simulation results. The devices were coated with antibodies (anti-EpCAM) after UV modification and amine functionalization. The simulated CTCs (MCF-7 cells) were collected and spiked in ~40% hematocrit solutions of human red blood cells. The polymer high flow rate device captured the CTCs with an 80% average recovery rate at a flow rate of 750 μL/min.
A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate ... more A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate functional titer plate-based microfluidic platforms. Nickel, large area mold inserts were fabricated using an SU-8 based, UV-LIGA technique on 150 mm diameter silicon substrates. Prior to UV exposure, the prebaked SU-8 resist was flycut to reduce the total thickness variation to less than 5 μm. Excellent UV lithography results, with highly vertical sidewalls, were obtained in the SU-8 by using an UV filter to remove high absorbance wavelengths below 350 nm. Overplating of nickel in the SU-8 patterns produced high quality, high precision, metal mold inserts, which were used to replicate titer plate-based SPRI reactors using hot embossing of polycarbonate (PC). Optimized molding conditions yielded good feature replication fidelity and feature location integrity over the entire surface area. Thermal fusion bonding of the molded PC chips at 150°C resulted in leak-free sealing, which was verified in leakage tests using a fluorescent dye. The assembled SPRI reactor was used for simple, fast purification of genomic DNA from whole cell lysates of several bacterial species, which was verified by PCR amplification of the purified genomic DNA.
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Papers by Michael Murphy