Global Gene Expression of Cells Attached to a Tissue Engineering Scaffold

Biomaterials. Nov, 2004  |  Pubmed ID: 15159079

A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Little is known about how cells respond on a molecular level to tissue engineering scaffold materials. In this work we used oligonucleotide microarrays to interrogate gene expression profiles associated with cell-biomaterial interactions. We seeded collagen-glycosaminoglycan meshes, a widely used tissue engineering scaffold material, with human IMR-90 fibroblasts and compared transcript levels with control cells grown on tissue culture polystyrene. Genes involved in cell signaling, extracellular matrix remodeling, inflammation, angiogenesis and hypoxia were all activated in cells on the collagen-GAG mesh. Understanding the impact of a scaffold on attached cells will facilitate the design of improved tissue engineering materials.

Thermoplastic Microfluidic Device for On-chip Purification of Nucleic Acids for Disposable Diagnostics

Analytical Chemistry. Feb, 2006  |  Pubmed ID: 16448052

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.

Fibroblast Remodeling Activity at Two- and Three-dimensional Collagen-glycosaminoglycan Interfaces

Biomaterials. Aug, 2006  |  Pubmed ID: 16620959

Previously we demonstrated that high throughput gene expression experiments can yield novel information about how cells respond to a collagen-glycosaminoglycan (GAG) three-dimensional culture environment. The goal of the current study was to determine which of these differences result from culture in a three-dimensional construct versus those caused simply by the presence of the collagen-GAG biomaterial. To make this distinction, cells were cultured both in collagen-GAG scaffolds fabricated using a phase separation method and on thin two-dimensional coatings of the same material. Control cells were grown on standard tissue culture polystyrene (TCPS). Cell response was measured using histology and microarray analysis and select results were verified with real time polymerase chain reaction (RT-PCR) assays. Genes involved in matrix remodeling (matrix components, matrix metalloproteinases and growth factors) and angiogenesis (VEGF, HGF and HMOX) were shown to be differentially expressed between the treatment conditions. Several matrix metalloproteinases (MMPs) were up regulated in mesh grown cell while some of their inhibitors (TIMPs) were down regulated. These results suggest that the three-dimensional presentation of the collagen-GAG material to the cells is required to stimulate the observed increase in fibroblast remodeling behavior.

Nanomechanical Analysis of Bone Tissue Engineering Scaffolds

Journal of Biomedical Materials Research. Part A. Jun, 2007  |  Pubmed ID: 17187400

Copolymers of (2-hydroxyethyl methacrylate) (HEMA) and methacrylamide monomers conjugated with amino acids were synthesized and crosslinked with ethylene glycol dimethacrylate. The resulting library of copolymers was mineralized in vitro using two distinct methods. In the first mineralization method, the copolymers were polymerized in the presence of a sub-micron hydroxyapatite (HA) suspension. In the second method, copolymers were mineralized with HA using a urea-mediated process. The mechanical properties of all of the copolymers, both mineralized and not, were determined using nanoindentation under both load and displacement control. A power law fit to the initial unloading curve was used to determine a reduced elastic modulus for each material. Between 30 and 300 indentations were performed on each material, and ANOVA analysis was run to determine the statistical significance of differences in modulus between samples. Using nanoindentation, the 22 different samples had reduced modulus values ranging from 840 MPa to 4.14 GPa. Aspartic acid-methacrylate (Asp-MA) copolymers were not distinguishable from the pHEMA control material. Polymerization in the presence of HA created a more uniform material than the urea method of mineralization. Several challenges and solutions encountered in the nanomechanical testing of soft, heterogeneous materials are discussed. These results demonstrate that with proper experimental design, the mechanical properties of tissue engineering scaffold materials based on polymer-ceramic composite materials can be determined using small samples and nanoindentation techniques.

Mechanical and Chemical Analysis of Plasma and Ultraviolet-ozone Surface Treatments for Thermal Bonding of Polymeric Microfluidic Devices

Lab on a Chip. Jul, 2007  |  Pubmed ID: 17594007

Here we have demonstrated that radio frequency plasma and ultraviolet-ozone (UVO) surface modifications are effective treatments for enabling the thermal bonding of polymeric microfluidic chips at temperatures below the T(g) (glass transition temperature) of the polymer. The effects of UVO and plasma treatments on the surface properties of a cyclic polyolefin and polystyrene were examined with X-ray photoelectron spectroscopy (XPS), contact angle measurements, atomic force microscopy (AFM) surface roughness measurements and surface adhesion measurements with AFM force-distance data. Three-point bending tests using a dynamic mechanical analyzer (DMA) were used to characterize the bond strength of thermally sealed polymer parts and the cross-sections of the bonded microchannels were evaluated with scanning electron microscopy (SEM). The experimental results demonstrated that plasma and UVO surface treatments cause changes in the chemical and physical characteristics of the polymer surfaces, resulting in a decrease in T(g) at the surface, and thus allowing the microfluidic chips to be effectively bonded at temperatures lower than the T(g) of the bulk polymer without losing the intended channel geometry.

Peptide- and Collagen-based Hydrogel Substrates for in Vitro Culture of Chick Cochleae

Biomaterials. Mar, 2008  |  Pubmed ID: 18037163

The overall goal of this work is to improve the culture of the auditory organ of birds for the dual use of developing a hair cell regeneration model and charting a pathway to the eventual replacement of the hearing organ. In doing so, we develop a protocol for removing the auditory organ from its basement membrane in the inner ear, attach the organ to a series of artificial basement membranes, and conduct qualitative and quantitative analysis of how cell morphology, viability and function change with time. Native matrix cultures, where the epithelium was floating in media with the basement membrane and accessory structures attached, were used as a basis of comparison. PuraMatrix, collagen I, collagen I/chondroitin-sulfate and Matrigel were chosen to encompass a diverse range of mechanical properties and macromolecule moieties. Surprisingly, we find that PuraMatrix outperformed the other matrices as a scaffold for sensory organ culture. PuraMatrix a self-assembled peptide hydrogel, is a biochemically specific culture substrate that contains none of the extracellular matrix (ECM) molecules and growth factors contained in the inner ear's basement membrane. Rheological measurements reveal that PuraMatrix may be a closer approximation to the stiffness of the soft tissue supporting the auditory organ. Cell density on the PuraMatrix substrate is comparable to that of the native matrix cultures, despite the absence of the basement membrane and accessory structures. Further studies show that PuraMatrix supports the culture of functional hair cells over a 72 h period, with a significant increase in the number of functional hair cells in comparison to the organ cultured without a matrix. This is the first example of adhesion of the adult auditory epithelium to a biomaterial for an extended period of time. With further optimization, this system will enable the performance of many novel biophysical and pharmacological studies involving hair cells and supporting cells.

Time-dependent Mechanical Characterization of Poly(2-hydroxyethyl Methacrylate) Hydrogels Using Nanoindentation and Unconfined Compression

Journal of Materials Research. May, 2008  |  Pubmed ID: 19081812

Hydrogels pose unique challenges to nanoindentation including sample preparation, control of experimental parameters, and limitations imposed by mechanical testing instruments and data analysis originally intended for harder materials. The artifacts that occur during nanoindentation of hydrated samples have been described, but the material properties obtained from hydrated nanoindentation have not yet been related to the material properties obtained from macroscale testing. To evaluate the best method for correlating results from microscale and macroscale tests of soft materials, nanoindentation and unconfined compression stress-relaxation tests were performed on poly-2-hydroxyethyl methacrylate (pHEMA) hydrogels with a range of cross-linker concentrations. The nanoindentation data were analyzed with the Oliver-Pharr elastic model and the Maxwell-Wiechert (j = 2) viscoelastic model. The unconfined compression data were analyzed with the Maxwell-Wiechert model. This viscoelastic model provided an excellent fit for the stress-relaxation curves from both tests. The time constants from nanoindentation and unconfined compression were significantly different, and we propose that these differences are due to differences in equilibration time between the microscale and macroscale experiments and in sample geometry. The Maxwell-Wiechert equilibrium modulus provided the best agreement between nanoindentation and unconfined compression. Also, both nanoindentation analyses showed an increase in modulus with each increasing cross-linker concentration, validating that nanoindentation can discriminate between similar, low-modulus, hydrated samples.

Sample Preparation Module for Bacterial Lysis and Isolation of DNA from Human Urine

Biomedical Microdevices. Jun, 2009  |  Pubmed ID: 19130239

Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10(1)-10(5) colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 10(1) CFU/mL. Results were comparable to a commercial kit at higher concentrations and performed better at recovering DNA at lower concentrations.

Effect of Trypan Blue Staining on the Elastic Modulus of Anterior Lens Capsules of Diabetic and Nondiabetic Patients

Journal of Cataract and Refractive Surgery. Feb, 2009  |  Pubmed ID: 19185249

To determine whether trypan blue causes a significant difference in the biomechanical properties (stiffness) of diabetic and nondiabetic anterior lens capsules and to determine whether diabetes significantly alters the stiffness of anterior lens capsules.

Microfluidic Diagnostics: Time for Industry Standards

Expert Review of Medical Devices. May, 2009  |  Pubmed ID: 19419277

Microscale Sample Preparation for PCR of C. Difficile Infected Stool

Journal of Microbiological Methods. Aug, 2009  |  Pubmed ID: 19505511

In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0x10(-3) ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world.

Surface Detection Errors Cause Overestimation of the Modulus in Nanoindentation on Soft Materials

Journal of the Mechanical Behavior of Biomedical Materials. Aug, 2009  |  Pubmed ID: 19627837

The accuracy of mechanical properties from depth-sensing indentation, or nanoindentation, depends on the accuracy of the displacement measurement used to calculate these properties. Here, current nanoindentation techniques and analysis methods for accurate displacement measurements are reviewed. First, the ability of a commercial instrument to sense the surface of soft materials is examined. Second, methods of sample surface detection are reviewed. Finally, a case of overestimation of the elastic modulus of a compliant material using nanoindentation with incorrect displacement values is presented.

Differential Gene Expression Using MRNA Isolated on Plastic Microfluidic Chips

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2009  |  Pubmed ID: 19965139

Here we demonstrate the ability to perform differential gene expression experiments using messenger RNA (mRNA) isolated from crude cell lysates using a plastic microfluidic solid phase extraction column. The microfluidic columns (100microm by 100microm by 1.5 cm) were fabricated in a cyclic polyolefin by hot-embossing with an electroformed master-mold. The solid-phase consisted of a photopolymerized microporous monolith embedded with functional microparticles and covalently attached to the channel walls via photoinitiated grafting. For mRNA isolation from total RNA and direct mRNA isolation from cell lysates, oligo(dT) beads were embedded in the monolith. The extraction efficiency of the system is approximately 80% and the nucleic acid binding capacity of the silica solid-phase in this configuration is approximately 3.5 ng. The micro solid-phase was applied for the extraction and purification of mRNA from human liver total RNA and the isolation of mRNA from neonatal human dermal fibroblast cells (NHDF) and MCF7 breast cancer cell lysates. Differential gene expression between the two cell lines is demonstrated.

Low Cost and Manufacturable Complete MicroTAS for Detecting Bacteria

Lab on a Chip. Oct, 2009  |  Pubmed ID: 19967117

In this paper, we present a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (M. D. Kulinski, M. Mahalanabis, S. Gillers, J. Y. Zhang, S. Singh and C. M. Klapperich, Biomed. Microdevices, 2009, 11, 671-678).(1) The chip is made of Zeonex(R), a thermoplastic with a high melting temperature to allow PCR, good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence for fluorescence detection of the amplicon. We have built a prototype instrument to automate control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chip designs. To enable fluid control without including valves or pumps on the chip, we utilized a remote valve switching technique. To allow fluid flow rate changes on the valveless chip, we incorporated speed changing fluid reservoirs. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer; all integrated in the instrument. The chip seamlessly and automatically is mated to the instrument through an interface block that presses against the chip. The interface block aligns and ensures good contact of the chip to the temperature controlled region and the optics. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the isolated bacterial DNA.

Cell Lysis and DNA Extraction of Gram-positive and Gram-negative Bacteria from Whole Blood in a Disposable Microfluidic Chip

Lab on a Chip. Oct, 2009  |  Pubmed ID: 19967118

Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in noncoronary ICUs and the tenth leading cause of death in the United States. We have developed a microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics. The microfluidic chips are made of a robust thermoplastic and can be easily multiplexed for high throughput applications. Bacteria are lysed on-chip via hybrid chemical/mechanical method. Once lysed, the bacterial DNA is isolated using a microscale silica bead/polymer composite solid-phase-extraction (SPE) column. Lysis was confirmed using off-chip real time PCR. We isolated and detected both gram-negative (Escherichia coli) and gram-positive (Bacillussubtilis and Enterococcus faecalis) bacterial genomic DNA from microliter scale spiked whole human blood samples. The system performs better for gram-negative bacteria than it does for gram-positive bacteria, with limits of detection at 10(2) CFU/ml and 10(3)-10(4) CFU/ml, respectively. Total extraction times are less than one hour and can be further decreased by altering the channel geometry and pumping configuration.

A Novel Biocompatible Adhesive Incorporating Plant-derived Monomers

Journal of Biomedical Materials Research. Part A. Nov, 2009  |  Pubmed ID: 18980208

We describe a new class of biomaterials with potential for a variety of applications in tissue engineering, wound healing, and transdermal drug delivery. These materials are based on oleic methyl ester (OME), which is derived from various plant oils including soybean oil. The OME was acrylated (AOME) and subsequently copolymerized with methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) to form pressure sensitive adhesives (PSAs). We assessed the cytocompatibility of each PSA product using Alamar Blue and Live/Dead assays. It was found that after 2 h, human fibroblast cells attached on all four of the PSA polymers tested. After 24 h, cell spreading was seen on all materials with the exception of the polymerized AOME product (PAOME). Cells attached to the copolymer PSA products continued to proliferate for up to 2 weeks, as shown by fluorescent confocal microscopy imaging. Finally, a mechanical analysis of each of the copolymers is presented demonstrating that they have a range of mechanical properties and cell adhesiveness depending on the formulation, making them attractive candidates for use as bioactive adhesives.

An Integrated Disposable Device for DNA Extraction and Helicase Dependent Amplification

Biomedical Microdevices. Apr, 2010  |  Pubmed ID: 20066496

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

RNA Isolation from Mammalian Cells Using Porous Polymer Monoliths: an Approach for High-throughput Automation

Analytical Chemistry. Jun, 2010  |  Pubmed ID: 20443545

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

A New Method for Simulating the Motion of Individual Ellipsoidal Bacteria in Microfluidic Devices

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20532377

To successfully perform biological experiments on bacteria in microfluidic devices, control of micron-scale cell motion in the chip-sized environment is essential. Here we describe a new method for simulating the motion of individual bacterial cells in a microfluidic device using a one-way coupling Lagrangian approach combined with rigid body theory. The cell was assumed to be an elastic, solid ellipsoid, and interactions with solid wall boundaries were considered to occur in one of two collision modes, either a "standing" or "lying" collision mode on the surface. The ordinary differential equations were solved along the cell trajectory for the thirteen unknown variables of the translational cell velocity, cell location vector, rotational angular velocity, and four Euler parameters, using the Rosenbrock method based on an adaptive time-stepping technique. As selected applications, we show how this novel simulation method may be applied to the designs of efficient hydrodynamic cell traps in a microfluidic device for bacterial applications and for cell separations. Modeled designs include optimized U-shaped sieve arrays with a single aperture for the hydrodynamic cell trapping, and three kinds of staggered micropillars for cell separations.

Contributors to the Emerging Investigators Issue

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20717627

Rapid Point-of-care Concentration of Bacteria in a Disposable Microfluidic Device Using Meniscus Dragging Effect

Lab on a Chip. Dec, 2010  |  Pubmed ID: 20938505

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon™) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 µL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.

Plastic Microfluidic Chip for Continuous-flow Polymerase Chain Reaction: Simulations and Experiments

Biotechnology Journal. Nov, 2010  |  Pubmed ID: 21053333

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.

Programmed Trapping of Individual Bacteria Using Micrometre-size Sieves

Lab on a Chip. Mar, 2011  |  Pubmed ID: 21293825

Monitoring the real-time behavior of spatial arrays of single living bacteria cells is only achieved with much experimental difficulty due to the small size and mobility of the cells. To address this problem, we have designed and constructed a simple microfluidic device capable of trapping single bacteria cells in spatially well-defined locations without the use of chemical surface treatments. The device exploits hydrodynamics to slow down and trap cells flowing near a narrow aperture. We have modeled this system numerically by approximating the motion of Escherichia coli cells as rigid 3-D ellipsoids. The numerical predictions for the speed and efficiency of trapping were tested by fabricating the devices and imaging GFP expressing E. coli at a high spatio-temporal resolution. We find that our numerical simulations agree well with the actual cell flow for varying trap geometries. The trapped cells are optically accessible, and combined with our ability to predict their spatial location we demonstrate the ease of this method for monitoring multiple single cells over a time course. The simplicity of the design, inexpensive materials and straightforward fabrication make it an accessible tool for any systems biology laboratory.

Plastic Microfluidic Chip for Continuous-flow Polymerase Chain Reaction: Simulations and Experiments

Biotechnology Journal. Feb, 2011  |  Pubmed ID: 21298803

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.

Erratum To: An Integrated Disposable Device for DNA Extraction and Helicase Dependent Amplification

Biomedical Microdevices. Jun, 2011  |  Pubmed ID: 21369762

In the original manuscript, we reported the demonstration of an integrated microfluidic chip that performed helicase dependent amplification (HDA) on samples containing live bacteria. Bacterial lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter were incorporated into a disposable polymer cartridge format. We reported that the device was able to detect as few as 10 colony-forming units (CFU) of E. coli in growth medium. While the main conclusions of the original paper remain sound, the data presented in support of those conclusions contained errors that we detail, discuss and correct here. In short, we misidentified a non-specific product as a specific product of our HDA reaction. We incorrectly called reactions containing the non-specific product (length 70 bp) positive. Further investigation demonstrated that our primer set was faulty and not capable of amplifying the specific product. Here we redesigned primers, sequenced all of the products and reran all of the experiments reported previously to generate a new, verified dataset.

Clinically Relevant Microfluidic Magnetophoretic Isolation of Rare-cell Populations for Diagnostic and Therapeutic Monitoring Applications

Analytical Chemistry. Feb, 2012  |  Pubmed ID: 22240089

Cells of biomedical interest are, despite their functional significance, often present in very small numbers. Therefore the analysis and isolation of previously inaccessible rare cells, such as peripheral hematopoietic stem cells, endothelial progenitor cells, or circulating tumor cells, require efficient, sensitive, and specific procedures that do not compromise the viability of the cells. The current study builds on previous work on a rationally designed microfluidic magnetophoretic cell separation platform capable of throughputs of 240 μL min(-1). Proof-of-concept was first conducted using MCF-7 (1-1000 total cells) as the target rare cell spiked into high concentrations of Raji B-lymphocyte nontarget cells (∼10(6) total cells). These experiments lead to the establishment of a magnet-based separation for the isolation of 50 MCF-7 cells directly from whole blood. Results show an efficiency of collection greater than 85%, with a purity of over 90%. Next, resident endothelial progenitor cells and hematopoietic stem cells are directly isolated from whole human blood in a rapid and efficient fashion (>96%). Both cell populations could be simultaneously isolated and, via immunofluorescent staining, individually identified and enumerated. Overall, the presented device illustrates a viable separation platform for high purity, efficient, and rapid collection of rare cell populations directly from whole blood samples.

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

PloS One. 2012  |  Pubmed ID: 22457740

A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008-2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%,100%) and negative predictive value = 96%(CI 88%,98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96%(CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%,61%) sensitivity and 98%(CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 10³ copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.

Sample Concentration and Purification for Point-of-care Diagnostics

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2012  |  Pubmed ID: 23366407

The ability to increase the concentration of target analytes in a fixed sample volume can potentially lower the limit of detection for many biosensing techniques, and thus is key in sample preparation for infectious disease diagnosis. Concentration by evaporation is an effective method to achieve target enrichment. However, concentrating human samples, including blood and plasma, by evaporation-based methods is made challenging by high concentrations of proteins and electrolytes. Dehydration of the proteins causes the sample to turn into a gel, hindering further analysis. At the same time, decreasing the volume increases the overall concentration of electrolytes, causing bacterial or viral particle lysis, and making them more difficult to detect in affinity-based biosensors. Thus, we fabricated a microfluidic chip that incorporates both dialysis and concentration in a single design. The chip dialyzes the proteins from the plasma, while maintaining an appropriate concentration of electrolytes and concentrating the sample targets. The process to concentrate plasma or serum samples by a factor of 10 takes less than 30 minutes. As a proof-of-concept, we demonstrated the chip using a defective Human Immunodeficiency Virus (HIV). To distinguish patients on antiretroviral therapy who are failing therapy from those who are not, a diagnostic must be able to detect HIV in plasma down to at least 1000 particles per milliliter. For a number of technical reasons, it is difficult to get on-chip PCR reactions to reach this level of sensitivity, so concentration of HIV from lower viral load samples has the potential to improve the sensitivity of many types of molecular point-of-care viral load tests.

Purification of DNA/RNA in a Microfluidic Device

Methods in Molecular Biology (Clifton, N.J.). 2013  |  Pubmed ID: 23329456

Often, modern diagnostic techniques require the isolation and purification of nucleic acids directly from patient samples such as blood or stool. Many diagnostic tests are being miniaturized onto micro-sized platforms and integrated into microfluidic devices due to the economies resulting from smaller sample and reagent volumes. Often, these devices perform sample preparation in series with the diagnostic tests. The sample preparation steps are vital in order to purify the desired genetic material from potential inhibitors that can interfere with the outcome of the test. There are various techniques used to selectively capture the nucleic acids while washing away potential contamination (proteins, enzymes, lipids, etc.). Two of the most common forms of selective capture are based on nucleic acid binding to silica surface or on the precipitation of nucleic acids with or without the presence of a carrier species. Each of these methods can be performed in liquid phase or in a solid support such as an extraction column. Here we discuss both methods and address microfluidic applications.