a. Cell Sorting
        - Acoustic Differential Extraction 

    This project highlights the use of acoustic forces in a valveless microfluidic d evice to trap sperm cells in the presence of female epithelial DNA obtained from sexual assault evidence.  The device is comprised of two layers: a printed circuit board layer containing microtransducers, and a glass fluidic layer.  An ultrasonic frequency tuned specifically to the transducer characteristics and channel dimensions is applied to the device, and an acoustic standing wave is set up within the microchannel, producing a trapping zone at a pressure node.  This method exploits the density, volume, and compressibility differences between sperm cells and free DNA from epithelial cell lysate to create a force strong enough to retain the sperm cells at these nodes, while allowing the free DNA to pass through the device.  Laminar flow valving is implemented to direct the two fractions to separate outlets.

 

        - Enhanced Sperm Cell Recovery from Cotton Swabs for Rape Kit Analysis

    Regardless of the method utilized for the separation of vaginal and sperm cell DNA, the overall effectiveness of the procedure is ultimately dependent on the efficiency with which material can be eluted and recovered from a cotton swab. The issue is especially important with swab samples containing low numbers of sperm cells, where any loss makes it even more difficult to obtain a profile of the perpetrator.  This project focuses on the development of improved methods for cell elution from a cotton matrix. Several alternative methods of intact cell removal have been investigated; including the use of cellulase-based enzyme mixtures [1], as well as the exclusive use of detergents [2]. These methods can be utilized in conjunction with or to circumvent conventional differential extraction. 

1. Voorhees, J.C., Ferrance, J.P., Landers, J.P. Enhanced elution of sperm from cotton swabs via enzymatic   digestion for rape kit analysis. J Forensic Sci 2006; 51(3):574-9.
2. Norris, J.V., Manning, K., Linke, S.J., Ferrance, J.P.,Landers, J.P.  Expedited, chemically-enhanced sperm cell recovery from cotton swabs for rape kit analysis. J Forensic Sci 2007; 52(4):800-5.

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b. Solid Phase Extraction
        - New Monolith Stationary Phase for Microfluidic DNA Purification

    Today, solid-phase extraction (SPE) is the most popular preparation method for the extraction and preconcentration of analytes. To obtain a high loading capacity, a large surface area of the solid phase is desired. Porous polymer monoliths are a new category of materials developed during the last decade. These materials are prepared using a very simple process in which a mixture of monomers and porogenic solvent is polymerized within a closed tuber or other container under carefully controlled conditions. Thus, the monoliths could be prepared into any shape. The polymerization mixture typically contains monomers, free-radical initiator, and porogenic solvent which affords macroporous materials with both large through-pores with a pore size of 1 to 20 µm and small meso-pore in 100-1,000 nm size range. The pore size can also be controlled over a broad range by different ratio of porogenic solvents. Since all the mobile phase must flow through the monolith, the mass transport within the monolith is dominated very much by convection, and the monolithic materials performed very well at high flow rates.
    UV-induced photo-polymerization enables the accurate placement of monolithic matrices within the architecture of microscale devices, and the functional surface groups on the monomers allows for easy chemical modification of the surface. These methods have been used to create a high capacity, high efficiency silica-based DNA extraction monolithic column within a microfluidic device.

(A) High resolution of scanning electron micrograph of monolith internal micro-structure.
(B) A two parallel straight channel design illustrated the accurate localization of solid phase within a channel.

        - Microfluidic-based Nucleic Acid Purification in a Two-Stage, Dual-Phase Microchip

    The need for high DNA binding capacity is important in many clinical applications that rely on whole blood as a source of genomic DNA. Lysed whole blood contains nucleic acids, proteins, lipids, metabolites, and inorganic ions, some of which are known to inhibit PCR, a technique used in almost all genetic analyses.  However, the proteins in blood also bind to the solid phase limiting the DNA binding capacity.  To solve this problem, a two-stage, dual phase microdevice for DNA extraction from whole blood has been developed.  This device captures proteins using an in-line C18 phase, allowing the DNA to bind to the DNA extraction phase with significantly decreased protein competition.  Successful PCR amplification following purification of DNA from human whole blood illustrated the effectiveness of the method.  An added benefit of this method is the ability to remove the protein wash step normally required to remove proteins from the DNA extraction phase; this accelerates the analytical process, reduces the number of steps and eliminates potential sample contamination that may occur from switching syringes or tubing.  With the majority of the extracted DNA released in a small volume, this system is ideal for concentrating and purifying DNA from whole blood on integrated microfluidic devices.

(A) Integrated protein capture/DNA extraction in two-stage, dual-phase
microdevice for DNA purification from human whole blood.
(B) The channels are filled with dyes for better visualization of the protein and
nucliec acid capture phase regions within the microchannel architecture.
Chip dimensions: 3 cm (length) x 2.5 cm (width). C18 bed length: 10 mm; monolith length: 4 mm

    - Large Volume Reduction Solid Phase Extraction

    Often times in forensic cases large volumes are required to elute or solubilize a blood sample from various surfaces including clothing and walls. In contrast, microdevices are characterized by small sample volumes.  This raises the need for a sample processing step that would reduce the volume of the forensic sample, providing both a crude purification and sample concentration effect.  These investigations focus on a volume reduction microdevice utilizing various solid phases including chitosan-coated-silica particles (charge switch binding) and magnetic silica particles (hydrogen bonding).  A comparison of the extraction efficiency of the two phases will be investigated, in addition to optimizing the integration with a second, silica based SPE step.  Later work will also involve integration with other processing steps including PCR, ME, and fluorescence detection.

The figure above shows the microdevice used for integrating
vrSPE with subsequent SPE for the extraction of DNA.

    - Plastic SPE Microdevices

    Silica or sol-gel based solid phases in glass microdevices are normally employed for DNA extractions but have several disadvantages including the difficulty in reproducibly packing the channel and the cost of the glass devices.  Polymeric devices provide for a cost effective method to produce microdevices which could be disposable, and current fabrication methods allow for establishment of a polymeric matrix within a microchannel during the fabrication process. This project will investigate the designs utilized in generating a highly reproducible solid phase, and the derivatization of the phase for extraction of DNA from these polymeric devices.  This device will be used to perform extractions of samples including blood, cells, frozen tissue, and laser-microdissected histological tissue which can play a major role in cancer research.

    - RNA

    Messenger RNA expression analysis requires isolation of RNA from biological samples, followed by reverse transcription-PCR (RT-PCR) amplification and separation of target amplicon to identify the sample.  The method is based on the inherently variable mRNA expression from different cell types, producing gene-specific patterns which can be verified by the presence of a unique mRNA expression patterns.   Recently, Juusola et al. [1] described a method using mRNA expression to identify specific body fluids.  To obtain mRNA for the transcription and amplification necessary for gene expression analysis, RNA must first be isolated and purified from the biological source of interest.  Consequently, a robust system for purification of RNA will be essential as the development of messenger RNA expression analysis methods unfold.  A closed microfluidic, silica-based purification system would represent a significant improvement to current methodologies for RNA extraction, which often involve time- and reagent-consuming organic extractions, by decreasing the opportunity for introduction of contaminants and RNases, as well as reducing the amount of sample, reagents, and time required to perform this delicate isolation.  The application of a silica-based microchip solid-phase extraction method for purification of RNA as a precursor to mRNA profiling would be also be a promising step toward simultaneous DNA and RNA purification technology, permitting a more comprehensive genetic analysis from a single source.  In addition, development of a single-process RNA purification device represents the first step towards creation of an integrated micro-total analysis system (μ-TAS) capable of total mRNA profiling.  Work with silica and alternative phases to silica for extraction of RNA is underway for use in genetic analysis as well as clinical diagnostics.

1. Juusola, J.; Ballantyne, J. For Sci Internat 2003, 135, 85-96.

    - Testing of New Phases

    A well-characterized method for the adsorption of nucleic acids to silica surfaces has been established and exploited for the purification of DNA and RNA from biological samples in commercially-available macroscale systems.  Using a chaotropic salt to promote adsorption of DNA and RNA to the silica surface, contaminating proteins can be removed through a series of washes and the purified nucleic acids recovered for downstream genetic analysis.  This method has been translated for use in microfluidic systems for DNA purification, where reduced sample and reagent consumption, as well as a reduction in analysis time has been achieved. [1-5]  Currently, work is underway to explore alternative matrices for solid phase extraction of DNA from biological samples. 

1. Breadmore, M.C.; Wolfe, K.A.; Arcibal, I.G.; Leung, W.K.; Dickson, D.; Giordano, B.C.; Power, M.E.; Ferrance, J.P.; Feldman, S.H.; Norris, P.M.; Landers, J.P. Anal Chem 2003, 75, 1880-1886.
2. Easley, C.J.; Karlinsey, J.M.; Bienvenue, J.M.; Legendre, L.A.; Roper, M.G.; Feldman, S.H.; Hughes, M.A.; Hewlett, E.L.; Merkel, T.J.; Ferrance, J.P.; Landers, J.P. PNAS 2006, 103, 19272-19277.
3. Bienvenue, J.M.; Duncalf, N.; Marchiarullo, D.; Ferrance, J.P.; and Landers, J.P. J Forensic Sci 2006, 51, 266-273.
4. Tian, H.; Huhmer, A.F.R.; Landers, J.P. Anal Biochem 2000, 283, 175-191.
5. Wolfe, K.A.; Breadmore, M.C.; Ferrance, J.P.; Power, M.E.; Conroy, J.F.; Norris, P.M.; Landers, J.P. Electrophoresis 2002, 23, 727-733.

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c. PCR
        - Infrared PCR

        - Microwave PCR

    Dielectric heating is commonly used, most notably in the form of a microwave oven. Our group in collaboration with Dr. N. Scott Barker in UVA’s department of Electrical and Computer Engineering is developing a microfluidic microwave heating device for small volumes of solution. Using microstrip transmission lines focused microwave power can be delivered directly to a microliter chamber containing solution. The figure shows an example of our chip-based microwave heating device. With about 1 W of non-resonant microwave power (for comparison a microwave oven uses ~ 1000 W) we rapidly heat buffer to boiling and can vary temperature by adjusting the microwave frequency, or delivered power. The figure also shows an example of temperature control by varying the applied microwave frequency. We are currently applying this technology to the polymerase chain reaction which requires thermocycling between two or three temperatures for up to 30 cycles to amplify a desired DNA fragment.

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d. Integration
        -  Cell Sorting and Solid Phase Extraction

    This project focuses on the use of an integrated microdevice that combines sedimentation-based cell sorting and solid phase extraction (SPE) of DNA from the sorted cells. The microdevice (figure-left), fabricated using standard photolithographic techniques, is designed with a domain for cell sorting and two separate SPE regions. A mixture of sperm and epithelial cells are separated according to their physical properties (figure-right). Sperm cells are then lysed on-chip, followed by isolation and purification of its DNA fractions. Following cell separation and SPE on the microdevice, DNA amplification and separation are performed using conventional laboratory methods. This work represents a major step towards the development of a fully integrated microdevice capable of genetic DNA analysis.

        - DNA Extraction and PCR Amplification Performed in a Single Microfluidic Chamber

    Integrated microdevices provide the opportunity to encompass multiple analytical steps on a single device, with the possibility of automating the entire sample analysis process.  Current work being developed involves a glass microdevice that has been designed to perform solid phase extraction (SPE) of DNA and IR-PCR (infrared-mediated) amplification in a novel format – with both processes in the same chamber.  This provides an inherent advantage over any microchip-based DNA extraction described previously [1], in that all of the sample DNA is used for nanoliter amplification, improving detection limits by 1-2 orders of magnitude. A novel solid phase, chitosan-coated magnetic beads, has been developed that has high DNA recoveries (72% ± 6%) using a pH-induced DNA release technique [2], while eliminating the use of high salt solutions and organics (both potent PCR inhibitors).  A simple external magnet is used to control the location of the beads in the chamber, removing the need for etching structures (such as weirs or pillars) into the channels, increasing the simplicity of device design and fabrication.  While previous SPE phases have been composed of materials incompatible with PCR [1] (due to high protein adsorption) the chitosan coating does not affect the efficiency of PCR, allowing the beads to remain in the very same chamber used for PCR.

1. Legendre, L.A., Bienvenue, J.M., Roper, M.R., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 1444.
2. Cao, W., Easley, C.J., Ferrance, J.P., Landers, J.P. Anal Chem, 2006, 78, 7222-7228.

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a. Clinical
        - A Fully Integrated Microfluidic Genetic Analysis Device for the Detection of Blood Cancers

    We are developing a fully-integrated microdevice capable of DNA extraction, PCR amplification and the subsequent electrophoretic separation specifically for the detection of T-cell lymphoma (TCL).  While we have recently reported a fully-integrated device for the detection of bacteria [1], the interrogation of human genomic DNA from whole blood provides new challenges, especially when detecting gene rearrangements correlative with cancer.  Detection of TCL involves the PCR amplification of select sequences in the T-cell receptor gene that are likely to have undergone gene rearrangement.  These PCR fragments represent a polyclonal cell population in normal individuals and a monoclonal cell population in patients with lymphoma in a way that can be discriminated by electrophoretic separation. In clinical labs, the PCR that follows DNA extraction requires ~3 hours followed by a 40 min capillary electrophoresis separation under single-stranded conditions and utilizing 4-color detection.  While integration of processing steps is a large advantage over traditional methods, the reduction in the times associated with these lengthy processes is also an important benefit.

1. Easley, C.J.,  Karlinsey, J.M.,  Bienvenue, J.M., Legendre, L.A., Roper, M.G., Feldman, S.H., Hughes, M.A., Merkel, T.J., Ferrance, J.P., Landers, J.P. PNAS, 2006, 109(51), 19272-19277.

 
        - Electric Field-Flow Fractionation for DNA Concentration

    In many separation scenarios, a simple preconcentration step between purification steps is highly desired. For example, the elution step on a microchip solid phase extraction column dilutes the DNA and raises the threshold for downstream amplifications. To solve this problem, we are developing electric field-flow based glass/PDMS microdevices to recover concentrated DNA samples from upstream extraction/purification steps.

        - Acousto-Optic Tunable Fiber

    Laser-induced fluorescence (LIF) is the most common form of detection on microchips. Typically, a confocal setup is used, where incoming laser light is focused into a microchannel through an objective, and the fluorescence emission is collected and sent to a photomultiplier tube (PMT) for detection. Increasing the number of fluorophores to be detected in the sample creates a need for separating out different emission lines. We are using an acousto-optic tunable filter (AOTF) to select these emission lines. The AOTF acts as an electronically tunable spectral bandpass filter. It is made up of a single optically-active crystal bonded to a piezoelectric. By applying different radio frequencies to the piezoelectric, acoustic waves are propagated through the crystal, and a single wavelength of light can be separated from a multi-color source. This allows for a separation of multiple species spectrally, even without temporal resolution. We plan on using this device to perform multicolor detection of tagged DNA bases for clinical and forensic applications.

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b. Forensic
        - STR Typing

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a. Spaceflight Therapeutic Drug Monitoring

    Astronauts have been taking medications during spaceflight since the first Mercury mission. Common ailments requiring treatment include space motion sickness, sleeplessness, congestion, and pain.  The rigors of spaceflight and microgravity induce a number of physiologic changes that affect drug metabolism and, therefore, the efficacy of medications in some cases. In collaboration with the Pharmacotherapeutics Laboratory at Johnson Space Center we are developing a portable device based on a microfluidic platform for therapeutic drug monitoring that can be taken on missions to the space station and beyond.
    This device will analyze biofluids (blood, urine and saliva) taken from astronauts during spaceflight allowing for proper dosing and to give insight into the effects of microgravity on drug metabolism. Solid phase extraction using commercially available sorbents is being developed on-chip for sample cleanup and preconcentration and will be integrated with an electrophoretic separation and appropriate detection method. The figure shows an envisioned integrated device using fluorescence detection.

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b. MEKC Drugs

    Micellar electrokinetic chromatography (MEKC) is a well established separation technique used to separate neutral molecules in an electric field using a pseudostationary phase such as SDS. We are applying MEKC to the separation of drugs and metabolites on a microchip. This work is being done in conjunction with the Spaceflight Therapeutic Drug Monitoring project with the goal of integrating the MEKC separation with SPE of small molecules from biofluids.

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c. Microchip Isoelectric Focusing

    Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS–glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids [1].

1. Guillo, C.; Karlinsey, J.; and Landers, J.P. On-chip pump for pressure mobilization of the focused zones following microchip isoelectric focusing. Lab Chip, 2007, 7, 112 - 118.

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d. Protein Separations

    The overall goal of this project is to develop microfluidic devices for rapid protein detection, purity determination, and charge heterogeneity.  Microdevices have proven useful in analytical and clinical chemistry providing much faster analyses, with sensitivity similar to conventional methods.  Application of these devices to electrophoretic separations has been well studied, but has mostly focused on DNA separations.  Protein separations on microchips have been more limited.  First, UV absorbance detection is normally used with proteins, but is not easily coupled with microdevices.  The development of microchip MS interfaces provides a new detection method for proteins, which has renewed interest in protein separations on microchips, although significant challenges remain.
    One of the biggest challenges to the use of capillary electrophoresis for protein separation is the propensity of the charged proteins to adsorb onto the negatively charged capillary walls.  This sticking may be eliminated via SDS-protein separations, however, when coated with negatively-charged SDS, all proteins have the same charge to mass ratio, giving them all the same electrophoretic mobility.  Sieving gels are therefore required to perform separations in the presence of SDS. Unfortunately, with this method information related to the charge or mass heterogeneity of the proteins is lost.  A major subgoal of this project is to evaluate the use of both semi-permanent and dynamic type coatings to determine which provide the adsorption prevention necessary to enable a free zone (CZE) separation, thus retaining information on the protein charge and mass heterogeneity. The coatings will also be evaluated for their compatibility with MS detection, for eventual incorporation into microdevice CE-MS systems.

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Dr. N. Scott Barker (Electrical & Computer Engineering, U.Va.)

Dr. Annelise Barron (Bioengineering, Stanford University, Stanford, CA)

Dr. Daniel T. Chiu (University of Washington) 

Dr. Sanford Feldman (Comparative Medicine, U.Va.)

Dr. Susan Greenspoon (Virginia Division of Forensic Science)

Dr. Doris Haverstick (Pathology, U.Va.)

Dr. Erik Hewlitt (Infectious Disease, U.Va.)

Dr. Molly Hughes (Infectious Disease, U.Va.)

Dr. Thomas Laurell (Lund University, Lund, Sweden) 

Dr. Rebecca McClure, Mayo Clinic, Division of Hematopathology, Rochester, MN

Dr. Tod Merkel (Center for Biologics Evaluation and Research, FDA)

Dr. Christopher Moskaluk (Pathology, U.Va.)

Dr. Johan Nilsson  (Lund University, Lund, Sweden)

Dr. Robin Patel (Division of Infectious Disease, Mayo Clinic, Rochester, MN)

Dr. Lakshmi Putcha (NASA Pharmacotherapeutics Laboratory at Johnson Space Center)

Dr. Marcel Utz (Mechanical and Aerospace Engineering, U.Va.)