Biomedical subjects

Robin L Garrell

Publications and source records attributed to Robin L Garrell.

6 recordsLinked to original sources

PubMed2006

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS.

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs--proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.

Angiotensin II↗

PubMed2006

Droplet-based microfluidics with nonaqueous solvents and solutions.

In droplet-based ("digital") microfluidics, liquid droplets in contact with dielectric surfaces are created, moved, merged and mixed by applying AC or DC potentials across electrodes patterned beneath the dielectric. We show for the first time that it is possible to manipulate droplets of organic solvents, ionic liquids, and aqueous surfactant solutions in air by these mechanisms using only modest voltages (<100 V) and frequencies (<10 kHz). The feasibility of moving any liquid can be predicted empirically from its frequency-dependent complex permittivity, epsilon*. The threshold for droplet actuation in air with our two-plate device configuration is /epsilon*/>8x10(-11). The mechanistic implications of these results are discussed, along with the greatly expanded range of applications for digital microfluidics that these results suggest are now feasible.

Algorithms↗

PubMed2005

Digital microfluidics with in-line sample purification for proteomics analyses with MALDI-MS.

An in-line sample purification method for MALDI-MS, which relies on the electrowetting-on-dielectric (EWOD)-based technique for digital microfluidics, is reported. In this method, a droplet containing peptides and impurities is moved by EWOD and deposited onto a Teflon-AF surface. A droplet of water is subsequently moved over the spot, where it dissolves and removes the impurities. A droplet containing MALDI matrix is then moved to the spot, which is analyzed by MALDI-MS. This purification method reduces the number of salt adduct peaks caused by low concentrations of impurities (e.g., 20 mM sodium phosphate), and reduces or eliminates the catastrophic effects of high concentrations of impurities (e.g., 8 M urea). The method was used to purify spots made by depositing multiple droplets of contaminated peptides. Spectra from the purified spots showed an increase in the S/N ratio as a function of the number of droplets deposited; when not purified, the S/N ratio remained constant regardless of the number of droplets. Finally, the method was used to purify protein digests for peptide mass fragment (PMF) searches, and was shown to be more efficient than the conventional method of purification with reversed-phase-packed pipet tips. We anticipate this new, in-line sample purification technique for EWOD-MALDI-MS will enable development of integrated high-throughput proteomics analysis methodologies.

Analytic Sample Preparation Methods↗

PubMed2004

Electrowetting-based microfluidics for analysis of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry.

A new technique for preparing samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The technique relies on electrowetting-on-dielectric (EWOD) to move droplets containing proteins or peptides and matrix to specific locations on an array of electrodes for analysis. Standard MALDI-MS reagents, analytes, concentrations, and recipes are demonstrated to be compatible with the technique. Mass spectra are comparable to those collected by conventional methods. Nonspecific adsorption of analytes to device surfaces is demonstrated to be negligible. The results suggest that EWOD may be a useful tool for automating sample preparation for high-throughput proteomics and other applications of MALDI-MS.

Equipment Design↗

PubMed2004

G-factor analysis of protein secondary structure in solutions and thin films.

The biological activity of proteins is structure dependent. In this discussion, we describe development of g-factor analysis for characterizing the secondary structure of proteins in solutions and films. In g-factor analysis, experimental circular dichroism (CD) and UV absorbance spectra are converted to dimensionless g-factor spectra by dividing the differential absorbance of circularly polarized light (AL - AR) by the UV absorbance (A) at each wavelength. The spectra can be deconvolved and the secondary structure estimated without information on the protein molecular weight, sample concentration, or sample path length. We have refined g-factor spectral acquisition and deconvolution parameters to improve the precision and accuracy of experimental g-factor spectra and the subsequent secondary structure estimations. In general, slower scan rates and longer response times are required to obtain high quality g-factor spectra than to obtain ordinary CD spectra, particularly for data at wavelengths longer than 230 nm. The spectral acquisition and deconvolution procedures have been validated for aqueous bovine serum albumin (BSA) and poly(L-proline). The secondary structures of fibronectin and laminin in buffer solutions are predicted. When BSA and poly(L-proline) form films on quartz, their secondary structures change significantly: 13% for BSA and 32% for poly(L-proline). By contrast, the secondary structure of fibronectin is the same in solution and films. The g-factor method is an easy, rapid, accurate and precise method for determining secondary structure and structural changes in protein solutions and films. Potential applications range from proteomics and structure-based drug discovery, to the design and fabrication of biosensors, biomaterials and biofluidic devices.

Algorithms↗

PubMed2003

Precursor tissue analogs as a tissue-engineering strategy.

Natural tissues are composed of functionally diverse cell types that are organized in spatially complex arrangements. Organogenesis of complex tissues requires a coordinated sequential transformation process, with individual stages involving time-dependent expression of cell-cell, cell-matrix, and cell-signal interactions in three dimensions. The common theme of temporal-spatial patterning of these cellular interactions is also observed in other physiological processes, such as growth and development, wound healing, and tumor migration. The "precursor tissue analog" (PTA) applies the temporal-spatial patterning theme to tissue engineering. The goal of PTA in tissue engineering is not to fabricate the final transplantable tissue but rather to guide the dynamic organization, maturation, and remodeling leading to the formation of normal and functional tissues. We describe the critical design principles of PTA. First, structural, mechanical, and physiological requirements of the PTA as a temporary scaffold must be met by a fabrication method with flexibility. The fabrication potential incorporating biological materials such as living cells and plasmid DNA has been addressed. Second, the PTA concept is considered suitable for future tissue engineering in light of the use of undifferentiated stem cells, and may possess a capability to guide stem cells toward diverse differentiation characteristics in situ. To this end, the behavior of the engineered cell and tissue must be monitored in detail. The development of a practical phenotype monitoring system such as a DNA microarray may be integral to the fabrication strategies of PTA. Third, the microtopographical and microenvironmental control on the liquid-solid interaction may lead to a critical design for PTA to provide soluble factors, nutrients, and gases to the cells embedded within the scaffold. We suggest that the level set numerical simulation method may be utilized to engineer the consistent circulation of bioactive liquid throughout the PTA microenvironment.

Animals↗