Method for single-cell mass and electrophoretic mobility measurement

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 137-146).Analysis of single cells using flow cytometry techniques has created a wealth of knowledge about cellular phenomena that could not be obtained by population average measurements. As these techniques are integrated with others to increase the number of parameters that can be measured on single cells and these measurements are made more quantitative, their ability to discriminate between sub-populations of cells increases. Microfabricated sensors offer unique advantages in this area because their internal geometries can be engineered at a size scale comparable to the cell's, making them naturally well-suited for single-cell measurements. The suspended microchannel resonator (SMR) is a versatile flow cytometry platform which is capable measuring the mass of single cells with femtogram resolution. The net frequency shift of a resonant cantilever as the cell transits the fluid-filled microchannel running through it is proportional to the buoyant mass of the cell. The resonance frequency of the SMR is also highly sensitive to a cell's position along the cantilever's length. This thesis presents a new method which makes use of this property to accurately quantify the electrophoretic mobility (EPM) of cells transiting the SMR while subjected to oscillatory electric fields. Recorded resonance frequency time courses can be analyzed to extract both the buoyant mass and EPM of individual cells. This instrument has been used to simultaneously measure the EPM and buoyant masses of discrete polystyrene microspheres and Escherichia coli bacteria. As it has been applied to microspheres of known density, the integrated measurement makes it possible to compute the absolute mass and surface charge of individual microspheres. It has been shown that integrated single-microsphere mass and surface charge measurement enables differentiation of complex aqueous suspensions which is not possible by either measurement alone.by Philip Dextras.Ph.D

Integrated measurement of the mass and surface charge of discrete microparticles using a suspended microchannel resonator

Supporting Information Available: Detailed examinations of the algorithms that have been described in the manuscript for use in signal processing. (PDF) This information is available free of charge via the Internet at http://pubs.acs.org.Measurements of the mass and surface charge of microparticles are employed in the characterization of many types of colloidal dispersions. The suspended microchannel resonator (SMR) is capable of measuring individual particle masses with femtogram resolution. Here, we employ the high sensitivity of the SMR resonance frequency to changes in particle position, relative to the cantilever tip, to determine the electrophoretic mobility of discrete particles in an applied electric field. When a sinusoidal electric field is applied to the suspended microchannel, the transient resonance frequency shift corresponding to a particle transit can be analyzed by digital signal processing to extract both the buoyant mass and electrophoretic mobility of each particle. These parameters, together with the mean particle density, can be used to compute the size, absolute mass, and surface charge of discrete microspheres, leading to a true representation of the mean and polydispersity of these quantities for a population. We have applied this technique to an aqueous suspension of two types of polystyrene microspheres, to differentiate them based on their absolute mass and their surface charge. The integrated measurement of electrophoretic mobility using the SMR is determined to be quantitative, based on comparison with commercial instruments, and exhibits favorable scaling properties that will ultimately enable measurements from mammalian cells.National Cancer Institute (U.S.) (Platform Partnership Grant R01-CA119402)Institute for Collaborative Biotechnologie

Simple, robust methods for high-throughput nanoliter-scale DNA sequencing

We have developed high-throughput DNA sequencing methods that generate high quality data from reactions as small as 400 nL, providing an approximate order of magnitude reduction in reagent use relative to standard protocols. Sequencing of clones from plasmid, fosmid, and BAC libraries yielded read lengths (PHRED20 bases) of 765 ± 172 (n = 10,272), 621 ± 201 (n = 1824), and 647 ± 189 (n = 568), respectively. Implementation of these procedures at high-throughput genome centers could have a substantial impact on the amount of data that can be generated per unit cost

Fabrication and Characterization of an Integrated Microsystem for Protein Preconcentration and Sensing

We report on a fabrication and packaging process for a microsystem consisting of a mass-based protein detector and a fully integrated preconcentrator. Preconcentration of protein is achieved by means of a nanofluidic concentrator (NC), which takes advantage of fast nonlinear electroosmotic flow near a nanochannel-microchannel junction to concentrate charged molecules inside a volume of fluid on the order of 1 pL. Detection of preconcentrated protein samples is accomplished by passing them through a suspended microchannel resonator (SMR), which is a hollow resonant cantilever serially connected to the NC on the same device. The transit of a preconcentrated sample produces a transient shift in the cantilever's resonance frequency that is proportional to the density of the sample and, hence, the concentration of protein contained in it. A device containing both NC and SMR structures was produced using a novel fabrication process which simultaneously satisfies the separate packaging requirements of the two structures. The initial testing of this prototype device has demonstrated that the integrated SMR can accurately measure the concentration of a bovine serum albumin solution, that was preconcentrated using the integrated NC. Future improvements in the fabrication process will allow site-specific surface modification of the device and compatibility with separation methods, which will create opportunities for its application to immunoassays and universal detection.National Cancer Institute (U.S.) (Contract R01CA119402)National Institutes of Health (U.S.) (NIH Cell Decision Process Center P50-GM68762

This article has been accepted for inclusion in a future issue of this journal. Content is final as presented, with the exception of pagination. JOURNAL OF MICROELECTROMECHANICAL SYSTEMS 1 Fabrication and Characterization of an Integrated Microsystem for

Abstract—We report on a fabrication and packaging process for a microsystem consisting of a mass-based protein detector and a fully integrated preconcentrator. Preconcentration of pro-tein is achieved by means of a nanofluidic concentrator (NC), which takes advantage of fast nonlinear electroosmotic flow near a nanochannel–microchannel junction to concentrate charged mole-cules inside a volume of fluid on the order of 1 pL. Detection of preconcentrated protein samples is accomplished by passing them through a suspended microchannel resonator (SMR), which is a hollow resonant cantilever serially connected to the NC on the same device. The transit of a preconcentrated sample pro-duces a transient shift in the cantilever’s resonance frequency that is proportional to the density of the sample and, hence, the concentration of protein contained in it. A device contain-ing both NC and SMR structures was produced using a nove