A microfluidic “baby machine” for cell synchronization

Josephine W. Shaw, Kristofor Payer, Sungmin Son, William H. Grover and Scott R. Manalis, Lab on a Chip, in press.

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the “baby machine” was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing “baby machine” is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic “baby machine” in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic “baby machine” by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.

Measuring single-cell density

William H. Grover, Andrea K. Bryan, Monica Diez-Silva, Subra Suresh, John M. Higgins, and Scott R. Manalis, Proceedings of the National Academy of Sciences of the United States of America 108 (27), 10992-10996 (2011). Download

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g/mL. We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here we demonstrate this with four examples: identifying P. falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient’s own blood, identifying irreversibly-sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single cell density will provide valuable insights into cell state for a wide range of biological processes.

See also the MIT News story “How dense is a cell?”, which includes a short animation I made to explain the density measurement (right). The source code for this animation is also available.

This paper was highlighted in the “In This Issue” feature of PNAS on July 5, 2011.

Thanks to Leiden University’s Cell Observatory for choosing this as their publication of the week.

Thanks to National Institute of General Medical Sciences for featuring this in their Biomedical Beat.

Using buoyant mass to measure the growth of single cells

Michel Godin, Francisco Feijo Delgado, Sungmin Son, William H. Grover, Andrea K. Bryan, Amit Tzur, Paul Jorgensen, Kris Payer, Alan D. Grossman, Marc W. Kirschner, and Scott R. Manalis, Nature Methods 7, 387 - 390 (2010). Download

We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the “instantaneous” growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.

See also the MIT News story “MIT measures rate at which single cells accumulate mass”.

This paper was also mentioned in the “Mysteries of the Cell” series in Science.

Teflon films for chemically-inert microfluidic valves and pumps

William H. Grover, Marcio G. von Muhlen, and Scott R. Manalis, Lab on a Chip 8 (6) 913-918, 2008. Download

We present a simple method for fabricating chemically-inert Teflon microfluidic valves and pumps in glass microfluidic devices. These structures are modeled after monolithic membrane valves and pumps that utilize a featureless polydimethylsiloxane (PDMS) membrane bonded between two etched glass wafers. The limited chemical compatibility of PDMS has necessitated research into alternative materials for microfluidic devices. Previous work has shown that spin-coated amorphous fluoropolymers and Teflon-fluoropolymer laminates can be fabricated and substituted for PDMS in monolithic membrane valves and pumps for space flight applications. However, the complex process for fabricating these spin-coated Teflon films and laminates may preclude their use in many research and manufacturing contexts. As an alternative, we show that commercially-available fluorinated ethylene-propylene (FEP) Teflon films can be used to fabricate chemically-inert monolithic membrane valves and pumps in glass microfluidic devices. The FEP Teflon valves and pumps presented here are simple to fabricate, function similarly to their PDMS counterparts, maintain their performance over extended use, and are resistant to virtually all chemicals. These structures should facilitate lab-on-a-chip research involving a vast array of chemistries that are incompatible with native PDMS microfluidic devices.

Suspended microchannel resonators for ultralow volume universal detection

Sungmin Son, William H. Grover, Thomas P. Burg, and Scott R. Manalis, Analytical Chemistry 80 (12), 4757-4760, 2008. Download

Universal detectors that maintain high sensitivity as the detection volume is reduced to the subnanoliter scale can enhance the utility of miniaturized total analysis systems (μ-TAS). Here the unique scaling properties of the suspended microchannel resonator (SMR) are exploited to show universal detection in a 10 pL analysis volume with a density detection limit of similar to 1 μg cm3 (10 Hz bandwidth) and a dynamic range of 6 decades. Analytes with low UV extinction coefficients such as polyethylene glycol (PEG) 8 kDa, glucose, and glycine are measured with molar detection limits of 0.66, 13.5, and 31.6 μM, respectively. To demonstrate the potential for real-time monitoring, gel filtration chromatography was used to separate different molecular weights of PEG as the SMR acquired a chromatogram by measuring the eluate density. This work suggests that the SMR could offer a simple and sensitive universal detector for various separation systems from liquid chromatography to capillary electrophoresis. Moreover, since the SMR is itself a microfluidic channel, it can be directly integrated into μ-TAS without compromising overall performance.

Micropneumatic digital logic structures for integrated microdevice computation and control

Erik C. Jensen, William H. Grover, and Richard A. Mathies, Journal of Microelectromechanical Systems 16 (6), 1378-1385, 2007. Download

It is shown that microfabricated polydimethylsiloxane membrane valve structures can be configured to function as transistors in pneumatic digital logic circuits. Using the analogy with metal-oxide-semiconductor field-effect transistor circuits, networks of pneumatically actuated microvalves are designed to produce pneumatic digital logic gates (AND, OR, NOT, NAND, and XOR). These logic gates are combined to form 4- and 8-bit ripple-carry adders as a demonstration of their universal pneumatic computing capabilities. Signal propagation through these pneumatic circuits is characterized, and an amplifier circuit is demonstrated for improved signal transduction. Propagation of pneumatic carry information through the 8-bit adder is complete within 1.1 s, demonstrating the feasibility of integrated temporal control of pneumatic actuation systems. Integrated pneumatic logical systems reduce the number of off-chip controllers required for lab-on-a-chip and microelectromechanical system devices, allowing greater complexity and portability. This technology also enables the development of digital pneumatic computing and logic systems that are immune to electromagnetic interference.

Development and multiplexed control of latching pneumatic valves using microfluidic logical structures

William H. Grover, Robin H.C. Ivester, Erik C. Jensen, and Richard A. Mathies, Lab on a Chip 6 (5), 623-631, 2006. Download

Novel latching microfluidic valve structures are developed, characterized, and controlled independently using an on-chip pneumatic demultiplexer. These structures are based on pneumatic monolithic membrane valves and depend upon their normally-closed nature. Latching valves consisting of both three- and four-valve circuits are demonstrated. Vacuum or pressure pulses as short as 120 ms are adequate to hold these latching valves open or closed for several minutes. In addition, an on-chip demultiplexer is demonstrated that requires only n pneumatic inputs to control 2(n-1) independent latching valves. These structures can reduce the size, power consumption, and cost of microfluidic analysis devices by decreasing the number of off-chip controllers. Since these valve assemblies can form the standard logic gates familiar in electronic circuit design, they should be useful in developing complex pneumatic circuits.

Microfluidic serial dilution circuit

Brian M. Paegel, William H. Grover, Alison M. Skelley, Richard A. Mathies, Gerald F. Joyce, Analytical Chemistry 78 (21) 7522-7527, 2006. Download

In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipetting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

An integrated microfluidic processor for single nucleotide polymorphism-based DNA computing

William H. Grover and Richard A. Mathies, Lab on a Chip 5 (10) 1033-1040, 2005. Download

An integrated microfluidic processor is developed that performs molecular computations using single nucleotide polymorphisms (SNPs) as binary bits. A complete population of fluorescein-labeled DNA “answers” is synthesized containing three distinct polymorphic bases; the identity of each base (A or T) is used to encode the value of a binary bit (TRUE or FALSE). Computation and readout occur by hybridization to complementary capture DNA oligonucleotides bound to magnetic beads in the microfluidic device. Beads are loaded into sixteen capture chambers in the processor and suspended in place by an external magnetic field. Integrated microfluidic valves and pumps circulate the input DNA population through the bead suspensions. In this example, a program consisting of a series of capture/rinse/release steps is executed and the DNA molecules remaining at the end of the computation provide the solution to a three-variable, four-clause Boolean satisfiability problem. The improved capture kinetics, transfer efficiency, and single-base specificity enabled by microfluidics make our processor well-suited for performing larger-scale DNA computations.

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars

Alison M. Skelley, James R. Scherer, Andrew D. Aubrey, William H. Grover, Robin H.C. Ivester, Pascale Ehrenfreund, Frank J. Grunthaner, Jeffrey L. Bada, Richard A. Mathies, Proceedings of the National Academy of Sciences of the United States of America 102 (4), 1041-1046, 2005. Download

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment.

Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices

William H. Grover, Alison M. Skelley, Chung N. Liu, Eric T. Lagally, Richard A. Mathies, Sensors and Actuators B 89 (3), 315-323, 2003. Download

Monolithic elastomer membrane valves and diaphragm pumps suitable for large-scale integration into glass microfluidic analysis devices are fabricated and characterized. Valves and pumps are fabricated by sandwiching an elastomer membrane between etched glass fluidic channel and manifold wafers. A three-layer valve and pump design features simple non-thermal device bonding and a hybrid glass-PDMS fluidic channel; a four-layer structure includes a glass fluidic system with minimal fluid-elastomer contact for improved chemical and biochemical compatibility. The pneumatically actuated valves have less than 10 nl dead volumes, can be fabricated in dense arrays, and can be addressed in parallel via an integrated manifold. The membrane valves provide flow rates up to 380 nl/s at 30 kPa driving pressure and seal reliably against fluid pressures as high as 75 kPa. The diaphragm pumps are self-priming, pump from a few nanoliters to a few microliters per cycle at overall rates from 1 to over 100 nl/s, and can reliably pump against 42 kPa pressure heads. These valves and pumps provide a facile and reliable integrated technology for fluid manipulation in complex glass microfluidic and electrophoretic analysis devices.

Localized deposition of zinc oxide films by automated fluid dispensing method

Karel Domansky, Aimee Rose, William H. Grover, Gregory J. Exarhos, Materials Science and Engineering B 76 (2) 116-121, 2000. Download

Optically clear Au- and Ga-doped zinc oxide films have been locally deposited from solution precursors on silicon and silica substrates as micrometer size dots using an automated fluid dispensing method. Dot dimensions have been shown to be strongly dependent on the solution composition and substrate temperature. Solution-dispensed films have been characterized by means of optical transmission and reflectance spectroscopy, spectroscopic ellipsometry, and profilometry measurements. Trivalent Au- and Ga-doped zinc oxide features about 500 μm in diameter were deposited on chemically-sensitive field-effect transistors (CHEM-FET). Contrasting behavior in measured work function and film resistance was found for Au and Ga doped films upon exposure to hydrogen and ammonia.

Inverse derivative chronopotentiometry quartz crystal microgravimetry of solid conducting salt films

Kris M. Scaboo, William H. Grover, James Q. Chambers, Analytica Chimica Acta 380 (1) 47-54, 1999. Download

An instrument, built in-house, for the purpose of making derivative chronopotentiometric and quartz crystal microgravimetric measurements is described. The spreadsheet-based software allows for presentation of the raw or derivative data of both the chronopotentiometric and gravimetric measurements in either the time or potential domain. The system was used to study the nucleation-controlled growth of the phase transition associated with the TCNQ(0/-1) couple isolated from a thin film of the conducting salt 9-aminoacridinium tetracyanoquinodimethane. The dt/dE-E curves for a nucleation process are presented along with the Hillman-Bruckenstein analysis of the corresponding mass changes of the film.


Boston-area researchers interested in cell growth, check out the Cell Growth symposium I'm helping organize at MIT on June 11.

In July I'll be joining the faculty of the Department of Bioengineering in the Bourns College of Engineering at the University of California, Riverside!

Archived news