Programmable Modification of Cell Adhesion and Zeta Potential in Silica Microchips

Lab on a Chip. Feb, 2003  |  Pubmed ID: 15100798

Spatial patterning of thin polyacrylamide films bonded to self-assembled monolayers on silica microchannels is described as a means for manipulating cell-adhesion and electroosmotic properties in microchips. Streaming potential measurements indicate that the zeta potential is reduced by at least two orders of magnitude at biological pH, and the adhesion of several kinds of cells is reduced by 80-100%. Results are shown for cover slides and in wet-etched silica microchannels. Because the polyacrylamide film is thin and transparent, this film is consistent with optical manipulation of cells and detection of cell contents. The spatial patterning technique is straightforward and has the potential to aid on-chip analysis of single adherent cells.

Flow Injection Analysis in a Microfluidic Format

Analytical Chemistry. Feb, 2003  |  Pubmed ID: 12622393

A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation of a standard six-port, two-way valve used in conventional liquid chromatography and flow injection experiments. Analyte and carrier solutions continuously flow through this injection system allowing for measurements and sample changes to be performed rapidly and simultaneously. Injection volumes of 1.25 nL generated peak area reproducibility of better than 3% relative standard deviation. The flow injection device was evaluated with fluorescent dyes and demonstrated a detection limit of 400 zmol for fluorescein. A rudimentary sample selection system allowed calibration curves to be rapidly produced, often in less than 10 min. The hydrolysis of fluorescein diphosphate by alkaline phosphatase demonstrates that chemical assays can be carried out with this device in a manner characterized by short analysis times and low sample consumption.

Microfluidic Device for Single-cell Analysis

Analytical Chemistry. Jul, 2003  |  Pubmed ID: 14570213

We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

Electroosmotic Flow in a Poly(dimethylsiloxane) Channel Does Not Depend on Percent Curing Agent

Electrophoresis. Apr, 2004  |  Pubmed ID: 15095455

Poly(dimethylsiloxane) (PDMS) microfluidic devices were prepared from different ratios of "curing agent" (which contains silicon hydride groups) to "base" (which contains vinyl-terminated noncross-linked PDMS), to determine the effect of this ratio on electroosmotic flow (EOF). In fabricating devices for this purpose, a novel method for permanently enclosing PDMS channels was developed. As a supplement to the microfluidic method, the inner walls of capillaries were coated with PDMS formed from varying ratios of curing agent to base. EOF was found to be constant for PDMS formed with each ratio, which implies that the negative surface charges do not arise from chemical species present only in the base or the curing agent.

Electrowetting-based Microfluidics for Analysis of Peptides and Proteins by Matrix-assisted Laser Desorption/ionization Mass Spectrometry

Analytical Chemistry. Aug, 2004  |  Pubmed ID: 15307795

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.

Digital Microfluidics with In-line Sample Purification for Proteomics Analyses with MALDI-MS

Analytical Chemistry. Jan, 2005  |  Pubmed ID: 15649050

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.

Droplet-based Microfluidics with Nonaqueous Solvents and Solutions

Lab on a Chip. Feb, 2006  |  Pubmed ID: 16450028

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.

An Integrated Digital Microfluidic Chip for Multiplexed Proteomic Sample Preparation and Analysis by MALDI-MS

Lab on a Chip. Sep, 2006  |  Pubmed ID: 16929401

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.

Proteome-on-a-chip: Mirage, or on the Horizon?

Lab on a Chip. Nov, 2006  |  Pubmed ID: 17066164

Proteomics has emerged as the next great scientific challenge in the post-genome era. But even the most basic form of proteomics, proteome profiling, i.e., identifying all of the proteins expressed in a given sample, has proven to be a demanding task. The proteome presents unique analytical challenges, including significant molecular diversity, an extremely wide concentration range, and a tendency to adsorb to solid surfaces. Microfluidics has been touted as being a useful tool for developing new methods to solve complex analytical challenges, and, as such, seems a natural fit for application to proteome profiling. In this review, we summarize the recent progress in the field of microfluidics in four key areas related to this application: chemical processing, sample preconcentration and cleanup, chemical separations, and interfaces with mass spectrometry. We identify the bright spots and challenges for the marriage of microfluidics and proteomics, and speculate on the outlook for progress.

Bio-microarray Fabrication Techniques--a Review

Critical Reviews in Biotechnology. Oct-Dec, 2006  |  Pubmed ID: 17095434

Microarrays with biomolecules (e.g., DNA and proteins), cells, and tissues immobilized on solid substrates are important tools for biological research, including genomics, proteomics, and cell analysis. In this paper, the current state of microarray fabrication is reviewed. According to spot formation techniques, methods are categorized as "contact printing" and "non-contact printing." Contact printing is a widely used technology, comprising methods such as contact pin printing and microstamping. These methods have many advantages, including reproducibility of printed spots and facile maintenance, as well as drawbacks, including low-throughput fabrication of arrays. Non-contact printing techniques are newer and more varied, comprising photochemistry-based methods, laser writing, electrospray deposition, and inkjet technologies. These technologies emerged from other applications and have the potential to increase microarray fabrication throughput; however, there are several challenges in applying them to microarray fabrication, including interference from satellite drops and biomolecule denaturization.

Microcontact Printing-based Fabrication of Digital Microfluidic Devices

Analytical Chemistry. Nov, 2006  |  Pubmed ID: 17105183

Digital microfluidics is a fluid manipulation technique in which discrete droplets are actuated on patterned arrays of electrodes. Although there is great enthusiasm for the application of this technique to chemical and biological assays, development has been hindered by the requirement of clean room fabrication facilities. Here, we present a new fabrication scheme, relying on microcontact printing (microCP), an inexpensive technique that does not require clean room facilities. In microCP, an elastomeric poly(dimethylsiloxane) stamp is used to deposit patterns of self-assembled monolayers onto a substrate. We report three different microCP-based fabrication techniques: (1) selective etching of gold-on-glass substrates; (2) direct printing of a suspension of palladium colloids; and (3) indirect trapping of gold colloids from suspension. In method 1, etched gold electrodes are used for droplet actuation; in methods 2 and 3, colloid patterns are used to seed electroless deposition of copper. We demonstrate, for the first time, that digital microfluidic devices can be formed by microCP and are capable of the full range of digital microfluidics operations: dispensing, merging, motion, and splitting. Devices formed by the most robust of the new techniques were comparable in performance to devices formed by conventional methods, at a fraction of the fabrication time. These new techniques for digital microfluidics device fabrication have the potential to facilitate expansion of this technology to any research group, even those without access to conventional microfabrication tools and facilities.

Maze Exploration and Learning in C. Elegans

Lab on a Chip. Feb, 2007  |  Pubmed ID: 17268620

The soil dwelling nematode, Caenorhabditis (C.) elegans, is a popular model system for studying behavioral plasticity. Noticeably absent from the C. elegans literature, however, are studies evaluating worm behavior in mazes. Here, we report the use of microfluidic mazes to investigate exploration and learning behaviors in wild-type C. elegans, as well as in the dopamine-poor mutant, cat-2. The key research findings include: (1)C. elegans worms are motivated to explore complex spatial environments with or without the presence of food/reward, (2) wild-type worms exhibit a greater tendency to explore relative to mutant worms, (3) both wild-type and mutant worms can learn to make unconditioned responses to food/reward, and (4) wild-type worms are significantly more likely to learn to make conditioned responses linking reward to location than mutant worms. These results introduce microfluidic mazes as a valuable new tool for biological behavioral analysis.

Flow of Microgel Capsules Through Topographically Patterned Microchannels

Lab on a Chip. Jul, 2007  |  Pubmed ID: 17594005

We investigated the flow dynamics of microgel capsules in topographically patterned microfluidic devices. For microgels flowing through channel constrictions, or orifices, we observed three phenomena: (i) the effect of confinement, (ii) the role of interactions between the microgels and the channel surface, and (iii) the effect of the velocities of microgels prior to their passage through an orifice. We studied negatively charged alginate microgels and positively charged alginate microgels coated with N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC). Aqueous dispersions of microgels were driven through poly(dimethyl siloxane) microchannels carrying a weak negative surface charge. The velocity of the continuous phase, and hence, the velocity of the microgels increased as they passed through topographically patterned orifices. Alginate microgels were observed to have a larger increase in velocity relative to HTCC-coated alginate microgels. This effect, which was attributed to electrostatic attraction or repulsion, was found to be strongest for orifices with dimensions close to the microgel diameter. For example, when 75 microm-diameter microgels flowed through a 76 microm orifice, alginate gels (negatively charged) experienced a 2x greater increase in velocity than HTCC-coated (positively charged) microgels. This effect was exaggerated at lower initial flow rates. For example, when 75 microm-diameter microgels flowed through an 80 microm orifice, a two-fold difference in the velocity changes of the two microgel types was observed when the initial flow rate was 275 microm s(-1), while a three-fold difference in velocity changes was observed when the initial flow rate was 130 microm s(-1). We speculate that these studies will be useful for modeling the flow of suspensions of cells or other biologically relevant particles for a wide range of applications.

Matrix-dependent Adhesion of Vascular and Valvular Endothelial Cells in Microfluidic Channels

Lab on a Chip. Dec, 2007  |  Pubmed ID: 18030398

The interactions between endothelial cells and the underlying extracellular matrix regulate adhesion and cellular responses to microenvironmental stimuli, including flow-induced shear stress. In this study, we investigated the adhesion properties of primary porcine aortic endothelial cells (PAECs) and valve endothelial cells (PAVECs) in a microfluidic network. Taking advantage of the parallel arrangement of the microchannels, we compared adhesion of PAECs and PAVECs to fibronectin and type I collagen, two prominent extracellular matrix proteins, over a broad range of concentrations. Cell spreading was measured morphologically, based on cytoplasmic staining with a vital dye, while adhesion strength was characterized by the number of cells attached after application of shear stresses of 11, 110, and 220 dyn cm(-2). Results showed that PAVECs were more well spread on fibronectin than on type I collagen (P < 0.0001), particularly for coating concentrations of 100, 200, and 500 microg mL(-1). PAVECs also withstood shear significantly better on fibronectin than on collagen for 500 microg mL(-1). PAECs were more well spread on collagen compared to PAVECs (P < 0.0001), but did not have significantly better adhesion strength. These results demonstrate that cell adhesion is both cell-type and matrix dependent. Furthermore, they reveal important phenotypic differences between vascular and valvular endothelium, with implications for endothelial mechanobiology and the design of microdevices and engineered tissues.

A Digital Microfluidic Approach to Homogeneous Enzyme Assays

Analytical Chemistry. Mar, 2008  |  Pubmed ID: 18220413

A digital microfluidic device was applied to a variety of enzymatic analyses. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in channel microfluidics. This approach is more easily reconfigured than a channel device, and the flexibility of these devices makes them suitable for a wide variety of applications. Alkaline phosphatase was chosen as a model enzyme and used to convert fluorescein diphosphate into fluorescein. Droplets of alkaline phosphatase and fluorescein diphosphate were merged and mixed on the device, resulting in a 140-nL, stopped-flow reaction chamber in which the fluorescent product was detected by a fluorescence plate reader. Substrate quantitation was achieved with a linear range of 2 orders of magnitude and a detection limit of approximately 7.0 x 10-20 mol. Addition of a small amount of a nonionic surfactant to the reaction buffer was shown to reduce the adsorption of enzyme to the device surface and extend the lifetime of the device without affecting the enzyme activity. Analyses of the enzyme kinetics and the effects of inhibition with inorganic phosphate were performed, and Km and kcat values of 1.35 microM and 120 s-1, respectively, agreed with those obtained in a conventional 384-well plate under the same conditions (1.85 microM and 155 s-1). A phototype device was also developed to perform multiplexed enzyme analyses. It was concluded that the digital microfluidic format is able to perform detailed and reproducible assays of substrate concentrations and enzyme activity in much smaller reaction volumes and with higher sensitivity than conventional methods.

Digital Microfluidics for Cell-based Assays

Lab on a Chip. Apr, 2008  |  Pubmed ID: 18369505

We introduce a new method for implementing cell-based assays. The method is based on digital microfluidics (DMF) which is used to actuate nanolitre droplets of reagents and cells on a planar array of electrodes. We demonstrate that this method is advantageous for cell-based assays because of automated manipulation of multiple reagents in addition to reduced reagent use and analysis time. No adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. A cytotoxicity assay using Jurkat T-cells was performed using the new method, which had approximately 20 times higher sensitivity than a conventional well plate assay. These results suggest that DMF has great potential as a simple yet versatile analytical tool for implementing cell-based assays on the microscale.

A Practical Interface for Microfluidics and Nanoelectrospray Mass Spectrometry

Electrophoresis. May, 2008  |  Pubmed ID: 18393343

We report a new method for fabricating nanospray ionization tips for MS, formed from glass substrates and the inert polymer, parylene-C. Using a single photolithography step, the emitters are formed contiguously with microchannels, such that no dead volumes are observed. In addition, because the devices are very thin (approximately 0.3 mm) and the tips are formed at rectangular corners, the Taylor cone volumes are small, which makes the method attractive for future integration with microfluidic separations. Device performance was demonstrated by evaluating diverse analytes, ranging from synthetic polymers, to peptides, to nucleic acids. For all analytes, performance was similar to that of conventional emitters (pulled-glass capillaries and the Agilent HPLC Chip) with the advantage of rapid, batch fabrication of identical devices.

All-terrain Droplet Actuation

Lab on a Chip. May, 2008  |  Pubmed ID: 18432335

Digital microfluidics has become a popular tool for biochemical and biomedical applications. However, its current format is restricted to actuation of droplets on a single plane. Here, we introduce a new method for fluid handling on flexible devices, which we have termed all-terrain droplet actuation (ATDA). We show that ATDA can be used to manipulate droplets across a wide range of geometries, including inclined, declined, vertical, twisted, and upside-down architectures. These new geometries enable flexible, straightforward integration of distinct physicochemical environments on monolithic devices. To illustrate this capacity, we developed temperature- and oxygen-sensitive colorimetric sensors, as well as an automated method for selective enrichment of DNA from a heterogeneous mixture. We anticipate that ATDA will be a useful new tool in the growing trend toward laboratory miniaturization.

Pluronic Additives: a Solution to Sticky Problems in Digital Microfluidics

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2008  |  Pubmed ID: 18481875

Digital microfluidics (DMF) is a promising technique for carrying out miniaturized, automated biochemical assays in which discrete droplets of reagents are actuated on the surface of an array of electrodes. A limitation for DMF is nonspecific protein adsorption to device surfaces, which interferes with assay fidelity and can cause droplets to become unmovable. Here, we report the results of a quantitative analysis of protein adsorption on DMF devices by means of confocal microscopy and secondary ion mass spectrometry. This study led us to a simple and effective method for limiting the extent of protein adsorption: the use of low concentrations of Pluronic F127 as a solution additive. This strategy has a transformative effect on digital microfluidics, facilitating the actuation of droplets containing greater than 1000-fold higher protein concentrations than is possible without the additive. To illustrate the benefits of this new method, we implemented a DMF-driven protein digest assay using large concentrations (1 mg/mL) of protein-substrate. The use of Pluronic additives solves a sticky problem in DMF, which greatly expands the range of applications that are compatible with this promising technology.

Soft Lithography: Masters on Demand

Lab on a Chip. Aug, 2008  |  Pubmed ID: 18651082

We report an ultra-rapid prototyping technique for forming microchannel networks for lab-on-a-chip applications, called masters on-demand. Channels are produced by replica molding on masters formed by laser printing on flexible copper printed circuit board (PCB) substrates. Masters of various designs and dimensions can be individually or mass produced in less than 10 minutes. Using this technique, we have fabricated channels as narrow as 100 microm with heights ranging between 9 microm and 70 microm. Multi-depth channel fabrication is also reported, using a two-step printing process. The functionality of devices formed in this manner is verified by performing in-channel electrophoretic separations and culture and analysis of primary mammalian cells.

Chemistry. Putting Electrowetting to Work

Science (New York, N.Y.). Oct, 2008  |  Pubmed ID: 18948529

Augmenting Microgel Flow Via Receptor-ligand Binding in the Constrained Geometries of Microchannels

Lab on a Chip. Jan, 2009  |  Pubmed ID: 19107286

We investigated the flow dynamics of biotin-conjugated microgel capsules in avidin-conjugated microchannel constrictions. Microgels were prepared using a microfluidic assembly approach. Biotinylated microgels passing through avidin-modified constrictions slowed relative to several control systems. This effect was observed below a critical velocity of the microgels in the channel-at-large. The reduction in microgel velocity in the constriction occurred for several different sizes of microgels and orifices. Soft compliant microgels showed a lower velocity in the constriction relative to rigid microgels with the same concentration of biotin on the surface, due to the ability of the softer microgels to deform in the orifice and maximize their surface area when in contact with the orifice wall.

A World-to-chip Interface for Digital Microfluidics

Analytical Chemistry. Feb, 2009  |  Pubmed ID: 19115860

Digital microfluidics (DMF) is a fluid handling technique that enables manipulation of discrete droplets on an array of electrodes. There is considerable enthusiasm for this method because of the potential for array-based screening applications. A limitation for DMF is nonspecific adsorption of reagents to device surfaces. If a given device is used to actuate multiple reagents, this phenomenon can cause undesirable cross-contamination. A second limitation for DMF (and all other microfluidic systems) is the "world-to-chip" interface; it is notoriously difficult to deliver reagents and samples to such systems without compromising the oft-hyped advantages of rapid analyses and reduced reagent consumption. We introduce a new strategy for digital microfluidics, in which a removable plastic "skin" is used to (a) eliminate cross-contamination and (b) bridge the world-to-chip interface. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots. This new method has the potential to transform DMF from being a curiosity for aficionados into a technology that is useful for biochemical applications at large.

Digital Microfluidic Method for Protein Extraction by Precipitation

Analytical Chemistry. Jan, 2009  |  Pubmed ID: 19117460

We present the first microfluidic method for extracting proteins from heterogeneous fluids by precipitation. The new method comprises an automated protocol for precipitation of proteins onto surfaces, rinsing the precipitates to remove impurities, and resolubilization in buffer for further analysis. The method is compatible with proteins representing a range of different physicochemical properties, as well as with complex mixtures such as fetal bovine serum and cell lysate. In all cases, the quantitative performance (measured using a fluorescent assay for % recovery) was comparable to that of conventional techniques, which are manual and require more time. Thus, this work represents an important first step in efforts to develop fully automated microfluidic methods for proteomic analyses.

Hybrid Microfluidics: a Digital-to-channel Interface for In-line Sample Processing and Chemical Separations

Lab on a Chip. Apr, 2009  |  Pubmed ID: 19350085

Microchannels can separate analytes faster with higher resolution, higher efficiency and with lower reagent consumption than typical column techniques. Unfortunately, an impediment in the path toward fully integrated microchannel-based labs-on-a-chip is the integration of pre-separation sample processing. Although possible in microchannels, such steps are challenging because of the difficulty in maintaining spatial control over many reagents simultaneously. In contrast, the alternative format of digital microfluidics (DMF), in which discrete droplets are manipulated on an array of electrodes, is well-suited for carrying out sequential chemical reactions. Here, we report the development of the first digital-channel hybrid microfluidic device for integrated pre-processing reactions and chemical separations. The device was demonstrated to be useful for on-chip labeling of amino acids and primary amines in cell lysate, as well as enzymatic digestion of peptide standards, followed by separation in microchannels. Given the myriad applications requiring pre-processing and chemical separations, the hybrid digital-channel format has the potential to become a powerful new tool for micro total analysis systems.

Gradient Elution in Microchannel Electrochromatography

Analytical Chemistry. May, 2009  |  Pubmed ID: 19438263

There is great interest in using microfluidic channels packed with a stationary phase for chemical separations of complex mixtures. A key advantage of such techniques is the use of electroosmotic flow (EOF), controlled simply by applying electrical potentials between reservoirs. A disadvantage for this technique, however, is a lack of compatibility with gradient elution separations. This limitation arises from the dependence of EOF velocity on run buffer content (including the concentration of organic modifier). Here, we introduce a method for implementing gradient elution in electrochromatography in which multiple run buffers are velocity-matched, such that the elution profile resembles that found in conventional HPLC. This method is driven entirely with EOF, meaning that pumps, valves, and pressure fittings are not required. The method was validated by application to separations of peptide standards and protein digests. These results suggest that microfluidic electrochromatography may be compatible with a wide range of applications that have previously been unexplored.

A Digital Microfluidic Approach to Proteomic Sample Processing

Analytical Chemistry. Jun, 2009  |  Pubmed ID: 19476392

A common characteristic for proteomic analyses is the need for extensive biochemical processing. Digital microfluidics (DMF), a technique characterized by the manipulation of discrete microdroplets (100 nL-10 microL) on an open array of electrodes, is a good match for carrying out rapid, automated solution-phase reactions. Here, we report a DMF-based method integrating several common processing steps in proteomics, including reduction, alkylation, and enzymatic digestion. Fluorogenic assays were used to quantitatively evaluate the kinetics and reproducibility of each reaction step, and MALDI-MS was used for qualitative confirmation. The method is fast, facile, and reproducible, and thus has the potential to be a useful new tool in proteomics.

Droplet-scale Estrogen Assays in Breast Tissue, Blood, and Serum

Science Translational Medicine. Oct, 2009  |  Pubmed ID: 20368154

Estrogen is a key hormone in human reproductive physiology, controlling ovulation and secondary sexual characteristics. In addition, it plays an important role in the pathogenesis of breast cancer. Indeed, estrogen receptor antagonists and aromatase inhibitors (which block estrogen biosynthesis) are primary drugs used for treatment and prevention in at-risk populations. Despite its importance, tissue concentrations of estrogen are not routinely measured because conventional techniques require large samples of biopsies for analysis. In response to this need, we have developed a digital microfluidic method and applied it to the extraction and quantification of estrogen in 1-microliter samples of breast tissue homogenate (as would be collected with fine-needle aspiration), as well as in whole blood and serum. This method may be broadly applicable to conditions requiring frequent analysis of hormones in clinical samples (for example, infertility and cancer).

Digital Bioanalysis

Analytical and Bioanalytical Chemistry. Jan, 2009  |  Pubmed ID: 18820902

Digital microfluidics has recently emerged as a new paradigm in the world of lab-on-a-chip technology. A wide variety of bioanalyses have been successfully implemented in this format. This paper reviews the various techniques that have been adapted to digital microfluidic systems, and the current state of the field.

A Microfluidic Platform for Complete Mammalian Cell Culture

Lab on a Chip. Jun, 2010  |  Pubmed ID: 20393662

We introduce the first lab-on-a-chip platform for complete mammalian cell culture. The new method is powered by digital microfluidics (DMF), a technique in which nanolitre-sized droplets are manipulated on an open surface of an array of electrodes. This is the first application of DMF to adherent cell culture and analysis, and more importantly, represents the first microfluidic platform capable of implementing all of the steps required for mammalian cell culture-cell seeding, growth, detachment, and re-seeding on a fresh surface. Three key innovations were required to implement complete cell culture on a microfluidic device: (1) a technique for growing cells on patterned islands (or "adhesion pads") positioned on an array of DMF actuation electrodes; (2) a method for rapidly and efficiently exchanging media and other reagents on cells grown on adhesion pads; and (3) a system capable of detachment and collection of cells from an (old) origin site and delivery to a (new) destination site for subculture. The new technique was applied to cells from several different lines which were seeded and repeatedly subcultured for weeks at a time in 150 nL droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. To illustrate an application for this system, a microfluidic method was developed to implement transient transfection-we propose that the combination of this technique with multigenerational culture allows for "on-demand" generation of transiently transfected cells. Broadly, we anticipate that the automated cell microculture technique presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

Immunoassays in Microfluidic Systems

Analytical and Bioanalytical Chemistry. Jun, 2010  |  Pubmed ID: 20422163

Immunoassays have greatly benefited from miniaturization in microfluidic systems. This review, which summarizes developments in microfluidics-based immunoassays since 2000, includes four sections, focusing on the configurations of immunoassays that have been implemented in microfluidics, the main fluid handling modalities that have been used for microfluidic immunoassays, multiplexed immunoassays in microfluidic platforms, and the emergence of label-free detection techniques. The field of microfluidic immunoassays is continuously improving and has great promise for the future.

Multilayer Hybrid Microfluidics: a Digital-to-channel Interface for Sample Processing and Separations

Analytical Chemistry. Aug, 2010  |  Pubmed ID: 20670000

Microchannels can separate analytes faster with higher resolution, higher efficiency and with lower reagent consumption than typical column techniques. Unfortunately, an impediment in the path toward fully integrated microchannel-based laboratories-on-a-chip is the integration of preseparation sample processing. In contrast, the alternative format of digital microfluidics (DMF), in which discrete droplets are manipulated on an array of electrodes, is well-suited for carrying out sequential chemical reactions such as those commonly employed in proteomic sample preparation. We recently reported a new paradigm of "hybrid microfluidics," integrating DMF with microchannels for in-line sample processing and separations. Here, we build on our initial efforts, introducing a second-generation hybrid microfluidic device architecture. In the new multilayer design, droplets are manipulated by DMF in the two-plate format, an improvement that facilitates dispensing samples from reservoirs, as well as droplet splitting and storage for subsequent analysis. To demonstrate the capabilities of the new method, we implemented an on-chip serial dilution experiment, as well as multistep enzymatic digestion. Given the myriad applications requiring preprocessing and chemical separations, the hybrid digital-channel format has the potential to become a powerful new tool for micro total analysis systems.

Let's Get Digital: Digitizing Chemical Biology with Microfluidics

Current Opinion in Chemical Biology. Oct, 2010  |  Pubmed ID: 20674472

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications in chemical biology. In DMF, nL-mL droplets containing samples and reagents are controlled (i.e., moved, merged, mixed, and dispensed from reservoirs) by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquid and solid) in heterogeneous systems with no need for complex networks of microvalves. Here, we review the state-of-the-art in DMF as applied to a wide range of applications in chemical biology, including proteomics, enzyme assays and immunoassays, applications involving DNA, cell-based assays, and clinical applications.

Synchronized Synthesis of Peptide-based Macrocycles by Digital Microfluidics

Angewandte Chemie (International Ed. in English). Nov, 2010  |  Pubmed ID: 20715231

Durable, Region-specific Protein Patterning in Microfluidic Channels

Biomaterials. Jan, 2010  |  Pubmed ID: 19800682

We present a straightforward, accessible method to covalently pattern proteins in poly(dimethyl siloxane) (PDMS) microchannels. Our approach includes (i) region-specific photografting of a layer of poly(acrylamide) (PAAm) and (ii) bioconjugation of PAAm with a desired protein. The method produces symmetric protein patterns on all channel walls, which have high specificity and pattern fidelity, are compatible with a variety of geometries and exhibit excellent longevity under shear stresses of up to 1 dyn/cm. We demonstrate the generality of the method by creating multi-protein gradients within microfluidic microchannels and by in-situ patterning of islands of multiple proteins. Protein activity was observed by the digestion of BODIPY-casein using channels patterned with trypsin.

Technique for Real-time Measurements of Endothelial Permeability in a Microfluidic Membrane Chip Using Laser-induced Fluorescence Detection

Analytical Chemistry. Feb, 2010  |  Pubmed ID: 20050596

Characterizing permeability of the endothelium that lines blood vessels and heart valves provides fundamental physiological information and is required to evaluate uptake of drugs and other biomolecules. However, current techniques used to measure permeability, such as Transwell insert assays, do not account for the recognized effects of fluid flow-induced shear stress on endothelial permeability or are inherently low-throughput. Here we report a novel on-chip technique in a two-layer membrane-based microfluidic platform to measure real-time permeability of endothelial cell monolayers on porous membranes. Bovine serum albumin (a model protein) conjugated with fluorescein isothiocyanate was delivered to an upper microchannel by pressure-driven flow and was forced to permeate a poly(ethylene terephthalate) membrane into a lower microchannel, where it was detected by laser-induced fluorescence. The concentration of the permeate at the point of detection varied with channel flow rates in agreement to less than 1% with theoretical analyses using a pore flow model. On the basis of the model, a sequential flow rate stepping scheme was developed and applied to obtain the permeability of cell-free and cell-bound membrane layers. This technique is a highly sensitive, novel microfluidic approach for measuring endothelial permeability in vitro, and the use of micrometer-sized channels offers the potential for parallelization and increased throughput compared to conventional shear-based permeability measurement methods.

A Circular Cross-section PDMS Microfluidics System for Replication of Cardiovascular Flow Conditions

Biomaterials. May, 2010  |  Pubmed ID: 20167361

Since the inception of soft lithography, microfluidic devices for cardiovascular research have been fabricated easily and cost-effectively using the soft lithography method. The drawback of this method was the fabrication of microchannels with rectangular cross-sections, which did not replicate the circular cross-sections of blood vessels. This article presents a novel, straightforward approach for the fabrication of microchannels with circular cross-sections in poly(dimethylsiloxane) (PDMS), using soft lithography. The method exploits the polymerization of the liquid silicone oligomer around a gas stream when both of them are coaxially introduced in the microchannel with a rectangular cross-section. We demonstrate (i) the ability to control the diameter of circular cross-sections of microchannels from ca. 40-100 mum; (ii) the fabrication of microchannels with constrictions, and (iii) the capability to grow endothelial cells on the inner surface of the microchannels.

Folded Emitters for Nanoelectrospray Ionization Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. Dec, 2010  |  Pubmed ID: 21072798

Electrospray ionization (ESI) has revolutionized mass spectrometry (MS), providing a facile method for the ionization of macromolecules for analysis by mass. The development of nanoESI-MS has further extended the utility of ESI-MS, permitting the analysis of small-volume samples with enhanced sensitivity over conventional ESI-MS. Traditional nanoESI-MS experiments use pulled-glass capillary emitters, which are expensive to purchase and require specialized instruments and training to fabricate in-house. Furthermore, these emitters suffer from problems including clogging, sample contamination, and irreproducible spray stability. Here, we report a new emitter for nanoESI-MS, made by folding small pieces of polyimide tape. In comparison with conventional pulled-glass capillary emitters, the new emitters are inexpensive and simple to make. Their low cost makes them disposable after a single use, such that sample contamination or clogging is never a problem. Emitter performance has been evaluated for diverse analytes encompassing a large mass range, including small molecules, peptides, proteins, and synthetic polymers. In all cases, the performance is similar to that of pulled-glass capillary emitters, with the advantages of low cost, ease of use, and disposability.

A Microfluidic Membrane Device to Mimic Critical Components of the Vascular Microenvironment

Biomicrofluidics. 2011  |  Pubmed ID: 21522499

Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.

A Digital Microfluidic Method for in Situ Formation of Porous Polymer Monoliths with Application to Solid-phase Extraction

Analytical Chemistry. May, 2011  |  Pubmed ID: 21524096

We introduce the marriage of two technologies: digital microfluidics (DMF), a technique in which droplets are manipulated by application of electrostatic forces on an array of electrodes coated by an insulator, and porous polymer monoliths (PPMs), a class of materials that is popular for use for solid-phase extraction and chromatography. In this work, circular PPM discs were formed in situ by dispensing and manipulating droplets of monomer solutions to designated spots on a DMF device followed by UV-initiated polymerization. We used PPM discs formed in this manner to develop a digital microfluidic solid-phase extraction (DMF-SPE) method, in which PPM discs are activated and equilibrated, samples are loaded, PPM discs are washed, and the samples are eluted, all using microliter droplets of samples and reagents. The new method has extraction efficiency (93%) comparable to that of pipet-based ZipTips and is compatible with preparative sample extraction and recovery for on-chip desalting, removal of surfactants, and preconcentration. We anticipate that DMF-SPE may be useful for a wide range of applications requiring preparative sample cleanup and concentration.

A New Angle on Pluronic Additives: Advancing Droplets and Understanding in Digital Microfluidics

Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2011  |  Pubmed ID: 21651299

Biofouling in microfluidic devices limits the type of samples which can be handled and the duration for which samples can be manipulated. Despite the cost of disposing fouled devices, relatively few strategies have been developed to tackle this problem. Here, we have analyzed a series of eight amphiphilic droplet additives, Pluronic coblock polymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO), as a solution to biofouling in digital microfluidics using serum-containing cell culture media as a model fluid. Our analysis shows that species with longer PPO chains are superior for enabling droplet motion and reducing biofouling. Two of the tested species, L92 and P105, were found to lengthen device lifetimes by 2-3 times relative to additives used previously when used at optimal concentrations. Pluronics with low PEO content such as L92 were found to be cytotoxic to an immortalized mammalian cell line, and therefore we recommend that Pluronic additives with greater or equal to 50% PEO composition, such as P105, be used for digital microfluidic applications involving cells. Finally, contact angle measurements were used to probe the interaction between Pluronic-containing droplets and device surfaces. Strong correlations were found between various types of contact angle measurements and the capacity of additives to reduce biofouling, which suggests that contact angle measurements may be useful as a tool for rapidly screening new candidates for the potential to reduce biofouling. We propose that this study will be useful for scientists and engineers who are developing digital microfluidic platforms for a wide range of applications involving protein-containing solutions, and in particular, for applications involving cells.

A Digital Microfluidic Method for Dried Blood Spot Analysis

Lab on a Chip. Oct, 2011  |  Pubmed ID: 21869989

Blood samples stored as dried blood spots (DBSs) are emerging as a useful sampling and storage vehicle for a wide range of applications. Unfortunately, the surging popularity of DBS samples has not yet been accompanied by an improvement in automated techniques for extraction and analysis. As a first step towards overcoming this challenge, we have developed a prototype microfluidic system for quantification of amino acids in dried blood spots, in which analytes are extracted, mixed with internal standards, derivatized, and reconstituted for analysis by (off-line and in-line) tandem mass spectrometry. The new method is fast, robust, precise, and most importantly, compatible with automation. We propose that the new method can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

A Switchable Digital Microfluidic Droplet Dye-laser

Lab on a Chip. Nov, 2011  |  Pubmed ID: 21901207

Digital microfluidic devices allow the manipulation of droplets between two parallel electrodes. These electrodes can act as mirrors generating a micro-cavity, which can be exploited for a droplet dye-laser. Three representative laser-dyes with emission wavelengths spanning the whole visible spectrum are chosen to show the applicability of this concept. Sub-microlitre droplets of laser-dye solution are moved in and out of a lasing site on-chip to down-convert the UV-excitation light into blue, green and red laser-pulses.

Integrated Microbioreactor for Culture and Analysis of Bacteria, Algae and Yeast

Biomedical Microdevices. Feb, 2011  |  Pubmed ID: 20838902

We introduce a micro-scale bioreactor for automated culture and density analysis of microorganisms. The microbioreactor is powered by digital microfluidics (DMF) and because it is used with bacteria, algae and yeast, we call it the BAY microbioreactor. Previous miniaturized bioreactors have relied on microchannels which often require valves, mixers and complex optical systems. In contrast, the BAY microbioreactor is capable of culturing microorganisms in distinct droplets on a format compatible with conventional bench-top analyzers without the use of valves, mixers or pumps. Bacteria, algae and yeast were grown for up to 5 days with automated semi-continuous mixing and temperature control. Cell densities were determined by measuring absorbances through transparent regions of the devices, and growth profiles were shown to be comparable to those generated in conventional, macro-scale systems. Cell growth and density measurements were integrated in the microbioreactor with a fluorescent viability assay and transformation of bacteria with a fluorescent reporter gene. These results suggest that DMF may be a useful new tool in automated culture and analysis of microorganisms for a wide range of applications.

A Feedback Control System for High-fidelity Digital Microfluidics

Lab on a Chip. Feb, 2011  |  Pubmed ID: 21038034

Digital microfluidics (DMF) is a technique in which discrete droplets are manipulated by applying electrical fields to an array of electrodes. In an ideal DMF system, each application of driving potential would cause a targeted droplet to move onto an energized electrode (i.e., perfect fidelity between driving voltage and actuation); however, in real systems, droplets are sometimes observed to resist movement onto particular electrodes. Here, we implement a sensing and feedback control system in which all droplet movements are monitored, such that when a movement failure is observed, additional driving voltages can be applied until the droplet completes the desired operation. The new system was evaluated for a series of liquids including water, methanol, and cell culture medium containing fetal bovine serum, and feedback control was observed to result in dramatic improvements in droplet actuation fidelity and velocity. The utility of the new system was validated by implementing an enzyme kinetics assay with continuous mixing. The new platform for digital microfluidics is simple and inexpensive and thus should be useful for scientists and engineers who are developing automated analysis platforms.

A Digital Microfluidic Approach to Heterogeneous Immunoassays

Analytical and Bioanalytical Chemistry. Jan, 2011  |  Pubmed ID: 21057776

A digital microfluidic (DMF) device was applied to a heterogeneous sandwich immunoassay. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in microchannels. Since droplets are manipulated on relatively generic 2-D arrays of electrodes, DMF devices are straightforward to use, and are reconfigurable for any desired combination of droplet operations. This flexibility makes them suitable for a wide range of applications, especially those requiring long, multistep protocols such as immunoassays. Here, we developed an immunoassay on a DMF device using Human IgG as a model analyte. To capture the analyte, an anti-IgG antibody was physisorbed on the hydrophobic surface of a DMF device, and DMF actuation was used for all washing and incubation steps. The bound analyte was detected using FITC-labeled anti-IgG, and fluorescence after the final wash was measured in a fluorescence plate reader. A non-ionic polymer surfactant, Pluronic F-127, was added to sample and detection antibody solutions to control non-specific binding and aid in movement via DMF. Sample and reagent volumes were reduced by nearly three orders of magnitude relative to conventional multiwell plate methods. Since droplets are in constant motion, the antibody-antigen binding kinetics is not limited by diffusion, and total analysis times were reduced to less than 2.5 h per assay. A multiplexed device comprising several DMF platforms wired in series further increased the throughput of the technique. A dynamic range of approximately one order of magnitude was achieved, with reproducibility similar to the assay when performed in a 96-well plate. In bovine serum samples spiked with human IgG, the target molecule was successfully detected in the presence of a 100-fold excess of bovine IgG. It was concluded that the digital microfluidic format is capable of carrying out qualitative and quantitative sandwich immunoassays with a dramatic reduction in reagent usage and analysis time compared to macroscale methods.

A Digital Microfluidic Platform for Primary Cell Culture and Analysis

Lab on a Chip. Jan, 2012  |  Pubmed ID: 22094822

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.

A Digital Microfluidic Method for Multiplexed Cell-based Apoptosis Assays

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22159547

Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.

Virtual Microwells for Digital Microfluidic Reagent Dispensing and Cell Culture

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22179581

Digital microfluidic (DMF) liquid handling includes active (electrostatic) and passive (surface tension) mechanisms for reagent dispensing. Here we implement a simple and straightforward Teflon-AF liftoff protocol for patterning hydrophilic sites on a two-plate device for precise passive dispensing of reagents forming virtual microwells - an analogy to the wells found on a microtitre plate. We demonstrate here that devices formed using these methods are capable of reproducible dispensing of volumes ranging from ∼80 to ∼800 nL, with CVs of 0.7% to 13.8% CV. We demonstrate that passive dispensing is compatible with DMF operation in both air and oil, and provides for improved control of dispensed nano- and micro- litre volumes when compared to active electrostatic dispensing. Further, the technique is advantageous for cell culture and we report the first example of reagent dispensing on a single-plate DMF device. We anticipate this method will be useful for a wide range of applications - particularly those involving adherent cell culture and analysis.