Microfluidic free-flow electrophoresis for proteomics-on-a-chip

A new free-flow electrophoresis microchip with integrated permeable membranes was developed, and different substances were separated by free-flow zone electrophoresis, free-flow isoelectric focusing and free-flow field step electrophoresis. This chip contained a new type of membranes enabling a stable carrier flow with a perpendicular electrical current. Due to this chip configuration, the device performance and efficiency were superior to recenty published alternative systems in terms of separation resolution and sample capacity. The results furthermore indicate that even better results are possible. Analytes were separated and focused within hundreds of milliseconds whereby only nanoliters of samples were consumed. In addition, a new sample steering method was demonstrated during free-flow zone electrophoresis, allowing the specific sorting of various components. As an alternative, a free-flow electrophoresis chip was developed with integated platinum electrodes, whereby the generation of gas bubbles caused by electrolysis was successfully suppressed by chemical means. Gas bubbles generated by electrolysis are major concern in free-flow electrophoresis systems in general leading to distorted separation. Based on the results, a fourth free-flow chip was developed with an integrated surface plasmon resonance gold detection region. Although fabrication was successful, certain hurdles, in particular surface chemistry issues still remain to be overcome to perform separation and real-time detection of biological samples within this hyphenated micro device. A strategy for proteomics-on-a-chip was developed aiming at the separation of antigens that play a role in autoimmune diseases. In addition two new continuous flow microfluidic chips were developed allowing for continuous biochemical reactions of surface patterning applications. These devices could be of further interest in future, in particular in more complex analytical systems related to proteomics-on-a-chip

Laminar Flow Microarray Patterning by Perpendicular Electrokinetic Focusing

This paper describes a method to pattern microarrays in a closed microfluidic device. Two perpendicular laminar flow streams can operate in terms to sequentially coat the surface of a flow-chamber with parallel lanes in two directions. Two perpendicular sample streams can be controlled in position and width by applying electrokinetic focusing, for which each of the two streams is sandwiched between two parallel laminar flow streams containing just a buffer solution. Electroosmotic flow allows a simple chip design without any moving parts being involved. With this device configuration it is possible to define an array of up to 169 spots on a surface area of 1 mm2

A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm2. This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.\ud \ud \ud \u

Proteomics-on-a-chip for Biomarker discovery

In proteomics research still two-dimensional gel electrophoresis (2D-GE) is currently used for biomarker discovery. We applied free flow electrophoresis (FFE) separation technology combined with biomolecular interaction sensing using Surface Plasmon Resonance (SPR) imaging in an integrated proteomics-on-a-chip device as a proof of concept for biomarker discovery

Microfluidic preparative free-flow isoelectric focusing in a triangular channel: System development and characterization

A preparative scale free-flow IEF device is developed and characterized with the aim of addressing needs of molecular biologists working with protein samples on the milligrams and milliliters scale. A triangular-shape separation channel facilitates the establishment of the pH gradient with a corresponding increase in separation efficiency and decrease in focusing time compared with that in a regular rectangular channel. Functionalized, ion-permeable poly(acrylamide) gel membranes are sandwiched between PDMS and glass layers to both isolate the electrode buffers from the central separation channel and also to selectively adjust the voltage efficiency across the separation channel to achieve high electric field separation. The 50×70 mm device is fabricated by soft lithography and has 24 outlets evenly spaced across a pH gradient between pH 4 and 10. This preparative free-flow IEF system is investigated and optimized for both aqueous and denaturing conditions with respect to the electric field and potential efficiency and with consideration of Joule-heating removal. Energy distribution across the functionalized polyacrylamide gel is investigated and controlled to adjust the potential efficiency between 15 and 80% across the triangular separation channel. The device is able to achieve constant electric fields high as 370±20 V/cm through the entire triangular channel given the separation voltage of 1800 V, enabling separation of five fluorescent pI markers as a demonstration example.United States. Army Research Office (Grant Number: W911NF-07-D-0004)National Institutes of Health (U.S.) (Grant Number: GM68762

Synchronized, continuous-flow zone electrophoresis

A new method for performing continuous electrophoretic separation of complex mixtures in microscale devices is proposed. Unlike in free-flow electrophoresis devices, no mechanical pumping is requiredboth fluid transport and separation are driven electrokinetically. This gives the method great potential for on-a-chip integration in multistep analytical systems. The method enables us to collect fractionated sample and tensfold purification is possible. The model of the operation is presented and a detailed description of the optimal conditions for performing purification is given. The chip devices with 10-μm-deep separation chamber of 1.5 mm × 4 mm in size were fabricated in glass. A standard microchip electrophoresis setup was used. Continuous separation of rhodamine B, rhodamine 6G, and fluorescein was accomplished. Purification was demonstrated on a mixture containing rhodamine B and fluorescein, and the recovery of both fractions was achieved. The results show the feasibility of the method

Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

In this paper, we are evaluating the strategy of sorting peptides/proteins based on the charge to mass without resorting to ampholytes and/or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.Korea Institute of Science and Technology. Intelligent Microsystems CenterMassachusetts Institute of Technology. Center for Environmental Health SciencesNational Institute of Environmental Health Sciences (Grant No. P30-ES002109)United States. National Institutes of Health (grant R21 EB008177

Rapid inoculation of single bacteria into parallel picoliter fermentation chambers

Probst C, Grünberger A, Braun N, et al. Rapid inoculation of single bacteria into parallel picoliter fermentation chambers. Analytical methods. 2015;7(1):91-98.Microfluidic single-cell cultivation devices have been successfully utilized in a variety of biological research fields. One major obstacle to the successful implementation of high throughput single-cell cultivation technology is the requirement for a simple, fast and reliable cell inoculation procedure. In the present report, an air-bubble-based cell loading methodology is described and validated for inoculating single bacteria into multiple picoliter sized growth chambers arranged in a highly parallel manner. It is shown that the application of the injected air bubble can serve as a reproducible mechanism to modify laminar flow conditions. In this way, convective flow was temporarily induced in more than 1000 cultivation chambers simultaneously, which under normal conditions operate exclusively under diffusive mass transport. Within an inoculation time of 100 s, Corynebacterium glutamicum cells were inoculated by convection at minimal stress level and single bacteria remain successfully trapped by cell-wall interactions. The procedure is easy, fast, gentle and requires only minimal fluidic control and equipment. The technique is well suited for microbial cell loading into commonly used microfluidic growth sites arranged in parallel intended for high throughput single-cell analysis

Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria

Probst C, Grünberger A, Wiechert W, Kohlheyer D. Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria. Micromachines. 2013;4(4):357-369.Microfluidics has become an essential tool in single-cell analysis assays for gaining more accurate insights into cell behavior. Various microfluidics methods have been introduced facilitating single-cell analysis of a broad range of cell types. However, the study of prokaryotic cells such as Escherichia coli and others still faces the challenge of achieving proper single-cell immobilization simply due to their small size and often fast growth rates. Recently, new approaches were presented to investigate bacteria growing in monolayers and single-cell tracks under environmental control. This allows for high-resolution time-lapse observation of cell proliferation, cell morphology and fluorescence-coupled bioreporters. Inside microcolonies, interactions between nearby cells are likely and may cause interference during perturbation studies. In this paper, we present a microfluidic device containing hundred sub-micron sized trapping barrier structures for single E. coli cells. Descendant cells are rapidly washed away as well as components secreted by growing cells. Experiments show excellent growth rates, indicating high cell viability. Analyses of elongation and growth rates as well as morphology were successfully performed. This device will find application in prokaryotic single-cell studies under constant environment where by-product interference is undesired

Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

Kampf J, Gerwig J, Kruse K, et al. Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability. mBio. 2018;9(5): e01464-18