Microfluidic dialysis cell for characterization of macromolecule interactions

Scrimgeour, Jan; Cho, Jae Kyu; Breedveld, Victor; Curtis, Jennifer E.

doi:10.1039/c1sm05125c

Cited by 13 publications

(11 citation statements)

References 39 publications

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“…Fluorescent labeling of HA was achieved using neurocan-G1 EGFP fusion protein (GFPn) expressed by HEK 293 EBNA cells, which were cultured at 37 C and 5% CO 2 in media (50% Dulbecco's modified eagle medium and 50% F12), with 10% FBS, 1% L-glutamine with 0.1% puromycin (EMD Biosciences, San Diego, CA) (28,29). The cells were a gift from Uwe Rauch (Dept.…”

Section: Gfpn Labeling Of Hyaluronanmentioning

confidence: 99%

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

Chang

McLane

Fogg

et al. 2016

Biophysical Journal

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The hyaluronan-rich pericellular matrix (PCM) plays physical and chemical roles in biological processes ranging from brain plasticity, to adhesion-dependent phenomena such as cell migration, to the onset of cancer. This study investigates how the spatial distribution of the large negatively charged bottlebrush proteoglycan, aggrecan, impacts PCM morphology and cell surface access. The highly localized pericellular milieu limits transport of nanoparticles in a size-dependent fashion and sequesters positively charged molecules on the highly sulfated side chains of aggrecan. Both rat chondrocyte and human mesenchymal stem cell PCMs possess many unused binding sites for aggrecan, showing a 2.5x increase in PCM thickness from ∼7 to ∼18 μm when provided exogenous aggrecan. Yet, full extension of the PCM occurs well below aggrecan saturation. Hence, cells equipped with hyaluronan-rich PCM can in principle manipulate surface accessibility or sequestration of molecules by tuning the bottlebrush proteoglycan content to alter PCM porosity and the number of electrostatic binding sites.

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Section: Gfpn Labeling Of Hyaluronanmentioning

confidence: 99%

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

Chang

McLane

Fogg

et al. 2016

Biophysical Journal

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“…We expect that HMM-RaD will enable a broad range of new studies in a variety of disciplines, with examples including provoking the responses of cells, embryos, organisms, and microbes to morphogens, chemokines, signaling proteins, drugs, or toxins [31]; modifying colloids and surfaces (e.g., for sample sorting, binding assays, or fundamental surface studies) [70]; and provoking the transient response of complex and soft materials [71].…”

Section: Rapid Dialysismentioning

confidence: 99%

Microfluidic Microdialysis: Spatiotemporal Control over Solution Microenvironments Using Integrated Hydrogel Membrane Microwindows

et al. 2013

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We present a powerful and versatile technique that enables exquisite spatial and temporal control over local solution chemistry in microfluidic devices. Using a microscope and a UV lamp, we use projection lithography to photopolymerize thin (10-25 m) hydrogel membrane ''microwindows'' (HMMs) into standard microfluidic devices. These microwindows are permeable to solute and solvent diffusion and to electric fields, yet act as rigid walls from the standpoint of fluid flow. Reservoirs of solution may thus be rapidly imposed, switched, and maintained on one side of a HMM using standard microfluidic techniques, provoking changes in solution conditions on the other side without active mixing, stirring, or diluting. We highlight three paradigmatic experimental capabilities enabled by HMMs: (1) rapid dialysis and swapping of solute and/or solvent, (2) stable and convection-free localized concentration gradients, and (3) local electric permeability. The functional versatility of hydrogel microwindow membranes, coupled with the ease and speed of their fabrication and integration into simple microchannels or multilayer devices, will open a variety of novel applications and studies in a broad range of fields.

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“…Because of the advantages accrued by avoiding direct flow in the measurement region, permeable-membrane devices have been developed for a variety of soft matter, chemical, or biological studies. [4][5][6][7][8][9][10] However, they have not previously been developed for single-molecule manipulation instruments.…”

Section: Introductionmentioning

confidence: 99%

“…The former geometry leads to wide channels stacked vertically and separated by a membrane that can potentially be very thin. 5,7 This has the benefit of a large contact surface and a short diffusion distance between chambers, thereby reducing the equilibration time upon buffer exchange. However, this vertically stacked geometry can cause problems with the high-resolution optical analysis needed for single-molecule measurement: the membrane, which is typically not perfectly transparent, intersects the optical axis, leading to scattering of transmitted light.…”

Section: Introductionmentioning

confidence: 99%

“…4,6 Permeable membranes in this geometry have been fabricated by cross-linking poly(ethylene glycol) diacrylate (PEG-DA) with patterned UV light. [7][8][9][10] This horizontal geometry does not hinder highresolution optical methods because the membranes do not obstruct transmitted light. Also, with this design, one can easily array several separate lanes; particularly, one can surround a central analysis channel with two flow channels.…”

Section: Introductionmentioning

confidence: 99%

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A thin permeable-membrane device for single-molecule manipulation

Park

Jacobson

Nguyen

et al. 2016

Review of Scientific Instruments

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Single-molecule manipulation instruments have unparalleled abilities to interrogate the structure and elasticity of single biomolecules. Key insights are derived by measuring the system response in varying solution conditions; yet, typical solution control strategies require imposing a direct fluid flow on the measured biomolecule that perturbs the high-sensitivity measurement and/or removes interacting molecules by advection. An alternate approach is to fabricate devices that permit solution changes by diffusion of the introduced species through permeable membranes, rather than by direct solution flow through the sensing region. Prior implementations of permeable-membrane devices are relatively thick, disallowing their use in apparatus that require the simultaneous close approach of external instrumentation from two sides, as occurs in single-molecule manipulation devices like the magnetic tweezer. Here, we describe the construction and use of a thin microfluidic device appropriate for single-molecule studies. We create a flow cell of only ∼500 μm total thickness by sandwiching glass coverslips around a thin plastic gasket and then create permeable walls between laterally separated channels in situ through photo-induced cross-linking of poly(ethylene glycol) diacrylate hydrogels. We show that these membranes permit passage of ions and small molecules (thus permitting solution equilibration in the absence of direct flow), but the membranes block the passage of larger biomolecules (thus retaining precious samples). Finally, we demonstrate the suitability of the device for high-resolution magnetic-tweezer experiments by measuring the salt-dependent folding of a single RNA hairpin under force.

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Microfluidic dialysis cell for characterization of macromolecule interactions

Cited by 13 publications

References 39 publications

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

Microfluidic Microdialysis: Spatiotemporal Control over Solution Microenvironments Using Integrated Hydrogel Membrane Microwindows

A thin permeable-membrane device for single-molecule manipulation

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