D.S. and C.E.C. were supported, in part, by grant AI053531 (NIAID, NIH); S.S and P.S.C. were supported by grant N00014-14-1-0792 (ONR).

The authors have nothing to disclose.

Each PIP variant, albeit at low concentrations, is present on the cytosolic surface of specific organelles where they contribute to the establishment of a unique physical composition and functional specificity of the organellar membrane1. One of the most important uses of PIPs is as a specific docking platform for the multitude of proteins requiring specific subcellular localization and/or activation6,7. Due to their role in cellular physi...

Myriad interactions and biochemical processes take place on two-dimensionally fluid membrane surfaces. Membrane-enclosed organelles in eukaryotic cells are unique not only in biochemical processes and their associated proteome but also in their lipid composition. One exceptional class of phospholipids is phosphoinositides (PIPs). Even though they comprise only 1% of the cellular lipidome, they play a crucial role in signal transduction, autophagy, and membrane trafficking, among others1,2,3,4. Dynamic phosphorylation of the inositol head group by cellular PIP kinases gives rise to seven PIP headgroups that are mono-, bis-, or tris-phosphorylated5. Additionally, PIPs define the subcellular identity of membranes and serve as specialized membrane docking sites for proteins/enzymes containing one or more phosphoinositide-binding domains, for example, Pleckstrin Homology (PH), Phox Homology (PX), and epsin N-terminal Homology (ENTH)6,7. One of the best-studied PIP-binding domains is phospholipase C (PLC)-&#948;1 PH domain that specifically interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within a high nanomolar-low micromolar range affinity8,9,10,11.
A variety of qualitative and quantitative in vitro methods have been developed and used to study the mechanism, thermodynamics, and specificity of these interactions. Among the most commonly used PIP-binding assays are surface plasmon resonance (SPR), isothermal calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, liposome flotation/sedimentation assay, and lipid-blots (Fat-blots/PIP-strips)12,13. Even though these are extensively utilized, they all have many disadvantages. For example, SPR, ITC, and NMR require large amounts of sample, expensive instrumentation, and/or trained personnel12,13. Some assay formats such as antibody-based lipid-blots utilize water soluble forms of PIPs and present them in a nonphysiological manner12,14,15,16. In addition, lipid-blots cannot be quantitated reliably and they have often resulted in false positive/negative observations12,17,18. To overcome these challenges and improve upon the current tool set, a new label-free method was established based on a supported lipid bilayer (SLB) in the context of a microfluidic platform, which was successfully applied to the study of protein-PIP interactions (Figure 1)19.
The strategy employed for detecting protein-PIP interactions is based on pH modulation sensing. This involves a pH-sensitive dye that has ortho-Sulforhodamine B (oSRB) directly conjugated to phosphatidylethanolamine lipid head group20. The oSRB-POPE probe (Figure 2A) is highly fluorescent at low pH and quenched at high pH with a pKa around 6.7 within 7.5 mol% PI(4,5)P2-containing SLBs (Figure 5B). PLC-&#948;1 PH domain has been used extensively for validating protein-PIP-binding methodologies due to its high specificity towards PI(4,5)P2 (Figure 5A)21,22,23,24,25.Hence, we reasoned that the PLC-&#948;1 PH domain can be used to test its binding to PI(4,5)P2 through the PIP-on-a-chip assay. The PH domain construct used in this study has a net positive charge (pI 8.4), and thus attracts OH- ions (Figure 5C). Upon binding to PI(4,5)P2-containing SLBs, the PH domain brings the OH- ions to the membrane surface, which in turn modulates the interfacial potential and shifts the protonation state of oSRB-POPE (Figure 5C)26. As a function of the PH domain concentration, the fluorescence is quenched (Figure 6A). Finally, the normalized data is fit to a binding isotherm to determine the affinity of the PH domain-PI(4,5)P2 interaction (Figure 6B, 6C).
In this study, a detailed protocol is provided to perform protein binding to PIP-containing SLBs within a microfluidic platform. This protocol takes the reader from assembling the microfluidic device and vesicle preparation to SLB formation and protein binding. In addition, directions for data analysis to extract affinity information for the PLC-&#948;1 PH domain-PI(4,5)P2 interaction are provided.

<table border="0" cellpadding="0" cellspacing="0" width="1638">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Coverslip</td>
			<td style="width:156px;"></td>
			<td style="width:123px;"></td>
			<td style="width:997px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glass Coverslips: Rectangles</td>
			<td>Fisher Scientific</td>
			<td>12-544B</td>
			<td>22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass</td>
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		<tr height="20">
			<td height="20" style="height:20px;">7X Cleaning Solution</td>
			<td>MP Biomedicals</td>
			<td>976670</td>
			<td>Detergent</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PYREX Crystallizing Dish</td>
			<td>Corning</td>
			<td>3140-190</td>
			<td>Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sentry Xpress 2.0</td>
			<td>Paragon Industries</td>
			<td>SC-2</td>
			<td>Kiln</td>
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			<td height="20" style="height:20px;">PDMS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning&#160;</td>
			<td>4019862</td>
			<td>Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PYREX Desiccator</td>
			<td>VWR</td>
			<td>89134-402</td>
			<td>Vacuum Rated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biopsy punch</td>
			<td>Harris</td>
			<td>15110-10</td>
			<td style="width:997px;">Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative</td>
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			<td height="21" style="height:21px;">Name</td>
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			<td height="20" style="height:20px;">Device</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma Cleaning System</td>
			<td>PlasmaEtch</td>
			<td>PE25-JW</td>
			<td>2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hot Plate</td>
			<td>Benchmark</td>
			<td>H3760-H</td>
			<td>Purchased through Denville Scientific (Cat. No. 1005640)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Frosted Micro Slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144</td>
		</tr>
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			<td height="21" style="height:21px;">Name</td>
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			<td height="20" style="height:20px;">Mold</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">AutoCAD</td>
			<td>Autodesk</td>
			<td>v.2016</td>
			<td>Drafting software for the photomask design</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomask</td>
			<td>CAD/Art Services</td>
			<td>N/A</td>
			<td>Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Wafers</td>
			<td>University Wafer</td>
			<td>1575</td>
			<td>Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU-8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SUV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850457C</td>
			<td>POPC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840045X</td>
			<td>PI4P</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td>PI(4,5)P2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850757C</td>
			<td>POPE; Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)</td>
			<td>ThermoFisher Scientific</td>
			<td>L-20</td>
			<td>Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>In-house Synthesis (Huang D. et al. 2013)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Scintillation Vial</td>
			<td>VWR</td>
			<td>66022-065</td>
			<td>20 mL volume capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aquasonic 250D</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>Ultrasonic Water Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclepore Track-Etched Membranes</td>
			<td>Whatman</td>
			<td>110605</td>
			<td>Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>VWR</td>
			<td>CX1054-6</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIPEX Extruder</td>
			<td>Transferra Nanosciences</td>
			<td>T.001</td>
			<td>LIPEX 10 mL Thermobarrel Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viscotek 802 DLS</td>
			<td>Malvern Instruments</td>
			<td>N/A</td>
			<td>Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>v.6</td>
			<td>Curve-fitting software for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axiovert 200M Epifluorescence Microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioCam MRm Camera</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Camera</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">X-Cite 120</td>
			<td>Excelitas Technologies</td>
			<td>N/A</td>
			<td>Light Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 568 Filter Set</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Ex/Em 576/603 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioVision LE64 v.4.9.1.0 Software</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Image Processing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>10034-132</td>
			<td>200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>53509-070</td>
			<td>10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion Star A321 pH meter</td>
			<td>Thermo Scientific</td>
			<td>STARA3210</td>
			<td>pH meter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion micro pH probe</td>
			<td>Thermo Scientific</td>
			<td>8220BNWP</td>
			<td>micro pH probe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-(2-Hydroxyethyl)-Piperazine-N&#39;-(2-Ethanesulfonic Acid)</td>
			<td>VWR</td>
			<td>VWRB30487</td>
			<td>HEPES, Free Acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>VWR</td>
			<td>BDH8014-2.5KGR</td>
			<td>NaCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Allied Wire &#38; Cable</td>
			<td>TFT-200-24 N</td>
			<td>Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrogen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxygen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
	</tbody>
</table>

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

We used the pH modulation assay to study the PLC-&#948;1 PH domain-PI(4,5)P2 interaction within a PIP-on-a-chip microdevice (Figure 1). Through a detailed protocol, we demonstrated how to prepare and assemble microfluidic device components, make small unilamellar vesicles (SUVs) (Figure 2), form SLBs within a device (Figure 3), and tested protein binding to PIP-containing SLBs. A flowchart...

Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

pip-on-chip-label-free-study-protein-phosphoinositide

Research

JoVE Journal

Bioengineering

9.4K Views.  The Pennsylvania State University. Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Video: PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...