Name: membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution
Uploaded: 2016-08-16T13:00:00
Description: Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

We thank Barbara Windschiegl for her help in establishing SOPs; Dennis Remme for his work on the NanoCalcFX software and Alina Kollmannsperger, Markus Braner and Milan Gerovac for helpful suggestions on the manuscript. The German-Israeli Project Cooperation (DIP) provided by the DFG and the Federal Ministry of Education and Research to R.T., as well as the Federal Ministry of Economics and Technology (ZIM R&amp;D Project) to R.T. and Nanospot GmbH supported this work.

1. Preparation of Large Unilamellar Vesicles (LUVs)
<ol>
	<li>Clean a round bottom flask (10 ml volume) with nitrogen gas to remove any dust particles. Rinse the round bottom flask with ethanol. 
	Note: Residual ethanol can be tolerated and does not disturb subsequent steps.</li>
	<li>Wash a 1 ml glass syringe 4x with chloroform, to remove any contamination. Add 4 ml SoyPC20 (25 mg/ml in chloroform) to the round bottom flask and dope the lipid solution with an additional DOPEATTO390 to a fina...

<ol>
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The technique presented here allows a highly parallel analysis of membrane protein transport. Reconstituted membrane protein systems can directly be applied to the biochip, making the adaption of theoretically every membrane transporter or channel possible. Transport analysis is only limited by the establishment of a fluorescent read-out system, either via direct fluorescence change (translocation of fluorophores or fluorescently labeled substrates) or indirect fluorescence change (pH-sensitive dyes, secondary enzymatic ...

The analysis of membrane proteins has become of increasing interest for basic and pharmaceutical research in the past 20 years. The development of novel drugs depends on the identification and detailed characterization of new targets, currently being one of the limiting factors. The fact that about 60% of all drug targets are membrane proteins1, makes the development of techniques to elucidate their function most important.
In the past, techniques for the study of electrogenic channels and transporters have been developed in multitude2-4. Non-electrogenic substrates in contrary present a more challenging task. They are however of special interest as prime drug targets, as they control the flux of solutes and nutrients across the cell membrane and function as key receptors in signaling cascades5.
Considerable effort has been put into the development of techniques to study the function of membrane transport proteins6,7. Systems using solid-supported membranes have emerged as most promising tools in this field8-10, including solid supported lipid bilayers, tethered bilayers11,12, microblack lipid membranes13,14 and native vesicle arrays15,16 to name a few. Some of them are even available as commercial setups17,18. Some examples have been published combining the ability to study single membrane proteins in a highly parallel manner14,19, a prerequisite for screening applications. However, these methods rarely bridge from basic research to the industrial environment. The difficulties often lie in the ability of the system to be automatable, the cost-intensive production and/or laborious preparation. An approach overcoming all the above mentioned obstacles is the final aim.
The technique presented here was developed to study membrane channels and transporters in vitro in a controlled environment on the single protein level20-22. Reconstitution of purified membrane proteins into LUVs is much more established than comparable approaches for GUVs23-26 or black lipid membranes27. They can directly be applied to the chip surface, where bilayer formation is taking place via a self-assembly process. The glass-bottom design of the nanoporous chip (Fig. 1) allows for air microscopy, which permits the straightforward automation of the system. In combination with a motorized stage multiple chips can be measured at the same time, with each field of view containing thousands of sealed cavities for analysis.
<img alt="Figure 1" src="/files/ftp_upload/53373/53373fig1.jpg" /> 
Figure 1. Design of multiplexed nanopore biochips. A) A silicon-on-insulator (SOI) wafer is structured by reactive-ion etching. Approximately 1,150 individual chips are fabricated from each wafer with identical properties and quality. B) Each chip comprises 250,000 individual microcavities with nano apertures. Scale bar: 200 &#181;m. C) Each cavity is addressable via multi-spectral fluorescence read-out. An intransparent top layer blocks the fluorescent signals from the buffer reservoir, making the biochip compatible with inverted fluorescence microscopes. D) Atomic force microscopy (AFM) imaging reveals evenly arranged pore openings and surface roughness of the silicon dioxide layer of 3.6 nm (n = 40) optimal for vesicle fusion. Scale bar: 5 &#181;m. E) Scanning electron microscopy (SEM) image shows a cross-section through the nanopore allowing access to the femtoliter cavities inside the silicon chip. This figure was reused with permission from 21. <a href="https://www.jove.com/files/ftp_upload/53373/53373fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
All data analysis is performed using freeware to guarantee unrestricted access for end users. Time series are analyzed using free image processing software and a custom build curve analysis software enabling batch processing and straightforward correlation of large datasets with multiple fluorescent channels and thousands of curves.
The model protein used in this protocol is the mechanosensitive channel of large conductance (MscL) channel protein derived from E. coli. It functions as a valve to release osmotic shock in nature, but was modified in such a way that rationally designed synthetic functionalities can covalently be attached to the channels constriction side. Via charge-repulsion of the covalently bound activator (MTSET) the channel is triggered to open, creating a nano-valve. Small molecules like ions, water, small proteins, but also small fluorophores can permeate through the channel. Here, the protein is used as a model to demonstrate the ability of the system to detect protein-mediated translocation.

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			<td height="21" style="height:21px;width:389px;">Reagent</td>
			<td style="width:232px;"></td>
			<td style="width:305px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">[2-(Trimethylammonium)ethyl] 
			methanethiosulfonate</td>
			<td style="width:232px;">Toronto Research Chemicals Inc.</td>
			<td style="width:305px;">T792900</td>
			<td>MTSET; hydrolized by water. Keep as dry pouder aliquot at -80 &#176;C. Use immediately (30 minutes) after solubilization in buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">1 ml gas-tight syringe</td>
			<td style="width:232px;">Hamilton</td>
			<td style="width:305px;">#1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">10 ml round flask</td>
			<td style="width:232px;">Schott Duran</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2.7 mm glas beads</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">N032.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">2-Propanole</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">9866.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">30 cm Luer-Lock Extension Tube</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Acetone</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">5025.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Bio-Beads SM-2 Adsorbent</td>
			<td style="width:232px;">Bio-Rad</td>
			<td style="width:305px;">152-3920</td>
			<td>need to be activated before first use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:389px;">CaCl2 Dihydrat</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">HN04.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Calcein</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:305px;">C0875</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Chloroform reagent grade</td>
			<td style="width:232px;">VWR Chemicals</td>
			<td style="width:305px;">22711324</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:389px;">DOPEATTO390</td>
			<td style="width:232px;">ATTO-TEC</td>
			<td style="width:305px;">AD 390-165</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Ethanol absolute</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">32205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt Single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">460 60 51V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Injekt-F single-use syringe</td>
			<td style="width:232px;">Braun</td>
			<td style="width:305px;">91 66 017V</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:389px;">Keck clips</td>
			<td style="width:232px;">Schott</td>
			<td style="width:305px;">KC29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">L-&#945;-Phosphatidylcholine, 20% (Soy)</td>
			<td style="width:232px;">Avanti Polar Lipids</td>
			<td style="width:305px;">5416016</td>
			<td>store under inert gas at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">NaCl 99.5% p.a.</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">3957.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nanopore E100 wafer/chips</td>
			<td style="width:232px;">Micromotive (Mainz/Germany)</td>
			<td style="width:305px;"></td>
			<td>available on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Nucleopore Track-Etch Membrane 0.4 &#181;m</td>
			<td style="width:232px;">Whatman</td>
			<td style="width:305px;">800282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oregon Green Dextran 488 (70 kDa)</td>
			<td style="width:232px;">life Technologies</td>
			<td style="width:305px;">D-7173</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Oy647</td>
			<td style="width:232px;">Luminartis (M&#252;nster/Germany)</td>
			<td style="width:305px;">OY-647-T-1mg</td>
			<td>store dark at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Rotilabo-syringe filtration, unsterile, pore-size 0.22 &#181;m</td>
			<td style="width:232px;">Roth</td>
			<td style="width:305px;">P 818.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Sephadex G-50</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:305px;">G5080</td>
			<td>column material for size exclusion chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Silastic MDX4-4210</td>
			<td style="width:232px;">Dow Corning</td>
			<td style="width:305px;"></td>
			<td>curing agent for chip fixation onto cover glass support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">sticky-Slide 8-well</td>
			<td style="width:232px;">ibidi</td>
			<td style="width:305px;">80828</td>
			<td>multi-well chamber for the mounting onto glass slides (chip holder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:389px;">Three-way stopcock blue</td>
			<td style="width:232px;">Sarstedt</td>
			<td style="width:305px;">744410001</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td height="21" style="height:21px;width:389px;">Name</td>
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			<td style="width:305px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;width:389px;">Software</td>
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membrane transport processes analyzed by a highly parallel nanopore chip system at single protein resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

Pore-spanning membranes can easily be created on the nanostructured chip surface in a self-assembled manner. However the underlying process is still delicate and influenced by many parameters like liposome size, monodispersity of the liposome population, lamellarity, lipid- and salt-concentration and chemical surface properties. Most of these parameters have been carefully characterized and standardized in the above protocol. Other parameters however should be checked during every new pre...

Watch this Scientific Journal Video about Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution at JoVE.com

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

The overall goal of this nano-structure chip-based platform is to analyze membrane protein function on the single molecular level in a highly parallel manner. Through its ability to resolve single proteins, new insights into membrane transport and flux are achieved. This method can help answer key questions in the membrane protein field, such as the conductivity or transport behavior of single channels or transporters, specially such with non-electrogenic cargo.The main advantage of this technique is its high parallelism derived from nano-structure chip design, featuring hundreds of thousand of nanopores and the fluorescent read out of transport events. The implications of this technique extend to what's creating best assays of effective libraries for membrane proteins. Because the chip-based platform enables highly parallel and automatic transport recordance.Generally, individuals new to this method will quickly learn how to handle the presented technique. The focus was always to create a user-friendly assay so that everyone interested can adopt it. This membrane transport assay utilizes a silicon on insulated wafer, engineered using reaction ion etching.About 1, 000 assay chips are fabricated from each wafer. Each chip contains 250, 000 microcavities. These cavities get sealed by lipid bilayers containing the transport proteins of interest.The membrane constitutes the sensor interface. Initially, fluorescent materials are contained to one side of the lipid bilayer, corresponding to the interior of the liposomes from which they are composed. Each cavity is addressable via a multispectral fluorescence readout.An opaque top layer blocks the fluorescent signals from the buffer reservoir, making the bio-chip compatible with inverted fluorescence microscopes. Using atomic force microscopy, the evenly arranged pore openings and surface roughness of the silicon-dioxide layer was measured at 3.6 nanometers, optimal for vesicle fusion. Scanning Electron Microscopy shows a cross-section through a nanopore, which accesses the femtoliter cavities inside the silicon chip.This video shows the preparation of the proteoliposomes, the preparation of the chips for the assay, and the basic assay execution. Connect an ethanol-washed round-bottom flask to a rotary evaporator. Dry the lipid film for 40 minutes at 300 millibar, 30 degrees Celsius water bath temperature, and maximal rotation speed to form a homogeneous lipid film on the glass wall of the flask.Then, using a high-vacuum pump, dry the lipid film for an additional 30 minutes at room temperature. Next, add five milliliters of buffer and eight ethanol-washed glass beads to the flask to make the multilamellar vesicles. Connect the flask to the rotary evaporator and rotate without vacuum at 50 degrees Celsius at maximum speed.After 45 minutes, the solution will appear milky, with no residual lipid film on the walls. Transfer the flask under an inert gas to an ultrasonic bath, and sonicate it for four minutes at room temperature. Sonication disrupts the liposomes making them unilamellar.Now, split the LUV solution into one milliliter aliquots, and perform five freeze thaw cycles using liquid nitrogen and a 40 degrees Celsius water bath. This leads to rupturing, rearrangement, and growth of the liposomes. At the last freezing step, store all samples at minus 80 degrees Celsius.Before reconstituting an LUV aliquot, extrude the liposomes by passing the aliquot through a 400 nanometer polycarbonate filter membrane 17 times, using a mini-extruder. Then, destabilize the LUVs by adding 4%Triton X-100 for 0.25%in solution. Incubate the mixture for five minutes at 50 degrees Celsius.Now, mix one part purified, modified MscL into 20 parts of the detergent-saturated liposomes, and continue the incubation for 30 minutes. After the incubation, take a 100 microliter aliquot, and add one volume of 30 millimolar calcine. Incubate this mixture for five minutes at 50 degrees Celsius.Now, remove the detergent from the calcine-treated and untreated sample. Add wet bio-beads in Tris buffer to each, and incubate them overnight at four degrees Celsius on a rotating plate. Before the experiment, test a preparation using an activity assay.To begin, mix elastomer base into the curing agent, and fully desiccate the mixture so it is aghast. The adhesive must be used within 30 minutes when ready. Meanwhile, taking precautions, clean a cover glass slide using five milliliters of chloroform, applied from a glass syringe, and let the slide dry.Now, using a 10 microliter pipette, deposit the prepared adhesive onto the cover glass. Then, carefully remove a chip from the wafer board using fine tweezers. With the coated side of the chip up, deposit it onto the adhesive, and gently rub it into the slide to evenly distribute the adhesive.Do not let air bubbles get trapped below, or any adhesive get above onto the chip. Also, make the assembly so it fits into the eight-chamber slide holder. Now desiccate the assemblies for two hours at 65 degrees Celsius in a cabinet dryer.Later, remove the protective layer on the chips with a few solvent washes at 54 degrees Celsius. First place the mounted chips into a slide holder. Then, incubate them in three solvent baths for 10 minutes each.Isopropanol, acetone, and isopropanol. Next, carefully and thoroughly wash the chips with ultra pure water. Complete the process by drying the chips under a gentle stream of nitrogen gas.Now, prepare an eight-well sticky slide and put it backwards on the bench. Then, carefully lift and press the cover slides onto the sticky slide with the chips facing the wells. Let the assembly rest for a few minutes before proceeding.Before proceeding with the translocation assay, wash the chip assembly thoroughly, as described in the text protocol, finishing with a Tris buffer bath. Next, exchange the bath solution with DOPEd Tris buffer, containing five millimolar calcium chloride, and one micromolar Oy647 dye. Next, add proteoliposomes to the chip chamber for a final concentration of one milligram per milliliter, and wait one hour.After an hour, rinse the chips four times with 400 microliters of calcium-free Tris buffer. Now, add the controlled dye to each well to five micromolar, and proceed with the assay. Next, mount the chip holder to the epifluorescence microscope, and focus on the rim of the nanopore chip to find the microcavities.Once the microcavities are in focus, scan the nanopore array for a region that displays the highest ceiling ratio. Details are provided in the text protocol. Liposomes were prepared and checked for their size distribution and dispersion.The prepared liposomes were larger than the pore diameter for the assay, which is essential. Monodispersed liposomes are also desirable. Before the experiment, the modified MscL protein in the liposome's preparation was checked using a fluorescence dequenching assay.The ratio of liposomes harboring active modified MscL was adequate to proceed with the translocation experiment. On the biochip, solute translocation of a fluorescent target via the modified MscL channel was observed. The chemical modulator, MTSET, was used to open the engineered MscL channels via electrostatic repulsion.The subsequent decrease of fluorescence inside the cavity space was monitored. This process proved to be highly reproducible, with very consistent efflux events. The resulting efflux curves clustered in two distinct populations, corresponding to single channel, or multi channel, translocation events.The system was able to follow up to three uniquely fluorescing dyes with real-time analysis. This facilitates precise discrimination of positive results, mono-exponential efflux events, complex kinetics, lipid intrusions, and membrane ruptures. Once mastered, this technique can be executed in consecutive steps, namely chip preparation, sample preparation, imaging, and analysis, making the entire protocol modular.

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