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

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.