Nanomed

NANO PATCH-CLAMP (NPC)

DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM BASED ON PLANAR PATCH-CLAMP

"Patch clamping is a marvelous technique. It allows you to have access to currents across a cell in two ways: one is to look at very few channels, very few molecules, or to look at the whole cell with high resolution." Francisco Bezanilla of the University of California.

The Patch Clamp is nawdays the electrophysiological technique of election to investigate ion channels. Ion channels are transmembrane proteins involved in nearly all physiological processes and in many human diseases, still today a membrane protein class in continuous exploration. The scientific interest for these proteins is manifested by base search, to the aim to comprise its structure, function and pharmacology, and by applied search biomedical and farmaceutical, for the screening of new drugs.

Even if traditional patch-clamp method has transformed both neuroscience and biophysics helping usher in a new branch of drug-development research focused on ion channels, these proteins remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of the technique of election. However, traditional patch approache work well in academia, where only researchers perform individual experiments. Industry, where streamlining and throughput are important, as well as functional genomics projects or screenings for neuroscience and pharmaceutical research, require assays with a much higher throughput.
Several companies have released or are planning to release commercial implementations of a new high-throughput variation on the technique, called planar patch clamping. In these automatic devices like the planar population array patch-clamp, PAPC, the micropipettes are replaced by pores in planar supports. Our primary intention was to achieve a high-throughput system based on PAPC. We rely on a Nano Patch-Clamp (NPC) device based on poly(dimethylsiloxane) (PDMS) planar chips because of high processability and low cost of that alternative material used instead of glass, quartz or silicon.

Figure 2. Schematic drawing. Establishing the whole-cell recording configuration with the prototype planar Patch-Clamp chamber. The whole procedure of preparing the whole-cell configuration is summarized into 3 states: in bath, sealed cell and whole-cell

Our microfabrication and processing technology is sufficiently mature to produce a consistent single-channel prototype of NPC system. Planar polymeric chips, obtained combinig soft-lithography and air-molding, and altered to be hydrophilic by rapid oxygen plasma treatment, have been developed. PDMS Chips will be used in a plexiglass bench-top device. Scanning Electron Microscopy (SEM) and precise Focused Ion Beam (FIB) systems are used to determine the characteristics of the PDMS cell-pore interface. Our device has been validated using both suspended and adherent cells. We are able to obtein gigaselas and we are currently in the process of realizing whole-cell configuration by using perforine proteins. The nearly future of application development is the realization of a relatively low-cost PDMS array support to perform simultaneous parallel automated recordings.

Fabrication of Silicon Masters
The FIB system uses a Ga+ ion beam to raster over the surface of a silicon master. The ion beam allows the milling of small holes in the sample at well localized sites _Fig. 3.

Figure 3.SEM images. Creation of different micropore conformations with FIB, after the Si-master is fixed on a pyrex support.

Soft Lithography
Soft lithography is a process which allows a pattern on a master to be embossed on soft substrate: polydimethylsiloxane, PDMS. The stamp on the master defines the shape of the final PDMS structure. The soft lithography is especially powerful because it is inexpensive and quick to make many structures from the same master _ Fig. 4.

Figure 4. Using Soft-Lithography the master is covered with PDMS prepolymer and spinned to have 10 um thick film. After curation the PDMS replica is peeled off from the Si-master coating it to a perfrated PDMS disch.

Air Molding
An experimental set-up for implementing the air molding technique in a highly flexible way has been designed, developed and tested. This apparatus uses a nitrogen stream, channeled through the micro-perforated silicon master, to shape a smooth micrometric pore in the PDMS thin film _ Fig. 5.

Figure 5. A: The PDMS film is perforated by air-molding then cured using an halogen lamp.

Oxygen Plasma
This surface treatment removes organic residues and chemically reacts with the surface to form strong covalent carbon-oxygen bonds, which are much more polar and more reactive than the initial carbon-hydrogen bonds. Before making an experiment the PDMS replica is exposed to oxygen plasma surface treatment, to change its surface from hydrophobic to hydrophilic.

Experimental Patch-Clamp prototype chamber
A PDMS replica is positioned between top and bottom compartments of the chamber _Fig. 6. 10 ml of lymphocytes CEM cell suspension in extracellular working solution (±104 cells) is added to the external well. Experiments start when cells are attached to the PDMS-chip surface, within 5'-10'.

Figure6. Patch-Clamp prototype chamber and PDMS replica fixed on PDMS disch.

Experimental Results
One of the recorded current in oscilloscope establishing an high resistence seal:

Patrizia Guida is a staff researcher, responsible of the Nano-Patch-Clamp project.
The team is directed by Professor Ugo Valbusa of the Physics Department of the University of Genova. Dr. Luca Repetto and Dr. Giuseppe Firpo realized the FIB fabrication and SEM imaging activity. The project directly involves Dr. Alessandro Bosca of the Italian Institute of Technology (IIT) of Genova.




	NANO PATCH-CLAMP (NPC) DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM BASED ON PLANAR PATCH-CLAMP "Patch clamping is a marvelous technique. It allows you to have access to currents across a cell in two ways: one is to look at very few channels, very few molecules, or to look at the whole cell with high resolution." Francisco Bezanilla of the University of California. The Patch Clamp is nawdays the electrophysiological technique of election to investigate ion channels. Ion channels are transmembrane proteins involved in nearly all physiological processes and in many human diseases, still today a membrane protein class in continuous exploration. The scientific interest for these proteins is manifested by base search, to the aim to comprise its structure, function and pharmacology, and by applied search biomedical and farmaceutical, for the screening of new drugs. Figure 1. Schematic view of an eukaryotic cell and its ion channels. Even if traditional patch-clamp method has transformed both neuroscience and biophysics helping usher in a new branch of drug-development research focused on ion channels, these proteins remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of the technique of election. However, traditional patch approache work well in academia, where only researchers perform individual experiments. Industry, where streamlining and throughput are important, as well as functional genomics projects or screenings for neuroscience and pharmaceutical research, require assays with a much higher throughput. Several companies have released or are planning to release commercial implementations of a new high-throughput variation on the technique, called planar patch clamping. In these automatic devices like the planar population array patch-clamp, PAPC, the micropipettes are replaced by pores in planar supports. Our primary intention was to achieve a high-throughput system based on PAPC. We rely on a Nano Patch-Clamp (NPC) device based on poly(dimethylsiloxane) (PDMS) planar chips because of high processability and low cost of that alternative material used instead of glass, quartz or silicon. Figure 2. Schematic drawing. Establishing the whole-cell recording configuration with the prototype planar Patch-Clamp chamber. The whole procedure of preparing the whole-cell configuration is summarized into 3 states: in bath, sealed cell and whole-cell Our microfabrication and processing technology is sufficiently mature to produce a consistent single-channel prototype of NPC system. Planar polymeric chips, obtained combinig soft-lithography and air-molding, and altered to be hydrophilic by rapid oxygen plasma treatment, have been developed. PDMS Chips will be used in a plexiglass bench-top device. Scanning Electron Microscopy (SEM) and precise Focused Ion Beam (FIB) systems are used to determine the characteristics of the PDMS cell-pore interface. Our device has been validated using both suspended and adherent cells. We are able to obtein gigaselas and we are currently in the process of realizing whole-cell configuration by using perforine proteins. The nearly future of application development is the realization of a relatively low-cost PDMS array support to perform simultaneous parallel automated recordings. MAKING THE PDMS PLANAR CHIPS Fabrication of Silicon Masters The FIB system uses a Ga+ ion beam to raster over the surface of a silicon master. The ion beam allows the milling of small holes in the sample at well localized sites _Fig. 3. Figure 3.SEM images. Creation of different micropore conformations with FIB, after the Si-master is fixed on a pyrex support. Soft Lithography Soft lithography is a process which allows a pattern on a master to be embossed on soft substrate: polydimethylsiloxane, PDMS. The stamp on the master defines the shape of the final PDMS structure. The soft lithography is especially powerful because it is inexpensive and quick to make many structures from the same master _ Fig. 4. Figure 4. Using Soft-Lithography the master is covered with PDMS prepolymer and spinned to have 10 um thick film. After curation the PDMS replica is peeled off from the Si-master coating it to a perfrated PDMS disch. Air Molding An experimental set-up for implementing the air molding technique in a highly flexible way has been designed, developed and tested. This apparatus uses a nitrogen stream, channeled through the micro-perforated silicon master, to shape a smooth micrometric pore in the PDMS thin film _ Fig. 5. Figure 5. A: The PDMS film is perforated by air-molding then cured using an halogen lamp. Oxygen Plasma This surface treatment removes organic residues and chemically reacts with the surface to form strong covalent carbon-oxygen bonds, which are much more polar and more reactive than the initial carbon-hydrogen bonds. Before making an experiment the PDMS replica is exposed to oxygen plasma surface treatment, to change its surface from hydrophobic to hydrophilic. Experimental Patch-Clamp prototype chamber A PDMS replica is positioned between top and bottom compartments of the chamber _Fig. 6. 10 ml of lymphocytes CEM cell suspension in extracellular working solution (±104 cells) is added to the external well. Experiments start when cells are attached to the PDMS-chip surface, within 5'-10'. Figure6. Patch-Clamp prototype chamber and PDMS replica fixed on PDMS disch. Experimental Results One of the recorded current in oscilloscope establishing an high resistence seal: "WHO'S WHO" OF THE PROJECT Patrizia Guida is a staff researcher, responsible of the Nano-Patch-Clamp project. The team is directed by Professor Ugo Valbusa of the Physics Department of the University of Genova. Dr. Luca Repetto and Dr. Giuseppe Firpo realized the FIB fabrication and SEM imaging activity. The project directly involves Dr. Alessandro Bosca of the Italian Institute of Technology (IIT) of Genova.

How to reach us Last update 7/15/10